Background: Identifying traits that reproductively isolate species, and the selective forces underlying their divergence, is a central goal of evolutionary biology and speciation research. There is growing recognition that postcopulatory sexual selection, which can drive rapid diversification of interacting ejaculate and female reproductive tract traits that mediate sperm competition, may be an engine of speciation. Conspecific sperm precedence (CSP) is a taxonomically widespread form of reproductive isolation, but the selective causes and divergent traits responsible for CSP are poorly understood.
Results: To test the hypothesis that postcopulatory sexual selection can generate reproductive isolation, we expressed green (GFP) or red fluorescent protein (RFP) in sperm heads of recently diverged sister species, Drosophila simulans and D. mauritiana, to enable detailed resolution of species-specific sperm precedence mechanisms. Between-species divergence in sperm competition traits and mechanisms prompted six a priori predictions regarding mechanisms of CSP and degree of cross asymmetry in reproductive isolation. We resolved four distinct mechanisms of CSP that were highly consistent with predictions. These comprise interactions between multiple sex-specific traits, including two independent mechanisms by which females exert sophisticated control over sperm fate to favor the conspecific male.
Conclusions: Our results confirm that reproductive isolation can quickly arise from diversifying (allopatric) postcopulatory sexual selection. This experimental approach to "speciation phenotypes" illustrates how knowledge of sperm precedence mechanisms can be used to predict the mechanisms and extent of reproductive isolation between populations and species.
Remate
Sperm numbers in space and time after second mating. Variables: Cross - Species identity of first and second male to mate with a D. simulans
female, where "s" indicates D. simulans and "m" indicates D. mauritiana, such that "ss" indicates that both males were D. simulans, "ms" indicates that the first male was D. mauritiana and the second male was D. simulans, etc. MinASM – time after the start of mating at which female was frozen, in minutes. Remate – day after first mating (day 0) on which female remated. Cop1 – duration in minutes of the first copulation. Cop2 – duration in minutes of the second copulation. PriorProg – number of progeny produced by female before remating. Matings - number of successful matings by that female. ThoraxF - female thorax length, in arbitrary units. ThoraxM1 - thorax length of the first male, in arbitrary units. ThoraxM2 - thorax length of the second male, in arbitrary units. Bursa1 – number of first-male sperm in the bursa. Bursa2 – number of second-male sperm in the bursa. SR1 – number of first-male sperm in the SR. SR2 – number of second-male sperm in the SR. Sp1 – number of first-male sperm in the spermathecae. Sp2 – number of second-male sperm in the spermathecae
Virgin
Data on sperm transfer and storage after single mating of D. simulans female with D. mauritiana male. Variables: Treat – treatment, storage or transfer. Cop – copulation duration in minutes. ThoraxF - female thorax length, in arbitrary units. ThoraxM - thorax length of the second male, in arbitrary units. Bursa – number of sperm in the bursa. SR – number of sperm in the seminal receptacle. Sp – number of sperm in the spermathecae
Ejection1
Timing and number of sperm ejected after a single mating of a D. simulans female with a D. mauritiana male. Variables: CopDur - copulation duration in minutes. EjTime - minutes after start of copulation when female ejected sperm mass. Sperm – number of sperm in the ejected sperm mass
Ejection2
Timing and numbers of first- and second-male sperm ejected after remating, and numbers of first- and second-male sperm remaining in the female reproductive tract after ejection. Variables: Cross – Species identity of first and second male to mate with a D. simulans female, where "s" indicates D. simulans and "m" indicates D. mauritiana, such that "ss" indicates that both males were D. simulans, "ms" indicates that the first male was D. mauritiana and the second male was D. simulans, etc. Remate – day after first mating (day 0) on which female remated. Eject - 1 if female ejected, 0 if she did not eject. EjStart - Minutes after start of mating when female began ejecting a sperm mass (sometimes it got stuck). EjEnd - Minutes after start of mating when female finished ejecting a sperm mass. PriorProg - presence of prior progeny, where 0 = none, 1 = a few, 2 = a lot. Eject1 – number of first-male sperm in ejected sperm mass. Eject2 - number of second-male sperm in ejected sperm mass. ThoraxF - female thorax length, in arbitrary units. ThoraxM - thorax length of the second male, in arbitrary units. Bursa1 – number of first-male sperm in the bursa. Bursa2 – number of second-male sperm in the bursa. SR1 – number of first-male sperm in the SR. SR2 – number of second-male sperm in the SR. Sp1 – number of first-male sperm in the spermathecae. Sp2 – number of second-male sperm in the spermathecae
Velocity
Sperm velocity and sperm numbers in the SR. Variables: Treat - sxm indicates single mating between D. simulans female and D. mauritiana male, NR indicates "no remate" treatment, other treatments are rematings defined by the species identity of first and second male to mate with a D. simulans female, where "s" indicates D. simulans and "m" indicates D. mauritiana, such that "ss" indicates that both males were D. simulans, "ms" indicates that the first male was D. mauritiana and the second male was D. simulans, etc. Region – region of the female reproductive tract, bursa or SR. Male – species and color tag of male whose sperm velocity is recorded. Order - first or second male to mate. Remate - for remating treatments, day after first mating (day 0) on which female remated. Velmps – instantaneous linear velocity in microns per second, averaged for each sperm and for all sperm recorded in a movie. N – number of sperm tracked per movie. SR.G – number of GFP sperm in the SR. SR.R – number of RFP sperm in the SR.
mxs
Numbers of sperm transferred and stored for D. mauritiana females mating with D. simulans males. Variables: Cross - mxs = D. mauritiana female mating once with a D. simulans male; mxsm = D. mauritiana female mating first with a D. simulans male followed by a D. mauritiana male; mxms = D. mauritiana female mating first with a D. mauritiana male followed by a D. simulans male. Cop - copulation duration in minutes, for second copulation for mxsm and mxms crosses. Bursa1 – number of first-male sperm in the bursa. Bursa2 – number of second-male sperm in the bursa. SR1 – number of first-male sperm in the SR. SR2 – number of second-male sperm in the SR. Sp1 – number of first-male sperm in the spermathecae. Sp2 – number of second-male sperm in the spermathecae
P2
Paternity and sperm count data. Variables: Cross - Species identity of first and second male to mate with a D. simulans female, where "s" indicates D. simulans and "m" indicates D. mauritiana, such that "ss" indicates that both males were D. simulans, "ms" indicates that the first male was D. mauritiana and the second male was D. simulans, etc.
Treat – time after mating for which paternity and sperm numbers in the female reproductive tract were quantified. ThoraxF - female thorax length, in arbitrary units. ThoraxM1 - thorax length of the first male, in arbitrary units. ThoraxM2 - thorax length of the second male, in arbitrary units. Cop1 – duration in minutes of the first copulation. Cop2 – duration in minutes of the second copulation. Remate – day after first mating (day 0) on which female remated. PriorProg – number of progeny produced by female before remating. P2Prog1 - number of progeny produced after remating sired by the first male. P2Prog2 - number of progeny produced after remating sired by the second male. P2 – proportion of progeny produced after remating sired by the second male. Bursa1 – number of first-male sperm in the bursa. Bursa2 – number of second-male sperm in the bursa. SR1 – number of first-male sperm in the SR. SR2 – number of second-male sperm in the SR. Sp1 – number of first-male sperm in the spermathecae. Sp2 – number of second-male sperm in the spermathecae.