To characterize the role of LSEs in the evolution of the respective classes of paralogous proteins in each lineage, we devised the expansion coefficient (EC), which is the ratio of the number of proteins in LSCs to the total membership of the given class of paralogs in a given proteome. The EC is a measure of the fraction of a given paralogous class that has evolved through LSE after the divergence of the given lineage from the closest sister lineage included in the analysis. LSCs with EC=1 are those families that have been invented de novo and proliferated thereafter in a particular lineage. The relative abundance of LSCs in the EC range between 0 and 0.9 is roughly constant for all taxa considered here, with slightly >5% of the LSCs in each of the bins of size 0.1 in this range (Fig. (Fig.4).4). Notably, ~40% (on average) of the LSCs present in a given proteome were in the EC range of 0.9 to 1 (Fig. (Fig.4).4). Thus, nearly one-half of the paralogous protein clusters encoded in eukaryotic genomes have been generated almost entirely through LSE. This applied to the full range of evolutionary distances explored here and there was no obvious dependence on the evolutionary depth at which LSEs were identified; the fraction of paralogous classes contained in these exclusive LSCs was even greater in the yeast S. cerevisiae than it was in A. thaliana (Fig. (Fig.4).4). This observation, together with the correlations between proteome size and different parameters of LSEs (Fig. (Fig.2),2), suggests that the ancestral core set of proteins inherited by the crown-group lineages from their last common ancestor contained few paralogs compared with the extant proteomes. Subsequent to the divergence of the individual lineages, many genes inherited from the common ancestor as well as gene families invented de novo have undergone one or more rounds of duplication. This process seems to have been particularly active in the generation of the large proteomes of multicellular eukaryotes and probably provided them with the raw material for their cellular differentiation. In principle, it could be argued that the ancestor had as many paralogous families as the most complex of the extant genomes or even more, and the appearance of LSE had been created by lineage-specific gene loss, which is common in the evolution of at least some eukaryotic lineages (Aravind et al. 2000; Braun et al. 2000). However, apart from the general implausibility of a highly complex common ancestor for the crown group, this mechanism for the evolution of apparent LSEs would necessarily entail independent gene losses in the same paralogous family in multiple lineages, as opposed to a single expansion. Therefore, the lineage-specific duplication scenario is more parsimonious than the scenario based on the lineage-specific losses.

Although LSEs occurred in most biological functional classes, LSCs with predicted organism-specific functions, such as pathogen and stress response, transcription regulation, controlled protein degradation mediated by the ubiquitin system, protein modification, signal transduction, chemoreception, and small molecule metabolism were most abundant (Tables 1 and 2). A typical example of an expansion related to an organism-specific function is that of the C. elegans collagens, which are required for cuticle formation, a characteristic adaptation of the nematodes (Johnstone 2000). Similarly, in D. melanogaster and Arabidopsis, prominent LSEs are, respectively, the insect cuticular proteins (Andersen et al. 1995) and pectin/cellulose biosynthesis enzymes (Willats et al. 2001), both of which are critical for the formation of morphological features unique to these lineages. Typically, these proteins are required in large amounts as structural components of the respective organisms; hence, these lineage-specific expansions could principally help in increased production of these proteins. Extending this analogy, it is possible that several of the LSCs with no detectable homologs elsewhere could represent as yet uncharacterized, but abundant, lineage-specific structural proteins (Table 2).

Many of the identified LSCs had predicted biochemical characteristics that pointed to roles in stress and pathogen response. Particularly striking in this category was the expansion of proteases of the pepsin-like and subtilisin-like families in A. thaliana, trypsin-like proteases in D. melanogaster, and Zn-metalloproteases in C. elegans (Table 2). All of these proteases are predicted to be secreted molecules, and their repeated, independent expansion suggests that they are widely utilized either for direct degradation of pathogen proteins or as components of stress-triggered proteolytic cascades broadly analogous to the vertebrate complement and clotting systems (Bouchard and Tracy 2001; Southan 2001). Alternatively, in the case of plants, they could aid in protein digestion in the process of germination. Better-understood cases of similar lineage-specific expansions related to stress/pathogen-response components include the massive proliferation of apoptotic (AP-) ATPases and the accompanying moderate expansion of metacaspases in plants, and the parallel expansion of caspases in vertebrates (Aravind et al. 2001; Holub 2001). These proteins are either known or predicted to participate in multiple pathways associated with apoptosis or hypersensitive response. In this context, also of interest are the expansions of molecules containing modules functioning in extracellular adhesion. Prominent examples of these include the C-type lectins (D. melanogaster, C. elegans), PR1 proteins (C. elegans, A. thaliana), CUB domain proteins (C. elegans), and the bulb-lectin domain (A. thaliana). As with the immunoglobulin domain protein, that are highly expanded in vertebrates, these molecules probably participate in the recognition and binding of specific pathogens as a part of defense mechanisms of the corresponding organisms (Table 2).

Earlier analysis of the LSEs involving transcription factors had suggested that they included proteins regulating critical aspects of the development of the organism (Aravind and Koonin 1999; Riechmann et al. 2000; Lander et al. 2001). For example, the proteins belonging to the POZ and SAZ-type Myb domain expansions in D. melanogaster (Table 2) regulate as diverse functions as maintenance of the antero-posterior Hox gene expression pattern, neurite outgrowth and pathfinding, and organogenesis (Aravind and Koonin, 1999; Lander et al. 2001). Thus, it appears that proliferation of new transcription factor families, followed by their recruitment as upstream or downstream regulators with respect to core conserved developmental pathways, have contributed substantially to the evolution of morphological diversity in animals. The generality of this observation was reinforced by the evidence of massive, lineage-specific expansion and diversification of various transcription-factor families in the plant A. thaliana (Table 2). Many of these include well-characterized DNA-binding proteins, such as the MADS box and MYB domain proteins, that have been shown previously to participate in plant-specific functions, including development of flowers and other structures, meristemal differentiation, and organ-specific gene expression (Riechmann et al. 2000). In this study, we detected certain unexpected expansions of DNA-binding proteins in plants that might point to previously unrecognized transcription regulators. Examples include the proteins homologous to the mitochondrial transcription termination factor, which, in other eukaryotes, is present in a single copy that functions in the mitochondrion (Fernandez-Silva et al. 1997). The additional paralogs in plants have probably acquired different transcription-related functions because they form a tight cluster, distinct from the ancestral mitochondrial version. Plants also show an expansion of the DNA-binding replication factor A (RF-A), with >40 copies in A. thaliana, in contrast to the one-three copies observed in other eukaryotes. The expansion and divergence of RF-A in plants suggest that the plant-specific paralogs are probably utilized as transcription factors rather than in their usual capacity in replication (Wold 1997). These and other such examples (Table 2) illustrate that transcription factors are recruited from a wide variety of pre-existing sources and diversify to occupy new functional niches via LSE.

We observed a major role of LSE in the elaboration of the ubiquitin pathway, which is involved in the degradation and regulatory modifications of proteins (Hershko and Ciechanover 1998). Evidence of LSE was obtained for several components of the ubiquitin system, in particular, E3 subunits of ubiquitin ligases containing the F-box domain (Kipreos and Pagano 2000) (A. thaliana and C. elegans) and the RING-finger (A. thaliana). Because the E3 proteins are specificity determinants that are involved in targeting the conserved ubiquitin-ligation machinery system to specific substrates (Jackson et al. 2000), their diversification through LSE probably provides a means of harnessing an otherwise conserved system to regulate the degradation of diverse sets of targets. In a similar vein, both nematodes and plants also show independent LSEs of the MATH domain. This domain, which tends to form fusions to ubiquitin carboxy-terminal hydrolases or RING-finger E3s (Aravind et al. 1999; Polekhina et al. 2002), might serve as an additional adaptor that mediates de/ubiquitination of specific targets. A. thaliana has a prominent proliferation of the adenovirus-like thiol protease superfamily whose members (e.g., Smt4/Ulp1) in yeast and in vertebrates, remove ubiquitin-like proteins from their targets (Li and Hochstrasser 2000; Nishida et al. 2000). Thus, in plants, this LSC probably contributes to further diversification of the regulation of ubiquitin-dependent protein degradation. Targeting of proteins for degradation has been shown to occur through the recognition of hydroxyproline by ubiquitin ligase complexes (Ivan et al. 2001). Thus, the LSE of 2-oxoglutarate-dependent prolyl hydroxylases (Aravind and Koonin 2001) detected in D. melanogaster and A. thaliana could represent another case in which the range of the core ubiquitination pathway is expanded via diversification of the terminal effectors.

The role of LSE in the diversification of proximal components of signal transduction systems, receptors, had been noticed previously in the cases of independent expansions of odorant receptors/7-transmembrane chemoreceptors seen in different animal lineages (Dryer 2000; Glusman et al. 2001) and plant receptor kinases containing extracellular leucine-rich repeats, bulb lectin, or EGF-like extracellular domains (Shiu and Bleecker 2001). Here, we detected other analogous expansions of upstream signaling proteins, such as potassium channels, innexin family channels (both in C. elegans), and tetraspanins and degenerin-type channels in D. melanogaster (similar LSEs of K-channels and tetraspanins are also seen in humans). The proteins involved in these expansions are linked to the organism's responses to external as well as internal homeostatic stimuli. Thus, such expansions could serve as the raw material for the behavioral and physiological adaptation of organisms to their specific environments. Lineage-specific expansions are also seen in a range of protein-modifying enzymes of different signal transduction cascade, such as protein kinase families in most lineages, SET-domain protein-methylases in D. melanogaster, and PP2C phosphatases in plants. As with the ubiquitin system, these appear to be a means of linking well-conserved stems of signaling pathways to distinct sets of terminal targets.

Another aspect of the involvement of LSEs in the evolution of signal-transduction networks is the extensive proliferation of families of proteins containing adaptor domains. Along with their expansion, many adaptor domains have also recombined with a variety of other domains, probably allowing the emergence of new networks of interactions. A striking example is the major expansion of proteins containing the small Ca-binding octicosapeptide (OOP) module (Ponting 1996) in A. thaliana. Some OOP modules are fused to VIV1-like plant-specific DNA-binding proteins and a specialized class of GAF domains, suggesting that they link transcription regulation and small molecule interactions to Ca-dependent signaling. Another notable case is a novel adaptor domain, typified by the amino-terminal domain of the Caspase-1A isoform, which so far was detected only in C. elegans. Altogether, the C. elegans genome encodes >40 members of this domain family, which, in addition to the caspase fusion, also form multidomain proteins with SET-domain methylases, PHD fingers, and kinases. Given the α-helical structure predicted for this domain, and enrichment in charged residues, it probably functions as a protein–protein interaction module.

We thank I. King Jordan and Kira Makarova for their help in developing the procedures for identifying the LSCs.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.