* Section of Integrative Biology, University of Texas-Austin
Section of Evolution and Ecology, University of California-Davis
Correspondence: E-mail: aholloway{at}mail.utexas.edu.
Abstract
To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.
Key Words: accessory gland protein Drosophila gene duplication gene expression molecular evolution selection
At least three classes of models have been proposed to explain the evolutionary processes for the retention and subsequent divergence of gene duplicates. Lynch and Force (2000) suggest that ancestral genes with multiple functions in different tissues or developmental stages may have high rates of retention of duplicates under mutation-selection balance. In this model, degenerative mutations result in subfunctionalization, which favors retention and subsequent evolution of duplicates. A second class of models invokes fixation of duplications by genetic drift (e.g., Lynch and Conery 2003; Walsh 2003). Finally, a third class of models relies on new, beneficial mutations driving adaptive divergence (and thus retention) of duplicates (Hughes 1994). One would expect new duplicates from classes of proteins under chronic directional selection to have unusually high fixation probabilities because a higher proportion of new mutations may be beneficial in such genes. For example, if reproduction-related proteins experience directional selection more frequently than other proteins (Civetta and Singh 1998; Nurminsky et al. 1998; Swanson and Vacquier 2002; Ranz et al. 2003), then perhaps a large number of duplicate reproduction-related genes spread through populations and diverge under directional selection.
We investigated duplication and divergence in reproduction-related accessory gland proteins genes (Acps) in Drosophila. Acps are male-specific seminal fluid proteins that affect multiple aspects of female physiology and behavior (for review see Wolfner 1997). We carried out Blast comparisons of the 13 annotated Acps (see Methods) to the D. melanogaster reference sequence (Flybase Consortium 2003). These Blast analyses suggested that two genes, Acp29AB and Acp53Ea, are members of small multigene families.
E-values returned from the tBlastN search (default parameters) with Acp29AB as the query sequence were 1.5 x 1047 and 2.6 x 1035 for Lectin29Ca and Lectin30A, respectively. Intraspecific paralogous protein divergence was, on average, 31% between Acp29AB and Lectin29Ca, 35.5% between Acp29AB and Lectin30A, and 38% between Lectin29Ca and Lectin30A. Lectin29Ca is 356 bases distal to Acp29AB and Lectin30A is approximately 1 Mbase distal to these tandem duplicates. Acp29AB is 234 amino acids, while Lectin29Ca and Lectin30A are 236 and 223 amino acids long, respectively. Each gene is composed of a single exon. Our analysis of Lectin30A and comparison to its paralogs suggested that the 5' end was incorrectly annotated. We confirmed this hypothesis by RACE, and we used our annotation in all analyses. The three members of the Acp29AB family are predicted to be lectin galactose binding proteins (Theopold et al. 1999) and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997). The tBlastN search returned several other more distantly related putative Acp29AB paralogs, primarily lectins (Lectin21Cb, Lectin24Db, Lectin22C, Lectin 21Ca, Lectin24A, Lectin28C, and CG15818). However, we will not present data from these genes in this report.
E-values returned from the tBlastN search with Acp53Ea as the query sequence were 2.1 x 105 for CG8626 and 9.4 x 104 for CG15616. CG8626 and CG15616 will hereafter be referred to as Acp53C14a and Acp53C14b, respectively, based on putative function, genomic location, and gene structure. Another more highly diverged putative duplicate identified by B. Wagstaff (personal communication) did not appear in our Blast results. However, this gene (Acp53C14c) appears to be another tandem duplicate and shows male-limited expression (B. Wagstaff, personal communication). Intraspecific paralogous protein divergence was 48.5% between Acp53Ea and Acp53C14a, 42.5% between Acp53Ea and Acp53C14b, and 45% between Acp53C14a and Acp53C14b. The divergence of Acp53C14c from other putative Acp53Ea duplicates was >65%. These genes are tandem duplications, with Acp53C14a located 423 bp proximal to Acp53C14b, Acp53Ea 487 bases distal to Acp53C14b, and Acp53C14c 519 bp distal to Acp53Ea. Acp53Ea, Acp53C14a, Acp53C14b, and Acp53C14c are predicted to be 120, 121, 132, and 124 amino acids long, respectively. Each is composed of two exons with a 5060 nt intron roughly 40 bases from the initiation codon. All genes are predicted to be peptide hormones and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997).
The high levels of silent and replacement divergence among putative paralogs suggest that the duplication events predate the split of D. yakuba from the D. melanogaster/D. simulans lineage. Nevertheless, the conserved gene structures, expression patterns, presence of predicted signal peptides, and, for most cases, tandem organization all indicate that we have correctly identified paralogous genes.
Acp29AB and Acp53Ea are expressed only in male accessory glands (Wolfner et al. 1997). Our RT-PCR experiments showed that the only detectable expression of Lectin29Ca, Acp53C14a, and Acp53C14b is in accessory glands (fig. 1). We were unable to detect an RT-PCR product from Lectin30A. However, given that our RACE products were derived from male cDNA, we are certain the gene is expressed in males (perhaps at low levels). The fact that the Acp duplicates identified here share accessory-gland enriched expression further supports the inference of paralogy and suggests that subfunctionalization, at least with respect to gene expression (sensu Lynch and Force 2000), cannot explain fixation of Acp duplicates. Levels of protein polymorphism and divergence for these Acp genes and putative duplicates (table 1) were higher than those typically seen in D. simulans and D. melanogaster genes, as was the case for previous surveys of Acp variation (Begun et al. 2000; Swanson et al. 2001). There was, however, a major exception in the Acp29AB family.
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Methods
Amino acid sequences of 13 annotated Acps from D. melanogaster (Acp26Aa, Acp26Ab, Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp70A, Acp76A, Acp95EF, and Acp 98AB) were compared to the D. melanogaster reference sequence (Genome Release 3.0, Flybase Consortium 2003) by tBlastN searches using default parameters. Sequences were aligned by manual curation and molecular population genetics of putative duplicates with <50% nucleotide divergence from a known Acp were investigated. Nucleotide divergence (or uncorrected pairwise-distance) was calculated as a measure of the number of mismatched nucleotides/total number of nucleotides at 1st and 2nd codon positions. Duplicate genes are and considered putative Acps but are referred to as Acps for convenience.
Candidate Acp duplicates were subjected to RT-PCR to determine whether their expression was restricted to accessory glands. mRNA was extracted from four tissues of D. melanogaster: male accessory glands, testes, male carcasses, and whole females. First strand synthesis was carried out using an oligo-dT primer and Superscript Reverse Transcriptase (Invitrogen, San Diego, Calif.). RT-PCR was carried out using gene-specific primers on RNA/DNA heteroduplex isolated from each tissue.
D. simulans population genetic data are from inbred lines established from flies collected at the Wolfskill Orchard in Winters, Calif. (Begun and Whitley 2000). D. melanogaster population data are from isochromosomal lines derived from the Wolfskill Orchard or from isofemale lines from Malawi, Africa. D. yakuba sequences are from an isofemale line. Some D. simulans Acp29AB sequences are from Begun et al. (2000). In most cases DNA sequencing was carried out directly on PCR products. For cases in which inbred lines were not available, PCR products were cloned prior to sequencing.
Sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI) and manually curated in MacClade 4.0 (Maddison and Maddison 2000). Alignments are available upon request from the authors. Summary statistics and tests of the neutral equilibrium model were carried out using DnaSP version 3.53 (Rozas and Rozas 1999). SignalP version 2.0 was used to predict presence/absence of signal peptides characteristic of Acps and other secreted proteins (Nielsen et al. 1997). Sequences were submitted to GenBank under accession numbers AY635196-AY635290.
Acknowledgements
We thank A. Kern and S. Joseph for useful comments on the manuscript. Grants from the University of Texas Graduate Program in Evolution, Ecology, and Behavior to A.K.H. and the NIH (GM55298) and NSF (DEB-0327049) to D.J.B supported this work.
Footnotes
Lauren McIntyre, Associate Editor
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