**3. Phylogenetic relationships of SERA genes**

The categorization of SERA genes into four groups, Group I to IV, was based on phylogenetic analysis (Arisue et al., 2007, 2011). SERA amino acid sequences from 18 *Plasmodium* species were aligned using CLUSTAL W program (http://clustalw.ddbj.nig.ac.jp/top-j.html) under default options with manual corrections. Unambiguously aligned amino acid positions corresponding to the putative pro-enzyme domain, enzyme domain and cysteine rich conserved domain were selected and used for the phylogenetic analysis. Maximum likelihood tree was inferred using the PROML program in PHYLIP version 3.69 (Felsenstein, 1996). Except for the number of genes and number of amino acid sequences included in the analysis, the same method was used to infer the phylogenetic tree shown in Fig. 4 and 5.

A simplified maximum likelihood tree inferred from 134 SERA genes with 392 amino acid positions is shown in Fig. 4. Bootstrap proportion values were placed only on the common ancestor branch of each group. The monophyletic grouping of Group I SERA genes was supported with a bootstrap value of 100%. The long internal branch separating Group I from Groups II to IV suggests that the root of the tree is located on the branch leading to the common ancestor of Group I SERA genes. It is thus likely that Group I genes have appeared early in the evolution of the SERA gene family. *P. gallinaceum* SERA1 branches at the common ancestor of Group II to IV, suggesting that gene duplication events which produced Groups II, III and IV likely occurred after the divergence of *P. gallinaceum* from the common ancestral lineage of *Plasmodium*.

Clues to Evolution of the SERA Multigene Family in the Genus *Plasmodium* 323

Group IV SERA genes which diverged after the cysteine to serine substitution in the catalytic site were further divided to five monophyletic sub-Groups: (i) *P. falciparum* and *P. reichenowi*, (ii) *P. ovale*, (iii) rodent *Plasmodium* species, (iv) *P. vivax* and *P. vivax*-related monkey parasite species, and (v) *P. malariae*. These indicate that genes have duplicated independently in each of the sub-group lineages. To increase the resolution of the tree, the long branched Group I genes were excluded from the dataset and the maximum likelihood tree was re-constructed from 115 SERA genes categorized into Group II to Group IV. The

Interestingly, Group IV SERA genes of *P. vivax* and *P. vivax*-related monkey parasites (10 species) were further categorized into six orthologous gene groups, namely, Clade 1 to Clade 6; and each clade has 5 (Clade 6) to 10 (Clade 5) parasite species. The number of SERA genes analyzed varied from 5 (*P. fragile*) to 12 (*P. vivax*). This does suggest that a common ancestor of *P. vivax* and related monkey malaria parasites had at least 6 SERA genes of Group IV; and that gene duplications and gene deletions occurred in each lineage. Orthologous relationships between SERA gene members and their relative arrangement are

Fig. 6. Organization and phylogenetic relationship of SERA gene in 18 *Plasmodium* species.

resultant tree is shown in Fig. 5.

shown in Fig. 6.

Fig. 4. Phylogenetic tree based on the alignment of 134 SERA genes from 18 *Plasmodium* species.

Fig. 5. Phylogenetic tree based on the alignment of 115 SERA genes from 18 *Plasmodium* species.

Fig. 4. Phylogenetic tree based on the alignment of 134 SERA genes from 18 *Plasmodium*

Fig. 5. Phylogenetic tree based on the alignment of 115 SERA genes from 18 *Plasmodium*

(i) *P. falciparum + P. reichenowi* 

species.

*P. falciparum*

species.

Group IV SERA genes which diverged after the cysteine to serine substitution in the catalytic site were further divided to five monophyletic sub-Groups: (i) *P. falciparum* and *P. reichenowi*, (ii) *P. ovale*, (iii) rodent *Plasmodium* species, (iv) *P. vivax* and *P. vivax*-related monkey parasite species, and (v) *P. malariae*. These indicate that genes have duplicated independently in each of the sub-group lineages. To increase the resolution of the tree, the long branched Group I genes were excluded from the dataset and the maximum likelihood tree was re-constructed from 115 SERA genes categorized into Group II to Group IV. The resultant tree is shown in Fig. 5.

Interestingly, Group IV SERA genes of *P. vivax* and *P. vivax*-related monkey parasites (10 species) were further categorized into six orthologous gene groups, namely, Clade 1 to Clade 6; and each clade has 5 (Clade 6) to 10 (Clade 5) parasite species. The number of SERA genes analyzed varied from 5 (*P. fragile*) to 12 (*P. vivax*). This does suggest that a common ancestor of *P. vivax* and related monkey malaria parasites had at least 6 SERA genes of Group IV; and that gene duplications and gene deletions occurred in each lineage. Orthologous relationships between SERA gene members and their relative arrangement are shown in Fig. 6.

Fig. 6. Organization and phylogenetic relationship of SERA gene in 18 *Plasmodium* species.

Clues to Evolution of the SERA Multigene Family in the Genus *Plasmodium* 325

both primate lineages showed similar transcription pattern of SERA gene which might

Fig. 8. Representation summary of SERA gene transcription analyses in primate and rodent *Plasmodium* spp. Abundantly transcribed SERA genes are differentiated using enlarged solid

In general, duplicated genes undergo either (i) concerted evolution or (ii) birth-and-death evolution (Nei & Rooney, 2005). In the concerted evolution model, all members of a gene family evolve as a unit rather than independently. When a mutation occurs in a gene, mutation spreads to all other gene members by unequal crossover or gene conversion. As a result, all members of the gene family show identical sequence to each other. The evolution of rRNA multigene families in vertebrates is a classic example of concerted evolution. Analysis of MHC genes in mammals (Hughes & Nei, 1989; Nei et al., 1997; Nei & Hughes, 1992), other immune system related genes (Hughes & Nei, 1990; Ota & Nei, 1994) and disease-related genes (Zhang et al., 2000) show a quite different evolutionary pattern. The birth-and-death evolution model was proposed to explain differential duplication/independent diversification processes that result to subsequent loss or maintenance of genes in a multigene family. Thus, some duplicated genes are maintained in the genome for a long time while others are deleted or became pseudogenes through deleterious mutations. This model applies to rRNAs of *Plasmodium* species in marked contrast to the concerted evolution of rRNAs in most organisms. The model aptly explains the observation that rRNA genes in *Plasmodium* were structurally and functionally distinct

The observed gene duplication and gene deletion found in the *Plasmodium* SERA genes are clearly in concordance with the birth-and-death model, although traits of gene conversion are detected in a few of Group IV SERA genes. The birth-and-death model has, likewise, been recently proposed for gene duplication/gene deletion of merozoite surface protein 7, an immune target parasite surface antigen gene (Garzón-Ospina et al., 2010). It, thus, seem

circles.

**5. Duplication in the multigene family** 

(Rooney, 2004; Nishimoto et al., 2008).

suggest a possible relationship between SERA function and host specificity.

In addition to several gene members characterizing Group IV, there are multiple SERA gene fragments and pseudogenes containing multiple stop codons. Taken together, these extensive gene duplications, gene deletions as well as pseudogenization/truncation are evident only in the serine type SERA gene (Group IV) of *P. vivax* and related monkey malaria parasites.

### **4. Transcription analyses of SERA genes**

Transcription analyses revealed, likewise, some discordance among *Plasmodium* species. Transcription profile of the SERA gene family was analyzed first by Aoki et al. (2002) in *P. falciparum*. Genes were most actively transcribed at the late trophozoite to schizont stages of the parasite with SERA5 predominantly transcribed among the family (Fig. 7.).

Fig. 7. The relative abundance of mRNA for each *P. falciparum* SERA gene during late trophozoite to schizont stages of the parasite.

Similar to *P. falciparum*, multiple number of SERA genes were transcribed at late trophozoite to schizont stages when transcription analysis was done for the SERA gene family in other *Plasmodium* parasites: human parasite *P. vivax* (Palacpac et al., 2006); rodent parasite *P. berghei* (Arisue et al., 2011); and three monkey parasites *P. knowlesi*, *P. cynomolgi* and *P. coatneyi* (Arisue et a.l, 2011). A representative summary of SERA gene transcription analysis is shown in Fig. 8. In malaria parasite species infecting humans, one of Group IV SERAs of *P. falciparum* (SERA5) and *P. vivax* (SERA4) showed the highest transcription level among other gene members. In the rodent malaria parasite *P. berghei*, Group III SERA gene (SERA3) was predominantly expressed. In three monkey malaria parasites, the abundantly expressed genes are members of Group IV Clade 3: SERA3 and SERA5, in *P. cynomolgi*; SERA3 in *P. coatneyi*; and SERA2 in *P. knowlesi*. These results show that SERA genes were differently expressed between rodent and primate parasites. Based on the malaria parasite mitochondrial genome, *P. falciparum* belongs to the primate parasite group 1 lineage whereas *P. vivax* and the three macaque parasites belong to primate parasite lineage 2. Phylogenetic analysis showed that these two lineages are not closely related; and the rodent parasite lineage is positioned between them (Hayakawa et al., 2008). Note, however, that

In addition to several gene members characterizing Group IV, there are multiple SERA gene fragments and pseudogenes containing multiple stop codons. Taken together, these extensive gene duplications, gene deletions as well as pseudogenization/truncation are evident only in the serine type SERA gene (Group IV) of *P. vivax* and related monkey

Transcription analyses revealed, likewise, some discordance among *Plasmodium* species. Transcription profile of the SERA gene family was analyzed first by Aoki et al. (2002) in *P. falciparum*. Genes were most actively transcribed at the late trophozoite to schizont stages of

the parasite with SERA5 predominantly transcribed among the family (Fig. 7.).

Fig. 7. The relative abundance of mRNA for each *P. falciparum* SERA gene during late

Similar to *P. falciparum*, multiple number of SERA genes were transcribed at late trophozoite to schizont stages when transcription analysis was done for the SERA gene family in other *Plasmodium* parasites: human parasite *P. vivax* (Palacpac et al., 2006); rodent parasite *P. berghei* (Arisue et al., 2011); and three monkey parasites *P. knowlesi*, *P. cynomolgi* and *P. coatneyi* (Arisue et a.l, 2011). A representative summary of SERA gene transcription analysis is shown in Fig. 8. In malaria parasite species infecting humans, one of Group IV SERAs of *P. falciparum* (SERA5) and *P. vivax* (SERA4) showed the highest transcription level among other gene members. In the rodent malaria parasite *P. berghei*, Group III SERA gene (SERA3) was predominantly expressed. In three monkey malaria parasites, the abundantly expressed genes are members of Group IV Clade 3: SERA3 and SERA5, in *P. cynomolgi*; SERA3 in *P. coatneyi*; and SERA2 in *P. knowlesi*. These results show that SERA genes were differently expressed between rodent and primate parasites. Based on the malaria parasite mitochondrial genome, *P. falciparum* belongs to the primate parasite group 1 lineage whereas *P. vivax* and the three macaque parasites belong to primate parasite lineage 2. Phylogenetic analysis showed that these two lineages are not closely related; and the rodent parasite lineage is positioned between them (Hayakawa et al., 2008). Note, however, that

malaria parasites.

**4. Transcription analyses of SERA genes** 

trophozoite to schizont stages of the parasite.

both primate lineages showed similar transcription pattern of SERA gene which might suggest a possible relationship between SERA function and host specificity.

Fig. 8. Representation summary of SERA gene transcription analyses in primate and rodent *Plasmodium* spp. Abundantly transcribed SERA genes are differentiated using enlarged solid circles.
