A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes.

BACKGROUND: Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes.
RESULTS: We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism. Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes.
CONCLUSIONS: The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes. The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms.

Background

Comparative analysis of genomes from distant species provides new insights into gene functions, genome evolution and phylogeny. In particular, the comparative genomics of prokaryotes has revealed previously underappreciated major trends in genome evolution, namely, extensive lineage-specific gene loss and horizontal gene transfer (HGT) [1-7]. To efficiently extract functional and evolutionary information from multiple genomes, rational classification of genes based on homologous relationships is indispensable. The two principal classes of homologs are orthologs and paralogs [8-11]. Orthologs are defined as homologous genes that evolved via vertical descent from a single ancestral gene in the last common ancestor of the compared species. Paralogs are homologous genes, which, at some stage of evolution, have evolved by duplication of an ancestral gene. Orthology and paralogy are intimately linked because, if a duplication (or a series of duplications) occurs after the speciation event that separated the compared species, orthology becomes a relationship between sets of paralogs, rather than individual genes (in which case, such genes are called co-orthologs).

Correct identification of orthologs and paralogs is of central importance for both the functional and evolutionary aspects of comparative genomics [12,13]. Orthologs typically occupy the same functional niche in different organisms; in contrast, paralogs evolve to functional diversification as they diverge after the duplication [14-16]. Therefore, robustness of genome annotation depends on accurate identification of orthologs. A clear demarcation of orthologs and paralogs is also required for constructing evolutionary scenarios, which include, along with vertical inheritance, lineage-specific gene loss and HGT [5,7].

In principle, orthologs, including co-orthologs, should be identified by means of phylogenetic analysis of entire families of homologous proteins, which is expected to define orthologous protein sets as clades [17-19]. However, for genome-wide protein sets, such analysis remains extremely labor-intensive, and error-prone as well. Accordingly, procedures have been developed for identifying sets of likely orthologs without explicit referral to phylogenetic analysis. These procedures are based on the notion of a genome-specific best hit (BeT), that is, the protein from a target genome that is most similar (typically in terms of similarity scores computed using BLAST or another sequence-comparison method) to a given protein from the query genome [20,21]. The assumption central to this approach is that orthologs have a greater similarity to each other than to any other protein from the respective genomes. When multiple genomes are analyzed, pairs of probable orthologs detected on the basis of BeTs are combined into orthologous clusters represented in all or a subset of the analyzed genomes [20,22]. This approach, amended with additional procedures for detecting co-orthologous protein sets and for treating multidomain proteins, was implemented in the database of Clusters of Orthologous Groups (COGs) of proteins [20,23,24]. The current COG set includes approximately 70% of the proteins encoded in 69 genomes of prokaryotes and unicellular eukaryotes [25]. The COGs have been used for functional annotation of new genomes [26-29], target selection in structural genomics [30-32], identification of potential drug targets [33,34] and genome-wide evolutionary studies [4,13,35-38]. Sonnhammer and co-workers independently developed a similar methodology for identification of co-orthologous protein sets from pairwise genome comparisons and applied it to the sequenced eukaryotic genomes [39].

A central notion introduced in the context of the COG analysis is that of a phyletic pattern, that is, the pattern of representation (presence-absence) of analyzed species in each COG [13,20]. Similar concepts have been independently developed and applied by others [40,41]. The COGs show a remarkable scatter of phyletic patterns, with only a small minority represented in all sequenced genomes. A recent quantitative study showed that parsimonious evolutionary scenarios for most COGs involve multiple events of gene loss and HGT [7]. Both similarity and complementarity among the phyletic patterns of COGs, in conjunction with other information, such as conservation of gene order, have been successfully employed to predict gene functions [13,42,43]. The comparison of phyletic pattern has been formalized in set-theoretical algorithms and systematically applied to the computational and experimental analysis of bacterial flagellar systems, which demonstrated the considerable robustness of this approach [44].

We recently extended the system of orthologous protein clusters to complex, multicellular eukaryotes [25]. Here, we examine the phyletic patterns of KOGs in connection with known and predicted protein functions. In-depth analysis of some of these KOGs resulted in prediction of previously uncharacterized, but apparently essential, conserved eukaryotic protein functions. We also reconstruct the parsimonious scenario of evolution of the crown-group eukaryotes by assigning the loss of genes (KOGs) and emergence of new genes to the branches of the phylogenetic tree and explicitly delineate the minimal gene sets for various ancestral forms. To our knowledge, this is the first systematic, genome-wide examination of the sets of orthologous genes in eukaryotes.

Results and discussion

KOGs for seven sequenced eukaryotic genomes: functional and evolutionary implications of phyletic patterns

Eukaryotic KOGs were constructed on the basis of the comparison of proteins encoded in the genomes of three animals (Homo sapiens [45], the fruit fly Drosophila melanogaster [46] and the nematode Caenorhabditis elegans [47]), the green plant Arabidopsis thaliana (thale cress) [48], two fungi (budding yeast Saccharomyces cerevisiae [49] and fission yeast Schizosaccharomyces pombe [50]) and the microsporidian Encephalitozoon cuniculi [51]. The procedure for KOG construction was a modification of the one previously used for COGs [20,24] and is described in greater detail elsewhere ([25]; see also Materials and methods). An important difference stems from the fact that complex eukaryotes encode many more multidomain proteins than prokaryotes and, furthermore, orthologous eukaryotic proteins often differ in domain composition, with additional domains accrued in more complex forms [3,45]. Accordingly, and unlike the original COG construction procedure, probable orthologs with different domain architectures were assigned to one KOG and were not split if they shared a common core of domains. In addition to the KOGs, which consisted of at least three species, clusters of putative orthologs from two species (TWOGs) and lineage-specific expansions (LSEs) of paralogs from each of the analyzed genomes were identified ([25,52]; see also Materials and methods). In most of the analyses discussed below, KOGs and TWOGs are treated together, unless otherwise specified.

Figure 1 shows the assignment of the proteins from each of the analyzed eukaryotes to KOGs with different numbers of species, TWOGs and LSEs. The fraction of proteins assigned to KOGs tends to decrease with the increasing genome size, from 81% for S. pombe to 51% for the largest, the human genome. (For reasons that remain unclear, but might be related to its intracellular parasitic lifestyle, E. cuniculi has a relatively small fraction of conserved proteins that belonged to KOGs: approximately 60%.) The contribution of LSEs shows the opposite trend, being the greatest in the largest genomes, that is, human and Arabidopsis, and minimal in the microsporidian (Figure 1). A notable difference was observed between eukaryotes in terms of their representation in KOGs found in different numbers of species. While the three unicellular organisms are represented mainly in the highly conserved seven- or six-species KOGs, a much larger fraction of the gene set in animals and Arabidopsis is accounted for by LSEs, and by KOGs found in three or four genomes. These include animal-specific genes and genes that are shared by plants and animals but not by fungi and the microsporidian (Figure 1). The large number of KOGs in the latter group (700 KOGs represented in Arabidopsis and at least two animal species) is notable and probably results from massive, lineage-specific loss of genes during eukaryotic evolution (see below).

Figure 1

Assignment of proteins from each of the seven analyzed eukaryotic genomes to KOGs with different numbers of species and to LSEs. 0, Proteins without detectable homologs (singletons); 1, LSEs. Species abbreviations: Ath, Arabidopsis thaliana; Cel, Caenorhabditis elegans; Dme, Drosophila melanogaster; Ecu, Encephalitozoon cuniculi; Hsa, Homo sapiens; Sce, Saccharomyces cerevisisae; Spo, Schizosaccharomyces pombe.

The phyletic patterns of KOGs reveal both the existence of a conserved eukaryotic gene core and substantial diversity. The 'pan-eukaryotic' genes, which are represented in each of the seven analyzed genomes, account for around 20% of the KOGs, and approximately the same number of KOGs include all species except for the microsporidian, an intracellular parasite with a highly degraded genome [51]. Among the remaining KOGs, a large group includes representatives of the three analyzed animal species (worm, fly and humans) but a substantial fraction (approximately 30%) are KOGs with unexpected patterns, for example, one animal, one plant and one fungal species (see [53] and examples in Table 1).

Table 1

KOGs and TWOGs with unexpected phyletic patterns (examples)

KOG/TWOG number	Phyletic pattern*	(Predicted) structure and function	Prokaryotic homologs	Comments
TWOG0892	---H--E	Discoidin domain protein, potential regulator of proteasome activity	Detected in a few phylogenetically scattered bacteria, no COG so far [69]
TWOG0263	A-----E	ATP/ADP translocase	ATP/ADP translocases of chlamydia, rickettsia, Xylella fastidiosa	ATP/ADP translocase is a hallmark of intracellular parasites and symbionts, which allows them to scavenge ATP from the host cell; chloroplast protein in plants. Could be acquired by plants and microsporidia via independent HGT from bacteria. [58]
TWOG0689	---HY--	Uncharacterized protein essential for propionate metabolism	PrpD protein of several bacteria and archaea (COG2079)	The yeast and human (and the orthologs from other vertebrates) proteins show the greatest similarity to different subsets of bacterial orthologs, which might suggest independent HGT events.
TWOG0871	---H-P-	Uncharacterized conserved protein, probably enzyme	COG4336, sporadic representation in several bacterial lineages	The human (and mouse) protein has an additional domain conserved in the archaeon Pyrococcus. Human and S. pombe proteins are most similar to different subsets of bacterial homologs, which suggests the possibility of independent HGT events.
TWOG0788	A----P-	Urease	Ureases of many bacterial species	Highly conserved enzyme present in plants and many fungi but not S. cerevisiae. Plant and fungal ureases have a common domain architecture distinct from that of bacterial orthologs, which suggests monophyletic origin. Might have evolved via early HGT from bacteria (proto-mitochondria?) with subsequent loss in animals and some fungi.
4751	A--H--E	Recombination repair protein BRCA2, contains varying number of BRCA2 repeats	None	Although sequence conservation is limited to the BRC repeats [101] the number of which varies substantially, statistical significance of the observed sequence similarity and the absence of other homologs suggests that the proteins in this KOG are true orthologs. Apparent orthologs of BRCA2 are detectable also in other species from the taxa represented in the KOGs (mosquito Anopheles gambiae, fungus Ustilago maydis) [102] and in early-branching eukaryotes (Leishmania, Trypanosoma; E.V.K., unpublished work), suggesting that evolution of BRCA2 involved multiple gene losses
4597	A--H--E	TATA-binding protein 1-interacting protein	None	Probable multiple gene losses
4486	A--H--E	3-methyl-adenine DNA glycosylase	Orthologs in many bacteria (COG2094)	The plant protein and those from mammals and microsporidia show the greatest similarity to different subsets of bacterial orthologs. Evolution might have included a combination of gene loss and independent HGT events
1594	A-D-Y--	Predicted epimerase related to aldose 1-epimerase	Bacterial orthologs, primarily proteobacteria (COG0676)	Eukaryotic proteins are more closely related to each other than to bacterial orthologs, indicating monophyletic origin. Function remains unknown; might be involved in a distinct and still uncharacterized pathway of polysaccharide biosynthesis. LSE in Arabidopsis (seven paralogs).
4141	---HYPE	Rad52/22, protein involved in double-strand break repair	None	Probable gene loss in plants, insects and nematodes
4528	-CDH--E	Uncharacterized predicted enzyme, possibly a polynucleotide kinase (structure of the ortholog from the bacterium Thermotoga maritima has been determined - pdb code 1j5u)	Conserved in all archaea and several bacteria (COG1371)	Context analysis of archaeal and bacterial genomes suggests functional interaction between proteins of KOG5324 and KOG4246, RNA 3'-terminal phosphate cyclase (KOG4398, COG0430), and tRNA/rRNA cytosine C5-methylase (KOG1299/COG0144) ([103] and E.V.K., unpublished observations). Taken together, the observations appear to implicate KOG5324 and KOG4246 in a still uncharacterized pathway of rRNA and/or tRNA processing and modification. Conservation of these proteins in archaea and early-branching eukaryotes suggests lineage-specific gene loss in plants and fungi.
3833	-CDH--E	Uncharacterized predicted enzyme, possibly a polynuclotide phosphatase	Conserved in all archaea and several bacteria (COG1690)	See comment for KOG5324

*Abbreviations: A, thale cress A. thaliana; C, nematode C. elegans; D, fruit fly D. melanogaster; E, microsporidian Encephalitozoon cuniculi; H, Homo sapiens; S, budding yeast S. cerevisiae; P, fission yeast S. pombe; a letter indicates the presence of the respective species in the given KOG and a dash indicates its absence.

During the manual curation of the KOG set, the KOGs with unexpected patterns were scrutinized in an effort to detect potential highly diverged members from one or more of the analyzed genomes. Some of these unexpected patterns might indicate that a gene is still missing in the analyzed set of protein sequences from one or more of the species included; reports of newly discovered genes have appeared since the release of the initial reports on genome sequences of complex eukaryotes, for example, as a result of massive sequencing of human cDNAs [54], exhaustive annotation of the Drosophila genome [55] and comparative analysis of closely related yeast genomes [56]. The unexpected phyletic patterns seem, however, largely to reflect the extensive, lineage-specific gene loss that is characteristic of eukaryotic evolution [57]; on many occasions, this scenario is supported by the presence of orthologs in other eukaryotic lineages and/or in prokaryotes (Table 1). However, interesting exceptions to the multiple loss explanation might exist as exemplified by the ATP/ADP-translocase, which is present in Arabidopsis and Encephalitozoon and could have evolved via independent HGT from intracellular bacterial parasites ([58] and Table 2).

Table 2

KOGs represented by exactly one ortholog in seven analyzed eukaryotic genomes (examples)

KOG number	(Predicted) function	Multiprotein complex	Functional class*	Prokaryotic homologs	Fitness class†		Comments
					Yeast‡	Worm§
Genes experimentally or computationally characterized previously
0392	SNF2 family DNA-dependent ATPase	TBP-DNA complex		Many bacteria and archaea (COG0553)	0	1	Involved in regulation of transcription from POL II promoters [104]
0121	Nuclear cap-binding protein complex, subunit CBP20 (RRM-domain-containing RNA-binding protein)	Cap-binding complex	A	Several bacteria (COG0724)	1	X	RRM-domain proteins show scattered presence in bacteria and might have been horizontally transferred from eukaryotes
0213	U2-snRNP associated splicing factor 3b, subunit 1	Spliceosome	A	None	0	0
0227	snRNA-associated protein, splicing factor 3a, subunit b (Prp11p)	Spliceosome	A	None	0	0
2268	Predicted nucleic-acid-binding protein kinase of the RIO1 family; 40S ribosomal subunit biogenesis/18S rRNA processing	Pre-40S subunit	A	Orthologs in most archaea but not in bacteria (COG0478)	0	X	One of the very small number of protein kinases that show a clear-cut orthologous relationship between all eukaryotes and most archaea, and, apparently, the only one containing a helix-turn-helix nucleic-acid-binding domain. [105] Associated with yeast pre-40S subunit and required for its maturation. [106]
3031	Protein required for 60S ribosomal subunit biogenesis; [107] contains the IMP4 domain, which is involved in rRNA processing [108]; paralog of KOG3095 and KOG3292, which are also represented in all analyzed genomes.	Processosome	A	Distantly related to COG2136, represented by orthologs in most archaea, but not in bacteria (KSM, unpublished)	0	X	The COG2136 proteins appear to be subunits of the predicted archaeal exosome [109]. Apparently, this gene has undergone at least two ancient duplications in eukaryotes
3045	Predicted RNA methylase involved in rRNA processing	Processosome?	A	Distantly related to numerous Rossmann-fold methylases but prokaryotic orthologs could not be confidently identified	1	1	This protein (Rrp8p in yeast) has been shown to participate in the processing of rRNA and sequence analysis reveals the presence of a Rossmann-fold methylase domain [110]. Therefore Rrp8p probably methylates either snoRNA or rRNA itself.
3064	RNA-binding nuclear protein containing a distinct C4 Zn-finger; implicated in the biogenesis of 60S ribosomal subunits [111]	Processosome	A	None	0	0	Initially identified in yeast as the MAK16 protein required for dsRNA virus reproduction [112]
0291, 0302, 0306, 310, 0319, 0650, 1272	WD40-repeat proteins, subunits of rRNA processing complexes [69,70]	Processosome	A	WD40-repeat proteins are present in several bacterial lineages and are particularly abundant in cyanobacteria but are missing in most archaea; none of them appear to be obvious orthologs of this protein (COG2319)	all 0	X,X,1,X,1,1,1
0284	Polyadenylation factor I complex, subunit PFS2, WD40-repeat protein	Poly-adenylation complex	A	Same as above (COG2319)	0	X
0337	RNA helicase involved in 28S rRNA processing	Processosome	A	Most of the archaea and bacteria (COG0513)	0	X
0343	RNA helicase involved in 28S rRNA processing	Processosome	A	Most of the archaea and bacteria (COG0513)	0	X
1069	3'-5' exoribonuclease (RNAse PH), exosome subunit Rrp46	Exosome	A	Most bacteria and archaea (COG0689)	0	1
1070	Exosome subunit Rrp5 (RNA-binding S1 domain fused to TPR repeats)	Exosome	A	Most bacteria (COG0539, COG0457)	0	1
1135	mRNA cleavage and polyadenylation complex subunit CFT2 (CPSF)	Cleavage and polyadenylation complex	A	Most archaea and some bacteria (COG1236)	0	0
1914	mRNA cleavage and polyadenylation factor I complex, subunit RNA14	Cleavage and polyadenylation complex	A	None	0	X
1975	RNA (guanine-7-) methyltransferase (capping enzyme subunit)	Capping enzyme	A	Numerous methyltrans-ferases (COG0500) but no ortholog	0	1
2051	Nonsense-mediated mRNA decay complex, subunit 2	NMD complex	A	None	1	X
2554	Pseudouridylate synthase	?	A	Most archaea and bacteria (COG0101)	1	1
2613	Upf1p-interacting protein, NMD complex subunit Nmd3p	NMD complex	A	All archaea, no bacteria (COG1499)	0	X
2771	tRNA-specific adenosine-34 deaminase subunit Tad3p	Heterodimeric RNA-specific deaminase	A	Most bacteria and some archaea (COG0590)	0	X
2780	Protein involved in ribosomal large subunit assembly (RPF1), contains IMP4 domain	Processosome	A	Most archaea, no bacteria (COG2136)	0	1
2781	Subunit of the small (ribosomal) subunit (SSU) processosome (snoRNP), IMP4	Processosome	A	Most archaea, no bacteria (COG2136)	0	1
2874	Protein involved in rRNA processing and ribosomal assembly	?	A	All archaea, no bacteria (COG1094)	0	1	Predicted RNA-binding protein containing KH domain
3013	Exosome subunit Rrp4	Exosome	A	Most archaea, on bacteria (COG1097)	0	X
3031	Protein involved in large ribosome subunit assembly and 28S rRNA processing (Rrf2)	Processosome	A	None	0	X	Contains the BRIX domain
3322	RNAse P/MRP subunit, involved in processing of pre-tRNAs and the 5.8S rRNA	RNAse P/MRP holoenzyme	A	None	0	1
3448	Predicted snRNP core protein	Spliceosome	A	All archaea, no bacteria (COG1958)	0	1
3482	Small nuclear ribonucleoprotein (snRNP) SMF subunit	Spliceosome	A	All archaea, no bacteria (COG1958)	0	0
2463	Predicted RNA-binding protein, consisting of a PIN domain and a Zn-ribbon. Involved in 26S proteasome assembly	26S proteasome, pre-40S subunit	A,O	Represented by orthologs in all archaea but no bacteria (COG1349)	0	X	PIN domain has been detected in exosome subunits and is thought to have RNA-binding properties or even nuclease activity [113,114]. The demonstration of the role of this protein (Nob1p) in proteasome assembly [115], 40S ribosome subunit assembly, and the processing of 18S rRNA 3'-end [116] supports the connection between degradation of RNA and proteins that seems to have been established already in archaea [109].
3273	Predicted RNA-binding protein containing KH domain, interacts with Nob1p	26S proteasome, pre-40S subunit	A,O	Orthologs in all archaea but no bacteria (COG1094)	0	0	This is the second predicted RNA-binding protein involved in proteasome assembly, [115] which emphasizes the aforementioned link between RNA and protein processing
1831	Deadenylating 3'-5' exonuclease, negative regulator of PolII transcription	CCR4-NOT core complex	AK	None	0	0
1159	NADP-dependent flavoprotein reductase, probably sulfite reductase subunit	?	CL	Many bacteria (COG0369)	0	X	Genetic evidence of a role in DNA replication [117]
1800	Ferredoxin/adrenodoxin reductase	?	C	Most bacteria and some archaea (COG0493)	0	X
1173	Anaphase-promoting complex (APC), Cdc16 subunit (TPR-repeat protein)	APC	D	Most of archaea and bacteria have TPR-repeat proteins (COG0457) but no orthologs of Cdc16	0	0
3437	Anaphase-promoting complex (APC), subunit 10	APC	D	None	1	1
1358	Serine palmitoyltransferase	?	I	Most bacteria and some archaea (COG0156)	0	0
1511	Mevalonate kinase	?	I	Most archaea and some bacteria (COG1577)	0	X
3059	N-acetylglucosaminyltransferase complex, subunit PIG-C/GPI2, involved in phosphatidylinositol biosynthesis	N-acetylglucos-aminyltransferase complex	I	None	0	1
0467	Translation elongation factor 2 paralog (GTPase)	?	J	All (COG0480)	0	X	Involved in 60S ribosomal subunit maturation [118]
1147	Glutamyl-tRNA synthetase	Multispecificity aminoacyl-tRNA synthetase complex	J	All (COG0008)	0	X
2784	Phenylalanyl-tRNA synthetase, beta subunit	Heterodimeric phenylalanyl-tRNA synthetase	J	All (COG0016)	0	X
3123	Diphtamide synthase (methyltransferase)	?	J	All archaea, no bacteria (COG1798)	1	1
0261	RNA polymerase III, largest subunit	RNAPIII holoenzyme	K	All (COG0086)	0	X
0262	RNA polymerase I, largest subunit	RNAPI holoenzyme	K	All (COG0086)	0	X
0215	RNA polymerase III, second largest subunit	RNAPIII holoenzyme	K	All (COG0085)	0	X
0216	RNA polymerase I, second largest subunit	RNAPI holoenzyme	K	All (COG0085)	0	X
1063	RNA polymerase II elongator complex, subunit ELP2, WD repeat protein	RNA polymerase II elongator complex	K	WD40-repeat proteins are present in several bacterial lineages and are particularly abundant in cyanobacteria but are missing in most archaea; none of them appear to be obvious orthologs of this protein (COG2319)	1	X
1131	RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, 5'-3' helicase subunit RAD3	RNAPII holoenzyme	K	Most archaea and bacteria (COG1199)	0	X
1920	RNA polymerase II Elongator subunit	RNAP II elongator complex	K	None	1	X
1932	TBP-associated factor (Taf2p)	TFIID complex	K	None	0	X
2009	Transcription initiation factor TFIIIB, Bdp1 subunit (Myb domain)	TFIIIB	K	None	0	0
2076	RNA polymerase III transcription factor TFIIIC, TPR-repeat-containing protein	TFIIIC	K	Most of archaea and bacteria have TPR-repeat proteins (COG0457) but no orthologs of TFIIC	0	X
2487	RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB4	TFIIH	K	None	0	1
2691	RNA polymerase II subunit 9	RNAP II holoenzyme	K	Most archaea, no bacteria (COG1594)	1	X
2807	RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, SSL1 subunit	TFIIH	K	No orthologs although von Willebrand A domains are present in a variety of prokaryotic proteins	0	0	Consists of a von Willebrand A domain most closely related to those in the proteasome subunit RPN10 [119] and a Zn-finger domain
2907	RNA polymerase I transcription factor TFIIS, subunit A12.2/RPA12	TFIIS	K	All archaea, no bacteria (COG1594)	1	0
3169	RNA polymerase II transcriptional regulation mediator	Mediator complex [120]	K	None	0	X
3233	RNA polymerase III subunit C34	RNAP III holoenzyme	K	None	0	1
3297	RNA polymerase III subunit C25	RNAP III holoenzyme	K	All archaea, no bacteria (COG1095)	0	0
3438	Subunit common to RNA polymerases I (A) and III (C); Rpc19p	RNAP I and III holoenzymes	K		0	1
3471	RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB2	TFIIH	K	None	0	X
3490	Transcription elongation factor SPT4, Zn-ribbon protein	Chromatin-associated transcription complexes	K	None	1	1
3497	RNA polymerase II subunit; Rpb10p	RNAP II holoenzyme	K	All archaea, no bacteria (COG1644)	0	X
3901	Transcription initiation factor IID subunit (Taf13p)	TFIID	K	None	0	X
3949	RNA polymerase II elongator complex, subunit ELP4	RNAP II elongator complex	K	None	1	1
4086	SOH1 protein potentially involved in Pol II transcription regulation and repair	SMCC complex [121]	K	None	1	X
1532	Predicted GTPase of the XAB1 family [122]	TBP-free TAF(II) complex	L	All archaea and several bacteria (COG1100)	0	0	XP-A-binding protein in humans, thus implicated in repair ([122] and references therein).
1533	Predicted GTPase of the XAB1 family (paralog of KOG1757) [122]	TBP-free TAF(II) complex?	L	All archaea and several bacteria (COG1100)	0	X	Might have a function in repair given the paralogous relationship with KOG1757.
1625	DNA polymerase α processivity subunit, inactivated phosphatase	DNA polymerase α holoenzyme	L	Small subunit of archaeal DNA polymerase II (COG1311)	0	0	The small, regulatory subunit of DNA polymerase α also forms a pan-eukaryotic KOG3044, which is a paralog of KOG0861 (the only recent duplication in KOG3044 is seen in vertebrates). In contrast, another paralog, the small subunit of DNA polymerase ε, is represented in animals, fungi and the early-branching protozoan Plasmodium, but not in plants or Microsporidia. Thus, the history of this polymerase subunit apparently involved inactivation of the phosphatase (or nuclease) inherited from archaea, with subsequent duplications at early stages of eukaryotic evolution [123]
0479	DNA replication licensing factor MCM3	Pre-replication complex	L	All archaea, no bacteria (COG1241)	0	X
0481	DNA replication licensing factor MCM5	Pre-replication complex	L	All archaea, no bacteria (COG1241)	0	X
0482	DNA replication licensing factor MCM7	Pre-replication complex	L	All archaea, no bacteria (COG1241)	0	0
0964	Structural maintenance of chromosome protein 3 (cohesin subunit SMC3)	Sister chromatid cohesion complex	L	Many archaea and bacteria (COG1196)	0	X
0979	Structural maintenance of chromosome protein 5 (cohesin subunit SMC5)	Sister chromatid cohesion complex	L	Many archaea and bacteria (COG1196)	0	X
1942	TBP-interacting protein TIP49 (DNA helicase)	chromatin remodeling complex	L	Most of the archaea, no bacteria (COG1224)	0	0
1979	DNA mismatch repair ATPase, MLH1	Mismatch repair complex	L	Most bacteria and some archaea (COG0323)	1	1
2267	DNA primase, large subunit	DNA polymerase α:primase complex	L	All archaea, no bacteria (COG2219)	0	0
2299	Ribonuclease HI	Replisome	L	All archaea, most bacteria (COG0164)	1	X
2310	DNA repair exonuclease MRE11	MRN complex involved in double-strand break repair	L	All archaea, most bacteria (COG0420)	1	1
2929	Origin recognition complex, subunit 2 (ORC2)	ORC	L	None	1	1
0179	20S proteasome, regulatory subunit beta type PSMB1/PRE7 (paralog of KOG0185)	20S proteasome	O	All archaea but only actinomycetes among bacteria (COG0638)	0	0
0185	20S proteasome, regulatory subunit beta type PSMB4/PRE4 (paralog of KOG0179)	20S proteasome	O	All archaea but only actinomycetes among bacteria (COG0638)	0	0
2708	Predicted metalloprotease with chaperone activity (RNAse H/HSP70 fold) [124]	Putative complex involved in translation regulation [125]	O	Represented by orthologs in all archaea and bacteria (COG0533)	0	X	One of the few remaining uncharacterized proteins that are universally conserved in all cellular life forms. The only experimentally demonstrated activity is that of sialoglycoprotease but fusion with a distinct protein kinase in several archaea and analysis of gene neighborhood suggest a fundamental role in signal transduction, possibly translation regulation. [125]
0301	Protein required for normal rates of ubiquitin-dependent proteolysis, contains WD40 repeats	Proteasome?	O	Same as above (COG2319)	1	X
0358	Chaperonin complex component, TCP-1 delta subunit (CCT4)	TCP-1	O	All archaea and nearly all bacteria (COG0459)	0	0
0363	Chaperonin complex component, TCP-1 beta subunit (CCT2)	TCP-1	O	All archaea and nearly all bacteria (COG0459)	0	0
0687	26S proteasome regulatory complex, subunit RPN7/PSMD6	26S proteasome	O	None	0	0
1299	Vacuolar sorting protein VPS45/Stt10 (Sec1 family)	t-SNARE complex	O	None	1	X	Involved in t-SNARE complex assembly [126]
1349	GPI-anchor transamidase complex, GPI8 subunit	GPI-anchor transamidase complex	O	Distantly related proteases in some bacteria (no COG)	0	1
1943	Beta-tubulin folding cofactor D, involved in chromosome segregation	?	O	None	1	1
2015	NEDD8-activating complex, UBA3 subunit	NEDD8-activating complex	O	Most bacteria and some archaea (COG0476)	1	1
2126	Phosphoethanolamine N-methyltransferase involved in GPI-anchor biosynthesis	?	O	Several bacteria and archaea (COG1524)	0	X
2884	26S proteasome regulatory complex, subunit RPN10/PSMD4	26S proteasome regulatory complex	O	No orthologs although von Willebrand A domains are present in a variety of prokaryotic proteins	1	1	Contains von Willebrand A domain
2908	26S proteasome regulatory complex, subunit RPN9/PSMD13	26S proteasome regulatory complex	O	None	0	0	Contains PINT domain
0209	Endoplasmic reticulum membrane P-type ATPase	?	P	Many bacteria and some archaea (COG0474)	1	X
3379	Uncharacterized member of the histidine triad superfamily of nucleotide hydorlases	?	R	Most archaea and bacteria (COG0537)	1	X	Only biochemical function predicted.
2635	Coatomer (COPI) complex delta subunit	COPI complex	U	None	0	0
2927	Membrane component of ER protein translocation apparatus (Sec62)	Sec complex	U	None	0	1
2978	Dolichol-phosphate mannosyltransferase	?	U	All archaea, most bacteria (COG0463)	0	X
3198	Signal recognition particle, subunit Srp19	Signal recognition particle	U	All archaea, no bacteria (COG1400)	0	X
3315	Subunit of the targeting complex (TRAPP) involved in ER to Golgi trafficking	TRAPP	U	None	0	X
3369	Subunit of the targeting complex (TRAPP) involved in ER to Golgi trafficking	TRAPP	U	None	0	X
1992	Nuclear export receptor CSE1/CAS (importin beta)	?	YU	None	0	X
New functional predictions
2316	PP-loop family ATP pyrophosphatase domain, which in fungi, plants and insects is fused to a duplicated translation inhibitor domain. The fusion, along with the phyletic pattern of the PP-ATPase domain, suggests an essential function in translation regulation	?	A	Orthologs of the PP-loop domain are present in all archaea (COG2102) but not in bacteria. Orthologs of the translation inhibitor domain are found in most bacteria and several archaea (COG0251)	1	X	PP-loop ATPases have been previously implicated in base thiolation in various RNAs [127] and proteins in this K/COG might have a similar function, which is likely to be conserved in eukaryotes and archaea. However, the fusion with translation inhibitor, which has been reported to have endoribonuclease activity [128] is a eukaryote-specific feature
2523	Predicted RNA-binding protein containing a PUA domain, probable role in RNA modification [129]	Putative novel RNA modification complex	A	Orthologs present in all archaea (COG2016) but not in bacteria	1	X	Several of the archaeal orthologs of this protein form fusions with a PP-loop ATPase domain implicated in base thiolation [127]. Thus, the proteins of this KOG might interact with those of KOG2840 (pan-eukaryotic, duplications in Arabidopsis and worm) or KOG2594 (missing in humans and microsporidia) to form a novel enzymatic complex involved in RNA modification
0270, 0271, 1539	WD40-repeat proteins	Processosome	A	WD40-repeat proteins are present in several bacterial lineages and are particularly abundant in cyanobacteria but are missing in most archaea; none of them appear to be obvious orthologs of this protein (COG2319)	all 0	X,1,X	By analogy with other conserved WD40-repeat proteins, predicted to be subunits of rRNA processing/ribosome assembly complexes
2321	Nucleolar protein, contains WD40 repeats	rRNA processosome?	A	WD40-repeat proteins are present in several bacterial lineages and are particularly abundant in cyanobacteria but are missing in most archaea; none of them appear to be obvious orthologs of this protein (COG2319)	0	1	Probable subunit of an rRNA-processing complex
1763	Uncharacterized conserved protein containing a CCCH Zn-finger; possible role in RNA processing or splicing	?	A	None	1	1	CCCH fingers have been shown to bind 3' untranslated regions in various mRNAs [130,131]
2837	Protein containing a U1-type, RNA-binding C2H2 Zn-finger. Probable role in RNA splicing/processing	Spliceosome?	A	None	0	0	U1-type fingers are essential for the assembly of U1 RNP [132]
3073	Predicted RNA-binding protein containing PIN domain and involved in 18S rRNA processing	Pre-40S subunit	A	Most archaea, no in bacteria (COG1412)	0	1	Interacts with Nop14p and is required for 40S subunit biogenesis and 18S rRNA maturation (11694595). The presence of the PIN domain suggests RNA-binding and, possibly, RNAse activity
3154	Uncharacterized protein with potential function in translation or ribosomal biogenesis	Pre-40S subunit?	A?	Most archaea, no bacteria (COG2042)	1	X	The general functional prediction stems from the observation that the gene for this protein forms a predicted conserved operon with the gene for ribosomal protein L40E in several archaeal genomes
3214	Small protein containing a Zn-ribbon, possibly RNA-binding; potential role in RNA processing or transcription regulation	?	A?	Conserved in Crenarchaeota (COG4888)	1	1
3800	Predicted E3 ubiquitin ligase containing RING finger, subunit of transcription/repair factor TFIIH and CDK-activating kinase assembly factor	TFIIH	KO	None	0	X
3176	Predicted α-helical protein, possibly involved in replication/repair; paralog of KOG3636	A novel complex with PCNA involved in replication?	L?	Conserved in most (possibly all) archaea but not in bacteria (COG1711)	0	X	A function in DNA replication/repair and/or transcription is suggested by the analysis of the genome context of archaeal orthologs which form an evolutionarily conserved association with the genes for replication sliding clamp (PCNA ortholog) (K.S.M. and E.V.K., unpublished work)
3303	Predicted α-helical protein, possibly involved in replication/repair transcription; paralog of KOG3508	A novel complex with PCNA involved in replication?	L?	Conserved in most (possibly all) archaea but not in bacteria (COG1711)	0	0	A function in DNA replication/repair and/or transcription is suggested by the analysis of the genome context of archaeal orthologs which form an evolutionarily conserved association with the genes for replication sliding clamp (PCNA ortholog) (K.S.M. and E.V.K., unpublished.work)
0396	Predicted E3 ubiquitin ligase	Ub ligase	O	None	1	1	The proteins in this KOG contain a modified RING domain, which might not be capable of metal-binding similarly to the U-box domain [133] that has been shown to function as E3 [134]
1443	Multitransmembrane protein, predicted drug/metabolite transporter	?	R	Most archaea and bacteria (COG0697)	1	X
2647	Multitransmembrane protein, potential transporter	?	R	Most bacteria and some archaea (COG0628)	0	1
2488	Predicted N-acetyltransferase	?	R	Most archaea and bacteria (COG0454)	1	X	Putative role in ribosomal maturation?
3347	Predicted nucleotide kinase; nuclear protein (Fap7p)	?	R	Conserved in all archaea but not in bacteria (COG1936)	0	1	Involved in oxidative stress reponse in yeast [135]
3974	Predicted sugar kinase	Putative novel complex with KOG2585 proteins	R	All archaea and most bacteria (COG0063)	1	1	Based on fusions seen in prokaryotes, predicted to interact functionally and, possibly, physically with uncharacterized proteins of KOG2585 (represented in all eukaryotes but includes paralogs in some species)
No functional prediction
2318	Uncharacterized conserved protein	?	S	None	0	1
3237	Uncharacterized conserved protein containing coiled-coil domain	?	S	None	0	1	Coiled-coil domains are often involved in complex assembly; this could be an uncharacterized component of the chromatin or the spliceosome

*Abbreviations for the functional categories are as in Figure 3. †0, essential gene (lethal knockout); 1, non-essential gene (non-lethal knockout); X indicates that no data is available for the given gene. ‡Data from [85]. §Data from [86].

Common phyletic patterns of genes that otherwise were not suspected to be functionally linked might suggest the existence of such connections and prompt additional analysis leading to concrete functional predictions [42,59-61]. The pair of KOG5324 and KOG4246 is a case in point that has not been described previously. The initial observation that these KOGs share the same unusual pattern of presence-absence in eukaryotes, and have similar phyletic patterns in prokaryotes, with a ubiquitous presence in archaea, prompted a more detailed examination of the multiple alignments of the respective proteins and the conservation of the (predicted) operon organization in archaea and bacteria (Table 2 and data not shown). The combination of clues from these analyses suggests that the two proteins interact in a still uncharacterized pathway of RNA processing, which also includes RNA 3'-phosphate cyclase (KOG3980)) [62] and cytosine-C5-methylase (NOL1/NOP2 in eukaryotes; KOG1122). The proteins in KOG3833 and KOG4528 are likely to represent novel enzyme families, possibly a kinase-phosphatase pair (E.V.K. and L. Aravind, unpublished data). Notably, these predicted new enzymes are present in animals and E. cuniculi but not in Arabidopsis or yeasts. In contrast, KOG3980 is present in all analyzed eukaryotic genomes except for Arabidopsis, whereas KOG1122 is pan-eukaryotic. These differences in the phyletic patterns of the components of the predicted pathway are concordant with the patterns in eukaryotes in that.

Figure 2 shows the distribution of known and predicted functions of eukaryotic proteins among 20 functional categories for the entire set of KOGs and, separately, for KOGs represented in six or seven species and the animal-specific KOGs. Compared to the functional breakdown of prokaryotic COGs [25], the prevalence of signal transduction is notable among eukaryotes. This feature is particularly prominent in animal-specific KOGs, whereas the highly conserved set is comparatively enriched in proteins that are involved in translation, transcription, chaperone-like functions, cell cycle control and chromatin dynamics (Figure 2). The large number of KOGs for which only general functional prediction was feasible, and those whose functions remain unknown, even among the subset that is represented in six or seven eukaryotic species, emphasizes that our current understanding of eukaryotic biology is seriously lacking with even in respect of the functions of highly conserved genes.

Figure 2

Distribution of the KOGs by the number of paralogs in each of the analyzed eukaryotic genomes. The species abbreviations are as in Figure 1.

The distribution of KOGs by the number of paralogs in each genome is shown in Figure 3. The preponderance of lineage-specific duplication of conserved genes, that is, intra-KOG LSEs, in multicellular eukaryotes is obvious. Cases when a single gene in yeast or, particularly, Encephalitozoon, has two or more co-orthologs in animals and/or plants are most common in KOGs, whereas the reverse situation is rare. These observations support the notion of the major contribution of LSE to the evolution of eukaryotic complexity [52]. However, 131 KOGs are represented by a single ortholog in all genomes compared (Table 2) and a substantial number of KOGs have one member from a majority of the genomes (data not shown). Recent theoretical modeling of the evolution of paralogous families has suggested that, in general, ancient protein families tend to have multiple paralogs [5,63]. Therefore, whenever a KOG has a single member in all or most species, this should be attributed to selection against duplication of this particular gene. A prominent cause of such selection could be the involvement of the respective gene products in essential multisubunit complexes, such that imbalance between subunits leads to deleterious effects [64].

Figure 3

Functional breakdown of the KOGs. Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell-cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism; I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, membrane and cell wall structure and biogenesis; O, post-translational modification, protein turnover, chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking and secretion; Y, nuclear structure; Z, cytoskeleton; R, general functional prediction only (typically, prediction of biochemical activity), S, function unknown. This breakdown is only for KOGs that included at least three species.

Known and new functions of single-member, pan-eukaryotic KOGs

We examined in greater detail the 131 KOGs that are represented by a single gene in each of the seven genomes (Table 2). As can be envisaged from their presence in diverse eukaryotic taxa, including the 'minimal' genome of Encephalitozoon, and as shown by comparison with the knockout phenotype data (Table 2 and see below), these pan-eukaryotic KOGs are of particular biological importance. For the great majority of these KOGs (113 of the 131), the function has been experimentally determined or confidently predicted to a varying degree of detail using computational methods (Table 2). However, around 20 KOGs from this set remained uncharacterized at the time of this analysis and, for all but two of these, substantial functional inferences could be drawn through a combination of sequence-profile analysis, structure prediction and genomic-context analysis of prokaryotic homologs (Table 2). Some of these predicted new functions are variations on well-known themes, such as two predicted PP-loop ATPases, which are probably involved in novel, essential RNA modifications (KOGs 2522 and 2316) or two predicted E3 components of ubiquitin ligases (KOGs 0396 and 3800). Other predicted functions appear to be completely new, such as proteins in KOG3176 and 3303 which are likely to be essential components of eukaryotic replication and/or repair systems. Each of these uncharacterized but ubiquitous and largely essential eukaryotic genes is an attractive target for experimental studies.

Examination of the experimentally characterized and predicted functions of pan-eukaryotic, single-member KOGs leads to interesting conclusions. Nearly all the functionally characterized KOGs in this set consist of proteins that are subunits of known multiprotein complexes (Table 2). The most prominent of these are the complexes involved in rRNA processing and ribosome assembly, such as the recently discovered rRNA processosome and the pre-40S subunit, as well as the spliceosome, and various complexes involved in transcription (Table 2). Accordingly, this set of KOGs is markedly enriched for proteins involved in various forms of RNA processing, assembly of ribonucleoprotein (RNP) particles and transcription. In addition, KOGs in the single-member pan-eukaryotic set include subunits of molecular complexes that are not directly related to RNA processing, such as the proteasome, the TCP-1 chaperonin complex [65] and the TRAPP complex involved in protein trafficking [66]. Altogether, more than 80% of the yeast proteins in the pan-eukaryotic, single-member KOGs belong to known macromolecular complexes included in the MIPS database [67], as compared to around 64% for all yeast proteins in the KOGs, which is a moderate but statistically highly significant excess (data not shown). This preponderance of multiprotein complex formation among the single-member pan-eukaryotic KOGs is fully compatible with the balance hypothesis [64].

The most unexpected observation regarding the single-member, pan-eukaryotic KOGs, is probably that in 14 of these proteins, the only detectable domain was the WD40 repeat (Table 2). This is particularly notable because WD40-repeat proteins, which are extremely abundant in eukaryotes and are present in several prokaryotic lineages as well [68], are not generally known to form well-defined, one-to-one orthologous relationships. The WD40 proteins in the pan-eukaryotic KOGs listed in Table 2 are exceptions, which is probably due to their unique and essential roles in the assembly of RNA-processing complexes. It has recently been demonstrated that, in S. cerevisiae, seven of these proteins are subunits of the 18S rRNA processosome, or at least are involved in ribosomal assembly [69,70]. Taking these results together with the unusual phyletic pattern, it seems possible to predict with considerable confidence that those WD40 proteins in the 131-KOG set that remain uncharacterized belong to the same or similar RNA-processing complexes (Table 2).

With some notable exceptions, such as the WD40 proteins, the KOGs in the single-member, pan-eukaryotic set show remarkable patterns of evolutionary conservation: they are either (nearly) ubiquitous in the three kingdoms of life, for example, RNA polymerase subunits, or are universally conserved in eukaryotes and archaea but missing in bacteria, such as most of the proteins implicated in RNA processing (Table 2). Thus, it appears that elaborate molecular machines central to the functioning of the eukaryotic cell have evolved, largely from ancestral archaeo-eukaryotic components, at the onset of eukaryotic evolution, and both loss and duplication of the respective genes have been strongly selected against throughout the rest of eukaryotic evolution.

Variation of evolutionary rates among KOGs

Genome-wide analysis of protein evolutionary rates shows a broad range of variation [71]. Here, we investigate the variation of evolutionary rates among the ubiquitous KOGs represented in all seven analyzed genomes and the connection between the evolutionary rate and protein function in the KOG set. The characteristic evolutionary rate of each KOG, which included a member(s) from Arabidopsis, was determined by measuring the mean evolutionary distance from Arabidopsis (the outgroup in the phylogenetic tree; see below) to the other species. Even among the KOGs that include all seven species and, accordingly, appear to represent the conserved core of eukaryotic genes, the evolutionary rates differ by a factor of 20 between the fastest- and the slowest-evolving KOGs. Excluding 5% of the KOGs from each tail of the distribution still leaves almost a fourfold difference in evolutionary rates (Figure 4a).

Figure 4

Variation of amino-acid substitution rates among KOGs. (a) Probability-density function for the distribution of evolutionary rates among the set of KOGs including all seven analyzed eukaryotic species. (b) Distribution functions for the evolutionary rates in different functional categories of KOGs. The designations of functional categories are as in Figure 3.

We then compared the distributions of evolutionary rates for different functional categories of KOGs (Tables 3,4 and Figure 4b). Although all the distributions substantially overlapped, there was a statistically highly significant difference between the evolutionary rates for proteins with different functions (Tables 3,4 and Figure 4b). The slowest-evolving proteins are those involved in translation and RNA processing, the fastest-evolving ones are involved in cellular trafficking and transport, whereas components of replication and transcription systems have intermediate evolutionary rates (Tables 3,4 and Figure 4b).

Table 3

Evolutionary rates in KOGs with different functions: evolutionary rates for different functional categories of KOGs*

Functional category	Number of KOGs	Mean rate, substitutions per site	Standard deviation
J	227	0.98	0.37
H	62	0.98	0.30
A	167	1.01	0.36
C	140	1.01	0.43
O	307	1.01	0.40
F	50	1.05	0.34
E	130	1.07	0.38
L	139	1.11	0.38
B	56	1.13	0.33
Z	64	1.13	0.46
K	209	1.15	0.42
G	115	1.16	0.43
I	110	1.16	0.32
T	200	1.18	0.39
D	111	1.19	0.40
R	415	1.23	0.42
M	33	1.26	0.47
U	196	1.27	0.42
Q	30	1.27	0.37
P	69	1.28	0.45
N	2	1.30	0.78
S	348	1.40	0.41
All	3203	1.16	0.42

*Only the KOGs that included a member(s) from Arabidopsis were analyzed; the evolutionary rates are the average distances between the Arabidopsis representative in the given KOG and the proteins from other species (see Material and methods for details). The functional categories are designated as in Figure 5.

Table 4

Statistical significance of differences in evolutionary rates between selected functional categories of KOGs (t-test)

	J	L	U	S
J	-
L	3 × 10-3	-
U	1 × 10-12	3 × 10-4	-
S	7 × 10-33	5 × 10-13	2 × 10-4	-

A parsimonious scenario of gene loss and emergence in eukaryotic evolution and reconstruction of ancestral eukaryotic gene sets

Assuming a particular species tree topology, methods of evolutionary parsimony analysis can be used to construct a parsimonious scenario of evolution, that is, mapping of different types of evolutionary events onto the branches of the tree. With prokaryotes, the problem is confounded by the major contributions from both lineage-specific gene loss and HGT to genome evolution, with the relative likelihoods of these events remaining uncertain [5,7]. The possibility of substantial HGT between major lineages of eukaryotes can apparently be safely disregarded, providing for an unambiguous most parsimonious scenario that includes only gene loss and emergence of new genes as elementary events.

Some crucial aspects of the phylogenetic tree of the eukaryotic crown group remain a matter of contention. The consensus of many phylogenetic analyses appears to point to an animal-fungal clade and clustering of microsporidia with the fungi. However, a major uncertainty remains with respect to the topology of the animal tree: the majority of studies on protein phylogenies support a coelomate (chordate-arthropod) clade [72-74], whereas rRNA phylogeny and some protein family trees point to the so-called ecdysozoan (arthropod-nematode) clade [75-78]. We treated the phyletic pattern of each KOG as a string of binary characters (1 for the presence of the given species and 0 for its absence in the given KOG) and constructed the parsimonious scenarios of gene loss and emergence during evolution of the eukaryotic crown group for both the coelomate and the ecdysozoan topologies of the phylogenetic tree. For the purpose of this reconstruction, the Dollo parsimony approach was adopted [79]. Under this approach, gene loss is considered irreversible; thus, a gene (a KOG member) can be lost independently in several evolutionary lineages but cannot be regained. This assumption is justified by the implausibility of HGT between eukaryotes (the Dollo approach is not valid for reconstruction of prokaryotic ancestors).

In the resulting parsimonious scenarios, each branch was associated with both gene loss and emergence of new genes, with the exception of the plant branch and the branch leading to the common ancestor of fungi and animals, to which gene losses could not be assigned with the current set of genomes (Figure 5a,b). There is little doubt that, once genomes of early-branching eukaryotes are included, gene loss associated with these branches will become apparent. The principal features of the reconstructed scenarios include massive gene loss in the fungal clade, with additional elimination of numerous genes in the microsporidian; emergence of a large set of new genes at the onset of the animal clade; and subsequent substantial gene loss in each of the animal lineages, particularly in the nematodes and arthropods (Figure 5a,b). The estimated number of genes lost in S. cerevisiae after its divergence from the common ancestor with the other yeast species, S. pombe, closely agreed with a previous estimate produced by a different approach [57]. The switch from the coelomate topology of the animal sub-tree to the ecdysozoan topology resulted in relatively small changes in the distribution of gains and losses: the most notable difference was the greater number of genes lost in the nematode lineage and the smaller number of genes lost in the insect lineage under the ecdysozoan scenario compared to the coelomate scenario (Figure 5a,b).

Figure 5

Parsimonious scenarios of loss and emergence of genes (KOGs) in eukaryotic evolution. (a) The coelomate topology of the phylogenetic tree of the eukaryotic crown group. (b) The ecdysozoan topology of the phylogenetic tree of the eukaryotic crown group. The numbers in boxes indicate the inferred number of KOGs in the respective ancestral forms. The numbers next to branches indicate the number of gene gains (emergence of KOGs) (numerator) and gene (KOG) losses (denominator) associated with the respective branches; a dash indicates that the number of losses for a given branch could not be determined. Proteins from each genome that did not belong to KOGs as well as LSEs were counted as gains on the terminal branches. The species abbreviations are as in Figure 1.

The parsimony analysis described above involves explicit reconstruction of the gene sets of ancestral eukaryotic genomes. Under the Dollo parsimony model, which was used for this analysis, an ancestral gene (KOG) set is the union of the KOGs that are shared by the respective outgroup and each of the remaining species. Thus, the gene set for the common ancestor of the crown group includes all the KOGs in which Arabidopsis co-occurs with any of the other analyzed species. Similarly, the reconstructed gene set for the common ancestor of fungi and animals consists of all KOGs in which at least one fungal species co-occurs with at least one animal species. These are conservative reconstructions of ancestral gene sets because, as already indicated, gene losses in the lineages branching off the deepest bifurcation could not be detected. Under this conservative approach, 3,413 genes (KOGs) were assigned to the last common ancestor of the crown group (Figure 5a,b). More realistically, it appears likely that a certain number of ancestral genes have been lost in all, or all but one, of the analyzed lineages during subsequent evolution, such that the gene set of the eukaryotic crown group ancestor might have been close in size to those of modern yeasts. In terms of the functional composition, the reconstructed core gene set of the crown-group ancestor resembled more the highly conserved KOGs than the animal-specific KOGs (Figure 3) in being enriched in housekeeping functions such as translation, transcription and RNA processing (data not shown).

The functional profiles of the gene sets that were lost in different lineages showed substantial differences (Table 5). Thus, for example, in the lineage leading to the common ancestor of the animals, the greatest loss among genes assigned to functional categories was seen in amino acid and coenzyme metabolism; in contrast, in the fly and the nematode, more substantial degradation was observed among transcription factors and proteins with chaperone-like functions. Genes for proteins involved in RNA processing and translation are, in general, not heavily affected by loss except in the highly degraded parasite E. cuniculi. On many occasions, the switch from the coelomate to the ecdysozoan topology replaces two independent, parallel losses in the insect and nematode clades with a single loss at the base of the ecdysozoan branch, although, on the whole, trees based on gene content support the coelomate topology [74]. In particular, the ecdysozoan topology, unlike the coelomate topology, implies early loss of several genes involved in translation, transcription and repair (Table 6). Notably, a large fraction of genes lost in each lineage has only a general functional prediction or no prediction at all (Table 5). This emphasizes the paucity of our current understanding of lineage-specific gene sets.

Table 5

Functional profiles of genes lost in different eukaryotic lineages

Functional category	Lost genes (KOGs)
	Hs*	Dm*	Coelomates/ Ecdysozoa	Ce*	Animals	Sc	Sp	Yeasts	Ec	Fungi-Ec
Total	162/114	520/369	37/188	541/751	193	299	202	55	1,969	802
RNA processing and modification	2/3	9/8	1/2	10/11	4	15	7	1	88	32
Translation	3/3	16/11	0/5	13/10	9	9	6	3	122	10
Transcription	5/2	16/12	0/4	29/33	2	16	9	4	83	40
Replication and repair	4/5	28/14	1/15	29/14	2	9	7	3	60	16
Chromatin structure and dynamics	1/1	8/6	0/2	8/6	0	5	3	1	29	11
Energy production and conversion	7/10	9/10	5/4	12/10	7	6	13	1	110	37
Cell cycle control and mitosis	3/3	11/6	0/5	15/11	3	12	3	1	61	16
Amino acid metabolism and transport	5/6	16/9	1/8	15/7	38	6	9	0	110	18
Nucleotide metabolism and transport	3/3	6/3	0/3	8/5	5	0	3	1	38	9
Carbohydrate metabolism and transport	3/3	13/10	1/4	18/14	8	10	16	3	70	41
Coenzyme metabolism	0/2	5/5	2/2	14/12	11	1	1	0	51	12
Lipid metabolism	1/5	27/19	4/12	18/6	4	9	19	2	74	33
Membrane and cell wall structure and biogenesis	5/4	10/10	2/2	9/11	7	5	3	0	37	15
Post-translational modification, protein turnover, chaperone functions	3/5	22/15	2/9	44/40	8	29	21	4	167	69
Inorganic ion transport and metabolism	2/4	8/8	2/2	8/7	9	2	6	4	50	14
Secondary metabolites biosynthesis, transport and catabolism	1/2	6/5	1/2	5/3	2	4	1	0	23	5
Signal transduction	5/3	32/22	0/10	30/37	4	16	7	3	110	52
Intracellular trafficking and secretion	4/3	10/8	0/2	14/14	3	5	11	0	116	22
Nuclear structure	0/0	3/3	0/0	5/6	0	1	0	0	16	5
Cytoskeleton	0/0	2/2	0/0	6/8	0	9	0	3	44	6
General functional prediction only (typically, prediction of biochemical activity)	14/13	79/55	5/29	88/72	30	55	24	11	241	134
Function unknown	91/34	184/128	10/66	143/414	37	75	33	10	269	205

*For each of the animals, the numerator indicates the number of genes lost under the coelomate topology of the species tree and the denominator indicates the number of genes lost under the ecdysozoan topology of the tree.

Table 6

Groups of functionally linked genes co-eliminated during evolution of different eukaryotic lineages

Functional group/ complex	Lost KOGs*
	Hs	Dm	Ce	Coelomates/ Ecdysozoa	Animals	Yeasts	Fungi-Ec
Mitochondrial ribosomal proteins	3331, 3435/ 3331, 3435	3505, 4600, 4612/ None	3505, 4122, 4600, 4612/ 4122	None/ 3505, 4600, 4612	0899, 0938, 1740, 3254, 3278, 4844		0408,1686, 1708, 4707
Spliceosome, including putative associated proteins		1847, 1960/ 1847	1902, 1960, 2991, 3414	None/ 1960			0105, 0107, 0117, 1365, 1588, 1676, 1847, 1996, 2191, 2242, 2548, 2991, 4207, 4211
Exosome		1004, 1613
Replication origin-recognition complex			2228, 2538, 4557				4557
Mismatch repair system		0218, 0220, 221, 1977	0218, 1977, 4120	None/ 0218, 1977
Ubiquitin system/ proteasome-signalosome components		0170, 0428, 1814, 4116, 4185, 4412	0168, 0170, 0320, 0421, 0423, 1364, 1571, 1645, 1871, 1873, 1887, 2561, 2932, 3061, 3250, 3268, 4146, 4159, 4275, 4412, 4413, 4414, 4692, 4761	None/ 0170, 4412		0823, 1645, 1734	0311, 0423, 0427, 0827, 0895, 1100, 1139, 1464, 1571, 1812, 1887, 2561, 2932, 3011, 3050, 3268, 4185, 4248, 4265, 4275, 4413, 4414, 4427, 4642, 4692, 4761
NADH-ubiquinone oxido-reductase/ NADH dehydro-genase							2865, 2870, 3256, 3300, 3365, 3382, 3389, 3426, 3446, 3456, 3458, 3466, 3468, 4009, 4662, 4668, 4669, 4770, 4845

*For each of the animals, the numerator indicates the KOGs lost under the coelomate topology of the species tree, and the denominator indicates KOGs lost under the ecdysozoan topology.

As noticed previously during the analysis of the genes lost in S. cerevisiae after its divergence from the common ancestor with S. pombe, functionally connected genes tend to be co-eliminated during evolution [57]. The present study generalizes this conclusion as many functionally coherent groups of co-eliminated KOGs become apparent (Table 5). Importantly, different branches of the same complex systems tend to be eliminated in parallel in different lineages, for example, largely non-overlapping sets of genes for proteins of the ubiquitin-proteasome-signalosome systems are lost in the fungal-microsporidial lineage and in the nematodes (Table 6). It seems likely that elimination of these genes reflects independent trends for simplification of regulatory processes in these lineages.

An interesting trend seen in these data is the deterioration of the mitochondrial ribosome, which occurred in several eukaryotic lineages and appears to have been partly parallel (as it occurred independently in fungi-microsporidia and in animals) and partly consecutive: early loss in the ancestral animal line was followed by elimination of additional genes for ribosomal proteins in individual lineages (Table 6). C. elegans has one of the shortest mitochondrial rRNAs and might have a 'minimal' mitochondrial ribosome [80]; the present analysis details the stages leading to this ultimate degradation of the mitochondrial ribosome.

An exhaustive analysis of the patterns of gene loss is beyond the scope of this work. It seems clear that it has potential of improving our understanding of eukaryotic evolution and functional predictions through examination of co-eliminated gene groups.

Evolutionary relationships between eukaryotic and prokaryotic orthologous gene sets

The prokaryotic COGs and eukaryotic KOGs were identified in separate genome comparisons, although an overlap existed because both sets included the unicellular eukaryotes, namely two yeasts and the microsporidian. To identify the prokaryotic counterparts of the KOGs, the sequences of the eukaryotic proteins included in the KOGs were compared using the RPS-BLAST program to the position-specific scoring matrices (PSSMs) constructed for all prokaryotic COGs ([81] see Materials and methods for details). The results were checked manually and also by comparing the assignment of proteins from unicellular eukaryotes to each of the orthologous gene sets. Altogether, probable orthologous relationships were established between 2,456 eukaryotic KOGs and TWOGs (44% of the total) and 1,516 prokaryotic COGs. A more detailed breakdown of the relationships between eukaryotic and prokaryotic orthologous gene clusters could reveal important evolutionary trends. Figure 6a compares the occurrence of prokaryotic counterparts for the entire set of eukaryotic KOGs and its subsets conserved at different levels. Clearly, the reconstructed gene set of the common ancestor of the crown group and, particularly, the pan-eukaryotic KOGs are enriched in ancient KOGs (those with prokaryotic counterparts) as compared to the full KOG collection. In contrast, among KOGs that are inferred to have evolved in individual lineages within the crown group, a significantly lower fraction has detectable prokaryotic counterparts (Figure 6a).

Figure 6

Correspondence between eukaryotic and prokaryotic orthologous gene sets. (a) Representation of prokaryotic counterparts in different subsets of KOGs. CGA, crown group ancestor; non-CGA, KOGs not represented in the crown group ancestor; MSP, metazoa-specific KOGs. (b) Evidence of ancient duplications of eukaryotic genes revealed by the KOGs against COGs comparison. The connections between KOGs and COGs detected by using RPS-BLAST (see text) were analyzed by single linkage clustering.

Early evolution of eukaryotes is known to have involved duplication of ancient genes inherited from prokaryotes [82], and this was apparent in the KOGs against COGs comparison. Although one-to-one relationships were predominant, in around 30% of cases, two or more eukaryotic KOGs corresponded to the same prokaryotic COG (Figure 6b). This indicates extensive duplication of ancestral genes at early stages of eukaryotic evolution; moreover, a substantial fraction of these genes have undergone repeated duplications, resulting in a one-to-many relationship between prokaryotic and eukaryotic orthologs (Figure 6b).

An in-depth analysis of the relationships between eukaryotic and prokaryotic orthologous gene clusters should include an attempt to decipher their evolutionary history, that is, classification of the C/KOGs represented both in eukaryotes and prokaryotes into: those that have been inherited from the last universal common ancestor; the archaeo-eukaryotic subset; and those that are shared because of HGT between bacteria and eukaryotes at various stages of eukaryotic evolution. This analysis is beyond the scope of the present work. Perhaps the principal message to stress here is that, using a fairly sensitive sequence comparison method, prokaryotic homologs could be detected for only some 44% of the eukaryotic KOGs, and this fraction increased to around 54% for those genes that could be traced to the last common ancestor of the crown group (Figure 6a). This observation emphasizes the major amount of innovation that accompanied the emergence and early evolution of eukaryotes; even those KOGs for which prokaryotic counterparts will be eventually identified through more sensitive sequence and structure comparison apparently experienced rapid evolution during the prokaryote-eukaryote transition.

Phyletic patterns of KOGs and dispensability of yeast and worm genes

There are 860 KOGs with at least one representative from each of the seven analyzed genomes. In accord with the 'knockout rate' hypothesis [83], which has been largely supported by recent, genome-wide analysis of gene conservation [38,84], it could be expected that these highly conserved genes were essential for the survival of eukaryotic organisms. This appears particularly plausible given the near-minimal eukaryotic gene complement of the microsporidian. The prediction was put to the test using the recently published functional profile of the yeast S. cerevisiae genome, which includes the data on the growth rates of homozygous deletion strains for 96% of the open reading frames (ORFs) in the yeast genome [85]. Growth rates have been previously interpreted as a measure of fitness [84].

When the phyletic patterns of the KOGs were superimposed on the data on gene dispensability (with essential genes operationally defined as those whose deletion had a lethal effect in a rich medium) [85], it was found that 45% of the essential genes were conserved in all seven species and 25% were represented in six species (typically with the exception of E. cuniculi); 15% of the essential yeast genes had no orthologs in the other analyzed genomes (Figure 7a). In a striking contrast, among non-essential genes, only 16.5% were represented in all compared genomes and 28.5% had no detectable orthologs (Figure 7a). The reciprocal comparison is equally illustrative: essential genes composed 18.5% of the entire set of yeast genes but 35% of the genes (KOGs) represented in all seven species. This translates into a statistically highly significant dependence between a gene's (in)dispensability and conservation over long evolutionary distances. The probability of the set of highly conserved genes being so enriched for essential genes as a result of chance was estimated at <<10-10. Notably, an even greater enrichment for essential genes was seen among the KOGs that were represented by one, and only one, ortholog in each of the seven analyzed genomes: of the 131 such KOGs, 98 (75%) included an essential yeast gene (Table 2). Such preponderance of essential genes could be expected because, in this set of KOGs, the indispensability of the respective function could not have been masked by the presence of paralogs.

Figure 7

Gene dispensability in yeast and worm and phyletic patterns of the respective KOGs. (a) Distribution of essential and non-essential genes among different size classes of KOGs and LSEs in yeast Saccharomyces cerevisiae. (b) Distribution of essential and non-essential genes among different size classes of KOGs and LSEs in the nematode C. elegans. The number of species in the KOGs and LSEs is color-coded as indicated to the right of each plot.

For an additional set of around 15% non-essential yeast genes, knockout results in a measurable retardation of growth [85]. Unexpectedly and in contrast to the result obtained with the essential genes, we failed to observe a correlation between the magnitude of a gene's knockout effect on yeast growth and the phyletic pattern (data not shown). This seems to indicate that the measured effect on yeast growth might not translate into an effect on fitness that the loss of the ortholog of the given gene has in distant species.

In C. elegans, much as in yeast, essentiality of genes appears to correlate with strong evolutionary conservation, as already noticed in the recent genome-wide study on inhibition of worm gene expression by RNA interference (RNAi) [86]. We compared this dataset, which covers around 86% of C. elegans genes, to the phyletic patterns of the respective KOGs. Of the essential worm genes, 38% were conserved in all seven compared species and 19% were conserved in six species (Figure 7b). In contrast, only 6% of the non-essential C. elegans genes were represented in seven species and 7% were conserved in six species (Figure 7b). Thus, there seems to be a strong and robust connection between a gene's essentiality and its tendency to be conserved in evolution over a wide span of taxa; this connection was established using two independent datasets from biologically extremely different model organisms.

Domain accretion in orthologous sets of eukaryotic proteins

As noticed previously, the complexity of domain architecture of proteins in some orthologous sets increases with increasing organismic complexity; this phenomenon has been dubbed domain accretion [3]. With the KOG set in hand, we sought to assess the extent of accretion quantitatively by using the data on the presence of domains from the CDD (conserved domain alignments database) collection in each of the KOG members. The results summarized in Table 7 show a relatively small but statistically significant excess of domains in proteins from multicellular organisms compared to the orthologs from unicellular organisms. Furthermore, among the multicellular eukaryotes, human proteins have the greatest complexity of domain architectures, followed by Drosophila and Arabidopsis (Table 6), in agreement with preliminary results reported previously. Among the unicellular eukaryotes, Encephalitozoon had by far the least complex domain architectures (Table 6), which reflects the general genome reduction in this intracellular parasite.

Table 7

Domain accretion in complex eukaryotes

	Hsa	Dme	Ath	Cel	Sce	Spo	Ecu
Hsa		<1 × 10-10	<1 × 10-10	<1 × 10-10	<1 × 10-10	<1 × 10-10	<1 × 10-10
	470
Dme	3214		2 × 10-1	<1 × 10-10	<1 × 10-10	<1 × 10-10	<1 × 10-10
	805
	327	354
Ath	2224	2085		3 × 10-1	<1 × 10-10	<1 × 10-10	<1 × 10-10
	530	403
	347	428	334
Cel	2986	2962	2052		1 × 10-8	<1 × 10-10	<1 × 10-10
	880	650	376
	149	161	183	197
Sce	1789	1704	1769	1715		1 × 10-2	<1 × 10-10
	504	411	374	336
	100	123	135	150	158
Spo	1880	1807	1886	1808	2360		<1 × 10-10
	549	426	388	359	216
	10	17	12	14	13	19
Ecu	700	738	739	748	816	835
	332	254	235	244	158	140

For a given pair of species the numbers in each cell below the diagonal represent, from top to bottom: the number of KOGs in which the average number of detected domains from the CDD collection (cut-off E = 10-3) in the proteins from the species to the left is greater than that for the species to the right; the number of KOGs with equal average number of domains; the number of KOGs in which the average number of domains is greater for the species to the right (for example, D. melanogaster has a greater number of detected domains than H. sapiens in 470 KOGs, the same number in 3,214 KOGs, and a smaller number in 805 KOGs). The numbers above the diagonal are the statistical significance of the difference, P(χ2).

Conclusions

The present analysis of KOGs provides quantitative backing for many trends in the evolution of eukaryotic genomes that previously have been noticed on the general, qualitative level. The important quantities reported here include the size of the conserved core of eukaryotic genes, the conservative reconstructions of ancestral gene sets, the numbers of genes that appear to have been lost and gained in individual eukaryotic lineages, and the extent of correlation between gene dispensability and evolutionary conservation, which is reflected in phyletic patterns. In addition, we evaluated the range of variation of evolutionary rates of genes in different functional categories and obtained statistical support for the important evolutionary phenomenon of domain accretion. Furthermore, we observed that only a minority of eukaryotic KOGs have readily detectable prokaryotic counterparts, which emphasizes the extent of innovation linked to the origin of eukaryotes and subsequent major transitions in eukaryotic evolution, such as the origin of multicellularity and the origin of animals.

The case study of the KOGs that are represented by just one member in all eukaryotic genomes compared shows the potential of KOGs for functional prediction by inferring the probable functions for almost all KOGs in this set that had remained uncharacterized. This analysis also revealed unexpected facets of evolution of widespread and essential eukaryotic proteins, such as the counterintutitive preponderance of WD40-repeat proteins among the single-member pan-eukaryotic KOGs.

The current KOG set includes proteins from seven genomes whose sequences were available as of 1 July, 2002. The genomes of the mouse [87], the fugu fish [88], the Anopheles mosquito [89], the urochordate Ciona instestinalis [90] and the malarial parasite Plasmodium falciparum [91] have become available since then but were not included, partly because of problems with protein annotation for some of these genomes, and partly due to the time-consuming and labor-intensive nature of KOG analysis. Inclusion of these and other newly sequenced genomes should proceed at a faster rate once the system itself is established, and will enable further, deeper studies into the functional and evolutionary patterns of eukaryotic life.

Materials and methods

Construction and annotation of KOGs

A more detailed description of the procedures employed for this purpose is presented elsewhere [25]. The protein sets for all eukaryotic species, with the exception of C. elegans and H. sapiens, were from the genome division of the National Center for Biotechnology Information (NCBI). The protein sequences for C. elegans were from the WormPep67 database and the human sequences were from NCBI build 30. Briefly, the KOG construction protocol included: First, the detection and masking of common, repetitive domains using the RPS-BLAST program and the PSSMs for the respective domains from the CDD collection [81]; second, all-against-all comparison of protein sequences from the analyzed genomes using the BLASTP program [92], with masking of low sequence complexity regions using the SEG program [93]; third, identification of triangles of mutually consistent BeTs; merging triangles of BeTs with a common side to form preliminary KOGs; forth, adding members of co-orthologous sets missed at previous step using the COGNITOR procedure [24]; fifth, manual examination of each candidate KOG, aimed at eliminating the false positives incorporated into the KOGs by the automatic procedure and inclusion of false negatives that were missed originally; sixth, assignment of proteins containing promiscuous domains masked at the first step to Fuzzy Orthologous Groups (FOGs), named after the respective domains (when a sequence assigned to a KOG contained one or more masked domains, the sequences of these domains were restored); and finally, examination of the largest preliminary KOGs, which included numerous proteins from all or several genomes by using phylogenetic trees, cluster analysis with the BLASTCLUST program [94], comparison of domain architectures, and visual inspection of alignments. As a result, some of these preliminary KOGs were split into two or more smaller final KOGs.

Annotation of KOGs included critical assessment of the annotations available through GenBank, other public databases and the primary literature and additional, in-depth sequence analysis aimed at detection of previously unnoticed homologous relationships. The annotated functions of KOGs were classified into 23 categories (see legend to Figure 3), which were adapted from the functional classification previously used for COGs [24] by including several specific eukaryotic categories.

Other sequence analysis procedures

During KOG annotation, proteins that are currently annotated as 'hypothetical' or 'unknown', or otherwise had a vague or suspect annotation, were subject to additional sequence analysis, which included iterative sequence similarity searches with the PSI-BLAST program [92], RPS-BLAST searches for conserved domains [80], and additional domain architecture analysis using the SMART system [95]. To estimate sequence evolution rates, multiple alignments of KOGs were constructed using the MAP program [96] and the pairwise evolutionary distances were calculated with the maximum likelihood method under the PAM model by using the PROTDIST program of the PHYLIP package [97]. When a KOG included more than one member from the given species, the paralog with the greatest average similarity to proteins from other organisms was selected to represent the species in the given KOG. Since A. thaliana is the most likely outgroup species for the analyzed set of eukaryotes, distances from the Arabidopsis representative to proteins from all other species were averaged to estimate the characteristic evolutionary distance for the given KOG. Data from KOGs with excessive variability of the distances between A. thaliana and other species (standard deviation to mean ratio > 0.5) were discarded. As the divergence times for all KOGs are presumed to be the same (and equal to the time elapsed since the last common ancestor for the eukaryotic crown group), the mean evolutionary distance in a KOG is a measure of the KOG's evolutionary rate.

The parsimonious evolutionary scenario, which included gene losses and emergence of KOGs mapped to the branches of the eukaryotic phylogenetic tree, was constructed by using the DOLLOP program of the PHYLIP package [97]; this program is based on the Dollo parsimony method, which assumes irreversibility of character loss [79].

For the analysis of domain accretion, conserved domains from the NCBI CDD database were detected in the eukaryotic proteins that belonged to the KOGs by using the RPS-BLAST program [81] with an E-value cut-off of 0.001. Domains with biased amino acid sequence composition, which tend to produce a high false-positive rate in RPS-BLAST searches, were excluded from the analysis.

The eukaryotic KOG set is accessible at [98] and via ftp at [99]. The reconstructed ancestral gene sets are available at [100].