ABSTRACT TRAUTWEIN, MICHELLE DENEE. Multi-gene Phylogenetics to Resolve Key Areas of the Fly Tree of Life. (Under the direction of Brian M. Wiegmann.) FLYTREE (an NSF Assembling the Tree of Life project) is a large collaborative project aimed at reconstructing relationships among major lineages of Diptera. Previous morphological and molecular work, along with preliminary analyses of phylogenomic and total evidence data sets from FLYTREE, have provided evidence that while much of the fly tree of life can be confidently resolved, some regions remain challenging to decipher. Flies are a species-rich lineage of insects that originated more than 240 MYA in the Mesozoic. Ancient radiations, particularly if they occurred rapidly, can be difficult to resolve, even with large amounts of data. Phylogenetic inference can be misled by both systematic and stochastic error. The reliance on rigorous data exploration to decipher phylogenetic signal from noise can be crucial to the accurate recovery of evolutionary relationships. This study utilizes multiple nuclear genes and data exploration to address three persistently problematic regions of dipteran evolution. The first chapter evaluates the relationships of the lower brachyceran superfamily Asiloidea, the putative sister-group to Eremoneura (Cyclorrhapha + Empidoidea). CAD + 28S support traditional asiloid clades and recover multiple hypotheses for the sister group to higher flies, primarily due to the indeterminate placement of the family Bombyliidae (bee flies) and the enigmatic genus Hilarimorpha. The genus Apystomyia is strongly supported as sister to Cyclorrhapha. Taxon stability and the effects of additional genes are explored. The second chapter addresses the phylogenetics of the subfamilies of Bombyliidae by analyzing CAD + 28S alone and with morphology. The monophyly of 8 of 15 subfamilies are confirmed along with the polyphyly of Bombyliinae. A hypothesis for the interrelationships of bee fly subfamilies is presented. Topological incongruence and the effect of the removal of conflict- inducing taxa are explored. The third chapter relies on six-nuclear genes to identify the sister-group of Diptera by resolving the phylogeny of Holometabola. Traditional supraordinal groupings are confirmed. Mecoptera+Siphonaptera are sister to Diptera. Strepsiptera, previously hypothesized as the closest relative of Diptera, is confidently placed as the sister-group to Coleoptera. A thorough exploration to rule out the effects of long-branch attraction is presented. Multi-gene Phylogenetics to Resolve Key Areas in the Fly Tree of Life by Michelle Denee Trautwein A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fufillment of the requirements for the Degree of Doctor of Philosophy Entomology Raleigh, North Carolina 2009 APPROVED BY: ! ! BIOGRAPHY Michelle Trautwein was born in Philadelphia, Pennsylvania in 1976 and relocated to Austin, Texas six months later. She began her studies at the University of Texas in 1994 as an art major. Three years later, after an entomology class and a summer catching frogs in Costa Rica, she changed her major to science and graduated in 1999 with a BS in Biology. Before focusing on insects, she assisted with research on pigeon guillemots in Alaska, prairie dogs in Utah, and bottlenose dolphins in Florida. An internship at the Smithsonian studying Diptera brought her back to insects and reintroduced her to flies. Michelle began her graduate studies at North Carolina State University in Raleigh in 2003. Her work and interests are focused on the evolutionary relationships of flies and phylogenomics in general. ! ""! ! ! ACKNOWLEDGMENTS Thank you to my professors and mentors who introduced me to entomology (flies in particular), evolution, and phylogenetics: Riley Nelson, Wayne Mathis, Amnon Friedberg, Lewis Deitz and Brian Wiegmann. Thank you for your time, your wisdom and your friendship. Additional thanks go to the other students and post-docs in the Wiegmann lab who have supported my work and made my years at NC State memorable. ! """! ! ! TABLE OF CONTENTS Page LIST OF TABLES…………………………………………………………………….….v LIST OF FIGURES ……………………………………………………………………..vi Chapter 1: A multi-gene phylogeny of the superfamily Asiloidea (Insecta: Diptera): exploring the effects of taxon sampling and the resolving power of additional genes…….………………………………………………………………1 Abstract.………………………………………………………………………….2 Introduction….…………………………………………………………………...3 Methods and Materials….………………………………………………………7 Results ………………………………………………………………………….14 Discussion………………………………………………………………………20 Conclusions ..………………………………………………………………......28 Acknowledgments ……………………….………………………………......29 References ……………………………………………………………………..30 Chapter 2: The evolutionary relationships of bee fly subfamilies: short branches, long branches and topological incongruence …………………….52 Abstract…………………………………………………………………………53 Introduction……………………………………………………………………..54 Materials and Methods………………………………………………………..55 Results and Discussion……………………………………………………….61 Conclusions …………………………………………………………………...70 ! "#! ! ! Acknowledgments ..……………………….………………………………......72 References ……………………………………………………………………..73 Chapter 3: Identifying the sister-group to Diptera: a multi-gene phylogeny of the holometabolous insects …………………………………………………….92 Abstract ………………………………………………………….……………..93 Introduction …………………………………………………….………………94 Results and Discussion ………………………………………….…………...97 Materials and Methods ..….………………………………………………….105 Acknowledgments ..……………………….………………………………....109 References ……………………………………………………………………110 ! #! ! ! LIST OF TABLES Chapter 1 Table 1. Sampled taxa ……………………………………………………….38 Table 2. Clade recovery results with varying methods of analysis, treatment of data and taxon inclusion …………………………..40 Table 3. Leaf stability values of all taxa, stable taxa and reduced taxa…41 Table 4. Testing of a priori hypotheses in parsimony ……………..………43 Chapter 2 Table 1. Sampled taxa ………………………….…………………..……….80 Chapter 3 Table 1. Taxa, genes and genbank numbers for Holometabola and out- groups………………………………………………………….….118 Table 2. Clade recovery results from ML analyses with varied taxon and character inclusion used to counter LBA ………………………121 ! #"! ! ! LIST OF FIGURES Chapter 1 Figure 1. Strict consensus of 6 maximum parsimony trees of 28S+CAD (bp 1+2). All taxa included …………………………………..…….45 Figure 2. Bayesian tree of 28S+CAD (bp 1+2). All taxa included ………46 Figure 3. Maximum- likelihood tree of 28S+CAD (bp 1+2). All taxa included ……………………………………………………….……..47 Figure 4. Maximum parsimony analysis of 28S, CAD, TPI, and CO1 …..48 Figure 5. Comparison of MP bootstrap consensus tree including all taxa and stable taxa only ……………………………………………….. 49 Figure 6. MP, ML, and BI congruent topology of reduced taxa …………..50 Figure 7. Four-cluster likelihood mapping image testing the support for a monophyletic Asiloidea ……………………………………….........51 Chapter 2 Figure 1. Strict consensus of 7 maximum parsimony trees of 28S+CAD (bp 1+2). All taxa included ………………………………………85 Figure 2. Maximum- likelihood tree of 28S+CAD (bp 1+2). All taxa included …………………………………………………………….86 Figure 3. Bayesian tree of 28S+CAD (bp 1+2). All taxa included ……….87 ! #""! ! ! Figure 4. Consensus network showing conflict between MP, ML, and BI topologies including all taxa ……………………………………...88 Figure 5. Consensus network revealing largely congruent topologies from MP, ML and BI when conflict-inducing taxa are removed. …….89 Figure 6. Bayesian tree of 28S+CAD (bp 1+2) with 5 conflict-inducing taxa removed …………………………………………………………….90 Figure 7. Total evidence tree. Bayesian analysis of 28S+CAD (bp 1+2) plus155 morphological characters . .……………………………..91 Chapter 3 Figure 1. The phylogeny of the holometabolous insects based on 6 nuclear protein-coding genes …………………………………….122 Figure 2. The congruent ML and BI topology with branch lengths and support values ……………………………………………….…….123 Figure 3a-b. Likelihood-mapping images showing the strength of our phylogenetic signal and the conflicting data supporting the placement of Strepsiptera ………………………………….…….124 Figure 4. Neighbor-Nets showing conflicting splits with all taxa included and with Strepsiptera excluded ……………………………...…..126 ! #"""! ! ! Chapter 1: A multi-gene phylogeny of the superfamily Asiloidea (Insecta: Diptera): exploring the effects of taxon sampling and the resolving power of additional genes ! $! ! ! Abstract Asiloidea are a group of nine lower brachyceran fly families made up of generally large-sized flower visitors, parasitoids and aerial predators of other insects. Traditionally, the Asiloidea has been viewed as a monophyletic assemblage and the closest relative to the large, successful dipteran radiation Eremonuera (Cyclorrhapha+Empidoidea). The evidence for asiloid monphyly is limited, and previous morphological and molecular studies demonstrate that this region of fly evolution is marked by very few characters delimiting the relationships between the presumed families of Asiloidea and Eremoneura. Adding to the phylogenetic complexity are the enigmatic ‘asiloid’ genera Hilarimorpha and Apystomyia, currently united in the family Hilarimorphidae, that retain morphological characters of both asiloids and higher flies. In this study we use the nuclear protein-coding gene CAD and 28S rDNA to test the monophyly of the Asiloidea and resolve its relationship to the Eremoneura. To this end, we also explore the effects of taxon sampling on support values and topological stability, the resolving power of additional