BACKGROUND
the set domain  is the catalytic center of lysine methyltransferases with a conserved sequence of ~ <dig> amino acid residues, initially identified at the c- terminus of three regulatory factors 3- <dig>  e and trithorax) in drosophila accounting for its name  <cit> .
currently, proteins containing the conserved set domain can be found in organisms ranging from virus to all three domains of life   <cit> .
in plants, baumbusch et al.
<cit>  first identified  <dig> putative arabidopsis set genes, and divided them into four distinct classes:  e homologues;  ash <dig> homologues and related genes;  trx homologues and related genes; and  su homologues and related genes.
subsequently, springer et al.
<cit>  added  <dig> maize set genes to those of  <dig> arabidopsis, and divided them into five classes based on phylogenetic relationships and domain organization; among these, the su homologues and related genes were designated as class v. recently, ng et al.
<cit>  established two additional plant set-gene classes, i.e.
class vi composed of the set genes  <cit>  and vii composed of the putative rubisco genes  <cit> ; however, these recent classes lack typical set domain, either interrupted in the set-i region of set domain or truncated.
among the seven classes of plant set genes, class v contains significantly more members relative to other classes and possess the preset domain  in their proteins  <cit> ; for example, from class i to vii, arabidopsis contains  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> members, respectively.
numerous copies in class v may complicate the evolutionary process of this class of plant set genes.
previous studies  <cit>  demonstrated that the class v set proteins can be further divided into seven orthologous groups  and two major types  based on their phylogenetic relationship and domain organization.
the suvh proteins consist of orthologous groups v- <dig>   <dig>   <dig> and  <dig>  and have an additional evolutionarily conserved sra domain  upstream of the preset domain.
the suvr proteins are composed of the remaining v- <dig>   <dig> and  <dig> orthologous groups and lack the sra domain.
baumbusch et al.
<cit>  and springer et al.
<cit>  noted that the majority of suvh members in arabidopsis and maize lacked introns, and supposed that these intronless suvh members probably originated from ancient retrotransposition events.
in arabidopsis, there are ten suvh and five suvr genes, in which five suvhs and three suvrs have been characterized functionally .
suvh <dig>   <dig>   <dig>   <dig> and  <dig> have been shown to control heterochromatic silencing by the hmtase activity  <cit> , and suvr <dig>   <dig>   <dig> were mainly localized in the nucleolus or nuclear bodies, suggestive of involvement in regulation of rrna expression  <cit> .
in contrast to suvh proteins, suvr <dig> acts as a dimethyltransferase with preference for mono-methylated h3k <dig> as substrate, suggesting that suvhs and suvrs can act in concert in achieving various functional h3k <dig> methylation states.
it has also been found that the sra domain of the suvh proteins may be involved in heterochromatin formation mediated by h3k <dig> methylation  <cit> .
suvrs, however, were once supposed to lack a shared n-terminal domain, although a novel conserved n-terminal domain, wiyld , was recently identified in a few members of the v- <dig> orthologous group, such as the arabidopsis suvr <dig>   <dig>  and  <dig>  <cit> .
here, we sampled from ten representatives of land plants to investigate the phylogeny and evolution of plant su3- <dig> set genes.
this is the first analysis of these genes covering the range of land plants.
we performed phylogenetic analysis using the combined datasets from the sequences of the conserved preset- and set-domain regions to increase phylogenetic resolution.
on the basis of phylogenetic analyses, we tracked the evolution of domain organizations and gene structures of plant su3- <dig> set genes in land plants; in turn, these domain organizations and gene structures were used as synapomorphies  to confirm the phylogenetic relationships.
finally, we explored the relationships between evolutionary patterns and functional diversification by combining the phylogenetic results with available literature for functions of plant su3- <dig> set genes; the results of our study would lay the foundation for the design of future experimental studies.
RESULTS
plant su3- <dig> set genes
arabidopsis thaliana and oryza sativa contained  <dig> and  <dig> full-length su3- <dig> set protein sequences, respectively.
to undertake an evolutionary analysis of su3- <dig> set genes in land plants, three other completely sequencing plant genomes and one algal genome were searched using multiple representatives of su3- <dig> set proteins in arabidopsis thaliana as queries.
by conducting tblastn searches against the jgi genome database, we obtained  <dig>   <dig>   <dig> and  <dig> su3- <dig> set protein sequences from populus trichocarpa , selaginella moellendorfii , physcomitrella patens  and chlamydomonas reinhardtii , respectively.
seven cdna sequences of pinus taeda  were obtained from tigr plant indices.
in addition,  <dig>   <dig> and  <dig> su3- <dig> set protein sequences were also obtained from nicotiana tabacum , vitis vinifera  and ricinus communis , respectively.
in total,  <dig> candidate set sequences were collected from ten species, and the detailed information is provided in additional file  <dig> and  <dig>  protein sequences lacking preset domain were not used for the further study even when they have very low e values in the blast searches.
the arabidopsis sdg <dig>  was also not used because it is likely a pseudogene  <cit> .
phylogenetic analysis
alignment of the combined dataset from preset and set domains resulted in a matrix with length of  <dig> sites after removing ambiguous regions and autapomorphic insertions .
the wag model  <cit>  was selected as the best-fit evolutionary model under the aic criterion  <cit>  with specific improvements .
a maximum-likelihood  analysis produced an optimal tree with an inl score of - <dig> .
the nj analyses recovered trees with almost identical topologies and support values to those of ml analyses.
most of differences between ml and nj trees were distributed on extremely short branches .
the ml tree is presented in figure  <dig> with bootstrap percentages at the node of the branch.
our analysis recovered all orthologous groups previously identified  <cit>  .
in the present investigation, we broadened these orthologous groups , hereafter) based on internal support  or conserved domain organization and gene structure , thus resulting in the inclusion of more members in each group.
in the current study, we used the definition of groups previously identified  <cit>  mainly for the purpose of comparison, and it is possible that some groups we have designated as a single group might actually represent multiple groups because of sampling limitations.
our tree showed that all members could be divided into two clades when the member of chlamydomonas reinhardtii  was designed as the outgroup.
the smaller clade was moderately supported, including v- <dig> group and other two members; it is worth noting that the v- <dig> group only contained angiosperm members excluding rice ; the larger clade was strongly supported, including all the remaining members, which was named as csuvhr 3- <dig> homologues &related genes) clade in our analysis .
within the csuvhr clade, the subclade including v- <dig>   <dig>   <dig>  and  <dig> groups was strongly supported and named here as the suvh clade  because all members possess a characteristic sra domain at the n terminus  <cit> ; this result was consistent with a previous hypothesis that all suvh genes had a common ancestor  <cit> .
the v- <dig> group within the csuvhr clade appears to be sister to the suvh clade, but only with low support.
the v- <dig> group within the csuvhr clade was placed at the basal position that did not appear to have a clear relationship with other groups.
plus sign  and minus sign  indicate presence and absence of a subtype, respectively; domain and species abbreviations are listed in figure 1; xxx indicates the protein sequence regions without predicted domain.
within the suvh clade, the v- <dig>  v- <dig> and v- <dig> groups plus several orphan members  formed a subclade with low support and only seed plant members; in contrast, the v- <dig> group was strongly supported and contained all representative land plants, with usually one copy in each species ; the suvh clade also contained several other orphan members , and their relationships with other members of this clade were uncertain .
the v- <dig> group contained members from both seed plants and moss, but not selaginella moellendorfii , and was characterized by the wiyld domain at the n-terminal region of protein sequences  <cit> .
the v- <dig> group contained members from each major land plant groups with one or several copies in each species, and possessed a characteristic znf_c2h <dig> domain   <cit>  at the n-terminal region of protein sequences.
an orphan member smsdg <dig> possessed a unique tpr_ <dig> domain  at the n-terminal regions of its protein sequences.
domain organization
to trace their evolutionary history in land plants, we predicted the domain organization of candidate su3- <dig> set proteins.
the candidate proteins could be classified into five types  and ten subtypes  based on their domain organization, with the major differences lying in their n-terminal regions.
type a contained a characteristic sra domain at the n-terminus , which was identified as the ydg_sra domain  in the pfam platform  <cit> .
the subtype a <dig>  which only existed in v- <dig> group, had an additional n-terminal domain, at_hook , a small dna-binding motif that functions in the transcription regulation of genes containing or in close proximity to at-rich regions  <cit> .
in contrast, the subtypes a <dig> and a <dig> were broadly distributed in v- <dig>   <dig>  and  <dig> and v- <dig> groups.
it was also worth noting that all members in v- <dig> group lack the postset domain  at their c-terminal regions.
type b contains one or more znf_c2h <dig> domain at its n-terminus  and was only distributed in the v- <dig> group.
the znf_c2h <dig> domain is one type of the c2h2-type zinc fingers , very common dna-binding motifs found extensively in eukaryotic and prokaryotic transcription factors  <cit> .
type c contains one wiyld domain at its n-terminus and was only found in the v- <dig> group .
type d lacked a typical preset domain and contains instead the aws domain  , a subdomain of preset domain.
this domain organization might have arisen recently because it was only found in angiosperms in the present study.
the aws domain was often found in association with the set domain, suggesting a role in methylation of lysine residues in histones and other proteins  <cit> .
type e refers to remaining domain organizations that were mostly from orphan members, either lacking identifiable n-terminal domains or having unique n-terminal domains; subtype e <dig> and e <dig> might be the ancestral domain organization due to their extensive distribution in eukaryotes .
gene structure
in the present study, the structures of only  <dig> plant su3- <dig> set genes  were analyzed due to the lack of the corresponding genomic sequences in other su3- <dig> set genes.
we found that the number of intron is highly variable in plant su3- <dig> set genes, ranging from  <dig> in v- <dig>  - <dig>  and - <dig> groups to  <dig> in smsdg <dig>  a total of  <dig> introns were present in  <dig> analyzed genes, an average of  <dig>  introns per gene; the average number of introns per gene also varied among groups, ranging from  <dig>  in v- <dig> group to  <dig> in v- <dig> group .
among the  <dig> introns,  <dig>  were in phase  <dig>   <dig>  in phase  <dig>  and  <dig>  in phase  <dig> , similar to the previous reports of  <dig> % for phase  <dig>   <dig> % for phase  <dig>  and  <dig> % for phase  <dig> in  <dig>  rice genes  <cit> .
to trace the evolutionary pattern of gene structure, the current study mainly focused on the most conserved preset and set domain regions.
figure  <dig> presents the gene structures of these two regions.
our result showed that at least three classes of gene structures  were formed probably through frequent inron loss and gain before the divergence of physcomitrella patens from other land plants.
in these three groups, the ancestral gene structures might be similar to ppsdg <dig>  ppsdg <dig> and ppsdg <dig> , respectively.
the sequence similarity between introns was not analyzed because their lengths were highly variable.
within the v- <dig> group, all introns maintained identical phases and positions, indicating a high degree of structural conservation during the evolution of land plants.
in contrast, the v- <dig> and - <dig> groups were less conserved; for example, in v- <dig> the intron sliding occurred in the last intron  .
also in v- <dig> ppsdg <dig> had only one conserved intron  compared to other genes.
atsdg <dig>  smsdg <dig> and ppsdg <dig> had a common intron , together with the low support on the relationship among them in phylogenetic tree, suggesting that v- <dig> group might have a common ancestor with these two orphan members.
most members in the suvh clade were intronless except for the v- <dig> group.
previous studies found that most arabidopsis genes in this clade were intronless, and suggested that these intronless members may have originated from one or a few retroposition events, followed by tandem duplication  <cit> .
if this hypothesis is correct, the donor genes of the retroposition might also be in the v- <dig> group, because the descendent retrogene and the donor genes should cluster together in the phylogenetic tree just as in figure  <dig>  in the suvh clade, intronless members formed three independent subclades, each with weak bp support values .
if multiple retroposition events occurred in donor gene lineage, the donor gene lineage would cluster with these retrogenes arranged paraphyletically in phylogenetic tree .
owing to the low support values in the current data, we are still unable to determine whether these three intronless branches originated independently or had a common ancestor.
discussion
the presence of gene families is one of the characteristics of eukaryotes  <cit> .
since the genes within families are initially redundant in molecular function, they likely have undergone evolutionary selection processes, and eventually formed multiple orthologous groups to carry out different functions  <cit> .
the current research first presented the phylogeny and evolution of plant su3- <dig> set gene family in land plants.
our analyses identified a novel phylogenetic relationship, that is, the csuvhr clade that includes most members analyzed except for the v- <dig> group and a few orphan members .
in addition, our results support the following evolutionary scenario of this gene family: multiple gene duplications had occurred independently before the split of physcomitrella patens  from other land plants, and since then each of orthologs experienced molecular divergence by mutations, domain acquisition and gene structure changes, resulted in different orthologous groups.
we suggested that the sar, znf_c2h <dig> and wiyld domains were early integrated into primordial preset/set/postset domain organization to form different evolutionary groups  because the type a, b and c domain organization in table  <dig> were all found in physcomitrella patens.
in contrast to previous reports  <cit> , our analyses showed that the postset domain was present in most plant su3- <dig> set proteins, but not in the v- <dig> group.
in the light of the parsimony rule of evolution, we propose that the ancestral plant su3- <dig> set proteins might have possessed the postset domain, which was lost in some members during the subsequent evolution .
the plant su3- <dig> set gene family exhibits a large diversity of gene structures, even in the conserved preset and set domains , implying frequent gain and/or loss of introns during evolution  <cit> .
for plant su3- <dig> set gene family, such frequent gain/loss of introns might have occurred during early evolution of land plants because the gene structure of the v- <dig>   <dig> and  <dig> groups had appeared before the divergence of physcomitrella patens .
because introns might have regulatory functions  <cit> , the gain or loss of introns may have contributed to functional divergence between paralogs, such as subfunctionalization, either directly by introducing regulatory differences or by facilitating exon shuffling.
in our study, the v- <dig> group demonstrated strict conservation of gene structure, indicating that this group may have evolved under high selective pressures and is functionally important; in contrast, v- <dig> and v- <dig> may have evolved under relatively relaxed selective pressures .
as in previous studies  <cit> , our study showed that the shared variations in gene structure can be used for the classification of paralogous genes into different evolutionary groups ; accordingly we further suggest that v- <dig> group be expanded to include two orphan members, smsdg <dig> and ppsdg <dig>  because these two genes have a common intron  with atsdg <dig> of v- <dig> group .
in the suvh clade , the majority of the genes, except the v- <dig> group, were intronless.
we suppose that their last common ancestor might possess introns and the intronless genes  originated from the lineages leading to the v- <dig> group by retroposition because all  <dig> genes analyzed including from basal evolutionary groups  and an outgroup gene  from chlamydomonas reinhardtii , also possessed introns .
many retrogenes have been identified in plant gene families  <cit> .
it is generally believed that most retrogenes become non-functional because they lack the regulatory elements required for expression  <cit> .
however, several recent studies have demonstrated that functional genes can occasionally be generated from retrogenes and that these processed genes take on a non-redundant functional role  <cit> .
in the plant su3- <dig> set gene family, transcripts of many intronless arabidopsis, oryza and maize genes have been detected in rt-pcr and/or microarray analyses  <cit> , suggesting that some retrogenes of the su3- <dig> set family might have gained regulatory elements and became functional.
we found that arabidopsis su3- <dig> set genes of different groups or clades have different functions , suggesting that they interact with the different substrates.
suvh <dig>   and suvh <dig>  <cit>  play major roles in arabidopsis histone h3k <dig> methylation modification; in contrast, the loss of suvh <dig>   <cit>  and suvh <dig>   <cit>  or suvh <dig>  <cit>  results in only minor reductions in global h3k <dig> methylation levels.
suvr <dig> , suvr <dig>  and suvr <dig>  proteins have been studied in detail  <cit> .
they are localized to the nucleolus or non-condensed nuclear bodies, which differs from suvh proteins localizing to heterochromatin region.
in vitro suvr <dig> acts as efficient dimethyltransferase specifically adding the second methyl group to monomethylated h3k9; in contrast, in vitro suvh <dig> , suvh <dig>  and suvh <dig>  proteins are very efficient monomethyltransferases but moderately efficient dimethyltransferase  <cit> .
the localization of the suvr proteins suggests that these proteins are not involved in heterochromatic gene silencing, and may function as a repressor of rdna gene clusters in the decondensed part of the nucleolus.
the suvr <dig>  and suvr <dig>  genes were the only arabidopsis representative of su3- <dig> set genes in the v- <dig> and v- <dig> groups , respectively, and their functions are unknown and will need to be investigated in the future.
CONCLUSIONS
our study provides novel phylogenetic relationship and new insights into the evolution of plant su3- <dig> set gene family in land plants, which includes most members analyzed except for the v- <dig> group and a few orphan members.
we found that the postset is not a common domain in plant su3- <dig> set proteins; it might be an ancestral characteristic of this gene family, which was lost in some members during the evolution.
we propose that the sar, znf_c2h <dig> and wiyld domains were integrated into primordial domain organization, preset/set/postset, during the early evolution of land plant and resulted in evolutionary differentiation.
plant su3- <dig> set genes exhibit a diversity of structures, even in the conserved preset and set domain regions.
at least three classes of gene structures in the v- <dig>  v- <dig> and v- <dig> groups had appeared before the divergence of physcomitrella patens from other land plants through frequent inron loss and gain.
in the suvh clade, the majority of the members were intronless retrogenes, probably originated from the ancestral genes leading to v- <dig> group with introns.
our results revealed the structural differences among evolutionary groups of plant su3- <dig> set genes with different functions, and further predicted that the function of arabidopsis suvr <dig>  and suvr <dig>  genes belonging to the v- <dig> and v- <dig> groups, respectively, are different from other arabidopsis su3- <dig> set genes.

 the set domain is the catalytic center of lysine methyltransferases with a conserved sequence of ~ amino acid residues, initially identified at the c- terminus of three regulatory factors 3- e and trithorax) in drosophila accounting for its name. currently, proteins containing the conserved set domain can be found in organisms ranging from virus to all three domains of life. in plants, baumbusch et al. first identified putative arabidopsis set genes, and divided them into four distinct classes: e homologues; ash homologues and related genes; trx homologues and related genes; and su homologues and related genes. subsequently, springer et al. added maize set genes to those of arabidopsis, and divided them into five classes based on phylogenetic relationships and domain organization; among these, the su homologues and related genes were designated as class v. recently, ng et al. established two additional plant set-gene classes, i.e. class vi composed of the set genes and vii composed of the putative rubisco genes; however, these recent classes lack typical set domain, either interrupted in the set-i region of set domain or truncated. among the seven classes of plant set genes, class v contains significantly more members relative to other classes and possess the preset domain in their proteins; for example, from class i to vii, arabidopsis contains and members, respectively. numerous copies in class v may complicate the evolutionary process of this class of plant set genes. previous studies demonstrated that the class v set proteins can be further divided into seven orthologous groups and two major types based on their phylogenetic relationship and domain organization. the suvh proteins consist of orthologous groups v- and and have an additional evolutionarily conserved sra domain upstream of the preset domain. the suvr proteins are composed of the remaining v- and orthologous groups and lack the sra domain. baumbusch et al. and springer et al. noted that the majority of suvh members in arabidopsis and maize lacked introns, and supposed that these intronless suvh members probably originated from ancient retrotransposition events. in arabidopsis, there are ten suvh and five suvr genes, in which five suvhs and three suvrs have been characterized functionally. suvh and have been shown to control heterochromatic silencing by the hmtase activity, and suvr were mainly localized in the nucleolus or nuclear bodies, suggestive of involvement in regulation of rrna expression. in contrast to suvh proteins, suvr acts as a dimethyltransferase with preference for mono-methylated h3k as substrate, suggesting that suvhs and suvrs can act in concert in achieving various functional h3k methylation states. it has also been found that the sra domain of the suvh proteins may be involved in heterochromatin formation mediated by h3k methylation. suvrs, however, were once supposed to lack a shared n-terminal domain, although a novel conserved n-terminal domain, wiyld, was recently identified in a few members of the v- orthologous group, such as the arabidopsis suvr and. here, we sampled from ten representatives of land plants to investigate the phylogeny and evolution of plant su3- set genes. this is the first analysis of these genes covering the range of land plants. we performed phylogenetic analysis using the combined datasets from the sequences of the conserved preset- and set-domain regions to increase phylogenetic resolution. on the basis of phylogenetic analyses, we tracked the evolution of domain organizations and gene structures of plant su3- set genes in land plants; in turn, these domain organizations and gene structures were used as synapomorphies to confirm the phylogenetic relationships. finally, we explored the relationships between evolutionary patterns and functional diversification by combining the phylogenetic results with available literature for functions of plant su3- set genes; the results of our study would lay the foundation for the design of future experimental studies.  plant su3- set genes arabidopsis thaliana and oryza sativa contained and full-length su3- set protein sequences, respectively. to undertake an evolutionary analysis of su3- set genes in land plants, three other completely sequencing plant genomes and one algal genome were searched using multiple representatives of su3- set proteins in arabidopsis thaliana as queries. by conducting tblastn searches against the jgi genome database, we obtained and su3- set protein sequences from populus trichocarpa, selaginella moellendorfii, physcomitrella patens and chlamydomonas reinhardtii, respectively. seven cdna sequences of pinus taeda were obtained from tigr plant indices. in addition, and su3- set protein sequences were also obtained from nicotiana tabacum, vitis vinifera and ricinus communis, respectively. in total, candidate set sequences were collected from ten species, and the detailed information is provided in additional file and protein sequences lacking preset domain were not used for the further study even when they have very low e values in the blast searches. the arabidopsis sdg was also not used because it is likely a pseudogene. phylogenetic analysis alignment of the combined dataset from preset and set domains resulted in a matrix with length of sites after removing ambiguous regions and autapomorphic insertions. the wag model was selected as the best-fit evolutionary model under the aic criterion with specific improvements. a maximum-likelihood analysis produced an optimal tree with an inl score of -. the nj analyses recovered trees with almost identical topologies and support values to those of ml analyses. most of differences between ml and nj trees were distributed on extremely short branches. the ml tree is presented in figure with bootstrap percentages at the node of the branch. our analysis recovered all orthologous groups previously identified. in the present investigation, we broadened these orthologous groups, hereafter) based on internal support or conserved domain organization and gene structure, thus resulting in the inclusion of more members in each group. in the current study, we used the definition of groups previously identified mainly for the purpose of comparison, and it is possible that some groups we have designated as a single group might actually represent multiple groups because of sampling limitations. our tree showed that all members could be divided into two clades when the member of chlamydomonas reinhardtii was designed as the outgroup. the smaller clade was moderately supported, including v- group and other two members; it is worth noting that the v- group only contained angiosperm members excluding rice; the larger clade was strongly supported, including all the remaining members, which was named as csuvhr 3- homologues &related genes) clade in our analysis. within the csuvhr clade, the subclade including v- and groups was strongly supported and named here as the suvh clade because all members possess a characteristic sra domain at the n terminus; this result was consistent with a previous hypothesis that all suvh genes had a common ancestor. the v- group within the csuvhr clade appears to be sister to the suvh clade, but only with low support. the v- group within the csuvhr clade was placed at the basal position that did not appear to have a clear relationship with other groups. plus sign and minus sign indicate presence and absence of a subtype, respectively; domain and species abbreviations are listed in figure 1; xxx indicates the protein sequence regions without predicted domain. within the suvh clade, the v- v- and v- groups plus several orphan members formed a subclade with low support and only seed plant members; in contrast, the v- group was strongly supported and contained all representative land plants, with usually one copy in each species; the suvh clade also contained several other orphan members, and their relationships with other members of this clade were uncertain. the v- group contained members from both seed plants and moss, but not selaginella moellendorfii, and was characterized by the wiyld domain at the n-terminal region of protein sequences. the v- group contained members from each major land plant groups with one or several copies in each species, and possessed a characteristic znf_c2h domain at the n-terminal region of protein sequences. an orphan member smsdg possessed a unique tpr_ domain at the n-terminal regions of its protein sequences. domain organization to trace their evolutionary history in land plants, we predicted the domain organization of candidate su3- set proteins. the candidate proteins could be classified into five types and ten subtypes based on their domain organization, with the major differences lying in their n-terminal regions. type a contained a characteristic sra domain at the n-terminus, which was identified as the ydg_sra domain in the pfam platform. the subtype a which only existed in v- group, had an additional n-terminal domain, at_hook, a small dna-binding motif that functions in the transcription regulation of genes containing or in close proximity to at-rich regions. in contrast, the subtypes a and a were broadly distributed in v- and and v- groups. it was also worth noting that all members in v- group lack the postset domain at their c-terminal regions. type b contains one or more znf_c2h domain at its n-terminus and was only distributed in the v- group. the znf_c2h domain is one type of the c2h2-type zinc fingers, very common dna-binding motifs found extensively in eukaryotic and prokaryotic transcription factors. type c contains one wiyld domain at its n-terminus and was only found in the v- group. type d lacked a typical preset domain and contains instead the aws domain, a subdomain of preset domain. this domain organization might have arisen recently because it was only found in angiosperms in the present study. the aws domain was often found in association with the set domain, suggesting a role in methylation of lysine residues in histones and other proteins. type e refers to remaining domain organizations that were mostly from orphan members, either lacking identifiable n-terminal domains or having unique n-terminal domains; subtype e and e might be the ancestral domain organization due to their extensive distribution in eukaryotes. gene structure in the present study, the structures of only plant su3- set genes were analyzed due to the lack of the corresponding genomic sequences in other su3- set genes. we found that the number of intron is highly variable in plant su3- set genes, ranging from in v- - and - groups to in smsdg a total of introns were present in analyzed genes, an average of introns per gene; the average number of introns per gene also varied among groups, ranging from in v- group to in v- group. among the introns, were in phase in phase and in phase, similar to the previous reports of % for phase % for phase and % for phase in rice genes. to trace the evolutionary pattern of gene structure, the current study mainly focused on the most conserved preset and set domain regions. figure presents the gene structures of these two regions. our result showed that at least three classes of gene structures were formed probably through frequent inron loss and gain before the divergence of physcomitrella patens from other land plants. in these three groups, the ancestral gene structures might be similar to ppsdg ppsdg and ppsdg, respectively. the sequence similarity between introns was not analyzed because their lengths were highly variable. within the v- group, all introns maintained identical phases and positions, indicating a high degree of structural conservation during the evolution of land plants. in contrast, the v- and - groups were less conserved; for example, in v- the intron sliding occurred in the last intron. also in v- ppsdg had only one conserved intron compared to other genes. atsdg smsdg and ppsdg had a common intron, together with the low support on the relationship among them in phylogenetic tree, suggesting that v- group might have a common ancestor with these two orphan members. most members in the suvh clade were intronless except for the v- group. previous studies found that most arabidopsis genes in this clade were intronless, and suggested that these intronless members may have originated from one or a few retroposition events, followed by tandem duplication. if this hypothesis is correct, the donor genes of the retroposition might also be in the v- group, because the descendent retrogene and the donor genes should cluster together in the phylogenetic tree just as in figure in the suvh clade, intronless members formed three independent subclades, each with weak bp support values. if multiple retroposition events occurred in donor gene lineage, the donor gene lineage would cluster with these retrogenes arranged paraphyletically in phylogenetic tree. owing to the low support values in the current data, we are still unable to determine whether these three intronless branches originated independently or had a common ancestor. discussion the presence of gene families is one of the characteristics of eukaryotes. since the genes within families are initially redundant in molecular function, they likely have undergone evolutionary selection processes, and eventually formed multiple orthologous groups to carry out different functions. the current research first presented the phylogeny and evolution of plant su3- set gene family in land plants. our analyses identified a novel phylogenetic relationship, that is, the csuvhr clade that includes most members analyzed except for the v- group and a few orphan members. in addition, our results support the following evolutionary scenario of this gene family: multiple gene duplications had occurred independently before the split of physcomitrella patens from other land plants, and since then each of orthologs experienced molecular divergence by mutations, domain acquisition and gene structure changes, resulted in different orthologous groups. we suggested that the sar, znf_c2h and wiyld domains were early integrated into primordial preset/set/postset domain organization to form different evolutionary groups because the type a, b and c domain organization in table were all found in physcomitrella patens. in contrast to previous reports, our analyses showed that the postset domain was present in most plant su3- set proteins, but not in the v- group. in the light of the parsimony rule of evolution, we propose that the ancestral plant su3- set proteins might have possessed the postset domain, which was lost in some members during the subsequent evolution. the plant su3- set gene family exhibits a large diversity of gene structures, even in the conserved preset and set domains, implying frequent gain and/or loss of introns during evolution. for plant su3- set gene family, such frequent gain/loss of introns might have occurred during early evolution of land plants because the gene structure of the v- and groups had appeared before the divergence of physcomitrella patens. because introns might have regulatory functions, the gain or loss of introns may have contributed to functional divergence between paralogs, such as subfunctionalization, either directly by introducing regulatory differences or by facilitating exon shuffling. in our study, the v- group demonstrated strict conservation of gene structure, indicating that this group may have evolved under high selective pressures and is functionally important; in contrast, v- and v- may have evolved under relatively relaxed selective pressures. as in previous studies, our study showed that the shared variations in gene structure can be used for the classification of paralogous genes into different evolutionary groups; accordingly we further suggest that v- group be expanded to include two orphan members, smsdg and ppsdg because these two genes have a common intron with atsdg of v- group. in the suvh clade, the majority of the genes, except the v- group, were intronless. we suppose that their last common ancestor might possess introns and the intronless genes originated from the lineages leading to the v- group by retroposition because all genes analyzed including from basal evolutionary groups and an outgroup gene from chlamydomonas reinhardtii, also possessed introns. many retrogenes have been identified in plant gene families. it is generally believed that most retrogenes become non-functional because they lack the regulatory elements required for expression. however, several recent studies have demonstrated that functional genes can occasionally be generated from retrogenes and that these processed genes take on a non-redundant functional role. in the plant su3- set gene family, transcripts of many intronless arabidopsis, oryza and maize genes have been detected in rt-pcr and/or microarray analyses, suggesting that some retrogenes of the su3- set family might have gained regulatory elements and became functional. we found that arabidopsis su3- set genes of different groups or clades have different functions, suggesting that they interact with the different substrates. suvh and suvh play major roles in arabidopsis histone h3k methylation modification; in contrast, the loss of suvh and suvh or suvh results in only minor reductions in global h3k methylation levels. suvr, suvr and suvr proteins have been studied in detail. they are localized to the nucleolus or non-condensed nuclear bodies, which differs from suvh proteins localizing to heterochromatin region. in vitro suvr acts as efficient dimethyltransferase specifically adding the second methyl group to monomethylated h3k9; in contrast, in vitro suvh, suvh and suvh proteins are very efficient monomethyltransferases but moderately efficient dimethyltransferase. the localization of the suvr proteins suggests that these proteins are not involved in heterochromatic gene silencing, and may function as a repressor of rdna gene clusters in the decondensed part of the nucleolus. the suvr and suvr genes were the only arabidopsis representative of su3- set genes in the v- and v- groups, respectively, and their functions are unknown and will need to be investigated in the future.  our study provides novel phylogenetic relationship and new insights into the evolution of plant su3- set gene family in land plants, which includes most members analyzed except for the v- group and a few orphan members. we found that the postset is not a common domain in plant su3- set proteins; it might be an ancestral characteristic of this gene family, which was lost in some members during the evolution. we propose that the sar, znf_c2h and wiyld domains were integrated into primordial domain organization, preset/set/postset, during the early evolution of land plant and resulted in evolutionary differentiation. plant su3- set genes exhibit a diversity of structures, even in the conserved preset and set domain regions. at least three classes of gene structures in the v- v- and v- groups had appeared before the divergence of physcomitrella patens from other land plants through frequent inron loss and gain. in the suvh clade, the majority of the members were intronless retrogenes, probably originated from the ancestral genes leading to v- group with introns. our results revealed the structural differences among evolutionary groups of plant su3- set genes with different functions, and further predicted that the function of arabidopsis suvr and suvr genes belonging to the v- and v- groups, respectively, are different from other arabidopsis su3- set genes. 
putative orthologs of plant su3- set protein sequences were retrieved from major representatives of land plants. the structural differences among evolutionary groups of plant su3- set genes with different functions were described, contributing to the design of further experimental studies. one or multiple retroposition events might have occurred among suvh genes with the donor genes leading to the v- orthologous group. we also found that the majority of suvh members were intronless and formed three subclades within the suvh clade. although there have been studies on the evolution of plant su3- set genes, a systematic evolutionary study including major land plant groups has not been reported. a novel clustering that included most members analyzed, henceforth referred to as core su3- homologues and related gene clade, was identified as well as all orthologous groups previously identified. a detailed phylogenetic analysis of the plant su3- set genes was performed. our analysis showed that plant su3- set proteins possessed a variety of domain organizations, and can be classified into five types and ten subtypes. at least three classes of gene structures had been formed before the divergence of physcomitrella patens from other land plants. plant su3- set genes also exhibit a wide range of gene structures among different paralogs within a family, even in the regions encoding conserved preset and set domains. additional domains such as sar, znf_c2h and wiyld were early integrated into primordial preset/set/postset domain organization.
 plants contain numerous su3- homologues and related genes, some of which await functional characterization. although there have been studies on the evolution of plant su3- set genes, a systematic evolutionary study including major land plant groups has not been reported. large-scale phylogenetic and evolutionary analyses can help to elucidate the underlying molecular mechanisms and contribute to improve genome annotation.  putative orthologs of plant su3- set protein sequences were retrieved from major representatives of land plants. a novel clustering that included most members analyzed, henceforth referred to as core su3- homologues and related gene clade, was identified as well as all orthologous groups previously identified. our analysis showed that plant su3- set proteins possessed a variety of domain organizations, and can be classified into five types and ten subtypes. plant su3- set genes also exhibit a wide range of gene structures among different paralogs within a family, even in the regions encoding conserved preset and set domains. we also found that the majority of suvh members were intronless and formed three subclades within the suvh clade.  a detailed phylogenetic analysis of the plant su3- set genes was performed. a novel deep phylogenetic relationship including most plant su3- set genes was identified. additional domains such as sar, znf_c2h and wiyld were early integrated into primordial preset/set/postset domain organization. at least three classes of gene structures had been formed before the divergence of physcomitrella patens from other land plants. one or multiple retroposition events might have occurred among suvh genes with the donor genes leading to the v- orthologous group. the structural differences among evolutionary groups of plant su3- set genes with different functions were described, contributing to the design of further experimental studies. 