Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice-mated females, using transgenic Drosophila simulans and D. mauritiana populations with fluorescently-labeled sperm heads. Comparisons among these two species and D. melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination, (2) sperm storage, and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer, (2) sperm displacement, (3) sperm ejection, and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within-population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.
Virgin.sim
Data on sperm transfer and storage after first mating for D. simulans. Variables: Treat – treatment, transfer or storage; Male – transgenic line by sperm label, GFP or RFP; Cop – copulation duration in minutes; Bursa – number of sperm in the bursa; Prox – number of sperm in the proximal half of the seminal receptacle (SR); Dist – number of sperm in the distal half of the SR; Caps – number of sperm in the paired spermathecal caps combined; Ducts – number of sperm in the paired spermathecal ducts combined
Virgin.maur
Data on sperm transfer and storage after first mating for D. mauritiana. Variables: Treat – treatment, transfer or storage; Male – transgenic line by sperm label, GFP or RFP; Cop – copulation duration in minutes; Bursa – number of sperm in the bursa; Prox – number of sperm in the proximal half of the seminal receptacle (SR); Dist – number of sperm in the distal half of the SR; Caps – number of sperm in the paired spermathecal caps combined; Ducts – number of sperm in the paired spermathecal ducts combined
Remate.sim
Sperm numbers in space and time after second mating for D. simulans. Variables: MinASM – time after the start of mating at which female was frozen; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; 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; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined
Remate.maur
Sperm numbers in space and time after second mating for D. mauritiana. Variables: MinASM – time after the start of mating at which female was frozen; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; 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; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined
Remate.mel
Sperm numbers in space and time after second mating for D. melanogaster. Variables: Block - different experimental blocks, A and B; MinASM – time after the start of mating at which female was frozen; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; Remate – day after first mating (day 0) on which female remated; Cop2 – duration in minutes of the second copulation; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined
Ejection.sim
Timing and number of sperm ejected after mating for D. simulans. Variables: Block – different experimental blocks, A and B; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; Mates – number of times each female mated; 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; EjTime1 –Ejection time after first mating, in minutes after the start of the mating; EjTime2 – Ejection time after second mating, in minutes after the start of mating; PriorProg – number of progeny produced by female before remating; Eject1 – number of first-male sperm in ejection after first mating; EjMale1 – number of first-male sperm in ejection after second mating; EjMale2 – number of second-male sperm in ejection after second mating; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined
Ejection.maur
Timing and number of sperm ejected after mating for D. mauritiana. Variables: Block – different experimental blocks, A and B; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; Mates – number of times each female mated; 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; EjTime1 –Ejection time after first mating, in minutes after the start of the mating; EjTime2 – Ejection time after second mating, in minutes after the start of mating; PriorProg – number of progeny produced by female before remating; Eject1 – number of first-male sperm in ejection after first mating; EjMale1 – number of first-male sperm in ejection after second mating; EjMale2 – number of second-male sperm in ejection after second mating; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined
Velocity.sim
Sperm velocity and sperm numbers in the SR for D. simulans. Variables: Fem – identity assigned to individual female; Movie – name of movie file from which data were collected; Treat – treatment; A = virgin mating, B = remating, C = no remate; Region – region of the female reproductive tract, bursa or SR; Male – first- or second-male sperm; 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; SR1 – number of first-male sperm in the SR; SR2 – number of second-male sperm in the SR
Velocity.maur
Sperm velocity and sperm numbers in the SR for D. mauritiana. Variables: Fem – identity assigned to individual female; Movie – name of movie file from which data were collected; Treat – treatment; A = virgin mating, B = remating, C = no remate; Region – region of the female reproductive tract, bursa or SR; Male – first- or second-male sperm; 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; SR1 – number of first-male sperm in the SR; SR2 – number of second-male sperm in the SR
Velocity.mel
Sperm velocity and sperm numbers in the SR for D. melanogaster. Variables: Fem – identity assigned to individual female; Movie – name of movie file from which data were collected; Treat – treatment (A = virgin mating, B = remating, C = no remate); Region – region of the female reproductive tract, bursa or SR; Male – first- or second-male sperm; 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; SR1 – number of first-male sperm in the SR; SR2 – number of second-male sperm in the SR
P2.sim
Paternity and sperm count data for D. simulans. Variables: Treat – time after mating for which paternity and sperm numbers in the female reproductive tract were quantified; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; 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; P2.24 – proportion of progeny sired by the second male (P2) for the first 24 hrs after mating; P2.48 – P2 for the first 48 hrs after mating; P2.72 - P2 for the first 72 hrs after mating ; P2 – P2 over entire time period; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined; SR1 – number of first-male sperm in the entire SR; SR2 – number of second-male sperm in the entire SR; Sp1 – number of first-male sperm in both spermathecae combined; Sp2 – number of second-male sperm in both spermathecae combined
P2.maur
Paternity and sperm count data for D. mauritiana. Variables: Block – for maur only, different experimental blocks, A and B; Treat – time after mating for which paternity and sperm numbers in the female reproductive tract were quantified; FirstM – transgenic line of first male by sperm label, GFP or RFP. Second male is automatically the reciprocal label; 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; P2.24 – proportion of progeny sired by the second male (P2) for the first 24 hrs after mating; P2.48 – P2 for the first 48 hrs after mating; P2.72 - P2 for the first 72 hrs after mating; P2 – P2 over entire time period; Bursa1 – number of first-male sperm in the bursa; Bursa2 – number of second-male sperm in the bursa; Prox1 – number of first-male sperm in the proximal half of the SR; Prox2 – number of second-male sperm in the proximal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist1 – number of first-male sperm in the distal half of the SR; Dist2 – number of second-male sperm in the distal half of the SR; Caps1 – number of first-male sperm in the two spermathecal caps combined; Caps2 – number of second-male sperm in the two spermathecal caps combined; Ducts1 – number of first-male sperm in the two spermathecal ducts combined; Ducts2 – number of second-male sperm in the two spermathecal ducts combined; SR1 – number of first-male sperm in the entire SR; SR2 – number of second-male sperm in the entire SR; Sp1 – number of first-male sperm in both spermathecae combined; Sp2 – number of second-male sperm in both spermathecae combined
P2.mel
Paternity and sperm count data for D. melanogaster. Variables: Treat – time after mating for which paternity and sperm numbers in the female reproductive tract were quantified; P2 – P2 over entire time period; SR1 – number of first-male sperm in the entire SR; SR2 – number of second-male sperm in the entire SR; Sp1 – number of first-male sperm in both spermathecae combined; Sp2 – number of second-male sperm in both spermathecae combined
Sperm.Length
Sperm lengths in mm for D. simulans and D. mauritiana. Variables: Species – sim or maur; Thorax – thorax length as a measure of body size, in an aritrary unit, to be entered as a covariate in analyses. Thorax length should be correlated with SR length but not sperm length.; Sperm1-5 – length in mm for each of 5 sperm measured per male. Average sperm lengths for each male are calculated from the four longest measurements.
SR.Length
Seminal receptacle (SR) lengths in mm for D. simulans and D. mauritiana. Variables: Species – sim or maur; SR – SR length in mm; Thorax – thorax length as a measure of body size, in an aritrary unit, to be entered as a covariate in analyses. Thorax length should be correlated with SR length but not sperm length.; Sperm1-5 – length in mm for each of 5 sperm measured per male. Average sperm lengths for each male are calculated from the four longest measurements.