Life 2015, 5, 949-968; doi:10.3390/life5010949 OPEN ACCESS life ISSN 2075-1729 www.mdpi.com/journal/life Article Phylogeny and Taxonomy of Archaea: A Comparison of the Whole-Genome-Based CVTree Approach with 16S rRNA Sequence Analysis

Guanghong Zuo 1, Zhao Xu 2 and Bailin Hao 1,*

1 T-Life Research Center and Department of Physics, Fudan University, 220 Handan Road, Shanghai 200433, China; E-Mail: [email protected] 2 Thermo Fisher Scientific, 200 Oyster Point Blvd, South San Francisco, CA 94080, USA; E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-21-6565-2305.

Academic Editors: Roger A. Garrett, Hans-Peter Klenk and Michael W. W. Adams

Received: 9 December 2014 / Accepted: 9 March 2015 / Published: 17 March 2015

Abstract: A tripartite comparison of Archaea phylogeny and taxonomy at and above the rank order is reported: (1) the whole-genome-based and alignment-free CVTree using 179 genomes; (2) the 16S rRNA analysis exemplified by the All-Species Living Tree with 366 archaeal sequences; and (3) the Second Edition of Bergey’s Manual of Systematic Bacteriology complemented by some current literature. A high degree of agreement is reached at these ranks. From the newly proposed archaeal phyla, Korarchaeota, Thaumarchaeota, Nanoarchaeota and Aigarchaeota, to the recent suggestion to divide the class Halobacteria into three orders, all gain substantial support from CVTree. In addition, the CVTree helped to determine the taxonomic position of some newly sequenced genomes without proper lineage information. A few discrepancies between the CVTree and the 16S rRNA approaches call for further investigation.

Keywords: Archaea; phylogeny and taxonomy; 16S rRNA analysis; whole-genome comparison; alignment free; CVTree Life 2015, 5 950

1. Introduction

Prokaryotes are the most abundant and diverse creatures on Earth. The recognition of Archaea as one of the three main domains of life [1,2] was a milestone in the development of biology and a great success of using the 16S rRNA sequences as molecular clocks for prokaryotes, as suggested by Carl Woese and coworkers [3,4]. The Second Edition of Bergey’s Manual of Systematic Bacteriology [5] (hereafter, the Manual), a magnificent work of more than 8000 pages, took 12 years (2001–2012) to complete and is being considered by many microbiologists as the best approximation to an official classification of prokaryotes [6]. As stated in the Preface to vol. 1 of the Manual, these volumes “follow a phylogenetic framework based on analysis of the nucleotide sequence of the small ribosomal subunit RNA, rather than a phenotypic structure.” However, the “congruence” of phylogeny and taxonomy on the basis of 16S rRNA sequence analysis raises a question of principle, namely the necessity of cross-verification of whether the present classification is capable of providing a natural and objective demarcation of microbial organisms. The answer comes with the advent of the genomic era. A whole-genome-based, alignment-free, composition vector approach to prokaryotic phylogeny, called CVTree [7–12], has produced robust phylogenetic trees that agree with prokaryotic taxonomy almost at all taxonomic ranks, from domain down to genera and species, and more importantly, many apparent disagreements have disappeared, with new taxonomic revisions appearing. In fact, all published taxonomic revisions for prokaryotes with sequenced genomes have added to the agreement of CVTree with taxonomy. A recent example from the domain Archaea was the reclassification of Thermoproteus neutrophilus to Pyrobaculum neutrophilum [13]. In this paper, we study Archaea phylogeny across many phyla. This is distinct from the phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large or, as Cavalier-Smith [14] put it, with “mega-classification” of prokaryotes, focusing on taxonomy of higher ranks. Although in taxonomy, the description of a newly discovered organism necessarily starts from the lower ranks, higher rank assignments are often incomplete or lacking. At present, the ranks above class are not covered by the Bacteriological Code [15,16]. The number of plausible microbial phyla may reach hundreds, and archaeal ones are among the least studied. According to the 16S rRNA analysis, the major archaeal classes and their subordinate orders have been more or less delineated. Therefore, in order to carry out the aforementioned cross-verification, we make an emphasis on higher ranks, such as phyla, classes and orders. A study using 179 Archaea genomes provides a framework for the further study of lower ranks.

2. Material and Method

Publicly available Archaea genome sequences are the material for this study. At present, more than 30,000 prokaryotic genomes have been sequenced [17], among which, about 16,000 have been annotated [18]. These numbers keep growing and make whole-genome approaches more than ever feasible. Life 2015, 5 951

As of the end of 2014, there were 165 Archaea genomes released on the NCBI FTP site [19]. These genomes with corresponding lineage information from NCBI taxonomy were part of the built-in database of the CVTree web servers [20,21]. A search of NCBI databases revealed 14 more archaeal genomes; these were uploaded to the web server at run time. Archaea genomes listed in the EBI Genome Pages [22] were all included. A full list of these 179 genomes with accession numbers is given in the Appendix. A whole-genome-based phylogeny avoids the selection of sequence segments or orthologous genes. It must be alignment-free, due to the extreme diversity of prokaryotic genome size and gene content. Our way of implementing alignment-free comparison consists of using K-peptide counts in all protein products encoded in a genome to form a raw “composition vector” (CV). The raw CV components then undergo a subtraction procedure in order to diminish the background caused by neutral mutations, hence to highlight the shaping role of natural selection [23]. Using whole genomes as input data also helps to circumvent the problem of lateral gene transfer (LGT), as the latter is merely a mechanism of genome evolution together with lineage-dependent gene loss. Being a nightmare for single- or few-protein-based phylogeny, LGT may even play a positive role in whole-genome approaches, as it takes place basically in shared ecological niches [24] and among closely-related species [25]. Plasmid genomes were excluded from our input data, thus further reducing plasmid-mediated LGT. Using whole genome input and the alignment-free method also makes CVTree a parameter-free approach. In other words, given the genomes, phylogenetic trees are generated without any adjustment of the parameters or the selection of sequence segments. As the CVTree methodology has been elucidated in many previous publications (see, e.g., [7–12]) and a web server was released twice in 2004 [26] and 2009 [20], we will not discuss the methodological aspects of CVTree here. However, it should be understood that the peptide length K, though looking like a parameter, does not function as a parameter. For a discussion on the role of K and why K = 5, 6 leads to the best results, we refer to a recent paper [27]. All CVTree figures shown in this paper were generated at K = 6. In this paper, the term CVTree is used to denote the method [7–12,27], the web server [20,21,26] and the resulting tree; see, e.g., [28]. Traditionally, a newly generated phylogenetic tree is subject to statistical re-sampling tests, such as bootstrap and jackknife. CVTree does not use sequence alignment. Consequently, there is no way to recognize informative or non-informative sites. Instead, we take all of the protein products encoded in a genome as a sampling pool for carrying out bootstrap or jackknife tests [7]. Although it was very time-consuming, CVTrees did pass these tests well [11]. However, successfully passing of statistical re-sampling tests only informs about the stability and self-consistency of the tree with respect to small variations of the input data. It is by far not a proof of the objective correctness of the tree. Direct comparison of all branchings in a tree with an independent taxonomy at all ranks would provide such a proof. The 16S rRNA phylogeny cannot be verified by Bergey’s taxonomy, as the latter follows the former. However, the agreement of branchings in CVTree with Bergey’s taxonomy would provide much stronger support to the tree, as compared to statistical tests. This is the strategy we adopt for the CVTree approach. There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that the mutation rate remains more or less a constant Life 2015, 5 952 across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study, like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without the indication of branch lengths and bootstrap values. Branching order in a tree by itself does not bring about taxonomic ranks, e.g., class or order. The latter can be assigned only after comparison with a reference taxonomy, which is not a rigid framework, but a modifiable system. Though there is a dissimilarity measure in the CVTree algorithm, it is not realistic to delineate taxa by using this measure, at least for the time being. Even if defined in the future, it must be lineage dependent. For example, it cannot be expected that the same degree of dissimilarity may be used to delineate classes in all phyla. In addition, monophyly is a guiding principle in comparing branching order with taxonomy. Here, monophyly must be understood in a pragmatic way, restricted to the given set of input data and the reference taxonomy. If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic. In order to effectively deal with several thousands of genomes in a run, we have parallelized the CVTree algorithm and moved the web server to a computer cluster with 64 cores. The new CVTree3 web server [21] is capable of producing trees with several thousands of leaves in a few minutes for a range of K-values, say for K = 3 to 7. In addition, the CVTree3 web server has the following advanced features: (1) CVTree3 is equipped with an interactive tree display, which allows collapsing or expanding the tree branches at the disposal of the user. The user may concentrate on an interested taxon by submitting an enquiry; only the neighborhood of the taxon is expanded and all of the rest collapsed properly, keeping the topology unchanged. Here, “collapsing” means replacing a whole branch by a single leaf. Usually, a collapsed branch is labeled by the name of the highest common taxon followed by the number of strains it represents. For example, Methanococci{12} denotes a class-level monophyletic branch containing 12 leaves. If a taxon name is seen in two (or more) collapsed branches, such as Classname{3/12} and Classname{9/12}, then the taxonomically monophyletic class does not correspond to a single branch in the collapsed tree. (2) The web server reports “convergence statistics” of all tree branches, i.e., a list of all monophyletic and non-monophyletic taxa at all taxonomic ranks for every K-value. For example, the first two lines of the report read:

Archaea{165} − − K5K6K7− Bacteria{2707} − − K5K6 − −

(Numerals in curly brackets tell the number of organisms present in a collapsed branch.) Therefore, the two domains Archaea and Bacteria are both well defined as monophyletic branches at K = 5 and 6. We note that in the statistics, only genomes with complete lineage information are counted. The example project referred to in this paper contained, in addition, 14 archaeal and 143 bacterial genomes with one or more “unclassified” rank in the lineage. Therefore, in total {165+14} = 179 Archaea and {2707+243} = 2850 Bacteria genomes were used. The {m+n} convention is useful for looking for incomplete lineages in CVTree branches. Life 2015, 5 953

(3) The lineage information of an organism is given in one line with labels ,

, , , , and , standing for the ranks domain, phylum, class, order, family, genus and species. The sTrain label does not appear in lineage information, but may be seen in a leaf. The original lineage information of the built-in genomes was taken from the NCBI taxonomy. The lineage information of user’s genomes was provided at uploading. Users are allowed to make lineage modifications and to see new statistics after doing re-collapsing. (4) When displaying a tree, the user may pull down a lineage modification window and enter a trial lineage in the form “old_lineage new_lineage”. For example, the initial lineage for Caldiarchaeum_cryptofilum_OPF8_uid58601 put it in phylum Thaumarchaeota, but there is evidence that it belongs to a new phylum, Aigarchaeota, so the modification may look like:

Thaumarchaeota ··· Caldiarchaeum

Aigarchaeota ··· Caldiarchaeum The modification line is not required to contain all ranks, but the written part must be uniquely recognizable. By submitting the lineage modification, the user performs “re-collapsing” and gets a new report of “convergence statistics”. (5) The user may select any part of a CVTree and produce a print-quality figure in SVG, EPS, PDF or PNG format.

All of these useful features help to reveal the agreement and discrepancy of a large tree with taxonomy.

3. Outline of Archaea Taxonomy at and above the Rank Order

The taxonomy of Archaea was described in Volume 1 of the Manual, which appeared in 2001 [29], thus being somewhat outdated. Two phyla, the Crenarchaeota and the Euryarchaeota, were listed there. The Crenarchaeota contained only one class, Thermoprotei. According to the latest information provided in the List of Prokaryotic Names with Standing in Nomenclature (LPSN [30]), the class Thermoprotei contains five orders: Thermoproteales, Desulfococcales, Sulfolobales, Acidilobales and Fervidicoccales, the last two being proposed in 2009 [31] and 2010 [32], respectively. Originally, the phylum, Euryarchaeota, contained seven classes: Methanobacteria, Methanococci, Halobacteria, Thermoplasmata, Thermococci, Archaeoglobi and Methanopyri; all comprising one order, except for Methanococci, which contained three orders. Later on, in a revised roadmap of the Manual [33], the class Methanococci was left with only one order; the other two orders became part of the newly proposed class, Methanomicrobia. A third order, Methanocellales, in the last class was proposed in 2008 [34]. Very recently, there appeared a proposal [35] to divide the single-order class, Halobacteria, into three orders. Over the past 15 years, a few new archaeal phyla have been proposed: Korarchaeota [36,37], Thaumarchaeota [38–40], Nanoarchaeota [41–43], Aigarchaeota [44], Parvarchaeota [45] and Bathyarchaeota [46]. All but the last three phyla have been listed in LPSN [30]. We will not touch on Parvarchaeota and Bathyarchaeota, due to a lack of well-annotated genome data. The main focus of the present study is to check and compare the positions of these high-rank taxa in CVTree and to compare them with the 16S rRNA sequence analysis where some results obtained by other authors are available. Life 2015, 5 954

4. Results and Discussion

4.1. 16S rRNA Archaeal Phylogeny According to All-Species Living Tree

An authoritative reference to the 16S rRNA phylogeny is the All-Species Living Tree Project (LTP) [47–49]. LTP is an ambitious project to construct a single 16S rRNA tree based on all available type strains of hitherto named species of Archaea and Bacteria. The latest release, LTPs115 [50], of March, 2014, was based on 366 archaeal and 9905 bacterial 16S rRNA sequences. However, the 104-page PDF of the tree is hard to comprehend, especially when it comes to comparing the tree branchings with classification at various taxonomic ranks. We fetched the treeing and lineage information files LPTs115_SSU_tree.newick and LTPs115_SSU.csv from the LTP web site [50] and then collapsed the fully-fledged tree into various taxonomic ranks where possible. We first obtained the Archaea branch containing 366 leaves and collapsed basically to the rank class without doing lineage modification (figure not shown). In fact, it was cut from the original “All-Species Living Tree” LTPs115 [50] based on all 366 archaeal and 9905 bacterial 16S rRNA sequences. There was a line Methanomicrobia{71/72} indicating that an outlier violated the monophyly of the branch. By inspecting the figure, the outlier turned out to be:

Unclassified_Methanomicrobia ··· HQ896499 ··· Unclassified_Methanomicrobia

It was located next to the monophyletic Thermoplasmata{8}. Therefore, it does not look like an “Unclassified_Methanomicrobia”, but might be a miss-classified Thermoplasmata. Judging by its close neighborhood, we may temporarily modify the lineage to:

ThermoplasmataThermoplasmatalesThermoplasmataceaeMethanomassiliicoccus···

After making the lineage modification, we get Figure1. The branchings in Figure1 fully agree with the taxonomy of Archaea, as outlined in Section3, at the phylum and class ranks. In particular, the eight classes of Euryarchaeota all behave as well-defined monophyletic branches. Further more, if one expands the class Methanomicrobia, its three subordinate orders, Methanocellales{3}, Methanosarcinales{31} and Methanomicrobiales{37}, all appear as monophyletic branches (not shown in Figure1). The definition of orders within Thermoprotei, the only class in Crenarchaeota, is somehow problematic (more on this point near the end of Subsection 4.2). This kind of agreement should be expected, as the archaeal taxonomy is largely based on the 16S rRNA sequence analysis. However, as by design, the LTP is restricted to type strains with validly published names, one cannot check the positions of the newly proposed phyla and those strains lacking a definite lineage. The whole-genome-based CVTree approach may complement these aspects of phylogeny, since the criterion for inclusion of a strain into the tree is the availability of a sequenced genome, independent of its standing in nomenclature. In Subsection 4.3, the CVTree results are compared with 16S rRNA analyses done by other authors. Life 2015, 5 955

CrenarchaeotaThermoprotei{48}

Methanopyri ... AE009439__Methanopyrus_kandleri__Methanopyraceae{1}

MethanococciMethanococcales{12}

ThermoplasmataThermoplasmatales{9}

Thermococci ... Thermococcaceae{34}

MethanobacteriaMethanobacteriales{42}

Archaeoglobi ... Archaeoglobaceae{8}

Methanomicrobia{71}

Halobacteria ... Halobacteriaceae{141}

Figure 1. The Archaea branch in the All-Species Living Tree based on 366 16S rRNA sequences. The tree has been collapsed to the rank class (), and only one lineage modification has been made. Numerals in curly brackets indicate the number of sequences contained in a collapsed branch. The collapsing and lineage modification was performed by using a web server similar to CVTree3. This Living Tree Viewer is accessible to all users [51].

4.2. The Whole-Genome-Based CVTree Phylogeny

CVTrees based on 179 Archaea, 2850 Bacteria and eight Eukarya genomes were generated by using the improved version CVTree3 [21] of the web server [20]. We show the Archaea part of a big CVTree in Figure2. When inspecting the figure, we pay more attention to the newly proposed phyla and those taxa with incomplete or suspicious lineage information.

Halobacteria ... Halobacteriaceae{28+1}

Nanoarchaeota ... Nanoarchaeum_equitans_Kin4_M_uid58009.NCBI{0+1} ThermoplasmataThermoplasmatales{4}

Euryarchaeota{0+3} Methanomicrobia{24} Archaeoglobi ... Archaeoglobaceae{6} Aciduliprofundum{0+2} Thermococci ... Thermococcaceae{20} Methanopyri ... Methanopyrus_kandleri_AV19_uid57883.NCBI{1} MethanococciMethanococcales{18} MethanobacteriaMethanobacteriales{12}

Thaumarchaeota ... Nitrososphaerales{2+5} Unclassified ... Caldiarchaeum_subterraneum_uid227223.NCBI{0+1}

Korarchaeota ... Korarchaeum_cryptofilum_OPF8_uid58601.NCBI{0+1} ThermofilaceaeThermofilum{2} Thermoprotei{48/50}

Figure 2. The 179-genome Archaea branch of CVTree obtained by using the CVTree3 web server [21] without making lineage modifications. It has been collapsed to the rank class where possible. The branching order is to be compared with taxonomy, but does not scale the branch lengths.

In what follows, the non-monophyletic branches are summarized and possible lineage modifications are suggested. Life 2015, 5 956

(1) The first line of Figure2 Halobacteriaceae{28+1} informs that among the 29 genomes, there was one without proper lineage information. In fact, it was Halophilic_archaeon_DL31_uid72619, a name not validly published and not following the basic rule for a binomen. Its NCBI lineage from phylum down to genus was “unclassified”. However, by expanding this line, the strain is seen to be located deeply inside the class Halobacteria (see Figure4). As at present, the class consists of only one order, which, in turn, is made of one family [33], it is safe to assign this strain to a yet unspecified genus. This modification would yield a monophyletic branch, Halobacteria{29}. (2) The fourth line of Figure2

Euryarchaeota{0+3} represents a cluster obtained by collapsing three strains (not explicitly written in the figure):

• Thermoplasmatales_archaeon_BRNA1_uid195930, with NCBI lineage ThermoplasmataUnclassifiedUnclassified; • Candidatus_Methanomethylophilus_alvus_Mx1201_uid196597, with NCBI lineage UnclassifiedUnclassifiedUnclassified, • Methanomassiliicoccus_sp_Mx1_Issoire_uid207287, with NCBI lineage MethanomicrobiaUnclassifiedUnclassified.

If the NCBI lineage would be accepted, two of the above strains must violate the monophyly of the classes Thermoplasmata{4/5} and Methanomicrobia{24/25}. However, the fact that these three strains, taken together, make a monophyletic branch hint of the possibility to assign them to a yet unspecified class. This modification would restore the monophyly of the two classes Methanomicrobia{24} (Line 5 in Figure2) and Thermoplasmata{4} (Line 3 in Figure2), as seen in Figure2. (3) The newly proposed phylum, Thaumarchaeota, appears to be non-monophyletic, as an outlying strain, Candidatus Caldiarchaeum subterranum, was assigned to this phylum according to the NCBI taxonomy. The NCBI assignment might reflect its position in some phylogenetic tree based on concatenated proteins, e.g., Figure 2 in [52]. However, in the original paper reporting the discovery of this strain [44] and in recent 16S rRNA studies, e.g., [46], Candidatus Caldiarchaeum subterranum was proposed to make a new phylum, Aigarchaeota. CVTrees support the introduction of this new phylum. A lineage modification of Candidatus Caldiarchaeum subterranum from Thaumarchaeota to Aigarchaeota would lead to a monophyletic Thaumarchaeota. (4) The Candidatus genus, Aciduliprofundum, is considered a member of the DHEV2 (deep-sea hydrothermal vent euryarchaeotic 2) phylogenetic cluster. No taxonomic information was given in the original papers [53,54]. The NCBI taxonomy did not provide definite lineage information for this taxon at the class, order and family ranks. According to [53], the whole DHEV2 cluster was located close to Thermoplasmatales in a maximum-likelihood analysis of 16S rRNA sequences. A similar placement was seen in [52], where a Bayesian tree of the archaeal domain based on concatenation of 57 ribosomal proteins put a lonely Aciduliprofundum next to Thermoplasmata. Life 2015, 5 957

However, in CVTrees, constructed for all K-values from three to nine, Aciduliprofundum is juxtaposed with the class Thermococci{18}. An observation in [54] that this organism shares a rare lipid structure with a few species from Thermococcales may hint to its possible association with the latter. If we temporarily presume a lineage:

ThermococciUnclassifiedUnclassifiedAciduliprofundum ···

one might have a monophyletic class Thermococci{20}. Since none of the 13 DHEV2 members listed in [53] have a sequenced genome so far, CVTree cannot tell the placement of the DHEV2 cluster as a whole for the time being. It remains an open problem whether DHEV2 is close to Thermoplasmata or to Thermococci or if a new class is needed to accommodate DHEV2. (5) The new phylum, Korarchaeota, violates the monophyly of the phylum, Crenarchaeota, by drawing to itself the family, Thermofilaceae. However, in an on-going study of ours (not published yet) using a much larger dataset, this violation no longer shows up; both Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes. However, it is worth noting that the whole lower cluster of Figure2 supports a recent proposal for a new “TACK” superphylum [55], made of Thaumarchaeota, Aigarchaeota, Crenarchaeota and Korarchaeota.

After making all of the aforementioned lineage modifications, the resulting CVTree (not shown) looks much like Figure2 with minor changes of some labels. All eight classes of Euryarchaeota, as listed in Section3, are well-defined on their own. In addition, a new class might be introduced for the three archaeons without detailed lineage information, collapsed as

Euryarchaeota{0+3}. The last point cannot be checked in the All-Species Living Tree without extending it to cover organisms without validly published names. Now, it comes to inspect the orders in the single-class phylum, Crenarchaeota. There is no a priori reason to expect that 16S rRNA sequence analysis and the CVTree approach should lead to identical tree branchings. Though all being assigned to Crenarchaeota, the forty eight 16S rRNA sequences in the All-Species Living Tree and the 50 genomes in the CVTree do not belong to the same set of organisms. One can only compare those in common. Two orders, Sulfolobales and Thermoproteales, are monophyletic in both CVTree and 16S rRNA trees, putting aside the insertion of the single-species, Korarchaeota, into Thermoproteales in CVTree. The introduction of the new orders, Acidilobales in 2009 [31] and Fervidicoccales in 2010 [32], violated the monophyly of the so-far monophyletic order, Desulfurococcales (the genus, Acidilobus, was considered part of Desulfurococcaceae before 2009). A main criterion to distinguish species of the new order from that in Desulfurococcales was indicated in [31] as acidophily, a point that might require further verification. Life 2015, 5 958

The CVTree results summarized above were a continuation and extension of a similar study [56] based on 62 Archaea genomes available at the beginning of 2010. The fact that, five years apart and with 117 more genomes added, the results remain consistent informs of the robustness of the CVTree approach.

4.3. Phylum Distribution in Other Phylogenies

The conclusions drawn above concerning the positions of the newly proposed phyla and organisms with uncertain lineage information cannot be directly compared with the All-Species Living Tree Project [47–49], as by design, LTP only includes strains with validly published names and standing in nomenclature. To this end, one must look for other published studies. An effective way of comprehending a tree with many leaves consists of collapsing the tree branches to appropriate taxonomic ranks, as we did in Figures1 and2. For published results of other authors, we collapsed their trees manually. Figure3 shows four such trees collapsed to the phylum level from corresponding trees in [44] and [52]. Figure3a is a maximum likelihood tree of concatenated SSU and LSU rRNAs using 3063 nucleotide positions; Figure3b is a maximum likelihood tree of 45 concatenated ribosomal proteins and nine RNA polymerase subunits using 5993 aligned amino acids; and Figure3c is a maximum likelihood tree from translation EF2 proteins based on 590 residues. All of these three subfigures were obtained by collapsing Figure 4 in [44]. Figure3d was collapsed from a Bayesian tree based on concatenation of 67 ribosomal proteins from 89 genomes (Figure 2 in [52]).

K(1) K(1) K(1) A(1) A(1) A(1) A(1) T(3) T(4) T(2) T(5) C(24) C(16) C(17) C(16) K(1) N(1) N(1) N(1) N(1) P(2) E(33) E(33) E(5/33) E(9/67) E(28/33) E(58/67) (a) (b) (c) (d) Figure 3. Archaea trees collapsed to phyla. Abbreviations: A = Aigarchaeota,C = Crenarchaeota, E = Euryarchaeota, K = Korarchaeota, P = Parvarchaeota, N = Nanoarchaeota, T = Thaumarchaeota.(a–c) Obtained by collapsing Figure 4 in [44]; (d) obtained by collapsing Figure 2 in [52]. Numerals in parentheses indicate the number of species represented in each phylum. For details, see the text and the cited papers.

The interrelationship among phyla deduced from a limited number of representatives in a tree is subject to further changes when more data become available. In 2001, when there was only one genome from each of the bacterial phyla, Aquificae and Thermotogae, there was speculation that these phyla would make a clade [57,58]. A decade later, it was observed that, though remaining in a big cluster, many other phyla have gotten inserted in between Aquificae and Thermotogae; see, e.g., [10]. This point concerns especially the archaeal phyla with only one representative genome for the time being. By comparing our Figure2 with trees in Figure3, we see: Life 2015, 5 959

(1) The newly proposed phyla, Thaumarchaeota, Korarchaeota and Aigarchaeota, are supported in many phylogenies; especially the superphylum “TACK” is supported in most phylogenies, with “TAC” being a persistent core. (2) The nano-sized archaean symbiont, Nanoarchaeum equitans, has a highly reduced genome (490,885 bp [42]). It is the only described representative of a newly proposed phylum, Nanoarchaeota, and it cuts into the otherwise monophyletic phylum, Euryarchaeota. We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees; see, e.g., Figure 4 in a 2009 paper [59], as well as (c) and (d) in Figure3. It has been known that tiny genomes of endosymbiont microbes often tend to move towards the baseline of a tree and distort the overall picture. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny; see, e.g., [28] and a note on the home page of the CVTree web server [20]. In the present case, we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly-reduced genome.

So far, we have concentrated on “mega-classification” [14] of Archaea species, mainly their taxonomy at the rank order and above. Quite recently, there appeared a proposal [35] to split the single-order class, Halobacteria, into three orders: Haloferacales, Natrialbales and Halobacteriales. In order to check whether CVTree supports this proposal or not, an expansion of the class, Halobacteria{29}, the first line in Figure2, is given in Figure4. Indeed, the three main branches are clearly seen in Figure4, corresponding to the three proposed orders, except for a single genus, Halakalicoccus, which did not take a definite position, even in trees obtained by different methods in [35]. Being supported by the previous predictive power of CVTree, we anticipate that the position of Halakalicoccus in Figure4 may better reflect the reality, a point verifiable in the future.

HaloquadratumHaloquadratum_walsbyi{2} Halobacterium{3}

Unclassified ... Halophilic_archaeon_DL31_uid72619.NCBI{0+1} Halorubrum ... Halorubrum_lacusprofundi_ATCC_49239_uid58807.NCBI{1} Halogeometricum ... Halogeometricum_borinquense_DSM_11551_uid54919.NCBI{1} Haloferax{2} Natronomonas{2} Halorhabdus{2} Halomicrobium ... Halomicrobium_mukohataei_DSM_12286_uid59107.NCBI{1} Haloarcula{3} Salinarchaeum ... Salinarchaeum_sp_Harcht_Bsk1_uid207001.NCBI{1} Halalkalicoccus ... Halalkalicoccus_jeotgali_B3_uid50305.NCBI{1} Halovivax ... Halovivax_ruber_XH_70_uid184819.NCBI{1} Halostagnicola ... Halostagnicola_larsenii_XH-48{1} Natrialba ... Natrialba_magadii_ATCC_43099_uid46245.NCBI{1} Natronobacterium ... Natronobacterium_gregoryi_SP2_uid74439.NCBI{1} Natronococcus ... Natronococcus_occultus_SP4_uid184863.NCBI{1} Natrinema{2} Halopiger ... Halopiger_xanaduensis_SH_6_uid68105.NCBI{1} Haloterrigena ... Haloterrigena_turkmenica_DSM_5511_uid43501.NCBI{1}

Figure 4. The class, Halobacteria, expanded to the genus level.

5. Conclusions

The CVTree approach to prokaryotic phylogeny distinguishes itself from the 16S rRNA sequence analysis, both in the input data (genomes instead of RNA sequences) and in the methodology (K-peptide Life 2015, 5 960 counting versus sequence alignment). The agreement of the two approaches makes the results more objective and convincing, whereas a few discrepancies call for further study. A phylogenetic study across many phyla naturally places emphasis on building a robust backbone for classification. At taxonomic rank order and above, whole-genome approaches are essentially simpler, as the only prerequisite is having the genomes at hand. Sooner or later, phylogenetic information and taxonomic placement will become by-products of genome analyses. The cost of sequencing a prokaryotic genome will drop below the average expense of carrying out conventional phenotyping experiments. To this end, a crucial factor is the availability of reliable, convenient and easy-to-use tools, such as the CVTree web server. The technique of collapsing and expanding tree branches with an interactive display, as well as automatic reporting of comparison results at all taxonomic ranks makes large-scale studies more feasible. The experience accumulated in this study on 179 archaeal strains will be instructive for carrying out similar studies on Bacteria, which would cover hundred-fold more strains. The 16S rRNA sequence analysis will remain an indispensable tool in microbiology. The number of sequenced genomes can never catch up with that of rRNA sequences. Although the CVTree method adds more agreement than discrepancy to the 16S rRNA results, the difference between the two approaches certainly deserves in-depth scrutiny. In addition, since high resolution power at the species level and below is a prominent advantage of CVTree as compared to 16S rRNA sequence analysis [12,60], we will elaborate on this aspect in the future when the amount of sequenced archaeal genomes will have increased substantially.

Acknowledgments

The authors thank the support of the National Basic Research Program of China (973 Project Grant No. 2013CB34100) and of The State Key Laboratory of Applied Surface Physics, as well as the Department of Physics, Fudan University. An early discussion with Jiandong Sun on problems raised in this study is also gratefully acknowledged. The authors thank the three anonymous reviewers for making essential comments and suggestions to improve the manuscript.

Author Contributions

Bailin Hao designed the study and wrote the manuscript. Guanghong Zuo and Zhao Xu built and maintained the web server, collected data and carried out the calculation. Guanghong Zuo and Bailin Hao performed the analysis. All authors have read and approved the final manuscript.

Appendix: List of Genomes Used in This Study

All of the 179 genomes used in the present study are listed in the following table, together with their accession number and approximate proteome size (in 106 amino acids). The 165 genomes from the NCBI FTP site [19] come with uid numbers, but the uploaded ones appear without uid. We note that in the EBI list of Archaea [22], there are 176 species. Excluding a tiny one, 175 genomes remain. The four genomes present at NCBI, but absent at EBI, are Nos. 31, 40, 106 and 137. Life 2015, 5 961

Table A1. list of genomes used in this study.

Proteome Accession No. Name of Strain Size (106 AA) Number 1 Acidianus hospitalis W1 uid66875 0.62 NC_015518 2 Acidilobus saccharovorans 345 15 uid51395 0.45 NC_014374 3 Aciduliprofundum boonei T469 uid43333 0.47 NC_013926 4 Aciduliprofundum sp. MAR08 339 uid184407 0.45 NC_019942 5 Aeropyrum camini SY1 JCM 12091 uid222311 0.47 NC_022521 6 Aeropyrum pernix K1 uid57757 0.49 NC_000854 7 Archaeoglobus fulgidus DSM 4304 uid57717 0.67 NC_000917 8 Archaeoglobus fulgidus DSM 8774 0.69 CP006577 9 Archaeoglobus profundus DSM 5631 uid43493 0.48 NC_013741 10 Archaeoglobus sulfaticallidus PM70 1 uid201033 0.61 NC_021169 11 Archaeoglobus veneficus SNP6 uid65269 0.56 NC_015320 12 Caldisphaera lagunensis DSM 15908 uid183486 0.44 NC_019791 13 Caldivirga maquilingensis IC 167 uid58711 0.60 NC_009954 14 Candidatus Caldiarchaeum subterraneum uid227223 0.51 NC_022786 15 Candidatus Korarchaeum cryptofilum OPF8 uid58601 0.48 NC_010482 16 Candidatus Methanomethylophilus alvus Mx1201 uid196597 0.49 NC_020913 17 Candidatus Nitrosopumilus koreensis AR1 uid176129 0.47 NC_018655 18 Candidatus Nitrosopumilus sp. AR2 uid176130 0.49 NC_018656 19 Candidatus Nitrososphaera evergladensis SR1 0.82 CP007174 20 Candidatus Nitrososphaera gargensis Ga9 2 uid176707 0.77 NC_018719 21 Methanomassiliicoccus sp. Mx1 Issoire uid207287 0.56 NC_021353 22 Cenarchaeum symbiosum A uid61411 0.62 NC_014820 23 Desulfurococcus fermentans DSM 16532 uid75119 0.40 NC_018001 24 Desulfurococcus kamchatkensis 1221n uid59133 0.40 NC_011766 25 Desulfurococcus mucosus DSM 2162 uid62227 0.39 NC_014961 26 Ferroglobus placidus DSM 10642 uid40863 0.66 NC_013849 27 Ferroplasma acidarmanus fer1 uid54095 0.57 NC_021592 28 Fervidicoccus fontis Kam940 uid162201 0.38 NC_017461 29 Halalkalicoccus jeotgali B3 uid50305 0.83 NC_014297 30 Haloarcula hispanica ATCC 33960 uid72475 1.00 NC_0159432 31 Haloarcula hispanica N601 uid230920 0.98 NC_0230102 32 Haloarcula marismortui ATCC 43049 uid57719 0.97 NC_0063972 33 Halobacterium salinarum R1 uid61571 0.60 NC_010364 34 Halobacterium sp. DL1 0.83 CP007060 35 Halobacterium sp. NRC 1 uid57769 0.59 NC_002607 36 Haloferax mediterranei ATCC 33500 uid167315 0.84 NC_017941 37 Haloferax volcanii DS2 uid46845 0.82 NC_013967 38 Halogeometricum borinquense DSM 11551 uid54919 0.82 NC_014729 39 Halomicrobium mukohataei DSM 12286 uid59107 0.90 NC_013202 40 Halophilic archaeon DL31 uid72619 0.81 NC_015954 41 Halopiger xanaduensis SH 6 uid68105 1.05 NC_015666 42 Haloquadratum walsbyi C23 uid162019 0.77 NC_017459 43 Haloquadratum walsbyi DSM 16790 uid58673 0.78 NC_008212 Life 2015, 5 962

Table A1. Cont.

Proteome Accession No. Name of Strain Size (106AA) Number 44 Halorhabdus tiamatea SARL4B uid214082 0.79 NC_021921 45 Halorhabdus utahensis DSM 12940 uid59189 0.91 NC_013158 46 Halorubrum lacusprofundi ATCC 49239 uid58807 0.93 NC_0120292 47 Halostagnicola larsenii XH-48 0.78 CP007055 48 Haloterrigena turkmenica DSM 5511 uid43501 1.09 NC_013743 49 Halovivax ruber XH 70 uid184819 0.91 NC_019964 50 Hyperthermus butylicus DSM 5456 uid57755 0.45 NC_008818 51 Ignicoccus hospitalis KIN4 I uid58365 0.40 NC_009776 52 Ignisphaera aggregans DSM 17230 uid51875 0.54 NC_014471 53 Metallosphaera cuprina Ar 4 uid66329 0.54 NC_015435 54 Metallosphaera sedula DSM 5348 uid58717 0.64 NC_009440 55 Methanobacterium formicicum strain BRM9 0.67 CP006933 56 Methanobacterium sp. AL 21 uid63623 0.72 NC_015216 57 Methanobacterium sp. MB1 complete sequence uid231690 0.56 NC_023044 58 Methanobacterium sp. SWAN 1 uid67359 0.66 NC_015574 59 Methanobrevibacter ruminantium M1 uid45857 0.76 NC_013790 60 Methanobrevibacter smithii ATCC 35061 uid58827 0.56 NC_009515 61 Methanobrevibacter sp. AbM4 uid206516 0.50 NC_021355 62 Methanocaldococcus fervens AG86 uid59347 0.44 NC_013156 63 Methanocaldococcus infernus ME uid48803 0.41 NC_014122 64 Methanocaldococcus jannaschii DSM 2661 uid57713 0.48 NC_000909 65 Methanocaldococcus sp. JH146 0.47 CP009149 66 Methanocaldococcus sp. FS406 22 uid42499 0.51 NC_013887 67 Methanocaldococcus vulcanius M7 uid41131 0.49 NC_013407 68 Methanocella arvoryzae MRE50 uid61623 0.89 NC_009464 69 Methanocella conradii HZ254 uid157911 0.70 NC_017034 70 Methanocella paludicola SANAE uid42887 0.86 NC_013665 71 Methanococcoides burtonii DSM 6242 uid58023 0.69 NC_007955 72 Methanococcus aeolicus Nankai 3 uid58823 0.44 NC_009635 73 Methanococcus maripaludis C5 uid58741 0.51 NC_009135 74 Methanococcus maripaludis C6 uid58947 0.51 NC_009975 75 Methanococcus maripaludis C7 uid58847 0.51 NC_009637 76 Methanococcus maripaludis KA1 DNA 0.55 AP011526 77 Methanococcus maripaludis OS7 DNA 0.52 AP011528 78 Methanococcus maripaludis S2 uid58035 0.49 NC_005791 79 Methanococcus maripaludis X1 uid70729 0.51 NC_015847 80 Methanococcus vannielii SB uid58767 0.49 NC_009634 81 Methanococcus voltae A3 uid49529 0.51 NC_014222 82 Methanocorpusculum labreanum Z uid58785 0.52 NC_008942 83 Methanoculleus bourgensis MS2T uid171377 0.77 NC_018227 84 Methanoculleus marisnigri JR1 uid58561 0.72 NC_009051 85 Methanohalobium evestigatum Z 7303 uid49857 0.63 NC_014253 86 Methanohalophilus mahii DSM 5219 uid47313 0.59 NC_014002 87 Methanolobus psychrophilus R15 uid177925 0.87 NC_018876 88 Methanomethylovorans hollandica DSM 15978 uid184864 0.69 NC_019977 89 Methanoplanus petrolearius DSM 11571 uid52695 0.83 NC_014507 Life 2015, 5 963

Table A1. Cont.

Proteome Accession No. Name of Strain Size (106AA) Number 90 Methanopyrus kandleri AV19 uid57883 0.50 NC_003551 91 Methanoregula boonei 6A8 uid58815 0.73 NC_009712 92 Methanoregula formicicum SMSP uid184406 0.81 NC_019943 93 Methanosaeta concilii GP6 uid66207 0.84 NC_015416 94 Methanosaeta harundinacea 6Ac uid81199 0.73 NC_017527 95 Methanosaeta thermophila PT uid58469 0.51 NC_008553 96 Methanosalsum zhilinae DSM 4017 uid68249 0.61 NC_015676 97 Methanosarcina acetivorans C2A uid57879 1.42 NC_003552 98 Methanosarcina barkeri str Fusaro uid57715 1.12 NC_007355 99 Methanosarcina mazei Go1 uid57893 1.02 NC_003901 100 Methanosarcina mazei Tuc01 uid190185 0.82 NC_020389 101 Methanosphaera stadtmanae DSM 3091 uid58407 0.49 NC_007681 102 Methanosphaerula palustris E1 9c uid59193 0.82 NC_011832 103 Methanospirillum hungatei JF 1 uid58181 1.01 NC_007796 104 Methanothermobacter marburgensis str Marburg uid51637 0.49 NC_014408 105 Methanothermobacter thermautotrophicus str Delta H uid57877 0.53 NC_000916 106 Methanothermobacter thermautotrophicus CaT2 DNA 0.51 AP011952 107 Methanothermococcus okinawensis IH1 uid51535 0.45 NC_015636 108 Methanothermus fervidus DSM 2088 uid60167 0.38 NC_014658 109 Methanotorris igneus Kol 5 uid67321 0.51 NC_015562 110 Nanoarchaeum equitans Kin4 M uid58009 0.15 NC_005213 111 Natrialba magadii ATCC 43099 uid46245 1.05 NC_013922 112 Natrinema pellirubrum DSM 15624 uid74437 1.06 NC_019962 113 Natrinema sp. J7 2 uid171337 1.05 NC_018224 114 Natronobacterium gregoryi SP2 uid74439 1.04 NC_019792 115 Natronococcus occultus SP4 uid184863 1.12 NC_019974 116 Natronomonas moolapensis 8 8 11 uid190182 0.82 NC_020388 117 Natronomonas pharaonis DSM 2160 uid58435 0.78 NC_007426 118 Nitrosopumilus maritimus SCM1 uid58903 0.49 NC_010085 119 Nitrososphaera viennensis EN76 0.73 CP007536 120 Palaeococcus pacificus DY20341 0.56 CP006019 121 Picrophilus torridus DSM 9790 uid58041 0.47 NC_005877 122 Pyrobaculum aerophilum str IM2 uid57727 0.66 NC_003364 123 Pyrobaculum arsenaticum DSM 13514 uid58409 0.61 NC_009376 124 Pyrobaculum calidifontis JCM 11548 uid58787 0.61 NC_009073 125 Pyrobaculum islandicum DSM 4184 uid58635 0.53 NC_008701 126 Pyrobaculum neutrophilum V24Sta uid58421 0.53 NC_010525 127 Pyrobaculum oguniense TE7 uid84411 0.71 NC_016885 128 Pyrobaculum sp. 1860 uid82379 0.73 NC_016645 129 Pyrococcus abyssi GE5 uid62903 0.54 NC_000868 130 Pyrococcus furiosus COM1 uid169620 0.57 NC_018092 131 Pyrococcus furiosus DSM 3638 uid57873 0.59 NC_003413 132 Pyrococcus horikoshii OT3 uid57753 0.55 NC_000961 133 Pyrococcus sp. NA2 uid66551 0.57 NC_015474 134 Pyrococcus sp. ST04 uid167261 0.52 NC_017946 135 Pyrococcus yayanosii CH1 uid68281 0.51 NC_015680 Life 2015, 5 964

Table A1. Cont.

Proteome Accession No. Name of Strain Size (106AA) Number 136 Pyrolobus fumarii 1A uid73415 0.54 NC_015931 137 Salinarchaeum sp. Harcht Bsk1 uid207001 0.91 NC_021313 138 Staphylothermus hellenicus DSM 12710 uid45893 0.46 NC_014205 139 Staphylothermus marinus F1 uid58719 0.46 NC_009033 140 Sulfolobus acidocaldarius DSM 639 uid58379 0.63 NC_007181 141 Sulfolobus acidocaldarius N8 uid189027 0.62 NC_020246 142 Sulfolobus acidocaldarius Ron12 I uid189028 0.64 NC_020247 143 Sulfolobus acidocaldarius SUSAZ uid232254 0.59 NC_023069 144 Sulfolobus islandicus HVE10 4 uid162067 0.76 NC_017275 145 Sulfolobus islandicus L D 8 5 uid43679 0.77 NC_013769 146 Sulfolobus islandicus L S 2 15 uid58871 0.76 NC_012589 147 Sulfolobus islandicus LAL14 1 uid197216 0.71 NC_021058 148 Sulfolobus islandicus M 14 25 uid58849 0.74 NC_012588 149 Sulfolobus islandicus M 16 27 uid58851 0.76 NC_012632 150 Sulfolobus islandicus M 16 4 uid58841 0.75 NC_012726 151 Sulfolobus islandicus REY15A uid162071 0.72 NC_017276 152 Sulfolobus islandicus Y G 57 14 uid58923 0.78 NC_012622 153 Sulfolobus islandicus Y N 15 51 uid58825 0.77 NC_012623 154 Sulfolobus solfataricus 98 2 uid167998 0.72 NC_017274 155 Sulfolobus solfataricus P2 uid57721 0.84 NC_002754 156 Sulfolobus tokodaii str 7 uid57807 0.76 NC_003106 157 Thermococcus barophilus MP uid54733 0.62 NC_014804 158 Thermococcus eurythermalis strain A501 0.60 CP008887 159 Thermococcus gammatolerans EJ3 uid59389 0.64 NC_012804 160 Thermococcus kodakarensis KOD1 uid58225 0.64 NC_006624 161 Thermococcus litoralis DSM 5473 uid82997 0.67 NC_022084 162 Thermococcus nautili strain 30 1 0.61 CP007264 163 Thermococcus onnurineus NA1 uid59043 0.56 NC_011529 164 Thermococcus sibiricus MM 739 uid59399 0.55 NC_012883 165 Thermococcus sp. 4557 uid70841 0.61 NC_015865 166 Thermococcus sp. AM4 uid54735 0.63 NC_016051 167 Thermococcus sp. CL1 uid168259 0.58 NC_018015 168 Thermococcus sp. ES1 0.58 CP006965 169 Thermofilum pendens Hrk 5 uid58563 0.54 NC_008698 170 Thermofilum sp. 1910b uid215374 0.52 NC_022093 171 Thermogladius cellulolyticus 1633 uid167488 0.41 NC_017954 172 Thermoplasma acidophilum DSM 1728 uid61573 0.45 NC_002578 173 Thermoplasma volcanium GSS1 uid57751 0.45 NC_002689 174 Thermoplasmatales archaeon BRNA1 uid195930 0.44 NC_020892 175 Thermoproteus tenax Kra 1 uid74443 0.55 NC_016070 176 Thermoproteus uzoniensis 768 20 uid65089 0.59 NC_015315 177 Thermosphaera aggregans DSM 11486 uid48993 0.40 NC_014160 178 Vulcanisaeta distributa DSM 14429 uid52827 0.71 NC_014537 179 Vulcanisaeta moutnovskia 768 28 uid63631 0.67 NC_015151 Life 2015, 5 965

Conflicts of Interest

The authors declare no conflict of interest.

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ISSN 2075-1729 www.mdpi.com/journal/life Peer-Review Record: Phylogeny and Taxonomy of Archaea: A Comparison of the Whole-Genome-Based CVTree Approach with 16S rRNA Sequence Analysis

Guanghong Zuo, Zhao Xu and Bailin Hao

Life 2015, 5, 949-968, doi:10.3390/life5010949

Reviewer 1: Anonymous Reviewer 2: Anonymous Editor: Roger A. Garrett, Hans-Peter Klenk and Michael W. W. Adams (Guest Editor of Special Issue “Archaea: Evolution, Physiology, and Molecular Biology”)

Received: 9 December 2014 / Accepted: 9 March 2015 / Published: 17 March 2015

First Round of Evaluation

Round 1: Reviewer 1 Report and Author Response

In the manuscript titled “Phylogeny and Taxonomy of Archaea: A Comparison of Whole-Genome-Based CVTree Approach with 16S rRNA Sequence Analysis”, Zuo et al. are describing a comprehensive analysis of archaeal phylogeny with a genome-based alignment free method, and then comparing the findings to 16S rRNA based phylogenies. The idea to use whole genome data for archaeal phylogenies is interesting, as the 16S rRNA phylogenies can be poor in resolving relationships among archaeal lineage due to GC content bias in hyperthermophilic Archaea. However, I am personally not convinced that whole genomes bring additional advantages, or if we are better off by just using a conserved set of proteins. Perhaps an additional discussion point would be the advantages of using CVtree approach with regular concatenated proteins. Leaving that opinion aside, I think the alignment-free methodology is interesting, however I would like to see a comparison with at least one regular alignment/treeing method, based on the same genomes the authors used, and not just a visual topological comparison with other published trees. Response: Multi-alignment of concatenated protein (or DNA) segments is a genome-scale, but not whole-genome approach. Its applicability depends on the scope of the phylogenetic study. When dealing with not-too-distantly related species it may yield more or less useful result. However, in a study covering many phyla it is very difficult, if not impossible, to collect a common set of conserved proteins. R2

Moreover, the concatenation method can never lead to very convincing conclusion, as give or take a few proteins may change the result. The phylogenomics people have noticed this problem, see, e.g., O. Jeffroy, H. Brinkman, F. Delsuc, H. Philippe (2008) Phylogenomics: the beginning of incongruence? Trends in Genetics, 22(4): 225–231. An example from the Bacteria domain is the relationship of the closely related Shigella and Escherichia coli strains. Concatenation of different number of genes led to different way of mixing-up of the two groups, but CVTree gave unambiguous separation of the strains as different species in the same genus Escherichia, see: G.-H. Zuo, Z. Xu, B.L. Hao (2013) Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia. Genomics Proteomics Bioinformatics, 11: 61–65. In order to carry out multi-alignment of concatenated sequences, a postdoc or well-trained PhD student equipped with the corresponding software is required. In contrast, with genome sequencing becoming a common practice in many labs it costs no additional work for a bench-microbiologist to get phylogenetic and taxonomic information by using a convenient and publically available tool such as the CVTree web serve. Well, we would be glad to see comparison of CVTree phylogeny with multi-alignment of concatenated proteins if anyone finds a way to do it for so many diverse phyla, but we do not consider it as a doable job. As far as I can see, statistical support on the branches is missing, so I have no way of assessing if this branching order is valid. Bootstrapping or jackknifing are by no means the final word on the significance of branches, however an explanation as to how the user should assess the significance of braches would be good, i.e., branch length. Response: These points were discussed in the “Material and Method” section added at the suggestion of Reviewer 2. The following was copied from the manuscript: “Traditionally a newly generated phylogenetic tree is subject to statistical re-sampling tests such as bootstrap and jackknife. CVTree does not use sequence alignment. Consequently, there is no way to recognize informative or non-informative sites. Instead we take all the protein products encoded in a genome as a sampling pool for carrying out bootstrap or jackknife tests (citing our 2004 paper). Although it was very time-consuming, CVTrees did have well passed these tests (citing our 2010 paper). However, successfully passing statistical re-sampling tests only tells about the stability and self-consistency of the tree with respect to small variations of the input data. It is by far not a proof of objective correctness of the tree. Direct comparison of all branchings in a tree with an independent taxonomy at all ranks would provide such a proof, The 16S rRNA phylogeny cannot be verified by the Bergey's taxonomy, as the latter follows the former. However, agreement of branchings in CVTree with the Bergey's taxonomy would provide much stronger support to the tree as compared to statistical tests. This is the strategy we adopt for the CVTree approach.” “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate R3

remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values”. The fact that Thermofilum is placed outside Crenarchaeota in Figures 3 and 4 is a little disturbing. I haven’t come across such a placement in other phylogenetic analyses of Archaea, for example in Brochier-Armanet et al 2008 Nat Rev Microb, or Rinke et al. 2013 Nature. I believe this needs to better explained in the manuscript, rather than just saying “this fact is noted”. Response: Yes, this is an apparent discrepancy of CVTree from 16S (and 23S) analysis for the given set of 179 archaeal genomes. However, in an on-going study of ours (not published yet) using a much larger data set this violation no longer shows up; both Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes. I am also not very convinced with the placement of Nanoarchaeota. It seems like this phylum is moving around with the addition of new sequence data (for example in Rinke et al. Figure 2 tree, they are on an entirely different branch than Euryarchaeota). Though the authors also rightfully point out that the reduced genome size may have something to do with this placement. Response: Highly degenerated genomes of many symbiont organisms tend to move around, in particular, to the baseline of a tree and thus distorts the overall structure of the tree. Therefore, it is better not to mix them with free-living organisms in a study. We rephrased the corresponding paragraph in the manuscript: “The nanosized archaean symbiont Nanoarchaeum equitans has a highly reduced genome (490,885 bp). It is the only described representative of a newly proposed phylum Nanoarchaeota and it cuts into the otherwise monophyletic phylum Euryarchaeota. We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees, see, e.g., Figure 4 in a 2009 microbial survey as well as (c) and (d) in our Figure 3. It has been known that tiny genomes of endosymbiont microbes often tend to move towards baseline of a tree and distort the overall picture. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny, see, e.g., (citing our 2010 paper) and a note in the home page of the CVTree Web Server. In the present case we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly reduced genome”. The placement of Halobacteria (due to interfering Nanoarchaeota, I presume) is also a little disturbing. I would recommend that the authors provide a discussion of this. Especially with regards to other archaeal trees. For instance, in the tree of Armanet et al 2011 that the authors also refer to, the placement of Halobacteria with respect to Nanoarchaeota is very different. R4

Response: Yes, there was certain disturbing effect of the tiny and lonely Nanoarchaeum genome, yet the Halobacteria is a very specific clade, forming a tightly connected group and moving around as a whole, mainly due to the biased acidity of their constituent amino acids. We anticipate that the relative placement of Halobacteria with respect to other groups may stabilize when more genomes are used to construct a tree. In summary, I find the piece interesting, but parts of the discussion are rather weak, therefore I am suggesting a major revision. Another reason for major revision is the style that the manuscript is written. I am not a native speaker, but given that I had to read sentences several times, I suspect the manuscript can benefit from an English language check. Response: We have done a major revision of the manuscript. A new “Material and Method” section has been added. Such issues as statistical resampling tests (bootstrap and jackknife), calibration of branch length, the meaning and choice of the peptide length K, etc., were discussed in the new section. Figures 1 and 2 were combined to a new Figure 1; Figures 3 and 4 were combined to become a new Figure 2. Figure captions were made more detailed. The whole text was checked for language flaws and many places were rephrased.

Round 1: Reviewer 2 Report and Author Response

Summary

Zuo et al. present a comparative analysis of the taxonomic classification of the Archaea domain. About 180 archaeal genomes were used to calculate a new tree topology using CVTree. The tree was compared with several 16S rRNA trees reported in the literature, and the differences were minor. Results also provide additional support to recently proposed archaeal phyla and halobacterial orders. Authors emend classification of some strains. Authors present a new interactive tree-visualization tool which enables direct validation of taxonomic groups according to their monophyly.

Evaluation

I found major concerns along this manuscript, and suggest a substantial revision. Authors should define well their objectives, also make sure that the conclusions are novel (which ones are novel, which are supportive to previous published work, etc.), and avoid redundancies in the text. Besides, would be desirable that authors provide more objective criteria for high taxa circumscription based on their methodology.

General Comments

The topic is relevant for microbial taxonomy. This kind of research should be encouraged further because old taxonomic paradigms must be systematically reviewed based on new genomic data. I would really appreciate a “Methods” part where the CVTree is explained shortly, and the tree-viewer is explained with more detail. The absence of branch lengths and bootstraps should be discussed here. Other technical aspects of the paper (e.g., sequence dataset), parameters, criteria ... all could be well organized in this part. R5

Response: A “Material and Method” section has been added where the CVTree algorithm, the interactive tree-viewer, statistical resampling tests (bootstrap, jackknife), calibration of branch lengths, etc., were discussed in slightly more detail. Archaeal phylogeny has already been studied in detail, with 16S and other marker genes, and with genomic approaches too. Some of the undersigning authors had already published on this before, although with smaller input datasets. Therefore, the fact that 16S topology is quite stable and comparable with other approaches is already known. Response: Yes, 16S rRNA phylogeny is quite stable and it almost defines the present taxonomy. We have given due credit for this. In general, CVTree does not challenge 16S rRNA analysis but complement it. Taxonomists have traditionally circumscribed the high taxa (specially orders and classes) with great subjectivity, i.e., without well accepted criteria. In terms of phylogenetic trees one premise has always been clear, a taxon must be monophyletic. This principle has been used in the present work to reconsider the status of some high taxa. However, authors do not explain objective criteria to properly interpret the rank of the clades, which impedes making a profound evaluation of the archaeal classification. Therefore, although authors have strong tools and dataset, they just achieved a small revision of the high taxa which is, indeed, quite biased by the underlying 16S guidelines. Response: A robust phylogenetic tree comes with a fixed branching order of leaves. One looks at the leaf names and their taxonomic lineage and tries to map the latter to the branches. To this end we added the following paragraphs in the “Material and Method” section. “There are two aspects of a phylogenetic tree: the branching order (topology) and the branch lengths. Branching order is related to classification and branch length to evolution time. Calibration of branch lengths is always associated with the assumption that mutation rate remains more or less a constant across all species represented in a tree, an assumption that cannot hold true in a large-scale phylogenetic study like the present one. Therefore, branching order in trees is of primary concern, whereas calibration of branch lengths makes less sense. Accordingly, all figures in this paper only show the branching scheme without indication of branch lengths and bootstrap values.” “Branching order in a tree by itself does not bring about taxonomic ranks, e,g, class or order. The latter can be assigned only after comparison with a reference taxonomy which is not a rigid framework but a modifiable system. Though a dissimilarity measure figures in the CVTree algorithm, it is not realistic to delineate taxa by using this measure at least for the time being. Even if defined in the future, it must be lineage-dependent. For example, it cannot be expected that the same degree of dissimilarity may be used to delineate classes in all phyla. In addition, monophyly is a guiding principle in comparing branching order with taxonomy. Here monophyly must be understood in a pragmatic way restricted to the given set of input data and the reference taxonomy. If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic.” R6

I have noted some lack of scientific rigor according to: many wrong taxonomic names and typos, scarce figure legends, few comments about the missing branch lengths or bootstraps (!), redundancy in text and figures and fragments which are really difficult to understand. Authors should pay attention to language, explanations and text organization. Response: We have reorganized the manuscript mainly by adding a new “Material and Method” section where discussions on branch length, statistical resampling, meaning and choice of K, etc., were given. The original Figure 1 was deleted with some related points explained in the text accompanying the original Figure 2. All figure captions have been rewritten for clarity. Authors shouldn't forget (particularly in conclusion) that the resolution power of 16S for high ranks (genus and above) is currently well accepted. And the number of non-redundant 16S entries available is much much larger than that of archaeal genomes. 16s data offer a much comprehensive view of the archaeal diversity, including deep branches. Response: A few more sentences were added in the “Conclusion” regarding the power and achievement of the 16S rRNA analysis. Introduction - L34–40: for me was difficult to follow this reasoning. Please rephrase. - L40 “Since at present (…) are not covered (...)” is a weak reason for choosing high ranks. I recommend to shortly summarize why high ranks are so important, and why do you choose order as the lowest considered rank. Response: We tried to rephrase the paragraph by changing, deleting, or adding a few words as follows: “In this paper we study Archaea phylogeny across many phyla. This is in contrast with phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large, or, as Cavalier-Smith (citing cavalier-smith 2002) put it, with “megaclassificaton” of prokaryotes. Although in taxonomy the description of a newly discovered organism necessarily starts from the lower ranks, higher rank assignments are often incomplete or lacking. At present the ranks above class are not covered by the Bacteriological Code. The number of plausible microbial phyla may reach hundreds and archaeal ones are among the less studied. According to the 16S rRNA analysis, the major archaeal classes and their subordinate orders have been more or less delineated. Therefore, in order to carry out the aforementioned cross verification we make emphasis on higher ranks such as phyla, classes, and orders. A study using 179 Archaea genomes should provide a framework for further study of lower ranks.” Part 3.1 The figure legends require more rigorous explanation. Would be interesting to remember that the branch lengths are not taken into account, or were omitted. Response: Branching order in a tree is directly related to taxonomy, while branch lengths have more to do with evolution. For large-scale phylogenetic study across many phyla the former is more important than the calibration of branch lengths. R7

The latter is based on the assumption that mutation rate is more or less constant. This assumption cannot hold when dealing with many phyla. In Line 80—authors write some explanations to understand the tree figures. I would suggest to put this text before the first tree figure. Response: This is done in the newly added “Material and Method” section. Figure 1 is a bit redundant. A short comment about the inclusion of that particular sequence into the Thermoplasmatales can be added into the legend of Figure 2. Response: The original Figure 1 was deleted and a few words added to the legend of the original Figure 2, now the new Figure 1. However, the reclassification of Methanomassiliicoccus into Thermoplasmataceae needs more explanation. To be objective, authors should address the following question, why in the same family and not in another new family? Response: Judging by the cluster labeled as Euryarchaeote{0+3} in Figure 2 Methanomassiliicoccus was not reclassified into Thermoplasmataceae but to an yet un-specified class. L106–107: maybe a bit inappropriate on that position. I would suggest to add a comment on the figure legend instead. Response: The sentence has been moved to the legend of Fig. 1 and slightly rephrased. Part 3.2 L109–118: Summarize and move to introduction. Those sentences are of general importance for the topic and not specific to part 3.2. Response: Done. L119–123: if the K issue is relevant to understand the text then please add a proper explanation. If not, then keep it simple and avoid entering into the K issue (L122–123, L132, L135, L139, 183, and also remove this K = 6 from Figure 3.) Response: the K issue is discussed in the newly added “Material and Method” section; so scattered mentioning of K has been deleted from the rest of text. Add more explanations in legend of Figure3. Response: Done in the caption of Figure 2. L143: Sounds clearer if you avoid mixing class and phylum, for example: “The placement of phylum Korarchaeota, as a closest neighbor of family Thermofilaceae, violates the monophyly of phylum Crenarchaeota.” Response: We have rewritten the paragraph as: “The new phylum Korarchaeota violates the monophyly of the phylum Crenarchaeota by drawing to itself the family Thermofilaceae. However, in an on-going study of ours (not published yet) using a much larger data set, this violation no longer shows up; both R8

Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes.” L146: No need to explain the 6+2 if it is properly explained in the figure legend. Figure 3 is redundant. I would recommend to avoid presenting different versions of the same tree; just the final tree is OK (use final/valid labels) and all important explanations in the text or legend. Perhaps the whole reasoning in Lines 142–180 is not so relevant for the current objective of comparing CVT, LTP, Bergeys? Or, perhaps, define this objective more clearly. Response: From the original Figures 3 and 4 only one has been kept and the legend rewritten. In fact, the whole paragraph changed to: “The newly proposed phylum Thaumarchaeota appears to be non-monophyletic as an outlying strain Candidatus Caldiarchaeum subterranum was assigned to this phylum according to the NCBI taxonomy. The NCBI assignment might reflect its position in some phylogenetic tree based on concatenated proteins, e.g., Figure 2 in […]. However, in the original paper reporting the discovery of this strain […] and in recent 16S rRNA studies, e.g., […], Candidatus Caldiarchaeum subterranum was proposed to make a new phylum Aigarchaeota. CVTrees support the introduction of this new phylum. A lineage modification of Candidatus Caldiarchaeum subterranum from Thaumarchaeota to Aigarchaeota would lead to a monophyletic Thaumarchaeota{7}.” L156–166. If I understood right, there was good support for Candidatus Aciduliprofundum as part of a clade called DHEV2, which is a sister clade of Thermoplasmatales. However in the present work the authors intend here to reclassify Aciduliprofundum into family Thermococcaceae of Thermococcales. This needs further explanation. Since the new affiliation is quite in disagreement with previous observations, and this is not properly justified in the results/discussion, the final statement “this modification would hold as long as no new facts challenge it” seems unacceptable. In addition, why should Aciduliprofundum be regarded as member of Thermococcaceae and not as another distinct family? Response: The problem of taxonomic placement of Aciduliprofundum is a good example to demonstrate how one extract information from CVTrees. In the Reysenbach et al. Nature 2006 paper it was taken as the first cultivated member of the DHEV2 (deep-sea hydrothermal euryarchaeate 2) clade based on a maximum-likelihood 16S rRNA tree. Unfortunately, all other 13 members of this clade were represented by 16S rRNA sequences only and no genome data are available so far. The NCBI taxonomy gave an incomplete lineage: Archaea; Euryrchaeota; unclassified Euryarchaeota; missing taxonomic assignment at the rank class and below. In order to make use of CVTree we must touch on the K-issue a little more. The alignment-free comparison of genomes in CVTree is implemented by counting the number of K-peptides in the protein products encoded in a genome followed by subtraction of a random background caused by neutral mutations. The peptide length K looks like a parameter, but it is actually not a parameter. Using a longer K emphasizes species-specificity, while a shorter K takes into account more common features with neighboring species. However, we never adjust K: a fixed K is used for all genomes to construct a tree, but one may construct a series of trees for K = 3, 4, 5, 6, 7, 8, … We have shown repeatedly that R9

K = 5 and 6 lead to best results in the sense of agreement with taxonomy, so usually only a K = 6 tree is given in publications. Let us look at a subtree, i.e., part of a tree, containing the organisms of interest. If the branching order in all trees built for different Ks turns out to be same, it would be a strong support to the branching order. In most cases the branching order varies with K: K = 3 and 4 make sense, K = 5 and 6 yield the best, K = 7 and 8 become slightly worse, etc. For too big a K, even if the closest strains remain grouped together the whole tree may tend to become a star-tree, i.e., every small clade stands in its own and their mutual placements become less meaningful. Therefore, inspection of trees for a range of K-values provides an additional dimension to evaluate the results. For Aciduprofumdum we have a stable pair (Thermococci{18}, Aciduprofumdum{2}) at K=3, 5, 6, 7. At K = 4 we have (Thermococci{18}, (Staphylothermus{2}, Aciduprofundum{2})) In all these cases Thermoplasmata stands farther away from the above pair. However, at K = 8 and 9, when the overall tree picture has been largely distorted, Aciduliprofundum does stand closer to Thermaplasmata. Putting together all the above results we tend to consider the pair (Thermococci{18}, Aciduliprofundum{2}) as reflecting a more probable relation. Confined to the available data for the time being one may assign Aciduliprofundum to Thermococci, e.g., to denote the pair as Thermococci{20}=(Thermococcaceae{18}, Aciduliprofundum{2}) leaving its family unclassified or assign it to a new family. Without further phenotypic and chemotaxonomic evidence it is better not to introduce new taxon names if the present naming scheme is capable to accommodate the leaves without conflict. This was why we wrote “this modification would hold as long as no new facts challenge it”. Anyway, taxonomy has always been a work in progress. One has to be prepared for modifications when new data appear. To make a long story short, we have rewritten the paragraph as: “The Candidatus genus Aciduliprofundum is considered a member of the DHEV2 (deap-sea hydrothermal vent euryarchaeotic 2) phylogenetic cluster. No taxonomic information was given in the original papers [55,56]. The NCBI Taxonomy did not provide definite lineage information for this taxon at the class, order, and family ranks. According to [55] the whole DHEV2 cluster was located close to Thermoplamatales in a maximum-likelihood analysis of 16S rRNA sequences. A similar placement was seen in [54] where a Bayesian tree of the archaeal domain based on concatenation of 57 ribosomal proteins put a lonely Aciduliprofundum next to Thermoplasmata. However, in CVTrees, constructed for all K-values from 3 to 9, Aciduliprofundum juxstaposes with the class Thermococci{18}. An observation in [56] that this organism shares a rare lipid structure with a few species from Thermococcales may hint on its possible association with the latter. If we temporarily presume a lineage ThermococciUnclassifiedUnclassifiedAciduliprofundum… R10

one might have a monophyletic class Thermococci{20}. Since none of the 13 DHEV2 members listed in [55] has a sequenced genome so far, CVTree cannot tell the placement of the DHEV2 cluster as a whole for the time being. It remains an open problem as whether DHEV2 is close to Thermoplasmata or to Thermococci, or a new class is needed to accommodate DHEV2.” L163: If I’m right the current observation actually does not support the previous work done by Brochier-Armanet. Response: No, we did not mean it. L167–173: The names are wrongly written (please check the original submission). Authors have to explain with more clarity, why is this clade of rank class. If that is the case, is it a single-order class? A single family order? etc. Response: We should first explain how these inappropriate names appeared. We have insisted to use the directory name at the NCBI FTP site as genome name. However, in November 2013 NCBI announced that they would not release genomes of different strains of the same species as before. In a period thereafter NCBI sometimes put several genomes in a directory and we had to extract the data and to assign a name from the “Source” line of the GenBank file. This caused some confusion. For example, as of February 27, 2015, a directory name at NCBI remained “archaeon_Mx1201_uid196597” and we had to change it to: Candidatus_Methanomethylophilus_alvus_Mx1201_uid196597 Now all “wrong names” as pointed out by the Reviewer no longer appear in figures. In the text we tried to refer to their names as complete as possible. L174–180: I don’t understand the reasoning along this paragraph. In addition “haolphiic_archaeon_DL31” is not well written, please be careful when copying names from other source. Also, a similar question about the objectiveness for detecting high taxa: why not to create new family? Response: The genome name at NCBI FTP site is “halophilic_archaeon_DL31_uid72619”. The uid number was dropped when mentioned in the text. We put it back and capitalized the first letter to “Halophilic”, still an illegal genus name. Figure 4: the reclassifications must be clearly justified in the text. Response: It is Figure 2 in the revised manuscript. We discussed it at some length. L191: organisms can't be validly published, but their names. Response: No, organism cannot be published. Thanks for correcting our mistake. Part 3.3 L221–222: Sure, but authors do not provide explanations about how do they know that a clade in a tree is a family, an order, a class, etc. There is a lack of criteria to reclassify the leaves.

R11

Response: One cannot tell the rank of a node/leaf in a tree by simply looking at it. A reference taxonomy is alwys needed. We put the following in the “Material and Method” section to explain it: “Branching order in a tree by itself does not bring about taxonomic ranks, e,g, class or order. The latter can be assigned only after comparison with a reference taxonomy which is not a rigid framework but a modifiable system. Though a dissimilarity measure figures in the CVTree algorithm, it is not realistic to delineate taxa by using this measure at least for the time being. Even if defined in the future, it must be lineage-dependent. For example, it cannot be expected that the same degree of dissimilarity may be used to delineate classes in all phyla. In addition, monophyly is a guiding principle in comparing branching order with taxonomy. Here monophyly must be understood in a pragmatic way restricted to the given set of input data and the reference taxonomy. If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic.” L224: I don’t understand “3063 identical nucleotide positions”. Why identical? Response: The phrase “3063 identical nucleotide positions” was copied from the caption of Figure 4 of the cited Nunoura et al. 2011 paper without much thinking. We simply deleted it. L239–244: hard to read, please rephrase. Response: The whole paragraph has been rewritten as: “The nanosized archaean symbiont Nanoarchaeum equitans has a highly reduced genome (490,885 bp [44]). It is the only described representative of a newly proposed phylum Nanoarchaeota and it cuts into the otherwise monophyletic phylum Euryarchaeota. We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees, see, e.g., Figure 4 in a 2009 paper [61] as well as (c) and (d) in our Figure 4. It has been known that tiny genomes of endosymbiont microbes often tend to move towards baseline of a tree and distort the overall picture. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny, see, e.g., [29] and a note in the home page of the CVTree Web Server [21]. In the present case we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly reduced genome.” Conclusion I disagree that CVTree approach is independent of 16S, because authors are using the current accepted classification (which is mainly 16S-based) to validate the observed clades. Response: As a method CVTree is independent of 16S rRNA analysis. First, it uses protein products in a genome instead of RNA segments in the genome. Second, it does not do sequence alignment. CVTree generates stable trees but cannot tell which branch corresponds to what taxon. Only after comparison with the existing classification and nomenclature one would be able to make connections with taxonomy. In this sense it does depend on 16S rRNA taxonomy. Anyway, CVTree does not challenge 16S rRNA analysis but makes it more convincing in most cases. The revealed discrepancies call for further study. R12

Why at higher ranks, genomic approaches are more effective? That needs more explanation. And authors should also consider the large benefits of 16S data availability, specially at high ranks (genus and above) where the 16S has good resolution. Response: In fact, genomic approaches are more effective at species level and below due to their high resolution power. At high ranks CVTree may be more effective in the sense that it does not require additional work. Suffice it to put genomes in CVTree web server and the branches come out, then compare them with a reference taxonomy.

Other corrections - L23: phynotypic → phenotypic — Done - L27: genomic era → the genomic era — Done - L39: branchings in trees are → branching order in trees is — Done - L40: classes → class — Done - L47: L48 and Fig5: Crearchaeota → Crenarchaeota — Done - L50: Fevridicoccales → Fervidicoccales — Done - L52: Thermoplasmat → Thermoplasmata — Done - L90: Thermopasmata → Thermoplasmata — Done - L92: Thermaplasmataceae → Thermoplasmataceae — Done - L105: on → of — Done - L134: monophyly or non-monophyly → monophyletic or non-monophyletic — Done - L164: rbosomal → ribosomal — Done - L206: Korarcgaeota → Korarchaeota — Done - L208: erenow → herenow — Even “herenow” does not seem to be an correct English word; we changed it to “so far”. - L212: was → were — Done - L225: aligned 5993 amino acids → 5993 aligned amino acids. — Done - L254: 15S → 16S — Done

Second Round of Evaluation

Round 2: Reviewer 1 Report and Author Response

I found the revised version of this manuscript quite good, and I thank the authors for responding thoroughly to all my comments. Response: We thank the Reviewer for the detailed comments/suggestions given in the previous report and the suggestion of doing spelling-check this time. We have gone through the final manuscript carefully once more.

R13

Round 2: Reviewer 2 Report and Author Response

Authors have substantially improved the article, including language corrections, and have provided extensive clarifications to all initial criticisms. I recommend acceptance for publication in Life Sciences journal. I had just a few minor suggestions. L35-38: I don’t get well the sentences which start from “This is in contrast ... ”. Please can you specify a bit more? Response: We changed “is in contrast with” to “is distinct from” and added a phrase “focusing on taxonomy of higher ranks” at the end of a sentence. Now the sentences read: “This is distinct from phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large, or, as Cavalier-Smith \cite{cavalier-smith2002} put it,with “megaclassificaton” of prokaryotes, focusing on taxonomy of higher ranks.” - L45: should provide → provides — Done - L47–49: move to conclusions? —Moved to the conclusion section and the first word “Though” replaced by “In addition, since” L81–85: I think this paragraph is interrupting a bit the text flow. I suggest deletion. Response: The whole paragraph has been deleted. This paragraph was added to the revised manuscript because one of the Reviewers asked “Does CVTree still require input genome data to be annotated to gene features, i.e., protein or CDS?” Well, this question reminds us that for many so-called “Permanent Draft” genomes it may be worthwhile returning to our early practice of using whole genome nucleotide sequences without distinguishing coding and non-coding segments. Although it did not lead to better results as compared with using translated protein products, but it is doable on un-annotated contigs. We will try this later. L244–245: I think the text within {} deviates the attention. I suggest delete that part, ending sentence with “monophyletic Thaumarchaeota” is also ok. Response: We have deleted all what appeared within the curly brackets and kept only “monophyletic Thaumarchaeota” as suggested. In fact, we could not tell how these words appeared there; there was none in our draft manuscript.

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