Independent origin of sperm heteromorphism in the Drosophilidae
Data files
May 06, 2026 version files 27.21 KB
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ComparativeTraitData.txt
3.28 KB
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DrosophilidaePhylogeny.nex
8.57 KB
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MaleProductionHeadLengthData_5May26.txt
2.78 KB
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MaleProductionMorphLengthData_5May26.txt
272 B
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README.md
7.35 KB
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SpermEjection_5May26.txt
797 B
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SperminFRT_5May26.txt
3.59 KB
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SpermMorphTotalLengthData_5May26.txt
575 B
Abstract
Sperm heteromorphism, the production multiple discrete sperm types (or "morphs"), occurs in diverse animal and plant taxa. This phenomenon represents a division of labor by male gametes, with one morph participating in fertilization and the other morph(s) specialized to perform some other reproductive function. One of the most intensively investigated systems is the Drosophila obscura species group, for which sperm heteromorphism evolved once in the common ancestor to the group, with all examined descendent species exhibiting sperm length heteromorphism. The absence of additional sperm heteromorphism origins among drosophilids has precluded comparative evolutionary analyses. Here, we report the discovery of a second origin within the Drosophilidae, with Chymomyza procnemis exhibiting the most extreme sperm length heteromorphism known (> 15-fold difference). Males produced and transferred to females approximately twice as many short sperm as long sperm. Shortly after insemination, females ejected from their reproductive tracts most of the short morph sperm, yet relatively few of the long morph sperm, resulting in a preponderance of long morph sperm occupying the females' sperm-storage organs. By the start of egg laying, few-to-no short morph sperm were detectable in females. Although C. procnemis have very long eusperm and the highest relative investment (Gonadosomatic Index; GSI) in testes for any drosophilid species, a comparative analysis of 149 Drosophila species revealed no significant coevolutionary relationship between sperm heteromorphism and either sperm length or GSI. Continued investigations with Chymomyza will advance our understanding of how sperm heteromorphism arises, persists, and diversifies across lineages.
Dataset DOI: 10.5061/dryad.crjdfn3m1
Description of the data and file structure
To quantify sperm morphometry and the production of heteromorphic sperm, the seminal vesicles of sexually mature males were dissected in phosphate-buffer saline (PBS) on subbed microscope slides. One seminal vesicle was then ruptured to release the contents, and fine pins were used to isolate individual sperm in the PBS. Preparations were then dried in a 60 °C oven, fixed in 3:1 methanol:acetic acid, treated with the blue-fluorescent DNA stain 4',6-diamidino-2-phenylindole (DAPI), then mounted with glycerol:PBS (9:1) with ProLong Gold Antifade (Thermo Fisher) under a glass coverslip and sealed with nail polish. Using an Olympus DP-71 camera mounted on an Olympus BX60 microscope (Olympus America Inc., Melville, NY), digital images of sperm heads were obtained using epifluorescence at 400X magnification and of individual sperm using darkfield optics at 200X magnification (note: several frames were required to capture entirety of individual long morph sperm). Sperm head and total lengths were measured using the line tool in the image analysis software Fiji.
The number of sperm morphs produced was determined by dissecting sperm from the seminal vesicles of well-fed males (N = 20) that were several days post-reproductive maturity, as described above. After establishing that there was a bimodal distribution (i.e., two discrete sperm length morphs; Fig. 2B in associated manuscript), we next quantified the proportional production of the different sperm morphs on a per male basis (N = 21 males) by counting the number of short and long sperm heads. Sperm morphs were classified based on head length, using the non-overlapping size distributions established from direct measurements.
After establishing a bimodal distribution of sperm heteromorphism in C. procnemis, we conducted a "spatiotemporal" experiment to assay the proportional representation of each sperm morph in ejaculates and to quantify morph-specific patterns of sperm storage and use by females. After completing the assay, flies were collected from a subsequent culture generation, and the entire experiment was replicated. For each replicate, 175 females were collected from culture bottles on the day of eclosion and randomly assigned to seven different time treatments: (1) immediately after copulation ending (T0), (2) 30 min post-mating (T0.5), (3) two hours post-mating (T2), (4) 6 hours post-mating (T6), (5) 24 hours post-mating (T24), (6) 48 hours post-mating (T48), and (7) 96 hours post-mating (T96).
In a companion "ejection" experiment, we assayed the number of each sperm morph ejected from the female reproductive tract with the copulatory plug. Once the plug is ejected, the bursa copulatrix tends to contain no sperm, as all stored sperm occupy the paird spermathecae in this species (Lüpold et al. 2013; Manier et al. 2010; Snook & Hosken 2004). Under conditions identical to the spatiotemporal sperm fate experiment, two replicate experiments were conducted (N = 15 females per replicate) in which females were transferred to individual deep well slides beneath a coverglass immediately after copulation ended and examined every 10 minutes under a stereomicroscope for the presence of an ejected mass. For each female, ejection time relative to the end of mating was recorded and each mass was placed in PBS on a subbed slide, dried, fixed, stained and mounted, and the number of each sperm morph was counted as described above.
The comparative trait data on sperm lengths and the presence or absence of heteromorphism was gathered using the methods described above for 149 species of fruit flies as part of a large, ambitious collaboration that seeks to understand the evolution of complex reproductive phenotypes; the phylogeny used to analyze these data is publicly available and also archived alongside both the discrete and continuous sperm data.
Files and variables
File: ComparativeTraitData.txt
Description:
Variables
- Species: The species sampled for sperm traits. Note the text string includes the first letter of the generic epithet (i.e., Genus name) and an underscore followed by the complete specific epithet (e.g., D_melanogaster).
- Heteromorphism: The absence ("0") or presence ("1") of sperm heteromorphism.
- SpermL: The total length of the fertilizing sperm morph, which is assumed to be the longer morph in all heteromorphic Drosophilidae sampled to date. Units are in millimeters.
File: DrosophilidaePhylogeny.nex
Description: Time-calibrated phylogeny in Nexus format for 149 Drosophilidae.
File: MaleProductionHeadLengthData_5May26.txt
Description: Tab-delimited text file with data on the production of sperm morphs in the fruit fly Chymomyza procnemis.
Variables
- male_id_production: Arbitrary identifier of the male specimen.
- head_length_um: Total length of the sperm head in microns.
File: MaleProductionMorphLengthData_5May26.txt
Description: Tab-delimited text file with data on the lengths of distinct sperm morphs in the fruit fly Chymomyza procnemis.
Variables
- male_id_production: Arbitrary identifier of the male specimen.
- long_morph: Total length in microns of long morph sperm.
- short_morph: Total length in microns of short morph sperm.
File: SpermEjection_5May26.txt
Description:
Variables
- Timepoint: The timepoint at which mated females were frozen for sperm assays.
- Type: The type of assay performed; either a spatiotemporal investigation of sperm in female sperm-storage or a study of ejected sperm.
- Vial ID: Identifier code of the fruit fly vial.
- LongMorphLength: Sperm head length of long sperm morph in microns.
- ShortMorphLength: Sperm head length of short sperm morph in microns.
- Cop_Duration(min): Duration of copulation in minutes.
- EjectionTime(min): The timing of sperm ejection in minutes.
File: SperminFRT_5May26.txt
Description:
Variables
- Timepoint: The timepoint at which mated females were frozen for sperm assays.
- Type: The type of assay performed; either a spatiotemporal investigation of sperm in female sperm-storage or a study of ejected sperm.
- Vial ID: Identifier code of the fruit fly vial.
- LongMorphLength: Head length in microns of long sperm morph.
- ShortMorphLength: Head length in microns of short sperm morph.
- Cop_Duration(min): Duration of copulation in minutes.
- Eggs: Number of eggs laid.
File: SpermMorphTotalLengthData_5May26.txt
Description:
Variables
- male_id_length: Arbitrary identifier of the male specimen.
- total.length_m: Total length of sperm.
- Morph: Either the short or the long sperm morph.
- Head_length_m: Total length of sperm head.
Code/software
The comparative phylogenetic analyses were conducted within R phytools version 2.4.19. All other statistical analyses of sperm production, sperm in female sperm-storage, and sperm ejection can be viewed using Microsoft Excel and were analyzed using R.
Access information
Other publicly accessible locations of the data: