The need for a phylogeny driven genomic encyclopedia of eukaryotes #SMBEEuks

The Need for a Phylogeny-Driven Genomic
Encyclopedia of Eukaryotes
Jonathan A. Eisen
@phylogenomics
University of California, Davis
Talk for SMBE-EUKS
Monday, April 29, 13

I: The Problem

Googling Sequenced Eukaryotic Genomes

Wikipedia On Sequenced Euks

More from Wikipedia

Better Source: GOLD
http://www.genomesonline.org/cgi-bin/GOLD/index.cgi

GOLD by Taxonomy

GOLD: Euks by Phylum
Phylum Count Percent
Korarchaeota 1 0
Nanoarchaeota 2 0
Thaumarchaeota 30 5
Crenarchaeota 142 25
Euryarchaeota 356 64
Unclassified 28 5
Caldiserica 1 0
Nitrospinae 1 0
Crenarchaeota 2 0
Chrysiogenetes 2 0
Dictyoglomi 2 0
Fibrobacteres 2 0
Armatimonadetes 3 0
Elusimicrobia 3 0
Lentisphaerae 3 0
Poribacteria 4 0
Gemmatimonadetes 6 0
Thermodesulfobacteria 7 0
Ignavibacteria 8 0
Deferribacteres 10 0
Chlorobi 14 0
Synergistetes 21 0
Euryarchaeota 23 0
Nitrospirae 24 0
Aquificae 24 0
Acidobacteria 30 0
Verrucomicrobia 41 0
Planctomycetes 42 0
Thermotogae 50 0
Chloroflexi 51 0
Fusobacteria 80 0
Deinococcus-Thermus 92 0
Chlamydiae 207 1
Cyanobacteria 245 1
Tenericutes 251 1
Spirochaetes 472 2
Bacteroidetes 762 4
Actinobacteria 2,065 10
Firmicutes 5,342 26
Proteobacteria 10,088 50
Unclassified 17 0
Phaeophyceae 1 0
Priapulida 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Blastocladiomycota 5 0
Echinodermata 6 0
Entomophthoromycota 9 0
Chytridiomycota 12 0
Neocallimastigomycota 12 0
Annelida 13 0
Eustigmatophyceae 13 0
Cnidaria 18 0
Bacillariophyta 21 0
Platyhelminthes 23 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Basidiomycota 976 18
Ascomycota 1,251 23
Unclassified 704 13
Back to GOLD
bution
Percent
0
0
0
0
0
0
0
Eukaryotic Phylum Distribution
Phaeophyceae 1 0
Priapulida 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
18/18 Family: 30/29 Genus: 103/118 Species: 340/673
jects over number of the classified subdivisions of this phylogenetic group.

GOLD: Euks by Phylum
Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23

Euks More Resolution
0.2
Bodomorpha minima
Lumbricus rubellus
Diplophrys
BOLA458
Chaunacanthida sp.
Labyrinthuloides minuta
Filamoeba nolandi
Chlamydaster sterni
RT7iin2
Phalansterium solitarium
Euglena gracilis
RT5iin20
BOLA383
Ulkenia profunda
LEMD267
Ammonia sp.
Oxymonas sp.
DH148EKB1
Diplonema ambulator
Minchinia teredinis
Pavlova salina
Glaucosphaera vacuolata
Cyanoptyche gloeocystis
OLI11305
Gromia oviformis
Cryptosporidium parvum
Breviata anathema
Achlya bisexualis
LEMD052
Phagomyxa odontellae
Raphidiophrys ambigua
Compsopogon coeruleus
BOLA212
Colpodella pontica
Uncultured eukaryote clone BOLA187
Jakoba libera
RT5iin2
CS.E036
Acrosphaera sp. CR6A
Acanthamoeba castellanii
AT1.3
Saccharomyces cerevisiae
OLI11150
Nuclearia simplex
RA000412.136
TCS 2002
BOLA868
Allogromia sp.
Monosiga brevicollis
RT5iin4
Plasmodiophora brassicae
RT5iin8
OLI51105
RA010412.17
BOLA515
OLI11032
RT 5iin25
AT4.11
Symphyacanthida
RT5iin44
CS.E045
Urosporidium crescens
Goniomonas truncata
Gymnophrys cometa
Podocoryne carnea
OLI11066
Reclinomonas americana
Reticulomyxa filosa
RT8n7
Oxytricha nova
AT4.50
C1.E027
Arthracanthida sp.
RT1n14cul
AT4.94
Telonema antarcticum
OLI11025
LKM30
LKM48
Filobasidiella neoformans
DH147EKD17
Mayorella sp.
C2.E026
Bacillaria paxillifer
Retortamonas sp.
OLI11059
Malawimonas jakobiformis
BOLA048
Streblomastix strix
Guillardia theta
Platyamoeba stenopodia
DH148EKD18
Cafeteria roenbergensis
Telonema subtilis RCC404.5
DH148EKD53
LKM74
Ciliophrys infusionum
Scherffelia dubia
Volvox carteri
CS.R003
Trypanosoma cruzi
BL010625.25
AT4.56
N-Por
Jakoba incarcerata
Sphaerozoum punctatum
Uncultured eukaryote clone BOLA366
Lecythium sp.
Acanthometra sp.
Loxophyllum utriculare
LKM101
Glaucocystis nostochinearum
OLI11056
BAQA072
Apusomonas proboscidea
Trimastix marina
C3.E012
Helianthus annuus
AT8.54
Ichthyobodo necator
CS.E022
RA001219.10
RT5in38
Paravahlkampfia ustiana
OLI11007
Telonema subtilis RCC358.7
Amastigomonas debruynei
Emiliania huxleyi
Leptomyxa reticulata
Hartmannella vermiformis
OLI11072
DH145EKD11
Noctiluca scintillans
Cyanophora paradoxa
Trimastix pyriformis
Naegleria gruberi
AT 4.96
Amoeba proteus
Gonyaulax spinifera
sp.
0.99/68
0.89/-0.40/-
0.87/-
0.88/-
0.88/-
0.84/-
0.78/59
0.66/61
0.55/-
0.89/-
Collodictyon triciliatum
Diphylleia rotans
Uncultured Collodictyonidae partial
1.0/77
-/84
1.0/63
1.0/56
0.99/-
1.0/-
0.96/-
0.99/-
0.95/-
0.99/-
0.99/68
1.0/63
1.0/62
0.69/-
0.63/- 0.83/-
0.79/75
0.69/57
0.79/-
0.87/-
0.59/-
0.68/-
1.0/-
0.57/50
0.63/-
1.0/78
0.53/-
SAR
Excavata
Diphyllatia
Amoebozoa
Opisthokonta
0.53/76
0.73/-
0.81/-
0.84/-
-/-
0.63/-
0.79/-
0.81/-
0.70/-
0.98/-
1.0/74
0.51/-
-/-
-/-
Haptophyta
Telonemia
Apusozoa
Centrohelida
Cryptophyta
Rhodophyta
Glaucophyta
Viridiplantae
FIG. 1. 18S rDNA phylogeny of the Diphyllatia species Collodictyon triciliatum (highlighted by black box) and Diphylleia rotans. The topology
was reconstructed by MrBayes v3.1.2 under the GTR þ GAMMA þ I þ covarion model. Posterior probabilities (PP) and ML bootstrap supports
(BP, inferred by RAxML v7.1.2 under GTR þ GAMMA þ I model) are shown at the nodes. Thick lines indicate PP . 0.90 and BP . 80%. Dashes
‘‘-’’ indicate PP , 0.5 or BP , 50%. A few long branches are shortened by 50% (/) or 75% (//).
Zhao et al. · doi:10.1093/molbev/mss001 MBE
1560
byguestonApril28,2013http://mbe.oxfordjournals.org/Downloadedfrom
Collodictyon—An Ancient Lineage in the Tree of Eukaryotes
Sen Zhao, ,1
Fabien Burki, ,2
Jon Bra˚te,1
Patrick J. Keeling,2
Dag Klaveness,1
and
Kamran Shalchian-Tabrizi*,1
1
Microbial Evolution Research Group, Department of Biology, University of Oslo, Oslo, Norway
2
Canadian Institute for Advanced Research, Botany Department, University of British Columbia, Vancouver, British Columbia,
Canada
These authors contributed equally to this work.
*Corresponding author: E-mail: kamran@bio.uio.no.
Associate editor: Herve´ Philippe
Abstract
The current consensus for the eukaryote tree of life consists of several large assemblages (supergroups) that are hypothesized to
describe the existing diversity. Phylogenomic analyses have shed light on the evolutionary relationships within and between
supergroups as well as placed newly sequenced enigmatic species close to known lineages. Yet, a few eukaryote species remain of
unknown origin and could represent key evolutionary forms for inferring ancient genomic and cellular characteristics of
eukaryotes. Here, we investigate the evolutionary origin of the poorly studied protist Collodictyon (subphylum Diphyllatia) by
sequencing a cDNA library as well as the 18S and 28S ribosomal DNA (rDNA) genes. Phylogenomic trees inferred from 124 genes
placed Collodictyon close to the bifurcation of the ‘‘unikont’’ and ‘‘bikont’’ groups, either alone or as sister to the potentially
contentious excavate Malawimonas. Phylogenies based on rDNA genes confirmed that Collodictyon is closely related to another
genus, Diphylleia, and revealed a very low diversity in environmental DNA samples. The early and distinct origin of Collodictyon
suggests that it constitutes a new lineage in the global eukaryote phylogeny. Collodictyon shares cellular characteristics with
Excavata and Amoebozoa, such as ventral feeding groove supported by microtubular structures and the ability to form thin and
broad pseudopods. These may therefore be ancient morphological features among eukaryotes. Overall, this shows that
Collodictyon is a key lineage to understand early eukaryote evolution.
Key words: 18S and 28S rDNA, Collodictyon, Diphyllatia, tree of life, phylogenomics, cDNA, pyrosequencing.
Introduction
Over the last few years, molecular sequence data have ad-
dressed some of the most intriguing questions about the
eukaryote tree of life. Phylogenomic analyses have con-
firmed the existence of several major eukaryote groups
(supergroups) as well as shown various levels of evidences
for the relationships among them (Burki et al. 2007; Parfrey
et al. 2010). Recently, two new large assemblages, SAR
(Stramenopila, Alveolata, and Rhizaria) and CCTH (Crypto-
phyta, Centrohelida, Telonemia, and Haptophyta), were
proposed to encompass a large fraction of the eukaryote
diversity, together with the other supergroups Opisthokon-
ta, Amoebozoa, Archaeplastida, and Excavata (Patron et al.
2007; Burki et al. 2009). Solid phylogenomic evidence
and complex genome histories (Simpson and Roger
2004; Parfrey et al. 2006; Roger and Simpson 2009).
Identification of sister lineages to these supergroups is
crucial for resolving the eukaryote tree and understanding
the early history of eukaryotes. If these key lineages exist,
they may be found among the few species that harbor dis-
tinct morphological features but are of unknown evolu-
tionary origin in single-gene phylogenies (Patterson 1999;
Shalchian-Tabrizi et al. 2006; Kim et al. 2011). Indications
that such enigmatic species can be placed in the eukaryote
tree come from recent phylogenomic analyses. For in-
stance, Ministeria (Opisthokonta), Breviata (Amoebozoa)
and Telonemia, Centroheliozoa, and Picobiliphyta have
been shown to constitute deep lineages within their re-
ResearcharticlebyguestonApril28,2013http://mbe.oxfordjournals.org/Downloadedfrom
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351787/

2010 PARFREY ET AL.—BROADLY SAMPLED TREE OF EUKARYOTIC LIFE 523
FIGURE 1. Most likely eukaryotic tree of life reconstructed using all 451 taxa and all 16 genes (SSU-rDNA plus 15 protein genes). Major
nodes in this topology are robust to analyses of subsets of taxa and genes, which include varying levels of missing data (Table 1). Clades in bold
are monophyletic in analyses with 2 or more members except in all:15 in which taxa represented by a single gene were sometimes misplaced.
Numbers in boxes represent support at key nodes in analyses with increasing amounts of missing data (10:16, 6:16, 4:16, and all:16 analyses; see
Table 1 for more details). Given uncertainties around the root of the eukaryotic tree of life (see text), we have chosen to draw the tree rooted with
the well-supported clade Opisthokonta. Dashed line indicates alternate branching pattern seen for Amoebozoa in other analyses. Long branches,
indicated by //, have been reduced by half. The 6 lineages labeled by * represent taxa that are misplaced, probably due to LBA, listed from
top to bottom with expected clade in parentheses. These are Protoopalina japonica (Stramenopiles), Aggregata octopiana (Apicomplexa), Mikrocytos
mackini (Haplosporidia), Centropyxis laevigata (Tubulinea), Marteilioides chungmuensis (unplaced), and Cochliopodium spiniferum (Amoebozoa).
byguestonApril28,2013http://sysbio.oxfordjournals.org/Downloadedfrom
Syst. Biol. 59(5):518–533, 2010
c The Author(s) 2010. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
DOI:10.1093/sysbio/syq037
Advance Access publication on July 23, 2010
Broadly Sampled Multigene Analyses Yield a Well-Resolved Eukaryotic Tree of Life
LAURA WEGENER PARFREY1
, JESSICA GRANT2
, YONAS I. TEKLE2,6
, ERICA LASEK-NESSELQUIST3,4
,
HILARY G. MORRISON3
, MITCHELL L. SOGIN3
, DAVID J. PATTERSON5
, AND LAURA A. KATZ1,2,∗
1Program in Organismic and Evolutionary Biology, University of Massachusetts, 611 North Pleasant Street, Amherst,
MA 01003, USA; 2Department of Biological Sciences, Smith College, 44 College Lane, Northampton, MA 01063, USA; 3Bay Paul Center for
Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA; 4Department of Ecology and
Evolutionary Biology, Brown University, 80 Waterman Street, Providence, RI 02912, USA; 5Biodiversity Informatics Group, Marine Biological
Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA; 6Present address: Department of Epidemiology and Public Health, Yale University School of
Medicine, New Haven, CT 06520, USA;
∗Correspondence to be sent to: Laura A. Katz, 44 College Lane, Northampton, MA 01003, USA; E-mail: lkatz@smith.edu.
Laura Wegener Parfrey and Jessica Grant have contributed equally to this work.
Received 30 September 2009; reviews returned 1 December 2009; accepted 25 May 2010
Associate Editor: Cécile Ané
Abstract.—An accurate reconstruction of the eukaryotic tree of life is essential to identify the innovations underlying the
diversity of microbial and macroscopic (e.g., plants and animals) eukaryotes. Previous work has divided eukaryotic diver-
sity into a small number of high-level “supergroups,” many of which receive strong support in phylogenomic analyses.
However, the abundance of data in phylogenomic analyses can lead to highly supported but incorrect relationships due
to systematic phylogenetic error. Furthermore, the paucity of major eukaryotic lineages (19 or fewer) included in these
genomic studies may exaggerate systematic error and reduce power to evaluate hypotheses. Here, we use a taxon-rich
strategy to assess eukaryotic relationships. We show that analyses emphasizing broad taxonomic sampling (up to 451 taxa
representing 72 major lineages) combined with a moderate number of genes yield a well-resolved eukaryotic tree of life.
The consistency across analyses with varying numbers of taxa (88–451) and levels of missing data (17–69%) supports the
accuracy of the resulting topologies. The resulting stable topology emerges without the removal of rapidly evolving genes
or taxa, a practice common to phylogenomic analyses. Several major groups are stable and strongly supported in these
analyses (e.g., SAR, Rhizaria, Excavata), whereas the proposed supergroup “Chromalveolata” is rejected. Furthermore, ex-
tensive instability among photosynthetic lineages suggests the presence of systematic biases including endosymbiotic gene
transfer from symbiont (nucleus or plastid) to host. Our analyses demonstrate that stable topologies of ancient evolutionary
relationships can be achieved with broad taxonomic sampling and a moderate number of genes. Finally, taxon-rich analy-
ses such as presented here provide a method for testing the accuracy of relationships that receive high bootstrap support
(BS) in phylogenomic analyses and enable placement of the multitude of lineages that lack genome scale data. [Excavata;
microbial eukaryotes; Rhizaria; supergroups; systematic error; taxon sampling.]
Perspectives on the structure of the eukaryotic tree
of life have shifted in the past decade as molecular
analyses provide hypotheses for relationships among
marks throughout to note groups where uncertaintie
remain. Moreover, it is difficult to evaluate the overal
stability of major clades of eukaryotes because phyloge
http://sysbio.oxfordjournals.org/content/59/5/518.full
Euks More Resolution

Syst. Biol. 59(5):518–533, 2010
c The Author(s) 2010. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
DOI:10.1093/sysbio/syq037
Advance Access publication on July 23, 2010
Broadly Sampled Multigene Analyses Yield a Well-Resolved Eukaryotic Tree of Life
LAURA WEGENER PARFREY1
, JESSICA GRANT2
, YONAS I. TEKLE2,6
, ERICA LASEK-NESSELQUIST3,4
,
HILARY G. MORRISON3
, MITCHELL L. SOGIN3
, DAVID J. PATTERSON5
, AND LAURA A. KATZ1,2,∗
1Program in Organismic and Evolutionary Biology, University of Massachusetts, 611 North Pleasant Street, Amherst,
MA 01003, USA; 2Department of Biological Sciences, Smith College, 44 College Lane, Northampton, MA 01063, USA; 3Bay Paul Center for
Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA; 4Department of Ecology and
Evolutionary Biology, Brown University, 80 Waterman Street, Providence, RI 02912, USA; 5Biodiversity Informatics Group, Marine Biological
Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA; 6Present address: Department of Epidemiology and Public Health, Yale University School of
Medicine, New Haven, CT 06520, USA;
∗Correspondence to be sent to: Laura A. Katz, 44 College Lane, Northampton, MA 01003, USA; E-mail: lkatz@smith.edu.
Laura Wegener Parfrey and Jessica Grant have contributed equally to this work.
Received 30 September 2009; reviews returned 1 December 2009; accepted 25 May 2010
Associate Editor: Cécile Ané
Abstract.—An accurate reconstruction of the eukaryotic tree of life is essential to identify the innovations underlying the
diversity of microbial and macroscopic (e.g., plants and animals) eukaryotes. Previous work has divided eukaryotic diver-
sity into a small number of high-level “supergroups,” many of which receive strong support in phylogenomic analyses.
However, the abundance of data in phylogenomic analyses can lead to highly supported but incorrect relationships due
to systematic phylogenetic error. Furthermore, the paucity of major eukaryotic lineages (19 or fewer) included in these
genomic studies may exaggerate systematic error and reduce power to evaluate hypotheses. Here, we use a taxon-rich
strategy to assess eukaryotic relationships. We show that analyses emphasizing broad taxonomic sampling (up to 451 taxa
representing 72 major lineages) combined with a moderate number of genes yield a well-resolved eukaryotic tree of life.
The consistency across analyses with varying numbers of taxa (88–451) and levels of missing data (17–69%) supports the
accuracy of the resulting topologies. The resulting stable topology emerges without the removal of rapidly evolving genes
or taxa, a practice common to phylogenomic analyses. Several major groups are stable and strongly supported in these
analyses (e.g., SAR, Rhizaria, Excavata), whereas the proposed supergroup “Chromalveolata” is rejected. Furthermore, ex-
tensive instability among photosynthetic lineages suggests the presence of systematic biases including endosymbiotic gene
transfer from symbiont (nucleus or plastid) to host. Our analyses demonstrate that stable topologies of ancient evolutionary
relationships can be achieved with broad taxonomic sampling and a moderate number of genes. Finally, taxon-rich analy-
ses such as presented here provide a method for testing the accuracy of relationships that receive high bootstrap support
(BS) in phylogenomic analyses and enable placement of the multitude of lineages that lack genome scale data. [Excavata;
microbial eukaryotes; Rhizaria; supergroups; systematic error; taxon sampling.]
Perspectives on the structure of the eukaryotic tree
of life have shifted in the past decade as molecular
analyses provide hypotheses for relationships among
the approximately 75 robust lineages of eukaryotes.
These lineages are defined by ultrastructural identities
(Patterson 1999)—patterns of cellular and subcellular
organization revealed by electron microscopy—and are
strongly supported in molecular analyses (Parfrey et al.
2006; Yoon et al. 2008). Most of these lineages now
fall within a small number of higher level clades, the
supergroups of eukaryotes (Simpson and Roger 2004;
Adl et al. 2005; Keeling et al. 2005). Several of these
clades—Opisthokonta, Rhizaria, and Amoebozoa—
marks throughout to note groups where uncertainties
remain. Moreover, it is difficult to evaluate the overall
stability of major clades of eukaryotes because phyloge-
nomic analyses have 19 or fewer of the major lineages
and hence do not sufficiently sample eukaryotic diver-
sity (Rodr´ıguez-Ezpeleta et al. 2007b; Burki et al. 2008;
Hampl et al. 2009), whereas taxon-rich analyses with
4 or fewer genes yield topologies with poor support at
deep nodes (Cavalier-Smith 2004; Parfrey et al. 2006;
Yoon et al. 2008).
Estimating the relationships of the major lineages
of eukaryotes is difficult because of both the ancient
age of eukaryotes (1.2–1.8 billion years; Knoll et al.
SYSTEMATIC BIOLOGY VOL. 59
uded all lin-
s additional
study (Table
rted, though
ed: i) Cerco-
Acantharea
adiolarians),
Plasmodio-
Fig. 3; Bass
a nematode-
lid amoebae
r to the plant
e SSU-rDNA
amoeba iso-
as Arachnula
nsistent with
trastructural
contaminant
omastix strix
6). Excavata
ause Malaw-
of Excavata
et al. 2009),
avata mem-
2006; Simp-
ests robustly
not have a
independent
tephanopogon
hin Heterolo-
Yubuki and
igmatic flag-
inia anisocys-
m this study
on sampling
nalyses pro-
d representa-
mbined with
FIGURE 5. Summary of major findings—the evolutionary relation-
ships among major lineages of eukaryotes. Clades have been collapsed
into those that we view to be strongly supported. The many poly-
tomies represent uncertainties that remain.
FUNDING
This work was made possible by the US National
byguestonApril28,2013http://sysbio.oxfordjournals.org/Downloadedfrom
http://sysbio.oxfordjournals.org/content/59/5/518.full
Euks More Resolution but Simpler

Mapping GOLD to Tree
Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
530 SYSTEMATIC BIOLOGY VOL. 59
a 97-taxon data set of Rhizaria that included all lin-
eages with previously published data plus additional
multigene data for 12 taxa added for this study (Table
S1). Three major clades are strongly supported, though
the relationships among them are unresolved: i) Cerco-
zoa, ii) Foraminifera plus Polycystinea and Acantharea
(formerly classified with Phaeodarea as radiolarians),
and (iii) the parasitic Haplosporidia and Plasmodio-
phorida with Gromia and vampyrellids (Fig. 3; Bass
et al. 2009). We show that Theratromyxa, a nematode-
eating soil amoeba, is related to vampyrellid amoebae
(Fig. 3; 100% BS), and together they are sister to the plant
parasites plasmodiophorids (100% BS). The SSU-rDNA
sequence for Theratromyxa is identical to an amoeba iso-
lated from Siberia where it was identified as Arachnula
impatiens (EU567294; Bass et al. 2009).
The topology within the Excavata is consistent with
previous hypotheses and clades with ultrastructural
identities (Simpson 2003; Fig. 4), when contaminant
EST data originally mislabeled as Streblomastix strix
are excluded (Slamovits and Keeling 2006). Excavata
is often polyphyletic in other analyses because Malaw-
imonas branches outside the other clades of Excavata
(Rodr´ıguez-Ezpeleta et al. 2007a; Hampl et al. 2009),
whereas in analyses of fewer genes Excavata mem-
bers fall into 2 or 3 clades (Parfrey et al. 2006; Simp-
son et al. 2006). Although Malawimonas nests robustly
within Excavata in our analyses, it does not have a
stable sister group and may represent an independent
lineage (Fig. 4). Our analyses confirm that Stephanopogon
(unplaced in Patterson 1999) branches within Heterolo-
bosea (Cavalier-Smith and Nikolaev 2008; Yubuki and
Leander 2008) and suggests that another enigmatic flag-
ellate, ATCC 50646 (tentatively named Soginia anisocys-
tis) is a basal member of Heterolobosea.
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic flag-
Soginia anisocys-

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-
Fungi
49%

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-
Animals
26%

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-
Green
algae
19%

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-
Apicomplexa
5%

Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-
A Very Biased Sampling

Solution to Biased Sampling?
Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
included all lin-
a plus additional
this study (Table
esolved: i) Cerco-
as radiolarians),
and Plasmodio-
Bass
a nematode-
pyrellid amoebae
sister to the plant
to an amoeba iso-
Arachnula
th ultrastructural
hen contaminant
). Excavata
Malaw-
),
s Excavata mem-
Simp-
t an independent
within Heterolo-
er enigmatic ﬂag-
Soginia anisocys-

Solution: Fill in the Tree
Priapulida 1 0
Phaeophyceae 1 0
Rotifera 1 0
Hemichordata 1 0
Pinguiophyceae 1 0
Ctenophora 1 0
Bolidophyceae 1 0
Chaetognatha 1 0
Porifera 2 0
Xanthophyceae 2 0
Tardigrada 2 0
Euglenida 2 0
Chromerida 3 0
Placozoa 3 0
Glomeromycota 3 0
Cryptomycota 4 0
Echinodermata 6 0
Annelida 13 0
Cnidaria 18 0
Mollusca 25 0
Microsporidia 31 1
Chlorophyta 77 1
Nematoda 110 2
Apicomplexa 264 5
Arthropoda 370 7
Chordata 626 12
Streptophyta 796 15
Ascomycota 1,251 23
SYSTEMATIC BIOLOGY
included all lin-
a plus additional
this study (Table
upported, though
esolved: i) Cerco-
a and Acantharea
as radiolarians),
and Plasmodio-
lids (Fig. 3; Bass
yxa, a nematode-
pyrellid amoebae
sister to the plant
). The SSU-rDNA
to an amoeba iso-
ified as Arachnula
is consistent with
th ultrastructural
hen contaminant
Streblomastix strix
g 2006). Excavata
s because Malaw-
ades of Excavata
ampl et al. 2009),
s Excavata mem-
et al. 2006; Simp-
nas nests robustly
does not have a
t an independent
hat Stephanopogon
within Heterolo-
2008; Yubuki and
er enigmatic flag-
d Soginia anisocys-
a.
FI G U R E 5. Summary of major findings
ships among major lineages of eukaryotes.Monday, April 29, 13

II: Filling in the Tree Example

Big Microbial Sequencing Projects
• Coordinated, top-down efforts
• Fungal Genome Initiative (Broad/Whitehead)
• Gordon and Betty Moore Foundation Marine Microbial Genome
Sequencing Project
• Sanger Center Pathogen Sequencing Unit
• NHGRI Human Gut Microbiome Project
• NIH Human Microbiome Program
• White paper or grant systems
• NIAID Microbial Sequencing Centers
• DOE/JGI Community Sequencing Program
• DOE/JGI BER Sequencing Program
• NSF/USDA Microbial Genome Sequencing
• Covers lots of ground and biological diversity

As of 2002

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40
phyla of
bacteria
As of 2002
Tree Based on Hugenholtz,
2002.
http://genomebiology.com/
2002/3/2/reviews/0003

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40
phyla of
bacteria
• Genome
sequences are
mostly from
three phyla
As of 2002
2002.
2002/3/2/reviews/0003

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40
phyla of
bacteria
• Genome
sequences are
mostly from
three phyla
• Some other
phyla are only
sparsely
sampled
As of 2002
2002.
2002/3/2/reviews/0003

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla
of bacteria
• Genome
sequences are
mostly from
three phyla
• Some other
phyla are only
sparsely sampled
• Solution I:
sequence more
phyla
• NSF-funded
Tree of Life
Project
• A genome
from each of
eight phyla
Eisen, Ward, Robb,
Nelson, et al
2002.
2002/3/2/reviews/0003

Phylum Species selected
Chrysiogenes Chrysiogenes arsenatis (GCA)
Coprothermobacter Coprothermobacter proteolyticus (GCBP)
Dictyoglomi Dictyoglomus thermophilum (GD T )
Thermodesulfobacteria Thermodesulfobacterium commune (GTC)
Nitrospirae Thermodesulfovibrio yellowstonii (GTY)
Thermomicrobia Thermomicrobium roseum (GTR )
Deferribacteres Geovibrio thiophilus (GGT)
Synergistes Synergistes jonesii (GSJ)
Organisms Selected

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
of bacteria
• Genome
sequences are
mostly from three
phyla
• Some other phyla
are only sparsely
sampled
• Still highly biased
in terms of the
tree
• NSF-funded
Tree of Life
Project
• A genome from
each of eight
phyla
Eisen & Ward, PIs Tree Based on Hugenholtz,
2002.
2002/3/2/reviews/0003

Major Lineages of Actinobacteria
2.5.1 Acidimicrobidae
2.5.1.1 Unclassified
2.5.1.2 "Microthrixineae
2.5.1.3 Acidimicrobineae
2.5.1.4 BD2-10
2.5.1.5 EB1017
2.5.2 Actinobacteridae
2.5.2.10 Ellin306/WR160
2.5.2.11 Ellin5012
2.5.2.12 Ellin5034
2.5.2.13 Frankineae
2.5.2.14 Glycomyces
2.5.2.15 Intrasporangiaceae
2.5.2.16 Kineosporiaceae
2.5.2.17 Microbacteriaceae
2.5.2.18 Micrococcaceae
2.5.2.19 Micromonosporaceae
2.5.2.2 Actinomyces
2.5.2.20 Propionibacterineae
2.5.2.21 Pseudonocardiaceae
2.5.2.22 Streptomycineae
2.5.2.23 Streptosporangineae
2.5.2.3 Actinomycineae
2.5.2.4 Actinosynnemataceae
2.5.2.5 Bifidobacteriaceae
2.5.2.6 Brevibacteriaceae
2.5.2.7 Cellulomonadaceae
2.5.2.8 Corynebacterineae
2.5.2.9 Dermabacteraceae
2.5.3 Coriobacteridae
2.5.3.2 Atopobiales
2.5.3.3 Coriobacteriales
2.5.3.4 Eggerthellales
2.5.4 OPB41
2.5.5 PK1
2.5.6 Rubrobacteridae
2.5.6.2 "Thermoleiphilaceae
2.5.6.3 MC47
2.5.6.4 Rubrobacteraceae
2.5 Actinobacteria
2.5.1 Acidimicrobidae
2.5.1.2 "Microthrixineae
2.5.1.3 Acidimicrobineae
2.5.1.3.1 Unclassified
2.5.1.3.2 Acidimicrobiaceae
2.5.1.4 BD2-10
2.5.1.5 EB1017
2.5.2 Actinobacteridae
2.5.2.10 Ellin306/WR160
2.5.2.11 Ellin5012
2.5.2.12 Ellin5034
2.5.2.13 Frankineae
2.5.2.13.2 Acidothermaceae
2.5.2.13.3 Ellin6090
2.5.2.13.4 Frankiaceae
2.5.2.13.5 Geodermatophilaceae
2.5.2.13.6 Microsphaeraceae
2.5.2.13.7 Sporichthyaceae
2.5.2.14 Glycomyces
2.5.2.15 Intrasporangiaceae
2.5.2.15.2 Dermacoccus
2.5.2.15.3 Intrasporangiaceae
2.5.2.16 Kineosporiaceae
2.5.2.17 Microbacteriaceae
2.5.2.17.2 Agrococcus
2.5.2.17.3 Agromyces
2.5.2.18 Micrococcaceae
2.5.2.19 Micromonosporaceae
2.5.2.2 Actinomyces
2.5.2.20 Propionibacterineae
2.5.2.20.2 Kribbella
2.5.2.20.3 Nocardioidaceae
2.5.2.20.4 Propionibacteriaceae
2.5.2.21 Pseudonocardiaceae
2.5.2.22 Streptomycineae
2.5.2.22.2 Kitasatospora
2.5.2.22.3 Streptacidiphilus
2.5.2.23 Streptosporangineae
2.5.2.23.2 Ellin5129
2.5.2.23.3 Nocardiopsaceae
2.5.2.23.4 Streptosporangiaceae
2.5.2.23.5 Thermomonosporaceae
2.5.2.3 Actinomycineae
2.5.2.4 Actinosynnemataceae
2.5.2.5 Bifidobacteriaceae
2.5.2.6 Brevibacteriaceae
2.5.2.7 Cellulomonadaceae
2.5.2.8 Corynebacterineae
2.5.2.8.2 Corynebacteriaceae
2.5.2.8.3 Dietziaceae
2.5.2.8.4 Gordoniaceae
2.5.2.8.5 Mycobacteriaceae
2.5.2.8.6 Rhodococcus
2.5.2.9 Dermabacteraceae
2.5.2.9.2 Brachybacterium
2.5.2.9.3 Dermabacter
2.5.3 Coriobacteridae
2.5.3.2 Atopobiales
2.5.3.3 Coriobacteriales
2.5.3.4 Eggerthellales
2.5.4 OPB41
2.5.5 PK1
2.5.6 Rubrobacteridae
2.5.6.2 "Thermoleiphilaceae
2.5.6.2.2 Conexibacter
2.5.6.2.3 XGE514
2.5.6.3 MC47
2.5.6.4 Rubrobacteraceae

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
of bacteria
• Genome
sequences are
mostly from three
phyla
are only sparsely
sampled
• Same trend in
Archaea
• NSF-funded
Tree of Life
Project
• A genome from
each of eight
phyla
2002.
2002/3/2/reviews/0003

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
of bacteria
• Genome
sequences are
mostly from three
phyla
are only sparsely
sampled
• Same trend in
Eukaryotes
• NSF-funded
Tree of Life
Project
• A genome from
each of eight
phyla
2002.
2002/3/2/reviews/0003

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
of bacteria
• Genome
sequences are
mostly from three
phyla
are only sparsely
sampled
• Same trend in
Viruses
• NSF-funded
Tree of Life
Project
• A genome from
each of eight
phyla
2002.
2002/3/2/reviews/0003

of bacteria
• Genome
sequences are
mostly from three
phyla
are only sparsely
sampled
• Solution: Really
Fill in the Trees
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroﬂexi
Proteobacteria
Chlorobi
Firmicutes
Fusobacteria
Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquiﬁcae
Thermotogae
TM6
OS-K
Termite Group
OP8
Marine GroupA
WS3
OP9
NKB19
OP3
OP10
TM7
OP1
OP11
Nitrospira
Synergistes
Deferribacteres
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
2002.
2002/3/2/reviews/0003

Filling in the Tree
Figure from Barton, Eisen et al. “Evolution”, CSHL Press based on Baldauf et al Tree

Filling in the Tree

Lots of Plants, Animals, Fungi

Exclude Plants, Animals, Fungi

A Genomic Encyclopedia of Microbes (GEM)

Just Say No to Eukaryotes

GEBA: A Genomic Encyclopedia
of Bacteria and Archaea

GEBA Pilot Project: Components
• Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan
Eisen, Eddy Rubin, Jim Bristow)
• Project management (David Bruce, Eileen Dalin, Lynne
Goodwin)
• Culture collection and DNA prep (DSMZ, Hans-Peter Klenk)
• Sequencing and closure (Eileen Dalin, Susan Lucas, Alla
Lapidus, Mat Nolan, Alex Copeland, Cliff Han, Feng Chen,
Jan-Fang Cheng)
• Annotation and data release (Nikos Kyrpides, Victor
Markowitz, et al)
• Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu,
Victor Kunin, Neil Rawlings, Ian Paulsen, Patrick Chain,
Patrik D’Haeseleer, Sean Hooper, Iain Anderson, Amrita Pati,
Natalia N. Ivanova, Athanasios Lykidis, Adam Zemla)
• Adopt a microbe education project (Cheryl Kerfeld)
• Outreach (David Gilbert)
• $$$ (DOE, Eddy Rubin, Jim Bristow)

rRNA Tree of Life
FIgure from Barton, Eisen et al.
“Evolution”, CSHL Press.
Based on tree from Pace NR, 2003.

rRNA Tree of BA
FIgure from Barton, Eisen et al.
“Evolution”, CSHL Press.
Based on tree from Pace NR, 2003.

GreenGenes

GEBA Pilot Project Overview
• Identify major branches in rRNA tree for which
no genomes are available
• Identify those with a cultured representative in
DSMZ
• DSMZ grew > 200 of these and prepped DNA
• Sequence and finish 200+
• Annotate, analyze, release data
• Assess benefits of tree guided sequencing
• 1st paper Wu et al in Nature Dec 2009

GEBA Pilot Target List
0
5
10
15
20
25
30
35
B:
Actinobacteria
(H
igh
G
C)
B:
Am
inanaerobia
B:
Aquificae
B:
Bacteroidetes
B:
Chloroflexi
B:
D
eferribacteres
B:
D
eferribacteres
B:
D
einococci
B:
D
elta
Proteobacteria
B:
Epsilon
Proteobacteria
B:
Firm
icutes
B:
Fusobacteria
B:
G
am
m
a
Proteobacteria
B:
G
em
m
atim
onadetes
B:
H
aloanaerobiales
B:
Planctom
ycetes
B:
Spirochaetes
B:
Therm
odesulfobacteria
B:
Therm
odesulfobia
B:
Therm
ovenabulae
A:
H
alobacteria
A:
Archaeoglobi
A:
M
ethanobacteria
A:
M
ethanom
icrobia
A:
Therm
ococci
A:
Therm
oprotei
Phyla
#ofGenomes
GEBA Initial Target List

Assess Benefits of GEBA
• All genomes have some value
• But what, if any, is the benefit of tree-
guided sequencing over other selection
methods
• Lessons for other large scale microbial
genome projects?

Lessons from GEBA

Lesson 1: rRNA PD IDs novel lineages
From Wu et al. 2009 Nature 462, 1056-1060

Concatenated Marker PD

Lesson 2: rRNA Tree is not perfect
Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026.
16s WGT, 23S

How Pick Novel Lineages for Euks?
• Molecular
• rRNA PD?
• Conserved markers by PCR?
• EST shotgun?
• Other data for phylogeny

Lesson 3: Improves annotation
• Took 56 GEBA genomes and compared results vs. 56
randomly sampled new genomes
• Better definition of protein family sequence “patterns”
• Greatly improves “comparative” and “evolutionary”
based predictions
• Conversion of hypothetical into conserved hypotheticals
• Linking distantly related members of protein families
• Improved non-homology prediction

Annotation for Euks?

Lesson 4 : Metadata Important

Lesson 5: Project management critical
• Tracking samples and status
• Getting permissions
• Shipping samples
• Contacting collaborators
• Data archiving and submission
• Communicating with core facilities
• and more

Lesson 6: Culture Collections Needed

Lesson 7: Data Publications

Lesson 8: Diversity Discovery
• Phylogeny-driven genome selection helps
discover new genetic diversity

Protein Family Rarefaction
• Take data set of multiple complete
genomes
• Identify all protein families using MCL
• Plot # of genomes vs. # of protein families

Wu et al. 2009 Nature 462, 1056-1060

Synapomorphies exist
Wu et al. 2009 Nature 462, 1056-1060

True for Euks?

Lesson 9: Improves metagenomics

Phylotyping
0
0.125
0.250
0.375
0.500
Alphaproteobacteria
Betaproteobacteria
G
am
m
aproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
C
yanobacteriaFirm
icutesActinobacteriaC
hlorobi
C
FB
C
hloroflexiSpirochaetesFusobacteria
Deinococcus-Therm
us
Euryarchaeota
C
renarchaeota
Sargasso Phylotypes
Weighted%ofClones
Major Phylogenetic Group
EFG EFTu
HSP70 RecA
RpoB rRNA
Venter et al., Science 304: 66-74. 2004
GEBA Project
improves
metagenomic
analysis

Eukaryotic Metagenomics?

GEBA Zoom

GEBA Now
• 300+ genomes
• Rich sampling of major groups of
cultured organisms
• Zoomed in sampling of haloarchaea,
cyanobacteria and more

GEBA Cyanobacteria
www.pnas.org/cgi/doi/10.1073/pnas.1217107110

Haloarchaeal GEBA-like
Lynch EA, Langille MGI, Darling A, Wilbanks EG, Haltiner C, et al. (2012) Sequencing of Seven Haloarchaeal
Genomes Reveals Patterns of Genomic Flux. PLoS ONE 7(7): e41389. doi:10.1371/journal.pone.0041389

88
Plan:
Sequence multiple Root Nodule Bacteria (RNBs) across the
planet. Pilot: 100 RNBs.
Alpha RNB
Bradyrhizobium
Mesorhizobium
Rhizobium
Beta RNB
Sinorhizobium
Cupriavidis
Burkholderia
Balneimonas-like
Devosia
Ochrobactrum
Phyllobacterium
Azorhizobium
Allorhizobium
Goal:
• Understand BioGeographical effects on species
evolution and understand host-specificity.
Rationale:
• N2 fixation by legume pastures and crops provides 65% of the
N currently utilized in agricultural production.
• Contributes 25 to 90 million metric tones N pa.
• Symbioses save $US 6-10 billion annually on N fertilizer.
• Grain and animal production enhanced by fixed nitrogen
supplied by the symbiosis.
Nikos Kyrpides
GEBA RNB

Phylotyping
0
0.125
0.250
0.375
0.500
Alphaproteobacteria
Betaproteobacteria
G
am
m
aproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
C
yanobacteriaFirm
icutesActinobacteriaC
hlorobi
C
FB
C
hloroflexiSpirochaetesFusobacteria
Deinococcus-Therm
us
Euryarchaeota
C
renarchaeota
Sargasso Phylotypes
Weighted%ofClones
Major Phylogenetic Group
EFG EFTu
HSP70 RecA
RpoB rRNA
But not a lot
Venter et al., Science 304: 66-74. 2004

Phylogenomics Future 1
• Need to adapt genomic and metagenomic
methods to make better use of data

Improving Metagenomic Analysis
• Methods
• More automation
• Better phylogenetic methods for short reads
and large data sets
• Improved tools for using distantly related
genomes in metagenomic analysis
• Data sets
• Rebuild protein family models
• New phylogenetic markers
• Need better reference phylogenies, including
HGT
• More simulations

Kembel Correction
Kembel, Wu, Eisen, Green. In press.
PLoS Computational Biology.
Incorporating 16S gene copy number
information improves estimates of
microbial diversity and abundance

alignment used to build the profile, resulting in a multiple
sequence alignment of full-length reference sequences and
metagenomic reads. The final step of the alignment process is a
PD versus PID clustering, 2) to explore overlap betw
clusters and recognized taxonomic designations, and
the accuracy of PhylOTU clusters from shotgun re
Figure 1. PhylOTU Workflow. Computational processes are represented as squares and databases are represented as cylinders in
workflow of PhylOTU. See Results section for details.
doi:10.1371/journal.pcbi.1001061.g001
Finding Meta
Sharpton TJ, Riesenfeld SJ, Kembel SW, Ladau J, O'Dwyer JP, Green JL, Eisen JA, Pollard KS. (2011)
PhylOTU: A High-Throughput Procedure Quantifies Microbial Community Diversity and Resolves Novel
Taxa from Metagenomic Data. PLoS Comput Biol 7(1): e1001061. doi:10.1371/journal.pcbi.1001061
PhylOTU

Phylosift/ pplacer
Aaron Darling, Guillaume Jospin, Holly Bik, Erik Matsen, Eric
Lowe, and others

Kembel Combiner
typically used as a qualitative measure because duplicate s
quences are usually removed from the tree. However, the
test may be used in a semiquantitative manner if all clone
even those with identical or near-identical sequences, are i
cluded in the tree (13).
Here we describe a quantitative version of UniFrac that w
call “weighted UniFrac.” We show that weighted UniFrac b
haves similarly to the FST test in situations where both a
FIG. 1. Calculation of the unweighted and the weighted UniFr
measures. Squares and circles represent sequences from two differe
environments. (a) In unweighted UniFrac, the distance between t
circle and square communities is calculated as the fraction of t
branch length that has descendants from either the square or the circ
environment (black) but not both (gray). (b) In weighted UniFra
branch lengths are weighted by the relative abundance of sequences
the square and circle communities; square sequences are weight
twice as much as circle sequences because there are twice as many tot
circle sequences in the data set. The width of branches is proportion
to the degree to which each branch is weighted in the calculations, an
gray branches have no weight. Branches 1 and 2 have heavy weigh
since the descendants are biased toward the square and circles, respe
tively. Branch 3 contributes no value since it has an equal contributio
from circle and square sequences after normalization.
Kembel SW, Eisen JA, Pollard KS, Green JL (2011) The Phylogenetic Diversity of Metagenomes. PLoS
ONE 6(8): e23214. doi:10.1371/journal.pone.0023214

NMF in MetagenomesCharacterizing the niche-space distributions of components
Sites
North American East Coast_GS005_Embayment
North American East Coast_GS002_Coastal
North American East Coast_GS011_Estuary
Eastern Tropical Pacific_GS021_Coastal
North American East Coast_GS006_Estuary
Polynesia Archipelagos_GS051_Coral Reef Atoll
Galapagos Islands_GS036_Coastal
Indian Ocean_GS117a_Coastal sample
Galapagos Islands_GS031_Coastal upwelling
Galapagos Islands_GS030_Warm Seep
Sargasso Sea_GS001c_Open Ocean
Eastern Tropical Pacific_GS022_Open Ocean
Indian Ocean_GS149_Harbor
Indian Ocean_GS123_Open Ocean
Caribbean Sea_GS016_Coastal Sea
Indian Ocean_GS148_Fringing Reef
Indian Ocean_GS112a_Open Ocean
Caribbean Sea_GS017_Open Ocean
Caribbean Sea_GS018_Open Ocean
Indian Ocean_GS108a_Lagoon Reef
Eastern Tropical Pacific_GS023_Open Ocean
Caribbean Sea_GS019_Coastal
Caribbean Sea_GS015_Coastal
Galapagos Islands_GS026_Open Ocean
Polynesia Archipelagos_GS049_Coastal
Polynesia Archipelagos_GS048a_Coral Reef
Component 1
Component 2
Component 3
Component 4
Component 5
0.1 0.2 0.3 0.4 0.5 0.6 0.2 0.4 0.6 0.8 1.0
Salinity
SampleDepth
Chlorophyll
Temperature
Insolation
WaterDepth
General
High
M edium
Low
NA
High
M edium
Low
NA
Water depth
>4000m
2000!4000m
900!2000m
100!200m
20!100m
0!20m
>4000m
2000!4000m
900!2000m
100!200m
20!100m
0!20m
(a) (b) (c)
Figure 3: a) Niche-space distributions for our ﬁve components (HT
); b) the site-
similarity matrix ( ˆHT ˆH); c) environmental variables for the sites. The matrices are
aligned so that the same row corresponds to the same site in each matrix. Sites are
ordered by applying spectral reordering to the similarity matrix (see Materials and
Methods). Rows are aligned across the three matrices.
Functional biogeography of ocean microbes
revealed through non-negative matrix
factorization Jiang et al. In press PLoS
One. Comes out 9/18.
w/ Weitz, Dushoff,
Langille, Neches,
Levin, etc

More Markers
Phylogenetic group Genome
Number
Gene
Number
Maker
Candidates
Archaea 62 145415 106
Actinobacteria 63 267783 136
Alphaproteobacteria 94 347287 121
Betaproteobacteria 56 266362 311
Gammaproteobacteria 126 483632 118
Deltaproteobacteria 25 102115 206
Epislonproteobacteria 18 33416 455
Bacteriodes 25 71531 286
Chlamydae 13 13823 560
Chloroflexi 10 33577 323
Cyanobacteria 36 124080 590
Firmicutes 106 312309 87
Spirochaetes 18 38832 176
Thermi 5 14160 974
Thermotogae 9 17037 684

Better Reference Tree
Morgan et al.
submitted

Sifting Families
Representative
Genomes
Extract
Protein
Annotation
All v. All
BLAST
Homology
Clustering
(MCL)
SFams
Align &
Build
HMMs
HMMs
Screen for
Homologs
New
Genomes
Extract
Protein
Annotation
Figure 1
Sharpton et al. submitted
A
B
C

Zorro - Automated Masking
cetoTrueTree
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
200 400 800 1600 3200
DistancetoTrueTree
Sequence Length
200
no masking
zorro
gblocks
Wu M, Chatterji S, Eisen JA (2012) Accounting For Alignment Uncertainty
in Phylogenomics. PLoS ONE 7(1): e30288. doi:10.1371/journal.pone.
0030288

• We have still only scratched the surface
of microbial diversity

rRNA Tree of Life
Figure from Barton, Eisen et al. “Evolution”, CSHL
Press. 2007.
Based on tree from Pace 1997 Science 276:734-740
Archaea
Eukaryotes
Bacteria

PD: Genomes

PD: Genomes + GEBA

PD: Isolates

PD: All

Uncultured Lineages: Methods
• Get into culture
• Enrichment cultures
• If abundant in low diversity ecosystems
• Flow sorting
• Microbeads
• Microfluidic sorting
• Single cell amplification

110
Number of SAGs from Candidate Phyla
OD1
OP11
OP3
SAR406
Site A: Hydrothermal vent 4 1 - -
Site B: Gold Mine 6 13 2 -
Site C: Tropical gyres (Mesopelagic) - - - 2
Site D: Tropical gyres (Photic zone) 1 - - -
Sample collections at 4 additional sites are underway.
Phil Hugenholtz
GEBA Uncultured

Uncultured Eukaryotes?

• Need Experiments from Across the Tree
of Life too

The need for a phylogeny driven genomic encyclopedia of eukaryotes #SMBEEuks

The need for a phylogeny driven genomic encyclopedia of eukaryotes #SMBEEuks

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