Molecular Evolution of Transferrin: Evidence for Positive Selection in Salmonids

Michael J. FordGo,

National Marine Fisheries Service, Northwest Fisheries Science Center, Conservation Biology Division, Seattle, Washington


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Transferrins are iron-binding proteins that are involved in iron storage and resistance to bacterial disease. Previous work has shown that nonsynonymous-to-synonymous-site substitution ratios (dn/ds ratios) between transferrin genes from some salmonid species were significantly greater than 1.0, providing evidence for positive selection at the transferrin gene. The purpose of the current study was to put these earlier results in a broader evolutionary context by examining variation among 25 previously published transferrin sequences from fish, amphibians, and mammals. The results of the study show that evidence for positive selection at transferrin is limited to salmonids—dn/ds ratios estimated for nonsalmonid lineages were generally less than 1.0. Within the salmonids, ~13% of the transferrin codon sites are estimated to be subject to positive selection, with an estimated dn/ds ratio of ~7. The three- dimensional locations of some of the selected sites were inferred by comparing these sites to homologous sites in the bovine lactoferrin crystallographic structure. The selected sites generally fall on the outside of the molecule, within and near areas that are bound by transferrin-binding proteins from human pathogenic bacteria. The physical locations of sites estimated to be subject to positive selection support previous speculation that competition for iron from pathogenic bacteria could be the source of positive selection.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Vertebrate transferrins (including lactoferrin and ovotransferrin) are iron-binding proteins found in blood serum, interstitial spaces, milk, tears, and egg whites (reviewed by Loehr 1989Citation ). Transferrins consist of two homologous lobes, each consisting of ~335 amino acids and containing a single highly conserved iron-binding site (Loehr 1989Citation ). Transferrins have high binding affinities for iron and keep free iron in low concentration in blood and other bodily fluids (Loehr 1989Citation ). Among other functions, iron binding by transferrin provides resistance to bacterial infection, because iron is often a limiting nutrient for bacterial growth (Guerinot 1994Citation ; Gray- Owen and Schryvers 1996Citation ). Bacterial species have a variety of mechanisms for obtaining transferrin-bound iron, including transferrin-binding proteins, metalloproteases, and siderophore-mediated systems (reviewed by Guerinot 1994Citation ). Competition for iron from bacterial pathogens could potentially be a strong source of natural selection on vertebrate transferrins. In some salmon populations, a specific transferrin genotype has been found to be associated with resistance to bacterial infection (Suzumoto, Schreck, and McIntyre 1977Citation ; Winter, Schreck, and McIntyre 1980Citation ). In mammals, transferrin genotype has been shown to influence fertility and mate choice, perhaps reflecting selection by bacteria for genetic variability (reviewed by Wedekind 1994Citation ).

In an earlier paper, Ford, Thornton, and Park (1999)Citation compared replacement (dn) and silent (ds) substitution rates among four salmonid species at the transferrin gene and found dn/ds ratios significantly greater than 1.0 in three of the six pairwise comparisons. This result suggested that positive selection for new replacement alleles has played a large role in the evolution of transferrin within at least some salmonid species. Iron competition with salmonid pathogens could be one selective mechanism. In this paper, the earlier salmonid results are put in a broader phylogenetic context through analysis of transferrin sequence variation from 25 vertebrates, including 7 additional recently published salmonid species (Lee et al. 1998Citation ; table 1 ). The specific goals of the study were to identify where in the history of its evolution vertebrate transferrin had been subject to positive selection and to use recently developed likelihood techniques (Goldman and Yang 1994Citation ; Nielsen and Yang 1998Citation ; Yang 1998Citation ) to identify specific positively selected sites. By mapping the sites that appear to be subject to positive selection onto specific functional regions of the protein, it should be possible to gain additional insight into the potential selective mechanisms responsible for the high dn/ds ratios among salmonid species.


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Table 1 Transferrin Sequences, Sources, and Accession Numbers

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Sequences and Alignment
All sequences were obtained from GenBank. Taxonomic information, accession numbers, and literature references are provided in table 1 . For several species, multiple sequences were available. In these cases, all available sequences were aligned and inspected for intraspecific variation. In each case, intraspecific variation appeared minor compared with interspecific variation, so only a single sequence from each species was used for further analysis. The coding regions of each sequence were conceptually translated, and the resulting inferred protein sequences were aligned using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ) with default alignment parameters (gap opening penalty 10.00; gap extension penalty 0.05; sequences >40% diverged delayed; BLOSUM protein weight matrix; residue-specific penalties on; hydrophilic penalties on). The alignments were then inspected and adjusted by eye using the GeneDoc alignment program (Nicholas and Nicholas 1997Citation ). The DNA sequences were then aligned using the same gap patterns. All subsequent analyses were performed on this set of aligned DNA sequences. A file containing the aligned sequences is available from the author upon request.

Estimation of dn/ds Ratios and Selected Sites
Several of the codon-based likelihood models of Nielsen and Yang (1998)Citation and Yang (1998)Citation were used to estimate dn/ds ratios for each branch of an estimated transferrin phylogeny. The likelihood models provide more accurate estimates of dn/ds ratios than do simpler approximation methods and allow specific selected sites to be identified (Yang and Nielsen 2000Citation ). In order to produce valid results, the models require an accurate phylogeny of the sequences involved. In this study, phylogenies were estimated from the aligned DNA sequences using maximum-likelihood, parsimony and neighbor- joining methods (Saitou and Nei 1987) as implemented in the DNAML, DNAPARS, and DNADIST programs in the PHYLIP computer package (Felsenstein 1993Citation ).

Several models of codon evolution were fitted either to the entire data set or to specific subsets of the data using the PAML computer package (Goldman and Yang 1994Citation ; Nielsen and Yang 1998Citation ; Yang 1998Citation ). All the models used maximum-likelihood methods for estimating the parameters of a transition matrix describing the substitution rates between pairs of codons, including dn/ds ratios, transition/transversion ratios, and branch lengths. The simplest model was called the single-ratio model. This model estimated a single average dn/ds ratio, {omega}, across every branch and every codon. The next model, called the free-ratio model, estimated a dn/ds ratio, {omega}b, for every branch b in the tree. The third model, called the neutral model, assumed a single dn/ds ratio for all branches in the tree but allowed for two different types of sites, each with a different {omega} value. Sites of the first type, in frequency p0, were subject to strong selection against replacement mutations and had a dn/ds ratio of {omega}0 = 0. Sites of the second type, in frequency p1, were neutral and had a dn/ds ratio of {omega}1 = 1. The fourth model, called the positive selection model, was the same as the neutral model except that there was a third class of sites, in frequency ps, that had a dn/ds ratio of {omega}s. This model only provided support for positive selection if the estimate of {omega}s was greater than 1.0. The probability that a particular codon site is positively selected can be estimated using the empirical Bayes' approach described by Nielsen and Yang (1998)Citation . For all models, the equilibrium codon frequencies were estimated from the products of the average observed nucleotide frequencies in the three codon positions (the f3x4 option in the PAML package). In order to determine if the model results were sensitive to assumptions about equilibrium codon frequencies, all models were also run under an assumption of equal codon frequencies (the 1/61 option in the PAML package). Both codon frequency assumptions produced very similar results, and only the results using the f3x4 assumption are reported here.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Phylogeny
A maximum-likelihood phylogeny was estimated for the entire data set (fig. 1 ), as well as for two different subsets: salmonids (fig. 2A ) and Eutherian mammals (fig. 3 ). In general, the phylogenies estimated for the transferrin gene are consistent with the generally accepted vertebrate phylogeny (Benton 1997Citation ) and with most previously published transferrin phylogenies (e.g., Baldwin 1993Citation ; Demmer et al. 1999Citation ). The maximum- likelihood phylogenies estimated for the two data subsets are consistent with the phylogeny estimated for the entire data set, with the exception of a minor change in the relationship between the Homo sapiens, Oryctolagus coniculus, and Rattus norvegicus transferrin genes (figs. 1 and 3 ). Phylogenetic relationships within the salmonids remain imperfectly known, and the maximum-likelihood phylogeny estimated for the salmonid sequences (figs. 1 and 2A ) differs somewhat from some previously published salmonid phylogenies (e.g., Stearley and Smith 1993Citation ; Lee et al. 1998Citation ) but is the same as or consistent with others (e.g., McKay, Devlin, and Smith 1996Citation ; Oakley and Phillips 1999Citation ). For the species included in this study, the differences among published phylogenies are primarily in whether amago salmon or rainbow trout is most basal within the Oncorhynchus clade and in the relationships among the three salmonid genera. The uncertainties within the Oncorhynchus clade were dealt with by employing two additional tree-building methods, parsimony and neighbor joining, and then including all plausible trees in subsequent analyses (fig. 2 ). The uncertainty of the relationships among the three salmonid genera has no effect on the present study because most of the analyses of salmonid sequences in this paper were based on unrooted trees (fig. 2 ).



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Fig. 1.—Maximum-likelihood phylogeny based on all sequences in the study. The tree topology was estimated using the DNAML program in the PHYLIP package (Felsenstein 1993Citation ). The branch lengths represent expected numbers of codon substitutions and were estimated using the PAML package (Goldman and Yang 1994Citation ; Yang 1998Citation ). Numbers next to the branches are the dn/ds ratios estimated under the free-ratio model (see Materials and Methods). Branches with dn/ds ratios estimated as "99" have a positive dn and a ds of 0. Note that the salmonid portion of the tree is drawn on a different scale than the rest of tree. See table 1 for species names

 


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Fig. 2.—Four alternative trees of the salmonid sequences used in this study. A, The maximum-likelihood tree and one of two most- parsimonious trees. B, The second of two most-parsimonious trees and the tree with the highest bootstrap values in a neighbor-joining analysis. Bootstrap values (out of 1,000 bootstraps) for the branches leading to the three genera, to the coho-chinook salmon clade, and to the brook- lake trout clade were all >90%. The bootstrap value for the sockeye-coho-chinook salmon clade was 61%; the value for the rainbow trout– sockeye salmon–coho salmo–chinook salmon clade was 52%. C and D, Additional trees with at least moderate neighbor-joining bootstrap values. Other details are the same as for figure 1

 


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Fig. 3.—Maximum-likelihood phylogeny of the Eutherian mammal sequences used in this study. Other details are the same as for figure 1

 
Free-Ratio Models
The purpose of the free-ratio models was to identify branches on the transferrin evolutionary tree where dn/ds ratios were particularly high or low and to determine if dn/ds ratios greater than 1.0 were limited to certain salmonid species or also occurred at other times during transferrin's evolutionary history. As shown in figure 1 , many branches within the salmonid clade had dn/ds ratios greater than 1.0, and most branches outside of the salmonids had dn/ds ratios substantially less than 1.0. Outside of the salmonids, there were only two branches with estimated dn/ds ratios greater than 1.0—a fairly basal branch in the mammalian transferrin lineage with a dn/ds ratio of 1.59, and a branch within the lactoferrin lineage with a ratio of 1.10. Within the salmonids, dn/ds ratios were particularly high in the lineages leading to chinook and coho salmon. As a point of comparison with the maximum-likelihood estimates, the average pairwise dn/ds ratios estimated for the salmonid sequences using the Nei and Gojobori (1986)Citation method were 1.06, 0.91, 1.06, and 1.54 for comparisons between all the sequences, the Salmo sequences, the Salvelinus sequences, and the Oncorhynchus sequences, respectively.

Inspection of the free-ratio model results clearly suggests different dn/ds ratios within the salmonids compared with other vertebrates. For this reason, and for reasons of computational efficiency, further analyses were applied to two smaller data sets: a data set consisting of all of the salmonid transferrin sequences and a data set consisting of the Eutherian mammal transferrin and lactoferrin sequences. The dn/ds ratios estimated under the free-ratio model for these smaller data sets were similar to the ratios estimated using the complete data set (figs. 2 and 3 ). The ratios estimated for the four alternative salmonid phylogenies were very similar to each other (fig. 2 ), showing that the results are robust to a variety of plausible phylogenies.

Neutral and Positive-Selection Models
Fitting the neutral and positive-selection models to the data is useful for two reasons. First, by comparing the fit of the two models, one can determine if the positive-selection model provides a significantly better fit to the data than the neutral model, and second, the positive-selection model can be used to identify specific sites that have been subject to positive selection. The relative fit of the two models can be evaluated using a likelihood ratio test (e.g., Nielsen and Yang 1998Citation and references therein). Under the hypothesis that the two nested models provide an equally good fit to the data, twice the log likelihood difference between the two models is expected to be approximately {chi}2 distributed with the number of degrees of freedom equal to the difference in the number of parameters between the models.

For the salmonid data set, the positive-selection model provided a much better fit to the data than did the neutral model (twice the log likelihood difference between the two models varied from 130.5 to 142.4 depending on the phylogeny used, df = 2, P < 0.001; table 2 ). The positive-selection model estimated that 13%–14% of the codons were subject to positive selection during their evolutionary history, with an average dn/ds ratio of 6.6–7.1 (table 2 )—clear evidence of strong positive selection. The estimates of the proportion of neutral sites and sites subject to strong constraint were 39%–41% and 46%–47%, respectively (table 2 ).


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Table 2 Results of Likelihood Models

 
Based on the results of the free-ratio model, there appears to be considerable variation in dn/ds ratios among branches within the salmonids (fig. 2 ). The statistical significance of this variation was tested by comparing the free-ratio model with a single-ratio model that assumed that all branches had the same dn/ds ratio. The free-ratio model provided a somewhat better fit to the data than the single-ratio model (twice the log likelihood difference = 19.9–26.5, df = 16, P < 0.05 to P < 0.25; table 2 ), suggesting that there may be differences in the rates of adaptive evolution among the salmonid lineages.

For the Eutherian mammal sequences, the selection model also provided a much better fit to the data than did the neutral model (twice the log likelihood difference = 430.54, df = 2, P < 0.001; table 2 ). The dn/ds ratio for selected sites under the selection model, however, was estimated to be less than 1.0, so even though the selection model provided a better fit to the data than did the neutral model, the selection model did not support a hypothesis of positive selection. Rather, the model was simply more realistic than the neutral model because it had three classes of sites instead of two, thus allowing for greater variation in selective constraint among sites.

Within the Eutherian mammal tree, two branches (labeled "a" and "b" in fig. 3 ) had estimated dn/ds ratios greater than 1.0 under the free-ratio model. In order to determine if the dn/ds ratios for these two branches were significantly greater than 1.0, two additional models were employed. The first additional model, the two-ratio model, assumed there were two dn/ds ratios within the Eutherian tree, one ratio for branches a and b, {omega}a,b, and another ratio for all other branches, {omega}0. The second additional model, the two-ratio constrained model, was the same as the first except that {omega}a,b was constrained to be equal to 1.0. The models differed by one degree of freedom, and a comparison of twice the log likelihood difference between the models indicated that the dn/ds ratios for the a and b branches were not significantly different from 1.0 (twice the log likelihood difference = 0.74, df = 1, P < 0.4; table 2 ).

Locations of Selected Sites
The selection model estimates the proportion of sites in a gene subject to positive selection, but does not identify which specific sites have actually been selected. An empirical Bayes' approach can be used along with the results of the selection model, however, to estimate the probability that specific sites are subject to selection (Nielsen and Yang 1998Citation ). Using this method, 29 sites in salmonid transferrin were estimated to be positively selected with posterior probability greater than 0.95 for at least one of the four alternative salmonid trees (table 3 ).


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Table 3 Positively Selected Sites

 
Physical interactions between bacterial transferrin- binding proteins and salmonid transferrins are a potential source of selection that could lead to high dn/ds ratios (e.g., Wedekind 1994Citation ; Ford, Thornton, and Park 1999Citation ). Salmonid pathogens utilize several mechanisms of iron acquisition (reviewed by Evelyn 1996Citation ), including mechanisms that require direct physical contact between bacterial proteins and host transferrin (e.g., Hirst and Ellis 1996Citation ; Mazoy and Lemos 1991Citation ). It therefore seems plausible to expect that areas on the surface of the transferrin molecule would be most subject to selection by bacterial pathogens.

The crystal structure of salmonid transferrin has not been determined, but the crystal structure of the N- and C-lobes of mammalian transferrins are very similar to each other (Anderson et al. 1989Citation ). The divergence time between the two lobes predates the divergence times between mammals and fish (e.g., Baldwin 1993Citation ), so it seems reasonable to assume that the three-dimensional structure of salmonid transferrin will be similar to that of the mammalian transferrin. The crystal structures of human transferrin, human lactoferrin, bovine lactoferrin, and rabbit transferrin are also all very similar (Moore et al. 1997Citation ), providing additional confidence that the general three-dimensional structure of transferrin is likely to be quite conserved among diverse species. Figure 4 shows the structure of diferric bovine lactoferrin (Moore et al. 1997Citation ), with the sites homologous to likely positively selected salmonid sites (table 2 ) shaded dark gray. Three of the 29 salmon sites identified as positively selected did not have homologous sites in the bovine sequence. Consistent with the bacterial-selection hypothesis, the 26 remaining sites that were estimated to be subject to positive selection in salmonids were all found near the outside of the molecule, and in several cases sites which were not close to each other in the primary sequence were physically close together in the folded molecule.



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Fig. 4.—Space-filling model of the diferric bovine lactoferrin molecule (Moore et al. 1997Citation ). For each view, the molecule is oriented with the N-lobe at the top and the C-lobe at the bottom. The angles of view are 0°, 90°, 180°, and 270° for the upper left, upper right, lower left, and lower right parts of the figure, respectively. Sites shown to be contacted by bacterial transferrin-binding proteins (Retzer, Yu, and Schryvers 1999Citation ) are shaded light gray. Sites homologous to those identified as being subject to positive selection in salmonids are identified by codon number (table 3 ) and are shaded dark gray. Salmon sites 149, 510, and 631 did not have homologous sites in the bovine sequence

 
Recently, Retzer, Yu, and Schryvers (1999)Citation identified sequences in human transferrin that are bound by the pathogenic bacteria Neisseria meningitidis and Moraxella catarrhalis transferrin-binding proteins B (TbpB). These binding domains are colored light gray in figure 4 . Fifteen of the 29 sites identified as positively selected fell in these binding areas, significantly more than would be expected under the hypothesis that the selected sites occur at random locations in the molecule (15/214, 14/ 428, {chi}2 = 4.17; P < 0.05). A possible explanation for the partial concordance of sites subject to positive selection in salmonids and the TbpB-binding domains is that competition for iron from pathogenic bacteria is responsible for the rapid evolution of salmonid transferrin, and these bacteria produce proteins which bind transferrin in locations similar to those in the binding proteins studied by Retzer, Yu, and Schryvers (1999)Citation . The identification of specific sites subject to positive selection suggests that experiments focusing on these sites (e.g., by directed mutagenesis) may provide considerable insight into the function of the transferrin protein and as well as the selective mechanisms acting upon it.

Why Are dn/ds Ratios Not High Among Mammals or Other Nonsalmonids?
If interaction with bacterial iron acquisition proteins is the mechanism leading to positive selection at transferrin, it is puzzling that the evidence for such selection is found only in salmonids. Transferrin plays a role in disease resistance in mammals (reviewed by Martinez, Delgado-Iribarren, and Baquero 1990Citation ), so if selection due to iron-binding competition from bacteria is a plausible selective mechanism in salmonids, it is also certainly a plausible selective mechanism in mammals. Furthermore, there is evidence that interactions between egg and sperm transferrin genotypes affect fertility in mammals (reviewed by Wedekind 1994Citation ), providing independent evidence that extant transferrin alleles are indeed under selection in at least some mammalian species. There are, however, at least four possible explanations for the lack of evidence for positive selection outside of the salmonids. The first possible explanation can be ruled out with the data in hand; the other three are speculative, and additional experiments will be required to test them.

First, the branch lengths outside of the salmonid clade of the transferrin tree are for the most part much longer than the branch lengths in the salmonid clade (fig. 1 ), suggesting the possibility that saturation of nonsynonymous substitutions could result in underestimation of dn/ds ratios outside of the salmonid clade. This possibility was tested by using the evolver program in the PAML package (see Materials and Methods) to simulate trees with branch lengths and topologies identical to the Eutherian mammalian tree in figure 3 but with a parametric dn/ds ratio of 1.15 (equal to the single-ratio estimate for the salmonids; table 2 ). The results of these simulations show that the model can adequately estimate dn/ds ratios similar to the salmonid ratios for trees with branch lengths equal to those estimated for the mammalian transferrin tree (the mean estimated dn/ds ratio for 10 simulations was 1.23, with a standard deviation of 0.056). Similar results were obtained using a tree of all of the nonsalmonid sequences (the mean estimated dn/ds ratio for 10 simulations was 1.22, with a standard deviation of 0.074). These simulation results show that longer branch lengths cannot explain the lack of high dn/ds ratios outside of the salmonid clade.

Second, some investigators have argued that fish rely more heavily on their nonadaptive immune systems than do higher vertebrates, perhaps leading to stronger selection on fish transferrin (e.g., Nonaka and Smith 2000). This explanation fails to explain why salmonid transferrins should be subject to greater positive selection than the transferrins of the nonsalmonid fishes in this study, however.

Third, the salmonid species in this study are all at least partially diadromous and therefore occupy a great variety of habitats throughout their life cycles (freshwater, estuarine, marine). This diverse lifestyle may result in exposure to a large variety of pathogens, thus imposing especially strong selection on transferrin.

Finally, the Salmonidae are ancestrally tetraploid (Sola, Cataudella, and Capanna 1981Citation ). There is evidence for duplicated transferrin genes in Atlantic salmon (Kvingedal, Rørvik, and Alestrøm 1993Citation ), and it is likely that other salmonid species contain two transferrin genes as well. It is possible that the genomewide duplication event that occurred in the lineage leading to the Salmonidae facilitated rapid evolution and adaptation of duplicated genes (e.g., Zhang, Rosenberg, and Nei 1998Citation ). Interestingly, the only two branches outside of the Salmonidae with dn/ds ratios greater than 1.0 (although not significantly so) occur soon after the duplication event leading to mammalian lactoferrin and transferrin (fig. 1 ).

In summary, within the salmonids, the selection model provides a significantly better fit to the data than the neutral model, with ~13% of the codons in the gene estimated to be subject to positive selection with a dn/ ds ratio of ~7. Of these selected codons, 29 could be identified with confidence. These selected sites code for residues on the outside of the transferrin molecule, and approximately half of them fall in areas bound by bacterial transferrin-binding proteins. In contrast to the salmonid results, there was no evidence for positive selection in the nonsalmonid sequences in this study.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
I thank Steven Kalinowski, Robin Waples, Maureen Purcell, Linda Park, and an anonymous reviewer for comments on earlier drafts of the manuscript.


    Footnotes
 
Antony Dean, Reviewing Editor

1 Keywords: positive selection likelihood disease resistance transferrin salmon evolution Back

2 Address for correspondence and reprints: Michael J. Ford, National Marine Fisheries Service, Northwest Fisheries Science Center, Conservation Biology Division, 2725 Montlake Boulevard East, Seattle, Washington 98112. mike.ford{at}noaa.gov Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
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Accepted for publication January 4, 2001.