ABSTRACT Hill, Hilary Nan. Phylogenetic Utility of Two New Nuclear Genes, Opsin and CAD, within the stiletto flies (Diptera: Therevidae) (Under the direction of Brian M. Wiegmann). A need for multiple phylogenetic markers to reconstruct evolutionary relationships is increasingly apparent within both the Therevidae and insect systematics as a whole, especially markers that can accommodate the immense species diversity that arose during the Mesozoic (~65-250mya). Phylogenetic trees are often best reconstructed using datasets from distinct sources and from character sets that have been under different selective pressures. Many recent insect systematics studies use various combinations of markers from multiple genomes, morphology, and biogeography. Nuclear genes, particularly protein-encoding genes that are highly conserved and low copy-number, are increasingly attractive as phylogenetic markers, and there is a recent concerted effort to find and increase access to new nuclear genes. Chapter one of this study investigates the phylogenetic utility of opsin, a new multiple copy nuclear protein- encoding gene in the dipteran family Therevidae. The present analyses of nucleotide sequence data of opsin reconstructed a therevid phylogeny that is partially concordant with previous classifications which demonstrates that opsin may have some limited utility as a phylogenetic marker within the Therevidae and closely related Diptera, but also shows that opsin may be better used in combination with other molecular markers or morphological data sets. Phylogenetic analyses of opsin at multiple levels within insects supports these results, but also reveals the significant obstacles in technical manipulation of the gene and in the interpretaion of ortholog/paralog relationships. In the second chapter two new nuclear, protein-encoding genes, opsin and CAD, in combination with EF-1α and 28S rDNA, are applied to reconstruct evolutionary relationships among the major lineages of Therevidae for which previous molecular evidence has been insufficient. The analyses of 28S rDNA, EF-1α, opsin, and CAD nucleotide sequences provide phylogenetic resolution that is highly concordant with the current therevid classification. The analyses also reveal that the phycine tribe Xestomyzini is monophyletic and could represent a major therevid clade. The new molecular dataset generated from this study can be used to build a larger dataset and can also be used in conjunction with a morphological dataset that is currently being generated within the therevid PEET project. PHYLOGENETIC UTILITY OF TWO NEW NUCLEAR GENES, OPSIN AND CAD, WITHIN THE STILETTO FLIES (DIPTERA: THEREVIDAE) by HILARY NAN HILL A thesis submitted to the Graduate Faculty of North Carolina State University In partial fulfillment of the Requirements for the Degree of Master’s of Science DEPARTMENT OF ENTOMOLOGY Raleigh 2003 APPROVED BY: _______________________________ _____________________________ Brian M. Wiegmann Lewis L. Deitz Chair of Advisory Committee ______________________________ Edward L. Vargo DEDICATION This dissertation is dedicated to my parents, Patrick and Carol Hill, who have supported and encouraged all of my choices and endeavors with love and understanding and, to my husband and best friend, Rob Ellison, without whom my life would be incomplete. ii BIOGRAPHY Hilary Hill was born February 29, 1972 in Baltimore, Maryland to Patrick E. Hill and Carol S. Hill. She grew up in Bel Air, MD and upon graduation from Bel Air High School, in 1990, Hilary spent two years at Harford Community College where she received an Associates Degree in Histotechnology. She completed the registry exam to become a board-certified histotechnologist, and started working for Corning Medical laboratories. After working for two years at the Maryland branch of Corning, Hilary transferred to the Phoenix, Arizona branch and took a few general courses at Arizona State University. Two years later Hilary decided to move back to Maryland and enrolled at Towson University (TU) as a full-time biology major while continuing to work as a histotechnologist part- time at the Greater Baltimore Medical Center. While pursuing her degree at TU, she took a course in entomology with Dr. Aubrey Scarbrough. This course spurred Hilary’s interest in entomology, and the following semester she enrolled in independent research with Dr. Scarbrough, learning the basic concepts of alpha taxonomy, through studies on robber flies (Diptera: Asilidae). That summer Hilary was selected to participate in the National Science Foundation’s Summer Undergraduate Research in Biology Program where she described new species and re-described previously known species of robber flies from Sri Lanka. After completing her B.Sc., Hilary began her Master’s program at North Carolina State University in the fall of 2000 under the supervision of Dr. Brian M. Wiegmann. Her research focused on the investigation of the phylogenetic utility of two new nuclear genes in stiletto flies (Diptera: Therevidae). Hilary met Rob Ellison in the summer of 1995, before she moved to Arizona. They married in June of 2001, during the second year of Hilary’s Master’s program. iii ACKNOWLEDGMENTS I sincerely thank Brian M. Wiegmann for his guidance and support throughout my research program. I thank Lewis L. Deitz and Edward L. Vargo for serving on my advisory committee and am grateful to Brian Cassel, Kevin Moulton, and Shaun Winterton for their aid, advice, and expertise in molecular techniques as well as dipteran systematics. Thanks to LongLong Yang for sharing his molecular data and to Dmitry Mukha for sharing his cloning expertise. I thank the entire therevid PEET team, especially: Martin Hauser, Kevin Holston, Mike Irwin, Gail Kampmeir, Donald Webb, and David Yeates. Thank you to Dr. Aubrey Scarbrough for his support and for encouraging me to pursue an advanced degree in entomology. This project was supported by funds from the National Science Foundation (DEB-9977958), North Carolina State University College of Agriculture and Life Sciences, and the North Carolina Agricultural Research Services. iv TABLE OF CONTENTS Page LIST OF TABLES............................................................................................................................. vii LIST OF FIGURES.......................................................................................................................... viii PHYLOGENETIC UTILITY OF TWO NEW NUCLEAR GENES, OPSIN AND CAD, WITHIN THE STILETTO FLIES (DIPTERA: THEREVIDAE)...................................1 CHAPTER 1: Molecular Evolution of Opsin in Therevidae and Their Close Relatives ............1 Abstract......................................................................................................................................2 Introduction ...............................................................................................................................3 Opsin: a New Gene for Insect Phylogenetics.............................................................4 Stiletto Flies (Diptera: Therevidae): Taxonomic Background & Natural History....7 Materials and Methods............................................................................................................10 Taxon Sampling.........................................................................................................10 Laboratory Methods ..................................................................................................10 Nucleotide Alignment and Phylogenetic Analysis...................................................12 Results .....................................................................................................................................13 Phylogenetic Analyses ..............................................................................................13 Published Insect Opsin Sequences..............................................................13 Published Insect Opsin Sequences + Therevidae and Close Relatives......14 Dipteran Opsin.............................................................................................15 Therevidae + Their Close Relatives (“lw” Copies Included).....................16 Therevidae + Their Close Relatives (“lw” Copies Excluded)....................17 Discussion ...............................................................................................................................18 Conclusions .............................................................................................................................22 Acknowledgments...................................................................................................................22 References ...............................................................................................................................23 CHAPTER 2: Therevid Phylogeny Based on Multiple Nuclear Genes.......................................48 Abstract....................................................................................................................................49 Introduction .............................................................................................................................50 Phylogenetic Markers................................................................................................51 Current Therevidae Classification ............................................................................53 Materials and Methods............................................................................................................54