BACKGROUND
the mitochondrion is the main intracellular site of energy production and is the evolutionary response to the main challenge that living organisms have to face: gaining energy from their environments to sustain their biological functions.
the mitochondrial production of atp is realised by the combination of the phosphorylation of adp into atp with an efficient chain of redox reactions, resulting in the so-called oxidative phosphorylation.
however, these two processes are not always efficiently coupled, and one reason is the presence in the inner membrane of a family of mitochondrial transporters: the uncoupling proteins .
ucp <dig> was first discovered and cloned in  <dig>  <cit>  and is involved in the non-shivering thermogenesis  activity of rodent's brown adipose tissue .
since then, the discovery of ucp genes has grown rapidly, ucp <dig> homologues being found across mammalian species  but also in other eukaryotes from plants to animals  <cit> .
most of the recent attention has been devoted to the evolutionary history of ucp <dig> since the discovery of ucp <dig> in ectotherm organisms like teleost fish  <cit>  and amphibians  <cit> .
the fact that organisms that do not show nst possess and express ucp <dig> raised the question of the exact evolutionary history of ucp <dig> and of its link with the apparition of thermoregulation.
this observation has stimulated an increasing number of phylogenetic studies on ucp  <cit>  to determine the origin of the physiological particularity  in the mammalian lineage  <cit> .
ucp <dig> and its close homologues  are thought to differ in the nature of their uncoupling activity  <cit>  and their potential physiological roles .
indeed, a rapid overview of the data collected on ucp <dig>   <dig> and  <dig> highlights how these proteins may be different.
first, while ucp <dig> tissue expression is localized  to bat, ucp <dig> is expressed  in a wider range of cell types  and ucp <dig> is mainly present in skeletal muscle .
also, the physiological role of ucp <dig> is restricted to thermogenesis, which is unlikely to be the case for ucp <dig> and  <dig> as shown by their respective knock-out models  <cit> .
ucp <dig> and  <dig> have been involved in a number of postulated functions in energy regulation, including regulation of insulin secretion  <cit>  or reactive oxygen species production and control of the immune response  <cit> .
however, accurate data on the mitochondrial activity of ucp <dig> and ucp <dig> are still lacking to determine the exact nature of their biological activity  <cit> .
therefore, despite the high sequence identity shared by ucp <dig>   <dig> and  <dig> , punctual amino acid replacement at key structural domains of the respective proteins may have evolved to allow functional specificity to take place.
interestingly, mutagenesis experiments have shown that single amino-acid replacement in ucp <dig> protein may change its proton permeability , its sensibility to fatty acid activation or nucleotide inhibition , or its transmembrane structure  <cit> .
the next step in the understanding of the biology of ucp is to determine whether the evolution of ucp genes and protein sequences may have been subjected to different selective pressures after duplication.
single copy genes are thought to evolve conservatively because of strong negative selective pressure.
gene duplications produce a redundant gene copy and thus release one or both copies from negative selection pressure.
there are a number of models for the fate of gene duplicates, the two most prominent of which are neofunctionilization and subfunctionalisation.
thus, duplications are thought to be an important precursor of functional divergence.
the increased availability of ucp sequences in the public databases allows the study of the molecular evolution of the ucp gene family and the evaluation of selection following duplication events.
in the present study, we will determine  the evolutionary history of the ucp gene family,  evaluate the changes in selection pressures following duplications, and  identify sites under positive darwinian selection.
RESULTS
sequence similarity searches and multiple alignment
two lizard sequences from anolis carolinensis were identified during similarity searches with high similarity to ucp <dig> and ucp <dig>  homologues of ucp <dig> were not found in the lizard scaffold genome.
table  <dig> outlines the sequences  used in the phylogenetic analyses.
it should be noted that additional ucp genes for eutherians and teleosts were identified.
inclusion of these did not improve the reliability of the phylogeny, and as the aim of this study was to determine the evolutionary history of the ucp gene family, only representatives from the major vertebrate clades were included.
sequence obtained from the february  <dig> draft assembly ) produced by the broad institute at mit and harvard,  sequences obtained from ensembl, * sequences from jgi.
phylogeny of the ucp gene family
the alignments were used to construct phylogenetic trees with maximum likelihood  and bayesian inference .
the different reconstruction methods provided poor support for basal nodes using the protein alignment .
the dna alignment showed support for the ucp <dig>  ucp <dig> and ucp <dig> clades, in particular when fewer distantly related outgroups are used perhaps as a consequence of systematic error .
the different reconstruction methods provided slightly different topologies.
most relationships could be resolved with confidence dividing the gene family into strongly supported clusters in most tree reconstructions.
ucp <dig> and ucp <dig> genes are sister clusters and the avian ucp gene is grouped within the ucp <dig> cluster.
the  <dig> different protein trees were reconciled against a species tree using genetree.
the protein ml topology required  <dig> duplications and  <dig> losses and the bi  <dig> duplications and  <dig> losses.
the high number of duplications and losses is a result of the basal topology of the gene tree and a number of incongruences between the gene and species trees.
however, in the ml protein phylogenies, the basal relationships have low bootstrap supports.
using the dna phylogeny, the ml tree required less duplications and losses  than the bi tree .
the higher number of duplications and losses in the bi reconstruction is mainly a result of duplications inferred through incongruence between the gene and species trees.
if as suggested by the protein phylogenies, the lamprey sequences are sister to the ucp <dig> clade and the takifugu ucp <dig> groups within the same clade, then the reconciliation infers  <dig> duplications  and  <dig> losses .
however, the results from the dna phylogenies suggest the lamprey sequences could have diverged before the duplication of ucp2/ <dig>  in this case, by removing losses and duplications inferred through mismatch of species and gene trees and losses due to incomplete genome sequences, the most parsimoniously reconciled tree shows  <dig> zebrafish specific duplication and two major duplications that occurred early in the vertebrate lineage .
one duplication is proposed to have occurred prior to the emergence of teleost fish resulting in two lineages which evolved into ucp <dig> and ucp2/ <dig> and probably took place early in vertebrate evolution due to the presence of ucp <dig> in lampreys, although further data are required to confirm the presence of ucp <dig> and ucp <dig> in lampreys.
a second duplication, also early in vertebrate evolution, resulted in ucp <dig> and ucp <dig>  further sequencing of a broader range of ancestral craniata is required to identify a more precise timing for the duplications.
interestingly, ucp <dig> and ucp <dig> have been independently lost from the avian lineage but further data are required to confirm the absence of ucp <dig> in lizards to be able to determine when the loss of ucp <dig> took place.
synonymous and non-synonymous substitution rate estimates
results using the dna dataset show that ucp genes are under varying selection pressures .
pairwise comparisons of human and mouse orthologs and human and platypus show that ucp <dig> has higher estimates of dn/ds ratio compared to ucp <dig> and ucp <dig> but suggest purifying selection in all three genes.
the lower substitution rates for ucp <dig> and ucp <dig> shows that they are under strong purifying selection.
substitution rates were estimated using yang and nielson  <cit>  method as implemented in yn <dig> in the paml package.
positive selection tests
more sophisticated codon-based substitution models were used to test for branch-specific selection.
the model was based on the assumption that selective constraints change following gene duplication.
we estimated ω as an average over all sites and branches and the ratio was substantially smaller than  <dig> .
the one-ratio model was compared with model r <dig>  and the lrt  indicated that there is a significant decrease in the rate of non-synonymous substitution following the duplication of ucp <dig> and ucp2/ <dig> .
the comparison of model r <dig> and r <dig> also showed that there was a significant difference in the selective pressure following the duplication of ucp <dig> and ucp <dig>  the branch specific model with three distinct rates of substitution , one for each ucp gene, is a significantly better fit than the one-ratio  and two-ratio  models according to the lrt .
this suggests significantly different selective pressures on ucp <dig>  ucp <dig> and ucp <dig>  however, none of the parameters estimated indicate positive darwinian selection.
the models were implemented in codeml from paml.
parameters in bold indicate positive selection.
significant tests are shown in bold.
the lrt of the one ratio model with m <dig> indicates that selective pressure is not uniform among sites .
only the m <dig> model indicates a site that is evolving under positive darwinian selection .
lrts  indicate that model m <dig> does not fit the data better than m <dig> whilst it does show that m <dig> fits the data better than m <dig>  which does not allow for positively selected sites .
branch-site models were also applied with ucp <dig> specified as the foreground branch, however the m <dig> model was the best fit to the data with a likelihood value of –  <dig> .
this model suggests that the variation in selection pressure is due to the evolution by positive selection of a single site, whilst the remaining sites are under strong purifying selection.
according to the alignment of the ucp sequences with the 3d structure of the bovine mitochondrial adp-atp carrier , the site under positive selection is in the mitochondrial matrix between the 5th and 6th alpha-helix .
as illustrated using hmm logos  <cit>  for each ucp gene, the site under positive selection follows a highly conserved  amino acid site present across the whole ucp gene family but the site under positive selection is not conserved within the different ucp groups .
the hmm logos also illustrate the high level of sequence conservation in the gene family.
discussion
in this study, we have sought to expand upon previous phylogenetic studies  <cit>  by focusing on the ucp gene families and incorporating sequences identified from completed genomes with a subset of cloned sequences, particularly those from non-mammalian species.
this study is the first to include lizard ucp genes.
the phylogenetic tree reconstruction of dna sequences gave well resolved topologies with stronger support values for basal relationships than using the protein data probably as a result of the highly conserved protein sequences.
these phylogenies provided a method to infer the evolutionary history of the ucp gene family.
the phylogeny of the ucp genes indicates that ucp <dig>  which is present in plants and arthropods, is the ancestral ucp as demonstrated in previous studies  <cit> .
ucp <dig> then duplicated prior to the divergence of vertebrates.
a second duplication of ucp <dig> and ucp <dig> also took place early in vertebrate evolution although the exact timing of the event  requires further genomic data to be gathered.
the multiple sequences of ucps found in the zebrafish, while termed ucp <dig> and ucp <dig> are both ucp <dig> orthologs and should be called ucp1a and ucp1b.
ucp <dig> is syntenic to gene a and gene b like other vertebrate ucp <dig> genes .
this could either be a zebrafish specific duplication, or the incomplete sequencing of cyprinus carpio could be hiding an additional paralog and the duplication may be a fish specific genome wide duplications hypothesised to have occurred during fish evolution  <cit> .
the latter is probably unlikely due to the lack of duplicates in the complete genome of takifugu rubripes.
importantly, the phylogenetic analyses suggest the independent loss of ucp <dig> and ucp <dig> from the avian lineage.
the absence of ucp <dig> in the lizard genome could be attributed to the incompleteness of the genome or could be the result of a loss of ucp <dig> in the sauropsid lineage.
ucp <dig> is the only uncoupling protein for which there is a scientific consensus concerning the nature of its physiological function .
the ucp <dig> knockout mice are able to maintain their body temperature, but suffer in pronounced cold exposure suggesting that ucp <dig> is principally involved in short-term adaptation to cold .
this adaptive evolution probably occurred after the divergence between eutherians and marsupials  <cit>  consistent with the fact that bat is only found in eutherians.
even though birds are lacking ucp <dig>  they are still able to respond to thermal challenges.
the loss of ucp <dig> and disappearance of bat in birds is likely due to the concomitant development of physiological adaptations which have replaced bat function.
as evidence, metabolic rate of birds increases in response to cold and body temperature can be maintained  <cit> .
indeed, induced uncoupling activity in the mitochondria has been found in the skeletal muscle of cold-acclimated birds  <cit>  and more recently the implication of ucp <dig>  has been suggested  <cit> .
these data lead to two non exclusive conclusions.
firstly, birds have evolved other mechanisms of thermoregulation  <cit>  before or after the loss of ucp <dig> and bat .
secondly, a fully demonstrated implication of ucp <dig>  in skeletal non-shivering thermogenesis in birds would suggest that ucp <dig>  which is not involved in thermoregulation in mammals  <cit> , has acquired a new function in birds.
in this case, the question is whether avianucp activity could also compensate for the loss of the ucp <dig> gene, implicated in mammalian immunity  <cit>  and glucose metabolism  <cit> .
this is an interesting point given the non pathologic high chronic glycemia of birds  <cit> .
the molecular evolution of ucp genes showed that they were under strong purifying selection with a significant change towards stronger purifying selection.
ucp <dig> has the highest dn/ds ratio followed by ucp <dig> and then ucp <dig>  this strong purifying selection highlights the importance of the function of this highly conserved gene family.
although highly variable regions of the sequence which were difficult to assign as homologous were removed from the analyses, the site models showed that adaptation has appeared at a single site located between the 5th and 6th α-helices.
the role of this positively selected site has yet to be determined but the amino acid site  immediately prior to it is highly conserved across mitochondrial carriers as are the transmembrane regions that follow the site.
additionally, saito et al.
<cit>  found that the two amino acid sites that follow this site are conserved in all eutherian mammal ucp <dig> genes.
based on studies conducted on ucp <dig>  the region delimited by the 5th and 6th α-helices is close to a site of regulation of ucp <dig> activity by nucleotides and thus could be implicated in the inhibitory control of ucp <dig> uncoupling effect  <cit> .
this region is also hypothesized to be implicated in the mechanism of transport of protons/free fatty acids  <cit>  in ucp <dig>  however, to date there is a gap in the knowledge of the relationship between amino acid sequence and structure for ucp <dig> and ucp <dig>  and we are unable to speculate on the particular role of this region in these ucp <dig> homologues.
unfortunately, shared evolutionary history and molecular selection alone cannot be used as the unique criterion to infer protein function, and the true nature of each ucp gene needs to be determined experimentally and independently.
therefore, this positively selected site may play an important functional role and could represent an interesting target site for future mutagenesis experiment thus facilitating our understanding of the structure-function relationships in ucp genes.
CONCLUSIONS
genomic data have provided an opportunity to gain a better understanding about the evolution of ucps using phylogenetic analyses.
the ucp gene family phylogeny shows that two duplications took place early in the evolution of vertebrates.
subsequent to these two duplications, ucp <dig> and ucp <dig> were lost from the avian lineage independently.
however, further genome projects on a greater diversity of evolutionary lineages are required to better understand the gene-duplication history.
evolutionary rate analysis shows purifying selection across branches and sites  suggesting that the function of the genes in the ucp gene family has been highly conserved after duplication events and over evolutionary time.
by considering the evolutionary history of the ucp gene family we provide insight into which amino acid residues might have undergone positive selection and could be targeted for site-directed mutagenesis.
however, the identification of a single site under positive selection requires supporting evidence from further studies with better algorithms for a more credible assessment of site-specific subfamily divergence.

 the mitochondrion is the main intracellular site of energy production and is the evolutionary response to the main challenge that living organisms have to face: gaining energy from their environments to sustain their biological functions. the mitochondrial production of atp is realised by the combination of the phosphorylation of adp into atp with an efficient chain of redox reactions, resulting in the so-called oxidative phosphorylation. however, these two processes are not always efficiently coupled, and one reason is the presence in the inner membrane of a family of mitochondrial transporters: the uncoupling proteins. ucp was first discovered and cloned in and is involved in the non-shivering thermogenesis activity of rodent's brown adipose tissue. since then, the discovery of ucp genes has grown rapidly, ucp homologues being found across mammalian species but also in other eukaryotes from plants to animals. most of the recent attention has been devoted to the evolutionary history of ucp since the discovery of ucp in ectotherm organisms like teleost fish and amphibians. the fact that organisms that do not show nst possess and express ucp raised the question of the exact evolutionary history of ucp and of its link with the apparition of thermoregulation. this observation has stimulated an increasing number of phylogenetic studies on ucp to determine the origin of the physiological particularity in the mammalian lineage. ucp and its close homologues are thought to differ in the nature of their uncoupling activity and their potential physiological roles. indeed, a rapid overview of the data collected on ucp and highlights how these proteins may be different. first, while ucp tissue expression is localized to bat, ucp is expressed in a wider range of cell types and ucp is mainly present in skeletal muscle. also, the physiological role of ucp is restricted to thermogenesis, which is unlikely to be the case for ucp and as shown by their respective knock-out models. ucp and have been involved in a number of postulated functions in energy regulation, including regulation of insulin secretion or reactive oxygen species production and control of the immune response. however, accurate data on the mitochondrial activity of ucp and ucp are still lacking to determine the exact nature of their biological activity. therefore, despite the high sequence identity shared by ucp and, punctual amino acid replacement at key structural domains of the respective proteins may have evolved to allow functional specificity to take place. interestingly, mutagenesis experiments have shown that single amino-acid replacement in ucp protein may change its proton permeability, its sensibility to fatty acid activation or nucleotide inhibition, or its transmembrane structure. the next step in the understanding of the biology of ucp is to determine whether the evolution of ucp genes and protein sequences may have been subjected to different selective pressures after duplication. single copy genes are thought to evolve conservatively because of strong negative selective pressure. gene duplications produce a redundant gene copy and thus release one or both copies from negative selection pressure. there are a number of models for the fate of gene duplicates, the two most prominent of which are neofunctionilization and subfunctionalisation. thus, duplications are thought to be an important precursor of functional divergence. the increased availability of ucp sequences in the public databases allows the study of the molecular evolution of the ucp gene family and the evaluation of selection following duplication events. in the present study, we will determine the evolutionary history of the ucp gene family, evaluate the changes in selection pressures following duplications, and identify sites under positive darwinian selection.  sequence similarity searches and multiple alignment two lizard sequences from anolis carolinensis were identified during similarity searches with high similarity to ucp and ucp homologues of ucp were not found in the lizard scaffold genome. table outlines the sequences used in the phylogenetic analyses. it should be noted that additional ucp genes for eutherians and teleosts were identified. inclusion of these did not improve the reliability of the phylogeny, and as the aim of this study was to determine the evolutionary history of the ucp gene family, only representatives from the major vertebrate clades were included. sequence obtained from the february draft assembly ) produced by the broad institute at mit and harvard, sequences obtained from ensembl, * sequences from jgi. phylogeny of the ucp gene family the alignments were used to construct phylogenetic trees with maximum likelihood and bayesian inference. the different reconstruction methods provided poor support for basal nodes using the protein alignment. the dna alignment showed support for the ucp ucp and ucp clades, in particular when fewer distantly related outgroups are used perhaps as a consequence of systematic error. the different reconstruction methods provided slightly different topologies. most relationships could be resolved with confidence dividing the gene family into strongly supported clusters in most tree reconstructions. ucp and ucp genes are sister clusters and the avian ucp gene is grouped within the ucp cluster. the different protein trees were reconciled against a species tree using genetree. the protein ml topology required duplications and losses and the bi duplications and losses. the high number of duplications and losses is a result of the basal topology of the gene tree and a number of incongruences between the gene and species trees. however, in the ml protein phylogenies, the basal relationships have low bootstrap supports. using the dna phylogeny, the ml tree required less duplications and losses than the bi tree. the higher number of duplications and losses in the bi reconstruction is mainly a result of duplications inferred through incongruence between the gene and species trees. if as suggested by the protein phylogenies, the lamprey sequences are sister to the ucp clade and the takifugu ucp groups within the same clade, then the reconciliation infers duplications and losses. however, the results from the dna phylogenies suggest the lamprey sequences could have diverged before the duplication of ucp2/ in this case, by removing losses and duplications inferred through mismatch of species and gene trees and losses due to incomplete genome sequences, the most parsimoniously reconciled tree shows zebrafish specific duplication and two major duplications that occurred early in the vertebrate lineage. one duplication is proposed to have occurred prior to the emergence of teleost fish resulting in two lineages which evolved into ucp and ucp2/ and probably took place early in vertebrate evolution due to the presence of ucp in lampreys, although further data are required to confirm the presence of ucp and ucp in lampreys. a second duplication, also early in vertebrate evolution, resulted in ucp and ucp further sequencing of a broader range of ancestral craniata is required to identify a more precise timing for the duplications. interestingly, ucp and ucp have been independently lost from the avian lineage but further data are required to confirm the absence of ucp in lizards to be able to determine when the loss of ucp took place. synonymous and non-synonymous substitution rate estimates results using the dna dataset show that ucp genes are under varying selection pressures. pairwise comparisons of human and mouse orthologs and human and platypus show that ucp has higher estimates of dn/ds ratio compared to ucp and ucp but suggest purifying selection in all three genes. the lower substitution rates for ucp and ucp shows that they are under strong purifying selection. substitution rates were estimated using yang and nielson method as implemented in yn in the paml package. positive selection tests more sophisticated codon-based substitution models were used to test for branch-specific selection. the model was based on the assumption that selective constraints change following gene duplication. we estimated ω as an average over all sites and branches and the ratio was substantially smaller than. the one-ratio model was compared with model r and the lrt indicated that there is a significant decrease in the rate of non-synonymous substitution following the duplication of ucp and ucp2/. the comparison of model r and r also showed that there was a significant difference in the selective pressure following the duplication of ucp and ucp the branch specific model with three distinct rates of substitution, one for each ucp gene, is a significantly better fit than the one-ratio and two-ratio models according to the lrt. this suggests significantly different selective pressures on ucp ucp and ucp however, none of the parameters estimated indicate positive darwinian selection. the models were implemented in codeml from paml. parameters in bold indicate positive selection. significant tests are shown in bold. the lrt of the one ratio model with m indicates that selective pressure is not uniform among sites. only the m model indicates a site that is evolving under positive darwinian selection. lrts indicate that model m does not fit the data better than m whilst it does show that m fits the data better than m which does not allow for positively selected sites. branch-site models were also applied with ucp specified as the foreground branch, however the m model was the best fit to the data with a likelihood value of –. this model suggests that the variation in selection pressure is due to the evolution by positive selection of a single site, whilst the remaining sites are under strong purifying selection. according to the alignment of the ucp sequences with the 3d structure of the bovine mitochondrial adp-atp carrier, the site under positive selection is in the mitochondrial matrix between the 5th and 6th alpha-helix. as illustrated using hmm logos for each ucp gene, the site under positive selection follows a highly conserved amino acid site present across the whole ucp gene family but the site under positive selection is not conserved within the different ucp groups. the hmm logos also illustrate the high level of sequence conservation in the gene family. discussion in this study, we have sought to expand upon previous phylogenetic studies by focusing on the ucp gene families and incorporating sequences identified from completed genomes with a subset of cloned sequences, particularly those from non-mammalian species. this study is the first to include lizard ucp genes. the phylogenetic tree reconstruction of dna sequences gave well resolved topologies with stronger support values for basal relationships than using the protein data probably as a result of the highly conserved protein sequences. these phylogenies provided a method to infer the evolutionary history of the ucp gene family. the phylogeny of the ucp genes indicates that ucp which is present in plants and arthropods, is the ancestral ucp as demonstrated in previous studies. ucp then duplicated prior to the divergence of vertebrates. a second duplication of ucp and ucp also took place early in vertebrate evolution although the exact timing of the event requires further genomic data to be gathered. the multiple sequences of ucps found in the zebrafish, while termed ucp and ucp are both ucp orthologs and should be called ucp1a and ucp1b. ucp is syntenic to gene a and gene b like other vertebrate ucp genes. this could either be a zebrafish specific duplication, or the incomplete sequencing of cyprinus carpio could be hiding an additional paralog and the duplication may be a fish specific genome wide duplications hypothesised to have occurred during fish evolution. the latter is probably unlikely due to the lack of duplicates in the complete genome of takifugu rubripes. importantly, the phylogenetic analyses suggest the independent loss of ucp and ucp from the avian lineage. the absence of ucp in the lizard genome could be attributed to the incompleteness of the genome or could be the result of a loss of ucp in the sauropsid lineage. ucp is the only uncoupling protein for which there is a scientific consensus concerning the nature of its physiological function. the ucp knockout mice are able to maintain their body temperature, but suffer in pronounced cold exposure suggesting that ucp is principally involved in short-term adaptation to cold. this adaptive evolution probably occurred after the divergence between eutherians and marsupials consistent with the fact that bat is only found in eutherians. even though birds are lacking ucp they are still able to respond to thermal challenges. the loss of ucp and disappearance of bat in birds is likely due to the concomitant development of physiological adaptations which have replaced bat function. as evidence, metabolic rate of birds increases in response to cold and body temperature can be maintained. indeed, induced uncoupling activity in the mitochondria has been found in the skeletal muscle of cold-acclimated birds and more recently the implication of ucp has been suggested. these data lead to two non exclusive conclusions. firstly, birds have evolved other mechanisms of thermoregulation before or after the loss of ucp and bat. secondly, a fully demonstrated implication of ucp in skeletal non-shivering thermogenesis in birds would suggest that ucp which is not involved in thermoregulation in mammals, has acquired a new function in birds. in this case, the question is whether avianucp activity could also compensate for the loss of the ucp gene, implicated in mammalian immunity and glucose metabolism. this is an interesting point given the non pathologic high chronic glycemia of birds. the molecular evolution of ucp genes showed that they were under strong purifying selection with a significant change towards stronger purifying selection. ucp has the highest dn/ds ratio followed by ucp and then ucp this strong purifying selection highlights the importance of the function of this highly conserved gene family. although highly variable regions of the sequence which were difficult to assign as homologous were removed from the analyses, the site models showed that adaptation has appeared at a single site located between the 5th and 6th α-helices. the role of this positively selected site has yet to be determined but the amino acid site immediately prior to it is highly conserved across mitochondrial carriers as are the transmembrane regions that follow the site. additionally, saito et al. found that the two amino acid sites that follow this site are conserved in all eutherian mammal ucp genes. based on studies conducted on ucp the region delimited by the 5th and 6th α-helices is close to a site of regulation of ucp activity by nucleotides and thus could be implicated in the inhibitory control of ucp uncoupling effect. this region is also hypothesized to be implicated in the mechanism of transport of protons/free fatty acids in ucp however, to date there is a gap in the knowledge of the relationship between amino acid sequence and structure for ucp and ucp and we are unable to speculate on the particular role of this region in these ucp homologues. unfortunately, shared evolutionary history and molecular selection alone cannot be used as the unique criterion to infer protein function, and the true nature of each ucp gene needs to be determined experimentally and independently. therefore, this positively selected site may play an important functional role and could represent an interesting target site for future mutagenesis experiment thus facilitating our understanding of the structure-function relationships in ucp genes.  genomic data have provided an opportunity to gain a better understanding about the evolution of ucps using phylogenetic analyses. the ucp gene family phylogeny shows that two duplications took place early in the evolution of vertebrates. subsequent to these two duplications, ucp and ucp were lost from the avian lineage independently. however, further genome projects on a greater diversity of evolutionary lineages are required to better understand the gene-duplication history. evolutionary rate analysis shows purifying selection across branches and sites suggesting that the function of the genes in the ucp gene family has been highly conserved after duplication events and over evolutionary time. by considering the evolutionary history of the ucp gene family we provide insight into which amino acid residues might have undergone positive selection and could be targeted for site-directed mutagenesis. however, the identification of a single site under positive selection requires supporting evidence from further studies with better algorithms for a more credible assessment of site-specific subfamily divergence. 
estimates of synonymous and nonsynonymous substitution rates and more complex branch and site models suggest that the duplication events were not associated with positive darwinian selection and that the ucp is constrained by strong purifying selection except for a single site which has undergone positive darwinian selection, demonstrating that the ucp gene family must be highly conserved. the uncoupling protein genes belong to the superfamily of electron transport carriers of the mitochondrial inner membrane. phylogenetic analysis was used to characterize the family evolutionary history by identifying two duplications early in vertebrate evolution and two losses in the avian lineage. we present a phylogeny describing the evolutionary history of the ucp gene family and show that the genes have evolved through duplications followed by purifying selection except for a single site in the mitochondrial matrix between the 5th and 6th α-helices which has undergone positive selection. members of the uncoupling protein family are involved in thermogenesis and determining the functional evolution of ucp genes is important to understand the evolution of thermo-regulation in vertebrates. sequence similarity searches of genome and scaffold data identified homologues of ucp in eutherians, teleosts and the first squamates uncoupling proteins.
 the uncoupling protein genes belong to the superfamily of electron transport carriers of the mitochondrial inner membrane. members of the uncoupling protein family are involved in thermogenesis and determining the functional evolution of ucp genes is important to understand the evolution of thermo-regulation in vertebrates.  sequence similarity searches of genome and scaffold data identified homologues of ucp in eutherians, teleosts and the first squamates uncoupling proteins. phylogenetic analysis was used to characterize the family evolutionary history by identifying two duplications early in vertebrate evolution and two losses in the avian lineage. estimates of synonymous and nonsynonymous substitution rates and more complex branch and site models suggest that the duplication events were not associated with positive darwinian selection and that the ucp is constrained by strong purifying selection except for a single site which has undergone positive darwinian selection, demonstrating that the ucp gene family must be highly conserved.  we present a phylogeny describing the evolutionary history of the ucp gene family and show that the genes have evolved through duplications followed by purifying selection except for a single site in the mitochondrial matrix between the 5th and 6th α-helices which has undergone positive selection. 