DOI: 10.5061/dryad.5x69p8d0j
title: Molecular evolution of the sex peptide network in Drosophila

description: Successful reproduction depends on interactions between numerous proteins
 beyond those involved directly in gamete fusion.   While
 such reproductive proteins evolve in response to sexual selection
 pressures, how networks of interacting proteins arise and evolve as
 reproductive phenotypes change remains an open question.  Here,
 we investigated the molecular evolution of the “sex peptide network” of
 Drosophila melanogaster, a functionally well-characterized reproductive
 protein network.  In this species, the peptide hormone sex
 peptide (SP) and its interacting proteins cause major changes in female
 physiology and behavior after mating.  In contrast, females of
 more distantly related Drosophila species do not respond to SP. 
 In spite of these phenotypic differences, we detected orthologs of all
 network proteins across 22 diverse Drosophila species and found evidence
 that most orthologs likely function in reproduction throughout the
 genus.  Within SP-responsive species, we detected the recurrent,
 adaptive evolution of several network proteins, consistent with sexual
 selection acting to continually refine network function.  We also
 found some evidence for adaptive evolution of several proteins along two
 specific phylogenetic lineages that correspond with increased expression
 of the SP receptor in female reproductive tracts or increased sperm
 length, respectively.  Finally, we used gene expression profiling
 to examine the likely degree of functional conservation of the paralogs of
 an SP network protein that arose via gene duplication.  Our
 results suggest a dynamic history for the SP network in which network
 members arose before the onset of robust SP-mediated responses and then
 were shaped by both purifying and positive selection.
description: We obtained the protein sequence for each SP
 network protein in <i>D. melanogaster</i> from FlyBase. For
 species for which protein annotations were available on FlyBase we
 obtained orthologous protein-coding DNA sequences using the FlyBase
 Orthologs feature. For species with sequenced genomes that lacked FlyBase
 protein annotations, we manually searched for gene orthologs using tBLASTn
 and the<i> D. melanogaster</i> protein sequence as the query.
 For genes expected to have introns, we looked in the unannotated species
 for the approximate location of the<i> D. melanogaster</i>
 intron, and used known intron border consensus sequences and six-frame
 translation to identify predicted intron borders and remove intronic
 sequences.  We used MUSCLE as implemented in MEGA
 6.06 to align amino acid sequences, then visually
 checked and edited each alignment for accuracy. Amino acid alignments were
 then back-translated in MEGA to obtain the cDNA alignment.
 (branch_alignments.txt) 
 To detect recombination, we used GARD as
 implemented in DataMonkey 2.0 Genes were partitioned at breakpoints
 evaluated as significant by the Kishino-Hasegawa test. 
 (Partitioned_alignments.txt)
 To infer a <i>Drosophila</i> consensus
 phylogeny based on all SP network proteins, we concatenated the amino acid
 alignments of all SP network proteins within each of the 22 species
 (master_alignment.phy). We used PROML in Phylip to infer an unrooted
 maximum-likelihood phylogeny (with random input order, slow analysis, and
 all other default parameters).
description: Gene alignments are in MEGA sequential format, for use in
 HyPhy. To use with PAML, simply move the trees to a separate
 file.   
 Branch_alignments.txt contains all the alignments
 for each gene evaluated in this study. Genes are denoted by ##.
  
 Partitioned_alignments.txt contains all the
 alignments for genes that were partitioned based on positive evidence of
 recombination.  
 Master_alignment.phy contains the concatenated
 amino acid alignments of all SP network proteins within each of the 22
 species, in PHYLIP format. Gaps in the alignment were used in cases in
 which a protein was not present in a particular species. Two branches were
 evaluated for lineage-specific adaptive evolution in this study denoted by
 'branch1', which leads to <i>D. ananassae</i> and
 <i>D. bipectinata</i>; and 'branch2', leading to the
 <i>melanogaster</i> group.