Abstract

フォルニカータ生物群の基部から分岐する新奇嫌気性生物PAP020株のミトコンドリア様オルガネラの代謝機能の推測／Putative function of the mitochondrion-related organelle in a novel eukaryote, stain PAP020, branched at the base of the Fornicata clade.

パラオ共和国のマングローブ林底泥サンプルから単細胞真核生物PAP020株が単離され、嫌気環境下でバクテリアを餌として培養、維持されている。PAP020株は2本の鞭毛をもつ楕円状の細胞である。PAP020株は、形態観察および小サブユニットリボソーマルDNA（SSU rDNA）を用いた系統解析では系統的位置を推定するには至らなかった。そこで大規模分子系統解析によってPAP020株の真核生物中での系統的位置を検証した。PAP020株の網羅的mRNA塩基配列を基盤に、PAP020株を含む83タクサ、148遺伝子のアミノ酸データセットを作成し、最尤法による系統解析を行った。その結果、PAP020株はGiardia含む寄生および嫌気性生物で構成されるフォルニカータ生物群の基部からBP100%で分岐することが示され、PAP020株の系統的位置を確定することができた。また、PAP020株は予備的な電子顕微鏡観察で細胞内に縮退したMitochondrial-Related Organelles（MRO）を持つことが示された。そこで、PAP020株の網羅的mRNAデータ中にミトコンドリア／MROに関連する可能性のある遺伝子を探索し、このMROの代謝機能を推測した。PAP020株で推測されたMRO機能を、これまでに最も知見が蓄積しているフォルニカータ生物Giardia intestinalisおよびパラバサリア類Trichomonas vaginalisのMROの機能と比較した。その結果、PAP020株のMROは、代謝的にGiardia MROよりもむしろTrichomonas MROに近いことが示された。これらの結果から、PAP020を取り巻く生物群でのMRO縮退進化を議論したい。

非光合成性珪藻類葉緑体における機能

珪藻類Nitzschia属には光合成能を喪失させ，従属栄養性へと進化した種が知られている。これまでにNitzschia属内において光合成能の喪失は比較的最近に複数回独立して起こり，それらの細胞内には未だ葉緑体および葉緑体ゲノムが残っていることが分かっている。しかし非光合成性Nitzschiaが何故未だに葉緑体を保持しているのかは不明であった。本研究では無色葉緑体内において機能している代謝経路を推定し，無色葉緑体の役割から珪藻類が光合成能喪失後も葉緑体を保持し続ける理由について考察する。Nitzschia sp. NIES3581株を用いた葉緑体ゲノムおよびRNA解析は，ルビスコを欠くカルビン回路，シキム酸経路，脂肪酸合成などが非光合成性珪藻類で働いていることを強く示唆した。これらの機能に関与するタンパク質配列は，珪藻類葉緑体タンパク質に特徴的なN末伸長配列を有していた。これらのN末端配列は光合成性珪藻類への遺伝子導入により葉緑体局在シグナルとして機能することが示されたため，上述した代謝経路は葉緑体に局在することが強く示唆された。種々の炭素代謝関連機能こそが，NIES3581株が光合成能喪失後も葉緑体を有している理由であると考えられる。

Rhopalodia科珪藻細胞内共生体の比較ゲノム解析．

Rhopalodia科珪藻は葉緑体やミトコンドリアに加え，spheroid bodyと呼ばれるシアノバクテリア由来の細胞内共生体を持つ。spheroid bodyは窒素固定を行い、その産物を宿主珪藻に供給する役割を担うと考えられており、宿主の世代を超えて受け継がれる。これまでの研究において，Rhopalodia科珪藻の一種Epithemia turgidaのspheroid bodyゲノムが解読され，窒素固定に関連する遺伝子は保持されている一方で，光合成関連遺伝子はほぼ全て失われていることが明らかとなった。このことからspheroid bodyは細胞内共生を通じて窒素固定に特化したゲノムへと縮退したと考えられる。本研究では，Rhopalodia科珪藻の多様性の中でspheroid bodyゲノムの縮退がどのように起きたかを検証するため，新たにEpithemia adnata，Rhopalodia gibberulaにおけるspheroid bodyの全ゲノムの解読を行ない，E. turgidaのspheroid bodyゲノムも含めた比較解析を行なった。Epithemia spp.およびR. gibberulaの全てのspheroid bodyゲノムにおいて，光化学系複合体タンパク質遺伝子がほとんど確認できなかったことから，光合成能の欠失はRhopalodia属，Epithemia属の分岐以前に起こったことが示唆された。一方、R. gibberula spheroid bodyゲノムにはEpithemia spp.のspheroid bodyからは既に失われたビタミンB₁₂生合成経路やクロロフィル生合成経路の酵素遺伝子が複数同定され，Epithemia spp.のspheroid bodyゲノムは，より縮退的であることが示された。さらに，Epithemia spp. spheroid bodyゲノムの遺伝子はR. gibberula spheroid bodyに対して比較的高い非同義置換率をもっていた。これらの結果から，spheroid bodyゲノムはRhopalodia科珪藻内でよく保存されているが、宿主珪藻の系統間において縮退の程度、遺伝子進化速度に違いが見られることが示された。

葉緑体を置換した渦鞭毛藻の葉緑体関連代謝系におけるEGTの傾向．

典型的な光合成性渦鞭毛藻の葉緑体は紅藻の二次共生に起源をもつ。ところが一部の渦鞭毛藻系統では、紅藻以外の真核藻類に由来する葉緑体を保持している。こうした系統では、真核藻を独立に葉緑体化して紅藻由来の二次葉緑体を置換したと解釈されている。葉緑体の置換が起きた渦鞭毛藻Lepidodinium chlorophorum（葉緑体は緑藻に由来）およびKarlodinium veneficum（葉緑体はハプト藻に由来）の宿主核ゲノムには、宿主渦鞭毛藻が元々もつ葉緑体関連遺伝子に加えて共生真核藻から水平転移（Endosymbiotic Gene Transfer, EGT）した葉緑体関連遺伝子が存在している。EGTは葉緑体置換過程の一部だと考えられているが、実際に両種がもつ葉緑体の主要な機能がどの程度EGT遺伝子に依存しているのか、全体像は明らかになっていない。

そこで本研究では全発現遺伝子データを基に両種のクロロフィルa(Chl a)合成系、イソプレノイド合成系（MEP/DOXP経路）およびヘム合成系を再構築し、各代謝系で発現している遺伝子の起源を系統解析により推測した。再現されたK. veneficumのChl a合成系は共生ハプト藻に由来するEGT遺伝子のみから構成され宿主由来遺伝子は検出されなかったことから、EGTがこの代謝系に及ぼした影響が大きいことが示唆された。同様の傾向はL. chlorophorumのChl a合成系においても見られたが、本種では緑藻由来のEGT遺伝子に加えて起源不明な遺伝子を多数検出した。Chl a合成系とは対照的に、両種ともイソプレノイド合成系およびヘム合成系においては宿主由来遺伝子の発現が保存されている傾向にあった。本発表では、葉緑体関連代謝系間でEGTの傾向が異なる生物学的原因について考察する。

Characterization of strain SRT308; a new heterotrophic flagellate basal to Euglenozoa.

We isolated a new heterotrophic flagellate, strain SRT308 from marine sediment sample collected in Republic of Palau on October 2013. The flagellate is round or oval shape with two long subequal flagella and shows unique rotating motion by beating the both flagella synchronously. Since the morphological combination of the flagellate is unique, the flagellate is apparently a novel lineage of eukaryotes. In molecular phylogenetic analysis using small subunit ribosomal RNA gene sequences, the flagellate shows no strong affinity with major eukaryotic lineages. Large scale phylogenetic analysis using 153 protein-coding genes placed the flagellate at the base of Euglenozoa with strong statistical support, suggesting that the flagellate is a previously undescribed member of the Discoba clade. Consistent with the position inferred from the phylogenomic analysis, the flagellate was found to share morphological characteristics, namely discoid mitochondrial cristae and parallel basal bodies, with euglenozoans. Furthermore, the flagellate has a euglenozoan-like tripartite flagellar root system, albeit the ventral root splits into two bands, which is similar to the R2 of other typical excavates. On the other hand, the flagellate lacks some englenozoan features, such as pellicle, paraxial rod, non-tubular mastigonemes, or feeding apparatus. Based on these morphological and ultrastructural features, the early character evolution of Englenozoa, as well as that of Discoba as a whole, will be discussed.

Trends in endosymbiotic gene transfer on plastid metabolic pathways in dinoflagellates with non-canonical plastids.

The major photosynthetic dinoflagellates possess red algal-derived plastids, but some minor lineages established non-canonical plastids derived from phylogenetically diverse eukaryotic algae. Dinoflagellates Karlodinium veneficum and Lepidodinium chlorophorum, which bear non-canonical plastids derived from haptophyte- and green algal endosymbionts, respectively, and their nuclear genomes contain genes encoding plastidal proteins that are likely transferred from the genomes of the endosymbiont algae (endosymbiotic gene transfer or EGT). Although EGT is generally considered to be an essential step in transforming an endosymbiotic alga into a plastid, it has yet to be fully understood to what extent metabolic functions in the Karlodinium and Lepidodinium plastids rely on endosymbiotically acquired proteins. We here surveyed nucleus-encoded plastidal proteins in Karlodinium and Lepidodinium, and investigated the origins of the proteins involved in two metabolic pathways localized in the plastid. Chlorophyll a biosynthetic pathway in the two species appeared to be reorganized in different ways. The pathway in Karlodinium was found to be occupied by proteins acquired from the haptophyte endosymbiont, while ‘laterally derived’ proteins, which were acquired from diverse eukaryotes rather than the green algal endosymbiont, comprise the pathway in Lepidodinium. In contrast, neither endosymbioticall or laterally acquired protein was detected in isoprene biosynthetic pathway in Karlodinium or Lepidodinium, suggesting that switch from the canonical to non-canonical plastids triggered no reorganization of this particular pathway in the two species. In this talk, we discuss biological reasons for the marked difference in the impact of gene transfer between the two metabolic pathways in Karlodinium and Lepidodinium.

The draft genome of Kipferlia bialata reveals that the gain of function contributes the massive reductive evolution in Metamonada.

Metamonada is a unicellular eukaryotic group known to consist of free-living and parasitic organisms. Almost all of metamonads have adapted to anaerobic or micro aerobic environments, and lost the several mitochondrial functions such as the oxidative phosphorylation. The biological pathways localized in those reduced mitochondria (so-called mitochondrion-related organelles or MROs) vary depending on the species. The nuclear genome of the model parasites such as Giardia intestinalis was also reduced in terms of the genome structure and the number of coding proteins, presumably throughout the adaptive evolution to the intra-cellular lifestyle. However, little is known how the genome reduction progressed in this parasite. Here, we present the draft genome sequence of the free-living Kipferlia bialata, which is a phylogenetic sister of G. intestinalis, and compare it to the genomes of the model metamonad parasites, G. intestinalis and Trichomonas vaginalis. Our data show that 1) K. bialata possesses two substrate-level phosphorylation pathways -- one is homologous to that in G. intestinalis and the other is to that in T. vaginalis, suggesting the once expansion of ATP synthesis pathways in the metamonad evolution to reach parasitic life style of G. intestinalis, and 2) no variant-specific surface protein (VSP), possibly an evasion mechanism of the host immunity in G. intestinalis, was detected from K. bialata genome, suggesting that the VSPs were acquired somehow on the line leading to G. intestinalis after the divergence of K. bialata. In sum, our results suggested that the gain of function/protein conversely contributed to the massive reductive evolution in metamonads.

Cyanobacterial genes in the nuclear genome of a diatom bearing N₂-fixing cyanobacterial endosymbionts: Potential factors involved in the host-endosymbiont partnership.

The evolution of mitochondria and plastids from bacterial endosymbionts were key events in the evolution of eukaryotes. While the ancient nature of these organelles preclude understanding the transition from a bacterium to an organelle (organellogenesis), the study of eukaryotic cells with recently evolved obligate endosymbiotic bacteria has the potential to provide important insights into the early events in the organellogenesis. Diatoms belonging to the family Rhopalodiaceae and their N₂-fixing cyanobacterial endosymbionts (spheroid bodies) are emerging as a useful model system in this regard. The experimental data accumulated to date suggest that the endosymbiont has been already integrated into the host cell during the endosymbiotic relationship. Our previous study on the genome sequence of the endosymbiont in a rhopalodeacean diatom provided insight into its reductive evolution and the metabolic dependency on the diatom host. However, it has yet to be elucidated how the host control the endosymbionts. In this study, to tackle this question, we obtained both genome and transcriptomic data of a rhopalodiacean diatom, Epithemia adnata, as well as the genome data of its cyanobacterial endosymbiont. Phylogenetic analyses showed that the nuclear genome encodes protein-coding genes of cyanobacterial origin, which are not seen in other diatom genomes. Some of these ‘cyanobacterial genes’ likely encode enzymes involved in the metabolism of peptidoglycan wall, which is a feature exclusively associated with the endosymbiont in the E. adnata cell. We will overview the cyanobacterial genes found in the diatom genome, and discuss their possible contributions to the host-endosymbiont partnership.

窒素固定はじめました － Rhopalodia科珪藻に見る細胞内共生進化.

炭素固定能（光合成能）および窒素固定能は，生態系に炭素・窒素を取り込む上で重要な代謝反応であるが，もともとはいずれも原核生物に限られた能力である．真核生物の祖先はこれらの能力を持たなかったと考えられるが，細胞内共生を通じた葉緑体の獲得によって，真核生物の系統にも炭素固定能がもたらされた．一方，細菌との共生を通じて間接的に窒素固定を行なう例は知られるものの，窒素固定能を細胞機能として定着させた真核生物は報告されていなかった．

Rhopalodia科に属する珪藻は，葉緑体とは別に窒素固定性のシアノバクテリアに由来する構造を細胞内に持ち，窒素固定を行なう．この構造はカルチャー内の全ての細胞に見られ，宿主が分裂する際には2つの娘細胞に受け継がれる．我々はRhopalodia科珪藻が，窒素固定シアノバクテリアとの細胞内共生の末，細胞の一部として定着させた “窒素固定を行なう真核生物”であると考え，その細胞内共生進化の解明を試みている．本発表ではバクテリアを取り込むことによって新たに “窒素固定をはじめた”本生物のユニークな進化について，最新のゲノム研究結果を交えて紹介する．

真核生物進化の空白を埋める！分子系統解析が解き明かすプロティストの系統関係．

古細菌の一群を起源とする原始真核生物が現存する真核生物群へ多様化した過程の解明は、生物学における重要な未解明課題の一つである。この真核生物の大系統という“パズル”を完成するには、真核生物の真の多様性、つまり“パズルのピース”がどのくらい存在するかを把握する必要がある。真核生物の多様性の大部分を原生生物（プロティスト）が占めることは認識されつつあるが、近年盛んな環境サンプルから抽出した核酸サンプルを対象にしたメタゲノム解析では、これまで我々が実態を把握していない多数の新奇プロティスト系統の存在が示唆されている。つまり真核生物の大系統というパズルを構成する多数のピースが、未知のプロティスト系統として自然環境中に手つかずのまま放置されているのである。近年、大規模な遺伝子データを迅速かつ安価に入手することが可能となり、真核生物系統（パズル）のどこに新奇プロティスト（パズルのピース）が“嵌る”のかを精度よく推測することができる。今回はパラオ共和国の環境サンプルから単離された新奇プロティストの解析結果を中心に、真核生物大系統の解明に向けた我々の研究成果の一部を紹介する。

微好気性鞭毛虫Dysnectes brevisのゲノムデータから推定するミトコンドリア関連オルガネラの機能．

フォルニカータ生物群は、嫌気・微好気性の単細胞生物で構成され、ミトコンドリアが機能的に縮退したと考えられるミトコンドリア関連オルガネラ（MRO）を保持している。この生物群に属するヒト寄生虫G. intestinalisのMROであるマイトソームは非常に縮退しており、寄生生活に適応していく中で、TCA回路や酸化的リン酸化によるATP合成系を失ったものと考えられている。しかしミトコンドリアからマイトソームへの縮退過程はいまだ解明されておらず、その解明にはミトコンドリアとマイトソームの中間段階にあるMROの解析が必要である。そこで本研究では、フォルニカータに属するDysnectes brevisという微好気性で自由生活性の鞭毛虫に注目した。D. brevisは、フォルニカータの系統樹上でG.intestinalis よりも早期に分岐しており、形態的にもマイトソームとミトコンドリアの中間程度の大きさのMROを保持している。今回はD. brevisのゲノム、トランスクリプトームデータからMRO機能を推定し、ミトコンドリアおよび既存のデータのある近縁生物のMRO機能と比較した。その結果MROの縮退過程において水素産生型ATP合成機能がMROから細胞質へと局在を変えた可能性が示唆された。

Ungulate malaria parasites.

Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.

窒素固定シアノバクテリア共生体をもつ珪藻の核ゲノム解析: 核にコードされる共生体制御遺伝子の探索.

Rhopalodia科珪藻は葉緑体に加え，spheroid bodyと呼ばれるシアノバクテリア由来の共生体を細胞内にもつ。Spheroid bodyは細胞内小器官と同様に細胞分裂時に受け継がれ，生活環を通して細胞内に維持される。Rhopalodia科珪藻はspheroid bodyがもつ窒素固定能を利用し，窒素ガスを窒素源として利用していると考えられている。我々の先行研究においてspheroid bodyゲノムが解読され，窒素固定関連遺伝子は保持されている一方で，光合成関連遺伝子はほぼ全て失われていることが明らかとなった。このことからspheroid bodyが珪藻細胞と不可分な状態であることが示されたが，珪藻細胞がどのようにspheroid bodyの代謝や分裂を制御しているのかは不明であった。本研究において，我々はRhopalodia科珪藻Epithemia adnataにおける核ゲノム解析・RNAseq解析を行い，spheroid bodyの制御に関わる遺伝子の探索を試みた。その結果，ペプチドグリカン（PG）代謝に関わる酵素が珪藻核ゲノム内に複数コードされ，発現していることが明らかとなった。PGはバクテリア由来であるspheroid bodyの細胞壁を除いて，珪藻細胞内には存在しないと考えられることから，Rhopalodia科珪藻はこれらの遺伝子を用いてspheroid bodyの成長・分裂を制御している可能性がある。

A phylogenomic study placed a previously undescribed eukaryote, strain SRT308, at the base of the Euglenozoa clade.

A novel unicellular flagellate, strain SRT308, was isolated from a marine sediment sample collected from the Republic of Palau in 2013, and has been maintained in the laboratory. We firstly explored the position of this flagellate using the maximum-likelihood (ML) phylogenetic analysis of small subunit ribosomal DNA (SSU rDNA) sequences. In the SSU rDNA tree, SRT308 showed no strong affinity to any eukaryotes or eukaryotic lineages, suggesting that this flagellate represents an unprecedented eukaryotic lineage. Therefore, to determine the accurate phylogenetic position of strain SRT308, we conducted a ML analysis based on 153 nucleus-encoded gene sequences, which included a part of the transcriptomic data of this flagellate. Our phylogenomic analyses robustly placed SRT308 at the base of the clade of kinetoplastids, euglenids, and diplonemids, collectively called the Euglenozoa. The intimate affinity between SRT308 and Euglenozoa is consistent with the discoidal mitochondrial cristae observed in the electron microscopy of SRT308. The clade of SRT308 + Euglenozoa further grouped with Heterolobosa, Jakobida, and Tsukubamonas globosa, forming the Discoba clade. Finally, we will evaluate two possibilities — (1) this flagellate is a very early-branching euglenozoan or (2) represents a novel lineage, which is closely related to, but distinct from Euglenozoa — by comparing the ultrastructural characteristics between SRT308 and euglenozoans.

Plastid comparative genomics elucidates multiple independent losses of photosynthesis in Cryptomonas (Cryptophyta).

In addition to photosynthesis, plastids are the site of a variety of biochemical processes such as fatty acid, isoprenoid, and amino acid biosyntheses. This explains the persistence of non-photosynthetic plastids in various eukaryotes, e.g., the malaria parasite Plasmodium. The unicellular algal genus Cryptomonas (Cryptophyta) contains both photosynthetic and non-photosynthetic members, the latter having recently evolved on at least three separate occasions. In order to elucidate the evolutionary mechanisms underlying the loss of photosynthesis in Cryptomonas, we sequenced the plastid genomes of two non-photosynthetic strains, Cryptomonas sp. M1634B and SAG977-2f, as well as the photosynthetic strain Cryptomonas curvata CCAP979/52. Together with the previously sequenced plastid genome of the non-photosynthetic C. paramecium CCAP977/2a, we carried out a four-way comparison of genome size, coding capacity, pseudogene content, and genome synteny. Here we discuss how the non-photosynthetic plastid evolved in these strains.

Phylogenetic positions of marine gregarines Selenidium terebellae and Lecudina tuzetae, and molecular evidence of their remnant nonphotosynthetic plastids (apicoplasts).Gregarines are a diverse group of apicomplexan parasites that mostly infect the intestines of insects and marine invertebrates. Because some marine gregarines have traits inferred to reflect the most recent apicomplexan ancestor, improved knowledge of these parasites is important for understanding the early evolution of apicomplexans as a whole. However, molecular data, with the exception of small subunit ribosomal RNA (SSU rRNA) genes, has not been widely gathered for gregarines as a whole. The SSU rRNA gene sequences that have been gathered for this group have been used to discriminate different species from one another and in assessing the deeper phylogenetic relationship among gregarines, as well as the relationship between these groups and non-gregarine apicomplexan parasites. Unfortunately, these data are unable to resolve the deepest relationships among apicomplexans because of the divergent nature of gregarine SSU rRNA genes. In the present study, we generated RNA-seq data from two species of marine gregarines, Selenidium terebellae and Lecudina tuzetae, that belong to different subgroups of gregarines, archigregarines and eugregarines, respectively. The results of a phylogenomic analysis using a dataset comprised of 76 protein sequences indicated that S. terebellae and L. tuzetae formed a clade. This gregarine clade formed the sister group to a clade of non-gregarine apicomplexan parasites. The monophyly of gregarines and the Apicomplexa as a whole were robustly supported by maximum-likelihood analyses. Our RNA-seq data also enabled us to establish evidence for the presence of nonphotosynthetic plastids in S. terebellae and L. tuzetae. In both gregarine RNA-seq data, we found transcripts encoding putative proteins bearing specific phylogenetic affinities to plastid proteins in other photosynthetic organisms. The potential plastid proteins included enzymes for fatty acid metabolism, pyruvate metabolism, and transporters for phosphorylated carbohydrates. Noteworthy, some of the potential plastid proteins were predicted to have signal peptides, which has been established as a part of the apicoplast-targeting signal in non-gregarine apicomplexans. Therefore, we conclude that both S. terebellae and L. tuzetae retain metabolically active, but non-photosynthetic plastids. Based on the results presented here, we propose that the common ancestor of archigregarines, eugregarines, and non-gregarine apicomplexan parasites evolved from a single ancestral cell bearing a nonphotosynthetic plastid.

Differential impacts of plastid replacement on plastidal biosynthetic pathways in dinoflagellates with non-canonical plastids, Karlodinium veneficum and Lepidodinium chlorophorum.

The common ancestor of dinoflagellates most likely established the plastid through a red algal endosymbiosis, and the vast majority of proteins work in the plastids (plastidal proteins) are encoded in their nuclear genomes. Unlike any other eukaryotic algal groups, multiple independent lineages in dinoflagellates replaced the original plastids by those acquired through the endosymbioses of phylogenetically diverse eukaryotic algae. Previously published studies on the dinoflagellates with non-canonical plastids demonstrated that a portion of the original set of plastidal proteins was replaced by the homologues acquired from the endosymbionts. In this study, we focused on the evolutions of Chlorophyll a (Chl-a) biosynthetic pathway and MEP/DOXP pathway for isopentenyl-diphosphate (IPP) biosynthesis in two dinoflagellates, Karlodinium veneficum bearing a haptophyte-derive plastid and Lepidodinium chlorophorum bearing a green alga-derive plastid. As the two pathways have been localized in the plastid beyond the plastid replacements, it is intriguing to evaluate the impact of plastid replacement on the two metabolic pathways in the dinoflagellates bearing non-canonical plastids. We surveyed the genes encoding the proteins of interest in the transcriptomic data of the two dinoflagellates, and assessed their evolutionary origins individually by maximum-likelihood phylogenetic analyses. Most or all of the proteins involved in MEP/DOXP pathway in Karlodinium and Lepidodinium appeared to share the evolutionary ancestries with the homologues in dinoflagellates bearing typical plastids, suggesting that plastid replacement gave no large impact on this particular pathway. On the other hand, Chl-a biosynthetic pathway in Karlodinium was most likely reorganized during plastid replacement. The Karlodinium pathway appeared to be occupied by ‘haptophyte proteins,’ which were most likely acquired from the haptophyte endosymbiont that gave rise to the current non-canonical plastid in this species. Similarly, the same pathway in Lepidodinium may have been also reorganized, but in a different manner from the Karlodinium pathway. The Lepidodinium pathway seemingly comprises the proteins acquired from phylogenetically diverse eukaryotes, rather than green algae that are close relatives of the endosymbiont engulfed by the ancestral Lepidodinium. In this presentation, we will propose a hypothesis to explain why plastid replacement reorganized Chl-a pathway intensely in both Karlodinium and Lepidodinium, while little/weak impact on MEP/DOXP pathway was observed in either of the two dinoflagellates.

Gregarine-like apicomplexan parasite isolated from the intestinal tract of a centipede Scolopocryptops rubiginosus.

Gregarines are a group of apicomplexan parasites that inhabit in the digestive tracts or body cavities of diverse invertebrates. Because of their morphological characteristics and phylogenetic positions inferred mainly from small subunit rRNA gene (SSU rDNA) sequences, gregarines are anticipated to provide novel insights into the early evolution of the Apicomplexa. Some of us have already gathered RNA-seq data of two gregarines parasitizing marine invertebrates (marine gregarines), and examined their phylogenetic positions as well as the presence/absence of remnant nonphotosynthetic plastids (apicoplasts) (see the presentation of Wakeman & Nakayama et al.). On the other hand, gregarines parasitizing terrestrial invertebrates (terrestrial gregarines) have not been subjected to RNA-seq analysis. Due to the lack of large-scale transcriptomic data of terrestrial gregarines, their phylogenetic positions amongst apicomplexan parasites (including marine gregarines) or the presence/absence of apicoplasts remain controversial. In this study, we isolated gregarine-like parasites from the digestive tract of a centipede Scolopocryptops rubiginosus, as the first step toward large-scale transcriptomic analyses of terrestrial gregarines. We amplified and determined the SSU rDNA sequence of the gregarine-like parasite, and the resultant sequence matched none of the known SSU rDNA sequences in NCBI nr database with high nucleotide identity. In the maximum-likelihood tree inferred from a SSU rDNA alignment, the parasite in S. rubiginosus grouped with non-gregarine apicomplexans, Cryptosporidium spp., albeit this grouping received little statistical support. Combining the results described above, we conclude that the gregarine-like parasite in S. rubiginosus is a novel, potentially early-branching member of the Apicomplexa. We will also report preliminary results from the RNA-seq analysis of this apicomplexan parasite currently underway.

Loss of the Calvin Benson cycle in non-photosynthetic plastids.

Regardless of the important role of plastids in the synthesis of various amino acids, fatty acids, heme, and isoprenoid in addition to ATP and sugars, some eukaryotes have experienced loss of photosynthesis. However, loss of photosynthesis did not always result in loss of plastids, as non-photosynthetic plastids have been found in a number of secondarily non-photosynthetic eukaryotes. Non-photosynthetic plastids still retain some functions and are indispensable for cell viability. However, how these non-photosynthetic organelles lose functions after loss of photosynthesis remained to be fully understood. In this study, we characterized the non-photosynthetic plastids of the apochlorotic diatom, Nitzschia sp. NIES-3581 by transcriptome analyses, cell biological experiments, and phylogenetic analyses. We found these plastids retained various pathways beyond photosynthesis, which include amino acid biosynthesis and the Calvin Benson cycle lacking RuBisCO. By comparative analyses, we discuss why the Calvin Benson cycle unable to fix CO₂ is still retained in non-photosynthetic plastids.

Evolutionary roles of SL-trans-splicing in the primary endosymbiosis.

Spliced leader (SL) trans-splicing, which adds short non-coding RNA sequences (20-30 bases) to the 5’ end of mRNAs by splicing in trans, is sporadically observed in the diverse eukaryotic lineages, including metazoa, kinetoplastida and dinoflagellata. Although this peculiar RNA maturation process is essential in those trans-splicing organisms, it remains largely unknown how those splicing systems have evolved in their lineages. Here we report a novel finding that SL trans-splicing occurs in a photosynthetic rhizarian organism, Paulinella chromatophora. This organism has a unique photosynthetic organelle termed cyanelle, which was derived from the cyanobacterial endosymbiosis that had occurred about 0.1 billion years ago. Considering that the cyanobacterial endosymbiosis that led the evolution of chloroplasts occurred 1.0-1.5 billion years before, P. chromatophora nuclear-organelle genome system is thought to be still young, thus suitable for studying the genome-transcriptome features in the initial stage of the primary endosymbiosis. In this presentation, we demonstrate the characteristics of the SL-trans-splicing in this unique organism, and discuss its possible roles and advantages for endosymbiotic evolution.