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 1995
). The McDonald and Kreitman (MK; 1991)
, Tajima's (1989)
D, and Fu and Li (1993)
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 1988
; Hughes 1991
; Metz, Robles-Sikisaka, and Vacquier 1998
; Yang 1998
).
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 1981
; Prestwich, Du, and Laforest 1995
; Vogt 1995
; Laue and Steinbrecht 1997
). 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 1995
; Bianchet et al. 1996
). 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 1991
). Vogt, Rybczynski, and Lerner (1991)
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 1999
). 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 1982
). 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 1992
; Sperling and Hickey 1994
; Harvey 1996
).
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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)
. 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)
. 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 1999
). 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 1998
). An exhaustive search was used to find the most parsimonious tree for the coding regions of the PBPs. Bremer (1988)
support indices were calculated using the program TreeRot (Sorenson 1996
) 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 1999
), 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)
D and Fu and Li's (1993)
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 1999
).
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 1995
). 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. 1998
). 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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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 1999
), 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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Tajima's (1989)
D test and Fu and Li's (1993)
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 1999
) 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 (
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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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 1989
; Hallberg, Hansson, and Steinbrecht 1994
). 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-proteincoupled receptor proteins that are thought to be involved in pheromone discrimination in moths (Laue and Steinbrecht 1997
).
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
1 1Present address: Marine Biology Research Division-0202, Scripps Institution of Oceanography, University of California San Diego.
2 Keywords: Choristoneura,
pheromone-binding proteins
sequence variation
protein evolution
adaptation
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
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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