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Finding the nearest relatives of Nasonia (Hymenoptera: Pteromalidae) Roger Burks University of California, Riverside Department of Entomology 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 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 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) 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 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 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 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? 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 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 Combined 28S and Wingless molecular results, Parsimony (PAUP) black = Pteromalinae red = other Pteromalids * = Wolbachia positive Numbers indicate bootstrap support (1000 replicates) Agrees with simple POY run in topology 1176 steps in PAUP rci = 0.209 ri = 0.403 Combined 28S and Wingless molecular results, Mr. Bayes 3.1 black = Pteromalinae red = other Pteromalids * = Wolbachia positive 6 parameters, 4 chains, partitioned by gene region, 1 million generations Numbers indicate posterior probability Combined 28S and Wingless molecular results, Likelihood black = Pteromalinae red = other Pteromalids * = Wolbachia positive model: GTR+I+G program: PAUP Testing hypotheses not present in the optimum maximum likelihood tree (500 total sampled trees for test) constraint tree with: au test p value sh test p value Nasonia+ Urolepis clade 0.81 1.00 Nasonia + Trichmalopsis + Urolepis paraphyly 0.50 0.85 monophyletic Pteromalinae 0.38 0.80 Nasonia + Trichomalopsis clade 0.23 0.79 monophyletic Trichomalopsis 0.07 0.70 0.01** 0.50 paraphyletic Nasonia au = approximately unbiased test (Shimodaira 2002) sh = Shimodaira-Hasegawa test (Shimodaira & Hasegawa 1999) 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 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 They differ by only one base pair in 28S?? Trichomalopsis sarcophagae Nasonia vitripennis 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 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