DOI: 10.5061/dryad.8cz8w9gm8
title: Data from: Quantitative proteomics reveals rapid divergence in the postmating response of female reproductive tracts among sibling species

description: Fertility depends, in part, on interactions between male and female
 reproductive proteins inside the female reproductive tract (FRT) that
 mediate postmating changes in female behavior, morphology, and physiology.
 Coevolution between interacting proteins within species may drive
 reproductive incompatibilities between species, yet the mechanisms
 underlying postmating-prezygotic isolating barriers remain poorly
 resolved. Here, we used quantitative proteomics in
 sibling Drosophila species to investigate the
 molecular composition of the FRT environment and its role in
 mediating species-specific postmating responses. We found that
 (1) FRT proteomes in D. simulans and D.
 mauritiana virgin females express unique combinations of secreted
 proteins and are enriched for distinct functional categories, (2) mating
 induces substantial changes to the FRT proteome in D.
 mauritiana but not in D. simulans, and (3)
 the D. simulans FRT proteome exhibits limited postmating
 changes irrespective of whether females mate with conspecific or
 heterospecific males, suggesting an active female role in mediating
 reproductive interactions. Comparisons with similar data in the closely
 related outgroup species D. melanogaster suggest that
 divergence is concentrated on the D. simulans lineage.
 Our study suggests that divergence in the FRT extracellular environment
 and postmating response contribute to previously described patterns
 of postmating-prezygotic isolation and the maintenance
 of species boundaries.
description: <strong><em>D. simulans </em>and <em>D.
 mauritiana</em> FRT proteins</strong> FRT
 samples were collected from the following five
 conditions: <i>Drosophila simulans</i> virgins
 (<i>sim</i> virgins), <i>D. mauritiana </i>virgins
 (<i>mau </i>virgins), <i>D. simulans </i>females
 mated to<i> D. simulans </i>males
 (<i>sim </i>× <i>sim</i>), <i>D.
 mauritiana </i>females mated to <i>D.
 mauritiana </i>males
 (<i>mau </i>× <i>mau</i>), and <i>D.
 simulans </i>females mated to <i>D. mauritiana </i>males
 (<i>sim </i>× <i>mau</i>). Mated samples were
 collected 6h after the end of a successful
 copulation. FRTs (i.e., bursa, oviduct, seminal receptacle, spermathecae, parovaria, and associated fat body) of ~100 females from each condition were dissected, pooled in PBS per replicate, and solubilized in 1M HEPES with 2% SDS and 5% b-mercaptoethanol. Two replicates were collected per condition resulting in ten samples. Samples were labelled with TMT isobaric tags and analyzed by LC-MS/MS. Peptides were quantified using a synchronous precursor selection MS3 method. Mass spectra were searched against the <i>D. simulans </i>protein database (dsim-all-translation-r2.02, FlyBase.org) using PEAKS X (Bioinformatics Solutions Inc.). Reporter ion intensities were calculated by summing the centroided reporter ions, and protein abundances were calculated by summing the reporter ion intensities in each channel. Peptide identifications were accepted if the false discovery rate (FDR) &lt; 1% based on the decoy-fusion approach, and protein identifications were accepted if the FDR &lt; 1%. <i>D. simulans </i>FlyBase gene (FBgn) identifiers were converted to their orthologous <i>D. melanogaster </i>FBgn identifiers using the FlyBase Drosophila Orthologs database. To compare the mating-induced abundance changes of FRT proteins, we identified and removed putative male-derived proteins. Proteins were classified as male-derived if they had been previously identified as either <i>D. melanogaster </i>sperm proteins or <i>D. melanogaster </i>SFPs, or if they showed signatures of being male-derived ejaculate proteins unique to <i>D. simulans </i>and/or <i>D. mauritiana</i>. Raw protein abundances were log<sub>2</sub>-transformed and median-normalized using the MSnbase package within BioConductor in R. Protein abundances were highly correlated between replicates, with Pearson’s r &gt; 0.97 for all pairwise comparisons. Differential protein abundances were evaluated with empirical Bayes moderated t-tests using the LIMMA package in BioConductor.  <strong><em>D. melanogaster</em> FRT proteins</strong> FRT samples were collected from virgin and mated <em>D. melanogaster </em>females. Consistent with the <i>simulans/mauritiana</i> samples, mated <i>D. melanogaster </i>samples were collected 6h after copulation. Two replicates were collected per condition. FRTs from ~150 females were dissected and pooled in PBS per replicate, solubilized in 2× Laemmli buffer with 10% TCEP, and analyzed by LC-MS/MS. Mass spectra were searched against the <i>D. melanogaster </i>protein database (dmel-all-translation-r6.30, FlyBase.org) in PEAKS X. Proteins that had previously identified as sperm proteins or SFPs were removed. Protein abundances were quantified as spectral counts corrected for protein length, and analyzed using identical methods in BioConductor.  <strong>Molecular evolutionary rate analysis</strong> Molecular evolutionary rate analysis between <i>D. simulans</i>, and <i>D. mauritiana </i>were based on established orthology relationships from FlyBase. Protein-coding nucleotide sequences were aligned using the linsi algorithm in MAFFT and reverse translated in frame. We estimated pairwise values of dN (non-synonymous substitution rate) and dS (synonymous substitution rate) using the Yang and Nielsen method as implemented by PAML. 
description: <strong>Table 1: <em>D.
 simulans </em>and <em>D. mauritiana</em> FRT
 proteins</strong> Final dataset of the 3287
 <em>D. simulans </em>and <em>D. mauritiana</em> 
 proteins used in the analyses
 Table1_sim_mau_FRT_proteins.csv  
 <strong>Table 2: <em>D. melanogaster </em>FRT
 proteins</strong> Final dataset of the 1909
 <em>D. melanogaster</em> FRT proteins used in the
 analyses Table2_mel_FRT_proteins.csv
   <strong>Table 3: dNdS
 estimates</strong> Estimates of non-synonymous
 (dN) and synonymous (dS) substitution rates for pairwise comparisons
 between <em>D. simulans </em>and <em>D.
 mauritiana</em> Table3_dNdS.csv
   <strong>Female
 list</strong> Raw protein abundance estimates
 (i.e., before transformation and normalization) for the
 <em>simulans/mauritiana </em>dataset
 female_list.csv  
 <strong>R code for normalization and differential
 expression analysis</strong> R code for
 normalizing the data and conducting the differential expression analyses
 on the <em>simulans/mauritiana </em>dataset
 sim_mau_analyses.R