Evidence for Directional Selection Acting on Pheromone-Binding Proteins in the Genus Choristoneura

Christopher S. Willett1,Go,

Department of Ecology and Evolutionary Biology, Cornell University

Abstract

Patterns of nucleotide variation consistent with the action of natural selection have been discovered at a number of different gene loci. Here, pheromone-binding proteins (PBPs) are examined to determine if selection has acted to fix amino acid changes in PBPs in lineages in which pheromone changes have occurred. PBPs from five different species of moths in the genus Choristoneura were sequenced, along with the PBP of Argyrotaenia velutinana, which serves as an outgroup. Three independent major pheromone changes are represented within this group of five Choristoneura species. Two different lineages show evidence for selection based on polymorphism and divergence comparisons and comparisons of rates of replacement evolution to silent and noncoding evolution. Along one of these lineages, leading to Choristoneura fumiferana, there has been a change to an aldehyde pheromone from an acetate pheromone. The second branch does not appear to be associated with a major pheromone change. Other branches in the tree show a trend toward greater replacement fixation than expected under neutrality. This trend could reflect undetected selective events within this group of PBPs. Selection appears to have acted to fix amino acid changes in the PBP of moths from the genus Choristoneura, but it is not clear that this selection is due to pheromone changes between species.

Introduction

One approach to studying selection at the molecular level is to start with a gene that could be involved in adaptation and to determine whether there is evidence for selection acting on this gene. A number of statistical tests have been developed to determine if there is evidence for selection acting on a particular gene based on the examination of nucleotide sequence variation (Kreitman and Akashi 1995Citation ). The McDonald and Kreitman (MK; 1991)Citation , Tajima's (1989)Citation D, and Fu and Li (1993)Citation tests are based on comparisons of polymorphism within species with neutral expectations. Another approach for detecting selection on a protein is to compare rates of nonsynonymous (dN) and synonymous (dS) substitution for a particular gene between species (Hughes and Nei 1988Citation ; Hughes 1991Citation ; Metz, Robles-Sikisaka, and Vacquier 1998Citation ; Yang 1998Citation ).

In this study, I examine pheromone-binding proteins (PBPs) in the genus Choristoneura (Lepidoptera: Tortricidae) to determine if selection has acted on these genes. PBPs are small, abundant proteins found in male moth antennae that appear to be involved in binding, transport, and possibly clearing of pheromone molecules within the pheromone-sensing organs of the male antennae (Vogt and Riddiford 1981Citation ; Prestwich, Du, and Laforest 1995Citation ; Vogt 1995Citation ; Laue and Steinbrecht 1997Citation ). PBPs are members of a gene family of odorant-binding proteins that also includes the Lepidopteran general odorant-binding proteins (GOBPs). PBPs and GOBPs are not homologous to either odorant receptor proteins or mammalian odorant-binding proteins (Pelosi and Maida 1995Citation ; Bianchet et al. 1996Citation ). Comparisons of orthologous PBPs and GOBPs between species show that PBPs have a faster rate of replacement evolution than the GOBPs (Vogt, Rybczynski, and Lerner 1991Citation ). Vogt, Rybczynski, and Lerner (1991)Citation suggested that this higher rate of replacement evolution in PBPs may be caused by selection on PBPs imposed by the relatively frequent pheromone changes between Lepidopteran species.

A previous study of PBPs of the European and Asian corn borers (Ostrinia nubilalis and Ostrinia furnacalis) showed that their PBPs have not diverged in amino acid sequence despite pheromone differences within and between these species (Willett and Harrison 1999Citation ). This study suggested that PBPs do not always change in response to pheromone changes but did not show whether other types of pheromone changes could lead to selection on PBPs for amino acid differentiation.

Pheromones of many different species of Choristoneura have been characterized. The predominant pheromone components in the Tortricidae (particularly in the tribe Archipini) are (E)- and (Z)-11-tetradecenyl acetate (E- and Z11-14:OAc) (Roelofs and Brown 1982Citation ). Three species included in this study use E- and Z11-14:OAc as their major pheromone components, Choristoneura rosaceana, Choristoneura pinus, and the outgroup species Argyrotaenia velutinana, also a member of the tribe Archipini (see table 1 ). Given the widespread distribution of the E- and Z11-14:OAc pheromone and its occurrence in the outgroup, E- and Z11-14:OAc pheromones are assumed to represent the ancestral major pheromone components. The three other species utilize three different pheromones representing changes in functional groups and chain length. The pheromone of Choristoneura fumiferana is an aldehyde pheromone and that of Choristoneura parallela predominantly an alcohol pheromone, while Choristoneura murinana uses a 12-carbon compound as its major pheromone component (table 1 ). The proposed relationships of these taxa are depicted in figure 1 , with the changes in major pheromone components mapped on the appropriate branches (Dang 1992Citation ; Sperling and Hickey 1994Citation ; Harvey 1996Citation ).


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Table 1 Pheromone Components

 


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Fig. 1.—Proposed relationships of Choristoneura taxa and changes in pheromones. The changes assume that E- and Z11-14:OAc represent the ancestral pheromone components and that there have been changes in an alcohol-predominated pheromone (OH), in an aldehyde pheromone (Ald), and in a chain length of 12 (Chain length). Relationships come from a combination of the relationships proposed by Dang (1992)Citation , Sperling and Hickey (1994)Citation , and Harvey (1996)Citation . In this tree, Choristoneura murinana's relationship to the other taxa is left unresolved, because Dang (1992)Citation proposed a relationship with the fumifera/pinus clade, while Harvey (1996)Citation had C. murinana falling basal to the other four taxa. Argyrotaenia velutinana is assumed to be an outgroup to the other Choristoneura taxa

 
I sequenced a PBP from each of these Choristoneura species and used both population genetics–based tests and the ratio of silent to replacement substitutions to look for evidence of selection associated with presumed pheromone changes. There has been selection associated with the change to an aldehyde pheromone in C. fumiferana, while there is no detectable selection associated with the switch to an alcohol pheromone in C. parallela. There has been a great deal of replacement change along the C. murinana lineage associated with the chain length difference of the pheromone of this species, but it is not clear that this replacement change has been driven by selection. There also appears to have been selection acting on the PBP of C. pinus, although no major pheromone change occurs along this branch. It appears that there have been episodes of selection acting on PBPs from moths in the genus Choristoneura, but it is unclear whether pheromone changes are causing this selection.

Materials and Methods

Species Collection
The moths C. rosaceana in this study were obtained from W. Roelofs in June 1996 from a culture maintained in Geneva, N.Y. W. Roelofs also supplied the A. velutinana. S. Polavarapu of Rutgers University provided the C. parallela used in this study. These moths came from a laboratory population derived from moths collected from cranberry bogs near Chatsworth, N.J. M. Kenis (CABI Bioscience, Delémont, Switzerland) collected C. murinana at Guebwiller, Alsace, France, in June 1998 and shipped them preserved in ethanol. J. McNeil (Laval University, Sainte-Foy, Quebec) supplied C. fumiferana in August 1997 from cultures maintained by the Canadian Forestry Lab in Sault Ste. Marie, Ontario. I collected C. pinus from stands of red pine near Ithaca, N.Y., in late July 1997. J. Franclemont aided in the identification of these moths.

Obtaining PBP DNA Sequences
The PBP of C. rosaceana was first identified by a 3' RACE method similar to the method described in Willett and Harrison (1999)Citation . Details of the procedure followed and the specific primers developed to amplify the PBP region from the Choristoneura species can be found in Willett (1999)Citation . DNA isolations were done using the QiaAmp tissue kit (QIAGEN, Chatsworth, Calif.). Three alleles from C. rosaceana and two alleles from C. parallela were sequenced from cloned PCR products (a minimum of two clones were sequenced for each allele). PCR products were cloned and sequenced for five alleles from three individuals from C. fumiferana and six alleles from three individuals from C. pinus. Eight alleles from four C. murinana individuals were sequenced directly from their PCR fragments (this species had fewer insertions and deletions in its noncoding regions).

Argyrotaenia velutinana required a new set of primers to amplify its PBP, because noncoding regions of this species were significantly diverged from the Choristoneura species. Two new degenerate primers, DF2 (5'-GCRGAYTTCTAYAACTTCTGG-3') and DR4 (5'-GGATYTCSRYYTTRAAGCACTTGGC-3'), designed to match conserved regions of PBP from a range of species, were used to obtain an initial fragment of this species PBP. Species-specific primers were developed to amplify the PBP gene region, and three individuals were sequenced directly from these PCR products (Willett 1999Citation ). AvelE was sequenced for the entire PBP region, while AvelC and AvelD were completely sequenced for the coding region only.

Sequence Alignment and Analysis
Sequence alignment and editing were performed using the set of programs in the Lasergene package (DNASTAR, Madison, Wis.). Initial alignment was done in the program MegAlign using the Clustal algorithm with a gap penalty of 15 and a gap length penalty of 5. Minor adjustments to this alignment were made by eye. Phylogenetic analyses were performed using the program PAUP*, version 4.0 (Swofford 1998Citation ). An exhaustive search was used to find the most parsimonious tree for the coding regions of the PBPs. Bremer (1988)Citation support indices were calculated using the program TreeRot (Sorenson 1996Citation ) in conjunction with PAUP*. The bootstrap values were calculated with PAUP* using branch- and-bound searches with 1,000 replicates.

The program DnaSP, version 3.0 (Roza and Roza 1999Citation ), was used to calculate nucleotide diversity measures. These values were calculated after first excluding all regions of alignment gaps and missing data. Heterozygotes representing polymorphisms between the two alleles of an individual were randomly assigned to the two alleles for each individual for regions for which haplotype phase had not been determined. DnaSP, version 3.0, was also used to calculate values of Tajima's (1989)Citation D and Fu and Li's (1993)Citation D* and F*.

Nucleotide changes were mapped onto trees by a parsimony approach. Argyrotaenia velutinana was assumed to root the changes. For some changes (particularly in the noncoding regions), A. velutinana was not informative, and these changes were rooted using the C. murinana sequence (this taxon was found to be basal to the other Choristoneura species in phylogenetic analyses presented later). The significance of the MK tests and other homogeneity tests were assessed with Fisher's exact test using the program Fish6, version 1.21 (B. Engels, Madison, Wis.). Likelihood analysis of rate variation was carried out using the Codeml program of the PAML package (Yang 1999Citation ).

Results

Choristoneura PBPs
An alignment of the PBP amino acid sequences is shown in figure 2 for the coding regions of the PBPs from each of the five Choristoneura taxa and the outgroup A. velutinana. The six conserved cysteine residues and extensive sequence similarity to other PBPs identify these proteins as members of the PBP gene subfamily (Pelosi and Maida 1995Citation ). The mature protein portion of the C. rosaceana PBP is 70% identical in amino acid sequence to the PBP of O. nubilalis, but only 50% identical to Lymantria dispar PBP (Merritt et al. 1998Citation ). Another class of antennal proteins from moths, the GOBPs, are only about 35% identical in amino acid sequence to the C. rosaceana PBP. There is considerable amino acid variation among the PBPs from Choristoneura and Argyrotaenia; however, this set of PBPs are more similar to one another than to any other odorant-binding proteins, strongly suggesting that they are orthologous. The introns and flanking regions of these PBP sequences do not show any apparent sequence similarity to the same regions of PBP loci from species from other families of Lepidoptera.



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Fig. 2.—Alignment of amino acid sequences of PBPs from the Choristoneura species and Argyrotaenia velutinana. Standard one-letter abbreviations are used for amino acids. Taxon names are abbreviated by using the first letter of the genus name followed by the first three letters of the species name. The first 23 amino acids of the protein correspond to the signal peptide seen in PBPs from other taxa (Vogt, Rybczynski, and Lerner 1991Citation ). The start of the predicted mature protein is indicated with "MP." Three sites are polymorphic for amino acids, and these are included with both amino acids at a single site

 
The relationships of the five Choristoneura PBPs were examined by building a phylogeny using nucleotide data from the coding region. When the five Choristoneura PBPs are rooted with the A. velutinana PBP, a single most- parsimonious tree is obtained (fig. 3 ). This tree appears to have good support based on both bootstrap values and Bremer support indices. It is consistent with the relationships for the genus Choristoneura proposed by Harvey (1996)Citation based on variation at allozyme loci. The concordance of the gene tree of these PBPs with proposed species relationships also argues for the orthology of these PBPs.



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Fig. 3.—Phylogram depicting relationships among PBPs of the Choristoneura rooted with Argyrotaenia velutinana. A single tree of 168 steps was obtained when an exhaustive search was performed on the coding regions of the PBPs. Numbers to the right of the branches are the Bremer (1988)Citation support indices, a measure of the number of changes supporting each branch. Numbers to the left are the bootstrap values calculated from 1,000 bootstrap replicates

 
Nucleotide and Insertion/Deletion Variation
The entire 2-kb region including the PBP was sequenced for 24 alleles from the five different Choristoneura species (alignment in Willett [1999Citation ] or at http://www.smbe.org/journal/supp;lldata.html). A region of the same size was sequenced for one A. velutinana individual (Avel3), but only portions of the noncoding sequences of A. velutinana PBP sequence could be aligned with the Choristoneura PBP sequences. There are 64 insertions or deletions fixed between the five Choristoneura taxa. There have also been 25 deletions and 8 insertions that are polymorphic in the five Choristoneura taxa. Insertions and deletions vary widely in size, from a single nucleotide to 200 bp.

I sequenced between two and eight alleles for each of the six taxa, all of which show extensive nucleotide polymorphism in both coding and noncoding regions (table 2 ). Over the entire locus, the average pairwise difference between any two alleles from one species ranges from 0.54% in C. murinana to 1.8% in C. rosaceana. There are only four replacement changes among the polymorphisms, three of which fall within the signal peptide (see fig. 2 ). The overall levels of variation observed in the PBPs of Choristoneura are of the same order of magnitude as those observed in the PBP of O. nubilalis. At synonymous sites, O. nubilalis had an average pairwise difference of 3.6% (Willett and Harrison 1999Citation ), while the values in Choristoneura range from 0.7% for C. pinus to 2.6% for C. rosaceana (A. velutinana is even more variable, at 5.0%).


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Table 2 Nucleotide Diversity

 
In contrast to the few replacement polymorphisms, there has been a great deal of replacement change fixed between the PBPs of the six taxa. Replacement differences were mapped on the PBP tree, along with synonymous and noncoding differences (fig. 4 ). These changes were placed on the tree using a parsimony approach with A. velutinana as the outgroup. The changes for C. murinana are not assigned to a specific branch, but are shown in figure 4 summed over the two branches on which they could fall, because the corresponding noncoding regions of A. velutinana could not be aligned with the other species' noncoding regions. Changes that can be assigned to one of the two branches include 11 replacement, 10 synonymous, and 2 noncoding changes along the branch leading to C. murinana and 3 replacement, 5 synonymous, and 1 noncoding change on the branch connecting C. murinana to the other four Choristoneura species, leaving 3 silent and 87 noncoding changes unrooted. I calculated ratios of nonsynonymous substitution to synonymous/noncoding substitution and synonymous substitution alone (fig. 4 ). Including the noncoding sites gives 10 times as many sites as the number of synonymous sites alone; however, these noncoding sites include an unknown number of sites potentially subject to functional constraint, such as promoters and splicing signals. The branch leading to C. pinus is greater than 1 for both measures, while the branch to C. fumiferana is greater than 1 for the value including noncoding substitutions. These ratios suggest that selection has occurred on the C. pinus branch and perhaps also on the C. fumiferana branch.



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Fig. 4.—Changes placed on tree using parsimony. Fixed differences are shown to the right of the branches that they have been inferred to fall upon, while polymorphisms are shown to the right of taxon names. "R" refers to replacement differences, "S" to synonymous differences, and "NC" to noncoding differences. The ratio of replacement changes per site to synonymous/noncoding changes per site is given in parentheses, followed by the ratio of replacement to synonymous changes alone. For interior branches, the number of sites was estimated by taking the average of the values for the two terminal taxa (given in table 2 ). For both Choristoneura murinana and Argyrotaenia velutinana, the changes include those on the branch ancestral to the rest of the Choristoneura and not simply those on the lineage leading to these taxa. Several noncoding differences could not be mapped on specific branches and are shown above the node of the two branches upon which they could fall. Eight non-coding differences between Choristoneura parallela/rosaceana and C. murinana fall into deleted regions of the other taxa, and their locations cannot be determined

 
Tests for Selection
Table 3 shows the results of 2-by-2 contingency tables comparing the changes along each of the three branches on which I postulated major pheromone changes with the changes along the other Choristoneura branches. Choristoneura fumiferana has a significantly higher ratio of replacement to synonymous/noncoding fixation along its branch than occurs along branches with no major pheromone changes. However, neither C. parallela nor C. murinana show a significantly elevated ratio. Synonyomous/noncoding sites do not show evidence for rate variation along these lineages or the other lineages. Comparisons of lineages with equal lengths of divergence do not differ significantly in either number of synonymous fixations alone or total number of synonymous/noncoding fixations along each branch (tested for departures from equality using binomial test results not shown).


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Table 3 Replacement/Synonymous and Noncoding Fixed Difference Homogeneity Tests

 
The MK test results also point to nonneutral evolution along the C. fumiferana and C. pinus branches (table 4 ). The polymorphism data for each taxon were compared with the replacement changes fixed along the branch directly leading to that taxon; therefore, the significant results for C. pinus and C. fumiferana are independent of one another. If we assume that synonymous and noncoding sites are not under selection, then both of these significant results are consistent with an excess of replacement fixation or lack of replacement polymorphism. Combined with the fact that both C. fumiferana and C. pinus have higher rates of nonsynonymous substitution than synonymous/noncoding substitution, this suggests that both of these branches have undergone selection for fixation of replacement changes.


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Table 4 McDonald/Kreitman Tests on Six Taxa

 
Although the C. murinana PBP does not show a significant excess of replacement fixation, the trend is in the direction of an excess of replacement fixation over replacement polymorphism. This excess is seen for the six species PBPs when the replacement and synonymous polymorphisms are summed over all six and compared with the replacement and synonymous fixations summed over the tree (table 5 ). This excess of replacement fixation is significant even if the changes both along the C. pinus and C. fumiferana branches and polymorphisms within these species are excluded (P = 0.002). The number of synonymous fixations along the A. velutinana branch and other long branches is likely to be an underestimate of the true number, because multiple hits have probably occurred at many of these synonymous sites given the large number of observed changes (54 out of 103 sites for A. velutinana). The Jukes and Cantor correction can be used to account for multiple hits, and if this correction is applied to the synonymous sites in this study, it suggests that there have been an additional 13 unobserved synonymous fixations. Adding this number to the number of synonymous fixations in table 5 still gives a significant result (P = 0.003).


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Table 5 Pooled Homogeneity Tests

 
To examine whether the synonymous sites are evolving differently than the noncoding sites, I looked at the ratio of synonymous polymorphism to fixation compared with the ratio of noncoding polymorphism to fixation for the species in the genus Choristoneura (table 5 ). The result is significant at the P = 0.05 level. Noncoding and synonymous sites do not show different patterns of fixation along the lineages of Choristoneura species. Contingency tables (data not shown) for fixation of these two categories of changes along equivalent branches are not significantly heterogeneous (e.g., noncoding vs. synonymous fixations along the C. rosaceana and C. parallela branches). Taken together, these data suggest that neither depressed synonymous fixation nor increased synonymous polymorphism can explain the difference between synonymous and replacement variation seen in table 5 .

Tajima's (1989)Citation D test and Fu and Li's (1993)Citation test were used to test for departures from neutrality in the distribution of polymorphism within species for the four taxa from which four or more alleles were sequenced. For C. fumiferana, C. pinus, and A. velutinana, the values for both of these tests are negative but not significant. Negative values of these tests are caused by too many rare polymorphisms, which could be caused by recent selective sweeps near or at a locus or by population bottlenecks. For C. murinana, Tajima's D is 2.01, Fu and Li's D* is 1.58, and Fu and Li's F* is 1.86, all of which are significant at the P = 0.05 level. The small number of haplotypes sequenced for these species decreases the power of these frequency-based tests and may also lead to deviations due to sampling error. The positive values seen for C. murinana suggest an excess of intermediate frequency variants, which can result from balancing selection or population expansion. If there is balancing selection acting on the PBP locus of C. murinana, it is not acting directly on the amino acid sequence of this protein because there are no replacement polymorphisms in the PBP of this species.

Likelihood analysis of the PBP data reveals many of the same patterns uncovered by a parsimony-based method. The Codeml program (Yang 1999Citation ) was used to estimate dN/dS ratios on the different branches of the PBP tree. This approach estimates the branch lengths and transition/transversion ratios along each branch of a tree given an alignment of coding sequences and their relationships. Comparing the likelihood of a model assuming a single rate over all branches (one-ratio Ln(l) = -1,368.86) to a model allowing the ratio to vary between branches (free-ratio Ln(l) = -1,354.53), it can be shown that there is significant variation in dN/dS ratios between branches ({chi}2 = 28.7 df = 8; P < 0.005). figure 5 shows the estimates of dN/dS ratios obtained under the free-ratio model and the estimates of the numbers of replacement and synonymous changes along each of the branches. Again, both the C. fumiferana and the C. pinus branches appear to have elevated dN/dS ratios, although the estimate for C. fumiferana is less than 1. The branch connecting C. murinana to the rest of the Choristoneura species also shows a high dN/dS ratio in this likelihood analysis but involves relatively few changes. The parsimony approach estimated a minimum of two replacement and five synonymous changes on this branch and does not show an elevated dN/dS ratio for this branch (dN/dS = 0.01 based on the fixed changes inferred by the parsimony approach).



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Fig. 5.—Likelihood estimates of dN/dS and numbers of replacement and silent changes for branches of the tree. Estimation was done under the free-ratio model with the program Codeml of the PAML, version 2.0, package (Yang 1999Citation ). The tree used is that presented in figure 3 , and the data include the nucleotide data for coding regions. The two branches with dN/dS > 20 approach infinity due to the lack of synonymous substitution estimated to have occurred on these branches

 
Discussion

The pheromone-binding proteins of moths in the genus Choristoneura have undergone at least two detectable selective episodes among this sample of six taxa. The change to an aldehyde pheromone in C. fumiferana was accompanied by an excess of replacement substitution in its PBP. The PBP of C. pinus appears to have undergone a similar acceleration in replacement substitution, but one that was not accompanied by a major pheromone change. The branches leading to both of these taxa show rates of nonsynonymous substitution greater than the synonymous/noncoding substitution rate. Both C. pinus and C. fumiferana give significant MK test results consistent with an excess of replacement fixation over replacement polymorphism. These MK results indicate that there has apparently not been a relaxation of functional constraint on both of these lineages that could explain the high rate of replacement fixation. Choristoneura pinus and C. fumiferana have levels of replacement polymorphism that are at least an order of magnitude lower than the level of synonymous/noncoding polymorphism (table 2 ). If functional constraint on protein sequence had been relaxed completely, the levels of replacement and synonymous/noncoding polymorphism should be equal under neutrality.

Selection on PBP along the branch leading to C. fumiferana could be explained by the association of this selection with an apparent change from an acetate to an aldehyde pheromone. Without binding studies, it is impossible to determine if this association between selection on the PBP and pheromone change is the result of selection for greater affinity of the PBP for the aldehyde pheromone component. Selection on the branch leading to C. pinus cannot be explained in the same manner, because there does not appear to have been a major pheromone change along the C. pinus branch. A possible explanation for selection on the C. pinus PBP is that interactions with odorants other than pheromones have led to the excess of amino acid substitution along this lineage. An example of another class of compounds recognized by the male antennae (in many cases by the same receptors that recognize pheromones) is antagonist compounds (Glover, Perez, and Roelofs 1989Citation ; Hallberg, Hansson, and Steinbrecht 1994Citation ). PBPs could also be interacting with other molecules than odorants, and it is possible that these interactions could lead to selection on the PBP. One potential interaction for PBPs could be with uncharacterized membrane-bound G-protein–coupled receptor proteins that are thought to be involved in pheromone discrimination in moths (Laue and Steinbrecht 1997Citation ).

Neither the C. murinana nor the C. parallela branches show a significantly elevated rates of nonsynonymous substitution nor significant MK test results despite the pheromone changes that appear to have occurred along these branches. Choristoneura murinana is using components with a different chain length in its pheromone (Z9-12:OAc and 11-12:OAc). Choristoneura parallela uses E11-14:OH as its major pheromone component rather than the E- or Z11-14:OAc major components used by the other Choristoneura. It is possible that the scenario of a single major pheromone change along each of these branches and no pheromone change on the other branches is overly simplistic. These species use many of the same components, but often in very different proportions. If PBPs must also have the ability to bind minor pheromone components, then the pattern of pheromone change that could be important for PBP evolution is likely to be different from that proposed here.

In the five Choristoneura species, there are 20 replacement changes along the branches where there has been no major pheromone change and another 19 replacement changes on the three branches with major changes. Only for C. pinus and C. fumiferana branches do the tests of selection give significant results. Are the replacement substitutions on the other Choristoneura branches neutral with no consequence for PBP function? Neither the MK test nor the rate homogeneity test are very powerful for detecting selective events involving few changes or selective events that are not very recent. A dN/dS ratio greater than 1 for an entire protein is also not likely to be observed when the selection event occurred some time ago and only involved a few residues in a protein. Therefore, the fact that the tests for selection have not yielded significant results for some of the branches in the PBP gene tree does not necessarily exclude selection having fixed some of these amino acid substitutions or the possibility that some of these substitutions could affect the binding affinities of the PBPs.

The trend in the overall data for an excess of amino acid fixations (table 5 ) could reflect undetected selective events on the PBP in the history of this protein in this group of moths. The excess is present even when the two branches that appear to be under selection are excluded. The comparison of synonymous and noncoding variation (table 5 ) suggests that these two categories of variation are behaving significantly differently, but the direction of the deviation strengthens the case for excess amino acid fixation. There appear to have been either more synonymous fixations or fewer synonymous polymorphisms than expected under neutrality (if noncoding sites are assumed to reflect neutral evolution), either of which would amplify the difference between replacement and synonymous sites (table 5 ).

The results from this study, combined with those from the corn borers, suggest that PBPs are not changing with every pheromone change. Given the fact that only one of three pheromone changes in this group of moths from Choristoneura is associated with detectable selection on the PBP, and other episodes of selection are not associated with pheromone change, it is not clear that pheromone changes cause selection on PBPs in general. It is clear that selection has driven amino acid substitution in PBPs from moths of the genus Choristoneura several times. Perhaps other undiscovered interactions are responsible for this selection. It is also possible that pheromone changes are driving selection, but these pheromone changes involve interactions between both major and minor pheromone components. Additional taxa reflecting independent pheromone changes could help to determine the relationship between pheromone change and selection on the PBP. For example, are other switches to aldehyde pheromones from acetate pheromones associated with selection on the PBP?

Acknowledgements

I would like to thank the members of my graduate committee, Charles Aquadro, Wendell Roelofs, and especially my advisor, Richard Harrison, for their assistance and advice on this project. I am indebted to Wendell Roelofs, Kathy Poole, Marc Kenis, Jeremy McNeil, and Sridhar Polavarapu, who provided moths for this work. John Franclemont gave helpful advice on collecting and identifying moths; Tim Carr and Scott Stanley helped me to collect. I would also like to thank the other members of the Harrison lab, especially Steve Bogdanowicz. D. Irwin and two anonymous reviewers provided helpful comments on the manuscript. The work was supported by National Science Foundation grant IBN-9700704 to Richard G. Harrison and myself, and I was supported by an NSF predoctoral fellowship and an NIH traineeship through the field of Genetics and Development.

Footnotes

David Irwin, Reviewing Editor

1 1Present address: Marine Biology Research Division-0202, Scripps Institution of Oceanography, University of California San Diego. Back

2 Keywords: Choristoneura, pheromone-binding proteins sequence variation protein evolution adaptation Back

3 Address for correspondence and reprints: Christopher S. Willett, Marine Biology Research Division-0202, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202. E-mail: cwillett{at}ucsd.edu Back

supplemental materials

    Accession numbers are as follows: C. fumiferana, AF177642-AF177644; C. rosaceana, AF177652, AF177653, and AF177655; C. parallela, AF177648-AF17749; C. murinana, AF177645-AF177647 and AF177662; C. pinus, AF177650, AF177651, and AF177653; and A. velutinana, AF177639-AF177641.

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Accepted for publication December 13, 1999.