Figures
Abstract
In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. Here, we investigate the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. We document a heretofore underappreciated rapid evolutionary rate for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation.
Citation: Takashima YA, Majane AC, Begun DJ (2023) Evolution of secondary cell number and position in the Drosophila accessory gland. PLoS ONE 18(10): e0278811. https://doi.org/10.1371/journal.pone.0278811
Editor: Arnar Palsson, University of Iceland, ICELAND
Received: November 23, 2022; Accepted: April 25, 2023; Published: October 25, 2023
Copyright: © 2023 Takashima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: within manuscript and/or Supporting information files.
Funding: This work was supported by the National Institutes of Health grant NIGMS R35 GM134930 to David J. Begun and a National Science Foundation Graduate Research Fellowship to Alex C. Majane. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. https://www.nigms.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interest exist.
Introduction
Along with sperm, Drosophila males transfer seminal fluid proteins (Sfps) to females. Transfer of molecules from male to female provides a fascinating example of male-female co-operation and conflict [1]. Female reproduction requires seminal fluid, yet this requirement exposes females to male adaptations that may subvert their reproductive interests [2–7]. Seminal fluid may also mediate competitive interactions between the sperm of multiple males in the female reproductive tract [8–10]. Significant polymorphism of genes affecting seminal fluid function is evident in the rapid laboratory evolution of male toxicity toward females and the rapid evolution of male sperm displacement phenotypes in experimental populations of Drosophila melanogaster [2, 3, 11]. The outcome of sperm competition is also influenced by female genetic variation and the interaction of genetic variation in the two sexes [12–14]. Drosophila Sfps are produced in the accessory gland (AG), ejaculatory duct, and ejaculatory bulb. The molecular products of the seminal fluid producing organs exhibit rapid evolution, as expected given their role in sexual conflict [15, 16] Sfp protein sequences often evolve very quickly [17], sometimes under the influence of directional selection [18–21]. Expression levels of AG-biased genes may vary widely even between closely related populations and species [22–25], and the AG transcriptome exhibits rapid turnover due to gene presence/absence variation [26–28] or the expression of different homologues in the AG of different species [23, 29–31].
In most Drosophila species the AG is composed of two major cell types [32]. In D. melanogaster, main cells constitute about 96% of the cells, which are the primary producers of the secreted proteins required for fertility, and induce both short- and long-term changes to female post-copulatory physiology and behavior [8, 33–38]. The secondary cells (estimated to be 4% of the cells) are clustered in the distal portion of the organ (reviewed in [39]). Their secreted products appear to play an important role in maintaining female post-copulatory responses [39–41]. Secondary cells (SC) play a role in the secretion of extracellular vesicles (i.e., exosomes), which bind to sperm, interact with the female reproductive tract epithelium, and play a role in the long-term mating response of females [42].
Recent RNA-seq analysis of secondary cells has revealed that their transcriptome is distinct from that of main cells and also evolves in a distinct manner [43, 44]. Given that male reproductive proteins and transcriptomes evolve quickly and that mating systems vary dramatically across Drosophila [45], whether most basic conclusions about the AG as described in the D. melanogaster model apply generally to Drosophila is an open question. A report by [32], which focused on AG main cell binucleation in multiple Drosophila species, provided incidental data (their Fig 1) strongly suggesting that Drosophila secondary cell number evolves, perhaps quickly. Given their inherent biological interest and relative ease of measurement, we set out to quantitatively investigate secondary cell number variation in a collection of Drosophila species.
Phylogeny of eight Drosophila species and estimates of secondary cell number for each. Number of glands analyzed for each species are indicated next to the species name; mean and standard deviation of secondary cell number per species are shown to the right.
Materials and methods
Fly stocks
D. melanogaster RAL 517, RAL 399, RAL 360 [46] (Mackay et al. 2012), D. yakuba Tai18E2 [47], D. simulans w501 [47], D. hydei, D. virilis (gift of S. Lott, UC Davis), D. mojavensis (Drosophila Species Stock Center #s 0218.15 and 0218.17), D. ananassae 11–4 (gift of B. Cooper, University of Montana), D. pseudoobscura SLC 10 (gift of S. Schaeffer, Pennsylvania State University). All stocks were maintained in a 25C incubator on a 12:12 hr light:dark cycle.
The phylogenetic relationships of these species is depicted in Fig 1. Four of these species (D. melanogaster, D. simulans, D. yakuba, and D. ananassae) are from the melanogaster group; two (D. hydei and D. mojavensis) are from the repleta group; one (D. pseudoobscura) is from the obscura group, and one (D. virilis) is from the virilis group. The species were sampled to provide information about shorter and longer time scale evolutionary change. On a long timescale, the most recent common ancestor of all eight species existed about 40–50 million years ago [48, 49]. On a shorter timescale, divergence between D. melanogaster and D. simulans is about 2–3 million years [50]. Three inbred lines of D. melanogaster were used to investigate the possibility of genetic variation affecting SC number in this species.
Dissecting, fixing, and staining glands
Virgin males were collected and then dissected two days after their estimated age of sexual maturity, which varies among species [45]. D. melanogaster, D. simulans, and D. yakuba were dissected two days post-eclosion. D. hydei were dissected 10 days post eclosion. D. mojavensis, D. pseudoobscura, D. ananassae, and D. virilis were dissected eight days post eclosion.
Dissected accessory glands were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and then washed with 1X PBS. The glands were treated with a 1:100 dilution of 10mg/ml RNAse in PBT for 30 minutes at room temperature and stained using propidium iodide at a concentration of 1:1000 for 30 minutes at room temperature [32]. Glands were then mounted on glass slides with spacers—Scotch tape layered and cut into squares that would mark where the corners of the 0.5mm coverslip would lie. Spacers were necessary to prevent compression of the gland. One gland of the two was imaged using a Leica confocal microscope at 20X (dry) and 40X (oil) magnification with 75–100 z-stacks per gland. The Z step size was approximately 1.8–2.0μm. Images were stitched together using the Pairwise Stitching of Images [51] or were merged at the Leica confocal upon image acquisition using the tilescan function with 10% overlap.
Secondary cell quantification
Due to the nature of the propidium iodide staining and the structure of the cell itself, SCs were easily distinguished from main cells by their characteristic large, dark vacuoles (Fig 3). SCs were counted manually across z-stack images using the Cell Counter Plugin in FIJI [52]. After initializing a .tif file in this plugin, the “show all” function allowed us to track and count each SC throughout the z plane. Two arbitrary counters, which were conveniently different colors, were used to tally the different secondary cells to properly ensure that double counting did not occur. One counter tracked one side of the epithelial layer, and after going through the lumen of the gland; the second counter was used to track cells on the other epithelial layer. The plugin then tallied the number of times the counter was used. Totals from the two counters were summed to estimate the number of secondary cells in a gland. Distinguishing between each epithelial layer was possible due to the spacers on the slide to prevent the compression of the tissue.
Accessory gland length and area
AG gland measurements were done using FIJI [52]. Gland length measurements were done by using the freehand tool and drawing a line directly down the middle of the gland spanning from the top of the ejaculatory duct and the tip of the gland. The measurements were taken in pixels following [53, 54]. AG area was estimated by using the lasso tool on FIJI to draw perimeters around the glands, after which, measurements were made and the area in microns generated following [53, 54].
Statistics and phylogeny
Statistics were done using R version 4.2.2; R Core Team 2020 /Rstudio version 2022.02.3 Build 492. "Prairie Trillium" Release (1db809b8, 2022-05-20) for Windows [55]. For ANOVA and Tukey’s Honest Significant Difference Test, the package ‘multicomp’ was used [56]. Violin plots were created using ggplot2 version 3.4 [57]. The phylogeny was constructed using package ‘ape’ version 5.6.2 in R/Rstudio [58].
Results and discussion
Fig 1 presents our estimates of SC number in eight species. Previous estimates of SC number in D. melanogaster were roughly 43 SC per gland [59]. Our results provide a similar, though slightly greater estimate of SC number per gland, and also provide a quantitative measure of variation among genotypes. The three D. melanogaster inbred lines we assayed, which originated in Raleigh, North Carolina [46], were not significantly heterogeneous for SC number (S1 Fig). Thus, while there may be genetic variation for SC number currently segregating in D. melanogaster, we observed no evidence for this in our small sample. It is worth noting that a range of SC numbers were observed within each inbred RAL genotype (e.g., for RAL 517 the range across individuals was 29 to 56). While some of this variation could represent measurement error, developmental variation in the process leading to SC differentiation could also contribute. D. simulans, the sister species to D. melanogaster, had significantly fewer SC (Figs 1 and 2). This provides strong evidence that SC numbers can evolve substantially, even on short timescales. Of course, the observation of species differences provides primae facie evidence for genetic variation affecting SC number. While our estimate of D. yakuba SC number is greater than the estimates from D. melanogaster and D. simulans, it is not significantly different from D. melanogaster. Thus, the most parsimonious explanation for the observed variation in SC number in the melanogaster subgroup is a recent decrease in SC number in D. simulans. All samples from the three melanogaster subgroup species exhibited the well documented spatial patterning previously observed in D. melanogaster, with all SC distributed in the distal tip of the gland (Fig 3).
Violin plot showing distributions of number of secondary cells in each species. Red bar = mean. Asterisks indicate p-values from ANOVA. * < 0.05, ** < 0.01, *** < 0.001.
(A-C) Accessory glands imaged at 40X, tilescan with 10% overlap, max projection confocal images of D. melanogaster, D. simulans, D. yakuba. (A’- C’) is a 40X image of the distal tip of those species. (D-H) are 40X tilescan images, 10% overlap, max projection confocal images of D) D. ananassae, E) D.pseudoobscura, F) D.hydei, G) D. mojavensis, H) D.virilis.
D. ananassae is the nearest outgroup for the melanogaster subgroup in our sample. D. ananassae mean SC number was significantly lower than the mean for the melanogaster subgroup (Fig 2), but without formal phylogenetic modeling of the trait we cannot infer the ancestral SC number of the melanogaster group and thus the direction of evolution in the branch leading to D. ananassae and the branch leading to the melanogaster subgroup. Despite the similar number of SC cells in D. ananassae and the melanogaster subgroup, there is evidence of qualitative differences in their spatial distribution in the gland. D. ananassae differs from the three melanogaster subgroup species in that the SC are not restricted to the distal tip of the gland. Instead they appear to be roughly homogeneously distributed throughout the distal half of the gland (Fig 3).
D. pseudoobscura, our only representative of the obscura group, is sister to the melanogaster group. It exhibits fewer SC than any melanogaster subgroup species, though the difference is not significant. Much like the melanogaster subgroup, the secondary cells are enriched in the distal tip of the gland. The D. pseudoobscura accessory gland was clearly shorter and about the same width (mean length and width, 546 microns and 191 microns, respectively) compared to the melanogaster subgroup (mean length and width, 1426 microns and 189 microns, respectively).
D. hydei and D. mojavensis, sister species in our sample, belong to the repleta group, a diverse clade of primarily cactophilic flies that originated about 20–30 MYA, most likely in South America [60, 61]. The reproductive biology of these flies differs from several other Drosophila species in multiple ways. For example, D. mojavensis and D. hydei share high re-mating rates compared to most other Drosophila species, with D. hydei apparently having a particularly high rate [45]. D. hydei also has very long sperm and testis compared to most flies [62]. We observed that these two species have extraordinarily high numbers of SC per gland, roughly six times as many as observed in the melanogaster group (Fig 1). Given that the D. hydei gland is only about four times the size of D. melanogaster (Table 1), it appears that the species differences in SC number cannot be explained solely by gland size variation, which is driven primarily by gland length differences. Supporting this conclusion, in striking contrast to the melanogaster group, repleta group flies exhibit SC distributed homogeneously throughout the gland (Fig 3). Interestingly, the repleta group appears to have lost the sex-peptide gene [63, 64], which codes for a protein critical for proper sperm use in D. melanogaster [65]. Whether the absence of sex-peptide, the increase in number of SC, and the homogeneous physical distribution of SC in the AG in the repleta group are functionally related is an interesting question.
D. virilis, which is sister to the repleta group (Fig 1), also exhibits substantially greater numbers of SC than the Sophophora species, though many fewer than repleta group flies (Fig 4). Thus, data from the sampled taxa are suggestive of greater numbers of SC per gland for the Drosophila group than for the Sophophora group (Fig 1), though additional sampling would be required to be confident of this inference. Similar to the repleta group species, D. virilis SC appear to be distributed homogeneously throughout the gland (Fig 3). In general, our data support the idea that restriction of the SC to the distal tip of the accessory gland in the D. melanogaster model system is a highly derived trait and not characteristic of most Drosophila lineages. The developmental basis of SC number and position, as well as their functional consequences and evolutionary processes driving divergence of these traits, are unknown.
Violin plot showing the distribution of secondary cells among the melanogaster, repleta, virilis, and pseudoobscura groups. Red bars = mean number of secondary cells. Asterisks indicate significance p-values from ANOVA: * = 0.05, ** = 0.01, *** = 0.001.
Conclusion
While phylogenetic comparative analysis of SC number and position in the accessory gland would be necessary to make strong, specific inferences on rates of phenotypic evolution of SC number on particular branches of the Drosophila phylogeny, the results reported here leave no doubt that SC number evolves quickly in the genus. Additionally, the physical distribution of SC in the gland also evolves, as most species do not appear to share the stereotypical clustering of SC in the tip of the gland seen in the D. melanogaster model. Further investigation of the patterns presented here promise to reveal new functional and evolutionary attributes of accessory gland diversification and its broader connections to mating system biology in the genus.
Supporting information
S1 Fig. Summary of SC number intraspecies.
A summary of the secondary cell numbers within the 3 RAL lines examined (517, 360, 390). Red bars = mean.
https://doi.org/10.1371/journal.pone.0278811.s001
(TIF)
Acknowledgments
We thank Ben Hopkins, Juan Hurtado, the editor, and an anonymous reviewer for comments, Didem Sarikaya for microscopy and image analysis advice and feedback, Noelia Carrasquila and Maria Florencia Ercoli for assistance with the confocal microscope, and the UC Davis Department of Plant Pathology for access to it. Kiichiro Taniguchi generously shared his propidium iodide staining protocol.
References
- 1. Wolfner MF. Precious essences: female secretions promote sperm storage in Drosophila. PLoS Biol. 2011;9(11):e1001191. pmid:22087072
- 2. Rice WR. Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature. 1996;381(6579):232–4. pmid:8622764
- 3. Holland B, Rice WR. Experimental removal of sexual selection reverses intersexual antagonistic coevolution and removes a reproductive load. Proc Natl Acad Sci U S A. 1999;96(9):5083–8. pmid:10220422
- 4. Lung O, Wolfner MF. Drosophila seminal fluid proteins enter the circulatory system of the mated female fly by crossing the posterior vaginal wall. Insect Biochem Mol Biol. 1999;29(12):1043–52. pmid:10612039
- 5. Civetta A, Clark AG. Correlated effects of sperm competition and postmating female mortality. Proc Natl Acad Sci U S A. 2000;97(24):13162–5. pmid:11078508
- 6. Fiumera AC, Dumont BL, Clark AG. Sperm competitive ability in Drosophila melanogaster associated with variation in male reproductive proteins. Genetics. 2005;169(1):243–57. pmid:15466425
- 7. Sirot LK, Wong A, Chapman T, Wolfner MF. Sexual conflict and seminal fluid proteins: a dynamic landscape of sexual interactions. Cold Spring Harb Perspect Biol. 2014;7(2):a017533. pmid:25502515
- 8. Harshman LG, Prout T. Sperm Displacement without Sperm Transfer in Drosophila Melanogaster. Evolution. 1994;48(3):758–66. pmid:28568282
- 9. Clark AG, Aguade M, Prout T, Harshman LG, Langley CH. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster. Genetics 1995; 139(1): 189–201.
- 10. Fiumera AC, Dumont BL, Clark AG. Associations between sperm competition and natural variation in male reproductive genes on the third chromosome of Drosophila melanogaster. Genetics. 2007;176(2):1245–60. pmid:17435238
- 11. Hollis B, Koppik M, Wensing KU, Ruhmann H, Genzoni E, Erkosar B, et al. Sexual conflict drives male manipulation of female postmating responses in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2019;116(17):8437–44. pmid:30962372
- 12. Clark AG, Begun DJ. Female genotypes affect sperm displacement in Drosophila. Genetics. 1998;149(3):1487–93. pmid:9649536
- 13. Clark AG, Begun DJ, Prout T. Female x male interactions in Drosophila sperm competition. Science. 1999;283(5399):217–20. pmid:9880253
- 14. Giardina TJ, Beavis A, Clark AG, Fiumera AC. Female influence on pre- and post-copulatory sexual selection and its genetic basis in Drosophila melanogaster. Mol Ecol. 2011;20(19):4098–108. pmid:21902747
- 15. Swanson WJ, Vacquier VD. The rapid evolution of reproductive proteins. Nat Rev Genet. 2002;3(2):137–44. pmid:11836507
- 16. Findlay GD, MacCoss MJ, Swanson WJ. Proteomic discovery of previously unannotated, rapidly evolving seminal fluid genes in Drosophila. Genome Res. 2009;19(5):886–96. pmid:19411605
- 17. Coulthart MB, Singh RS. High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans. Mol Biol Evol. 1988;5(2):182–91. pmid:3130539
- 18. Begun DJ, Whitley P, Todd BL, Waldrip-Dail HM, Clark AG. Molecular population genetics of male accessory gland proteins in Drosophila. Genetics. 2000;156(4):1879–88. pmid:11102381
- 19. Swanson WJ, Clark AG, Waldrip-Dail HM, Wolfner MF, Aquadro CF. Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc Natl Acad Sci U S A. 2001;98(13):7375–9. pmid:11404480
- 20. Wagstaff BJ, Begun DJ. Molecular population genetics of accessory gland protein genes and testis-expressed genes in Drosophila mojavensis and D. arizonae. Genetics. 2005;171(3):1083–101. pmid:16085702
- 21. Haerty W, Jagadeeshan S, Kulathinal RJ, Wong A, Ravi Ram K, Sirot LK, et al. Evolution in the fast lane: rapidly evolving sex-related genes in Drosophila. Genetics. 2007;177(3):1321–35. pmid:18039869
- 22. Begun DJ, Lindfors HA. Rapid evolution of genomic Acp complement in the melanogaster subgroup of Drosophila. Mol Biol Evol. 2005;22(10):2010–21. pmid:15987879
- 23. Ahmed-Braimah YH, Unckless RL, Clark AG. Evolutionary Dynamics of Male Reproductive Genes in the Drosophila virilis Subgroup. G3 (Bethesda). 2017;7(9):3145–55. pmid:28739599
- 24. Garlovsky MD, Evans C, Rosenow MA, Karr TL, Snook RR. Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation. Mol Ecol. 2020;29(22):4428–41. pmid:32939895
- 25. Cridland JM, Contino CE, Begun DJ. Selection and Geography Shape Male Reproductive Tract Transcriptomes in Drosophila Melanogaster. Genetics. 2023. pmid:36869688
- 26. Wagstaff BJ, Begun DJ. Comparative genomics of accessory gland protein genes in Drosophila melanogaster and D. pseudoobscura. Mol Biol Evol. 2005;22(4):818–32. pmid:15601888
- 27. Mueller JL, Ravi Ram K, McGraw LA, Bloch Qazi MC, Siggia ED, Clark AG, et al. Cross-species comparison of Drosophila male accessory gland protein genes. Genetics. 2005;171(1):131–43. pmid:15944345
- 28. Begun DJ, Lindfors HA, Thompson ME, Holloway AK. Recently evolved genes identified from Drosophila yakuba and D. erecta accessory gland expressed sequence tags. Genetics. 2006;172(3):1675–81. pmid:16361246
- 29. Cridland JM, Majane AC, Sheehy HK, Begun DJ. Polymorphism and Divergence of Novel Gene Expression Patterns in Drosophila melanogaster. Genetics. 2020;216(1):79–93. pmid:32737121
- 30. Cridland JM, Majane AC, Zhao L, Begun DJ. Population biology of accessory gland-expressed de novo genes in Drosophila melanogaster. Genetics. 2022;220(1). pmid:34791207
- 31. Hurtado J, Almeida FC, Belliard SA, Revale S, Hasson E. Research gaps and new insights in the evolution of Drosophila seminal fluid proteins. Insect Mol Biol. 2022;31(2):139–58. pmid:34747062
- 32. Taniguchi K, Kokuryo A, Imano T, Minami R, Nakagoshi H, Adashi-Yamada T. Bi-nucleation of the Drosophila adult male accessory gland cells increases plasticity of organ size for effective reproduction. Biological Systems Open Access 2012; 1(1).
- 33. Manning A. The control of sexual receptivity in female Drosophila. Anim Behav. 1967;15(2):239–50. pmid:6030948
- 34. Kalb JM, DiBenedetto AJ, Wolfner MF. Probing the function of Drosophila melanogaster accessory glands by directed cell ablation. Proc Natl Acad Sci U S A. 1993;90(17):8093–7. pmid:8367469
- 35. Xue L, Noll M. Drosophila female sexual behavior induced by sterile males showing copulation complementation. Proc Natl Acad Sci U S A. 2000;97(7):3272–5. pmid:10725377
- 36. Neubaum DM, Wolfner MF. Mated Drosophila melanogaster females require a protein, Acp36DE, to store sperm efficiently. Genetics 1999; 153(2): 845–857.
- 37. Ram KR, Wolfner MF. Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins. PLoS Genet. 2007;3(12):e238. pmid:18085830
- 38. Ram KR, Wolfner MF. A network of interactions among seminal proteins underlies the long-term postmating response in Drosophila. Proc Natl Acad Sci U S A. 2009;106(36):15384–9. pmid:19706411
- 39. Wilson C, Leiblich A, Goberdhan DC, Hamdy F. The Drosophila Accessory Gland as a Model for Prostate Cancer and Other Pathologies. Curr Top Dev Biol. 2017;121:339–75. pmid:28057306
- 40. Sitnik JL, Gligorov D, Maeda RK, Karch F, Wolfner MF. The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster. Genetics. 2016;202(3):1029–41. pmid:26746709
- 41. Hopkins BR, Sepil I, Bonham S, Miller T, Charles PD, Fischer R, et al. BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions. Proc Natl Acad Sci U S A. 2019;116(49):24719–28. pmid:31740617
- 42. Corrigan L, Redhai S, Leiblich A, Fan SJ, Perera SM, Patel R, et al. BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol. 2014;206(5):671–88. pmid:25154396
- 43. Immarigeon C, Frei Y, Sofie Y, Delbare N, Gligorov D, Machado Almeida P, et al. Identification of a micropeptide and multiple secondary cell genes that modulate Drosophila male reproductive success. Proceedings of the National Academy of Sciences of the United States of America 2021; 118 (15). pmid:33876742
- 44. Majane AC, Cridland JM, Begun DJ. Single-nucleus transcriptomes reveal evolutionary and functional properties of cell types in the Drosophila accessory gland. Genetics. 2022;220(2). pmid:34849871
- 45.
Markow TA. Evolution of Drosophila Mating Systems. In Evolutionary Biology. Ed. Hecht Max K. Plenum Press, NY;1996.
- 46. Mackay TF, Richards S, Stone EA, Barbadilla A, Ayroles JF, Zhu D, et al. The Drosophila melanogaster Genetic Reference Panel. Nature. 2012;482(7384):173–8. pmid:22318601
- 47. Begun DJ, Holloway AK, Stevens K, Hillier LW, Poh YP, Hahn MW, et al. Population genomics: whole-genome analysis of polymorphism and divergence in Drosophila simulans. PLoS Biol. 2007;5(11):e310. pmid:17988176
- 48.
Powell JR. Progress and Progress in Evolutionary Biology: The Drosophila Model. Oxford University Press;1997.
- 49. Tamura K, Subramanian S, Kumar S. Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol Biol Evol. 2004;21(1):36–44. pmid:12949132
- 50. Obbard DJ, Maclennan J, Kim KW, Rambaut A, O’Grady PM, Jiggins FM. Estimating divergence dates and substitution rates in the Drosophila phylogeny. Mol Biol Evol. 2012;29(11):3459–73. pmid:22683811
- 51. Preibisch S, Saalfeld S, Tomancak P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics. 2009;25(11):1463–5. pmid:19346324
- 52. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. pmid:22743772
- 53. Bangham J., Chapman T., & Partridge L. Effects of body size, accessory gland and testis size on pre- and postcopulatory success in Drosophila melanogaster. Animal Behaviour. 2002; 64(6), 915–921.
- 54. Baker RH, Denniff M, Futerman P, Fowler K, Pomiankowski A, Chapman T. Accessory gland size influences time to sexual maturity and mating frequency in the stalk-eyed fly, Cyrtodiopsis dalmanni. Behavioral Ecology. 2003;14(5):607–11.
- 55.
R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.
- 56. Hothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. Biom J. 2008;50(3):346–63. pmid:18481363
- 57.
Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.
- 58. Paradis E, Schliep K. ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2018;35(3):526–8.
- 59. Bertram MJ, Akerkar GA, Ard RL, Gonzalez C, Wolfner MF. Cell type-specific gene expression in the Drosophila melanogaster male accessory gland. Mech Dev. 1992;38(1):33–40. pmid:1525037
- 60.
Patterson JT, Stone WS. Evolution in the Genus Drosophila. Macmillan;1952.
- 61. Oliveira DC, Almeida FC, O’Grady PM, Armella MA, DeSalle R, Etges WJ. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group. Mol Phylogenet Evol. 2012;64(3):533–44. pmid:22634936
- 62. Pitnick S, Markow TA. Large-male advantages associated with costs of sperm production in Drosophila hydei, a species with giant sperm. Proc Natl Acad Sci U S A. 1994;91(20):9277–81. pmid:7937755
- 63. McGeary MK, Findlay GD. Molecular evolution of the sex peptide network in Drosophila. J Evol Biol. 2020;33(5):629–41. pmid:31991034
- 64. Hopkins BR, Perry JC. The evolution of sex peptide: sexual conflict, cooperation, and coevolution. Biological Reviews. 2022;97(4):1426–48. pmid:35249265
- 65. Chen PS, Stumm-Zollinger E, Aigaki T, Balmer J, Bienz M, Bohlen P. A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell. 1988;54(3):291–8. pmid:3135120