Applied Microbiology and Biotechnology

https://doi.org/10.1007/s00253-018-8831-x

MINI-REVIEW

Two or three domains: a new view of tree of life in the genomics era
Zhichao Zhou 1,2 & Yang Liu 1,3 & Meng Li 1 & Ji-Dong Gu 2

Received: 29 November 2017 / Revised: 30 January 2018 / Accepted: 1 February 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
The deep phylogenetic topology of tree of life is in the center of a long-time dispute. The Woeseian three-domain tree theory, with
the Eukarya evolving as a sister clade to Archaea, competes with the two-domain tree theory (the eocyte tree), with the Eukarya
branched within Archaea. Revealed by the ongoing debate over the last three decades, sophisticated and proper phylogenetic
methods should necessarily be paid with more emphasis, especially these are focusing on the compositional heterogeneity of sites
and lineages, and the heterotachy issue. The newly emerging archaeal lineages with numerous eukaryotic-like features, such as
membrane trafficking and cellular compartmentalization, are phylogenetically the closest to eukaryotes currently. These findings
highlight the evolutionary history from an ancient archaeon to a more complex archaeon with protoeukaryotic-like features and
complex cellular structures, thus providing clues to understand eukaryogenesis process. The increasing repertoire of precise
genomic contents provides great advantages on understanding the deep phylogeny of tree of life and ancient evolutionary events
on Eukarya branching process.

Keywords Tree of life . Eocyte tree . Woeseian tree . Asgard superphylum . Eukaryotic-like features

Introduction origin of eukaryotes (Archibald 2008; Bapteste and Brochier

The rRNA-based phylogeny divides the bacteria, archaea, and
eukaryotes into three district groups (Woese and Fox 1977). This
has been adopted as the paradigm for the universal tree of life in
textbooks for years, but it has been always accompanied to the
debate of the eocyte tree hypothesis (Heinz and Domman 2017;
Lake 1988, 1990; Lake et al. 1984). The key argument concerns
the evolutionary positioning of these three biologically different
groups considering their vertically inherited core genetic compo-
nents, and deciphering the process of eukaryogenesis and the
Zhichao Zhou and Yang Liu contributed equally to this review and Yang
Liu is the co-first author.
* Meng Li
limeng848@szu.edu.cn
1
Institute for Advanced Study, Shenzhen University,
Shenzhen 518060, People’s Republic of China
2
Laboratory of Environmental Microbiology and Toxicology, School
of Biological Sciences, The University of Hong Kong, Pokfulam
Road, Hong Kong SAR, Hong Kong, People’s Republic of China
3
Key Laboratory of Optoelectronic Devices and Systems of Ministry
of Education and Guangdong Province, College of Optoelectronic
Engineering, Shenzhen University, Shenzhen 518060, People’s
Republic of China
2004; Cox et al. 2008; de Duve 2007; Guy and Ettema 2011;
Hug et al. 2016; Lake 1990, 2015; Pace et al. 2012; Philippe and
Forterre 1999; Rochette et al. 2014; Williams et al. 2013; Yang
and Roberts 1995). The major controversial issues are rooted on
the methods and applications of phylogenetic reconstruction of
tree of life by conserved proteins among the three groups, and the
interpretation on the evolutionary history of ancient
eukaryogenesis process in a genetically and metabolically rea-
sonable manner (Eme et al. 2017; Foster 2004; van der Gulik
et al. 2017; Williams and Embley 2014). The ancient phyloge-
netic signals could be obscured by few shared conservative pro-
teins between groups, inconsistent nucleotide mutation rate and
pattern among sites, lineages and evolving time, lateral gene
transfers, and gene duplications which blur evolutionary line.
Furthermore, improper application of evolutionary models will
also entangle the true topology of phylogenetic tree through
many ways, such as long branch attraction effect, artifact topol-
ogy through unbalanced sampling and heterotachy issue, and so
on (Archibald 2008; Foster 2004; Kolaczkowski and Thornton
2004; Lopez et al. 1999; Philippe and Forterre 1999). Together
with the enhancement of phylogenetic reconstruction methods
and applications, many other ways of thinking are suggested to
overcome the “phylogenomic impasse” in the recent decades
(Albani et al. 2010; de Duve 2007; Forterre 2015; Poole and
Neumann 2011; van der Gulik et al. 2017). Hypothetical

frameworks on the order of events of the eukaryogenesis process
have been proposed recently, with consensus agreed on the en-
dosymbiosis origin hypothesis, but still encouraging new evi-
dence on addressing the detail information (Eme et al. 2017;
Roger et al. 2017). New insights accompany the advances of
phylogenetic reconstruction approaches in the recent decades.
Increasingly available genomic data and analysis methods have
been used to delineate the ancient divergence process and the
deep phylogenetic relationship. In the era of genomics, the appli-
cation of using precise genomic contents reconstructed from gap-
bridging archaea lineages by advanced phylogenomic ap-
proaches has just brought new visions to the topic of tree of life
(Ettema 2016; Li et al. 2015; Liu et al. 2018; Spang et al. 2015;
Zaremba-Niedzwiedzka et al. 2017). This mini-review serves
as an up-to-date renewing summary in the era of genomics
to this old but still ongoing and essential topic. We
interpreted pros and cons of phylogenetic reconstruction
applications and clarified the key points on both sides of
debate and ongoing evidences on addressing issues of tree
topology, genomic interpretation, and hypothesis of
eukaryogenesis. More importantly, this review also pro-
vides some perspectives on progressively addressing these
issues according to the understanding on the current
studies.
Disputes concerning the tree topology
Since 1970s, divergent opinions on the tree topology are based
on the alignment processing methods and phylogenetic recon-
struction approaches (Table 1). For instance, a comprehensive
phylogenetic analysis of 51 proteins, including ribosomal pro-
teins, elongation factors, and polymerases involved in the rep-
lication, transcription, and translation, revealed that individual
protein-based analyses do not lead to a consistent tree topolo-
gy but, rather, suggest a trend favoring the three-domain tree;
on the other hand, the analysis of a concatenated set of these
proteins and 16S–23S rRNA genes strongly supports the two-
domain tree when advanced maximum-likelihood (ML) and
Bayesian phylogenetic modeling is used (Archibald 2008;
Cox et al. 2008). Opposite results were obtained when apply-
ing maximum parsimony or a compositional homogeneous
model on analyzing the alignments of LSU and small subunit
(SSU) rRNA, and 51 core proteins (Archibald 2008; Cox et al.
2008). Recent advances in sophisticated phylogenetic recon-
struction method allow a reevaluation of phylogeny taking
into consideration of compositional heterogeneity and vari-
able evolutionary rates for amino acids, nucleotide sites, and
branches of a tree. Furthermore, these methods should also be
introduced, to eliminate artificial tree topology, e.g., long
branch attraction effect (Foster 2004; Lake 1988, 1994;
Sidow and Wilson 1990; Tourasse and Gouy 1999; Yang
and Roberts 1995). Additionally, the validation of fitness of
Appl Microbiol Biotechnol
model to data via a posterior predictive simulation should be
employed for phylogenetic model testing (Cox et al. 2008).
The highly saturated scenario of mutational sites in the infor-
mative protein alignments (e.g. the alignments of elongation
factors, ATPases, etc.) provides few ancient phylogenetic sig-
nals, which might lead to the phylogeny highly sensitive to
differences of evolutionary rates. Furthermore, if this scenario
also combined with the effects of gene duplication, loss, and
lateral gene transfer (LGT), it will haunt the reliability of deep
phylogeny topologies (Archibald 2008; Lopez et al. 1999;
Philippe and Forterre 1999).
Current phylogenomic approaches that employ a heteroge-
neous compositional method can delay mutational saturation
by calculating the among-site rate variation, thus facilitating the
investigation of ancient phylogenetic events (Foster et al. 2009).
The rRNA genes are about to become saturated even when the
heterogeneous compositional model is applied, explaining why,
formerly, each rRNA sequence had to be described by two com-
position vectors for a better fitness of the model to data during
NDCH modeling to overcome the highly saturated scenario
(Cox et al. 2008; Foster et al. 2009). When combined with ad-
vanced reconstruction methods, the concatenated gene or protein
sequences increase the catalog of homologs across three domains
and have become a realistic solution to root tree of life
(Archibald 2008; Bapteste and Brochier 2004; Cox et al. 2008;
Foster et al. 2009; Guy and Ettema 2011; Tourasse and Gouy
1999). However, ancient LGT and reconstruction artifacts would
still blur the true phylogeny (Bapteste and Brochier 2004). The
order of pairwise sequence alignments in the early time and the
option to individually align single sequences before concatena-
tion also dictate the final tree topologies (Ciccarelli et al. 2006;
Katoh et al. 2001; Lake 1991). A dataset of discrete orthologous
protein sequence groups (DOGs) from archaeal lineages was
analyzed using a substitution-model-independent method, and
this generated a consistent phylogenomic tree topology with
different heterogeneous composition models (Kelly et al.
2011). Subsequently, Bayesian and ML approaches using homo-
logs of DOGs in eukaryotic genomes placed the intersection of
Eukarya and Archaea at the branching site of Thaumarchaeota
from the euryarchaeotal line, which is supporting the two-
domain tree (Kelly et al. 2011). A phylogenomic investigation
of 28 vertically inherited last eukaryotic common ancestor
(LECA) clades supported eukaryotes either branching deep
within Archaea or close to the root of Archaea, but separate from
Crenarchaeota and Euryarchaeota (Rochette et al. 2014).
Addressing the elusive deep phylogeny of the origin of
Eukarya by molecular phylogenetic reconstruction is difficult,
and it would constitute a “phylogenetic impasse” (Forterre
2015; Rochette et al. 2014). Several alternative approaches
have been suggested, to provide more evidence to approach
the ultimate truth, including the thinking of biological plausi-
bility, and comparative cell biology and fossil records (Albani
et al. 2010; de Duve 2007; Forterre 2015; Poole and Neumann
Appl Microbiol Biotechnol

Table 1 Primary comparison of the context, features, and pros and cons of the tree topology hypotheses

Three-domain theory Two-domain theory

Eukarya and Archaea are monophyletic, separated sister groups. Eukarya are contained within Archaea and share a common ancestor with
Bacteria form the roots of the tree of life. Crenarchaeota. Life is divided into two domains: Bacteria and Archaea.
Also called the “eocyte tree” theory.
Three-domain theory founder: Carl Woese (Woese and Fox 1977) Two-domain theory founder: James Lake (Lake et al. 1984) (Archaea,
(Archaea, Bacteria, and Eukarya) Bacteria)
Major points: Major points:
1. The living system comprises three aboriginal lines of descent 1. Eocytes (Crenarchaeota) and eukaryotes share a similar ribosome
(Woese and Fox 1977). structure (Lake et al. 1984).
2. The origin of life is on the bacterial line of descent (Woese 1987; 2. Evolutionary parsimony treeing algorithm places eocytes as a sister group
Woese and Fox 1977). of eukaryotes (Lake 1988).
3. Chloroplasts and mitochondria are of bacterial origin (Lang et al. 3. A tree based on a concatenated 53-gene alignment favors the eocyte tree
1999). topology (Cox et al. 2008).
4. The eukaryal nuclear line is primordial, not derived from Bacteria or 4. Eukarya might have originated from the ancestral Crenarchaeota or
Archaea (Woese et al. 1990). Thaumarchaeota (Kelly et al. 2011).
5. The essential apparatus of eukaryotic cell has originated from an archaeal
ancestor (Archibald 2008)
Pros: Pros:
1. Compositional statistics, an advanced method based on evolutionary 1. Specific operational genes of eukaryotes have bacterial origins, and this
parsimony, supports the three-domain tree based on the phylogeny may also be the case for the lipid biosynthesis machinery (Esser et al.
of DNA alignments of genes encoding the large subunit of RNA 2004; Rivera et al. 1998).
polymerase (Sidow and Wilson 1990). 2. Most informational genes involved in DNA replication, transcription, and
2. Protoeukaryotes before LECA might have evolved to acquire most translation in Archaea are related to their eukaryotic counterparts (Esser
features of the contemporary eukaryotic cell, including the et al. 2004; Rivera et al. 1998).
phagocytosis machinery, leading up to a subsequent mitochondrion 3. The components of the Crenarchaeal cell division machinery are
engulfment. The direct archaea-origin theory of LECA, which homologous to the components of the eukaryotic vesicle budding system
favors the eocyte tree, should need to explain the evolution and the (Lindås et al. 2008).
loss of a potential archaeal mechanism for engulfment (Poole and
Neumann 2011).
Cons: Cons:
1. Early time phylogenetic reconstruction of the three-domain tree is 1. Archaea have acquired glycerol-ether lipids, while bacteria and eukaryotes
based on parsimony and distance methods, which are unrealistic acquired glycerol-ester lipids. If, as posited by the eocyte hypothesis,
since they do not consider the compositional heterogeneity and protoeukaryotes are of an archaeal origin, they should have evolved to
variable evolutionary rate among sites (Lake 1994; Yang and replace their glycerol-ether lipids biosynthesizing pathway to a
Roberts 1995). bacterial-like pathway (Archibald 2008).
2. Evolutionary parsimony method, which reduces the long branch
attraction effect, supports the eocyte tree topology based on rRNA
alignments (Lake 1988).
3. Universal phylogenies of rRNA and RNA polymerase sequences are
affected by the variable rates of nucleotide and amino acid
substitution among sequences sites, resulting in long branch
attraction (Tourasse and Gouy 1999).
Common points
1. The ancestor of the mitochondrion is a proto-alphaproteobacterium, contributing many of the original bacterial genes to eukaryotes (Esser et al. 2004).
There is no evidence for the involvement of other bacterial lineages in the origin of Eukarya (Rochette et al. 2014).
2. LECA is necessarily a primordial ancestor that acquired the majority of the cellular biological features from extant eukaryotic cells (Poole and
Neumann 2011; Rochette et al. 2014).
3. Eukaryotic genome has archaea original, bacterial original, and eukaryote-specific gene clusters (Rochette et al. 2014).
4. Bacteria have diversified well before the eukaryogenesis, and all extant eukaryotes are derived from LECA, as they are monophyletic (López-García
and Moreira 2015).

2011). They are prone to support the Woeseian three-domain
tree topology hypothesis. With respect to the former, a dra-
matic transformation after the diversification of Archaea
evolving toward Eukarya seems highly unlikely (Table 1)
(Archibald 2008; Forterre 2015). Furthermore, eukaryotic-
like features in the last archaeal common ancestor (LACA)
were much more pronounced than in modern archaea, with
their selective loss during the development of the archaeal
route leading to the modern archaea (de Duve 2007; Forterre
2015). The selective loss theory could also be applied to
Woeseian three-domain tree hypothesis, in which these fea-
tures presented in the last common ancestor of Archaea and
Eukarya selectively lost in the streamlining process leading
the divergence of Eukarya and modern Archaea (Forterre

2015). With respect to the latter, the consensus on LECA to
acquire essential cell machineries has been achieved clearly
(Forterre 2015; Poole and Neumann 2011). LECA could have
originated well around three Gyr ago, supported by discover-
ing multicellular eukaryote fossil dating back to 2.1 billion
years ago (Albani et al. 2010; Forterre 2015). This also indi-
cates that the tempo of evolution of the central componentry
of molecular cell fabric decreased around three Gyr ago, prob-
ably after the divergence of three domains (Forterre 2015).
Key hypotheses in the battle of the two
theories
The origin of eukaryotes is crucial for placing the root of
Eukarya and disentangling the early evolutionary processes of
eukaryogenesis. Several hypotheses have been proposed, re-
volving around the two ways of non-vertical gene flow, endo-
symbiotic gene transfer (EGT) by mitochondrion engulfment,
and other forms of horizontal gene transfer (HGT). According
to the hydrogen hypothesis, an early mitochondrion hypothesis,
eukaryotes arose through endosymbiosis between a respiratory
alphaproteobacteria and a hydrogenotrophic archaeon (Martin
and Muller 1998; Sousa et al. 2016). Consensus has been
achieved as to the endosymbiosis origin hypothesis; however,
a key issue dictating the phylogenetic relationship between
Archaea and Eukarya concerns the determination of the receiv-
ing host. The mito-early hypothesis claims that mito-
endosymbiosis was the trigger driving eukaryogenesis, while
the mito-late hypothesis supports a functionally fledged
protoeukaryotic cell receiving the protomitochondrion late in
the eukaryogenesis (Degli Esposti 2016; Pittis and Gabaldón
2016; Rochette et al. 2014). Recent phylogenomic tests re-
vealed shorter phylogenetic distances between LECA protein
clades of alphaproteobacterial ancestry and proteins with mito-
chondrial localization, than proteins of different prokaryotic
origins or cellular localization, supporting the mito-late hypoth-
esis (Ettema 2016; Pittis and Gabaldón 2016). Nevertheless,
subsequent doubts on the mis-assignment of mitochondrial
proteins to other bacterial origin have weakened the statistics-
based conclusions of the mito-late theory (Degli Esposti 2016).
The slow-drip hypothesis, supporting the notion that the
early eukaryotic ancestors experienced several waves of
Fig. 1 Schematic diagram on
two-domain and three-domain
tree. This comparison highlights
the different tree topologies and
positions of process of
eukaryogenesis in two hypotheses
Appl Microbiol Biotechnol
massive HGT events, explains the origin of non-
mitochondrial bacteria-related proteins and is also endorsed
by phylogenomic analysis (Ettema 2016; Lester et al. 2006;
Rochette et al. 2014). However, the exact order of
eukaryogenesis events could not be delineated since extant
eukaryotes have diversified following the emergence of
LECA, with no intermediate forms existing in nature. In ad-
dition, neither early-mito nor late-mito hypotheses propose a
defined tree topology because the eukaryogenesis always oc-
curs on the eukaryotic stem from the ancestral archaea to
LECA under both two- or three-domain tree theories
(Fig. 1). By recovering protein clusters present in both
Archaea and Bacteria and with no interdomain LGT signals,
the predicted physiological features suggest that the last uni-
versal common ancestor (LUCA) was an anaerobic, H2-de-
pendent, thermophilic, diazotrophic autotroph with methyl
metabolism-centered Wood-Ljungdahl pathway, dwelling in
a hydrothermal setting (Martin et al. 2016; Weiss et al.
2016). This finding links the geochemical processes with the
potential physiology of LUCA reasonably well and converges
with the two-domain theory as Eukarya are not involved in the
origin of life (Martin et al. 2016; Weiss et al. 2016).
Revival of the two-domain tree and new
features introduced by gap-bridging archaea
The increasing microbial genome data and advanced evolu-
tionary reconstruction methods have driven the revival of two-
domain tree since the 1990s (Table 1). Since all the ancestor-
finding efforts were necessarily based on the investigation of
extant organisms, disentanglement of the deep phylogeny re-
quired data on the intermediate-stage life forms. Placing the
eukaryotes as the sister clade of the archaeal superphylum
TACK (including Thaum-, Aig-, Cren-, and Korarchaeota,
and currently expanding with Bathy-, Verstraetearchaeota,
and YNPFFA group archaea) in the two-domain tree is sup-
ported by an analysis of 26 universally conserved protein
clusters (Guy and Ettema 2011; He et al. 2016; Meng et al.
2014; Vanwonterghem et al. 2016). Proteins with eukaryotic
features (Eukaryotic-specific proteins, ESPs) are continually
discovered in the TACK superphylum; they play roles in ge-
netic information storage and processing, and mechanisms
Appl Microbiol Biotechnol
including cytokinesis, membrane remodeling, cell shaping,
division, and protein recycling (Ettema et al. 2011; Guy and
Ettema 2011; Küper et al. 2010; Makarova et al. 2010). These
results indicated their potential vertical inheritance from an
archaeal parent close to the TACK superphylum (Ettema
et al. 2011; Guy and Ettema 2011; Küper et al. 2010;
Makarova et al. 2010). This conclusion is largely compatible
with current studies (Raymann et al. 2015; Williams et al.
2013), as Rochette et al. claimed that eukaryotic stem is
deep-branched with current archaeal phyla (Rochette et al.
2014). Nevertheless, two scenarios still exist for this
branching relationship, since it is possible that the eukaryotic
clade is either close to the archaeal phyla and shares a short
stem with protoarchaea (three-domain tree topology) or basal
within archaeal phyla (two-domain tree topology) (Rochette
et al. 2014). It was also proposed that Eukarya is nested within
the TACK superphylum, close to Korarchaeota or emerging
as a sister group of the TACK superphylum (Williams et al.
2012). Not all ESP features are necessarily observed in a sin-
gle phylum but may be patchily distributed, which indicates
that potential gene loss from the shared ancestor and HGT
might have contributed to the formation of extant archaeal
and eukaryotic lineages (Forterre 2015; Guy and Ettema
2011; Williams et al. 2013).
The Asgard superphylum (including Loki-, Thor- Odin-,
and Heimdallarchaeota) discovered in the last 2 years is
phylogenomically closest to eukaryotes, based on conserved
marker proteins and rRNA genes (Seitz et al. 2016; Spang
et al. 2015; Zaremba-Niedzwiedzka et al. 2017; Liu et al.
2018). The majority of sophisticated Bayesian and ML phy-
logenetic analyses place eukaryotes in the closest sister branch
of Heimdallarchaeota, indicating a shared ancestry of the
Asgard archaea and eukaryotes, and a clear two-domain tree
topology (Fig. 2a) (Zaremba-Niedzwiedzka et al. 2017).
Many ESPs that are patchily distributed in the TACK
superphylum are encoded by the Lokiarchaeota genomes;
many of these proteins play vital roles in cellular structure
and compartmentalization, including membrane remodeling
and vesicular trafficking (Fig. 2b) (Klinger et al. 2016;
Spang et al. 2015). This lends support to the late-mito hypoth-
esis that the archaeal ancestor of eukaryotes had a rudimentary
cellular structure and/or potential phagocytosis functions
(Pittis and Gabaldón 2016; Roger et al. 2017; Spang et al.
2015). The ESPs identified in Lokiarchaeota have been also
identified in other genomes of the Asgard superphylum mem-
bers, including an expanded set of GTPases, RLC7 dynein
proteins, dynamic actin cytoskeleton proteins, ESCRT I-III
machineries, ubiquitin modifier system proteins, and
oligosaccharyl transferase complex proteins. New features,
not represented in Lokiarchaeota, have also been found in
the Asgard lineages, including cytoskeletal componentries,
epsilon DNA polymerase subunit, ribosomal protein
L28e/Mak16, etc. (Zaremba-Niedzwiedzka et al. 2017; Liu
et al. 2018). Thorarchaeota genomes encode several
orthologs of eukaryotic membrane trafficking machinery
components and putative eukaryotic-like coat proteins in-
volved in vesicle biogenesis (Zaremba-Niedzwiedzka et al.
2017; Liu et al. 2018). These findings expand the repertoire
of ESPs in archaea taxa, which are found especially abundant
in Asgard lineages, indicating that the protoeukaryote host cell
has already emerged some aspects of eukaryotic cellular com-
plexity before acquiring protomitochondrion (Eme et al.
2017). Beyond genomics, the complex endomembrane sys-
tem in the Crenarchaeota Ignicoccus hospitalis discovered
by micro-anatomy experiments also prompts this assumption
of archaea-origin of eukaryotic endomembrane system
(Heimerl et al. 2017). It consists of cytoplasmic protrusions,
and with secretory function, representing a primary
endomembrane system that possesses selective features to its
eukaryotic counterpart (Heimerl et al. 2017). A recent analysis
of gene family evolution patterns based on 31,236 archaeal
gene families places the archaeal root in the monophyletic
DPANN superphylum (Diapherotrites, Parv-, Aenigm-,
Nano-, and Nanohaloarchaeota) and other archaea
(Williams et al. 2017). The newly emerging archaeal lineages
bridge the existing gap between extant archaeal and eukaryot-
ic clades with respect to the complexity of cellular and phys-
iological organization, and it is encouraging to explore geno-
mic and functional properties of even closer archaeal lineages
to protoeukaryotes, which will push forward the emerging
time of the direct archaeal ancestor of protoeukaryote and
probably reveal more eukaryotic specific features within it
(Zaremba-Niedzwiedzka et al. 2017). To the opposite,
Patrick Forterre group raised claims that chimeric features of
elongation factor EF2 and RNA polymerase A of
Lokiarchaeota bins (from 2015 Loki castle) could lead to fault
tree topologies in the concatenated protein alignments (Da
Cunha et al. 2017). However, an updated response including
more Asgard superphylum bins used sophisticated methods
still holding that eukaryotes are branched within archaeal lin-
eage (Spang et al. 2017). It is still in hot debate on the
phylogenomic tree topology of these archaea and their inter-
mediate role on the evolutionary path toward protoeukaryotes,
thus highlighting significant evolutionary meanings (Da
Cunha et al. 2017; Nasir et al. 2016; Zaremba-Niedzwiedzka
et al. 2017).
The new tree of life and ongoing debate
The tree of life proposed by Hug et al. is the first comprehen-
sive phylogenomic tree since the advent of genome-resolved
metagenomic sequencing and analysis methods (Hug et al.
2016). One representative high-quality or complete genome
per genus (3083 organisms, out of which 1011 organisms are
novel) was used for phylogenomic reconstruction of this tree
 Appl Microbiol Biotechnol

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Heimdalarchaeota LC_3
Odinarchaeota LCB_4 
represented taxa with no pure Thorarchaeota AB_25 Asgard
Thorarchaeota SMTZ1−45
cultured archaeon reported. The Thorarchaeota SMTZ1−83 
LokiarchaeumVSGC14_75
nodes with bootstrap value within Lokiarchaeota CR_4
Hyperthermus butylicus DSM_5456 (Crenarchaeota 
the range of 80–100% were Ignicoccus hospitalis KIN4I (Crenarchaeota)
Caldisphaera lagunensis IC_154_DSM_15908 (Crenarchaeota)
labeled with black dots, and dot Acidianus hospitalis W1 (Crenarchaeota) 
YNPFFA SCGC_AAA471_L13_GBS_N_001_22
sizes were scaled to the values (a). YNPFFA SCGC_AAA471_B05
Thermofilum pendens Hrk_5 (Crenarchaeota)
The gray squares indicated the Korarchaeum cryptofilum23)Korarchaeota
Verstraetearchaeota V2
presence of ESPs, and the empty Verstraetearchaeota V1
Verstraetearchaeota V3 TACK
white squares indicated the Verstraetearchaeota V5 (Proteoarchaeota)
Verstraetearchaeota V4
absence. The heatmap bar Nitrosopumilus koreensis Ar1 (Thaumarchaeota)
Cenarchaeum symbiosum A (Thaumarchaeota)
indicated the changing trend of Nitrososphaera viennensis En76 (Thaumarchaeota)
Bathyarchaeota B24
ESP numbers (b) Bathyarchaeota AD8-1
Bathyarchaeota BA1
Bathyarchaeota B26-2
Caldiarchaeum subterraneum (Aigarchaeota)
Aigarchaeota SCGC_AAA471_F17_GBS_N_001_13
Aigarchaeota JGI_0000106_J15_GBS_C_001_288
Nanoarchaeota SCGC_AAA011_L22_DUSEL4_1
Nanoarchaeota SCGC_AAA011_G17_DUSEL_001_198
Pacearchaeota RBG_13_36_9
Woesearchaeota AR3
Woesearchaeota AR15
DPANN
Haloredivivus sp*Nanohaloarchaeota
Aenigmarchaeota AR5_gwa2
Micrarchaeum acidiphilum ARMAN2 (Parvarchaeota)
Diapherotrites SCGC_AAA011_E11_DUSEL_001_206
Diapherotrites AR10_gwc2
Pyrococcus furiosus'60BEuryarchaeota
Methanotorris igneus Kol_5 Euryarchaeota
Thermoplasmatales%51$Euryarchaeota
Haladaptatus paucihalophilus'60BEuryarchaeota Euryarchaeota
Archaeoglobus fulgidus'60BEuryarchaeota
Methanopyrus kandleri$V19Euryarchaeota
Methanobrevibacter smithii$TCC_35061 Euryarchaeota

(Hug et al. 2016). The SSU rRNA gene-based phylogeny 2016). This study highlights the expanding knowledge of di-
largely agrees with concatenated 16 ribosomal protein-based versity of life by genome-resolving approaches and the deep
phylogeny (the new tree of life by Hug et al.); however, the evolutionary history contained in the domain of bacteria (es-
former one shows a three-domain topology, while the latter pecially for the novel CPR) (Hug et al. 2016) and, neverthe-
one shows a two-domain topology, placing Eukarya sibling to less, does not offer conclusive opinions on deep tree topology.
Lokiarchaeota (Hug et al. 2016). In this tree, the proposed Once released, Norman Pace raised doubts about the foun-
Candidate Phyla Radiation (CPR) has symbiotic-like geno- dation of this tree, the methods used, and conclusions obtained
mic features with reduced metabolic capacity. The early emer- as follows (Pace 2016). First, Parametric evolutionary
gence of CPR and/or subjection to a symbiotic lifestyle could methods, including ML and Bayesian methods, compromise
potentially explain the high diversity and fast evolution of this the estimates of sites and lineages, simulating sequences by a
clade; however, the early emergence of CPR is only represent- homogeneous evolutionary process in general, ignoring the
ed by ribosomal proteins but not by SSU rRNA (Hug et al. heterotachy issue (evolutionary rates at specific sites change
Appl Microbiol Biotechnol
non-identically over time), and leading to highly aberrant trees
(Kolaczkowski and Thornton 2004; Lopez et al. 2002; Pace
2016). Moreover, a concatenated gene tree produces substan-
tial variation in single-gene histories, and when including ad-
ditional concatenated gene sets, its topology changes
(Kolaczkowski and Thornton 2004; Kubatko and Degnan
2007; Pace 2016). Secondly, because unbalanced sampling
between the Bacteria and Eukarya/Archaea domains would
cause the long branch attraction effect, the overloaded sam-
pling of Bacteria in the three-domain tree hampers the resolv-
ing of relationship between Eukarya and Archaea, which will
exacerbate the heterotachy effect (Pace 2016). Thirdly,
masking uncertain gaps and unique places not shared by three
domains is required as suggested (Pace 2016).
Recently, researchers also suggested to set aside the phylo-
genetic approach and use the functional division as the first
taxonomic division (van der Gulik et al. 2017). They
contended that the cytosolic ribosome and mitoribosome of
eukaryotes are different from their prokaryotic counterpart
from various aspects and argued that the eukaryotic cell is
not simply the ancestral archaeal host with minor medications
and the mitochondrion is not simply a reduced bacterium (van
der Gulik et al. 2017). They also pointed out that the appear-
ance of eukaryotic cells and their characteristics come out as a
result of mutual adaptations of two prokaryotes, in which the
framework of symbiogenesis is suitable for the general
scheme and dictates a three-domain model (Martin and
Muller 1998; van der Gulik et al. 2017).
Furthermore, recent genomic studies of giant viruses re-
vealed that giant viruses contain proteins characteristic for
cellular life, hypothetically regarded as remnant components
of a novel ancient cellular life, hence igniting the debate of the
fourth origin of life (Abrahão et al. 2017; Forterre 2010; Heinz
and Domman 2017; Koonin and Yutin 2010). However,
phylogenomic analysis of such proteins encoded by the re-
cently characterized Klosneuviruses genomes suggests that
their origin is similar to that of diverse eukaryotes, which is
contrary to the fourth domain hypothesis (Schulz et al. 2017).
Instead, giant viruses would derive from small viruses through
piecemeal acquisition of their host genes as proposed (Schulz
et al. 2017). Overall, it seems that the release of the latest tree
of life in the genomics era does not close the debate; on the
contrary, it instigates careful revisiting of the evolution and
diversity of extant biological lineages by sophisticated
methods and precise genomic contents.
Conclusions and perspectives
A consensus on the deep topology of the tree of life is still
lacking, and the origin of eukaryotes and precise stages of
eukaryogenesis remain elusive. However, by understanding
the constraints of and improvement of the phylogenomic
reconstruction methods and increasing the repertoire of pre-
cise genomic contents, a gradually clear prospect of the tree of
life is approaching in this era of genomics.
Future evolutionary modeling methods should include the
consideration on the various evolutionary rates during the
evolving history besides the compositional heterogeneity is-
sue of mutations over different sites and lineages, thus
avoiding tree topology artifacts. The posterior predictive sim-
ulation upon a model for testing its fitness for interpreting the
data should be included to enhance its reliability. Since that,
conservative proteins provide more stable phylogenetic sig-
nals among selective lineages; when solving branch place-
ments within lower level trees, more shared sets of proteins
or makers could be acquired, by which, an overall structure of
clearer sub-lineage tree topology will be achieved and pave a
way to offer details and initiating points to make a “return” to
full tree structure configuration (Eme et al. 2017).
The micro-anatomy experiment provides direct cell structure
evidence proving the complex endomembrane system in selec-
tive gap-bridging archaea (Heimerl et al. 2017); together with the
ongoing genomic evidence on archaeal ESPs for functions of
complex cellular structure and compartmentalization, now it be-
comes an integrated working path to address the theory of ar-
chaeal origin of eukaryotic endomembrane system. It is pointed
out that Planctomycete endomembrane system might represent
an analogous complex endomembrane structure which arises in
another evolutionary path (Heimerl et al. 2017). Planctomycete
is also an old bacterial lineage branching out from the other
bacteria at ancient time. It will be intriguing to study and com-
pare the evolutionary processes that build the convergent sys-
tems, which is essential to find out the mechanisms and details of
formation of endomembrane system. One specific species within
Planctomycete, Gemmata obscuriglobus, has developed a eu-
karyotic nuclear-like membrane structure enveloping the nucle-
oid, with primitive pores and other compartmentalized mem-
brane structures (Fuerst 2005). This, from another perspective,
provides a clue for explaining nuclear formation in
eukaryogenesis process that protoeukaryotic cell could have
evolved with a functionally fledged cell structure compartments
before the endosymbiotic event.
Endosymbiosis is a pivotal process and studies have come
into convergence to agree with the endosymbiosis theory for
eukaryote origin as a proto-alphaproteobacterium engulfed
into an archaeal host (Forterre 2015; Williams and Embley
2014). Endosymbiosis has also been proposed a paradigm to
address the origin of several organelles, e.g., plastid, mito-
chondria, and peroxisome (still in discussion) (Tabak et al.
2006; Timmis et al. 2004). The anammox bacteria, which is
a distinct and deeply branching group within Planctomycete,
is now subjected to endosymbiosis theory for addressing the
origin for its special anaerobic ammonium oxidation proto
“organelle” or single membrane-bounded compartment,
anammoxsome (Hong et al. 2014). Anammoxsome is

associated with biochemical process of anaerobically oxidiz-
ing ammonium with nitrite for ATP generation, which is spe-
cifically the only identified prokaryote-hosted organelle
possessing the same function parallel to mitochondrion in eu-
karyotes (Hong et al. 2014). Its origin was proposed by a
plausible theory, in which an ancient archaeon capable for
anaerobically oxidizing archaea is engulfed into a bacterial
host for mutual advantages with interdependent metabolisms
and stays stable through subsequently endosymbiotic fusion
(Hong et al. 2014). Genomic, cell biological, and metabolic
exploration on the endosymbiosis process of anammoxsome
in a bacterial host will service as a source of reference for
better understanding the endosymbiosis process forming
eukaryotes.
Dedicated efforts into the discovery of deep-branching ar-
chaea close to eukaryotes or proto forms of eukaryotes with
primary functions close to LECA are encouraging, and the
genomic properties are important for us to figure out the piece
of evidence that is lost in the eukaryogenesis process, as well
as the cell biological and physiological properties. It will pro-
vide additional insights into the ancient evolutionary history,
thus promoting the understanding of the phylogenetic and
physiological relationships between Eukarya and Archaea.
Beyond all the rapidly developing genomic approaches,
scientists suggest a revisiting on the biology and function of
the origin of eukaryotes (van der Gulik et al. 2017). The huge
biological function differences between eukaryote and pro-
karyote could not be simply addressed by searching for more
complex prokaryotes with advanced eukaryotic-specific fea-
tures which approaches a model of an archaeon host, but set-
ting aside a biological reality thinking (van der Gulik et al.
2017). It is proposed that eukaryotic features, including cell
structure formation, organelle formation, etc., should be
emerging from the process of symbiosis effects of mutual
adaptation of the merging participants (two or more) (van
der Gulik et al. 2017). The emerging properties are more than
putting two partners (or more) in together (van der Gulik et al.
2017).
Evidently, many exciting progresses have been reported
recently. Although many scientists are debating the topology
tree of life and origin of eukaryotes, we are now closer to the
frontline to answer the primary question of life and nature with
the progress of modern techniques and discovery of new life
forms.
Acknowledgments We thank for presentations, critiques, and comments
from Prof. Norman R. Pace, Prof. Jared R. Leadbetter, and valuable
discussions by the participants in the 2016 MBL microbial diversity sum-
mer course. We also thank Prof. William F. Martin for his suggestions on
this work.
Author contributions Z.Z., Y.L., M.L., and J.D.G. conceived the review.
Z.Z., Y.L., and M.L. wrote the original manuscript and prepared the tables
and figures. M.L. and J.D.G. reviewed and edited the final manuscript.
Appl Microbiol Biotechnol
Funding This project is supported by a postgraduate student fellowship
from HKU and Lorus J. & Margery J. Milne Scholarship from MBL to
Zhichao Zhou (ZZ) and The National Natural Science Foundation of
China (No. 31622002, 41506163) to Meng Li (ML).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants performed by any of the authors.
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