BUILDING THE TREE OF LIFE: RECONSTRUCTING THE EVOLUTION OF A RECENT AND MEGADIVERSE BRANCH (CALYPTRATAE: DIPTERA) SUJATHA NARAYANAN KUTTY (B.Tech) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 The great tragedy of Science - the slaying of a beautiful hypothesis by an ugly fact. - Thomas H. Huxley ii ACKNOWLEDGEMENTS We don't accomplish anything in this world alone... and whatever happens is the result of the whole tapestry of one's life and all the weavings of individual threads from one to another that creates something - Sandra Day O'Connor. The completion of this project would have been impossible without help from so many different quarters and the few lines of gratitude and acknowledgements written out in this section would do no justice to the actual amount of support and encouragement that I have received and that has contributed to making this study a successful endeavor. I am indebted to Prof. Meier for motivating me to embark on my PhD (at a very confusing point for me) and giving me a chance to explore a field that was quite novel to me. I express my sincere gratitude to him for all the guidance, timely advice, pep talks, and support through all the stages of this project and for always being patient while dealing with my ignorance. He has also been very understanding during all my non- academic distractions in the last two years. Thanks Prof.- your motivation and inspiration in the five years of my graduate study has given me the confidence to push the boundaries of my own capabilities. You should be acknowledged as the best supervisor one can have. I would also like to thank Dr. Thomas Pape for sending large amounts of specimens all of which have been invaluable for this study and for all his timely inputs, immaculate proof reading and important suggestions at various junctures of the project. I would also like to i thank Dr. Brian Wiegmann and his lab members especially Brain Cassel for my enriching stint at NCSU, Raleigh. Special thanks to Dr. Frederik Petersen, Dr. Adrian Pont, Dr. Marco Bernasconi and Dr. František Šifner for all their important and useful inputs in this study. I also thank all other collaborators who have directly and indirectly contributed to this study. I would like to thank Kathy, my first labbie along with whom I figured out the tricks of pcr-ing and Nalini for all the unwinding sessions and especially for her support and ‘motivation’ during the last phase of thesis writing. Thanks girls for not just being such wonderful colleagues but also lovely friends with whom I have spent some of the best times in evolab. Thanks to all the other members of the evolab (past and current) who have helped and supported me at different stages and many of whom I have seen begin and finish their projects during these years: Gaurav, Shiyang, Gwynne, Guanyang, Weisong, Yuchen, Farhan, Danwei, Denise, Dave, Nanthinee, Zeehan, Martin, Mirza, WaiKit and Huifung. Thanks Anu, Aparna, Nilofer and Rika for all the fun times, get-togethers and of course the brilliant dance sessions. Thanks to Suni for being my support system for almost a decade now, Janesh for always sticking by my side, Ettan, Chechi and Vishu for being my family away from home and my ii friends on the other side of the globe who have ‘virtually’ always supported me- Pavan and Venky. I have to thank Acha, Amma and Sumi, my ‘perfect’ family for pitching in whichever way they could, and my extended family especially my darling Meena and also my new family (in-law). Lastly and most importantly - Sunil, who switched roles from being the ‘crazy’ guy in my world to my fiancée and finally to my better half, all during the course of this dissertation. In your own quiet way, you have always been my pillar of strength and constant source of support over the years. Thank you so much for understanding and putting up with my daily crankiness, random cooking patterns and the late night writing sessions for the last couple of months. I owe you one! iii TABLE OF CONTENTS Acknowledgements i Table of contents iv Summary ix List of Tables xi List of Figures xii List of Publications xv Introduction 1 Chapter 1: Phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers 1.1 Introduction 7 1.1.1 Family portraits: Biology and Systematics 8 1.1.2 Hippoboscoidea Phylogenetics 11 1.2 Materials and Methods 14 1.2.1 Taxon sampling, DNA extraction and sequencing 14 1.2.2 Phylogenetic Analysis 17 1.3 Results 19 1.4 Discussion 23 1.4.1 Classificatory implications 26 1.4.2 Host-shifts and diversification in Hippoboscoidea 28 1.4.3 Morphological and life history evolution in Hippoboscoidea 32 1.5 Conclusions 34 iv Chapter 2: Sensitivity analysis, molecular systematics, and natural history evolution of Scathophagidae (Diptera:Cyclorrhapha:Calyptratae) 2.1 Introduction 36 2.1.1 Comparing alignment techniques 38 through a sensitivity analysis 2.2 Materials and Methods 40 2.2.1 Taxa and DNA extractions 40 2.2.2 DNA amplification and sequencing 40 2.2.3 Tree search strategies 42 2.2.4 Sensitivity analysis 43 2.2.5 Natural history 44 2.3 Results 49 2.3.1 Alignment techniques, indel treatment, character 49 transformation weighting 2.4 Discussion 56 2.4.1 Sensitivity analyses and Choice of alignment 56 2.4.2 Node support and Node stability 60 2.4.3 Systematic conclusions 61 2.4.4 Natural history evolution 64 2.5 Conclusions 68 v Chapter 3: The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes 3.1 Introduction 70 3.1.1 Fanniidae 71 3.1.2 Muscidae 72 3.1.3 Anthomyiidae 73 3.1.4 Scathophagidae 74 3.1.5 Interfamilial relationships 75 3.2 Materials and Methods 77 3.2.1 Taxa and DNA extractions 77 3.2.2 DNA amplification 77 3.2.3 Alignments 81 3.2.4 Tree search strategies 82 3.3 Results 83 3.3.1 Alignment techniques, indel treatment, character transformation weighting 3.4 Discussion 90 3.4.1 Comparison of tree hypotheses 91 3.4.2 Calyptrate monophyly 92 3.4.3 Superfamily monophyly and the relationships 92 between the calyptrate superfamilies 3.4.4 Interfamilial relationships within the muscoid grade 94 3.4.5 Family monophyly and relationships within families 95 3.4.6 Alignments of the ribosomal genes 98 3.5 Conclusions 108 vi Chapter 4: Molecular phylogeny of the Calyptratae (Diptera:Cyclorrhapha) with emphasis on the Oestroidea 4.1 Introduction 110 4.1.1 Oestroidea phylogenetics 111 4.1.2 Oestroidea family relationships 111 4.1.3 Oestroidea family portraits 113 4.1.4 Is the McAlpine’s fly a calyptrate? 120 4.1.5 Calyptratae phylogenetics 121 4.2 Materials and Methods 126 4.2.1 Taxa 126 4.2.2 DNA extractions 127 4.2.3 DNA amplification and sequencing 134 4.2.4 Alignments 135 4.2.5 Taxa selection strategy for analysis 136 4.2.6 Tree search strategies 137 4.3 Results 138 4.4 Discussion 147 4.4.1 Oestroidea monophyly 148 4.4.2 Placement of the McAlpine’s fly and M. zelandica in the 148 Oestroidea 4.4.3 Oestroidea family relationships 149 4.4.4 Higher-level systematics of the Calyptratae 152 4.5 Conclusions 155 vii Overall conclusions 156 References 159 viii SUMMARY The Calyptratae (Cyclorrhapha: Schizhophora) consists of about 18,000 described species representing about 12% of Diptera diversity. Traditionally, the Calyptratae were divided into three superfamilies, viz. the Hippoboscoidea, Muscoidea and the Oestroidea. In this study, 1.14 million base pairs of data from eight genes for 258 species and this molecular data are used to reconstruct the relationships within the Calyptratae. In the first chapter, the phylogenetic relationships within the superfamily Hippoboscoidea are reconstructed using maximum parsimony and Bayesian methods based on nucleotide sequences from four genes: 28S, CAD, and 16S and COI. Most of the presently recognized groups within Hippoboscoidea, including the superfamily as a whole, the Hippoboscidae and the Nycteribiidae are recovered as monophyletic. A single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates is confirmed and at least two host shifts from mammals to birds have occurred. The 60,000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life history traits, which range from saprophagy over phytophagy to parasitism and predation. In the second chapter, the Scathophagidae, a relatively small muscoid family that mirrors this diversity in natural history strategies is used for reconstructing the direction of change of larval habits in the group. The molecular data set utilizes data from seven genes (12S, 16S, Cytb, COI, 28S, Ef1-alpha, Pol II) and was subjected to an extensive sensitivity analysis and the performance of three different alignment strategies ix (manual, Clustal, POY) was compared. Phytophagy in the form of leaf mining is shown to be the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation have occurred while a second origin of predation is from a saprophagous ancestor. The monophyly of the Scathophagidae, its two constituent subfamilies, and most genera are confirmed. The reconstruction of the Muscoidea relationships in Chapter 3 demonstrates paraphyly. The Muscoidea comprises with approximately 7,000 described species a significant portion of the species-level Diptera diversity (ca. 5%). In this chapter, four mitochondrial genes (12S, 16S, COI, Cytb), and four nuclear genes (18S, 28S, Ef1a, CAD) are used to reconstruct the relationships within the Muscoidea using both maximum parsimony and likelihood techniques.