We used discovery, bottom up proteomics to provide the first in-depth accessory gland proteome in D. pseudoobscura. Computational bioinformatics identified Ͼ500 proteins in the secretory pathway of which 163 were annotated as extracellular., and therefore candidate, seminal fluid proteins. We further compared molecular rates of evolution between intra-and extracellular proteins, showing a hierarchy of rapid evolution with putative seminal fluid proteins evolving more rapidly than other secreted proteins, and those proteins evolving more rapidly than intra-cellular proteins. Graphical Abstract Highlights• First deep proteomic coverage of an accessory gland proteome in Drosophila.• Discovery proteomics identified Ͼ3000 proteins of the D. pseudoobscura accessory gland proteome.• Identified 132 putative novel seminal fluid proteins in this species.• Demonstrated the exoproteome as the most rapidly evolving subcellular component of the proteome.

Sperm morphology is incredibly diverse, even among closely related species, yet the coevolution between males and females of fertilization recognition systems is necessary for successful karyogamy (male and female pronuclear fusion). In most species, the entire sperm enters the egg during fertilization so sperm morphological diversity may impact the intracellular sperm–egg interactions necessary for karyogamy. We quantified morphological variation of sperm inside eggs prior to and following karyogamy in several species of Drosophila to understand whether evolution of sperm morphology could influence intracellular sperm–egg interactions (ISEIs). We measured seven parameters that describe ISEIs among species to determine whether these parameters varied both within a species across development and across species at the same developmental stage. We used heterospecific crosses to test the relative role of male origin, female origin, and interaction between the male and female in determining ISEIs. We found that sperm shape changed within a species as development proceeded and, at particular development stages, species varied in some ISEIs. Parental origin had an effect on some ISEIs, with a general trend for a stronger female effect. Overall, our findings identify conserved and variable ISEIs among species and demonstrate the potential to contribute understanding to gamete evolution and development.

13 14 15 Running title: Sexual selection and protein interaction networks 16 The abbreviations used are: 17 BLAST, Basic Local Alignment Search Tool 18 Dpse, Drosophila pseudoobscura 19 PCSS, postcopulatory sexual selection 20 SFPs, seminal fluid proteins 21 Dmel, D. melanogaster 22 SDS, sodium dodecylsulfate 23 SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis 24 MS, mass spectrometry 25 LC-MS/MS, liquid chromatography-MS/MS 26 AcgP, accessory gland proteome 27 FDRs, False Discovery Rates 28 AcgS, accessory gland secretome 29 exoP, exoproteome 30 LFQ, label-free quantitation 31 P, polyandry 32 M, monandry 33 GO, gene ontology 34 CC, cellular component 35 MF, molecular function 36 BP, biological process 37 STRING, Search Tool for the Retrieval of Interacting Genes/Proteins 38 DIOPT, DRSC Integrative Ortholog Prediction Tools 39 ER, endoplasmic reticulum Polyandry drives postcopulatory sexual selection (PCSS), resulting in rapid evolution 41 of male ejaculate traits. Critical to male and female fitness, the ejaculate is known to 42 contain rapidly evolving seminal fluid proteins (SFPs) produced by specialized male 43 secretory accessory glands. The evidence that rapid evolution of some SFPs is driven 44 by PCSS, however, is indirect, based on either plastic responses to changes in the 45 sexual selection environment or correlative macroevolutionary patterns. Moreover, 46 such studies focus on SFPs that represent but a small component of the accessory 47 gland proteome. Neither how SFPs function with other reproductive proteins, nor how 48 PCSS influences the underlying secretory tissue adaptations and content of the 49 accessory gland, has been addressed at the level of the proteome. Here we directly test 50 the hypothesis that PCSS results in rapid evolution of the entire male accessory gland 51 proteome and protein networks by taking a system-level approach, combining 52 divergent experimental evolution of PCSS in Drosophila pseudoobscura (Dpse), high 53 resolution mass spectrometry (MS) and proteomic discovery, bioinformatics and 54 population genetic analyses. We demonstrate that PCSS influences the abundance of 55 over 200 accessory gland proteins, including SFPs. A small but significant number of 56 these proteins display molecular signatures of positive selection. Divergent PCSS also 57 results in fundamental and remarkably compartmentalized evolution of accessory 58 gland protein networks in which males subjected to strong PCSS invest in protein 59 networks that serve to increase protein production whereas males subjected to relaxed 60 PCSS alters protein networks involved in protein surveillance and quality. These results 61 directly demonstrate that PCSS is a key evolutionary driver that shapes not only 62 individual reproductive proteins, but rewires entire reproductive protein networks. 63Polyandry, in which females mate with different males across a reproductive bout, generates 64 PCSS in which ejaculates compete for fertilization of a limited supply of ova and females 65 may choose w...