BACKGROUND
members of the cation diffusion facilitator  family have been shown to be important for maintenance of cation homeostasis in bacteria, yeast, plants, and mammals .
cdf proteins, in general, bind to and efflux such cations as zn from the cytoplasm through sequestration into internal compartments or through efflux from the cell.
this role in modulating cellular cation concentrations has been demonstrated to impact cation accumulation, cation tolerance, signal transduction cascades, oxidative stress resistance, and protein turnover  <cit> .
several research groups have analyzed the phylogenetic relationships of cdfs and found that this is an ancient gene family that pre-dates the origin of eukaryotes, as reflected in the grouping of sequences from diverse organisms within several branches of constructed phylogenetic trees.
plant cdf members, including  <dig> members from the sequenced genome of arabidopsis thaliana have been grouped into three or four lineages  <cit> .
however, these analyses were limited by sequence availability due to the lack of sequence genomes and available cdna libraries, which resulted in incomplete or weakly supported hypotheses about cdf family phylogeny within plants.
montanini, et al.
conducted global phylogenetic analysis on  <dig> cdfs from prokaryotes, eukaryotes, and archaea  <cit> .
based on a maximum parsimony analysis, variation across the gene family could be partitioned into three major groups, designated zn-cdfs, zn/fe-cdfs, and mn-cdfs based on the hypothesized or confirmed transported substrate of one or more group members.
for example, the mn-cdf group containes  <dig> sequences and, within this group, the plant members mtp <dig> and mtp <dig> have been characterized as mn transporters.
using vastly expanded sequence information and substrate-defined groups, an updated cdf signature sequence was derived as well as group-specific signature sequences.
the conserved residues comprising these signature sequences were the target of amino acid substitution, many of which were found to be critical residues for a fully functional protein.
recently, migeon et al.
expanded this analysis by incorporating cdf sequences from additional plant genomes with emphasis on phylogenetic and molecular characterization of metal transporters in populus trichocarpa  <cit> .
this analysis confirmed partitioning the sequences into three major functional groups.
grouping the sequences by predicted substrate specificity provides a useful hypothesis-generation tool for uncharacterized proteins within these broad groupings.
however, higher resolution analysis of plant-specific cdf sequences is likely to reveal informative relationships within the linage of land plants.
with the generation of full genome sequences for multiple eukaryotic organisms, a wealth of information is available from which to generate detailed phylogenomic relationships of gene families within and between organisms.
as genome sequences become available for more species, this "genomic" method of phylogenetic analysis should enable robust estimation of orthology and paralogy among related genes.
this high level resolution of familial evolution provides a powerful analytical tool from which to synthesize hypothesis about, among other things, the function of gene family members  <cit> .
the precision in functionally annotating an uncharacterized sequence based on sequence similarity to a characterized protein should increase if a detailed estimation of family phylogeny is known  <cit> .
once a sufficiently detailed map of the gene family structure and evolution are constructed, a more global understanding of the adaptive significance of the family dynamics through the course of evolution may become clearer and lead to testable hypotheses about the roles members play in organismal evolution.
genome sequencing of a red alga, cyanidioschyzon merolae, green algae, ostreococcus tauri, ostreococcus lucimarinus, and clammydomonas reinhardii, basal nonvascular and vascular land plants, physcomitrella patens  and selaginella moellendorffii, and representatives of angiosperm lineages have been completed  <cit> .
c. merolae is a non-motile unicellular red alga that lives in extreme environmental conditions, such as sulfate-rich hot springs and is estimated to have diverged from the lineage leading to true plant  approximately  <dig>  billion years ago  <cit> .
ostreococcus species are the smallest known eukaryotic organisms and belong to the prasinophyceae, an early diverging class in the lineage of the green algae  <cit> .
the algal model, c. reinhardii, is estimated to have shared a common ancestor with such species as a. thaliana  <dig>  billion years ago  <cit> .
p. patens and s. moellendorffii represent early land plant lineages of bryopsida and lycopsid, respectfully, which are estimated to have diverged from seed-bearing plants  approximately  <dig> million years ago  and  <dig> mya, respectively  <cit> .
within the more recent lineages of flowering plants , several genomes have been sequenced, including the monocotyledonous genomes of oryza sativa  and sorghum bicolor , and the eudicotyledonous genomes of a. thaliana, p. trichocarpa, and medicago truncatula <cit> .
the monocot lineage is predicted to have diverged from other angiosperms approximately  <dig> mya, and within eudicots, the a. thaliana and p. trichocarpa lineages are predicted to have diverged in the eurosid clade approximately  <dig> mya  <cit> .
collectively, the genomes of the six land plants contain information that allow for comparison of genome evolution throughout the approximately  <dig> million year history of land plants and inclusion of the genomes of red and green algae enables extension to  <dig>  billion years of plant evolution.
in this study we conduct a detailed phylogenic analysis of plant cdf family members to lay out a framework from which more informed hypotheses can be generated regarding the function of cdf proteins in plants.
RESULTS
plant cdf family member sequences
scanning the genomes of the taxonomically diverse set of organisms outlined in the introduction for cdf sequences identified or confirmed the following number of sequences: o. lucimarinus , o. tauri , c. merolae , c. reinhardii , p. patens , s. moellendorffii , o. sativa , s. bicolor , p. trichocarpa , and a. thaliana  .
the number of cdf sequences identified from c. reinhardii, c. merolae, s. moellendorffii, p. patens, s. bicolor, and a. thaliana, genomes agree with previous published studies  <cit> , however, the gene models may not be the same.
the number of p. trichocarpa cdfs was expanded to  <dig> from the previous estimate of  <dig>  <cit>  .
the expanded set includes a predicted pseudogene ptmtp <dig>  and previously unidentified ptmtp <dig> .
the number of cdf sequences in the o. sativa genome was expanded from  <dig> to  <dig> due to the inclusion of previously unidentified members osmtp <dig> and osmtp <dig>
plant cdf family structure
phylogenetic analysis of the cdf superfamily, including genomes from  <dig> archaea,  <dig> bacteria,  <dig> protozoa,  <dig> fungi,  <dig> red alga,  <dig> green algae,  <dig> land plants,  <dig> nematode, and  <dig> mammal can be grouped into three primary clades, as indicated by the colored branches  in figure  <dig>  these three primary clades are consistent with the previously defined zn-cdf, fe/zn-cdf, and mn-cdf groups based on functional evidence of resident members  <cit> , with one exception.
while previous analysis of the branch containing hsznt <dig> and atmtp <dig> had this branch of sequences as an orphaned, ungrouped branch, in this analysis it is included with the fe/zn-cdf group, therefore, this analysis suggests that these sequences be included into the fe/zn-cdf group.
cdf family members from viridiplantae and rhodophyta genomes were used to estimate the cdf family phylogeny in land plants.
the cdf sequences form  <dig> groups .
groups were defined as lineages originated prior to or at the time of land plant evolution , and group nomenclature was assigned based on annotated cdf sequences from a. thaliana.
nomenclature for genes with prior annotations were kept  <cit> .
at least one sequence from all six land plant genomes included in this study was maintained in each of the seven groups.
cdf members from algae c. reinhardii, c. merolae, o. lucimarinus, and o. tauri, are present within  <dig> of the  <dig> groups .
maintenance and in some cases expansion of these genes suggests that the cdf members from each group play important roles in plants.
group 1
group  <dig> originated prior to the evolution of the red alga c. merolae, and is maintained in diverse land plant genomes.
group i sequences are found in the both red and green algae, cmmtp <dig> and crmtp <dig>  respectively   <cit> .
the genomes of ostreococcus do not contain a sequence from group  <dig> indicating that this cdf member has been lost in these species.
both s. moellendorffii and p. patens genomes contain two group  <dig> sequences .
the p. patens duplication is predicted to have occurred after the mosses diverged from other vascular plant lineages .
however, placement of the two s. moellendorffii sequences supports an ancestral duplication event prior to that divergence  with subsequent propagation of one of the two genes.
while the branch support for this model was relatively weak, this topology was consistently supported by both bayesian and maximum likelihood methods of phylogenetic inference using multiple substitution models.
a second duplication event resulted in the formation of lineages containing mtp <dig> sequences and mtp1/2/ <dig> sequences .
this analysis supports the evidence that the origin of this duplication occurred prior to the monocot/eudicot divergence due to the presence of monocot and eudicot sequences within the mtp1/2/ <dig> clade.
the s. bicolor and o. sativa genomes lack mtp <dig> sequences, suggesting that the monocot lineage may have lost this gene.
a third duplication event occurring after the monocot/eudicot divergence produced a lineage containing mtp <dig> sequences and a lineage containing mtp1/ <dig> sequences .
more recent duplication events within p. trichocarpa and a. thaliana have generated numerous inparalogs reflecting the genome duplication events that occurred after the divergence of these plants.
the p. trichocarpa genome contains paralogs for all genes in group  <dig>  which could reflect the observation that the genome of p. trichocarpa is evolving at a six-time slower rate than that of the a. thaliana genome, and so might be expected to have a slower rate of loss of duplicated genes  <cit> .
therefore, group  <dig> paralogous genes in p. trichocarpa may be highly redundant.
crmtp <dig> contains multiple introns and the p. patens sequences, ppmtp <dig> and ppmtp <dig> , contain one and two introns, respectively .
the remaining sequences primarily contain only one 5' intron, with a few exceptions.
through searches of public databases, transcript support has been identified for each of the group  <dig> members, except for ptmtp <dig> and ppmtp <dig> .
missing transcript data from p. trichocarpa and p. patens may be due to incomplete transcript catalogues of these plants.
the transcriptional evidence suggests that the genes of group  <dig> are largely expressed in a variety of plants and algae, providing further evidence of this group's general importance in plants.
phylogenetic analysis indicates that the mtp1/ <dig> sequences and mtp <dig> sequences share a common ancestor some time after the monocot/eudicot divergence .
at the time of duplication, mtp1/ <dig> and mtp <dig> most likely shared identical redundant function in that ancestor.
the fate of the duplicated genes could take several different paths, including elimination, neofunctionalization, subfuctionalization, or even full/partial redundancy  <cit> .
the atmtp <dig> and atmtp <dig> dna sequences share  <dig> % sequence identity, and the proteins have similar predicted secondary structure with six transmembrane domains, cytoplasmically facing n-terminal and c-terminal ends, and a histidine-rich region  <cit> .
both proteins have been localized to the tonoplast membrane in yeast and plants, and both proteins have been shown to affect zn and possibly co tolerance and accumulation in yeast  <cit> .
however, the spatial, temporal, and responsive transcriptional regulation of each gene suggests that these proteins have different roles in plant zn homeostasis.
evidence from an a. thaliana relative, brassica juncea, suggests that bjmtp <dig> is expressed in secondary xylem parenchyma cells of the root while atmtp <dig> is expressed in root epidermal and cortical cells  <cit> .
also, atmtp <dig> and bjmtp <dig> transcription is not regulated by zn, while atmtp <dig> is activated by elevated zn influx  <cit> .
therefore, when mtp <dig> is expressed in conditions of high zn or low fe, accumulation of mtp <dig> and mtp <dig> could provide a continuous sequestration path in epidermal/cortical cell layers and xylem parenchyma cells limiting zn translocation to the shoot  <cit> .
spatial expression patterns of mtp <dig> and mtp <dig> are also different in vegetative and inflorescent shoot tissues  <cit> .
so, while the protein sequence, structure, location, and substrate are very similar, the expression patterns between atmtp <dig> and atmtp <dig> are unique.
therefore maintenance in the genome of the originally duplicated genes may be attributed to neofunctionalization/subfunctionalization via changes in expression patterns of the gene.
additionally, the genome of a. thaliana maintains a more recent  duplication event yielding sequences atmtp <dig> and atmtp <dig> .
comparing their gene expression metaprofiles across a database of microarrays suggests that they are not coexpressed   <cit> , which suggests that these paralogs are not redundant.
groups  <dig> and 9
this clade of the cdf superfamily tree contains cdf members from genomes of both red and green algae , and the presence of other prokaryote and eukaryote cdf sequences supports the ancient origin of this clade .
representing the viridiplantae sequences of this clade as rooted by the red algae sequence, cmmtp <dig>  indicates that a duplication event  within this branch of the cdf family occurred to produce two distinct groups, group  <dig> and group  <dig> .
the duplication event appears to have occurred prior to or coincident with early land plants due to the presence of p. patens and s. moellendorffii sequences in both groups.
the two monocot genomes each contain two group  <dig> sequences  and phylogenetic relationships between these sequences suggest that they are a product of a duplication event that occurred in a common ancestor of o. sativa and s. bicolor.
indeed, the mtp <dig> and mtp <dig>  sequences from rice and sorghum fall within syntenous blocks between their respective genomes, confirming duplication within the pre-grass ancestor  <cit> .
group  <dig> shows evidence of a duplication event prior to the moncot/eudicot split .
this duplication event produced two group  <dig> lineages in higher plants, both of which are maintained, and in some cases expanded, in representative genomes.
in the p. trichocarpa genome, groups  <dig> and  <dig> contain  <dig> cdf members .
amplification of these groups in p. trichocarpa is primarily the result of tandem gene replication of three of the group  <dig> sequences  and  <dig> of the group  <dig> sequences .
members of groups  <dig> and  <dig> have been functionally characterized as mn transporters.
the first member of these groups to be cloned was shmtp <dig>  from the mn hyperaccumulating legume, stylosanthes hamata.
the clone was identified from a screen for cdnas that enhanced mn tolerance in yeast  <cit> .
cdna sequences for three other group  <dig> and  <dig> members were also identified from the screen.
fluorescent tagging of shmtp <dig> suggested that the protein functions at the tonoplast where it was predicted to be involved in mn sequestration into the vacuole.
group  <dig> sequences, atmtp <dig>  ptmtp <dig> , and ptmtp <dig>  have also been characterized as mn transporters.
however, these proteins reside not within the vacuole, but within a punctate endomembrane compartment consistent with either trans-golgi or prevacuolar organelles  <cit> .
deletion of the atmtp <dig> gene product increased accumulation of mn in leaves of plants grown in vitro or hydroponically  <cit> .
deletion or reduction of atmtp <dig> transcripts makes the mutant plant sensitive to elevated mn, whereas ectopic over expression of atmtp <dig> increases resistance to elevated mn.
it is clear that cdfs from group  <dig> and  <dig> are important for mn homeostasis and the early bifurcation and subsequent expansion of these gene families implies an adaptively significant role for mn homeostasis in plants.
the intron-exon boundaries largely support the evolutionary relationships of these sequences.
in group  <dig>  two gene models, ptmtp <dig>  and ppmtp <dig> , do not conform to a seven-exon gene structure.
these loci have no associated ests, and when compared to their respective group  <dig> sequences, both loci have large truncations of 5' regions that eliminate large portions of the cation efflux domains.
this suggests that these loci are pseudogenes.
group  <dig> angiosperm sequences have very similar gene models .
the exon boundaries of the s. moellendorffii and p. patens sequence deviate slightly from those defined in the angiosperms, but a clear  <dig> exon pattern is evident for most group  <dig> sequences.
groups  <dig> and 12
group  <dig> and group  <dig> lineages derive from a common ancestor prior to the origin of land plants and within each lineage are sequences from prokaryotes and eukaryotes, thus showing that each group is of ancient origin .
unlike groups  <dig>   <dig>  and  <dig>  green algal sequences are absent from these groups.
however, a cdf sequence from red algae, cmmtp <dig>  which is distantly related to either group  <dig> or group  <dig>  falls within the larger grouping of these sequences in the superfamily tree , so this sequence was used as the root to estimate the phylogenetic relationships within these groups .
each group contains only one sequence from each of the included plant genomes, implying strict maintenance of a single gene copy within genomes, unlike in groups  <dig>   <dig>  and  <dig>  group  <dig> sequences are maintained as large single-exon genes, while the gene structure of group  <dig> sequences contain numerous intons with reasonably well maintained exon structure .
group  <dig> sequences are also relatively variable in size, and all but smmtp <dig> have associated cdna or est support.
only two members of group  <dig> are supported by ests, ptmtp <dig> and ppmtp <dig>  the sequences that make up group  <dig> are of note because the average sequence of these members is approximately twice the length of a typical cdf sequence.
the cation efflux domain starts at the center and extends toward the 3' end of the gene, while the 5' half of the gene  does not show clear homology to other genes or to known functional domains.
function evidence for the role of group  <dig> or group  <dig> genes in plants is limited.
the only functional data for these groups comes from the high throughput ionomic phenotyping database in which diverse plant accessions are screened for ionomic profiles  <cit> .
among the many mutant lines screened by this group was an ems induced mutation of atmtp <dig>  the ionomic profile of this mutant shows repeatable alterations in multiple ions in the mutant leaves including reduced levels of mo, mn, and mg and increased levels of k and zn.
these data suggest that atmtp <dig> has a role in regulating ion concentrations in a. thaliana under normal conditions.
groups  <dig> and 7
group  <dig> and group  <dig> plant sequences each belong to lineages that radiate from the base of the unrooted cdf superfamily tree and each lineage includes other cdf sequences from diverse organisms demonstrating that these groups are of ancient origin .
rooting group  <dig> with the branches leading to the p. patens sequences, ppmtp <dig> and ppmtp <dig> , and rooting group  <dig> with the branch leading to the ostreococcus sequences, olmtp <dig> and otmtp <dig>  produces the cladistic relationship among the sequences .
similar to groups  <dig> and  <dig>  plants have maintained only one copy of group  <dig> and  <dig> sequences in their genomes, and  <dig> of the  <dig> sequences from these groups are supported by ests or cdnas.
the genomes of the green and red algae representatives included in this analysis do not contain group  <dig> sequences, suggesting that the group  <dig> members in algae have been lost   <cit> .
the group  <dig> members are the only plant cdf sequences to fall into the zn/fe-cdf group, although no studies have been conducted on group  <dig> plant family members to confirm this substrate specificity  <cit> .
the only functional data for these groups comes from ionomic phenotyping  <cit> .
profiling of an a. thaliana line with a homozygous t-dna insertion into the coding region of atmtp <dig> shows consistent diverse alterations in the ionome with reduced levels of mg, mo, and ca and increased levels of na, k, mn, and cd.
the altered ion profile of the mtp <dig> mutant leaves suggests that group  <dig> sequences are required for the maintenance of the plant ionome under normal conditions.
the group  <dig> sequences were not placed into any of the three substrate-specific groups and no functional data are available for members of this group  <cit> .
CONCLUSIONS
studies in mammals, nematodes, yeast, bacteria, and plants suggest cdf proteins serve important roles in essential cation transport and homeostasis.
there is also evidence supporting other, more complex, roles in these organisms, such as involvement in oxidative stress resistance, interactions in signal transduction cascades, and proper functioning of the endoplasmic reticulum.
within plants only four members have been functionally characterized to any degree, and these studies show the importance of each member in essential cation accumulation, partitioning, and tolerance.
using phylogenomic analysis of complete cdf families from genomes of multiple, taxonomically diverse plants and algae, the plant cdf family is organized into seven primary groups that were present in ancestral genomes prior to or coincident with the origin of land plants.
within land plants, gene copy number expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome.
defining these cdf lineages contributes to the study of this family in four ways.
1) defining within group orthology/paralogy of particular genomes will help highlight potential redundant genes.
for example, the p. trichocarpa genome has six group  <dig> members, however these six sequences are actually three separate recent duplications of members in three different clades within group  <dig> .
this might predict that the protein products of the recently duplicated genes  may have redundant function, but the inparalogs  might not be redundant, but rather are subfunctionalized members similar to atmtp <dig> and atmtp <dig> .
2) defining the primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation when embarking upon functional studies of the members.
for example, plant sequences from groups  <dig>   <dig>   <dig>   <dig>  and  <dig> are likely monophyletic lineages derived within ancestral prokaryotes and largely maintained in extant organisms.
this suggests that comparisons with bacterial, archaeal, fungal, and mammalian homologues may be useful.
conversely, group  <dig> and  <dig> lineages most likely result from a duplication of an ancestral viridiplantae sequence.
therefore, sequences within at least one of these groups might have an altered functional role in plants as compared with the function of coorthologs in other organisms.
3) defining groups will allow for a group-specific sequence motif to be generated that will help define future cdf family sequences and aid in functional motif and critical residue identification in plants.
a cdf family signature sequence was defined that identifies cdf family members with only a 5% false identification rate, but this sequence is quite elaborate  <cit> .
the necessarily complex signature sequence may reflect the constraints inherent in encompassing all cdf family members and includes all variations within a diverse set of organisms.
by focusing specifically on plant cdf members, the sequence variability due to host genome diversity will be reduced leading to more accurate identification of group-specific sequence motifs and critical residues important in plant cdf proteins.
4) the plant-specific expansion resulting in groups  <dig> and  <dig> evolved prior to or coincident with the early primary radiation of plants onto land.
the primary siluro-devonian radiation of terrestrial plants necessitated development of physiological mechanisms that would allow pioneering plants to take advantage of new ecological niches on land.
in terms of the cdf family, the expansion from five to seven primary groups prior to or coincident with the divergence of bryophytes from the vascular plant lineage suggests the cdf family expansion provided an adaptive advantage before significant vascular development occurred in early land plants.

 members of the cation diffusion facilitator family have been shown to be important for maintenance of cation homeostasis in bacteria, yeast, plants, and mammals. cdf proteins, in general, bind to and efflux such cations as zn from the cytoplasm through sequestration into internal compartments or through efflux from the cell. this role in modulating cellular cation concentrations has been demonstrated to impact cation accumulation, cation tolerance, signal transduction cascades, oxidative stress resistance, and protein turnover. several research groups have analyzed the phylogenetic relationships of cdfs and found that this is an ancient gene family that pre-dates the origin of eukaryotes, as reflected in the grouping of sequences from diverse organisms within several branches of constructed phylogenetic trees. plant cdf members, including members from the sequenced genome of arabidopsis thaliana have been grouped into three or four lineages. however, these analyses were limited by sequence availability due to the lack of sequence genomes and available cdna libraries, which resulted in incomplete or weakly supported hypotheses about cdf family phylogeny within plants. montanini, et al. conducted global phylogenetic analysis on cdfs from prokaryotes, eukaryotes, and archaea. based on a maximum parsimony analysis, variation across the gene family could be partitioned into three major groups, designated zn-cdfs, zn/fe-cdfs, and mn-cdfs based on the hypothesized or confirmed transported substrate of one or more group members. for example, the mn-cdf group containes sequences and, within this group, the plant members mtp and mtp have been characterized as mn transporters. using vastly expanded sequence information and substrate-defined groups, an updated cdf signature sequence was derived as well as group-specific signature sequences. the conserved residues comprising these signature sequences were the target of amino acid substitution, many of which were found to be critical residues for a fully functional protein. recently, migeon et al. expanded this analysis by incorporating cdf sequences from additional plant genomes with emphasis on phylogenetic and molecular characterization of metal transporters in populus trichocarpa. this analysis confirmed partitioning the sequences into three major functional groups. grouping the sequences by predicted substrate specificity provides a useful hypothesis-generation tool for uncharacterized proteins within these broad groupings. however, higher resolution analysis of plant-specific cdf sequences is likely to reveal informative relationships within the linage of land plants. with the generation of full genome sequences for multiple eukaryotic organisms, a wealth of information is available from which to generate detailed phylogenomic relationships of gene families within and between organisms. as genome sequences become available for more species, this "genomic" method of phylogenetic analysis should enable robust estimation of orthology and paralogy among related genes. this high level resolution of familial evolution provides a powerful analytical tool from which to synthesize hypothesis about, among other things, the function of gene family members. the precision in functionally annotating an uncharacterized sequence based on sequence similarity to a characterized protein should increase if a detailed estimation of family phylogeny is known. once a sufficiently detailed map of the gene family structure and evolution are constructed, a more global understanding of the adaptive significance of the family dynamics through the course of evolution may become clearer and lead to testable hypotheses about the roles members play in organismal evolution. genome sequencing of a red alga, cyanidioschyzon merolae, green algae, ostreococcus tauri, ostreococcus lucimarinus, and clammydomonas reinhardii, basal nonvascular and vascular land plants, physcomitrella patens and selaginella moellendorffii, and representatives of angiosperm lineages have been completed. c. merolae is a non-motile unicellular red alga that lives in extreme environmental conditions, such as sulfate-rich hot springs and is estimated to have diverged from the lineage leading to true plant approximately billion years ago. ostreococcus species are the smallest known eukaryotic organisms and belong to the prasinophyceae, an early diverging class in the lineage of the green algae. the algal model, c. reinhardii, is estimated to have shared a common ancestor with such species as a. thaliana billion years ago. p. patens and s. moellendorffii represent early land plant lineages of bryopsida and lycopsid, respectfully, which are estimated to have diverged from seed-bearing plants approximately million years ago and mya, respectively. within the more recent lineages of flowering plants, several genomes have been sequenced, including the monocotyledonous genomes of oryza sativa and sorghum bicolor, and the eudicotyledonous genomes of a. thaliana, p. trichocarpa, and medicago truncatula. the monocot lineage is predicted to have diverged from other angiosperms approximately mya, and within eudicots, the a. thaliana and p. trichocarpa lineages are predicted to have diverged in the eurosid clade approximately mya. collectively, the genomes of the six land plants contain information that allow for comparison of genome evolution throughout the approximately million year history of land plants and inclusion of the genomes of red and green algae enables extension to billion years of plant evolution. in this study we conduct a detailed phylogenic analysis of plant cdf family members to lay out a framework from which more informed hypotheses can be generated regarding the function of cdf proteins in plants.  plant cdf family member sequences scanning the genomes of the taxonomically diverse set of organisms outlined in the introduction for cdf sequences identified or confirmed the following number of sequences: o. lucimarinus, o. tauri, c. merolae, c. reinhardii, p. patens, s. moellendorffii, o. sativa, s. bicolor, p. trichocarpa, and a. thaliana. the number of cdf sequences identified from c. reinhardii, c. merolae, s. moellendorffii, p. patens, s. bicolor, and a. thaliana, genomes agree with previous published studies, however, the gene models may not be the same. the number of p. trichocarpa cdfs was expanded to from the previous estimate of. the expanded set includes a predicted pseudogene ptmtp and previously unidentified ptmtp. the number of cdf sequences in the o. sativa genome was expanded from to due to the inclusion of previously unidentified members osmtp and osmtp plant cdf family structure phylogenetic analysis of the cdf superfamily, including genomes from archaea, bacteria, protozoa, fungi, red alga, green algae, land plants, nematode, and mammal can be grouped into three primary clades, as indicated by the colored branches in figure these three primary clades are consistent with the previously defined zn-cdf, fe/zn-cdf, and mn-cdf groups based on functional evidence of resident members, with one exception. while previous analysis of the branch containing hsznt and atmtp had this branch of sequences as an orphaned, ungrouped branch, in this analysis it is included with the fe/zn-cdf group, therefore, this analysis suggests that these sequences be included into the fe/zn-cdf group. cdf family members from viridiplantae and rhodophyta genomes were used to estimate the cdf family phylogeny in land plants. the cdf sequences form groups. groups were defined as lineages originated prior to or at the time of land plant evolution, and group nomenclature was assigned based on annotated cdf sequences from a. thaliana. nomenclature for genes with prior annotations were kept. at least one sequence from all six land plant genomes included in this study was maintained in each of the seven groups. cdf members from algae c. reinhardii, c. merolae, o. lucimarinus, and o. tauri, are present within of the groups. maintenance and in some cases expansion of these genes suggests that the cdf members from each group play important roles in plants. group 1 group originated prior to the evolution of the red alga c. merolae, and is maintained in diverse land plant genomes. group i sequences are found in the both red and green algae, cmmtp and crmtp respectively. the genomes of ostreococcus do not contain a sequence from group indicating that this cdf member has been lost in these species. both s. moellendorffii and p. patens genomes contain two group sequences. the p. patens duplication is predicted to have occurred after the mosses diverged from other vascular plant lineages. however, placement of the two s. moellendorffii sequences supports an ancestral duplication event prior to that divergence with subsequent propagation of one of the two genes. while the branch support for this model was relatively weak, this topology was consistently supported by both bayesian and maximum likelihood methods of phylogenetic inference using multiple substitution models. a second duplication event resulted in the formation of lineages containing mtp sequences and mtp1/2/ sequences. this analysis supports the evidence that the origin of this duplication occurred prior to the monocot/eudicot divergence due to the presence of monocot and eudicot sequences within the mtp1/2/ clade. the s. bicolor and o. sativa genomes lack mtp sequences, suggesting that the monocot lineage may have lost this gene. a third duplication event occurring after the monocot/eudicot divergence produced a lineage containing mtp sequences and a lineage containing mtp1/ sequences. more recent duplication events within p. trichocarpa and a. thaliana have generated numerous inparalogs reflecting the genome duplication events that occurred after the divergence of these plants. the p. trichocarpa genome contains paralogs for all genes in group which could reflect the observation that the genome of p. trichocarpa is evolving at a six-time slower rate than that of the a. thaliana genome, and so might be expected to have a slower rate of loss of duplicated genes. therefore, group paralogous genes in p. trichocarpa may be highly redundant. crmtp contains multiple introns and the p. patens sequences, ppmtp and ppmtp, contain one and two introns, respectively. the remaining sequences primarily contain only one 5' intron, with a few exceptions. through searches of public databases, transcript support has been identified for each of the group members, except for ptmtp and ppmtp. missing transcript data from p. trichocarpa and p. patens may be due to incomplete transcript catalogues of these plants. the transcriptional evidence suggests that the genes of group are largely expressed in a variety of plants and algae, providing further evidence of this group's general importance in plants. phylogenetic analysis indicates that the mtp1/ sequences and mtp sequences share a common ancestor some time after the monocot/eudicot divergence. at the time of duplication, mtp1/ and mtp most likely shared identical redundant function in that ancestor. the fate of the duplicated genes could take several different paths, including elimination, neofunctionalization, subfuctionalization, or even full/partial redundancy. the atmtp and atmtp dna sequences share % sequence identity, and the proteins have similar predicted secondary structure with six transmembrane domains, cytoplasmically facing n-terminal and c-terminal ends, and a histidine-rich region. both proteins have been localized to the tonoplast membrane in yeast and plants, and both proteins have been shown to affect zn and possibly co tolerance and accumulation in yeast. however, the spatial, temporal, and responsive transcriptional regulation of each gene suggests that these proteins have different roles in plant zn homeostasis. evidence from an a. thaliana relative, brassica juncea, suggests that bjmtp is expressed in secondary xylem parenchyma cells of the root while atmtp is expressed in root epidermal and cortical cells. also, atmtp and bjmtp transcription is not regulated by zn, while atmtp is activated by elevated zn influx. therefore, when mtp is expressed in conditions of high zn or low fe, accumulation of mtp and mtp could provide a continuous sequestration path in epidermal/cortical cell layers and xylem parenchyma cells limiting zn translocation to the shoot. spatial expression patterns of mtp and mtp are also different in vegetative and inflorescent shoot tissues. so, while the protein sequence, structure, location, and substrate are very similar, the expression patterns between atmtp and atmtp are unique. therefore maintenance in the genome of the originally duplicated genes may be attributed to neofunctionalization/subfunctionalization via changes in expression patterns of the gene. additionally, the genome of a. thaliana maintains a more recent duplication event yielding sequences atmtp and atmtp. comparing their gene expression metaprofiles across a database of microarrays suggests that they are not coexpressed, which suggests that these paralogs are not redundant. groups and 9 this clade of the cdf superfamily tree contains cdf members from genomes of both red and green algae, and the presence of other prokaryote and eukaryote cdf sequences supports the ancient origin of this clade. representing the viridiplantae sequences of this clade as rooted by the red algae sequence, cmmtp indicates that a duplication event within this branch of the cdf family occurred to produce two distinct groups, group and group. the duplication event appears to have occurred prior to or coincident with early land plants due to the presence of p. patens and s. moellendorffii sequences in both groups. the two monocot genomes each contain two group sequences and phylogenetic relationships between these sequences suggest that they are a product of a duplication event that occurred in a common ancestor of o. sativa and s. bicolor. indeed, the mtp and mtp sequences from rice and sorghum fall within syntenous blocks between their respective genomes, confirming duplication within the pre-grass ancestor. group shows evidence of a duplication event prior to the moncot/eudicot split. this duplication event produced two group lineages in higher plants, both of which are maintained, and in some cases expanded, in representative genomes. in the p. trichocarpa genome, groups and contain cdf members. amplification of these groups in p. trichocarpa is primarily the result of tandem gene replication of three of the group sequences and of the group sequences. members of groups and have been functionally characterized as mn transporters. the first member of these groups to be cloned was shmtp from the mn hyperaccumulating legume, stylosanthes hamata. the clone was identified from a screen for cdnas that enhanced mn tolerance in yeast. cdna sequences for three other group and members were also identified from the screen. fluorescent tagging of shmtp suggested that the protein functions at the tonoplast where it was predicted to be involved in mn sequestration into the vacuole. group sequences, atmtp ptmtp, and ptmtp have also been characterized as mn transporters. however, these proteins reside not within the vacuole, but within a punctate endomembrane compartment consistent with either trans-golgi or prevacuolar organelles. deletion of the atmtp gene product increased accumulation of mn in leaves of plants grown in vitro or hydroponically. deletion or reduction of atmtp transcripts makes the mutant plant sensitive to elevated mn, whereas ectopic over expression of atmtp increases resistance to elevated mn. it is clear that cdfs from group and are important for mn homeostasis and the early bifurcation and subsequent expansion of these gene families implies an adaptively significant role for mn homeostasis in plants. the intron-exon boundaries largely support the evolutionary relationships of these sequences. in group two gene models, ptmtp and ppmtp, do not conform to a seven-exon gene structure. these loci have no associated ests, and when compared to their respective group sequences, both loci have large truncations of 5' regions that eliminate large portions of the cation efflux domains. this suggests that these loci are pseudogenes. group angiosperm sequences have very similar gene models. the exon boundaries of the s. moellendorffii and p. patens sequence deviate slightly from those defined in the angiosperms, but a clear exon pattern is evident for most group sequences. groups and 12 group and group lineages derive from a common ancestor prior to the origin of land plants and within each lineage are sequences from prokaryotes and eukaryotes, thus showing that each group is of ancient origin. unlike groups and green algal sequences are absent from these groups. however, a cdf sequence from red algae, cmmtp which is distantly related to either group or group falls within the larger grouping of these sequences in the superfamily tree, so this sequence was used as the root to estimate the phylogenetic relationships within these groups. each group contains only one sequence from each of the included plant genomes, implying strict maintenance of a single gene copy within genomes, unlike in groups and group sequences are maintained as large single-exon genes, while the gene structure of group sequences contain numerous intons with reasonably well maintained exon structure. group sequences are also relatively variable in size, and all but smmtp have associated cdna or est support. only two members of group are supported by ests, ptmtp and ppmtp the sequences that make up group are of note because the average sequence of these members is approximately twice the length of a typical cdf sequence. the cation efflux domain starts at the center and extends toward the 3' end of the gene, while the 5' half of the gene does not show clear homology to other genes or to known functional domains. function evidence for the role of group or group genes in plants is limited. the only functional data for these groups comes from the high throughput ionomic phenotyping database in which diverse plant accessions are screened for ionomic profiles. among the many mutant lines screened by this group was an ems induced mutation of atmtp the ionomic profile of this mutant shows repeatable alterations in multiple ions in the mutant leaves including reduced levels of mo, mn, and mg and increased levels of k and zn. these data suggest that atmtp has a role in regulating ion concentrations in a. thaliana under normal conditions. groups and 7 group and group plant sequences each belong to lineages that radiate from the base of the unrooted cdf superfamily tree and each lineage includes other cdf sequences from diverse organisms demonstrating that these groups are of ancient origin. rooting group with the branches leading to the p. patens sequences, ppmtp and ppmtp, and rooting group with the branch leading to the ostreococcus sequences, olmtp and otmtp produces the cladistic relationship among the sequences. similar to groups and plants have maintained only one copy of group and sequences in their genomes, and of the sequences from these groups are supported by ests or cdnas. the genomes of the green and red algae representatives included in this analysis do not contain group sequences, suggesting that the group members in algae have been lost. the group members are the only plant cdf sequences to fall into the zn/fe-cdf group, although no studies have been conducted on group plant family members to confirm this substrate specificity. the only functional data for these groups comes from ionomic phenotyping. profiling of an a. thaliana line with a homozygous t-dna insertion into the coding region of atmtp shows consistent diverse alterations in the ionome with reduced levels of mg, mo, and ca and increased levels of na, k, mn, and cd. the altered ion profile of the mtp mutant leaves suggests that group sequences are required for the maintenance of the plant ionome under normal conditions. the group sequences were not placed into any of the three substrate-specific groups and no functional data are available for members of this group.  studies in mammals, nematodes, yeast, bacteria, and plants suggest cdf proteins serve important roles in essential cation transport and homeostasis. there is also evidence supporting other, more complex, roles in these organisms, such as involvement in oxidative stress resistance, interactions in signal transduction cascades, and proper functioning of the endoplasmic reticulum. within plants only four members have been functionally characterized to any degree, and these studies show the importance of each member in essential cation accumulation, partitioning, and tolerance. using phylogenomic analysis of complete cdf families from genomes of multiple, taxonomically diverse plants and algae, the plant cdf family is organized into seven primary groups that were present in ancestral genomes prior to or coincident with the origin of land plants. within land plants, gene copy number expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome. defining these cdf lineages contributes to the study of this family in four ways. 1) defining within group orthology/paralogy of particular genomes will help highlight potential redundant genes. for example, the p. trichocarpa genome has six group members, however these six sequences are actually three separate recent duplications of members in three different clades within group. this might predict that the protein products of the recently duplicated genes may have redundant function, but the inparalogs might not be redundant, but rather are subfunctionalized members similar to atmtp and atmtp. 2) defining the primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation when embarking upon functional studies of the members. for example, plant sequences from groups and are likely monophyletic lineages derived within ancestral prokaryotes and largely maintained in extant organisms. this suggests that comparisons with bacterial, archaeal, fungal, and mammalian homologues may be useful. conversely, group and lineages most likely result from a duplication of an ancestral viridiplantae sequence. therefore, sequences within at least one of these groups might have an altered functional role in plants as compared with the function of coorthologs in other organisms. 3) defining groups will allow for a group-specific sequence motif to be generated that will help define future cdf family sequences and aid in functional motif and critical residue identification in plants. a cdf family signature sequence was defined that identifies cdf family members with only a 5% false identification rate, but this sequence is quite elaborate. the necessarily complex signature sequence may reflect the constraints inherent in encompassing all cdf family members and includes all variations within a diverse set of organisms. by focusing specifically on plant cdf members, the sequence variability due to host genome diversity will be reduced leading to more accurate identification of group-specific sequence motifs and critical residues important in plant cdf proteins. 4) the plant-specific expansion resulting in groups and evolved prior to or coincident with the early primary radiation of plants onto land. the primary siluro-devonian radiation of terrestrial plants necessitated development of physiological mechanisms that would allow pioneering plants to take advantage of new ecological niches on land. in terms of the cdf family, the expansion from five to seven primary groups prior to or coincident with the divergence of bryophytes from the vascular plant lineage suggests the cdf family expansion provided an adaptive advantage before significant vascular development occurred in early land plants. 
first, when embarking upon functional studies of the members, defining primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation. members of the cation diffusion facilitator family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. within land plants, local expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome. third, the plant-specific expansion resulting in groups and evolved coincident to the early primary radiation of plants onto land, suggesting these families may have been important for early land colonization. second, defining groups will allow a group-specific sequence motif to be generated that will help define future cdf family sequences and aid in functional motif identification, which currently is lacking for this family in plants. among these, of the groups are likely to have originated at the base of the tree of life, and of groups appear to be derived from a duplication event prior to or coincident with land plant evolution. members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. the spectrum of cations known to be transported by proteins of the cdf family include zn, fe, co, cd, and mn. phylogenetic analysis suggests that the cdf family has expanded within plants, but a definitive plant cdf family phylogeny has not been constructed. representative cdf members were annotated from diverse genomes across the viridiplantae and rhodophyta lineages and used to identify phylogenetic relationships within the cdf family. bayesian phylogenetic analysis of cdf amino acid sequence data supports organizing land plant cdf family sequences into groups. cdf families range in size from nine members in selaginella moellendorffii to members in populus trichocarpa. the origin of the groups predates the emergence of land plants.
 members of the cation diffusion facilitator family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. the spectrum of cations known to be transported by proteins of the cdf family include zn, fe, co, cd, and mn. members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. cdf families range in size from nine members in selaginella moellendorffii to members in populus trichocarpa. phylogenetic analysis suggests that the cdf family has expanded within plants, but a definitive plant cdf family phylogeny has not been constructed.  representative cdf members were annotated from diverse genomes across the viridiplantae and rhodophyta lineages and used to identify phylogenetic relationships within the cdf family. bayesian phylogenetic analysis of cdf amino acid sequence data supports organizing land plant cdf family sequences into groups. the origin of the groups predates the emergence of land plants. among these, of the groups are likely to have originated at the base of the tree of life, and of groups appear to be derived from a duplication event prior to or coincident with land plant evolution. within land plants, local expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome.  defining the cdf gene family phylogeny contributes to our understanding of this family in several ways. first, when embarking upon functional studies of the members, defining primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation. second, defining groups will allow a group-specific sequence motif to be generated that will help define future cdf family sequences and aid in functional motif identification, which currently is lacking for this family in plants. third, the plant-specific expansion resulting in groups and evolved coincident to the early primary radiation of plants onto land, suggesting these families may have been important for early land colonization. 