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The Old and the New: Discovery Proteomics Identifies Putative Novel Seminal Fluid Proteins in Drosophila*

  • Timothy L. Karr
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    From the ‡Center for Mechanisms of Evolution, The Biodesign Institute, Arizona State University, Tempe, Arizona;
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  • Helen Southern
    Affiliations
    Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK;
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  • Matthew A. Rosenow
    Affiliations
    Caris Life Sciences, Phoenix, Arizona;
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  • Toni I. Gossmann
    Affiliations
    Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK;
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  • Rhonda R. Snook
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Department of Zoology, Stockholm University, Stockholm, Sweden
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  • Author Footnotes
    * Funding of the experimental evolution lines came from NSF (DEB-0093149), NERC (NE/B504065/1; NE/I014632/1), and EU (ITN-2008-213780 SPECIATION) to RRS. Funding and technical support for the proteomic work came from the University of Sheffield Biological Mass Spectrophotometry Facility (funded by Yorkshire Cancer Research (Shend01) and the Wellcome Trust). TIG was supported by a Leverhulme Early Career Fellowship (Grant ECF-2015-453) and a NERC grant (NE/N013832/1). Funding from the Kyoto Institute of Technology to TLK also supported this research.
    This article contains supplemental Figures and Tables.
Open AccessPublished:February 13, 2019DOI:https://doi.org/10.1074/mcp.RA118.001098
      Seminal fluid proteins (SFPs), the nonsperm component of male ejaculates produced by male accessory glands, are viewed as central mediators of reproductive fitness. SFPs effect both male and female post-mating functions and show molecular signatures of rapid adaptive evolution. Although Drosophila melanogaster, is the dominant insect model for understanding SFP evolution, understanding of SFP evolutionary causes and consequences require additional comparative analyses of close and distantly related taxa. Although SFP identification was historically challenging, advances in label-free quantitative proteomics expands the scope of studying other systems to further advance the field. Focused studies of SFPs has so far overlooked the proteomes of male reproductive glands and their inherent complex protein networks for which there is little information on the overall signals of molecular evolution. Here we applied label-free quantitative proteomics to identify the accessory gland proteome and secretome in Drosophila pseudoobscura, a close relative of D. melanogaster, and use the dataset to identify both known and putative novel SFPs. Using this approach, we identified 163 putative SFPs, 32% of which overlapped with previously identified D. melanogaster, SFPs and show that SFPs with known extracellular annotation evolve more rapidly than other proteins produced by or contained within the accessory gland. Our results will further the understanding of the evolution of SFPs and the underlying male accessory gland proteins that mediate reproductive fitness of the sexes.

      Graphical Abstract

      Male ejaculates typically consist of a sperm component and a nonsperm component, both of which are transferred to females during mating. The nonsperm component is seminal fluid, containing secreted peptides and proteins (SFPs)
      The abbreviations used are: SFPs, seminal fluid proteins; BLAST, Basic Local Alignment Search Tool; PCSS, postcopulatory sexual selection; SDS, sodium dodecylsulfate; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; MS, mass spectrometry; LC-MS/MS, liquid chromatography-MS/MS; AcgP, accessory gland proteome; FDRs, False Discovery Rates; AcgS, accessory gland secretome; exoP, exoproteome; LFQ, label-free quantitation; P, polyandry; M, monandry; GO, gene ontology; CC, cellular component; MF, molecular function; BP, biological process; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; DIOPT, DRSC Integrative Ortholog Prediction Tools; ER, endoplasmic reticulum.
      1The abbreviations used are: SFPs, seminal fluid proteins; BLAST, Basic Local Alignment Search Tool; PCSS, postcopulatory sexual selection; SDS, sodium dodecylsulfate; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; MS, mass spectrometry; LC-MS/MS, liquid chromatography-MS/MS; AcgP, accessory gland proteome; FDRs, False Discovery Rates; AcgS, accessory gland secretome; exoP, exoproteome; LFQ, label-free quantitation; P, polyandry; M, monandry; GO, gene ontology; CC, cellular component; MF, molecular function; BP, biological process; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; DIOPT, DRSC Integrative Ortholog Prediction Tools; ER, endoplasmic reticulum.
      , typically produced in the testes and specialized male exocrine glands (
      • Poiani A.
      Complexity of seminal fluid: a review.
      ,
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ). SFPs have profound effects on both male and female reproductive fitness (
      • Perry J.C.
      • Sirot L.
      • Wigby S.
      The seminal symphony: How to compose an ejaculate.
      ) and therefore significant attention has been focused on the role of SFPs in polyandrous species. Polyandry, where females mate with different males across a reproductive bout generating postcopulatory sexual selection, results in ejaculates that compete for fertilization of a limited supply of ova, and females may choose whose sperm will fertilize those limited ova (
      • Snook R.R.
      The evolution of polyandry 159–180.
      ). Polyandry also engenders sexual conflict, in which male and female reproductive interests differ, because of the disproportionate costs and benefits of mating between the sexes (
      • Arnqvist G.
      • Rowe L.
      ). In internally fertilizing species, postcopulatory sexual selection operates between the male ejaculate that is transferred to and stored in the female reproductive tract (
      • Chen P.S.
      The functional morphology and biochemistry of insect male accessory glands and their secretions.
      ). SFPs in these species may increase female fecundity, reduce female receptivity, decrease female life span, alter female hunger, and remodel female reproductive tract morphology (
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ,
      • Perry J.C.
      • Sirot L.
      • Wigby S.
      The seminal symphony: How to compose an ejaculate.
      ,
      • Mattei A.L.
      • Riccio M.L.
      • Avila F.W.
      • Wolfner M.F.
      Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract, obtained by micro-computed tomography scanning.
      ,
      • Avila F.W.
      • Wolfner M.F.
      Acp36DE is required for uterine conformational changes in mated Drosophila females.
      ).
      SFPs were first identified by their canonical signal peptide sequence that direct proteins to the secretory pathway (
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ). Cross-species comparative work has found that general classes of SFPs are conserved (e.g., proteases and protease inhibitors, lectins and prohormones) suggesting that their mechanisms of action are also conserved. However, individual SFPs can rapidly evolve with signals of accelerated rates of adaptive molecular evolution found in studies of coding sequence and male-biased gene expression observed across different animal taxa (e.g., mammals (
      • Dorus S.
      • Evans P.D.
      • Wyckoff G.J.
      • Choi S.S.
      • Lahn B.T.
      Rate of molecular evolution of the seminal protein gene SEMG2 correlates with levels of female promiscuity.
      ,
      • Ramm S.A.
      • McDonald L.
      • Hurst J.L.
      • Beynon R.J.
      • Stockley P.
      Comparative proteomics reveals evidence for evolutionary diversification of rodent seminal fluid and its functional significance in sperm competition.
      ); birds (
      • Harrison P.W.
      • Wright A.E.
      • Zimmer F.
      • Dean R.
      • Montgomery S.H.
      • Pointer M.A.
      • Mank J.E.
      Sexual selection drives evolution and rapid turnover of male gene expression.
      ); Drosophila (
      • Clark N.L.
      • Aagaard J.E.
      • Swanson W.J.
      Evolution of reproductive proteins from animals and plants.
      ,
      • Mueller J.L.
      • Ravi Ram K.
      • McGraw L.A.
      • Bloch Qazi M.C.
      • Siggia E.D.
      • Clark A.G.
      • Aquadro C.F.
      • Wolfner M.F.
      Cross-species comparison of Drosophila male accessory gland protein genes.
      ,
      • Panhuis T.M.
      • Clark N.L.
      • Swanson W.J.
      Rapid evolution of reproductive proteins in abalone and Drosophila.
      )). Sex-biased genes in general show faster rates of sequence and expression divergence that is consistent with predictions from sexual selection (e.g., (
      • Parsch J.
      • Ellegren H.
      The evolutionary causes and consequences of sex- biased gene expression.
      ) but see (
      • Harrison P.W.
      • Wright A.E.
      • Zimmer F.
      • Dean R.
      • Montgomery S.H.
      • Pointer M.A.
      • Mank J.E.
      Sexual selection drives evolution and rapid turnover of male gene expression.
      )).
      Despite these general patterns, there are limitations to understanding the evolution of SFPs and their function. For example, SFP identification and their role in influencing fitness is dominated by work in D. melanogaster,. This species is relatively highly polyandrous (
      • Markow T.A.
      Evolution of Drosophila mating systems.
      ) and studies identifying SFPs in species with different mating systems is necessary to understand the evolution of reproductive proteins and their fitness consequences. The advent of high throughput proteomics using LC-MS/MS should allow identification of SFPs, even in nonmodel organisms although tests of adaptive evolution may be restricted (
      • Bayram H.
      • Sayadi A.
      • Immonen E.
      • Arnqvist G.
      Identification of novel ejaculate proteins in a seed beetle and division of labour across male accessory reproductive glands.
      ).
      Moreover, SFPs function in a complex network of protein-tissue interactions (
      • Poiani A.
      Complexity of seminal fluid: a review.
      ,
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      )). However, the general focus on SFPs (a small subset of the accessory gland proteome) leaves open questions about the full complexity of the accessory gland proteome that supports the production of these critical reproductive proteins. Further, the larger role other accessory gland proteins play in postcopulatory sexual selection, and how the accessory gland proteome responds to such selection in toto, has been relatively ignored. For example, despite D. melanogaster, being a model system for this work, there is but a single study of its accessory gland proteome which is based on 2D gel electrophoresis (
      • Takemori N.
      • Yamamoto M.T.
      Proteome mapping of the Drosophila melanogaster male reproductive system.
      ). The recent advent of high throughput proteomics using LC-MS/MS should allow identification of not only SFPs but of the supporting proteins in the male accessory reproductive tissues. A proteomic study of the oriental fruit fly, Bactrocera dorsalis, identified ∼3000 male accessory gland proteins by LC- MS/MS (
      • Wei D.
      • Li H.M.
      • Tian C.B.
      • Smagghe G.
      • Jia F.X.
      • Jiang H.B.
      • Dou W.
      • Wang J.J.
      Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction.
      ) but focused only on the proteins with identified signal sequences and did not further study the entire proteome. Although a recent study used LC-MS/MS to determine both the male and female accessory gland proteome of the silk worm, Bombyx mori, no tests of molecular evolution of these proteins were performed (
      • Dong Z.
      • Wang X.
      • Zhang Y.
      • Zhang L.
      • Chen Q.
      • Zhang X.
      • Zhao P.
      • Xia Q.
      Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori.
      ). Previous studies have shown that the subcellular localization of a protein is a strong predictor of its evolutionary rate, and that extracellular proteins secreted from the cell evolve faster than intracellular proteins (
      • Sojo V.
      • Dessimoz C.
      • Pomiankowski A.
      • Lane N.
      Membrane proteins are dramatically less conserved than water-soluble proteins across the Tree of Life.
      ,
      • Feyertag F.
      • Berninsone P.M.
      • Alvarez-Ponce D.
      Secreted proteins defy the expression level-evolutionary rate anticorrelation.
      ,
      • Liao B.Y.
      • Weng M.P.
      • Zhang J.
      Impact of extracellularity on the evolutionary rate of mammalian proteins.
      ). Whether this pattern is observed in the male accessory gland requires testing.
      Here we aimed to address these limitations by using LC-MS/MS to characterize the accessory gland proteome of Drosophila pseudoobscura, whose mating system, although displaying lower levels of polyandry than the model species D. melanogaster, has nonetheless proven useful for experimental evolution studies documenting rapid sex-specific responses to across-generation changes of the mating system (
      • Crudgington H.S.
      • Fellows S.
      • Badcock N.S.
      • Snook R.R.
      Experimental manipulation of sexual selection promotes greater male mating capacity but does not alter sperm investment.
      ,
      • Crudgington H.S.
      • Beckerman A.P.
      • Brustle L.
      • Green K.
      • Snook R.R.
      Experimental removal and elevation of sexual selection: Does sexual selection generate manipulative males and resistant females?.
      ). Our comparative study also takes advantage the extensive genetic knowledgebase available in Drosophila melanogaster, and on SFP functional genomics and evolution. We further characterize the D. pseuddobscura, accessory gland proteome by constructing, using bioinformatics and gene ontology (GO), an accessory gland secretome (AcgS), exoproteome (exoP) and candidate SFPs. Finally, we compare rates of molecular evolution between these proteome subcomponents to test how subcellular protein localization impacts evolutionary rates in this system

      DISCUSSION

      We used label-free quantitative proteomics to describe the accessory gland proteome and its subcomponents, including identifying candidate SFPs, in D. pseudoobscura,. This species is less polyandrous than D. melanogaster, and patterns of evolution therefore may differ, given the role SFPs play in postcopulatory sexual selection. Indeed, the microevolutionary response of sex-biased gene expression to experimental sexual selection in this species is different than that of D. melanogaster, (
      • Crudgington H.S.
      • Fellows S.
      • Badcock N.S.
      • Snook R.R.
      Experimental manipulation of sexual selection promotes greater male mating capacity but does not alter sperm investment.
      ,
      • Hollis B.
      • Houle D.
      • Yan Z.
      • Kawecki T.J.
      • Keller L.
      Evolution under monogamy feminizes gene expression in Drosophila melanogaster.
      ,
      • Veltsos P.
      • Fang Y.
      • Cossins A.R.
      • Snook R.R.
      • Ritchie M.G.
      Mating system manipulation and the evolution of sex-biased gene expression in Drosophila.
      ). Here we identified 163 proteins that meet many criteria for being putative SFPs, 132 which were previously unknown for this species. GO term enrichment for biological processes for putative SFPs returned terms related to those expected to influence reproductive fitness including sperm competition. We found that only one third of the exoP overlapped with previously described D. melanogaster, SFPs. Four obvious but not mutually exclusive possibilities exist to explain the differences in the lists: (1) not all SFPs have yet been discovered in either species, (2) the different SFP discovery methods used in various studies will necessarily result in variable lists, (3) our exoP list contains false positives, and/or (4) although nearly all the proteins in the AcgS, from which we bioinformatically derived the exoP, were homologous with the D. melanogaster, genome, some of these proteins may have rapidly diversified to function as SFPs. From a discovery perspective, there is yet no AcgP and AcgS equivalent in D. melanogaster,. Such a resource would improve and extend the predictive abilities of identifying putative SFPs in related species and help understand the evolution of this tissue that generates proteins with profound fitness consequences on both sexes. Related to false positives and potential evolutionary recruitment, we computationally derived putative SFPs but reproductive proteins with extracellular function does not necessarily mean they will be transferred to females (
      • Sepil I.
      • Hopkins B.R.
      • Dean R.
      • Thézénas M.-L.
      • Charles P.D.
      • Konietzny R.
      • Fischer R.
      • Kessler B.M.
      • Wigby S.
      Quantitative Proteomics Identification of Seminal Fluid Proteins in Male Drosophila Melanogaster.
      ). Future work will require more downstream analyses of these putative SFPs that will also inform about recruitment. Such analyses include testing that these are transferred to females and functionally determining their effect on sex-specific fitness by taking advantage of the published genome of this species and the increasing use of sophisticated gene editing technology in previously nonmodel organisms (e.g., (
      • Gantz V.M.
      • Akbari O.S.
      Gene editing technologies and applications for insects.
      )). Putative SFPs that would be good targets for further investigation include the D. pseudoobscura, SFPs with protease function that were not identified in D. melanogaster,. We argue this because seminal fluid proteases in D. melanogaster, are well-known reproductive players, regulating proteolytic and post-mating reproductive processes in a variety of arthropod taxa including Drosophila (
      • Mueller J.L.
      • Ravi Ram K.
      • McGraw L.A.
      • Bloch Qazi M.C.
      • Siggia E.D.
      • Clark A.G.
      • Aquadro C.F.
      • Wolfner M.F.
      Cross-species comparison of Drosophila male accessory gland protein genes.
      ,
      • Dong Z.
      • Wang X.
      • Zhang Y.
      • Zhang L.
      • Chen Q.
      • Zhang X.
      • Zhao P.
      • Xia Q.
      Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori.
      ,
      • Sitnik J.L.
      • Francis C.
      • Hens K.
      • Huybrechts R.
      • Wolfner M.F.
      • Callaerts P.
      Neprilysins: an evolutionarily conserved family of metalloproteases that play important roles in reproduction in Drosophila.
      ,
      • Xu J.
      • Baulding J.
      • Palli S.R.
      Proteomics of Tribolium castaneum seminal fluid proteins: Identification of an angiotensin-converting enzyme as a key player in regulation of reproduction.
      ).
      In addition to our list of potentially novel candidate SFPs, several D. melanogaster, SFPs with known impacts on postcopulatory reproductive fitness were also found. For example, we identified nearly all SFP members of the canonical Sex Peptide network (
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ,
      • Findlay G.D.
      • MacCoss M.J.
      • Swanson W.J.
      Proteomic discovery of previously unannotated, rapidly evolving seminal fluid genes in Drosophila.
      ). In D. melanogaster, Sex Peptide bound to sperm is transferred to the female seminal receptacle during copulation and is required for both long-term female resistance to remating and for sperm release from storage (
      • Avila F.W.
      • Ravi Ram K.
      • Bloch Qazi M.C.
      • Wolfner M.F.
      Sex peptide is required for the efficient release of stored sperm in mated Drosophila females.
      ). We identified the gene duplicate pair lectins CG1652 and CG1656, aquarius (CG14061), intrepid (CG12558), antares (CG30488), seminase (CG10586), CG17575 (a cysteine-rich secretory protein), and CG9997 (a serine protease homolog) (
      • Findlay G.D.
      • Sitnik J.L.
      • Wang W.
      • Aquadro C.F.
      • Clark N.L.
      • Wolfner M.F.
      Evolutionary rate covariation identifies new members of a protein network required for Drosophila melanogaster female post-mating responses.
      ). CG9997 is processed in the female and males that do not produce this protein are unable to transfer the lectins, which are required to slow the rate at which CG9997 is processed in the female. All proteins identified in the D. melanogaster, SP network, except SP itself, were detected in our putative list of SFPs. Absence of detectable D. pseudoobscura, SP protein is consistent with the lack of a recognized SP ortholog in this species and raises the interesting possibility that either the D. pseudoobscura, SP ortholog has significantly diverged, or has been replaced by another gene. If indeed a bona fide D. pseudoobscura, SP gene exists, then further MS searches using algorithms to detect amino acid replacements (
      • Starkweather R.
      • Barnes C.S.
      • Wyckoff G.J.
      • Keightley J.A.
      Virtual polymorphism: finding divergent peptide matches in mass spectrometry data.
      ) may be useful in the search for this elusive SFP.
      One aim of this work was to extend the focus from solely SFPs to the functional complexity of other proteins in the accessory gland tissue. We generated a robust accessory gland proteome containing 3160 proteins, representing the first accessory gland proteome to be described in Drosophila. 96% of these proteins showed homology to D. melanogaster,. The AcgP proteins in D. pseudoobscura, were enriched for cellular components expected from a tissue whose primary function is secretory, including several cellular component GO terms related to membranes, extracellular regions, and peptidase complex. The top biological process GO terms clearly indicated a large investment in processes directly and indirectly related to protein synthesis, protein assembly, transport, and secretion. We then in silico, concatenated this list to include proteins with secretory signal sequences to identify 506 accessory gland secretome proteins, which were enriched for GO terms related to the biological processes of reproduction, behavior, and proteolysis, with these proteins heavily biased toward subcellular localizations in the plasma member or extracellular components, as predicted from proteins with secretory signals. As previously noted, describing the D. melanogaster, AcgP and AcgS, along with other related species, will provide the basis for evolutionary analyses required to understand how selection, particularly arising from postcopulatory selection pressures on males to influence female reproductive fitness, has acted on this tissue.
      Documenting the AcgP then allowed testing another aim of our work—determining signals of molecular evolution in different subcomponents of accessory gland proteins. Rapid evolution at the molecular level is common for reproductive proteins including SFPs (e.g., (
      • Clark N.L.
      • Aagaard J.E.
      • Swanson W.J.
      Evolution of reproductive proteins from animals and plants.
      ,
      • Mueller J.L.
      • Ripoll D.R.
      • Aquadro C.F.
      • Wolfner M.F.
      Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid.
      ,
      • Vicens A.
      • Borziak K.
      • Karr T.L.
      • Roldan E.R.S.
      • Dorus S.
      Comparative sperm proteomics in mouse species with divergent mating systems.
      ,
      • Wilburn D.B.
      • Swanson W.J.
      From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins.
      )). However, extracellular proteins in general exhibit more rapid molecular evolution than proteins restricted to functions within the cell (
      • Sojo V.
      • Dessimoz C.
      • Pomiankowski A.
      • Lane N.
      Membrane proteins are dramatically less conserved than water-soluble proteins across the Tree of Life.
      ,
      • Feyertag F.
      • Berninsone P.M.
      • Alvarez-Ponce D.
      Secreted proteins defy the expression level-evolutionary rate anticorrelation.
      ,
      • Liao B.Y.
      • Weng M.P.
      • Zhang J.
      Impact of extracellularity on the evolutionary rate of mammalian proteins.
      ). We therefore compared molecular evolution rates of D. pseudoobscura, with its close relatives across proteins from different subcomponents of the male reproductive accessory gland tissue that identified genes encoding SFPs with significantly faster rates of molecular evolution compared with both the AcgS (i.e., other secreted protein encoding genes that were not candidate SFPs) and to non secreted accessory gland proteome genes. Moreover, we also found that secretome genes showed higher rates of molecular evolution than nonsecreted proteins (i.e., the remainder of the AcgP). These results support not only previous work that SFP genes evolve faster than non-SFP genes, but also the more general finding that genes coding proteins which interact extracellularly evolve more rapidly than those that remain within the cytoplasm, irrespective of reproductive function (
      • Feyertag F.
      • Berninsone P.M.
      • Alvarez-Ponce D.
      Secreted proteins defy the expression level-evolutionary rate anticorrelation.
      ,
      • Liao B.Y.
      • Weng M.P.
      • Zhang J.
      Impact of extracellularity on the evolutionary rate of mammalian proteins.
      ).

      CONCLUSION

      Increasing emphasis on understanding how SFPs impact reproductive fitness across many different organisms requires not only identifying those proteins but also understanding the protein complexity of the SFP-producing tissues. Using organisms with different mating systems and testing the extent to which signatures of rapid molecular evolution are shared across taxa, and across the different environments in which proteins function (i.e., intra- versus, extra- cellular), will generate improved understanding of the causes and consequences of SFP evolution. Here we show that the use of label-free quantitative proteomics methods can address such questions and, specifically, will serve as the basis for more detailed work in this species on the role of postcopulatory sexual selection and reproductive protein evolution.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012545.

      Acknowledgments

      We thank the reviewers for their careful and thoughtful comments and criticisms that helped to make this work better. We also thank the many people who have contributed to the maintenance of the Snook experimental evolution lines.

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