Haldane’s rule in Carabus : interspecific mating between Carabus punctatoauratus and Carabus splendens using exper

1. Haldane’s rule in Carabus: interspecific mating between Carabus punctatoauratus and Carabus splendens using experimental tests and molecular markers Author: Carine Brouat Journal: The Netherlands Entomological Society SongZhao Department of Entomology, SCAU

2. 科 族 属 亚属 种 亚种 Carabidae Who are Carabus? Carabini Carabus Chrysocarabus auronitens punctatoauratus splendens

3. Carabus (coptolabrus) ignimetallus Bates,1888

4. Carabus (Zasiocoptolabrus）sunwukongImura,1993

5. Carabus (Apotomopterus）toulgoeti Deuve,1989

6. Carabus (Isiocarabus）miaorum Lassalle et Peunies,1993

7. Carabus (coptolabrus) lafossei Feisthame,1845

8. Carabus (coptolabrus) lafossei Feisthame,1845

9. Carabus (Apotomopterus） davidis Deyrolle et Fairmaire, 1878

10. Carabus (Chrysocarabus) punctatoauratus Germar Elytral primary rows is sharp Which are endemic to the Pyrenean spring-breeders predators of slugs and earthworms they overwinter as adults

11. Carabus (Chrysocarabus) splendens Olivier,1790 Elytral primary rows is absolescent; shape and colour of palpi and antennae Which are endemic to the Pyrenean spring-breeders predators of slugs and earthworms they overwinter as adults

12. What is Haldane’s rule? Haldane: the heterogametic sex (the male for carabids) is less fertile or has a reduced viability. 1922

13. Evolution, Species, Problem • Natural hybridization can change the genetic diversity, population dynamics, and interactions between species • lead to adaptive evolution and evolutionary diversification • Species; • Reproductive barriers:microspatial or microtemporal allopatry • BUT what will happen about sympatric insect species when we relaxing some under controlled conditions?

14. Why they are choiced? • The two species cluster in the same mitochondrial DNA clade showing that they are phylogenetically closely related. • They are in sympatry • ?

15. What questions we try to answer? • Does interspecific mating occur between C.splendens and C. punctatoauratus? • Is there a preferential hybridization direction? • Are hybrid individuals viable and fertile?

16. Sampling and Conditions • Sampling: April 2001, the males and females were kept in separate boxes at 5 ℃ • Experimental conditions: pairs: boxes (12×12×6.5 cm) with 2 cm soil .slugs and apple. eggs: boxes(2×2×2×2cm) with sterilized soil. larvae: cups (8.5 cm diameter and 11.5 cm high) with 7 cm soil, crushed slugs and apple adults: were reared individually for 21 days and then kept under winter conditions(5 ℃)

17. Obtaining virgin parents C. punctatoauratus×C. punctatoauratus C. punctatoauratus×C. splendens 8 mating pairs 20 mating pairs PP ps C. splendens×C. splendens C. splendens×C. punctatoauratus 10 mating pairs 10 mating pairs ss sp

18. How to assess the paternity of progeny? • Using four microsatellite loci :Cs2/10, Csol9/170, Cp4/63, and Cs1/134(how? beg) • The loci were sufficiently polymorphic to easily establish the paternity of any resulting offspring by simple exclusion • The extra-pair offspring were discarded (generalized linear models with Poisson,SAS)

19. Mating of inter- and intraspecific crosses F1(PP×SS, 10 pairs; SS×PP, 10 pairs) F2(SP×SP, 8 pairs PS×PS, 8 pairs) ppss sspp spsp psps Backcrosses(F1 hybrids to the parent species) (PP×PS, 8 pairs; SS×SP, 9 pairs; PS×SS, 10 pairs; SP×PP, 6 pairs) ppps sssp psss sppp Pure C. Splendens (SS×SS, 8 pairs) Pure C. Punctatoauratus (SS×SS,8pairs) ssss ?

20. Mating of inter- and intraspecific crosses X=42 ppss sspp spsp psps pssp sssp psss pppp? sppp ssss ppps ppsp pspp ssps spss spps

21. Result All F1 and backcross hybrids from our experiments had the C. punctatoauratus pattern of sharp elytral primary rows. Thus, the morphological trait used to distinguish the two species displays dominance and using only this trait to detect hybrids in natural populations is therefore inappropriate.

22. Extra-pair fertilization for the first cross-experiment Three of these females had a mixture of extra-pair offspring and offspring from the male used in the laboratory 280 individuals (no consider aboout Extra-pair offspring)

23. Why PP crosses had significantly fewer incompatible offspring than SP crosses？ C. punctatoauratus females can preferentially choose’ the sperm they use to fertilize their eggs, favouring conspecific sperm.

24. Why only C. punctatoauratus females produced progeny that did not originate from their experimental partner？ • some C. punctatoauratus females had been fertilized in the field before sampling, being able to store the sperm of their sexual partner for some weeks. • onset of the reproductive period may be later for C. splendens than for C. punctatoauratus

25. Why only C. punctatoauratus females produced progeny that did not originate from their experimental partner？ • C. splendens females are unable to store sperm • Interspecific mating may exist but could be inefficient in the case of multiple mating due to sperm precedence favouring conspecific sperm

26. Second cross-experiments Mean number of eggs,larvae, and adult individuals produced per pair in each type of cross • proportion of fertile pairs • mean number of eggs per female • percentage of fertile eggs per female • mean number of larvae/adults • percentage of emerged adult per hatching larva

27. Development time • Fertility estimates for the different types of crosses • Wald X2 tests or Tukey test, P<0.05

28. Discussion • We produced 114 and 168 hybrid individuals in the first and second laboratory experiments, • The hybrids were viable but only females were fertile（ third question）. • Introgression in nature between the two Carabus species can only proceed through females (first question).

29. Why easy in controlled conditions but difficult in natural conditions？ • Counter-selection of hybrids under natural environmental conditions • hybrid male sterility was the only trait associated with post-zygotic counter selection of hybrids.

30. Why easy in controlled conditions but difficult in natural conditions？ • But This alone cannot explain the low proportion of F1 individuals under natural conditions • Hybrid male sterility is an incomplete barrier because gene introgression between species can still occur through females by repeated backcrosses

31. Why number of F1 hybrids is so low in natural populations？ • Copulation may be impossible if the male and female genitalia of the two species do not fit together well • Unsuccessful mating attempts would damage female genitalia

32. Why number of F1 hybrids is so low in natural populations？ • Dissection of genitalia showed no sclerotized segment • No physical incompatibility in size or shape of the male and female genitalia between the two species.

33. Why number of F1 hybrids is so low in natural populations？ • If copulation does occur, it is possible that heterospecific sperm may not be adequately transferred, implying a limited viability of stored sperm, or an increase in gametic mortality • no significant differences in the number of descendants between SSSS cross-types and certain interspecific crosses

34. Why number of F1 hybrids is so low in natural populations？ • Specific attraction mechanisms exist: highly specific mate choice（second question） • C. punctatoauratus and C. splendens indicate highly specific mate choice in both species • differences in the seasonal or daily rhythms of the two species may prevent physical encounter and thus hybridization

35. Conclusion • pre-zygotic barriers may be sufficiently high to prevent complete homogenization of genomes of C. splendens and C. punctatoauratus, despite the fact that hybrids can be easily obtained under controlled laboratory conditions

36. Thanks for your attention!