Estimating the timing of early eukaryotic
diversification with multigene molecular clocks
Laura Wegener Parfreya,b,2, Daniel J. G. Lahra,b, Andrew H. Knollc,1, and Laura A. Katza,b,1
a
Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA 01003; bDepartment of Biological Sciences, Smith College,
Northampton, MA 01063; and cDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138
Contributed by Andrew H. Knoll, July 1, 2011 (sent for review February 9, 2011)
Although macroscopic plants, animals, and fungi are the most
familiar eukaryotes, the bulk of eukaryotic diversity is microbial.
Elucidating the timing of diversification among the more than 70
lineages is key to understanding the evolution of eukaryotes. Here,
we use taxon-rich multigene data combined with diverse fossils
and a relaxed molecular clock framework to estimate the timing
of the last common ancestor of extant eukaryotes and the diver-
gence of major clades. Overall, these analyses suggest that the last
common ancestor lived between 1866 and 1679 Ma, consistent with
the earliest microfossils interpreted with confidence as eukaryotic.
During this interval, the Earth’s surface differed markedly from to-
day; for example, the oceans were incompletely ventilated, with
ferruginous and, after about 1800 Ma, sulfidic water masses com-
monly lying beneath moderately oxygenated surface waters. Our
time estimates also indicate that the major clades of eukaryotes
diverged before 1000 Ma, with most or all probably diverging be-
fore 1200 Ma. Fossils, however, suggest that diversity within major
extant clades expanded later, beginning about 800 Ma, when the
oceans began their transition to a more modern chemical state. In
combination, paleontological and molecular approaches indicate
that long stems preceded diversification in the major eukaryotic
lineages.
microbial eukaryotes | Proterozoic oceans | taxon sampling |
origin of eukaryotes
T he antiquity of eukaryotes and the tempo of early eukaryotic
diversification remain open questions in evolutionary biology.
Proposed dates for the origin of the domain based on the fossil
record and molecular clock analyses differ by up to 2 billion years
(1). Microfossils attributed to eukaryotes occur at about 1800 Ma
(2) and putative biomarkers of early eukaryotes have been found
in 2700 Ma rocks (3). Such geological interpretations contrast
with both molecular clock studies that place the origin of
eukaryotes at 1250–850 Ma (4, 5), and a controversial hypothesis
that rejects the eukaryotic interpretation of all older fossils and
places eukaryogenesis at 850 Ma (6, 7).
Paleontologists generally agree that an unambiguous record
of eukaryotic microfossils extends back to ∼1800 Ma (2, 8, 9).
Microfossils of this age are assigned to eukaryotes because they
combine informative characters that include complex morphol-
ogy (e.g., the presence of processes and evidence for real-time
modification of vegetative morphology), complex wall ultra-
structure, and specific inferred behaviors (2, 9, 10). Despite being
interpreted as eukaryotic, the taxonomic affinities of these fossils
remain unclear (2). Eukaryotic fossils that can be assigned to
extant taxonomic groups begin to appear ∼1200 Ma (11) and
become more widespread, abundant, and diverse in rocks ∼800
Ma and younger (2, 12, 13).
Molecular estimation of divergence times has improved dra-
matically in recent years due the development of methods that
incorporate uncertainty from sources that include phylogenetic
reconstruction, fossil calibrations, and heterogeneous rates of
molecular evolution (1, 14, 15). Relaxed clock approaches ac-
count for heterogeneity in evolutionary rates across branches
and enable the use of complex models of sequence evolution
(reviewed in refs. 16 and 17), although debate continues as to the
best method for relaxing the clock (18–20). The process of cal-
13624–13629 | PNAS | August 16, 2011 | vol. 108 | no. 33
ibrating molecular clocks has also been greatly improved with
both the recognition that single calibration points are insufficient
(21, 22), and the availability of methods incorporate uncertainty
from the fossil record by specifying calibrations as time dis-
tributions rather than points (15, 16). Additional limitations in
previous molecular clock studies of eukaryotes stem from the
tradeoff between analyses of many taxa and calibration points
but only a single gene (4), and analyses of many genes but a small
number of taxa and calibrations (5, 23).
Molecular clock estimates rely on robust phylogenies. Recon-
structions of relationships among eukaryotes have begun to sta-
bilize in recent years with the increasing availability of multigene
data from diverse lineages (24–26). The majority of the >70 lin-
eages of eukaryotes fall within four major groups: Opisthokonta;
Excavata; Amoebozoa; and Stramenopiles, Alveolates, and Rhi-
zaria (SAR) (25, 26), while the placement of some photosynthetic
lineages remains controversial (25, 27, 28). Greater data avail-
ability also yields more accurate estimates of divergence times
because more nodes are available for calibration (29).
The availability of taxon- and gene-rich datasets coupled with
flexible molecular clock methods make this an ideal time to re-
visit the timing of early eukaryotic evolution. Here, broadly
sampled multigene trees are used to estimate dates, with rate
heterogeneity across the tree and among genes incorporated into
the model. We use 23 calibration points derived from diverse
fossils of Proterozoic and Phanerozoic age specified as prior
distributions (Table 1). The Proterozoic fossil record is sparse (2,
8, 9), and the taxonomic assignment of some Proterozoic fossils
has been called into question by a minority of researchers (6). In
the spirit of testing these ideas, we assess the impact of including
calibration constraints derived from Phanerozoic fossils alone
and Phanerozoic plus Proterozoic fossils. We also assess di-
vergence dates across analyses that varied in the position of the
root, and the number of taxa included, as well as across different
software platforms and models.
Results
Taxon-rich analyses of multiple genes reveal a stability in di-
vergence dates across the eukaryotic tree of life that is robust to
changing taxon inclusion, position of the root, molecular clock
model, and choice of calibration points (Phanerozoic only or
both Phanerozoic and Proterozoic fossils). Collectively, these
analyses provide a mean age for the root of extant eukaryotes
to 1866–1679 Ma in analyses including both Proterozoic and
Phanerozoic calibrations (“All” analyses; Fig. 1A and Table S1).
Varying the position of the root had little impact on divergence
Author contributions: L.W.P. and L.A.K. designed research; L.W.P. and D.J.G.L. performed
research; L.W.P., D.J.G.L., A.H.K., and L.A.K. analyzed data; and L.W.P., D.J.G.L., A.H.K.,
and L.A.K. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
To whom correspondence may be addressed. E-mail: aknoll@oeb.harvard.edu or lkatz@
smith.edu
2
Present address: Department of Chemistry and Biochemistry, University of Colorado,
Boulder, CO 80309.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1110633108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1110633108
 Table 1. Calibration constraints for dating the eukaryotic tree of life
Calibration†

Taxon Fossil Eon* Min Dist Ref(s).

Amniota Westlonthania Phan 328.3 4, 3 (54)

Angiosperms Oldest angio pollen Phan 133.9 2, 10 (55)
Ascomycetes Paleopyrenomycites Phan 400 4, 50 (56)
Coccolithophores Earliest Heterococcolith Phan 203.6 2, 8 (57)
Diatoms Earliest diatoms Phan 133.9 2, 100 (58)
Dinoflagellates Earliest gonyaulacales Phan 240 2, 10 (59)
Embryophytes Land plant spores Phan 471 2, 20 (60)
Endopterygota Mecoptera Phan 284.4 5, 5 (61)
Eudicots Eudicot pollen Phan 125 2, 1.5 (62, 63)
Euglenids Moyeria Phan 450 2, 40 (64)
Foraminifera Oldest forams Phan 542 2, 200 (65)
Gonyaulacales Gonyaulacaceae split Phan 196 2,10 (59)
Pennate diatoms Oldest pennate Phan 80 3, 5 (66)
Spirotrichs Oldest tintinnids Phan 444 2.5, 100 (67)
Trachaeophytes Earliest trachaeophytes Phan 425 4, 2.5 (68)
Vertebrates Haikouichthys Phan 520 3, 5 (69)
Animals LOEMs, sponge biomarkers Protero 632 2, 300 (70, 71)
Arcellinida Paleoarcella Protero 736 2, 300 (12)
Bilateria Kimberella Protero 555 2, 30 (72)
Chlorophytes Palaeastrum Protero 700 2.5, 300 (73)
Ciliates Gammacerane Protero 736 2.5, 300 (74)
Florideophyceae Doushantuo red algae Protero 550 2.5, 100 (75)
Red algae‡ Bangiomorpha Protero 1174 3, 250 (11)

*Eon: Phan, Phanerozoic; Protero, Proterozoic. Proterozoic calibrations are excluded from Phan analyses.
†
Calibration constraints are specified for BEAST using a gamma distribution with a minimum date in Ma based
on the fossil record parameters as indicated: min, minimum divergence data; dist, gamma prior distribution
(shape, scale). See Table S3 for details of PhyloBayes calibrations.
‡
In the All 720 analysis (c), the minimum age constraint for the red algae node is set to 720 Ma.

dates, especially for the estimated date of the root itself, which
generally changed by <100 million years (myr; Fig. 1A). Phylo-
bayes estimates generally showed more uncertainty than those
from BEAST analyses, but around similar means. Similarly,
estimates were robust to changing models (uncorrelated or
autocorrelated) and to the inclusion of only Phanerozoic (Phan)
or all calibrations (All) with one exception: under the auto-
age of 720 Ma to this constraint, representing the absolute
younger bound of the Hunting Formation, Canada, in which it is
found (SI Text) (11). In BEAST, placing the Bangiomorpha
constraint at 720 Ma shifted the estimated age of the root by only
95 myr toward the present (Fig. 1A and Fig. S3, analysis c).
The autocorrelated CIR model combined with the low number
of substitutions on deep branches of the eukaryotic tree appears

correlated Cox–Ingersoll–Ross (CIR) model, estimates are much
more recent in Phan analyses (1038 Ma and 1180 Ma; Fig. 1A).
Impact of Calibration Constraints on Estimates of the Origin of Extant
Eukaryotes. We assessed the impact of including Proterozoic
fossils, which are considered controversial by some (6, 7), by
analyzing datasets without these seven calibration constraints
(Phan analyses). In BEAST analyses, the exclusion of Proterozoic
fossils shifted estimated divergence times toward the present, but
not dramatically so: estimates for the mean age of root of extant
eukaryotes fall between 1506–1471 Ma in Phan analyses [95%
highest-probability density (HPD) range 1643–1347 Ma; Fig. 1A,
Figs. S1, S5, and S7, analyses b, f, and h] compared with 1837–
1717 Ma (95% HPD range 1954–1601 Ma; Figs. 1A and 2 and
Figs. S4 and S6; analyses a, e, and g) when Proterozoic fossils
were included (All analyses). Similar dates were recovered in
Phan and All PhyloBayes analyses when the uncorrelated gamma
model (UGAM) model (uncorrelated) of the molecular clock
was assumed (Fig. 1A, analyses i–l).
Of the seven Proterozoic calibration points used in our anal-
yses, only the Bangiomorpha point is controversial in terms of
either systematic attribution or age. The Bangiomorpha calibra-
tion constraint is more than 400 myr older than our other Pro-
terozoic constraints (Table 1). To determine whether this
calibration point drives results in analyses with All calibrations,
we assessed the age of the root with a much more conservative
estimate for the age of this red alga (All 720; Fig. 1, analysis c). A
number of factors place the age of Bangiomorpha ∼1200 Ma (SI
Text); however, given the importance of the fossil we assigned an
EVOLUTION
more sensitive to the distribution of calibration dates included in
these analyses. Under the CIR autocorrelated model, a consistent
age was estimated with All calibrations included (1798–1691 Ma;
Fig. 1A, analyses m and o), although confidence intervals are
greater in PhyloBayes analyses in general (Fig. 1A, analyses i–p).
However, excluding Proterozoic calibration points did cause es-
timated ages to shift more than 600 myr younger under the CIR
model (1180–1038 Ma; Fig. 1A, analyses n and p), pushing the
estimated age for the root of extant eukaryotes younger than
the widely accepted date for the Bangiomorpha fossils. Similarly,
the CIR analyses in PhyloBayes were sensitive to the age of the
Bangiomorpha constraint, shifting more than 500 myr younger to
1296 Ma and 1167 Ma in analyses with All calibration points
rooted with Opisthokonta and “Unikonta,” respectively (Dataset
S1). The necessity of using PhyloBayes to explore the differences
between autocorrelated and uncorrelated models introduces
confounding factors, as PhyloBayes requires both uniform dis-
tributions around calibration points and a fixed tree topology.
Given that calibration points are likely best represented by more
informative distributions, and that the topology of the tree is not
fully known, we focus the rest of our discussions on the results
from BEAST, although data from all PhyloBayes analyses are
available in Fig. 1A and Dataset S1.
Origin of Major Clades. In most analyses, the major clades of extant
eukaryotes diverged before 1200 Ma, with SAR, Excavata, and
Amoebozoa arising within a similar time frame, as evidenced by
overlapping 95% HPD ranges (Figs. 1 and 2, Figs. S1–S7, and
Dataset S1). The 95% HPD intervals are wider for clades with few

Parfrey et al. PNAS | August 16, 2011 | vol. 108 | no. 33 | 13625
A BEAST
2400
2200
2000
d i
1800 e k
a The molecular clock analyses presented here suggest that the last
c g
1600
b f h 1679 Ma when both Phanerozoic and Proterozoic fossils are
1400
1200
1000
800
uncorrelated
Root op op op op est est un un op op un un op op un un
Calibration All Ph 720 All All Ph All Ph All Ph All Ph All Ph All Ph
B 2400
2200
2000
1800
d
e much earlier than their first fossil appearance (2, 31). Thus, it is
1600 d a g
a c c de g
1400 e g b
f
h d
b a
1200
f h
1000
800
Fig. 1. Summary of mean divergence dates for the most recent common
ancestor of major clades of extant eukaryotes. Letters are at the mean di-
vergence time and denote analyses, as detailed in Table S1. Error bars rep-
resent 95% HPD for BEAST analyses (a–h) and the 95% confidence interval
for PhyloBayes (analysis i–p). (A) Estimated age of the root of extant
eukaryotes across analyses. Root position: Opis, root constrained to Opis-
thokonta; Uni, root constrained to “Unikonta”; Estim, root estimated by
BEAST. Calibration: All, all Phanerozoic and Proterozoic CCs; Phan, Phaner-
ozic CCs only; 720, All CCs with the minimum age of red algae set to 720 Ma.
d = 91 taxa. (B) Estimated ages of major clades from BEAST analyses.
calibration points, such as Excavata and Amoebozoa (Fig. 1B).
Estimates for the last common ancestor of extant Opisthokonta are
younger than the other clades, at 1389–1240 Ma in analyses with All
calibration constraints.
Exclusion of Proterozoic calibration constraints (Phan analy-
ses) shifted age estimates for the origins of major extant
eukaryotic clades younger by 200–300 myr (Fig. 1B). Differences
in divergence times are relatively small for nested clades—e.g.,
the 95% HPD for Alveolata shifts from 1445 to 1236 Ma in
analysis a (Fig. 2) to 1206–1020 Ma with only Phanerozoic cal-
ibration points (analysis b; Fig. S1). Not surprisingly, the differ-
ing calibration schemes had their most dramatic impact on the
estimated age of the red algae, which changes from 1285 to 1180
Ma 95% HPD (Fig. 2) to 959–625 Ma 95% HPD when Prote-
rozoic calibration points, including the constraint on red algae at
1174 Ma in accordance with the widely cited age for Bangio-
morpha, are excluded (Fig. S1). Estimated ages of major clades
were also much younger in analyses using the CIR model with
Phan calibrations (analyses n and p; Dataset S1).
The topology of the eukaryotic tree produced through coes-
timation of phylogeny and divergence times in BEAST is broadly
consistent with other analyses (SI Text) (25, 26). Hence, the
BEAST topology was also used for the PhyloBayes analyses, which
require a fixed topology. Though the relationships among the
photosynthetic eukaryotes remain uncertain (25), our analyses
suggest that many photosynthetic clades, such as red and green
13626 | www.pnas.org/cgi/doi/10.1073/pnas.1110633108
PhyloBayes algae, diverged within a similar time frame (Fig. 2). These results
imply an early acquisition of photosynthesis in eukaryotes, in ac-
cordance with both previous molecular clock estimates (30) and the
∼1200 Ma age assigned to the red algal fossil Bangiomorpha (11).
Discussion
m
o
j l
common ancestor of extant eukaryotes lived between 1866 and
considered. We favor these more-inclusive analyses as they should
reveal a more accurate picture of eukaryotic diversification, es-
p
pecially because the chosen fossils are widely accepted by pale-
n
ontologists, and calibration constraints were assigned in a
conservative manner that accounts for age uncertainties. Esti-
mated ages are younger when we remove Proterozoic calibration
autocorrelated constraints, though not dramatically so, with the notable excep-
tion of the autocorrelated model CIR as implemented in Phylo-
Bayes with only Phanerozoic calibrations. Thus, our results tend
to place the last common ancestor of extant eukaryotes deep
within the Proterozoic Eon.
Our estimates for the timing of the origin of extant eukaryotes
are in line with fossil evidence (2, 13), but reject the hypothesis
that eukaryotes originated only 850 Ma (6, 7). Fossils provide
minimum dates, leaving open the possibility that clades evolved
not surprising that divergence times for many eukaryotic clades
a
c are older than their first unambiguous fossil occurrence (Table
c e g b f h 2). The paleontological literature contains some references to
b f h eukaryotic fossils older than our estimate of the last common
ancestor. In some cases, these paleontological reports are in-
correct or ambiguous. For example, large carbonaceous fossils
assigned to the genus Grypania were originally reported to be
older than our molecular clock estimate (32), but more recent
radiometric dates indicate an age of 1874 ± 9 Ma (33), consistent
with the clock analyses presented here. Older still are the 50- to
300-μm spheroidal microfossils described from ∼3200 Ma rocks
by Javaux et al. (34), and proposed as possible eukaryotes by
Buick (35), and sterane biomarkers from 2700 Ma shales (3).
Whether these materials record Archean eukaryotes remains a
subject of debate (34, 36). Our molecular clock estimates suggest
that if these fossils do represent eukaryotes, they record stem
lineages—early representatives of eukaryotic groups that went
extinct—that were present before the emergence of extant eu-
karyotic clades.
The major lineages of extant eukaryotes (Opisthokonta, SAR,
Excavata, and Amoebozoa) are projected to have diverged from
one another by the Mesoproterozoic era (1600–1000 Ma), rela-
tively early in the history of the domain (Fig. 1 and Table 2).
This, in turn, suggests that these lineages were present for hun-
dreds of millions of years before the observed increase in the
abundance and diversity of eukaryotic microfossils beginning
∼800 Ma (2, 37–40). Our molecular clock estimates indicate that
stem groups were present well before recognizable members of
crown lineages—monophyletic groups consisting of living rep-
resentatives and their ancestors—diversified. A similar pattern of
long stems preceding diversification is seen in animal and plants
and may be a consistent pattern in evolution (38).
Fossils and our molecular clock analyses agree that eukaryotes
originated and diversified during a time when oceans differed
substantially from the modern seas. Increasingly, geochemical
data indicate that for much of the Proterozoic eon, mildly oxic
surface waters lay above an oxygen-minimum zone that was per-
sistently anoxic and commonly sulfidic (41, 42). Such conditions
are compatible with scenarios for eukaryogenesis that rely on
anaerobic methanogens in symbiotic partnership with faculta-
tively aerobic proteobacteria or sulfate reducers (see references
in ref. 43), because facultatively anaerobic mitochondria may
have enabled early eukaryotes to live in the sulfidic Proterozoic
oceans (44). Because sulfide interferes with the function of mi-
tochondria in aerobically respiring eukaryotes, the radiation of
diverse species within eukaryotic clades may have become pos-
Parfrey et al.
 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Heterocapsa rotundata
Alexandrium tamarense
Crypthecodinium cohnii
Karenia brevis
Oxyrrhis marina SAR
Perkinsus marinus
Theileria parva Alveolates
Plasmodium berghei
Toxoplasma gondii
Eimeria tenella
Stylonychia lemnae
Sterkiella histriomuscorum
Nyctotherus ovalis
Paramecium tetraurelia
Tetrahymena thermophila
Chilodonella uncinata
Reticulomyxa filosa
Ovammina opaca
Plasmodiophora brassicae Rhizaria
Bigelowiella natans
Gromia
Corallomyxa tenera
Heteromita globosa
Thalassiosira pseudonana
Phaeodactylum tricornutum
Aureococcus anophagefferens Stramenopiles
Heterosigma akashiwo
Ectocarpus siliculosus
Apodachlya brachynema
Phytophthora infestans
Isochrysis galbana
Emiliania huxleyi
Prymnesium parvum Haptophytes
Pavlova lutheri
Oryza sativa
Arabidopsis thaliana
Welwitschia mirabilis
Ginkgo biloba
Physcomitrella patens
Mesostigma viride
Volvox carteri
Chlamydomonas reinhardtii
Dunaliella salina
Green algae
Acetabularia acetabulum
Micromonas pusilla
Ostreococcus tauri
Goniomonas
Guillardia theta
Leucocryptos marina
Cryptomonads
Gracilaria changii
Chondrus crispus
Porphyra yezoensis Red algae
Cyanidioschyzon merolae
Glaucocystis nostochinearum Glaucocystophytes
Cyanophora paradoxa
Trypanosoma brucei
Leishmania major
Bodo saltans
Diplonema papillatum
Euglena longa
Euglena gracilis
Entosiphon sulcatum
Jakoba libera
Reclinomonas americana
Seculamonas ecuadoriensis
Naegleria gruberi Excavata
Sawyeria marylandensis
Trichomonas vaginalis
Giardia duodenalis
Spironucleus barkhanus
Carpediemonas membranifera
Monocercomonoides sp.
Streblomastix strix
Trimastix pyriformis
Malawimonas californiana
Malawimonas jakobiformis
Acanthamoeba castellanii
Hartmannella vermiformis
Arcella hemisphaerica
Rhizamoeba sp.
Entamoeba histolytica Amoebozoa
Mastigamoeba balamuthi
Dictyostelium discoideum
Physarum polycephalum
Capitella capitata
Aplysia californica
Schistosoma mansoni
Apis mellifera
Drosophila melanogaster
Caenorhabditis elegans
Gallus gallus
Homo sapiens

EVOLUTION
Branchiostoma floridae
Mnemiopsis leidyi
Oscarella carmela
Aphrocallistes vastus Opisthokonta
Nematostella vectensis
Monosiga brevicollis
Amoebidium parasiticum
Sphaeroforma arctica
Capsaspora owczarzaki
Candida albicans
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Phanerochaete chrysosporium
Ustilago maydis
Glomus intraradices
Allomyces macrogynus
Spizellomyces punctatus

2000 1750 1500 1250 1000 750 500 250 0

Fig. 2. Time-calibrated tree of extant eukaryotes using All calibration points, 109 taxa, and root constrained to Opisthokonta. Nodes are at mean divergence
times and gray bars represent 95% HPD of node age. (Upper) Geological time scale; (Lower) Absolute time scale in Ma. Thick vertical bars demarcate eras and
•
thin vertical lines denote periods, with dates derived from the 2009 International Stratigraphic Chart. Node calibrated with Phanerozoic fossils ( ); node
calibrated with Proterozoic fossils (◯). Estimated ages of calibrated nodes differ from calibration constraints (Table 1) because they have been modified by
relaxed clock analysis of sequence data.

sible only when sulfidic subsurface waters began to wane about
800 Ma (45). Alternatively, early eukaryotic evolution may have
occurred in coastal environments sheltered from the impact of
sulfidic waters or in freshwater systems, which are both poorly
sampled by the geologic record and not impacted by sulfidic
oceanic water masses (46). Consistent with this view, moderately
diverse assemblages of fossil eukaryotes occur in well-ventilated
lake deposits of the 1200 to 900 Ma Torridonian succession,
Scotland (47, 48), and in coastal marine deposits of the ∼1500 to
1400-Ma Roper Group, Australia (49).
Within Proterozoic oceans, low concentrations of biologically
available nitrogen may also have inhibited the diversification of
photosynthetic eukaryotes (50). Many cyanobacteria and other
photosynthetic bacteria are capable of nitrogen fixation, ame-
liorating the impact of nitrate and ammonia limitation on pri-
mary production. Eukaryotes, however, have no such capacity;
thus, it may not be a coincidence that biomarkers indicating an
expanding importance of algae in marine primary production
occur in conjunction with geochemical data recording the spread
of oxygen through later Neoproterozoic oceans (51). In our
analyses, the clade that contains extant photosynthetic taxa, in-
cluding green algae plus land plant and red algae, arose between
1670 and 1428 Ma, but diversification within these lineages oc-
curred later in the Neoproterozoic and may correspond to
a changing redox profile in the oceans (Fig. 2).

Parfrey et al. PNAS | August 16, 2011 | vol. 108 | no. 33 | 13627
Table 2. Comparison of major node ages to fossil dates
Major clade Estimated age, Ma Oldest fossil, Ma
Eukaryotes * 1800
Extant eukaryotes 1679–1866 1200
Amoebozoa 1384–1624 800
Excavata 1510–1699 450
Opisthokonta 1240–1481 632
Rhizaria 1017–1256 550
SAR 1365–1577 736
Estimated age is range of mean dates from All analyses.
*The age of the root of all eukaryotes is not estimated because molecular
clock studies can only inform the timing of extant clades.
Discrepancy Between These and Previous Molecular Clock Studies.
Previous molecular clock studies yielded vastly different dates for
the root of extant eukaryotes, ranging from 3970 to 1100 Ma (1). In
a recent analysis of small subunit ribosomal DNA (SSU-rDNA)
from 83 broadly sampled eukaryotes, Berney and Pawlowski (4)
placed the origin of eukaryotes at 1100 Ma, a conclusion that was
robust to changing the position of the root. They had numerous
Phanerozoic calibration constraints specified as either minimum or
maximum divergence dates (4), but they found that including
Proterozoic calibration points, such as Bangiomorpha at 1200 Ma,
shifted their estimates of the origin and diversification of eukar-
yotes by 1000–2500 Ma. The age discrepancy observed by Berney
and Pawlowski (4), when Proterozoic calibration constraints are
included, contrasts sharply with the relative stability of dates seen in
our analyses (Fig. 1A). We hypothesize that the increased gene and
taxon sampling, as well as the use of flexible prior distributions of
calibration points as implemented in BEAST, are major factors
contributing to the stability of molecular clock estimation in
our analyses.
Conclusion
Our molecular clock analyses yield a timeline of eukaryotic
evolution that is congruent with the paleontological record and
robust to varying analytical conditions. According to our analy-
ses, crown (extant) groups of eukaryotes arose in the Paleo-
proterozoic era (2500–1600 Ma) and began to diversify soon
thereafter, suggesting that early eukaryotic evolution was influ-
enced by anoxic and sulfidic water masses in contemporaneous
oceans. The stability in our analysis across a range of variables is
a welcome departure from the large age discrepancies reported
in earlier molecular analyses, reflecting improved paleontologi-
cal interpretation, advancements in molecular methods, and the
rapidly growing body of molecular data from diverse eukaryotes.
Materials and Methods
Alignments. Alignments are derived from the 15 protein-coding genes ana-
lyzed in Parfrey et al. (dataset 15:10 of ref. 25). Using this 88-taxon dataset
as a starting point, taxa were added to capture additional lineages, partic-
ularly those with fossil data available (Table S2). Rapidly evolving taxa (e.g.,
Encephalitozoon cuniculi) and orphans (e.g., Breviata anathema) were re-
moved to minimize rate heterogeneity for the clock analysis. The resulting
Ref.
109-taxon data matrix includes 5,696 characters, with each taxon having
(2) between three and 15 of the target genes (36% missing character data;
(11) Table S2; analyses a–c and e–p). A 91-taxon alignment was created by re-
(12) moving additional taxa with either long branches or high levels of missing
(64) data to ensure that our results were not driven by these potential sources of
(71) artifact (analysis d).
(65)
(74) Molecular Dating Analyses. Dating analyses were predominantly performed
in BEAST v1.5.4 (52), and we also assessed results obtained in PhyloBayes
3.2f (53) (see SI Text for analysis details). BEAST offers a number of desirable
features, including flexible specification of prior distributions that enable
the uncertainty of the fossil record to be realistically modeled, as well as the
ability to coestimate divergence times with topology (15). We compared
divergence dates for eukaryotes obtained from different models to assess
whether our conclusions were driven by the choice of a particular model (SI
Text, Fig. 1 and Table S1).
Calibration Constraints. Calibration constraints were specified with prior dis-
tributions to incorporate errors arising from age dating, stratigraphy, and
clade assignment (Table 1). The impact of Proterozoic fossils was assessed by
analyzing the data with only the 16 Phanerozoic calibration constraints
(Phan analyses b, f, h, j, l, n, and p) or with Phanerozoic and Proterozoic
calibration constraints (All analyses a, c–e, g, i, k, m, and o). Calibration
constraints were specified with prior distributions in BEAST using BEAUTi
v1.5.4 (52) and were derived from a conservative reading of the fossil record
(i.e., we err toward younger rather than older ages; SI Text). Distributions
were specified with long tails unless the fossil record provided minimum-
divergence information. Calibration constraints used for PhyloBayes had to
be specified as a uniform distribution (Table S3).
Assessing Impact of the Root on the Inferred Age of Eukaryotes. Molecular
clock analyses require a rooted tree. However, the position of the eukaryotic
root remains an open question; therefore, we compared age estimates from
molecular clock analyses with multiple positions for the root of extant
eukaryotes. First, the root was constrained to the branch leading to the
Opisthokonta or to Opisthokonta + Amoebozoa (“Unikonta”) in accordance
with current hypotheses (see SI Text for discussion of the position of the
eukaryotic root). In BEAST, the root was specified by constraining a mono-
phyletic ingroup. PhyloBayes requires the tree topology to be fixed, and we
used the tree in Fig. 2 rooted on either Opisthokonta or “Unikonta”. Finally,
for the third condition, the root was estimated by the molecular clock cri-
terion, as implemented in BEAST (SI Text), which yielded variable estimates
of the location of the root.
ACKNOWLEDGMENTS. We thank Ben Normark, Rob Dorit, and Sam Bowser
for useful discussions, and Jeff Thorne and Bengt Sennblad for helpful
discussions about molecular clock models. This manuscript has been improved
following the comments of Emmanuelle Javaux, Andrew Roger, and Heroen
Verbruggen. We thank Jessica Grant and Tony Caldanaro for technical help.
This research was supported by the National Aeronautics and Space Admin-
istration Astrobiology Institute (A.H.K.) and by National Science Foundation
Assembling the Tree of Life Grant 043115 and National Science Foundation
Systematics Grant 0919152 (to L.A.K). D.J.G.L. is supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico-Brazil Doutorado no Exterior Fel-
lowship 200853/2007-4.

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PNAS | August 16, 2011 | vol. 108 | no. 33 | 13629
Supporting Information
Parfrey et al. 10.1073/pnas.1110633108
SI Text
Calibration Constraints. Calibration constraints (CCs) were as-
signed from the fossil record and were set to take into account the
multiple sources of uncertainty that arise from using paleonto-
logical information to calibrate molecular clocks (1–3). The level
of informativeness of the priors varied among calibration con-
straints to reflect to the level of confidence in the timing of the
split. For many lineages (including all Proterozoic CCs) the fossil
record provides only a minimum divergence time, which is re-
flected as a very long tail in the prior probability that extends
back to ∼3500 Ma. In most cases, the CC was placed at the node
where the clade with an available fossil split from its sister group;
for example the first recorded angiosperm pollen (4) is used to
constrain the split of angiosperms from their gymnosperm an-
cestors (Table 1 and Fig. 2). In cases where the fossil falls within
the crown clade, the CC was placed at the base of the clade, as in
the Endopterygota where the first Mecoptera fossils constrain
the split between Apis and Drosophila (Table 1). Minimum dates
(offsets in BEAST) were assigned conservatively. We used ra-
diometric dates when available, and set the minimum constraint
to the youngest edge of the reported confidence interval. Thus,
the minimum age of the CC for Arcellinida is 736 Ma, because
arcellinid fossils are found in rocks older than 742 ± 6 Ma (Table
1) (5). For fossils assigned to geological stages, we used the upper
boundary of the stage according to the 2009 International Stra-
tigraphy Chart published by the International Commission on
Stratigraphy (http://www.stratigraphy.org/). For example, angio-
sperm pollen is first found in Valanginian rocks (4) and so was
constrained to a minimum date of 133.9 Ma.
Prior distributions were set in one of two ways depending on
the level of uncertainty. For clades with robust fossil records
where the maximum age of the clade is unlikely to be substantially
earlier than its first occurrence (e.g., angiosperms), the prior
distribution was set to include 95% of the probable age of the
clade. In contrast, Proterozoic records and fossils of groups with
a poor fossilization potential provide only minimum dates for
lineage origin and, commonly, no information on maximum clade
age (e.g., Arcellinida). In these cases the prior distribution was
specified with a very long tail, as assessed in BEAUTi, that ex-
tended back to ∼3500 Ma.
Selected CCs are discussed here (see Table 1 for details of the
remaining CCs). Fossils of the earliest red alga, Bangiomorpha,
occur in the lower section of the Hunting Formation, Canada,
which is bracketed by U-Pb radiometric dates on volcanic rocks
of 1267 ± 2 Ma and 723 ± 3 Ma. Direct Pb-Pb dates on carbo-
nates correlative with those containing the fossils yield a much
narrow constraint of 1198 ± 24 Ma (6), but this date remains
unpublished, and radiometric dating of carbonates can be
problematic. The true age of the Hunting Bangiomorpha fossils
may therefore lie closer to the lower U-Pb age constraint than
the upper, because of the sequence stratigraphic position of
fossiliferous strata relative to constraining volcanic rocks and
chemo- and biostratigraphic data consistent with a later Meso-
proterozoic (>1250 Ma) age (6). In most All 720, and Phan
analyses, the minimum date for the Bangiomorpha constraint was
set at 1174 Ma. Given the importance of the Bangiomorpha
calibration as potentially the oldest phylogenetically constraining
fossil by roughly 450 myr, we also ran the All 720, and Phan
analysis with the constraint for Bangiomorpha set at 720 Ma (the
minimum age for the Hunting Formation) for comparison
(analysis d). Because of the controversy surrounding Bangio-
morpha, we have placed the calibration on the base of the red
Parfrey et al. www.pnas.org/cgi/content/short/1110633108
algae rather than on a particular node within the clade to be
conservative.
The single CC in the Excavata is placed within the euglenids.
Although the Excavata generally have a poor fossil record, the
euglenid Moyeria is widely distributed in the Ordovician and
Silurian, with an earliest occurrence in the Caradocian (7), dated
at 450 Ma. Moyeria is thought to have been photosynthetic based
on the patterning of its pellicle, indicating an early acquisition
of the secondary green alga endosymbiont (8), thus the CC is
placed at the split between photosynthetic (Euglena) and het-
erotrophic (Entosiphon) euglenids in the tree (Fig. 1).
The calibration constraint for diatoms is based on the earliest
diatom fossils from the Valanginian to Hauterivian Myogok
Formation in Korea (9); a date of 133.9 Ma is used to represent
the upper Valanginian boundary. The CC of this node is younger
than in other clock analyses (10) because we do not rely on
Pyxidicula, a putative Toarcian diatom (11) for which the ma-
terial has been lost.
We include a CC for ciliates in the All 720, and Phan analyses
that is based on the presence of gammacerane in Neoproterozoic
sedimentary rocks (12). Tetrahymenol, the precursor of gam-
macerane, is commonly found in some ciliates, although it has
also been found in bacteria (13). Tetrahymenol production is
documented from the Oligohymenophorea and the Plagiopylea
(Trimyena), which are not included in this analysis, so the CC was
placed at the stem of the Oligohymenophorea (Tetrahymena and
Paramecium). This CC was included despite the possibility of
bacterial origin, because the 736 Ma constraint is much younger
than the date estimated for ciliates (∼1150 Ma) without this
constraint in the Phan analyses.
Root of the Eukaryotic Tree of Life. Although our goal was to
elucidate timing of major events in eukaryotic evolution, we also
explored the impact of changing the position of the root, because
rooted phylogenies are crucial for interpreting the evolutionary
events in the history of a lineage. A root must be either provided
or estimated for molecular clock analyses (14, 15). However, the
root of the eukaryotic tree of life is difficult to determine because
the common methods for rooting phylogenies are vulnerable to
artifacts caused by rate heterogeneity among lineages of eukar-
yotes and the vast distance between eukaryotes and archaea or
bacteria (16–18). Although numerous hypotheses have been
proposed (19–23), the position of the root remains an open
debate (16, 17, 24, 25). The most popular hypothesis of recent
years places the root of eukaryotes between the Opisthokonta +
Amoebozoa (unikonts) and the remaining eukaryotes (bikonts)
(19, 26), and previous molecular clock analyses of eukaryotes
rooted trees in this manner (10, 27, 28). However, several lines of
evidence contradict the unikont/bikont split (23, 24), and alter-
native roots have been suggested, including at the base of
Opisthokonta (23, 29), within Archaeplastida (21, 22), or along
the lineage leading to Euglenozoa (20). Rooting the tree of ex-
tant eukaryotes along the branch leading to Opisthokonta is
supported by ongoing gene-tree species-tree reconciliation work
by Gordon Burleigh (University of Florida).
Here, we assess the impact different positions of the root have
on estimates of the age of eukaryotes. The root is (i) estimated in
BEAST using the molecular clock criterion (30); (ii) placed be-
tween Opisthokonta and the rest of eukaryotes (23, 29); or (iii)
placed between “Unikonta” and the rest of eukaryotes (19).
PhyloBayes requires a fixed topology for molecular dating anal-
yses, hence those analyses were run rooted either on Opistho-
1 of 15
konta or “Unikonta”. No strongly supported alternative root
emerged from the analyses in which BEAST determined the root
based on the molecular clock. Rooting by the molecular clock
criterion uses information from branch lengths and has been
shown in simulation studies to work reasonably well in situations
where there is no outgroup available (31). The position of the
root varied across runs, falling between Excavata or Excavata +
unikonts and the rest of eukaryotes (analyses e and f). All trees
are available in SI Text (Figs. S1–S7 and Dataset S1).
Topology of the Eukaryotic Tree. The topology of the trees produced
by BEAST through coestimation of phylogeny and dates are
consistent with the broad outlines of eukaryotic topologies re-
covered in other analyses (32, 33). Of note, three relationships
emerge in the BEAST topologies that were not present in the
RAxML analyses (Dataset S1). RAxML analyses divide Excavata
into two clades when all major lineages of Excavata are included
(109-taxon set; Dataset S1); however, Excavata is monophyletic in
all BEAST analyses (unless the root of eukaryotes falls within
Excavata). Second, BEAST consistently places haptophytes as the
sister clade of SAR (Fig. 2 and Figs. S1–S7). Finally, cryptomo-
nads consistently branch as sister to kathablepharids and within
the clade of primary photosynthetic eukaryotes: red algae, green
algae (including plants), and glaucophytes (the Archaeplastida or
Plantae hypothesis; Fig. 2 and Figs. S1–S7).
Prior-Only Analyses. The differences in estimated node age between
the calibration sets (Phan vs. All) are driven by the sequence data,
rather than the prior distribution of calibration constraints. All
720 analysis conditions in Table S1 were assessed without the data
to determine the impact of priors on estimated divergence dates
(prior-only analyses). In BEAST analyses of priors alone, without
sequence data, yield dates for major nodes that are 200–800 myr
younger than analyses with data. The disparity was much greater
for Phan analyses: prior-only analyses yielded dates 500–800 myr
younger here. For example, the root age is 1478 Ma in the 109-
taxon Phan analysis b, but only 717 Ma when this analysis is run
without sequence data. In contrast, all PhyloBayes prior-only
analyses produce dates much older than analyses run with the
data with the root falling between 3817 Ma and an unreasonable
5047 Ma. These results demonstrate that the CCs chosen did not
determine our dates.
SI Materials and Methods
Phylogenetic Analysis. BEAST requires a reasonable starting tree
to analyze complex datasets so the initial topology was obtained in
RAxML. Two hundred bootstrap replicates followed by an ex-
haustive maximum likelihood search were done using the MPI
version of RaxML 7.0.4 with rapid bootstrapping and the WAG +
gamma model (34). The best-fitting amino acid substitution
matrix available in BEAST was WAG for all partitions as esti-
mated in ProtTest (35). This resulted in a highly supported to-
pology consistent with that found by RAxML analyses in Parfrey
et al. (33) (Dataset S1).
BEAST Model Conditions and Analyses. We ran preliminary analyses
in BEAST to assess the impact of several options, including type
of molecular clock and partitioning of genes. Analyses were run at
Smith College and on the freely available Oslo Bioportal (www.
bioportal.uio.no/). Parameters were deemed a poor fit for the
data if likelihood values did not converge across four runs of
10 million generations. Based on this criterion, we selected the
UCL relaxed clock model combined with unpartitioned genes for
subsequent BEAST analyses (analyses a–h).
A two-pronged approach was used to increase chain mixing, as
measured by estimated sample size in Tracer. First, four initial
chains of 10 million generations each were run with the best
RAxML tree as the starting tree and the remaining priors at
Parfrey et al. www.pnas.org/cgi/content/short/1110633108
default settings (excluding CCs). In the first phase, initial runs
were done with a RAxML starting tree that had branch heights
(ages) set to 360 so that all nodes were older than the CCs
assigned to them. Priors for all parameters (excluding CCs) were
left at default settings. Five million generations were removed
from each of these chains as burnin (as determined by conver-
gence of likelihood values in Tracer v1.5.4), and chains were
combined in LogCombiner v1.5.4 (distributed with BEAST) (36).
The final 1 million generations of the preliminary runs were used
to generate a starting tree for subsequent analyses that had a
robust tree topology with realistic branch heights. Trees were
annotated using TreeAnnotator v1.5.4 (36) using the mean node
heights and maximum clade credibility tree settings. In the sec-
ond phase of the analysis, eight runs of 10 million generations
each were conducted for each analysis (Table S1). Operator
values and prior distributions on substitution rates were in-
formed from the results of initial runs. One million generations
were removed from each chain as burnin, and the remaining
generations were combined from both log and tree files in
LogCombiner v1.5.4. Trees were annotated in TreeAnnotator
v1.5.4 and assessed in FigTree v1.3.1 (http://tree.bio.ed.ac.uk/
software/figtree/).
Model conditions for BEAST were determined in preliminary
analyses of four chains run for 10 million generations each. If
likelihood values did not converge across four runs of 10 million
generations, as assessed in Tracer v1.5 (distributed with BEAST
v1.5.4) (36), the model was deemed a poor fit for the data. The
strict clock and uncorrelated exponential molecular clock models
were both rejected based on this criterion, as was analyzing the
15 genes as a single partition. If competing models converged in
likelihood scores, the likelihoods were compared using Bayes
factors (37) as assessed in Tracer, although we did not rely on
this metric as the harmonic mean calculation of Bayes factors
has been demonstrated to be unreliable (38). In these cases, the
estimated divergence dates were compared between competing
models. Fixing tree topology to the most likely RAxML tree
(Dataset S1) resulted in lower likelihood scores. Further, al-
lowing BEAST to coestimate phylogeny and divergence dates
might yield better results for both, so for all subsequent analyses
the topology was estimated. All 720, and Phanowing BEAST to
modify tree topology resulted in a highly supported topology that
was broadly consistent with other analyses (32, 33, 39).
Each gene was analyzed as a separate partition for both site
models and molecular clock models because the analyses did not
converge when the 15 genes were analyzed as a single partition.
The WAG amino acid substitution matrix was used for all genes,
as it was the best-fitting model available in BEAST as determined
by PROTTEST (35). A model of amino acid substitution that
included gamma-distributed rate classes was found to be a better
fit for the data; however, this resulted in a 10-fold computational
cost, thus the gamma correction was used in only a few cases for
comparison and yielded similar dates and topologies compared
with analyses without a gamma correction. For example, adding
gamma correction to BEAST analyses of 109 taxa rooted on
opisthokonts with All and Phan CCs (analyses a and b) yielded
the same topology as Fig. 2 and Fig. S1, respectively, with the age
of the root shifted from 1774 Ma to 1668 Ma in analysis a and
1478 Ma to 1433 Ma in analysis b, where Proterozoic CCs were
excluded. These analyses are not included in Table S1 because
they were run only 5 million generations (rather than 10 million)
due to constraints on compute resources.
The UCL relaxed clock model was found to be the best clock
model available in BEAST for these data, as analyses using either
a strict molecular clock or an uncorrelated exponential relaxed
clock did not converge Only uncorrelated models are imple-
mented in BEAST (36). The UCL relaxed clock is expected to
perform better on datasets with deep divergences and rate het-
erogeneity across the tree, because the SD parameter captures
2 of 15
the variation in rates across the tree (30). The coefficient of
variation of the UCL clock ranges from 0.3 to 2.5 for different
genes, which indicates that rates vary between 30% and 250%
across the tree depending on the gene. Thus, these data are not
clock-like.
PhyloBayes Analyses. Analyses were run in PhyloBayes version 3.2f
(40). All chains were run for at least 1,000 cycles. Calibrations for
PhyloBayes were based on the calibrations in BEAST but spec-
ified as a date range as required by PhyloBayes with soft bounds
at the default setting (Table S3). The model of sequence evo-
lution was the same across all PhyloBayes analyses with a gen-
eralized time-reversible (GTR) amino acid substitution matrix
(-gtr), a Dirichlet mixture profile (-cat), and a Dirichlet process
modeling rates across sites (-ratecat). For each condition, two
replicate chains were run. Analyses were run with the tree to-
pology of Fig. 2, which was fixed and rooted either on
the Opisthokonta or “Unikonta”. Analyses were run under either
the CIR molecular clock model, in which rates across branches
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as burn-in for each analysis. The mean dates were averaged, and
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3 of 15
 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Heterocapsa_rotundata
Alexandrium_tamarense
Crypthecodinium_cohnii
Karenia_brevis SAR
Oxyrrhis_marina
Perkinsus_marinus
Theileria_parva Alveolates
Plasmodium_berghei
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Reticulomyxa_filosa
Ovammina_opaca
Plasmodiophora_brassicae
Rhizaria
Bigelowiella_natans
Gromia
Corallomyxa_tenera
Heteromita_globosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum Stramenopiles
Aureococcus_anophagefferens
Heterosigma_akashiwo
Ectocarpus_siliculosus
Apodachlya_brachynema
Phytophthora_infestans
Isochrysis_galbana
Emiliania_huxleyi Haptophytes
Prymnesium_parvum
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri
Chlamydomonas_reinhardtii Green algae
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Goniomonas
Guillardia_theta Cryptomonads
Leucocryptos_marina
Glaucocystis_nostochinearum Glaucocystophytes
Cyanophora_paradoxa
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Naegleria_gruberi
Sawyeria_marylandensis
Trichomonas_vaginalis
Jakoba_libera Excavata
Reclinomonas_americana
Seculamonas_ecuadoriensis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis
Arcella_hemisphaerica
Rhizamoeba_sp
Hartmannella_vermiformis
Acanthamoeba_castellanii
Entamoeba_histolytica Amoebozoa
Mastigamoeba_balamuthi
Dictyostelium_discoideum
Physarum_polycephalum
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Oscarella_carmela
Aphrocallistes_vastus
Mnemiopsis_leidyi Opisthokonta
Nematostella_vectensis
Monosiga_brevicollis
Amoebidium_parasiticum
Sphaeroforma_arctica
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus

2000 1750 1500 1250 1000 750 500 250 0

Fig. S1. Time-calibrated tree of eukaryotes using Phanerozoic calibration points, 109 taxa, rooted on Opisthokonta, and constructed in BEAST (analysis b).
Nodes are at mean divergence times, and gray bars represent 95% HPD of node age. (Upper) Geological time scale. (Lower) Absolute time scale (in Ma). Thick
vertical bars demarcate eras, and thin vertical lines denote periods, with dates derived from the 2009 International Stratigraphic Chart.

Parfrey et al. www.pnas.org/cgi/content/short/1110633108 4 of 15

 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Heterocapsa_rotundata
Alexandrium_tamarense
Crypthecodinium_cohnii
Karenia_brevis SAR
Oxyrrhis_marina
Perkinsus_marinus
Theileria_parva Alveolates
Plasmodium_berghei
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Reticulomyxa_filosa
Ovammina_opaca Rhizaria
Plasmodiophora_brassicae
Bigelowiella_natans
Gromia
Corallomyxa_tenera
Heteromita_globosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum Stramenopiles
Aureococcus_anophagefferens
Heterosigma_akashiwo
Ectocarpus_siliculosus
Apodachlya_brachynema
Phytophthora_infestans
Isochrysis_galbana
Emiliania_huxleyi
Prymnesium_parvum
Haptophytes
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri Green algae
Chlamydomonas_reinhardtii
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Goniomonas
Guillardia_theta
Leucocryptos_marina Cryptomonads
Glaucocystis_nostochinearum Glaucocystophytes
Cyanophora_paradoxa
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Naegleria_gruberi
Sawyeria_marylandensis
Trichomonas_vaginalis
Jakoba_libera Excavata
Reclinomonas_americana
Seculamonas_ecuadoriensis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis
Arcella_hemisphaerica
Rhizamoeba_sp
Acanthamoeba_castellanii
Hartmannella_vermiformis
Entamoeba_histolytica
Amoebozoa
Mastigamoeba_balamuthi
Dictyostelium_discoideum
Physarum_polycephalum
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Oscarella_carmela
Aphrocallistes_vastus
Mnemiopsis_leidyi Opisthokonta
Nematostella_vectensis
Monosiga_brevicollis
Amoebidium_parasiticum
Sphaeroforma_arctica
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus

2000 1750 1500 1250 1000 750 500 250 0

Fig. S2. Time-calibrated tree of eukaryotes using All (Proterozoic and Phanerozoic) calibration points with the Bangiomorpha CC set at 720 Ma, 109 taxa,
rooted on Opisthokonta, and constructed in BEAST (analysis c). Other notes as in Fig. S1.

Parfrey et al. www.pnas.org/cgi/content/short/1110633108 5 of 15

 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Crypthecodinium_cohnii
Alexandrium_tamarense
Heterocapsa_rotundata SAR
Karenia_brevis
Perkinsus_marinus Alveolates
Theileria_parva
Plasmodium_berghei
Eimeria_tenella
Toxoplasma_gondii
Sterkiella_histriomuscorum
Stylonychia_lemnae
Tetrahymena_thermophila
Paramecium_tetraurelia
Heteromita_globosa Rhizaria
Corallomyxa_tenera
Bigelowiella_natans
Ovammina_opaca
Reticulomyxa_filosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum Stramenopiles
Aureococcus_anophagefferens
Ectocarpus_siliculosus
Phytophthora_infestans
Emiliania_huxleyi
Isochrysis_galbana Haptophytes
Prymnesium_parvum
Pavlova_lutheri
Volvox_carteri
Chlamydomonas_reinhardtii
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri Green algae
Arabidopsis_thaliana
Oryza_sativa
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Gracilaria_changii
Chondrus_crispus Red algae
Porphyra_yezoensis
Glaucocystis_nostochinearum Glaucocystophytes
Cyanophora_paradoxa
Guillardia_theta
Goniomonas Cryptomonads
Jakoba_libera
Reclinomonas_americana
Seculamonas_ecuadoriensis
Sawyeria_marylandensis
Naegleria_gruberi
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Diplonema_papillatum Excavata
Bodo_saltans
Streblomastix_strix
Monocercomonoides_sp
Trimastix_pyriformis
Malawimonas_jakobiformis
Malawimonas_californiana
Arcella_hemisphaerica
Hartmannella_vermiformis
Acanthamoeba_castellanii
Dictyostelium_discoideum Amoebozoa
Physarum_polycephalum
Mastigamoeba_balamuthi
Entamoeba_histolytica
Drosophila_melanogaster
Apis_mellifera
Caenorhabditis_elegans
Capitella_capitata
Aplysia_californica
Homo_sapiens
Gallus_gallus
Branchiostoma_floridae
Mnemiopsis_leidyi
Oscarella_carmela
Nematostella_vectensis
Schistosoma_mansoni Opisthokonta
Monosiga_brevicollis
Capsaspora_owczarzaki
Sphaeroforma_arctica
Saccharomyces_cerevisiae
Candida_albicans
Schizosaccharomyces_pombe
Ustilago_maydis
Phanerochaete_chrysosporium
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus

2000 1750 1500 1250 1000 750 500 250 0

Fig. S3. Time-calibrated tree of eukaryotes using All calibration points, 91 taxa, rooted on Opisthokonta, and constructed in BEAST (analysis d). Other notes as
in Fig. S1.

Parfrey et al. www.pnas.org/cgi/content/short/1110633108 6 of 15

 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Alexandrium_tamarense
Heterocapsa_rotundata
Crypthecodinium_cohnii
Karenia_brevis
Oxyrrhis_marina
SAR
Perkinsus_marinus
Theileria_parva
Plasmodium_berghei
Alveolates
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Reticulomyxa_filosa
Ovammina_opaca Rhizaria
Plasmodiophora_brassicae
Bigelowiella_natans
Gromia
Corallomyxa_tenera
Heteromita_globosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum
Aureococcus_anophagefferens
Stramenopiles
Heterosigma_akashiwo
Ectocarpus_siliculosus
Apodachlya_brachynema
Phytophthora_infestans
Isochrysis_galbana
Emiliania_huxleyi
Prymnesium_parvum Haptophytes
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri
Chlamydomonas_reinhardtii Green algae
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Goniomonas
Guillardia_theta
Leucocryptos_marina Cryptomonads
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Glaucocystis_nostochinearum
Cyanophora_paradoxa Glaucocystophytes
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Oscarella_carmela
Aphrocallistes_vastus
Mnemiopsis_leidyi Opisthokonta
Nematostella_vectensis
Monosiga_brevicollis
Amoebidium_parasiticum
Sphaeroforma_arctica
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus
Acanthamoeba_castellanii
Hartmannella_vermiformis
Dictyostelium_discoideum
Physarum_polycephalum
Arcella_hemisphaerica Amoebozoa
Rhizamoeba_sp
Entamoeba_histolytica
Mastigamoeba_balamuthi
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Jakoba_libera
Reclinomonas_americana
Seculamonas_ecuadoriensis
Naegleria_gruberi
Sawyeria_marylandensis
Excavata
Trichomonas_vaginalis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis

2000 1750 1500 1250 1000 750 500 250 0

Fig. S4. Time-calibrated tree of eukaryotes using All calibration points, 109 taxa, root estimated by BEAST, and constructed in BEAST (analysis e). Other notes
as in Fig. S1.

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 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Alexandrium_tamarense
Heterocapsa_rotundata
Crypthecodinium_cohnii
Karenia_brevis
Oxyrrhis_marina
SAR
Perkinsus_marinus
Theileria_parva
Plasmodium_berghei
Alveolates
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Reticulomyxa_filosa
Ovammina_opaca Rhizaria
Plasmodiophora_brassicae
Bigelowiella_natans
Gromia
Corallomyxa_tenera
Heteromita_globosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum
Aureococcus_anophagefferens
Stramenopiles
Heterosigma_akashiwo
Ectocarpus_siliculosus
Apodachlya_brachynema
Phytophthora_infestans
Isochrysis_galbana
Emiliania_huxleyi Haptophytes
Prymnesium_parvum
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri
Chlamydomonas_reinhardtii Green algae
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Goniomonas
Guillardia_theta
Leucocryptos_marina Cryptomonads
Glaucocystis_nostochinearum
Cyanophora_paradoxa
Glaucocystophytes
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Oscarella_carmela
Aphrocallistes_vastus Opisthokonta
Mnemiopsis_leidyi
Nematostella_vectensis
Monosiga_brevicollis
Sphaeroforma_arctica
Amoebidium_parasiticum
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus
Acanthamoeba_castellanii
Hartmannella_vermiformis
Dictyostelium_discoideum
Physarum_polycephalum
Arcella_hemisphaerica Amoebozoa
Rhizamoeba_sp
Entamoeba_histolytica
Mastigamoeba_balamuthi
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Jakoba_libera
Reclinomonas_americana
Seculamonas_ecuadoriensis
Naegleria_gruberi
Sawyeria_marylandensis Excavata
Trichomonas_vaginalis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis

2000 1750 1500 1250 1000 750 500 250 0

Fig. S5. Time-calibrated tree of eukaryotes using Phanerozoic calibration points, 109 taxa, root estimated by BEAST, and constructed in BEAST (analysis f).
Other notes as in Fig. S1.

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 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Alexandrium_tamarense
Heterocapsa_rotundata
Crypthecodinium_cohnii
Karenia_brevis
Oxyrrhis_marina
SAR
Perkinsus_marinus
Theileria_parva
Plasmodium_berghei
Alveolates
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Thalassiosira_pseudonana
Phaeodactylum_tricornutum
Aureococcus_anophagefferens
Heterosigma_akashiwo Stramenopiles
Ectocarpus_siliculosus
Apodachlya_brachynema
Phytophthora_infestans
Reticulomyxa_filosa
Ovammina_opaca
Plasmodiophora_brassicae Rhizaria
Bigelowiella_natans
Gromia
Corallomyxa_tenera
Heteromita_globosa
Isochrysis_galbana
Emiliania_huxleyi
Prymnesium_parvum Haptophytes
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri
Chlamydomonas_reinhardtii Green algae
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Goniomonas
Guillardia_theta Cryptomonads
Leucocryptos_marina
Glaucocystis_nostochinearum
Cyanophora_paradoxa Glaucocystophytes
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Jakoba_libera
Reclinomonas_americana
Seculamonas_ecuadoriensis
Naegleria_gruberi Excavata
Sawyeria_marylandensis
Trichomonas_vaginalis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Aphrocallistes_vastus
Oscarella_carmela
Mnemiopsis_leidyi Opisthokonta
Nematostella_vectensis
Monosiga_brevicollis
Amoebidium_parasiticum
Sphaeroforma_arctica
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus
Acanthamoeba_castellanii
Hartmannella_vermiformis
Dictyostelium_discoideum
Physarum_polycephalum
Arcella_hemisphaerica Amoebozoa
Rhizamoeba_sp
Entamoeba_histolytica
Mastigamoeba_balamuthi

2000 1750 1500 1250 1000 750 500 250 0

Fig. S6. Time-calibrated tree of eukaryotes using All calibration points, 109 taxa, rooted on “Unikonta” and constructed in BEAST (analysis g). Other notes as
in Fig. S1.

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 Paleoproterozoic Mesoproterozoic Neoproterozoic Phanerozoic

Heterocapsa_rotundata
Alexandrium_tamarense
Crypthecodinium_cohnii
Karenia_brevis SAR
Oxyrrhis_marina
Perkinsus_marinus
Theileria_parva Alveolates
Plasmodium_berghei
Toxoplasma_gondii
Eimeria_tenella
Stylonychia_lemnae
Sterkiella_histriomuscorum
Nyctotherus_ovalis
Paramecium_tetraurelia
Tetrahymena_thermophila
Chilodonella_uncinata
Reticulomyxa_filosa
Ovammina_opaca
Plasmodiophora_brassicae
Bigelowiella_natans
Gromia Rhizaria
Corallomyxa_tenera
Heteromita_globosa
Thalassiosira_pseudonana
Phaeodactylum_tricornutum
Aureococcus_anophagefferens
Heterosigma_akashiwo
Ectocarpus_siliculosus
Stramenopiles
Apodachlya_brachynema
Phytophthora_infestans
Isochrysis_galbana
Emiliania_huxleyi Haptophytes
Prymnesium_parvum
Pavlova_lutheri
Oryza_sativa
Arabidopsis_thaliana
Welwitschia_mirabilis
Ginkgo_biloba
Physcomitrella_patens
Mesostigma_viride
Volvox_carteri
Chlamydomonas_reinhardtii Green algae
Dunaliella_salina
Acetabularia_acetabulum
Micromonas_pusilla
Ostreococcus_tauri
Gracilaria_changii
Chondrus_crispus
Porphyra_yezoensis Red algae
Cyanidioschyzon_merolae
Goniomonas
Guillardia_theta Cryptomonads
Leucocryptos_marina
Glaucocystis_nostochinearum Glaucocystophytes
Cyanophora_paradoxa
Trypanosoma_brucei
Leishmania_major
Bodo_saltans
Diplonema_papillatum
Euglena_longa
Euglena_gracilis
Entosiphon_sulcatum
Jakoba_libera
Reclinomonas_americana
Seculamonas_ecuadoriensis
Naegleria_gruberi
Sawyeria_marylandensis
Excavata
Trichomonas_vaginalis
Giardia_duodenalis
Spironucleus_barkhanus
Carpediemonas_membranifera
Monocercomonoides_sp
Streblomastix_strix
Trimastix_pyriformis
Malawimonas_californiana
Malawimonas_jakobiformis
Capitella_capitata
Aplysia_californica
Schistosoma_mansoni
Apis_mellifera
Drosophila_melanogaster
Caenorhabditis_elegans
Gallus_gallus
Homo_sapiens
Branchiostoma_floridae
Oscarella_carmela
Aphrocallistes_vastus
Mnemiopsis_leidyi Opisthokonta
Nematostella_vectensis
Monosiga_brevicollis
Amoebidium_parasiticum
Sphaeroforma_arctica
Capsaspora_owczarzaki
Candida_albicans
Saccharomyces_cerevisiae
Schizosaccharomyces_pombe
Phanerochaete_chrysosporium
Ustilago_maydis
Glomus_intraradices
Allomyces_macrogynus
Spizellomyces_punctatus
Acanthamoeba_castellanii
Hartmannella_vermiformis
Dictyostelium_discoideum
Physarum_polycephalum
Arcella_hemisphaerica Amoebozoa
Rhizamoeba_sp
Entamoeba_histolytica
Mastigamoeba_balamuthi

2000 1750 1500 1250 1000 750 500 250 0

Fig. S7. Time-calibrated tree of eukaryotes using Phanerozoic calibration points, 109 taxa, rooted on “Unikonta” and constructed in BEAST (analysis h). Other
notes as in Fig. S1.

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Table S1. Estimates of dates for the last common ancestor of extant eukaryotes across analyses
Root age, Ma

Analysis Taxa CCs Root Mean Range Model Program Tree

a 109 All Opis 1774 1632–1911 UCL BEAST Fig. 2

b 109 Phan Opis 1478 1362–1595 UCL BEAST Fig. S1
c 109 All 720 Opis 1679 1548–1797 UCL BEAST Fig. S2
d 91 All Opis 1837 1725–1954 UCL BEAST Fig. S3
e 109 All Estim 1784 1639–1939 UCL BEAST Fig. S4
f 109 Phan Estim 1506 1365–1643 UCL BEAST Fig. S5
g 109 All Uni 1717 1601–1819 UCL BEAST Fig. S6
h 109 Phan Uni 1471 1347–1604 UCL BEAST Fig. S7
i 109 All Opis 1866 1569–2235 UGAM PhyloBayes —
j 109 Phan Opis 1594 1288–1979 UGAM PhyloBayes —
k 109 All Uni 1810 1549–2161 UGAM PhyloBayes —
l 109 Phan Uni 1561 1268–1886 UGAM PhyloBayes —
m 109 All Opis 1798 1441–2133 CIR PhyloBayes —
n 109 Phan Opis 1038 889–1350 CIR PhyloBayes —
o 109 All Uni 1691 1048–2357 CIR PhyloBayes —
p 109 Phan Uni 1180 897–1839 CIR PhyloBayes —

Root age range is the 95% HPD for BEAST analyses and minimum and maximum ages of 95% confidence interval for PhyloBayes. See Table S2 for details of
taxon sampling, and Table 1 for calibration constraints. All trees are available in Dataset S1. All, 22 calibration points of Phanerozoic and Proterozoic age
included; All 720, Bangiomorpha CC set to 720 Ma; CCs, calibration constraints; CIR, autocorrelated CIR model; Estim, root estimated by BEAST; model,
molecular clock model; Opis, root constrained to Opisthokonta; Phan, calibration points of Phanerozoic age included; root, position of the root; UCL, un-
correlated log normal; UGAM, uncorrelated gamma model; Uni, root constrained to “Unikonta”.

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 Table S2. Details of gene and taxon sampling
Lineage Taxon* 14–3-3 40S Actin αtub βtub Ef1α Ef2 Enolase Grc5 Hsp70cyt Hsp90 MetK Rps22a Rps23a Tsec61 Sum

Alveolates Alexandrium tamarense 1 1 1 1 1 1 1 1 1 1 1 1 1 14

Alveolates Chilodonella uncinata 1 1 1 1 1 1 1 1 1 10
Alveolates Crypthecodinium cohnii 1 1 1 1 5
Alveolates Eimeria tenella 1 1 1 1 1 1 1 1 1 10
Alveolates Heterocapsa rotundata 1 1 1 1 1 6
Alveolates Karenia brevis 1 1 1 1 1 1 1 1 1 1 11
Alveolates Nyctotherus ovalis 1 1 1 1 1 1 1 1 1 1 1 12
Alveolates Oxyrrhis marina 1 1 1 1 1 6
Alveolates Paramecium tetraurelia 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Alveolates Perkinsus marinus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Alveolates Plasmodium berghei 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Alveolates Sterkiella histriomuscorum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Alveolates Stylonychia lemnae 1 1 1 1 1 6
Alveolates Tetrahymena thermophila 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16

Parfrey et al. www.pnas.org/cgi/content/short/1110633108

Alveolates Theileria parva 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Alveolates Toxoplasma gondii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Amoebozoa Acanthamoeba castellanii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Amoebozoa Arcella hemisphaerica 1 1 3
Amoebozoa Dictyostelium discoideum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Amoebozoa Entamoeba histolytica 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Amoebozoa Hartmannella vermiformis 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Amoebozoa Mastigamoeba balamuthi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Amoebozoa Physarum polycephalum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Amoebozoa Rhizamoeba sp. ATCC 50933 1 1 1 4
Animals Aphrocallistes vastus 1 1 1 1 5
Animals Apis mellifera 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Aplysia californica 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Animals Branchiostoma floridae 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Caenorhabditis elegans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Capitella capitata 1 1 1 1 1 1 1 1 1 1 11
Animals Drosophila melanogaster 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Gallus gallus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Homo sapiens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Mnemiopsis leidyi 1 1 1 1 1 1 1 1 1 1 1 12
Animals Nematostella vectensis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Animals Oscarella carmela 1 1 1 1 1 1 1 1 1 1 1 12
Animals Schistosoma mansoni 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Choanoflagellida Monosiga brevicollis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Cryptophyta Goniomonas† 1 1 1 1 1 1 1 1 1 1 11
Cryptophyta Guillardia theta 1 1 1 1 1 1 1 1 1 10
Euglenozoa Bodo saltans 1 1 1 1 1 1 1 8
Euglenozoa Diplonema papillatum 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Euglenozoa Entosiphon sulcatum 1 1 1 4
Euglenozoa Euglena gracilis 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Euglenozoa Euglena longa 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Euglenozoa Leishmania major 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16

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 Table S2. Cont.
Lineage Taxon* 14–3-3 40S Actin αtub βtub Ef1α Ef2 Enolase Grc5 Hsp70cyt Hsp90 MetK Rps22a Rps23a Tsec61 Sum

Euglenozoa Trypanosoma brucei 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16

Fornicata Carpediemonas membranifera 1 1 1 1 5
Fornicata Giardia duodenalis ATCC 50803 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Fornicata Spironucleus barkhanus 1 1 1 1 1 1 1 1 1 1 1 12
Fungi Allomyces macrogynus 1 1 1 1 1 1 1 1 1 1 1 1 13
Fungi Candida albicans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Fungi Glomus intraradices 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Fungi Phanerochaete chrysosporium 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Fungi Saccharomyces cerevisiae 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Fungi Schizosaccharomyces pombe 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Fungi Spizellomyces punctatus 1 1 1 1 1 1 1 1 1 1 1 12
Fungi Ustilago maydis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Glaucophytes Cyanophora paradoxa 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Glaucophytes Glaucocystis nostochinearum 1 1 1 1 1 1 1 1 1 1 1 1 13

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Haptophytes Emiliania huxleyi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Haptophytes Isochrysis galbana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Haptophytes Pavlova lutheri 1 1 1 1 1 1 1 1 1 1 1 1 13
Haptophytes Prymnesium parvum 1 1 1 1 1 1 1 1 1 1 1 1 13
Heterolobosea Naegleria gruberi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Heterolobosea Sawyeria marylandensis 1 1 1 1 1 1 1 1 1 1 1 1 13
Ichthyosporea Amoebidium parasiticum 1 1 1 1 1 1 1 1 1 1 11
Ichthyosporea Capsaspora owczarzaki 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Ichthyosporea Sphaeroforma arctica 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Jakodidae Jakoba libera 1 1 1 1 1 1 1 1 1 1 1 12
Jakodidae Reclinomonas americana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Jakodidae ‘Seculamonas ecuadoriensis’ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Kathablepharidae Leucocryptos marina 1 1 1 1 1 1 7
Malawimonas Malawimonas californiana 1 1 1 1 1 1 1 1 1 1 1 1 12
Malawimonas Malawimonas jakobiformis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Parabasalidea Trichomonas vaginalis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Preaxosytla Monocercomonoides sp. 1 1 1 1 1 6
Preaxosytla Streblomastix strix 1 1 1 1 5
Preaxosytla Trimastix pyriformis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Rhizaria Bigelowiella natans 1 1 1 1 1 1 1 1 1 1 1 12
Rhizaria Corallomyxa tenera 1 1 1 1 1 1 7
Rhizaria Gromia‡ 1 1 3
Rhizaria Heteromita§ 1 1 1 1 1 1 1 1 1 10
Rhizaria Ovammina opaca 1 1 1 4
Rhizaria Plasmodiophora brassicae 1 1 1 4
Rhizaria Reticulomyxa filosa 1 1 1 1 1 1 1 1 1 10
Red algae Chondrus crispus 1 1 1 1 1 1 1 1 1 1 1 1 13
Red algae Cyanidioschyzon merolae 1 1 1 1 1 1 7
Red algae Gracilaria changii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Red algae Porphyra yezoensis 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Stramenopiles Apodachlya brachynema 1 1 1 1 1 1 7
Stramenopiles Aureococcus anophagefferens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15

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 Table S2. Cont.
Lineage Taxon* 14–3-3 40S Actin αtub βtub Ef1α Ef2 Enolase Grc5 Hsp70cyt Hsp90 MetK Rps22a Rps23a Tsec61 Sum

Stramenopiles Ectocarpus siliculosus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16

Stramenopiles Heterosigma akashiwo 1 1 1 1 1 1 7
Stramenopiles Phaeodactylum tricornutum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Stramenopiles Phytophthora infestans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Stramenopiles Thalassiosira pseudonana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Green algae Acetabularia acetabulum 1 1 1 1 1 1 1 1 1 10
Green algae Arabidopsis thaliana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Green algae Chlamydomonas reinhardtii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Green algae Dunaliella salina 1 1 1 1 1 1 1 1 1 1 1 12
Green algae Ginkgo biloba 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Green algae Mesostigma viride 1 1 1 1 1 1 1 1 1 1 1 1 1 14
Green algae Micromonas pusilla 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Green algae Oryza sativa 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Green algae Ostreococcus tauri 1 1 1 1 1 1 1 1 1 1 1 1 13

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Green algae Physcomitrella patens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16
Green algae Volvox carteri 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15
Green algae Welwitschia mirabilis 1 1 1 1 1 1 1 1 1 1 1 1 1 14

*Taxa in bold are included in both the 91-taxon and 109-taxon analyses.
†
Composite of Goniomonas truncata and Goniomonas cf. pacifica.
‡
Composite of G. oviformis and Gromia sp. Antarctica.
§
Composite of. H. globosa and Heteromita sp. ATCC PRA-74.

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Table S3. PhyloBayes calibrations
Node specification Calibration†

Taxon* Species 1 Species 2 Max Min

Amniota Gallus gallus Homo sapiens 400 328.3

Angiosperms Arabidopsis thaliana Welwitschia mirabilis 425 133.9
Ascomycetes Schizosaccharomyces pombe Phanerochaete chrysosporium 1,000 400
Coccolithophores Emiliania huxleyi Isochrysis galbana 260 203.6
Diatoms Aureococcus anophagefferens Thalassiosira pseudonana 550 133.9
Dinoflagellates Karenia brevis Crypthecodinium cohnii 300 240
Embryophytes Mesostigma viride Oryza sativa 600 471
Endopterygota Apis mellifera Drosophila melanogaster 350 284.4
Eudicots Arabidopsis thaliana Oryza sativa 133.9 125
Euglenids Entosiphon sulcatum Euglena gracilis 3,000 450
Foraminifera Ovammina opaca Reticulomyxa filosa 3,000 542
Gonyaulacales Alexandrium tamarense Crypthecodinium cohnii 240 196
Pennate diatoms Phaeodactylum tricornutum Thalassiosira pseudonana 110 80
Spirotrichs Sterkiella histriomuscorum Stylonychia lemnae 3,000 444
Trachaeophytes Physcomitrella patens Arabidopsis thaliana 471 425
Vertebrates Branchiostoma floridae Homo sapiens 555 520
Animals Nematostella vectensis Capitella capitata 3,000 632
Arcellinida Arcella hemisphaerica Rhizamoeba sp 3,000 736
Bilateria Branchiostoma floridae Capitella capitata 630 555
Chlorophytes Acetabularia acetabulum Volvox carteri 3,000 700
Ciliates Paramecium tetraurelia Chilodonella uncinata 3,000 736
Florideophyceae Chondrus crispus Porphyra yezoensis 3,000 550
Red algae Cyanidioschyzon merolae Chondrus crispus 3,000 1,174

*Taxon is same as in Table 1; see Table 1 for other notes.

†
Calibrations in PhyloBayes are specified as a uniform distribution with minimum and maximum dates, and were run with soft bounds.

Other Supporting Information Files

Dataset S1 (XLS)

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