1. 1 Topping off: A mechanism of first- male sperm precedence in a vertebrate Adam G. Jones, Erika M. Adams, and Stevan J. Arnold

2. 2 Introduction Materials and Methods Result Discussion

3. 3 Introduction Materials and Methods Result Discussion

4. 4 Sexual selection is an important facet of the evolutionary process. -male male competition & female choice Many of key aspects of sexual selection occur before mating, the competition among sperm within a female’s reproductive tract.

5. 5 Sperm competition research in recent years has been to quantify the proportion of offspring sired by the second of two males mated sequentially to a female, a value known as P 2. Multiple distinct mechanisms can lead to the same value of P 2, so such an approach to the problem often says very little about the actual mechanism of sperm competition.

6. 6 Taricha granulosa

7. 7 Due to the three following reasons Females receive sperm during a short receptive period, then it lay eggs after several weeks to months, fertilizing them with stored sperm. Females lay large numbers of eggs, a characteristic that permits a description of the change in P 2 over time. The transfer of sperm in newts is indirect.

8. 8 Sperm competition appears to be rare or nonexistent in Taricha. (Halliday,1998) The extrapolation from laboratory observation may be premature! -no data from natural population

9. 9 Last-male precedence? If postinsemination period of amplexus represents mate guarding, then we might expect such a behavior to evolve in a species with last-male sperm precedence. Some aspects of the mating behavior of related newts of the genus Triturus imply that these species experience last-male precedence.

10. 10 Introduction Materials and Methods Result Discussion

11. 11 Paternity in Seminatural Breeding Assemblages collected unmated females newt on rainy night collected males by submerging plastic minnow trap 8 ♂ +8 ♀ 8 ♂ +3 ♀ Water-filled tanks ╳6╳6 ╳6╳6

12. 12 Each female given an injection of 10µl of a 0.5mg/ml LH-RH analog to induce egg laying. 57 of them laid eggs. They collected either hatching or containing well-developed embryo for microsatellite analysis.

13. 13 Mating Patterns in a Natural Population Field-mated female newts were collected from a natural population during the egg-laying phase by the trap. Each female given an injection of 10µl LH-RH. 30 of them laid eggs. Hatching for microsatellite analysis.

14. 14 Sperm Precedence Experiment Female was placed with male in a water-filled 38-liter aquarium, and were scored every 12h for insemination. They removed the males and waited until the sperm cap was no longer visible in the female’s cloaca, at which time a second male was placed in the tank with the female.

15. 15 Females successfully inseminated twice in isolation for at least 20 days and then injected them with 10 l of LH-RH. They collect eggs in temporal groups by checking a female’s tank daily for the presence of eggs. Eggs raised to hatching for microsatellite analysis.

16. 16 Microsatellite Analyses In first two experiments, we assayed a random sample of an average of 24 offspring from each female. Paternity was assigned for 99.1% of the offspring genotyped, resulting in a very precise estimate of multiple mating by females

17. 17 Natural Population: three loci :Tgr14, Tgr10, and Tgr06 computer program GERUDSIM1.0 Sperm Precedence Experiment: first assayed all males and females by using six microsatellite loci chose the loci that would differentiate the two experimental males and assayed all hatchlings

18. 18 Introduction Materials and Methods Result Discussion

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20. 20 This result prove that T. granulosa mate with multiple males during the course of the breeding season. The majority of multiply inseminated females appeared to have mated with just two males.

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23. 23 tested for a trend in P 2 over time linear model for categorical data (procedure CATMOD in SAS ) (P=0.09) failed to reject the null hypothesis that P 2 remained constant (P=0.09) no significant relationships between P 2 and other measured variables (insemination interval,first male size, second male size, male size difference)

24. 24 Introduction Materials and Methods Result Discussion

25. 25 clearly that the potential for sperm competition is very high in natura populations of T. granulosa The idea that each female typically receives sperm from a single male was based on an extrapolation from laboratory observations. Such extrapolations plainly should be treated with caution. Molecular markers should play a central role in future efforts to connect the laboratory observations.

26. 26 Female accepts sperm from her first mate, and, if additional space remains in her spermathecae, she until she has no need for further sperm. ‘‘ Topping off ’’ This interpretation is also consistent with anatomical observations of sperm storage in Notophthalmus and Triturus, close relatives of Taricha.

27. 27 Female should accept sperm only from high- quality males. Sperm from different males in her spermathecae should have equivalent value to her. There should be no selective pressure for a female to engage in cryptic female choice.

28. 28 The pattern of sperm competition that we have documented in T. granulosa differs from the patterns described for other vertebrate taxa. The closest vertebrate analog ‘‘passive sperm loss’’ (PSL) model. (birds)

29. 29 Same feather: passive and lack sperm stratification. Different feather: PSL model typically produces last-male advantage, whereas topping-off model produces first-male advantage. PSL model, the timing of inseminations has a profound effect on the outcome of sperm competition.

30. 30 Technique of assaying offspring in temporal groups provides far more information regarding the mechanisms of sperm usage than simply measuring P 2. This study is consistent with the idea that sexual dialectics may explain an important aspect of the evolution of rough-skinned newt reproductive ecology. Study contributes to debates regarding the importance of cryptic female choice.