sequence set consisting of both the computationally detected orthologs and the homologs was prepared. For these sequence sets, phylogenetic trees were constructed by the neighbor-joining (NJ) method (18). In detail, multiple amino acid sequence alignments and phylogenetic trees were constructed by ClustalW (19) with the options of bootstrap = 1000, seed = 1, kimura, tossgaps, bootlabels = node. Phylogenetic trees were examined by experts in the ﬁeld of molecular evolution, who attended the evolu- tionary annotation meetings described in the introduction. The trees were drawn by NJplot (http://pbil.univ-lyon1.fr/ software/njplot.html) and the default rooting was used. Discarding or re-rooting the tree was judged by the experts if necessary. All the ortholog pairs of human and other species detected by the computational analysis were examined (Figure 1). The primary principles of manual curation in Evola to be checked were as follows. [1] Phylogenetic topology between gene tree and species tree is consistent. As a gene tree, the minimum sub-clade including the pair (a part of the tree) was examined. As a species tree of reference, a phylogenetic tree indicating the trifurcation among primates, rodents and Laurasiatherian (dog, cow, etc.) species (20) was used, because the phylogenetic relationship has been controversial among them (21). In fact, we found that ((human–mouse)–dog) clades for some genes and ((human–dog)–mouse) clades for other genes. [2] Outgroup includes either two or more species that are phylogenetically distant from all the species in the sub-clade, or human and other species. In the latter case, human duplicate genes might exist. [3] Available bootstrap values of the corresponding three branches (one between the sub-clade and outgroup, and its two descendants) are all 900. The gene pairs consistent with all the principles were deﬁned as ‘manually curated orthologs’, otherwise their annotation status remained to be ‘computationally detected orthologs’. DATABASE CONTENTS Evola contains two ortholog datasets: (1) more compre- hensive set of orthologs (computational analysis); and (2) more reliable orthologs (computational analysis supported by manual curation). In the current Evola (release 4.1), orthology information for 18 968 human genes is available among 11 vertebrates: chimpanzee, macaque, mouse, rat, dog, cow, opossum, chicken, zebraﬁsh, Tetraodon and Fugu (Table 1). Manually curated orthologs occupied 25.4% of all computationally detected ortholog pairs (24 122/94 935) (release 4.1, 2007). Evola is a sub-database of H-InvDB (9,10,11), and orthology information in Evola is, as ‘Evolutionary annotation’, a part of the comprehensive human gene annotations in H-InvDB. Thus, orthology information can be utilized with close reference to other annotation in H-InvDB. For example, 2090 human genes with orthology information belonged to H-Inv protein similarity categories of ‘hypothetical proteins’ (similarity category IV–VI). Molecular functions of these hypothetical Nucleic Acids Research, 2008, Vol. 36, Database issue D789 Figure 1. An example of manually curated gene pair from H.sapiens (red underline) and Macaca.sp (blue underline). In this case, conditions of phylogenetic topologies, outgroup species (light gray background) and bootstrap values (two circles) are suﬃcient (refer to the text). Thus, the pair was deﬁned as a manually curated ortholog. Table 1. Number of orthologs provided in Evola (release 4.1, June 2007) Species Homo sapiens (Human) Pan troglodytes (Chimpanzee) Macaca sp. (Macaque)a Mus musculus (Mouse) Rattus norvegicus (Rat) Canis familiaris (Dog) Bos taurus (Cow) Monodelphis domestica (Opossum) Gallus gallus (Chicken) Danio rerio (Zebraﬁsh) Tetraodon nigroviridis (Tetraodon) Takifugu rubripes (Fugu) Genes 18 968 16 368 12 037 15 570 15 632 14 730 9375 13 201 9266 12 334 11 505 9738 Human genes – 15 615 12 352 14 574 14 302 13 916 10 181 13 588 10 738 10 468 9820 9459 Numbers of genes of both human and other species are listed. Owing to lineage-speciﬁc duplication or loss, the numbers are usually diﬀerent (for example, 15 570 mouse genes are orthologous to 14 574 human genes). 18 968 human genes have at least one ortholog among other 11 species. aMacaca mulatta, Macaca fascicularis, Macaca fuscata, etc. are included. proteins can be analyzed using model species. Moreover, cross references between Evola and other annotations in H-InvDB (protein–protein interaction (PPI), expres- sion, polymorphism, disease, etc.) can produce valuable information contributing to the comprehensive under- standing of the human genes. We aimed to develop user-friendly interfaces that provide easy access to a variety of orthology information in Evola. Users can search orthologs in the top page of Evola as well as in the search systems of H-InvDB [simple search, advanced search and navigation system (Navi)]. Users can download data for each human gene on the main page as well as all the data of Evola in the download page. On the main page of Evola (Figure 2),