Evolutionary history of the UCP gene family: gene duplication and selection
© Hughes and Criscuolo; licensee BioMed Central Ltd. 2008
Received: 27 March 2008
Accepted: 03 November 2008
Published: 03 November 2008
The uncoupling protein (UCP) 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 (excluding duplications within a species, excluding the losses due to incompletely sequenced taxa and excluding the losses and duplications inferred through mismatch of species and gene trees). Estimates of synonymous and nonsynonymous substitution rates (dN/dS) 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.
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, ). UCP1 was first discovered and cloned in 1986  and is involved in the non-shivering thermogenesis (NST) activity of rodent's brown adipose tissue (BAT, ). Since then, the discovery of UCP genes has grown rapidly, UCP1 homologues being found across mammalian species (UCP2, UCP3, [4, 5]) but also in other eukaryotes from plants to animals [6–8]. Most of the recent attention has been devoted to the evolutionary history of UCP1 since the discovery of UCP1 in ectotherm organisms like teleost fish  and amphibians . The fact that organisms that do not show NST possess and express UCP1 raised the question of the exact evolutionary history of UCP1 and of its link with the apparition of thermoregulation. This observation has stimulated an increasing number of phylogenetic studies on UCP [10–15] to determine the origin of the physiological particularity (cold-induced thermogenesis in BAT) in the mammalian lineage .
UCP1 and its close homologues (UCP2 and UCP3) are thought to differ in the nature of their uncoupling activity [16, 17] and their potential physiological roles (see ). Indeed, a rapid overview of the data collected on UCP1, 2 and 3 highlights how these proteins may be different. First, while UCP1 tissue expression is localized (and abundant) to BAT, UCP2 is expressed (in smaller quantities) in a wider range of cell types (like immune or pancreatic β-cells) and UCP3 is mainly present in skeletal muscle ([4, 5], see ). Also, the physiological role of UCP1 is restricted to thermogenesis, which is unlikely to be the case for UCP2 and 3 as shown by their respective knock-out models [20, 21]. UCP2 and 3 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 [20, 23, 24]. However, accurate data on the mitochondrial activity of UCP2 and UCP3 are still lacking to determine the exact nature of their biological activity [17, 25]. Therefore, despite the high sequence identity shared by UCP1, 2 and 3 (close to 60% in humans and mice), 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 UCP1 protein may change its proton permeability (nature of the mitochondrial transport), its sensibility to fatty acid activation or nucleotide inhibition (regulation of the activity, ), 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 (1) the evolutionary history of the UCP gene family, (2) evaluate the changes in selection pressures following duplications, and (3) identify sites under positive Darwinian selection.
Sequence similarity searches and multiple alignment
List of species and accession numbers for protein and DNA sequences
Anolis carolinensis (a)
Anolis carolinensis (a)
Phylogeny of the UCP gene family
The 2 different protein trees were reconciled against a species tree using GeneTree. The protein ML topology required 14 duplications and 47 losses and the BI 15 duplications and 51 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 (8 d + 2 l) than the BI tree (12 d + 42 l). 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.
Synonymous and non-synonymous substitution rate estimates
Synonymous (dS) and nonsynonynous (dN) substitution rates for all UCP genes
Positive selection tests
Parameter estimates for UCP genes under different branch models, site models and branch-site models
Parameters for branches
Positively selected sites
ω0 = 0.07649
ω0 = 0.0950
ω1 = 0.0660
ω0 = 0.0946
ω1 = 0.0845
ω2 = 0.0496
ω0 = 0.0657, ρ0 = 0.92529
ω1 = 1, ρ1 = 0.07471
ω0 = 0.06571, ρ0 = 0.9253
ω1 = 1, ρ1 = 0.07470
ω2 = 1.266, ρ2 = 0.00000
ω0 = 0.1109, ρ0 = 0.49068
(K = 3)
ω1 = 0.10086, ρ1 = 0.38265
ω2 = 0.32064, ρ2 = 0.12668
ρ = 0.50769 q = 4.86274
ρ0 = 0.99, p = 0.53223
224 K (P = 0.914)
q = 5.57248, ρ1 = 0.00373, ω1 = 1.69524
ρ0 = 0.9155, ρ1 = 0.04732,
In the foreground lineage:
ρ2a = 0.03529, ρ2b = 0.00182
180 H (P = 0.953), 220 L (P = 0.997), 235 M (P = 0.969)
ω2 = 0.06259
ρ0 = 0.49203, ρ1 = 0.43366,
In foreground lineage:
ρ2a = 0.03950, ρ2b = 0.03481
No significant sites
ω0 = 0.012, ω1 = 0.12812, ω2 = 0.52
In the background lineage:
no significant site
Likelihood ratio test statistics (2δ) for the test of model fit
One ratio versus H1
One ratio versus H2
H1 vs H2
LRTs of variable w's among sites
One ratio vs. M3
M1 vs M2
M7 vs M8
In this study, we have sought to expand upon previous phylogenetic studies [10, 15, 26] 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 UCP1, which is present in plants and Arthropods, is the ancestral UCP as demonstrated in previous studies [10, 15, 26]. UCP1 then duplicated prior to the divergence of vertebrates. A second duplication of UCP2 and UCP3 also took place early in vertebrate evolution although the exact timing of the event (before or after the divergence of lampreys) requires further genomic data to be gathered. The multiple sequences of UCPs found in the zebrafish, while termed UCP4 and UCP3 are both UCP1 orthologs and should be called UCP1a and UCP1b. UCP4 is syntenic to Gene A and Gene B like other vertebrate UCP1 genes (Figure 3). 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 [29, 30]. 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 UCP1 and UCP2 from the avian lineage. The absence of UCP1 in the lizard genome could be attributed to the incompleteness of the genome or could be the result of a loss of UCP1 in the sauropsid lineage.
UCP1 is the only uncoupling protein for which there is a scientific consensus concerning the nature of its physiological function (thermogenesis, ). The UCP1 knockout mice are able to maintain their body temperature, but suffer in pronounced cold exposure suggesting that UCP1 is principally involved in short-term adaptation to cold (Enerback et al. 1997). 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 UCP1, they are still able to respond to thermal challenges. The loss of UCP1 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 [33, 34] and more recently the implication of UCP3 (avianUCP) 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 UCP1 and BAT (e.g.: futile cycle of Ca2+ in bird skeletal muscle or greater adenine nucleotide translocase-catalysed proton conductance, [35, 36]). Secondly, a fully demonstrated implication of UCP3 (avianUCP) in skeletal non-shivering thermogenesis in birds would suggest that UCP3, which is not involved in thermoregulation in mammals [21, 37], has acquired a new function in birds. In this case, the question is whether avianUCP activity could also compensate for the loss of the ucp2 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. UCP1 has the highest dN/dS ratio followed by UCP3 and then UCP2. 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 (Y) 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 ucp1 genes. Based on studies conducted on UCP1, the region delimited by the 5th and 6th α-helices is close to a site of regulation of UCP1 activity by nucleotides and thus could be implicated in the inhibitory control of UCP1 uncoupling effect [15, 26]. This region is also hypothesized to be implicated in the mechanism of transport of protons/free fatty acids  in UCP1. However, to date there is a gap in the knowledge of the relationship between amino acid sequence and structure for UCP2 and UCP3, and we are unable to speculate on the particular role of this region in these UCP1 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, UCP1 and UCP2 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 (except for one single site with site specific positive selection) 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.
Sequences and sequence similarity searches
Amino acid and nucleotide sequences of UCP gene family members were obtained from GenBank for most species (see Table 1 for accession numbers). The sequences for the lizard (Anolis carolinensis) were obtained from the February 2007 draft assembly (Broad Institute AnoCar (1.0)) produced by the Broad Institute at MIT and Harvard . A total of 50 sequences for 27 species were used in the final analyses.
Multiple sequence alignment and phylogenetic analysis
Fifty protein sequences were aligned using MUSCLE  and gaps, which are problematic in phylogenetic analysis, were removed using Gblocks 0.91b . The final protein dataset was 274 amino acids long (Additional material 2). Uncoupling proteins from plants, insects, the sea squirt and the sea urchin were included. Nucleotide sequences were aligned using ClustalX  with the default parameters followed by manual alignment in Macclade  according to the amino acid translation. Regions before the starting codon were excluded from the analysis as well as regions poorly aligned due to uncertain homology (positions from the first nucleotide of the start codon: 64–66, 142–180, 331–375, 469–504, 931 to end). The final dataset was 810 nucleotides long (Additional material 3).
Phylogenetic trees were reconstructed using maximum likelihood (ML) implemented in PHYML and Bayesian inference (BI) in MrBayes. Phyml v2.4.4  was used with the online web server  for maximum likelihood analysis using the GTR+I+G substitution DNA model selected with ModelTest  and JTT substitution model selected with ModelGenerator for the protein analyses . The robustness of the trees were assessed by bootstrapping (500 pseudoreplicates) with PHYML. Bayesian analyses were conducted using the same model with MrBayes v3.1.2 . Node support was assessed as the posterior probability from two independent runs each with four chains of 200,000 generations (sampled at intervals of 100 generations with a burn-in of 1000 trees).
Reconciliation of gene and species trees
Gene trees of the UCP gene family were reconciled with a species tree using GeneTree . GeneTree attempts to resolve the incongruence between the gene and species trees by predicting duplications and losses . The species tree was based on the Tree of Life phylogeny  and NCBI taxonomy . The reconciled tree was edited to remove losses and duplications inferred due to mismatches of the species and gene trees.
Estimation of substitution rates and testing positive selection
Synonymous (dS) and non-synonymous (dN) substitution rates were estimated using the methods of Yang and Nielson  as implemented in yn00 in the PAML software . The two trees (ML and BI) were tested separately for positive selection. Using Codeml from PAML the branch specific models, One-ratio (R1) and Two-ratios (R2) were used to detect lineage-specific changes in selective pressure after the duplication events. The site specific models, Neutral (M1), Selection (M2), Discrete (M3) with 3 site classes, Beta (M7) and Beta&ω (M8) were also used to test for individual residues under positive selection. Likelihood ratio tests (LRT) were used to assess their goodness of fit, by comparing a model that does allow for dN/dS>1 against a model that does not (i.e. null model). Therefore, the branch specific LRT was R2 vs R1. The site specific LRTs were M3, M2 and M8 against their respective null models, M0, M1 and M7. Positively selected sites were listed. Because some of the models like M2 and M8 are noted to be prone to the problem of multiple local optima, we ran the program twice, once with a starting omega value <1 and a second time with a value >1. We used the results corresponding to the highest likelihood.
The authors wish to thank Barbara Mable for her suggestions and recommendations for the molecular selection analyses. We thank two anonymous reviewers for their comments on this manuscript. This work was conducted during the NERC grant NE/B000079/1 to JH.
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