CCS International Symposium 2018, University of Tsukuba
Oct. 15-16, 2018

Recent progress in understanding the early
evolution of eukaryotes based on
phylogenomic data-analyses

Yuji Inagaki
Center for Computational Sciences
University of Tsukuba

Outline of my talk Background/introduction
 3domains of Life
• Bacteria, Archaea and Eukaryotes

 Motivation of our study• Biodiversity, Data & Methods

 Our achievements Tsukubamonas globose
 Palpitomonas bilix
 CRuMs Fronicata

 Summary Acknowledgements

Diversity of organisms on the Earth Three domains of life Archaea, Bacteria &Eukarya

 Diversity of the threedomains are too far
underrepresented

http://blogs.discovermagazine.com/loom/files/2011/03/three-domains.jpg

Diversity of bacteria

Hug et al. A new view of the tree of Life. Nat Microbiol 1:16048

 We have overlooked a large
proportion of bacterial diversity

Diversity of archaea

 We have overlookeda large proportion ofarchaeal diversity
 Two domains, notthree domains
Zaremba-Niedzwiedzka et al. Asgard archaea illuminate the origin of eukaryotic cellularcomplexity. Nature 2017 541:353-358

Diversity of eukaryotes

 Diversity ofeukaryotes have been
underestimated Interested inreconstructing thetree of eukaryotes
 Number of majorgroups Branching order of themajor groups

http://blogs.discovermagazine.com/loom/files/2011/03/three-domains.jpg

Diversity of eukaryotes

 Diversity ofeukaryotes have been
underestimated Interested inreconstructing thetree of eukaryotes
 Number of majorgroups Branching order of the
major groups

http://blogs.discovermagazine.com/loom/files/2011/03/three-domains.jpg

Our goal is

Cover the diversityof eukaryotes

Well resolved treeof eukaryotes
Robust methodsfor phylogeny

Generate large
sequence dataImportance to understand the true eukaryotic diversity

Blind men (researchers who are investigating the tree of eukaryotes)
and the elephant (the diversity of eukaryotes)

International Journal of Computer Applications 140(3):6-14

Sequence data for phylogeny

hemoglobin α chains from four vertebrates
60
Human
mvlspadktnvkaawgkvgahageygaealermflsfpttktyfphf-dlshgsaqvkgh
dog
mvlspadktnikstwdkigghagdyggealdrtfqsfpttktyfphf-dlspgsaqvkah
chicken mvlsaadknnvkgiftkiaghaeeygaetlermfttypptktyfphf-dlshgsaqikgh
carp
mslsdkdkaavkglwakispkaddigaealgrmltvypqtktyfahwadlspgsgpvkkh* ** ****** * * *
* ***** * *** **
* *
120
human
gkkvadaltnavahvddmpnalsalsdlhahklrvdpvnfkllshcllvtlaahlpaeft
dog
gkkvadalttavahlddlpgalsalsdlhayklrvdpvnfkllshcllvtlachhpteft
chicken gkkvvaalieaanhiddiagtlsklsdlhahklrvdpvnfkllgqcflvvvaihhpaalt
carp
gkvimgavgdavskiddlvgglaalselhafklrvdpanfkilahnvivvigmlypgdfp
**
*
*
**
* ** *** ****** *** ***humandogchicken
carppavhasldkflasvstvltskyr
pavhasldkfftavstvltskyr
pevhasldkflcavgtvltakyr
pevhmsvdkffqnlalalsekyr* ** * **** ****identical residues among the four organisms

Amino acid residues vary among the four vertebrates⇒substitutions occurred during vertebrate evolution

Maximum-likelihood method for phylogeny
Xh : time reversible Markov process, independent evolution for each branch
Based on the formula of Chapmann-Kolmogorov,f (x1, x2, x3, x4|q )← probability for getting data of h’th sitewith a given Pij(t) and a given tree topology= SS P{X0 = i}P{Xt1 = x1, Xt2 = x2, Xt5 = j | X0 = i}i jx P{Xt5+t3 = x3, Xt5+t4 = x4 | Xt1 = x1, Xt2 = x2, Xt5 = j, X0 = i}=S{p Pix (t )Pix (t ) S Pij(t ) Pjx (t ) Pjx (t )}
i
1i

122

5

3

3

4

4

Transition rate matrix Q⇒ Transition
probability matrix
Pij(t) = etQj

pi : composition of the base (amino acid) i
S1 x 1t1t2S2 x 2On internal nods (ancestral species),
all possibilities for the base (amino acid)i or j are summed overIn case of base, i or j = A,C,G,T

x 3 S3ijt5

t3
t4x 4 S4

S1,…, S4x1,…, x4i, jt1,…, t5

: extant species: sequence data of h’th site
for extant species: sequence data for ancestral species: branch lengths

Importance of data size

 Larger the data, moreaccurate thephylogenetic inference Genome data Transcriptome data Technology is rapidlygrowing A single or few cells are
sufficient to generate
genome/transcriptomedataOutline of my talk Background/introduction
 3domains of Life
• Bacteria, Archaea and Eukaryotes

 Motivation of our study• Biodiversity, Data & Methods

 Our achievements Tsukubamonas globose
 Palpitomonas bilix
 CRuMs Fronicata

 Summary Acknowledgements

Major groups of eukaryotes

 8 major groups have been recognized “Orphans” can be members of the 8 groups or
represent novel groups

Major groups of eukaryotes

 8 major groups have been recognized5123

 “Orphans” can be members of the 8 groups orrepresent novel groups

Tsukubamonas globosa

 Represents a novel
lineage in Discoba
Photo by N. Yubuki

University of Tsukuba: Home of Tsukubamonas globosa

Image taken from Google Maps

 Isolated from Hyoutaro-pond
 Maintained in UR-YT medium at 20ᵒC since October
2002

Tsukubamonas: How it looks likeA fibreSinglet-root
associated fibre
10 mm

10 mm

10 mm

N, Nucleus; Fv, Food vacuole

Right root
I fibreSinglet root
Posteriorbasal body

B fibre0.5 mmPosteriorbasal body
Backward
Right root

Right root

Posteriorbasal body
Inner right root

Outer
Right root

Mitochondrion

2 mmYabuki et al. 2011 J Eukaryot Microbiol.

Singlet-root
associated fibre

B fibre0.5 mmTsukubamonas: phylogenomic analyses
157 genes, 41,372 aa positions
ML method, LG + Γ + F model
Bayesian method, CAT + Γ model Tg is basal toeuglenozoans +heteroloboseans Tg is a novel memberof Discoba
Jakobida
Tsukubamonas globosa

DISCOBA

Euglenozoa
Heterolobosea
Kamikawa et al. 2014 Genome Biol Evol

Palpitomonas bilix

 Represents the earliest
diverged lineage inCryptista

10 μm

Yabuki et al. 2010 Protist

Palpitomonas bilix: Yabuki, Inagaki, Ishida 2010 Protist 161:523-38
Image taken from Google Maps

Palau islands Isolated from seawater sampled on
Macharchar island, on July 2006
 Maintained in EMS medium at 20ᵒC

1 μm

Yabuki et al. 2010 Protist

10 μm

Palpitomonas: Phylogenomic analysis
157 genes, 41,372 aa positions
ML method, LG4X model
Bayesian method, CAT + Γ model Pb is a basal to a cladeof kathablepharids,goniomonads andcryptophytes Pb is a novel memberof Cryptistacryptophytes
goniomonadkathablepharid CRYPTISTA

Palpitomonas bilix

Yabuki et al. 2014 Sci Rep.

CRuMs: A novel group of eukaryotes

 Phylogenomic study
assembled multipleorphan species
into a group

Emergence of a new group tentatively named as ‘CRuMs’

 Diphylleids, Rigifila andMantamonas have beenclassified as “orphan” eukaryotes
 Phylogenomic study bycollaboration with institutes in
the US and Canada

Rigifila ramosa

Yabuki et al. 2013 Protist

Diphylleia rotans

diphylleids

Mantamonas
plastica

Collodictyon tricilatum

https://www.apollon.uio.no/english/articles/2012/microorganism.html
http://tolweb.org/Protists_of_uncertain_placement/2383

Glucksman et al. 2011 Protist

Emergence of a new group tentatively named as ‘CRuMs’
AMOEBOZOAAMORPHEAOBAZOA“CRuMs”

351 genes, 97,002 aa positions
ML method, LG +C60 + Γ + PMSF model
Bayesian method, CAT-GTR + Γ model

Brown et al. 2018 Genome Biol Evol

Fornicata: A group of mitochondrion-less organisms

 Fornicata is composedof mitochondrion-lessorganisms
 Parasitic species havebeen studies
 Free-living members
have been overlooked Possess reduced typesof mitochondria

Free-living fornicates Free-living species have been
isolated from various
anaerobic/microaerophilic
environments
Yubuki et al. J Eukaryot Microbiol 2007 57:191-200

Takishita et al. Extremphiles 2007 11:563-576

Kolisko et al. Environ Microbiol 2010 12:2700-2710

Takishita et al. Protist 2010 163:344-355

Relationship among fornicates To model how eukaryotes adapted toanaerobic/microaerophilic environments, the robust
relationship among fornicates Phylogenomic study by collaboration with institutes in
the US, Canada, Czech Republic and Sweden

Relationship among fornicates Fornicata tree was fully resolved
 Proposed the evolution of reduced mitochondria

159 genes, 39,089 aa positions
ML method, LG + Γ + F model
Bayesian method, CAT-GTR + Γ model
Legar et al. Nat Ecol Evol 2017 1:0092

Summary Identify new “orphans” in natural environments
 Clarify the placements of orphans by phylogenomic studies
 Challenge to resolve “the backbone”

Iterate the process from the field to the HPC

1

Sampling

Culturing2
https://www.forbes.com/

https://assets.illumina.com/

Sequencing
Phylogenomic analyses

3

Acknowledgements
科学研究費補助金

計算科学研究センター
学際共同利用• Phylogenomicデータによる真核生物大系統解析（稲垣）• 基盤B H15-17年度 複数遺伝子配列情報に基づく真核生物の初期進化の解明 （橋本）• 基盤C H18-19年度 カタブレファリス類配列を含む連結データ系統解
析によるクロミスタ仮説の検討 （稲垣）• 基盤B H21-24年度 ハプト・クリプト藻類を含む新奇巨大生物群の提唱とクロムアルベオラータ仮説の検証 （稲垣）• 基盤A H23-26年度 新型分割イントロン切り出し因子同定に基づく真核生物mRNA成熟機構進化の解明 （橋本）• 挑戦的萌芽 H22-23年度 原始真核生物を求めて：新奇真核微生物の形態・発現遺伝子・ミトコンドリアゲノム解析 （稲垣）• 新学術領域研究 計画研究 H23-27年度 ミトコンドリア・色素体以外の共生オルガネラ成立過程の解明 （稲垣）• 基盤B海外学術 H23-25年度 ミトコンドリアを持たない真核微生物群
フォルニカータの多様性の解明 （橋本）• 基盤B H26-28年度 新型分割イントロンのスプライシング機構と進化多様性の解明 （橋本）• 基盤B海外学術 H26-28年度 嫌気環境に生育する真核微生物の多様性の解明 （橋本）• 基盤B H28-30年度 渦鞭毛藻細胞内に発見された新たな共生体痕跡
核ゲノムの解読 （稲垣）• 国際共同研究強化B H30-H34年度 海洋原生生物に共生する細菌多様性の実態解明 （稲垣）大学からの研究経費• 特別経費プロジェクト 「生命の樹」研究機能の充実 －多様な非モデ
ル生物研究に基づく生命原理の再構築ー

Major groups of eukaryotes

 4 published works; highlighted in blue

 3 unpublished works; highlighted in green