BACKGROUND
the environmental stress response in all organisms as diverse as pro- and eukaryotes is generally coupled with a remarkable change in gene expression patterns and an enhanced synthesis of several ‘stress proteins’  <cit> .
because they were first described in drosophila melanogaster larvae that were accidentally exposed to elevated temperatures  <cit> , these stress-related proteins were called heat-shock proteins .
extensive research on hsps revealed also a constitutive expression of some members of these proteins, suggesting that they are also essential in maintaining the cellular functions under normal physiological conditions.
these members are therefore designated as heat-shock cognate proteins   <cit> .
furthermore, hsps respond not only to increased temperatures, but also chemicals, heavy metals, uv light, hypoxia and other stressors can induce their synthesis  <cit> .
some of the most important and well investigated hsps are the members of the  <dig> kda heat-shock protein family .
they belong to the highest conserved proteins and are present in almost all species, except for some archaea.
prokaryotic hsp <dig>  proteins share about 50% amino acid identity with eukaryotic hsp70s.
all known hsp <dig> proteins exhibit highly conserved amino acid sequences and domain structures, such as a conserved n-terminal atpase domain, a region with protease sensitive sites and a peptide binding domain at the c-terminal region .
due to this high conservation, hsp <dig> has been widely used as a suitable phylogenetic marker in molecular evolution.
it has been applied to deep phylogenetic relationships  or to confirm the monophyly of metazoa  <cit> , as well as relationships between archaea and gram-positive bacteria or gram-negative bacteria and eukaryotes  <cit> .
hsp <dig> genes and proteins have also been used for phylogenetic studies of different protozoan parasites such as trypanosoma or leishmania <cit> , as well as of non-parasitic protozoans such as euplotes or paramecium <cit> .
in eukaryotes, several of these hsp <dig> proteins that belong to four subfamilies are encoded by the nuclear genome .
the discrimination of the four hsp <dig> subfamilies corresponds to the intracellular localisation of the hsp <dig> proteins in the major compartments of the cell: the cytosol, the endoplasmic reticulum  and the organelles mitochondria and chloroplasts  <cit> .
these multigene family members emerged from numerous duplication events or replicative transpositions and evolved by a combination of birth-and-death processes, gene conversion events and purifying selection .
therefore, caution is called for when using hsp70s as phylogenetic marker, because paralogy can distort phylogenetic relationships  <cit> .
while the evolution of all hsp <dig> family members is not yet completely understood, the pathways for the evolution of the nucleus-encoded hsp70s of the cell organelles are well known.
the mitochondrial and the chloroplast homologs are derivatives from an endosymbiotic alphaproteobacterium and cyanobacterium, respectively, followed by a subsequent horizontal gene transfer of the hsp <dig> homologs to the nucleus.
further, the evolutionary relationship between the er- and cytosol-type hsp70s indicates that these genes have arisen very early in the common eukaryotic ancestor by gene duplication  <cit> .
all members of the hsp <dig> family carry out molecular chaperone functions that facilitate correct protein folding, as well as membrane translocation and the subsequent refolding of proteins  <cit> .
through the course of evolution, these multiple hsp <dig> family members, which can be found in pro- and eukaryotes, have acquired different chaperone tasks and functions .
for example, in all multicellular eukaryotes studied so far, some cytosol-type members show a constitutive expression under normal physiological conditions  while others are highly inducible  in consequence of several stress conditions .
detailed knowledge about the number, diversity and differential gene expression of the hsp <dig> family members in most unicellular eukaryotes is, however, scarce.
for example, the yeast genome of saccharomyces cerevisiae encodes fourteen hsp70-like genes  <cit>  comprising cytosol-type hsp70s that show constitutive expression  as well as heat-inducibility .
further, the genome sequencing projects of the ciliate species and model eukaryotes tetrahymena thermophila <cit>  and paramecium tetraurelia <cit>  unveiled several putative hsp <dig> genes with conserved hsp <dig> domains.
in paramecium, whole genome duplications and subsequent gene loss events have shaped the hsp <dig> gene family  <cit> , but it is still uncertain whether subfunctionalization or gene dosage constraints are the main forces for hsp <dig> duplicate retention.
while the major constitutively expressed and heat-inducible cytosol-type hsp70s have been identified for t. thermophila <cit> , the differential expression pattern of the hsp <dig> family in p. tetraurelia has remained unclear.
additionally, the evolutionary relationships of the hsp <dig> homologs from these closely related microbial eukaryotes are vague.
therefore, we have investigated the hsp <dig> multigene family of another paramecia species, paramecium caudatum, by sequencing an hsp <dig> cdna clone library.
based on the resultant sequence data, we performed phylogenetic and comparative sequence analyses to gain insight into the evolution of the hsp <dig> gene family in paramecium.
to disentangle the evolutionary relationships among ciliate hsp70s, which are in certain cases affected by whole genome duplications, we included not only a comprehensive set of homologous hsp70s of different pro- and eukaryotes, but also data from recent ciliate genome projects: tetrahymena, ichthyophthirius and oxytricha.
we further developed a target specific rt-qpcr assay for p. caudatum hsp70s in order to demonstrate an expected differential gene expression among the family members and to distinguish constitutively expressed and heat-induced hsp70s.
using these data, we tested for a hypothesised functional conservation of paramecium hsp <dig> orthologs via comparative est analyses and investigated the diversification of eukaryotic hsp70s to clarify the evolution of heat-inducibility within the  <dig> kda molecular chaperone family.
RESULTS
characterisation of p. caudatum hsp70s
sequencing of sixty putative hsp <dig> gene fragments, amplified from cdna with degenerated primers, revealed eleven different hsp <dig> nucleotide sequences  from the hsp70/dnak-family of paramecium caudatum.
when the majority of the obtained sequences became redundant, we stopped sequencing of further clones of our hsp <dig> cdna clone library.
therefore, we cannot exclude the possibility of additional homologs expressed in p. caudatum, but a sequence homology search  clearly assigned the eleven homologs to the three major hsp70-subfamilies: cytosol , endoplasmic reticulum  and mitochondria .
these analyses further revealed the expression of one mt, five cy and five er related hsp <dig> proteins in p. caudatum.
the sequences were designated according to their origin and sequence similarity as follows: pchsp70cy1a, pchsp70cy1b, pchsp70cy1c, pchsp70cy2a, pchsp70cy2b, pchsp70er1a, pchsp70er1b, pchsp70er2a, pchsp70er2b, pchsp70er2c, pchsp70mt1a.
the homologs pchsp70cy1a and pchsp70cy1b differed at only  <dig> nucleotide positions, and encode the same amino acid sequence.
the homologs pchsp70er1a and pchsp70er1b differed by only one ‘c’ to ‘t’ substitution, resulting also in the same amino acid sequence.
pchsp70er2a, pchsp70er2b and pchsp70er2c showed  <dig> to  <dig> nucleotide substitutions, but were indiscriminative at the amino acid level too .
because we used a proofreading polymerase for pcr amplification and a high fidelity rna-dependent dna polymerase for cdna synthesis, these nucleotide substitutions can be considered authentic.
the analyses of the deduced amino acid sequences revealed seven different homologous sequences, with an averaged pairwise distance of  <dig>  and a maximum pairwise distance of  <dig> .
the amplified fragments for all cy homologs as well as pchsp70er1a and pchsp70er1b encode  <dig> amino acids ; pchsp70er2a, pchsp70er2b and pchsp70er2c encode  <dig> aa and pchsp70mt1a encodes  <dig> aa.
all identified sequences shared two amino acid signature motifs , which are highly conserved regions and present in all eukaryotic hsp <dig> family members .
the consensus sequences are as follows: hsp <dig> family signature  <dig> --d-l-g-t-f-d--s--l and hsp <dig> family signature  <dig> --l-v-g---r--p-k---, where the square brackets show a list of acceptable amino acids at the given position and the numerical values between parentheses indicate repetition of the respective amino acid  <cit> .
another conserved signature, the putative atp/gtp-binding site motif a , could be identified in all eleven homologs, albeit with relatively high variation .
as expected, the putative bipartite nuclear localization signal, a targeting sequence essential for the selective translocation of nucleo-/cytoplasmatic proteins into the nucleus, could be detected for the cy homologs only  <cit> .
its consensus sequence is -k-x-l-r-l-r, where the symbol ‘x’ indicates a position of any acceptable amino acid .
additionally, all cy and er homologs possess the r-a--f-e-e-l consensus motif that serves as a potential signature for eukaryotic non-organellar hsp <dig> proteins  <cit> .
evolutionary relationships
as illustrated in the phylogenetic hsp <dig> tree in figure  <dig> showing the three major hsp70-subfamilies  among different eukaryotes, the ascertained p. caudatum sequences integrated well within the separate subfamilies with high support values.
in addition, all ciliate sequences clustered into clades in which the paramecium homologs formed monophyla.
these analyses also revealed the existence of different hsp70-groups within the different p. caudatum hsp <dig> subfamilies.
here, two distinct hsp70-groups within the cytosolic as well as the er-type subfamily could be detected.
each of these p. caudatum hsp70-groups from the same subfamily  showed a closer relationship to orthologous hsp <dig> sequences of paramecium tetraurelia than to another p. caudatum hsp70-group.
the group cy-a consisted of the three in-paralogs pchsp70cy1a, pchsp70cy1b and pchsp70cy1c; the group cy-b contained pchsp70cy2a and pchsp70cy2b; the group er-a comprised the genes pchsp70er1a and pchsp70er1b; and group er-b included pchsp70er2a, pchsp70er2b and pchsp70er2c.
the mitochondrial homolog pchsp70mt1a was determined as group mt for further analyses, even though we could detect only one mitochondrial hsp <dig> gene sequence.
since we cannot confirm complete locus homozygosity of the clonal p. caudatum strain that we used in this study, the detected within-group paralogs  might constitute alleles.
however, the amitotic division of the ciliate macronucleus  during vegetative growth can lead to an unequal partitioning of the alleles to daughter macs  <cit> , resulting in the expression of only one of the two alleles.
in conjunction with the detected low mutation rate in paramecium <cit>  and the comparatively high sequence divergence of the cy-b in-paralogs , we would argue in this case for in-paralogs rather than alleles.
the detection of eleven putative hsp <dig> homologs in p. caudatum is in concordance with former studies on the hsp <dig> multigene family in other ciliate species .
the p. tetraurelia genome project  <cit> , for example, revealed fifteen putative hsp <dig> sequences in mac dna, while only thirteen possess the typical conserved domain structures with an n-terminal atpase and a c-terminal peptide-binding domain.
however, our motif analyses revealed that only ten homologs exhibit all three hsp <dig> family signatures, indicating the existence of potential hsp <dig> pseudogenes in the p. tetraurelia mac genome.
in addition, genome analyses for tetrahymena thermophila revealed thirteen putative hsp <dig> genes featuring conserved hsp <dig> domains  <cit> .
similar to p. tetraurelia, only seven hsp <dig> homologs comprise the three hsp <dig> family signatures.
interestingly, while we could find all orthologous sequences of these seven t. thermophila hsp70s in its close relative t. malaccensis by utilizing the tetrahymena comparative database, we could not detect the heat-inducible ortholog of hsp70- <dig> in t. borealis .
furthermore, in ichthyophthirius multifiliis and oxytricha trifallax we could detect only one cy- and one er-type hsp <dig> homolog comprising all three hsp <dig> family signatures.
modes of evolution
the five putative hsp70-groups detected in paramecium showed consistent phylogenetic patterns according to the assumed evolutionary relationship between the species , thereby supporting the model of divergent evolution in the paramecium hsp <dig> gene family.
additionally, a codon-based z-test  <cit>  on all p. caudatum hsp70s revealed evidence for purifying selection, which is indicated by higher numbers of synonymous  to non-synonymous  nucleotide differences per site in pairwise comparisons .
while the paralogs between the two hsp70-groups per subfamily  show a comparatively high amino acid divergence of up to 19%, the potential paralogs within each group  show a rather low nucleotide diversity .
this high similarity of the in-paralogs could be indicative of very recent duplication events or might be explained by purifying selection  or gene conversion .
the results of a codon-based z-test for neutral evolution revealed that the null hypothesis of strict-neutrality  could not be rejected for the cy-b and er-type in-paralogs , while the sequence similarity among the cy-a in-paralogs seems to be caused by purifying selection .
a test for gene conversion events revealed significant evidence  for only two very short regions  between out-paralogs, which might in fact be misidentifications due to possible misalignment artefacts.
please note that the substitutions within the er and cy-a groups are very few and therefore do not allow for definite conclusions of the within-group comparisons; on the other hand, the discrepancy between the cy-a and cy-b group is obvious and implies different modes of evolution.
the number of synonymous differences per synonymous site   and of nonsynonymous differences per nonsynonymous site   for comparisons of eleven p. caudatum hsp <dig> homologs are shown.
analyses were conducted using the nei-gojobori model included in mega <dig>  all ambiguous positions were removed for each sequence pair with a total of  <dig> amino acid positions in the final dataset.
differential gene expression
the group classification with cy-a, cy-b, er-a, er-b and mt was used for the expression analyses, since rt-qpcr primers and mgb™-probes were specifically designed to amplify or bind the respective hsp <dig> sequences within one group.
the stability analysis of the reference genes gapdh and ef-1α using the genorm v. <dig>  applet  <cit>  revealed a high stability of both genes.
the averaged expression stability value was  <dig>  and the pairwise variation value  was below the proposed cut-off value of  <dig> .
we therefore used these two genes to normalise the hsp <dig> mrna levels in all further analyses.
as illustrated in figure 3a, the cy-a relative expression level  at optimum temperature  was the highest among all five hsp70-groups.
while the basal expression levels of the er-a, er-b and mt group were comparatively similar , the cy-b levels instead were considerably lower , indicating that these genes  were transcribed at low basal levels under normal physiological conditions.
after heat-shock exposure for two hours at 34°c, we could detect changes of the hsp <dig> transcript levels .
while the reference genes gapdh and ef-1α showed comparatively stable expression levels between 28°c  and 34°c , the heat shock provoked an induction of all different hsp70-groups.
after heat shock, the relative transcription level of cy-a  was higher than that of gapdh  and still showing the highest hsp <dig> mrna expression level.
the cy-b group, however, was highly induced , resulting in the second most expressed hsp70-group.
performing relative expression analyses using rest  <dig> unveiled a significant up-regulation of all hsp70-groups .
while the transcription level of the cy-a, er-a, er-b and the mt group were slightly up-regulated between  <dig> - and  <dig> -fold, cy-b was strikingly induced by thermal stress with an averaged up-regulation of  <dig> -fold.
this indicates that the cy-b genes  are the major inducible forms of the hsp <dig> multigene family in p. caudatum.
note that the fairly high standard errors as indicated in the whisker-box plots in figure  <dig> resulted from the inclusion of the pcr efficiency into the relative expression calculation of rest  <dig>
the screening of two p. tetraurelia est libraries for orthologous sequences of the detected five p. caudatum hsp70-groups revealed that all corresponding p. tetraurelia hsp70s seem to be up-regulated during heat exposure to 35°c, compared to normal physiological conditions at 27°c .
while est counts for cy-b related genes could not be detected at 27°c, these genes were highly abundant at 35°c.
therefore, the cy-b related homologs seem to represent the major heat inducible hsp <dig> genes of this multigene family in p. tetraurelia as well.
discussion
phylogenetic analyses based on an hsp <dig> amino acid alignment reliably assigned the detected p. caudatum hsp <dig> genes to the respective hsp70-subfamilies cytosol , endoplasmic reticulum  and mitochondria  .
these analyses further showed not only that these homologs can be divided into the three subfamilies, but they also revealed the existence of five putative hsp70-groups in p. caudatum: one mt group consisting of only one homolog, but two distinct groups within the cy-, as well as two groups within the er-type subfamily.
here, each cy- and er-type hsp70-group shows a closer relationship to putative orthologous sequences of p. tetraurelia than to the other p. caudatum hsp70-group of the same subfamily .
these findings suggest a gene duplication event before the speciation of p. caudatum and p. tetraurelia, but obviously after the divergence of paramecium and tetrahymena  since both paramecium cy- and er-type homologs form monophyletic clades .
therefore, the last common ancestor of paramecium and tetrahymena should have had one functional cy- and one er-type hsp <dig> homolog, while the common ancestor of p. caudatum and p. tetraurelia has possessed one functional gene of each of the four non-organellar hsp70-groups, meaning one cy-a, cy-b, er-a and er-b homolog.
using an rt-qpcr approach, we could show considerable differences among the five assigned hsp70-groups of paramecium caudatum.
these analyses revealed the group cy-a as the major constitutively expressed hsp70-group in p. caudatum at optimum physiological temperatures.
even though this study showed a significant up-regulation in mrna levels of the cy-a group members after heat shock , this does not necessarily cause the rejection of their hsc <dig>  affiliation, since many constitutively expressed hsp70s can be induced under specific conditions .
we have also seen that the hsp70-group cy-b holds a very low mrna abundance at optimum growth temperatures, but was highly induced after heat shock .
this induction resulted in the second most abundant hsp70s during heat stress and suggests that the two genes pchsp70cy2a and pchsp70cy2b of group cy-b represent the major heat-inducible homologs in p. caudatum.
as previously mentioned, the p. caudatum hsp70-groups cy-a and cy-b showed a closer relationship to orthologous hsp <dig> sequences of p. tetraurelia than to each other.
the comparative analysis of two p. tetraurelia est libraries  revealed comparable expression patterns between the p. caudatum and the p. tetraurelia hsp70-groups .
here, the cy-a group homologs are also highly expressed under normal physiological conditions, indicating constitutively expressed hsp <dig> genes, while the cy-b related homologs appear to represent the major heat-inducible hsp70s in p. tetraurelia as well.
this result suggests conserved functions and a general expression pattern of the paramecium hsp <dig> gene family, at least between p. caudatum and p. tetraurelia.
in this context it is interesting to note that tetrahymena features also constitutively expressed and heat-inducible cy-hsp70s  <cit> , which form together with the single ichthyophthirius multifiliis cy-hsp <dig> homolog a sister clade to all paramecium cy-homologs .
in this cytosolic ichthyophthirius–tetrahymena clade, the constitutively expressed t. thermophila hsp70- <dig> gene clusters together with orthologs of t. malaccensis, t. borealis and i. multifiliis by forming a sister clade to all other tetrahymena cy-homologs including the heat-inducible t. thermophila hsp70- <dig> gene.
furthermore, while heat-inducible and constitutively expressed cy-hsp70s are common in higher eukaryotes too, they obviously do not form clear separated clades .
in conjunction with our findings for ciliate hsp70s, this strongly indicates that heat-inducibility in cytosolic hsp70s evolved several times independently during eukaryote evolution; at least twice within closely related unicellular eukaryotes and within metazoans.
this result provides a striking example of convergent evolution during subfunctionalization among eukaryotes.
our relative expression analyses also demonstrated that the p. caudatum hsp70-groups er-a and er-b showed comparatively similar transcription levels at optimum temperatures, but the relative amount was considerably lower compared to the major constitutively expressed hsp70-group cy-a.
heat treating the p. caudatum cells significantly induced the mrna expression of both er-type groups, but only to a comparatively small amount of  <dig> -fold for er-a and  <dig> -fold for er-b.
our study, therefore, partially supports the findings of hori and fujishima  <cit> , who showed only trace amounts of er-type hsp <dig> mrna when p. caudatum cells were cultured at 25°c, but also an up-regulation of approx.
4-fold when cells were heat shocked at 35°c.
on the other hand, they suggested a predominant expression of er-type hsp70s because they could not detect cytosolic homologs  <cit> .
this is in contrast to our results showing that the cytosolic hsp70-group cy-a encompassed the major constitutively expressed hsp70s, while group cy-b covered the major inducible homologs.
further, the relative mrna levels of the er-groups were nearly 23% less of the relative cytosolic mrna transcript amount at optimum temperatures.
and after heat stress, the cytosolic hsp <dig> transcription level was about  <dig> -fold higher than that of all er-type homologs, mainly because of the striking induction of the cy-b group, but also due to the significant up-regulation of the cy-a group hsp70s.
nevertheless, studies on other eukaryotic organisms revealed that er-type hsp <dig> proteins, mostly designated as grp <dig> or bip, are abundant proteins in animal cells as well  <cit> .
they are primarily induced by glucose depletion  <cit> , and also due to cadmium exposure  <cit> , during apoptosis  <cit>  or due to the presence of unfolded or misfolded proteins in the er.
however, they are not significantly heat-inducible  <cit> , while p. caudatum er-type hsp70s seem to be.
therefore, experiments on the gene expression of these er-type hsp <dig> genes investigating the effect of endosymbionts  <cit>  or other stressors would be valuable in unveiling their role in the stress response of paramecium.
in this study, we further identified one hsp <dig> gene encoding for a mitochondrial hsp <dig> protein .
these proteins are required for the translocation of cytosolic preproteins across the mitochondrial membrane as well as the subsequent folding reactions in the mitochondrial matrix  <cit> .
in most organisms, organelle-specific hsp70s are generally encoded by a single gene  <cit> , but in saccharomyces cerevisiae three distinct mthsp <dig> genes are described, and p. tetraurelia seems to have at least two functional mthsp70s.
humans seemingly hold only one chaperone-active mthsp <dig> protein , while another highly similar protein  plays a major role in the import of preproteins into the mitochondria.
the single mthsp <dig> gene pchsp70mt1a that we have observed in p. caudatum showed a constitutive expression pattern , suggesting its essential role for mitochondria to function properly under normal physiological conditions.
the  <dig> -fold up-regulation in mrna levels after heat shock, which is comparable to s. cerevisiae or blastocladiella emersonii mthsp70s  <cit> , implies also its key role for proper mitochondrial function under heat stress.
though elevated temperatures can lead to enhanced oxidative stress, this might be overcome by an increased protein import into the mitochondria or an enhanced chaperone activity.
whether the mthsp <dig> gene in p. caudatum covers both chaperone- and preprotein translocation activity and whether the p. tetraurelia mthsp70s have undergone subfunctionalization after gene duplication should be determined by future studies addressing hsp <dig> protein interactions.
the phylogenetic and evolutionary patterns detected within the paramecium hsp <dig> gene family support the model of divergent evolution, while the different hsp70-groups seem to have evolved under functional and structural constraints.
on the other hand, in relation to the further investigated ciliate species and metazoans, these patterns indicate distinct evolutionary pathways and convergent evolution .
therefore, this multigene family seems to be differentially affected also in ciliates by gene duplication events and/or whole genome duplications, by gene loss and retention, subfunctionalization and/or pseudogenization.
the finding of a similar basal gene duplication pattern of the cy- and the er-type hsp70s in paramecium, for example, is indicative of a whole-genome duplication  event within the last common ancestor of p. caudatum and p. tetraurelia.
it also may point to an intermediary wgd that occurred within the genus paramecium, because the most recent wgd is supposed to coincide with rapid speciation events that gave rise to the p. ‘aurelia’ complex .
further, an old wgd is thought to have occurred before the divergence of paramecium and tetrahymena <cit> , but is still under question due to recent studies that failed to detect evidence for wgd in t. thermophila and on the ichthyophthirius/tetrahymena branch  <cit> .
the age of these wgd events would be of major significance to clarify speciation and radiation events in ciliates as well as to understand the dynamics of gene loss and retention or the metabolic adaptation after a wgd  <cit> .
for example, the dates of these duplications could be interesting in consideration of the phylogenetic relationships among the genus paramecium, whether these events occurred within the last common paramecium ancestor or before/after certain speciation events.
insights into the different scenarios would be of importance to potentially support the proposed subdivision of the genus paramecium into the four subgenera chloroparamecium, helianter, cypriostomum and paramecium <cit> .
here, further analyses of the hsp <dig> multigene family of at least three additional paramecium species  would be required.
however, due to a missing reliable molecular clock for ciliate divergence times  and recent findings on p. tetraurelia that indicate extraordinarily reduced dna mutation rates  <cit> , more genome data would be favourable to estimate the dates of paramecium species divergence or the timing of wgd events in the ciliate phylum.
assuming a wgd event within the last common ancestor of p. caudatum and p. tetraurelia, the lack of mt paralogs in p. caudatum has to be the result of pseudogenization and a rapid gene loss.
even though many of the duplicates of the recent wgd in p. tetraurelia are functionally redundant and are supposed to be progressively lost, most of the gene duplicates did not go through such a rapid elimination  <cit> .
on the other hand, the four putative mthsp70s in p. tetraurelia, which seem to comprise two functional genes and two pseudogenes, are suggestive of an intermediary wgd event causing the hsp70-group differentiation in paramecium.
since our study on p. caudatum was based on cdna and all the detected homologs show typical hsp <dig> family signatures and motifs as well as no premature stop codons or frameshifts, they can be considered as functional hsp <dig> genes.
therefore, we have not detected either potentially expressed or unexpressed hsp <dig> pseudogenes, which would represent a hallmark of the birth-and-death process after duplication events.
hence, to disentangle in more detail the different patterns of concerted and non-concerted evolution that shaped the p. caudatum hsp <dig> gene family, further gdna/genome analyses would be necessary to unveil potential hsp <dig> pseudogenes in p. caudatum.
moreover, comparative genome analyses can uncover the gene order and orientation of recent hsp70s to infer pre-duplication states and to predict patterns of duplicate loss and retention.
such analyses can further improve our understanding of the evolution of such gene families in paramecium and other microbial eukaryotes.
CONCLUSIONS
the results of this study demonstrate first, that the hsp <dig> multigene family of paramecium caudatum comprises several homologous genes that can be assigned to three major hsp70-subfamilies: one mitochondrial, five cytosolic and five endoplasmic-reticulum-related hsp70s.
phylogenetic analyses further revealed two separate hsp70-groups within the cytosolic as well as the er-type homologs, showing a closer relationship to paramecium tetraurelia orthologs than to each other.
these groups are derivatives of duplication events preceding speciation and have evolved in a conservative fashion under purifying selection.
second, our results revealed significant differences in the expression levels of these genes as well as significant heat-shock effects.
while we observed constitutively expressed hsp <dig> genes for all three cellular compartments, indicating the general importance of these proteins, our analyses also unveiled a major inducible hsp70-group.
this group was assigned as a cytosolic member with a low basal expression level, but a striking up-regulation of approx.
84-fold after heat shock.
a similar expression pattern could be found in p. tetraurelia via the analyses of different est libraries, which suggests conserved functions of orthologous hsp70s, at least between these two paramecium species.
on the other hand, the constitutively expressed and heat-induced cytosol-type hsp70s evolved independently in paramecium and in its close relative tetrahymena, but also in higher eukaryotes.
this fact provides evidence of convergent evolution during subfunctionalization of eukaryote hsp70s.
in addition, the herein developed rt-qpcr assay provides a crucial tool for future selective studies examining the effect of several stresses on the hsp <dig> gene expression in paramecium, a complex unicellular eukaryote that is an excellent model organism in cellular physiology, developmental biology, genome evolution and epigenetic inheritance.

 the environmental stress response in all organisms as diverse as pro- and eukaryotes is generally coupled with a remarkable change in gene expression patterns and an enhanced synthesis of several ‘stress proteins’. because they were first described in drosophila melanogaster larvae that were accidentally exposed to elevated temperatures, these stress-related proteins were called heat-shock proteins. extensive research on hsps revealed also a constitutive expression of some members of these proteins, suggesting that they are also essential in maintaining the cellular functions under normal physiological conditions. these members are therefore designated as heat-shock cognate proteins. furthermore, hsps respond not only to increased temperatures, but also chemicals, heavy metals, uv light, hypoxia and other stressors can induce their synthesis. some of the most important and well investigated hsps are the members of the kda heat-shock protein family. they belong to the highest conserved proteins and are present in almost all species, except for some archaea. prokaryotic hsp proteins share about 50% amino acid identity with eukaryotic hsp70s. all known hsp proteins exhibit highly conserved amino acid sequences and domain structures, such as a conserved n-terminal atpase domain, a region with protease sensitive sites and a peptide binding domain at the c-terminal region. due to this high conservation, hsp has been widely used as a suitable phylogenetic marker in molecular evolution. it has been applied to deep phylogenetic relationships or to confirm the monophyly of metazoa, as well as relationships between archaea and gram-positive bacteria or gram-negative bacteria and eukaryotes. hsp genes and proteins have also been used for phylogenetic studies of different protozoan parasites such as trypanosoma or leishmania, as well as of non-parasitic protozoans such as euplotes or paramecium. in eukaryotes, several of these hsp proteins that belong to four subfamilies are encoded by the nuclear genome. the discrimination of the four hsp subfamilies corresponds to the intracellular localisation of the hsp proteins in the major compartments of the cell: the cytosol, the endoplasmic reticulum and the organelles mitochondria and chloroplasts. these multigene family members emerged from numerous duplication events or replicative transpositions and evolved by a combination of birth-and-death processes, gene conversion events and purifying selection. therefore, caution is called for when using hsp70s as phylogenetic marker, because paralogy can distort phylogenetic relationships. while the evolution of all hsp family members is not yet completely understood, the pathways for the evolution of the nucleus-encoded hsp70s of the cell organelles are well known. the mitochondrial and the chloroplast homologs are derivatives from an endosymbiotic alphaproteobacterium and cyanobacterium, respectively, followed by a subsequent horizontal gene transfer of the hsp homologs to the nucleus. further, the evolutionary relationship between the er- and cytosol-type hsp70s indicates that these genes have arisen very early in the common eukaryotic ancestor by gene duplication. all members of the hsp family carry out molecular chaperone functions that facilitate correct protein folding, as well as membrane translocation and the subsequent refolding of proteins. through the course of evolution, these multiple hsp family members, which can be found in pro- and eukaryotes, have acquired different chaperone tasks and functions. for example, in all multicellular eukaryotes studied so far, some cytosol-type members show a constitutive expression under normal physiological conditions while others are highly inducible in consequence of several stress conditions. detailed knowledge about the number, diversity and differential gene expression of the hsp family members in most unicellular eukaryotes is, however, scarce. for example, the yeast genome of saccharomyces cerevisiae encodes fourteen hsp70-like genes comprising cytosol-type hsp70s that show constitutive expression as well as heat-inducibility. further, the genome sequencing projects of the ciliate species and model eukaryotes tetrahymena thermophila and paramecium tetraurelia unveiled several putative hsp genes with conserved hsp domains. in paramecium, whole genome duplications and subsequent gene loss events have shaped the hsp gene family, but it is still uncertain whether subfunctionalization or gene dosage constraints are the main forces for hsp duplicate retention. while the major constitutively expressed and heat-inducible cytosol-type hsp70s have been identified for t. thermophila, the differential expression pattern of the hsp family in p. tetraurelia has remained unclear. additionally, the evolutionary relationships of the hsp homologs from these closely related microbial eukaryotes are vague. therefore, we have investigated the hsp multigene family of another paramecia species, paramecium caudatum, by sequencing an hsp cdna clone library. based on the resultant sequence data, we performed phylogenetic and comparative sequence analyses to gain insight into the evolution of the hsp gene family in paramecium. to disentangle the evolutionary relationships among ciliate hsp70s, which are in certain cases affected by whole genome duplications, we included not only a comprehensive set of homologous hsp70s of different pro- and eukaryotes, but also data from recent ciliate genome projects: tetrahymena, ichthyophthirius and oxytricha. we further developed a target specific rt-qpcr assay for p. caudatum hsp70s in order to demonstrate an expected differential gene expression among the family members and to distinguish constitutively expressed and heat-induced hsp70s. using these data, we tested for a hypothesised functional conservation of paramecium hsp orthologs via comparative est analyses and investigated the diversification of eukaryotic hsp70s to clarify the evolution of heat-inducibility within the kda molecular chaperone family.  characterisation of p. caudatum hsp70s sequencing of sixty putative hsp gene fragments, amplified from cdna with degenerated primers, revealed eleven different hsp nucleotide sequences from the hsp70/dnak-family of paramecium caudatum. when the majority of the obtained sequences became redundant, we stopped sequencing of further clones of our hsp cdna clone library. therefore, we cannot exclude the possibility of additional homologs expressed in p. caudatum, but a sequence homology search clearly assigned the eleven homologs to the three major hsp70-subfamilies: cytosol, endoplasmic reticulum and mitochondria. these analyses further revealed the expression of one mt, five cy and five er related hsp proteins in p. caudatum. the sequences were designated according to their origin and sequence similarity as follows: pchsp70cy1a, pchsp70cy1b, pchsp70cy1c, pchsp70cy2a, pchsp70cy2b, pchsp70er1a, pchsp70er1b, pchsp70er2a, pchsp70er2b, pchsp70er2c, pchsp70mt1a. the homologs pchsp70cy1a and pchsp70cy1b differed at only nucleotide positions, and encode the same amino acid sequence. the homologs pchsp70er1a and pchsp70er1b differed by only one ‘c’ to ‘t’ substitution, resulting also in the same amino acid sequence. pchsp70er2a, pchsp70er2b and pchsp70er2c showed to nucleotide substitutions, but were indiscriminative at the amino acid level too. because we used a proofreading polymerase for pcr amplification and a high fidelity rna-dependent dna polymerase for cdna synthesis, these nucleotide substitutions can be considered authentic. the analyses of the deduced amino acid sequences revealed seven different homologous sequences, with an averaged pairwise distance of and a maximum pairwise distance of. the amplified fragments for all cy homologs as well as pchsp70er1a and pchsp70er1b encode amino acids; pchsp70er2a, pchsp70er2b and pchsp70er2c encode aa and pchsp70mt1a encodes aa. all identified sequences shared two amino acid signature motifs, which are highly conserved regions and present in all eukaryotic hsp family members. the consensus sequences are as follows: hsp family signature --d-l-g-t-f-d--s--l and hsp family signature --l-v-g---r--p-k---, where the square brackets show a list of acceptable amino acids at the given position and the numerical values between parentheses indicate repetition of the respective amino acid. another conserved signature, the putative atp/gtp-binding site motif a, could be identified in all eleven homologs, albeit with relatively high variation. as expected, the putative bipartite nuclear localization signal, a targeting sequence essential for the selective translocation of nucleo-/cytoplasmatic proteins into the nucleus, could be detected for the cy homologs only. its consensus sequence is -k-x-l-r-l-r, where the symbol ‘x’ indicates a position of any acceptable amino acid. additionally, all cy and er homologs possess the r-a--f-e-e-l consensus motif that serves as a potential signature for eukaryotic non-organellar hsp proteins. evolutionary relationships as illustrated in the phylogenetic hsp tree in figure  showing the three major hsp70-subfamilies among different eukaryotes, the ascertained p. caudatum sequences integrated well within the separate subfamilies with high support values. in addition, all ciliate sequences clustered into clades in which the paramecium homologs formed monophyla. these analyses also revealed the existence of different hsp70-groups within the different p. caudatum hsp subfamilies. here, two distinct hsp70-groups within the cytosolic as well as the er-type subfamily could be detected. each of these p. caudatum hsp70-groups from the same subfamily showed a closer relationship to orthologous hsp sequences of paramecium tetraurelia than to another p. caudatum hsp70-group. the group cy-a consisted of the three in-paralogs pchsp70cy1a, pchsp70cy1b and pchsp70cy1c; the group cy-b contained pchsp70cy2a and pchsp70cy2b; the group er-a comprised the genes pchsp70er1a and pchsp70er1b; and group er-b included pchsp70er2a, pchsp70er2b and pchsp70er2c. the mitochondrial homolog pchsp70mt1a was determined as group mt for further analyses, even though we could detect only one mitochondrial hsp gene sequence. since we cannot confirm complete locus homozygosity of the clonal p. caudatum strain that we used in this study, the detected within-group paralogs might constitute alleles. however, the amitotic division of the ciliate macronucleus during vegetative growth can lead to an unequal partitioning of the alleles to daughter macs, resulting in the expression of only one of the two alleles. in conjunction with the detected low mutation rate in paramecium and the comparatively high sequence divergence of the cy-b in-paralogs, we would argue in this case for in-paralogs rather than alleles. the detection of eleven putative hsp homologs in p. caudatum is in concordance with former studies on the hsp multigene family in other ciliate species. the p. tetraurelia genome project, for example, revealed fifteen putative hsp sequences in mac dna, while only thirteen possess the typical conserved domain structures with an n-terminal atpase and a c-terminal peptide-binding domain. however, our motif analyses revealed that only ten homologs exhibit all three hsp family signatures, indicating the existence of potential hsp pseudogenes in the p. tetraurelia mac genome. in addition, genome analyses for tetrahymena thermophila revealed thirteen putative hsp genes featuring conserved hsp domains. similar to p. tetraurelia, only seven hsp homologs comprise the three hsp family signatures. interestingly, while we could find all orthologous sequences of these seven t. thermophila hsp70s in its close relative t. malaccensis by utilizing the tetrahymena comparative database, we could not detect the heat-inducible ortholog of hsp70- in t. borealis. furthermore, in ichthyophthirius multifiliis and oxytricha trifallax we could detect only one cy- and one er-type hsp homolog comprising all three hsp family signatures. modes of evolution the five putative hsp70-groups detected in paramecium showed consistent phylogenetic patterns according to the assumed evolutionary relationship between the species, thereby supporting the model of divergent evolution in the paramecium hsp gene family. additionally, a codon-based z-test on all p. caudatum hsp70s revealed evidence for purifying selection, which is indicated by higher numbers of synonymous to non-synonymous nucleotide differences per site in pairwise comparisons. while the paralogs between the two hsp70-groups per subfamily show a comparatively high amino acid divergence of up to 19%, the potential paralogs within each group show a rather low nucleotide diversity. this high similarity of the in-paralogs could be indicative of very recent duplication events or might be explained by purifying selection or gene conversion. the results of a codon-based z-test for neutral evolution revealed that the null hypothesis of strict-neutrality could not be rejected for the cy-b and er-type in-paralogs, while the sequence similarity among the cy-a in-paralogs seems to be caused by purifying selection. a test for gene conversion events revealed significant evidence for only two very short regions between out-paralogs, which might in fact be misidentifications due to possible misalignment artefacts. please note that the substitutions within the er and cy-a groups are very few and therefore do not allow for definite conclusions of the within-group comparisons; on the other hand, the discrepancy between the cy-a and cy-b group is obvious and implies different modes of evolution. the number of synonymous differences per synonymous site and of nonsynonymous differences per nonsynonymous site for comparisons of eleven p. caudatum hsp homologs are shown. analyses were conducted using the nei-gojobori model included in mega all ambiguous positions were removed for each sequence pair with a total of amino acid positions in the final dataset. differential gene expression the group classification with cy-a, cy-b, er-a, er-b and mt was used for the expression analyses, since rt-qpcr primers and mgb™-probes were specifically designed to amplify or bind the respective hsp sequences within one group. the stability analysis of the reference genes gapdh and ef-1α using the genorm v. applet revealed a high stability of both genes. the averaged expression stability value was and the pairwise variation value was below the proposed cut-off value of. we therefore used these two genes to normalise the hsp mrna levels in all further analyses. as illustrated in figure 3a, the cy-a relative expression level at optimum temperature was the highest among all five hsp70-groups. while the basal expression levels of the er-a, er-b and mt group were comparatively similar, the cy-b levels instead were considerably lower, indicating that these genes were transcribed at low basal levels under normal physiological conditions. after heat-shock exposure for two hours at 34°c, we could detect changes of the hsp transcript levels. while the reference genes gapdh and ef-1α showed comparatively stable expression levels between 28°c and 34°c, the heat shock provoked an induction of all different hsp70-groups. after heat shock, the relative transcription level of cy-a was higher than that of gapdh and still showing the highest hsp mrna expression level. the cy-b group, however, was highly induced, resulting in the second most expressed hsp70-group. performing relative expression analyses using rest unveiled a significant up-regulation of all hsp70-groups. while the transcription level of the cy-a, er-a, er-b and the mt group were slightly up-regulated between - and -fold, cy-b was strikingly induced by thermal stress with an averaged up-regulation of -fold. this indicates that the cy-b genes are the major inducible forms of the hsp multigene family in p. caudatum. note that the fairly high standard errors as indicated in the whisker-box plots in figure  resulted from the inclusion of the pcr efficiency into the relative expression calculation of rest the screening of two p. tetraurelia est libraries for orthologous sequences of the detected five p. caudatum hsp70-groups revealed that all corresponding p. tetraurelia hsp70s seem to be up-regulated during heat exposure to 35°c, compared to normal physiological conditions at 27°c. while est counts for cy-b related genes could not be detected at 27°c, these genes were highly abundant at 35°c. therefore, the cy-b related homologs seem to represent the major heat inducible hsp genes of this multigene family in p. tetraurelia as well. discussion phylogenetic analyses based on an hsp amino acid alignment reliably assigned the detected p. caudatum hsp genes to the respective hsp70-subfamilies cytosol, endoplasmic reticulum and mitochondria. these analyses further showed not only that these homologs can be divided into the three subfamilies, but they also revealed the existence of five putative hsp70-groups in p. caudatum: one mt group consisting of only one homolog, but two distinct groups within the cy-, as well as two groups within the er-type subfamily. here, each cy- and er-type hsp70-group shows a closer relationship to putative orthologous sequences of p. tetraurelia than to the other p. caudatum hsp70-group of the same subfamily. these findings suggest a gene duplication event before the speciation of p. caudatum and p. tetraurelia, but obviously after the divergence of paramecium and tetrahymena since both paramecium cy- and er-type homologs form monophyletic clades. therefore, the last common ancestor of paramecium and tetrahymena should have had one functional cy- and one er-type hsp homolog, while the common ancestor of p. caudatum and p. tetraurelia has possessed one functional gene of each of the four non-organellar hsp70-groups, meaning one cy-a, cy-b, er-a and er-b homolog. using an rt-qpcr approach, we could show considerable differences among the five assigned hsp70-groups of paramecium caudatum. these analyses revealed the group cy-a as the major constitutively expressed hsp70-group in p. caudatum at optimum physiological temperatures. even though this study showed a significant up-regulation in mrna levels of the cy-a group members after heat shock, this does not necessarily cause the rejection of their hsc affiliation, since many constitutively expressed hsp70s can be induced under specific conditions. we have also seen that the hsp70-group cy-b holds a very low mrna abundance at optimum growth temperatures, but was highly induced after heat shock. this induction resulted in the second most abundant hsp70s during heat stress and suggests that the two genes pchsp70cy2a and pchsp70cy2b of group cy-b represent the major heat-inducible homologs in p. caudatum. as previously mentioned, the p. caudatum hsp70-groups cy-a and cy-b showed a closer relationship to orthologous hsp sequences of p. tetraurelia than to each other. the comparative analysis of two p. tetraurelia est libraries revealed comparable expression patterns between the p. caudatum and the p. tetraurelia hsp70-groups. here, the cy-a group homologs are also highly expressed under normal physiological conditions, indicating constitutively expressed hsp genes, while the cy-b related homologs appear to represent the major heat-inducible hsp70s in p. tetraurelia as well. this result suggests conserved functions and a general expression pattern of the paramecium hsp gene family, at least between p. caudatum and p. tetraurelia. in this context it is interesting to note that tetrahymena features also constitutively expressed and heat-inducible cy-hsp70s, which form together with the single ichthyophthirius multifiliis cy-hsp homolog a sister clade to all paramecium cy-homologs. in this cytosolic ichthyophthirius–tetrahymena clade, the constitutively expressed t. thermophila hsp70- gene clusters together with orthologs of t. malaccensis, t. borealis and i. multifiliis by forming a sister clade to all other tetrahymena cy-homologs including the heat-inducible t. thermophila hsp70- gene. furthermore, while heat-inducible and constitutively expressed cy-hsp70s are common in higher eukaryotes too, they obviously do not form clear separated clades. in conjunction with our findings for ciliate hsp70s, this strongly indicates that heat-inducibility in cytosolic hsp70s evolved several times independently during eukaryote evolution; at least twice within closely related unicellular eukaryotes and within metazoans. this result provides a striking example of convergent evolution during subfunctionalization among eukaryotes. our relative expression analyses also demonstrated that the p. caudatum hsp70-groups er-a and er-b showed comparatively similar transcription levels at optimum temperatures, but the relative amount was considerably lower compared to the major constitutively expressed hsp70-group cy-a. heat treating the p. caudatum cells significantly induced the mrna expression of both er-type groups, but only to a comparatively small amount of -fold for er-a and -fold for er-b. our study, therefore, partially supports the findings of hori and fujishima, who showed only trace amounts of er-type hsp mrna when p. caudatum cells were cultured at 25°c, but also an up-regulation of approx. 4-fold when cells were heat shocked at 35°c. on the other hand, they suggested a predominant expression of er-type hsp70s because they could not detect cytosolic homologs. this is in contrast to our results showing that the cytosolic hsp70-group cy-a encompassed the major constitutively expressed hsp70s, while group cy-b covered the major inducible homologs. further, the relative mrna levels of the er-groups were nearly 23% less of the relative cytosolic mrna transcript amount at optimum temperatures. and after heat stress, the cytosolic hsp transcription level was about -fold higher than that of all er-type homologs, mainly because of the striking induction of the cy-b group, but also due to the significant up-regulation of the cy-a group hsp70s. nevertheless, studies on other eukaryotic organisms revealed that er-type hsp proteins, mostly designated as grp or bip, are abundant proteins in animal cells as well. they are primarily induced by glucose depletion, and also due to cadmium exposure, during apoptosis or due to the presence of unfolded or misfolded proteins in the er. however, they are not significantly heat-inducible, while p. caudatum er-type hsp70s seem to be. therefore, experiments on the gene expression of these er-type hsp genes investigating the effect of endosymbionts or other stressors would be valuable in unveiling their role in the stress response of paramecium. in this study, we further identified one hsp gene encoding for a mitochondrial hsp protein. these proteins are required for the translocation of cytosolic preproteins across the mitochondrial membrane as well as the subsequent folding reactions in the mitochondrial matrix. in most organisms, organelle-specific hsp70s are generally encoded by a single gene, but in saccharomyces cerevisiae three distinct mthsp genes are described, and p. tetraurelia seems to have at least two functional mthsp70s. humans seemingly hold only one chaperone-active mthsp protein, while another highly similar protein plays a major role in the import of preproteins into the mitochondria. the single mthsp gene pchsp70mt1a that we have observed in p. caudatum showed a constitutive expression pattern, suggesting its essential role for mitochondria to function properly under normal physiological conditions. the -fold up-regulation in mrna levels after heat shock, which is comparable to s. cerevisiae or blastocladiella emersonii mthsp70s, implies also its key role for proper mitochondrial function under heat stress. though elevated temperatures can lead to enhanced oxidative stress, this might be overcome by an increased protein import into the mitochondria or an enhanced chaperone activity. whether the mthsp gene in p. caudatum covers both chaperone- and preprotein translocation activity and whether the p. tetraurelia mthsp70s have undergone subfunctionalization after gene duplication should be determined by future studies addressing hsp protein interactions. the phylogenetic and evolutionary patterns detected within the paramecium hsp gene family support the model of divergent evolution, while the different hsp70-groups seem to have evolved under functional and structural constraints. on the other hand, in relation to the further investigated ciliate species and metazoans, these patterns indicate distinct evolutionary pathways and convergent evolution. therefore, this multigene family seems to be differentially affected also in ciliates by gene duplication events and/or whole genome duplications, by gene loss and retention, subfunctionalization and/or pseudogenization. the finding of a similar basal gene duplication pattern of the cy- and the er-type hsp70s in paramecium, for example, is indicative of a whole-genome duplication event within the last common ancestor of p. caudatum and p. tetraurelia. it also may point to an intermediary wgd that occurred within the genus paramecium, because the most recent wgd is supposed to coincide with rapid speciation events that gave rise to the p. ‘aurelia’ complex. further, an old wgd is thought to have occurred before the divergence of paramecium and tetrahymena, but is still under question due to recent studies that failed to detect evidence for wgd in t. thermophila and on the ichthyophthirius/tetrahymena branch. the age of these wgd events would be of major significance to clarify speciation and radiation events in ciliates as well as to understand the dynamics of gene loss and retention or the metabolic adaptation after a wgd. for example, the dates of these duplications could be interesting in consideration of the phylogenetic relationships among the genus paramecium, whether these events occurred within the last common paramecium ancestor or before/after certain speciation events. insights into the different scenarios would be of importance to potentially support the proposed subdivision of the genus paramecium into the four subgenera chloroparamecium, helianter, cypriostomum and paramecium. here, further analyses of the hsp multigene family of at least three additional paramecium species would be required. however, due to a missing reliable molecular clock for ciliate divergence times and recent findings on p. tetraurelia that indicate extraordinarily reduced dna mutation rates, more genome data would be favourable to estimate the dates of paramecium species divergence or the timing of wgd events in the ciliate phylum. assuming a wgd event within the last common ancestor of p. caudatum and p. tetraurelia, the lack of mt paralogs in p. caudatum has to be the result of pseudogenization and a rapid gene loss. even though many of the duplicates of the recent wgd in p. tetraurelia are functionally redundant and are supposed to be progressively lost, most of the gene duplicates did not go through such a rapid elimination. on the other hand, the four putative mthsp70s in p. tetraurelia, which seem to comprise two functional genes and two pseudogenes, are suggestive of an intermediary wgd event causing the hsp70-group differentiation in paramecium. since our study on p. caudatum was based on cdna and all the detected homologs show typical hsp family signatures and motifs as well as no premature stop codons or frameshifts, they can be considered as functional hsp genes. therefore, we have not detected either potentially expressed or unexpressed hsp pseudogenes, which would represent a hallmark of the birth-and-death process after duplication events. hence, to disentangle in more detail the different patterns of concerted and non-concerted evolution that shaped the p. caudatum hsp gene family, further gdna/genome analyses would be necessary to unveil potential hsp pseudogenes in p. caudatum. moreover, comparative genome analyses can uncover the gene order and orientation of recent hsp70s to infer pre-duplication states and to predict patterns of duplicate loss and retention. such analyses can further improve our understanding of the evolution of such gene families in paramecium and other microbial eukaryotes.  the results of this study demonstrate first, that the hsp multigene family of paramecium caudatum comprises several homologous genes that can be assigned to three major hsp70-subfamilies: one mitochondrial, five cytosolic and five endoplasmic-reticulum-related hsp70s. phylogenetic analyses further revealed two separate hsp70-groups within the cytosolic as well as the er-type homologs, showing a closer relationship to paramecium tetraurelia orthologs than to each other. these groups are derivatives of duplication events preceding speciation and have evolved in a conservative fashion under purifying selection. second, our results revealed significant differences in the expression levels of these genes as well as significant heat-shock effects. while we observed constitutively expressed hsp genes for all three cellular compartments, indicating the general importance of these proteins, our analyses also unveiled a major inducible hsp70-group. this group was assigned as a cytosolic member with a low basal expression level, but a striking up-regulation of approx. 84-fold after heat shock. a similar expression pattern could be found in p. tetraurelia via the analyses of different est libraries, which suggests conserved functions of orthologous hsp70s, at least between these two paramecium species. on the other hand, the constitutively expressed and heat-induced cytosol-type hsp70s evolved independently in paramecium and in its close relative tetrahymena, but also in higher eukaryotes. this fact provides evidence of convergent evolution during subfunctionalization of eukaryote hsp70s. in addition, the herein developed rt-qpcr assay provides a crucial tool for future selective studies examining the effect of several stresses on the hsp gene expression in paramecium, a complex unicellular eukaryote that is an excellent model organism in cellular physiology, developmental biology, genome evolution and epigenetic inheritance. 
therefore, we have characterised the hsp gene family of paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. eleven putative hsp genes could be detected in p. caudatum comprising homologs of three major hsp70-subfamilies. phylogenetic analyses revealed five evolutionarily distinct hsp70-groups, each with a closer relationship to orthologous sequences of paramecium tetraurelia than to another p. caudatum hsp70-group. information about the hsp copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. this result indicates convergent evolution during hsp subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution. ciliateconvergent evolutiondnakgene duplicationheat-inducibilityheat-shock proteinsmolecular chaperonesparameciumrt-qpcrtemperature stress our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in paramecium and in its close relative tetrahymena, as well as within higher eukaryotes. in eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. heat-shock proteins of the kda family are essential chaperones required for key cellular functions. heat-shock treatments were performed to test for differential expression patterns among the five hsp70-groups as well as for a functional conservation within paramecium. comparative est analyses for p. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the hsp gene family in paramecium.
 heat-shock proteins of the kda family are essential chaperones required for key cellular functions. in eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. generally, multiple cytosol-type hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. information about the hsp copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. therefore, we have characterised the hsp gene family of paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility.  eleven putative hsp genes could be detected in p. caudatum comprising homologs of three major hsp70-subfamilies. phylogenetic analyses revealed five evolutionarily distinct hsp70-groups, each with a closer relationship to orthologous sequences of paramecium tetraurelia than to another p. caudatum hsp70-group. these highly diverse, paralogous groups resulted from duplications preceding paramecium speciation, underwent divergent evolution and were subject to purifying selection. heat-shock treatments were performed to test for differential expression patterns among the five hsp70-groups as well as for a functional conservation within paramecium. these treatments induced exceptionally high mrna up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. all other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. comparative est analyses for p. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the hsp gene family in paramecium.  our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in paramecium and in its close relative tetrahymena, as well as within higher eukaryotes. this result indicates convergent evolution during hsp subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution. ciliateconvergent evolutiondnakgene duplicationheat-inducibilityheat-shock proteinsmolecular chaperonesparameciumrt-qpcrtemperature stress 