- Research article
- Open Access
The evolutionary history of the SAL1 gene family in eutherian mammals
© Meslin et al; licensee BioMed Central Ltd. 2011
- Received: 4 March 2011
- Accepted: 28 May 2011
- Published: 28 May 2011
SAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions.
Here, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection.
This work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.
- Gene Conversion
- Olfactory Receptor
- Eutherian Mammal
- Concerted Evolution
- Mouse Lemur
The barriers that lead to divergence of species during the course of evolution were classified by Dobzhansky in two categories: prezygotic and postzygotic reproductive barriers . Postzygotic reproductive barriers concern all the events that occur after fertilization, such as reduced hybrid viability and fertility, while prezygotic reproductive barriers concern isolation of sexual partners via ecological, temporal or behavioral isolation. Pheromones play a key role in pre-mating recognition of sexual partners . These compounds are defined as substances released by an animal that are able to induce specific behavioral and/or endocrinological reactions in a sexual partner of the same species . Through these reactions, they could be involved in mate choice and sexual selection.
Odorant binding proteins (OBP) are small soluble proteins that are present in the olfactory apparatus as well as in biological fluids such as saliva, urine or vaginal discharge, and are able to bind pheromones (for review see ). OBP are assumed to be directly involved in chemical communication and in the pre-mating recognition process. Three hypotheses are proposed concerning their mechanism of action. The first is that olfactory receptors can recognize the OBP/pheromone complex, not just the pheromone alone. The second hypothesis is that the pheromone can be transferred to olfactory receptors only if assisted by the OBP. The third hypothesis is that the ligand can spontaneously dissociate from the complex with OBP and bind to the receptor as a "free pheromone" .
The role of saliva in chemical communication between males and females is well established in pig , like the role of urine in mouse . In pig, saliva contains the pheromonal steroids 5α-androst-16-en-3-one and 5α-androst-16-en-3α-ol, as well as abundant quantities of salivary lipocalin (SAL1), the most abundant OBP isolated from submaxillary glands of mature males. When extracted from its source, this protein is associated with both pheromonal steroids , and appears to play a key role in the standing reflex in the sow  and also in the boar's libido . SAL1 is also expressed in the nasal and vomeronasal area, but devoid of ligand [10, 11]. SAL1 exhibits a classical structure of lipocalins characterized by a fully conserved N-terminal -G-X-W- motif and the typical folding pattern of a nine-stranded antiparallel β-barrel forming an internal ligand binding site for small hydrophobic molecules , despite relatively low sequence similarity . SAL1 also possesses a glycosylation site on Asn53. Two natural variants have been identified in which in three residues differ (Val61, Ile64 and Ala89 of isoform A are respectively Ala, Val and Val in isoform B). Two residues (Val61 and Ala89) are located inside the β-barrel while the third residue (Ile64) is located next to the β-barrel, suggesting that these minor structural differences lead to ligand binding specificities .
Olfactory receptors are located on the olfactory sensory neurons of the main olfactory system in mammals and on the vomeronasal organ in rodents and other non-primate species . Several authors examined the evolution of olfactory receptors, but few studies of lipocalins and OBP have been performed. Ganfornina et al.  undertook phylogenetic analysis of prokaryotic and eukaryotic lipocalins and showed that this family appeared early and is composed of 13 monophyletic clades. These authors also showed that ancestral lipocalin clades in the phylogenetic tree are able to bind large ligands while more recent lipocalin clades, such as clades composed of OBP and MUP (Mouse Urinary Protein), bind smaller ligands. They also found that later clades had higher rates of amino acid substitution, more flexible protein structures and greater ligand-binding efficiency than more ancestral lipocalins.
Logan et al.  undertook an extensive study of the Mup cluster in the mouse genome. They identified 21 Mup genes and 21 Mup pseudogenes on chromosome 4. They also identified Mup gene expansion in rat (9 genes and 13 pseudogenes), in horse (3 genes) and in mouse lemur (2 genes and 1 pseudogene) in the same syntenic region. Orangutan, chimpanzee, dog, pig (with SAL1), bushbaby and rhesus monkey have only one Mup gene in the syntenic region. The inferred phylogeny, the accumulation of synonymous substitutions, and the genomic organization of the Mup loci suggest that gene expansion occurred independently in several species .
In the light of previous analyses, the aim of the present work was to study the evolution of SAL1 which is involved in pre-mating recognition in pig. We wanted to determine if selective pressures act on these proteins and to check if positive selection may play a role in binding specificity toward ligands or olfactory receptors.
Identification of SAL1 homologous genes, genomic localization and phylogenetic study
Evolution of paralogs in the SAL1 family
Interlocus gene conversion events
Number of sequences in the dataset (1)
Number of sequence involved in a gene conversion event (2)
Converted tract length (bp)
Parameter estimates and likelihood scores for branch-site models for paralogs
Estimates of parameters (2)
Positively selected sites (BEB)
ρ0 = 0.32, (ρ1 = 0.68), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.30, ρ1 = 0.66, (ρ2 = 0.05), ω0 = 0.23, (ω1 = 1), ω2 = ∞
1 site p > 99%: 36Y
Guinea pig ENSCPOG00000023399
ρ0 = 0.25, (ρ1 = 0.50), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.27, ρ1 = 0.55, (ρ2 = 0.18), ω0 = 0.23, (ω1 = 1), ω2 = 336.70
7 sites p > 99%: 24R, 27A, 113L, 118Q, 123T, 125T, 128T, 8 sites p > 95%: 31L, 25E, 26T, 85V, 114T, 117T, 122V, 126L
ρ0 = 0.22, (ρ1 = 0.47), ω0 = 0.23, (ω1 = 1)
ρ0 = 0.09, ρ1 = 0.19, (ρ2 = 0.72), ω0 = 0.23, (ω1 = 1), ω2 = 6.44
6 sites p > 95%: 81E, 97A, 123Q, 135K, 166K, 168F
ρ0 = 0.32, (ρ1 = 0.67), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.30, ρ1 = 0.66, (ρ2 = 0.04), ω0 = 0.23, (ω1 = 1), ω2 = ∞
2 sites p > 99%: 174K, 177F
ρ0 = 0.32, (ρ1 = 0.67), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.32, ρ1 = 0.66, (ρ2 = 0.02), ω0 = 0.24, (ω1 = 1), ω2 = ∞
1 site p > 99%: 135A
Positively selected sites in the SAL1 family and putative biological significance
Parameter estimates and likelihood scores for site models
Estimates of parameters (2)
Positively selected sites (BEB) (4)
ω = 0.92
ρ0 = 0.32
ρ0 = 0.24, ρ1 = 0.53, ρs = 0.23, ωs = 2.08
35.59 *** (M2a vs M1a)
1 site p > 99%: 72Y, 9 sites p > 95%: 9V, 10T, 62R, 73A, 75C, 86A, 90E, 159R, 162Q
p = 0.64, q = 0.24
ρ0 = 0.41, ρ1 = 0.59, p = 1.69, q = 3.62
ρ0 = 0.71, ρs = 0.29, p = 0.83, q = 0.44, ωs = 1.84
33.02 *** (M8 vs M8a)
9 sites p > 99%: 9V, 10T, 62R, 72Y, 73A, 75C, 86A, 90E, 162Q; 7 sites p > 95%: 6Q, 11S, 63K, 71F, 113G, 119L, 159R
Estimates of parameters (2)
Positively selected sites (BEB) (4)
p = 1.10, q = 1.73
p = 0.80, q = 2.56
5 sites p > 95%: 9V, 72Y, 73A, 90E, 163L
Positive selection events in marmoset, dog, guinea pig, horse and mouse clades
Parameter estimates and likelihood scores for branch- site models for 5 species
Estimates of parameters (2)
Positively selected sites (BEB)
ρ0 = 0.18, (ρ1 = 0.39), ω0 = 0.23, (ω1 = 1)
ρ0 = 0.30, ρ1 = 0.63, (ρ2 = 0.07), ω0 = 0.23, (ω1 = 1), ω2 = 60.38
2 sites p > 95%: 159R, 161F
ρ0 = 0.25, (ρ1 = 0.52), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.28, ρ1 = 0.58, (ρ2 = s0.14), ω0 = 0.23, (ω1 = 1), ω2 = 11.15
2 sites p > 95%: 14H, 144Y
ρ0 = 0.32, (ρ1 = 0.67), ω0 = 0.24, (ω1 = 1)
ρ0 = 0.27, ρ1 = 0.51, (ρ2 = 0.22), ω0 = 0.25, (ω1 = 1), ω2 = 3.94
3 sites p > 95%: 11S, 75C, 104V
ρ0 = 0.32, (ρ1 = 0.67), ω0 = 0.24, (ω1 = 1 )
ρ0 = 0.30, ρ1 = 0.61, (ρ2 = 0.09), ω0 = 0.24, (ω1 = 1), ω2 = 7.57
2 sites p > 99%: 75C, 119L; 1 site p > 95%: 144Y
ρ0 = 0.24, (ρ1 = 0.55), ω0 = 0.19, (ω1 = 1 )
ρ0 = 0.25, ρ1 = 0.58, (ρ2 = 0.17), ω0 = 0.19, (ω1 = 1), ω2 = 3.25
1 site p > 95%: 162Q
Phylogenomic analyses showed that the SAL1 family originated in eutherian mammals and that genes belonging to this family were duplicated after speciation events in five mammalian species. In certain living species, such as mouse lemur, bushbaby, orangutan and human, the gene has been lost, as it has in the Neanderthal genome. We can date the loss of the gene in hominid before the Neandertal-modern human split, 400,000 to 350,000 years ago . The number of duplication events varies greatly among species. In mouse and rat, massive cis-duplication events have occurred, with respectively 42 and 22 genes in the cluster, followed by gene loss with respectively 21 and 11 pseudogenes.
Gene duplication represents a source of new genetic material, and can lead to evolutionary novelties. The fate of duplicated genes can follow different models of evolution, with different selective pressures acting on the genes . We checked for a change in selective pressure in all paralogs identified in the SAL1 family and found that only a few paralogs underwent positive selection: one gene in cow, guinea pig, horse, mouse and rat. Moreover, few sites of these genes were identified. A large proportion (66%) of each gene evolved under neutrality and only a small proportion (2 to 5%) under positive selection. However, among the single genes identified as positively selected in guinea pig and horse, a larger proportion of sites evolved under positive selection (18 and 72%, respectively) and more sites were identified as being positively selected (15 sites in guinea pig and 6 sites in horse).
Because sequences of some paralogs share high similarity, we searched for gene conversion in our paralogous gene datasets and found extensive interlocus gene conversion events in mouse and guinea pig, and to a lesser extent in horse and rat. Karn and Laukaitis  compared the mouse Mup cluster with a gene tree published by Mudge et al.  and suggested that concerted evolution masked the common origin of the gene and neighboring pseudogenes . Our results confirmed this hypothesis, indicating extensive gene conversion in the mouse Mup cluster. This extensive gene conversion phenomenon led to sequence homogenization and is the cause of the concerted evolution of these genes. Such extensive concerted evolution suggests that, at least in mouse and guinea pig, both maintenance of sequence homogeneity and increased gene dosage are important for these species. The evolution of SAL1 paralogs resembles the evolution of the β-globin gene family. In this family, paralogous copies evolved under a process of functional divergence and there is evidence for two gene conversion events in mouse and goat clusters composed of β-globin duplicated genes There is also evidence for variable selective pressure among sites for β and γ-globin genes with 4 to 9% of sites evolving under positive selection .
By combining phylogenetic, gene conversion and selective pressure results on paralogs evolution, we can try to describe the fate of duplicated genes, in which duplication can be seen as an advantageous phenomenon for the species concerned, by combining two scenarios from Innan and Kondrashov . In the first scenario, one could consider the massive duplication in rat and mouse as a gene amplification where the increase in dosage of these genes is beneficial. This scenario of evolution corresponds to category IIa described by Innan and Kondrashov . In this model, if selection for the duplicated copy is weak, pseudogenization can occur if a null mutation is fixed, which is the case in both mouse and rat. The occurrence of gene conversions that maintain sequence similarity and promote conservation of gene copies could be consistent with that hypothesis, but the high frequency of gene conversion events is not restricted to mouse and rat. In fact, guinea pigs, which do not harbor large gene amplifications, have the highest frequency of conversion events per gene copy among the species tested. The beneficial increase in dosage has already been shown to apply to genes that mediate the interaction between the organism and the environment , as is true of genes of the SAL1 family. However, we also showed that among the many duplicates in rat, mouse and guinea pig, one gene per species is under positive selection so increased gene dosage and gene conversion events are not the only driving force of the evolution of these genes in these species. For these positively selected duplicates, it is the scenario of the category III  which fits, where a new copy can be fixed and preserved by positive selection, leading to the possible emergence of a new function for the positively selected gene.
To study selective pressure in the SAL1 family in more detail, we tested the amino acids changing occurring in the 12 branches supporting species as positively selected by PAML. Our results showed that marmoset, dog, guinea pig, horse and mouse branches underwent positive selection just after divergence. This evolutionary scenario likely reflects the ability of the SAL1 family to diverge and to adapt to new behaviour between sexual partners. A previous study on mouse and rat genes identified 32 sites as positively selected on rodent co-orthologs of SAL1 . In that study, mouse and rat genes were considered together, whereas in our study, mouse and rat genes were analyzed separately, this explains the difference between the two results. Indeed, we only identified one site under positive selection in mouse and no positive selection in rat. The difference between the two results is also due to a difference in the probability threshold chosen to determine whether a site is subject to positive selection or not. In Emes et al. , a site was said to be positively selected if the probability for one model is > 0.90 and > 0.50 for at least one other model. In our study, we chose to consider only sites whose probability was > 0.95 in order to minimize false positives.
Finally, we compared tests of variable selective pressures for the family using several PAML codon models. We found evidence for positive selection in a small proportion of sites. Because positive selection is known to play a role in the diversification of protein functions, we mapped all positively selected sites on the 3D structure, in order to assess their biological significance for the gene family as a whole and for each species independently. Apart from the three amino acids that were under positive selection and involved in ligand binding, the other amino acids identified by site models of PAML analyses projected out of the binding pocket. Moreover, the majority of these sites were exposed to solvent. If these sites were involved in the interaction with pheromones, they would be found preferentially in the hydrophobic core and would be buried. We thus propose that positive selection plays a role not only in the binding specificity but also in the interaction between the protein and its environment. We were not able to draw any conclusions concerning selective pressures on each site involved in ligand binding, because gaps in the multiple sequence alignment made these calculations impossible. Nevertheless, for the 16 amino acids involved in pheromone binding, we identified three sites that probably evolved under purifying constraints (87Y, 91N and 93F) and four sites that probably evolved under relaxed constraints (60F, 85V, 121E and 123Y). The three sites that evolved under purifying constraints may be essential for protein function, because they were well conserved during the evolution of the family. In rodent populations, Emes et al.  found that MUPs, which are co-orthologs of SAL1, exhibited amino acids under positive selection, and that these positively selected sites were located at the interface between MUPs and their receptors, probably V2R receptors on the vomeronasal organ. They also found evidence that olfactory receptors, such as V2Rs, underwent positive selection. The hypothesis they proposed is that this adaptation phenomenon is due to conspecific competition, resulting in well adapted pheromones, pheromone binding proteins such as MUP, and olfactory receptors . Our results allow us to extend this hypothesis because positive selection also drives the evolution of pheromone binding proteins in other eutherian mammals. So for all the family, and not just for rodents, there is an adaptive evolution of these proteins to their ligands and maybe their receptors, too. It would be interesting to test if V2R receptors are subject to positive selection, not only in rodents but also in other mammals. Several authors reported evidence for positive selection on other OR genes in mammals [35–39], with possible involvement of positively selected sites in the binding property of proteins. Moreno-Estrada  suggested that positive selection could be at the origin of a new ligand binding capability or the modification of odorant perception and could improve the overall degenerated OR gene repertoire, at least in human. In insects, co-evolution of the two enzymes involved in the pheromone biosynthetic pathway and in the pheromone receptor has been suggested to play a role in the speciation process . It would be interesting to test co-evolution of enzyme/receptor, pheromone/receptor and OBP/receptor in mammals.
In mice, MUPs are important for the delivery, via urine, of chemical signals conveying information about the sex and hormonal status of the animal who release the scent mark . In pig, SAL1 may be involved in pre-mating recognition by binding pig specific sex pheromones in saliva . In both species, these proteins are involved in conspecific recognition in the context of reproduction. When the genomes of marine mammals are completed, it will be interesting to search for SAL1 orthologs. Indeed, in such a different environment, chemical communication between sexual partners is probably not mediated by the same olfactory cues as in terrestrial mammals. If a SAL1 ortholog is found in marine mammal genomes, it will be interesting to discover if it evolved under relaxed constraints or positive selection.
It is well established that reproduction is a very competitive process, and that selective pressures on genes involved in the process are not rare (for a review, see ). Positive Darwinian selection is not atypical, especially for genes involved in sensory perception and mate choice . Our results demonstrated that (i) positively selected sites differ between genes and (ii) positively selected sites are involved in ligand binding and are putatively involved in receptor binding. Such a selective pressure on these proteins could be at the origin of a divergence process between species and thus contribute to the speciation phenomenon by reinforcing prezygotic barriers. To test this hypothesis, we performed in vitro mutagenesis experiments on SAL1, but the poor folding of the resulting proteins prevented further experimentation.
The SAL1 gene family originated in eutherian mammals and duplicated after speciation in cow, horse, guinea pig and rodents. Some duplicated genes underwent concerted evolution with extensive gene conversion. Others were subject to positive selection at different sites, and our knowledge of the 3D structure of this protein suggests that the selected sites are involved in pheromone binding and possibly in olfactory receptor binding. This result suggests a functional divergence between species because positively selected sites differ between species. All these data suggest that the evolution of the SAL1 family allows a species-specific strategy to transduce pheromonal signals in mammals, reinforcing species divergence through species-specific sexual behaviour.
Phylogenetic and syntenic analyses
The protein sequence of the pig salivary lipocalin (SAL1) was retrieved from GenBank (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/genbank/)  (NP_998979.1). Proteins from other species were searched by using TBLASTN with porcine protein sequence as the query against all mammalian genomes available on the NCBI (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/mapview/)  and ENSEMBL databases (http://www.ensembl.org/index.html) . Identified proteins were then located on genomes for syntenic analyses of the most recent genome sequence assemblies: pig (Sus scrofa: ENSEMBL Sscrofa9), cow (Bos Taurus: NCBI Btau5.2), horse (Equus caballus: NCBI EquCab2.0), dog (Canis familiaris: ENSEMBL CanFam2.0), guinea pig (Cavia porcellus: ENSEMBL cavPor3), rat (Rattus norvegicus: NCBI RGSC 3.4), mouse (Mus musculus: NCBIM37), rabbit (Oryctolagus cuniculus: ENSEMBL OryCun2), rhesus monkey (Macaca mulatta: NCBI Build 1.2), chimpanzee (Pan troglodytes: NCBI Build 2.1), gorilla (Gorilla gorilla: ENSEMBL gorGor3), marmoset (Callithrix jacchus: ENSEMBL C_jacchus3.2.1) and elephant (Loxodonta Africana: ENSEMBL loxAfr3). To improve homology assignment, we only included genes from the same syntenic region in the final dataset. Sequences with no syntenic information were discarded. No genes were identified in other available mammalian genomes, and existing genome assemblies did not allow us to identify the syntenic region. Multiple sequence alignments were performed using the Clustal W algorithm . The chimpanzee sequence was removed from the dataset in order to have the most possible informative sites. All alignment gap sites were removed before phylogenetic analyses. Phylogenetic trees were reconstructed using maximum likelihood (ML) in PhyML 3.0  in order to establish orthologous and paralogous relationships among the gene datasets. Bootstrap values  were estimated with 1000 replications and the tree was rooted using the midpoint rooting method. Orthology and paralogy relationships were inferred from the resulting phylogenetic tree.
The four clusters of paralogs identified for the guinea pig, horse, rat and mouse were tested for interlocus gene conversion, i.e. nonreciprocal transfer of genetic information between genes of the same locus, using GENECONV version 1.81 , which is a widely used method for detecting partial gene conversion . Each subset alignment was analyzed using the Clustal W algorithm  to search for pairs of sequences sufficiently similar to suggest gene conversion events. Three p-values were calculated and compared to assess the significance of the results. Evidence for gene conversion was strong when a fragment had a p-value < 0.05 for at least two different types of statistical tests. In each alignment, indels and missing data were treated as a single polymorphism. All polymorphic sites were tested for evidence of gene conversion using adjusted mismatch penalties of 0, 1 or 2, to enable detection of both ancient and recent gene conversion events.
To investigate selective pressure, we used the CODEML application in the PAML package version 4.4 , which allows the ratio dN/dS to vary across codons and estimates the probability for each codon to be under positive selection. The alignments resulted from Clustal W  and PAL2NAL .
Study of selective pressure in the SAL1 family
To determine if selective pressure varied among sites in the SAL1 family, we used site models implemented in PAML , which allows the ω ratio to vary among sites [52, 53]. Like for reconstruction of the phylogenetic tree, the chimpanzee sequence (the shortest sequence) was removed in order to have the most possible informative sites. We used three pairs of models including M1a (nearly neutral: 0 < ω 0 <1 and ω1 = 1) versus M2a (positive selection: 0 < ω 0 < 1, ω 1 = 1 and ω 2 >1) , M8a (beta & ω s = 1: 0 < ω < 1 and ω s = 1) versus M8  and MEC (a combined mechanistic-empirical model implemented in the Selecton server, http://selecton.tau.ac.il/index.html) [25, 55] versus M8a and the PhyML generated tree for the analysis. Likelihood ratio tests were used to compare log likelihood values for M1a vs. M2a and M8a vs. M8 . The Akaike information criterion (AICc score) was used to compare M8a and MEC . Bayes Empirical Bayes (BEB) method  implemented in PAML was used to estimate posterior probabilities of selection on each codon, probabilities > 0.95 were considered significant.
Study of selective pressure on species and paralogs
To determine whether different species underwent selective pressure, we used the branch-site models of PAML [27, 57], which estimate different dN/dS values among branches and among sites. These models can detect a short episode of positive selection if it occurs in a small fraction of amino acids. We tested 13 branches as the foreground branch (i.e. the branch for which positive selection is allowed), eight branches leading to a species (pig, dog, rabbit, macaque, human, gorilla, marmoset and elephant) and five internal branches situated after speciation and before duplication events (in cow, horse, guinea pig, rat and mouse). Figure 3 shows which branches on the phylogenetic tree were tested for positive species selection. We tested each individual branch that led to a paralog in order to detect selective pressures following duplication events. We also used the PhyML generated tree for the analysis. Two models were used to test for positive selection, one model called 'alternative' in which the foreground branch may have some sites under positive selection, and one model called 'null' in which the foreground branch may have different proportions of sites under neutral evolution than the background branch. For the 'alternative' model, three classes were defined: ω0: dN/dS < 1, ω1: dN/dS = 1 and ω2: dN/dS≥1, while in the 'null' model, ω2 was fixed to 1. Like for the site model, LRT  and BEB  were used.
Putative function of positively selected sites
To assess the functionality of positively selected sites, the sites were positioned on the SAL1 structure (PDB: 1GM6) and their positions evaluated against the accessible surface area (ASA) of amino acids in SAL1 as determined by ASAView . SAL1 androstenol and androstenone binding sites were previously determined by Spinelli et al. . These amino acids were positioned on the SAL1 structure. Molecular graphics images were produced using the UCSF Chimera package .
CM is funded by a MENRT PhD fellowship. This work was supported by INRA.
- Dobzhansky T: Genetics and the origin of species/by Theodosius Dobzhansky. 1964, New York: Columbia University PressGoogle Scholar
- Smadja C, Butlin RK: On the scent of speciation: the chemosensory system and its role in premating isolation. Heredity. 2008, 102 (1): 77-97. 10.1111/j.1601-5223.1985.tb00468.x.View ArticlePubMedGoogle Scholar
- Karlson P, Luscher M: Pheromones': a new term for a class of biologically active substances. Nature. 1959, 183 (4653): 55-56. 10.1038/183055a0.View ArticlePubMedGoogle Scholar
- Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, Ramoni R, Cambillau C: Mammalian odorant binding proteins. Biochimica et Biophysica Acta (BBA). Protein Structure and Molecular Enzymology. 2000, 1482 (1-2): 229-240. 10.1016/S0167-4838(00)00167-9.View ArticleGoogle Scholar
- Pelosi P: The role of perireceptor events in vertebrate olfaction. Cell Mol Life Sci. 2001, 58 (4): 503-509. 10.1007/PL00000875.View ArticlePubMedGoogle Scholar
- Signoret JP: Reproductive behaviour of pigs. J Reprod Fertil Suppl. 1970, 11 (11): Suppl 11:105+.Google Scholar
- Beynon RJ, Hurst JL: Multiple roles of major urinary proteins in the house mouse, Mus domesticus. Biochem Soc Trans. 2003, 31 (Pt 1): 142-146.View ArticlePubMedGoogle Scholar
- Marchese S, Pes D, Scaloni A, Carbone V, Pelosi P: Lipocalins of boar salivary glands binding odours and pheromones. Eur J Biochem. 1998, 252 (3): 563-568. 10.1046/j.1432-1327.1998.2520563.x.View ArticlePubMedGoogle Scholar
- Perry GC, Patterson RLS, Macfie HJH, Stinson CG: PIG COURTSHIP BEHAVIOR - PHEROMONAL PROPERTY OF ANDROSTENE STEROIDS IN MALE SUB-MAXILLARY SECRETION. Animal Production. 1980, 31 (OCT): 191-199.View ArticleGoogle Scholar
- Guiraudie G, Pageat P, Cain AH, Madec I, Meillour PN-L: Functional Characterization of Olfactory Binding Proteins for Appeasing Compounds and Molecular Cloning in the Vomeronasal Organ of Pre-pubertal Pigs. Chem Senses. 2003, 28 (7): 609-619. 10.1093/chemse/bjg052.View ArticlePubMedGoogle Scholar
- Scaloni A, Paolini S, Brandazza A, Fantacci M, Bottiglieri C, Marchese S, Navarrini A, Fini C, Ferrara L, Pelosi P: Purification, cloning and characterisation of odorant- and pheromone-binding proteins from pig nasal epithelium. Cell Mol Life Sci. 2001, 58 (5-6): 823-834.View ArticlePubMedGoogle Scholar
- Flower DR: The lipocalin protein family: structure and function. Biochem J. 1996, 318 (Pt 1): 1-14.View ArticlePubMedPubMed CentralGoogle Scholar
- Spinelli S, Vincent F, Pelosi P, Tegoni M, Cambillau C: Boar salivary lipocalin. Three-dimensional X-ray structure and androsterol/androstenone docking simulations. Eur J Biochem. 2002, 269 (10): 2449-2456. 10.1046/j.1432-1033.2002.02901.x.View ArticlePubMedGoogle Scholar
- Loebel D, Scaloni A, Paolini S, Fini C, Ferrara L, Breer H, Pelosi P: Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin. Biochem J. 2000, 350 (Pt 2): 369-379.View ArticlePubMedPubMed CentralGoogle Scholar
- Touhara K, Vosshall LB: Sensing Odorants and Pheromones with Chemosensory Receptors. Annual Review of Physiology. 2009, 71 (1): 307-332. 10.1146/annurev.physiol.010908.163209.View ArticlePubMedGoogle Scholar
- Ganfornina MD, Gutierrez G, Bastiani MSD: A Phylogenetic Analysis of the Lipocalin Protein Family. Mol Biol Evol. 2000, 17 (1): 114-126.View ArticlePubMedGoogle Scholar
- Logan DW, Marton TF, Stowers L: Species Specificity in Major Urinary Proteins by Parallel Evolution. PLoS ONE. 2008, 3 (9): e3280-10.1371/journal.pone.0003280.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang ZD, Frankish A, Hunt T, Harrow J, Gerstein M: Identification and analysis of unitary pseudogenes: historic and contemporary gene losses in humans and other primates. Genome Biol. 2010, 11 (3): R26-10.1186/gb-2010-11-3-r26.View ArticlePubMedPubMed CentralGoogle Scholar
- Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Li H, Zhai W, Fritz MH, et al: A draft sequence of the Neandertal genome. Science. 2010, 328 (5979): 710-722. 10.1126/science.1188021.View ArticlePubMedGoogle Scholar
- Flicek P, Aken BL, Ballester B, Beal K, Bragin E, Brent S, Chen Y, Clapham P, Coates G, Fairley S, et al: Ensembl's 10th year. Nucleic Acids Res. 2009, 38 (Database issue): D557-562.PubMedPubMed CentralGoogle Scholar
- Sawyer S: Statistical tests for detecting gene conversion. Mol Biol Evol. 1989, 6 (5): 526-538.PubMedGoogle Scholar
- Yang Z, Bielawski JP: Statistical methods for detecting molecular adaptation. Trends Ecol Evol. 2000, 15 (12): 496-503. 10.1016/S0169-5347(00)01994-7.View ArticlePubMedGoogle Scholar
- Yang Z, Swanson WJ: Codon-substitution models to detect adaptive evolution that account for heterogeneous selective pressures among site classes. Mol Biol Evol. 2002, 19 (1): 49-57.View ArticlePubMedGoogle Scholar
- Yang Z: PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007, 24 (8): 1586-1591. 10.1093/molbev/msm088.View ArticlePubMedGoogle Scholar
- Doron-Faigenboim A, Stern A, Mayrose I, Bacharach E, Pupko T: Selecton: a server for detecting evolutionary forces at a single amino-acid site. Bioinformatics. 2005, 21 (9): 2101-2103. 10.1093/bioinformatics/bti259.View ArticlePubMedGoogle Scholar
- Rost B, Sander C: Conservation and prediction of solvent accessibility in protein families. Proteins. 1994, 20 (3): 216-226. 10.1002/prot.340200303.View ArticlePubMedGoogle Scholar
- Yang Z, Nielsen R: Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol. 2002, 19 (6): 908-917.View ArticlePubMedGoogle Scholar
- Weaver TD, Roseman CC, Stringer CB: Close correspondence between quantitative- and molecular-genetic divergence times for Neandertals and modern humans. Proc Natl Acad Sci USA. 2008, 105 (12): 4645-4649. 10.1073/pnas.0709079105.View ArticlePubMedPubMed CentralGoogle Scholar
- Innan H, Kondrashov F: The evolution of gene duplications: classifying and distinguishing between models. Nat Rev Genet. 2010, 11 (2): 97-108.PubMedGoogle Scholar
- Karn RC, Laukaitis CM: The mechanism of expansion and the volatility it created in three pheromone gene clusters in the mouse (Mus musculus) genome. Genome Biol Evol. 2009, 1: 494-503.View ArticlePubMedPubMed CentralGoogle Scholar
- Mudge JM, Armstrong SD, McLaren K, Beynon RJ, Hurst JL, Nicholson C, Robertson DH, Wilming LG, Harrow JL: Dynamic instability of the major urinary protein gene family revealed by genomic and phenotypic comparisons between C57 and 129 strain mice. Genome Biol. 2008, 9 (5): R91-10.1186/gb-2008-9-5-r91.View ArticlePubMedPubMed CentralGoogle Scholar
- Aguileta G, Bielawski JP, Yang Z: Gene conversion and functional divergence in the beta-globin gene family. J Mol Evol. 2004, 59 (2): 177-189. 10.1007/s00239-004-2612-0.View ArticlePubMedGoogle Scholar
- Kondrashov FA, Rogozin IB, Wolf YI, Koonin EV: Selection in the evolution of gene duplications. Genome Biol. 2002, 3 (2): RESEARCH0008-View ArticlePubMedPubMed CentralGoogle Scholar
- Emes RD, Beatson SA, Ponting CP, Goodstadt L: Evolution and comparative genomics of odorant- and pheromone-associated genes in rodents. Genome Res. 2004, 14 (4): 591-602. 10.1101/gr.1940604.View ArticlePubMedPubMed CentralGoogle Scholar
- Gilad Y, Man O, Glusman G: A comparison of the human and chimpanzee olfactory receptor gene repertoires. Genome Res. 2005, 15 (2): 224-230. 10.1101/gr.2846405.View ArticlePubMedPubMed CentralGoogle Scholar
- Moreno-Estrada A, Casals F, Ramirez-Soriano A, Oliva B, Calafell F, Bertranpetit J, Bosch E: Signatures of selection in the human olfactory receptor OR5I1 gene. Mol Biol Evol. 2008, 25 (1): 144-154.View ArticlePubMedGoogle Scholar
- Nielsen R, Bustamante C, Clark AG, Glanowski S, Sackton TB, Hubisz MJ, Fledel-Alon A, Tanenbaum DM, Civello D, White TJ, et al: A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol. 2005, 3 (6): e170-10.1371/journal.pbio.0030170.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhuang H, Chien MS, Matsunami H: Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates. Proceedings of the National Academy of Sciences. 2009, 106 (50): 21247-21251. 10.1073/pnas.0808378106.View ArticleGoogle Scholar
- Shi P, Bielawski JP, Yang H, Zhang YP: Adaptive diversification of vomeronasal receptor 1 genes in rodents. J Mol Evol. 2005, 60 (5): 566-576. 10.1007/s00239-004-0172-y.View ArticlePubMedGoogle Scholar
- Roelofs WL, Rooney AP: Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera. Proc Natl Acad Sci USA. 2003, 100 (16): 9179-9184.View ArticlePubMedPubMed CentralGoogle Scholar
- Cavaggioni A, Mucignat-Caretta C: Major urinary proteins, alpha(2U)-globulins and aphrodisin. Biochim Biophys Acta. 2000, 1482 (1-2): 218-228. 10.1016/S0167-4838(00)00149-7.View ArticlePubMedGoogle Scholar
- Clark NL, Aagaard JE, Swanson WJ: Evolution of reproductive proteins from animals and plants. Reproduction. 2006, 131 (1): 11-22. 10.1530/rep.1.00357.View ArticlePubMedGoogle Scholar
- Horth L: Sensory genes and mate choice: evidence that duplications, mutations, and adaptive evolution alter variation in mating cue genes and their receptors. Genomics. 2007, 90 (2): 159-175. 10.1016/j.ygeno.2007.03.021.View ArticlePubMedGoogle Scholar
- Dorus S, Evans PD, Wyckoff GJ, Choi SS, Lahn BT: Rate of molecular evolution of the seminal protein gene SEMG2 correlates with levels of female promiscuity. Nat Genet. 2004, 36 (12): 1326-1329. 10.1038/ng1471.View ArticlePubMedGoogle Scholar
- Schwalie PC, Schultz J: Positive selection in tick saliva proteins of the Salp15 family. J Mol Evol. 2009, 68 (2): 186-191. 10.1007/s00239-008-9194-1.View ArticlePubMedGoogle Scholar
- Carson AR, Scherer SW: Identifying concerted evolution and gene conversion in mammalian gene pairs lasting over 100 million years. BMC Evol Biol. 2009, 9 (156): 156-View ArticlePubMedPubMed CentralGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22 (22): 4673-4680. 10.1093/nar/22.22.4673.View ArticlePubMedPubMed CentralGoogle Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52 (5): 696-704. 10.1080/10635150390235520.View ArticlePubMedGoogle Scholar
- Felsenstein J: Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution. 1985, 39 (4): 783-791. 10.2307/2408678.View ArticleGoogle Scholar
- Posada D: Evaluation of methods for detecting recombination from DNA sequences: empirical data. Molecular biology and evolution. 2002, 19 (5): 708-717.View ArticlePubMedGoogle Scholar
- Suyama M, Torrents D, Bork P: PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 2006, 34 (Web Server issue): W609-612.View ArticlePubMedPubMed CentralGoogle Scholar
- Nielsen R, Yang Z: Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics. 1998, 148 (3): 929-936.PubMedPubMed CentralGoogle Scholar
- Yang Z: Maximum likelihood estimation on large phylogenies and analysis of adaptive evolution in human influenza virus A. J Mol Evol. 2000, 51 (5): 423-432.PubMedGoogle Scholar
- Swanson WJ, Nielsen R, Yang Q: Pervasive adaptive evolution in mammalian fertilization proteins. Mol Biol Evol. 2003, 20 (1): 18-20.View ArticlePubMedGoogle Scholar
- Doron-Faigenboim A, Pupko T: A combined empirical and mechanistic codon model. Mol Biol Evol. 2007, 24 (2): 388-397.View ArticlePubMedGoogle Scholar
- Yang Z, Wong WS, Nielsen R: Bayes empirical bayes inference of amino acid sites under positive selection. Molecular biology and evolution. 2005, 22 (4): 1107-1118. 10.1093/molbev/msi097.View ArticlePubMedGoogle Scholar
- Zhang J, Nielsen R, Yang Z: Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol. 2005, 22 (12): 2472-2479. 10.1093/molbev/msi237.View ArticlePubMedGoogle Scholar
- Ahmad S, Gromiha M, Fawareh H, Sarai A: ASAView: database and tool for solvent accessibility representation in proteins. BMC Bioinformatics. 2004, 5 (51): 51-View ArticlePubMedPubMed CentralGoogle Scholar
- Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004, 25 (13): 1605-1612. 10.1002/jcc.20084.View ArticlePubMedGoogle Scholar
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