1. Finding the nearest relatives of Nasonia (Hymenoptera: Pteromalidae) Roger Burks University of California, Riverside Department of Entomology

2. What is Nasonia? Gregarious puparial parasitoids of calyptrate flies in bird nests and refuse Model system, better known than any other species of Chalcidoidea— genome project ongoing Three species, each infected by two unique strains of Wolbachia

3. The three species of Nasonia Females almost identical (Darling & Werren 1990) Males differ in degree of wing reduction Nasonia vitripennis worldwide, synanthropic N. giraulti in eastern North America, N. longicornis in western North America –specialized on flies in bird nests

4. Wolbachia basic background Bacteria infecting arthropods and filarial nematodes Transmitted vertically from mother to offspring (Binnington & Hoffmann 1989) Cause crossing incompatibility in Nasonia ( Breeuwer & Werren 1990) Phylogenetic congruence between bacteria and host usually absent– horizontal transmission? May cause rapid speciation in arthropods (Laven 1959, 1967; Breeuwer & Werren 1990)

5. How Wolbachia affects Nasonia Cytoplasmic Incompatibility ( Breeuwer & Werren 1990) –Causes death of offspring of mothers that do not have same Wolbachia strains as the father Incompatible crosses: –Uninfected female x infected male –Infected female x male infected by at least one different strain Infection rate near 100% in wild Nasonia –“Cured” colonies used to study Wolbachia effects in lab

6. Why Nasonia’s relationships still need studying Nasonia is a model system for evolutionary biology studies, yet… Ancestral states cannot be inferred with only three analyzed species! No agreement in classification of wasps in its family (Pteromalidae) Needed: means to reject some pteromalids as close Nasonia relatives

7. Pteromalidae is a scary taxon 587 genera in 31 subfamilies Pteromalinae with only 283 genera Parasitoids of various terrestrial arthropods No previous phylogenetic analysis using more than 10 pteromaline genera Previous analyses with either morphology only or 28S ribosomal sequences only

8. Pteromalinae molecular vs. morphological rates of evolution 283 genera of Pteromalinae, but... 28S D2 sequence divergence equal to that of the genus Aphelinus (Heraty 2004) Rapid morphological evolution or ribosomal constraints? Rapid evolution due to Wolbachia?

9. Tools for the search Morphology –105 morphological characters (work in progress) 28S D2-D5 ribosomal DNA, Wingless –Secondary structure alignment for 28S (Gillespie et al. 2005) to be compared with POY results Analysis with parsimony (PAUP, TNT, POY), maximum likelihood, Mr. Bayes Hypothesis testing with ML using CONSEL

10. Outgroup selection Based on Heraty lab matrix of Chalcidoidea –28S D2-D5, 18S E17-E35 ribosomal DNA –471 taxa (including outgroups) –All families, 84 total subfamilies represented Subfamilies Diparinae, Ormocerinae are legitimate outgroups for Pteromalinae

11. Combined 28S and Wingless molecular results, Parsimony (PAUP) Numbers indicate bootstrap support (1000 replicates) Agrees with simple POY run in topology 1176 steps in PAUP rci = 0.209 ri = 0.403 black = Pteromalinae red = other Pteromalids * = Wolbachia positive

12. Combined 28S and Wingless molecular results, Mr. Bayes 3.1 Numbers indicate posterior probability black = Pteromalinae red = other Pteromalids * = Wolbachia positive 6 parameters, 4 chains, partitioned by gene region, 1 million generations

13. Combined 28S and Wingless molecular results, Likelihood black = Pteromalinae red = other Pteromalids * = Wolbachia positive model: GTR+I+G program: PAUP

14. Testing hypotheses not present in the optimum maximum likelihood tree (500 total sampled trees for test) constraint tree with:au test p valuesh test p value Nasonia+ Urolepis clade0.811.00 Nasonia + Trichmalopsis + Urolepis paraphyly 0.500.85 monophyletic Pteromalinae 0.380.80 Nasonia + Trichomalopsis clade 0.230.79 monophyletic Trichomalopsis 0.070.70 paraphyletic Nasonia0.01**0.50 au = approximately unbiased test (Shimodaira 2002) sh = Shimodaira-Hasegawa test (Shimodaira & Hasegawa 1999)

15. Problem: Not enough variation to have statistical power Solution: Add a more rapidly evolving gene Candidates: Long-wavelength Rhodopsin—multiple copies? Pten—contains intron, but short Cytochrome Oxidase I & II—AT richness

16. Perspective Trichomalopsis sarcophagae 28S sequence (>1100 base pairs) differs from that of Nasonia vitripennis by only 1 base pair Sampling remains incomplete –Nasonia not well-surveyed in Palearctic region –Trichomalopsis with 54 species! Trichomalopsis microptera male

17. They differ by only one base pair in 28S?? Trichomalopsis sarcophagae Nasonia vitripennis

18. Further goals Sequence from more species of Trichomalopsis, other genera near Nasonia (>120 specimens to be sequenced) Finish morphological analysis Wolbachia survey across Pteromalinae, comparing bacteria and wasp phylogenies

19. Acknowledgments Advisory committee: John Heraty Richard Stouthamer Bob Luck Cheryl Hayashi Jack Werren Matt Yoder Doug Yanega Serguei Triapitsyn Lara Baldo James Russell Genet Tulgetske Danel Vickerman Heraty lab: Dave Hawks Johan Liljeblad James Munro Jeremiah George Jason Mottern Chrissy Romero Adena Why Jutta Burger Matt Buffington Funded by: NSF FIBR: 0328363