ABSTRACT WALLACE, CHARLES REID. A Molecular and Morphological Analysis of Ulidiidae (Diptera: Tephritoidea). (Under the direction of Brian M. Wiegmann). Ulidiidae (Diptera: Tephritoidea) is a relatively small family of true flies, called the “picture-winged flies” for their patterned wings. Its relationship to the other members of Tephritoidea, and its monophyly, has been well established, through both morphological and molecular analysis. Its subfamilial relationships, however, have received limited molecular analysis, having had a significant cross-section of the family’s taxa represented in only two molecular studies (Galinskaya et al. 2014, Han and Ro 2016). The most contemporary treatment of the classification of the family is by Kamenev and Korneyev (2006), according to which the Ulidiidae is divided into two subfamilies (Otitinae, Ulidiinae), each of which is further delineated into tribes (Cephaliini, Myennidini, Otitini; Lipsanini, Pterocallini, Seiopterini, Ulidiini, respectively), with an additional incertae sedis group of genera within the Otitinae. These classifications are based solely on morphological analysis, and there remain multiple outstanding questions of the placement and monophyly regarding the tribes or several large, diverse, or enigmatic genera. Here, using the next-generation sequencing method of anchored hybrid enrichment, I investigate the phylogeny of Ulidiidae with particular attention paid to the constituency and monophyly of hypothesized tribes, monophyly of the two sub-families, and placement of genera. Through maximum likelihood analysis, my results establish strong support for the separation of Ulidiidae into two monophyletic subfamilies, but challenges the assignment of Myennidini to the Ulidiidae, and potentially of Seiopterini to the Ulidiinae. It additionally suggests the paraphyly of the tribe Pterocallini with respect to both Myennidini and Lipsanini, but supports the monophyly of all other tribes. Ulidiidae is of additional interest due to the diversity of larval ecology within the family, as its members span the spectrum from absolute saprophagy, to true phytophagy as primary invaders of living plant tissue. However, the biology of many members of Ulidiidae, especially in the relatively understudied Neotropical area from which the majority of our taxa were sampled, remains unknown, undocumented, or understudied. Although our analysis supports the evolutionary isolation of true phytophagy within the family, a rigorous character mapping analysis is rendered impossible given the current availability of information on the family. This lack of information acts as one barrier to the study of Ulidiidae, another of which being the relative absence or disorganization of identification tools for the family. Many genera and species of Ulidiidae remain to be photographed or otherwise imaged, known exclusively from singular types held in museum collections, or described in untranslated papers unavailable except through physical copy. Although the full reparation of this issue is beyond the scope of a single MS thesis, progress has been made through the updating and photographic illustration of the “Otitidae” chapter in the Manual of Nearctic Diptera (Steyskal 1987). The text of Steyskal’s dichotomous key has been edited for clarity and illustrated with high-quality color photographs taken of pinned museum specimens located in the collection at North Carolina State University, the Florida State Collection of Arthropods at the University of Florida in Gainesville, and the National Museum of Natural History at the Smithsonian in Washington, DC., with additional images of Hiatus fulvipes Cresson, 1942 from the Academy of Natural Sciences in Philadelphia. © Copyright 2018 Charles Reid Wallace All Rights Reserved A Molecular and Morphological Analysis of the Picture-Winged Flies (Diptera: Tephritoidea: Ulidiidae) by Charles Reid Wallace A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Entomology Raleigh, North Carolina 2018 APPROVED BY: _______________________________ _______________________________ Brian M. Wiegmann Qiuyun Xiang Committee Chair _______________________________ _______________________________ Michael H. Reiskind Matthew A. Bertone DEDICATION To my parents, my best friend, and my cat. ii BIOGRAPHY Charles Wallace completed a Bachelor of Arts and Science at McGill University, double majoring in biology and linguistics. Between the module on ant systematics in a required biology course and Montreal’s Insectarium, he became interested in insect systematics. The fall after graduating from McGill, he enrolled at North Carolina State University in the Department of Entomology (later, The Department of Entomology and Plant Pathology), to study Diptera phylogenetics under the advisement of Dr. Brian Wiegmann, specifically to work on the phylogenetics of Ulidiidae, the “picture-winged flies.” He has one cat. iii TABLE OF CONTENTS LIST OF TABLES ………………………………………………………………………... v LIST OF FIGURES ……………………………………………………………………… vi A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF ULIDIIDAE (DIPTERA: TEPHRITOIDEA) …………………………………………………. 1 CHAPTER 1: Molecular Phylogeny of the Picture-Winged Flies (Diptera: Tephritoidea: Ulidiidae) Using Anchored Hybrid Enrichment ……………...... 1 Abstract ………………………………………………………………...….. 1 Introduction .……………………………………………………...………... 1 Materials and Methods …………………………………………….…...….. 5 Taxon Sampling ……………………………………………...……. 5 DNA extraction …………………………………………...……….. 7 Anchored Hybrid Enrichment ………………………...…………… 7 Orthology Assessment and Informatics Pipeline ……...…………... 8 Phylogenetic Analysis …………………………………...………… 9 Results Alignment …………………………………………………...…….. 9 Previously Unsequenced Taxa ……………………………………. 10 Phylogenetic Analysis ………………………………...………….. 10 Discussion ………………………………………………………...……… 12 Acknowledgments …………………………………………………...…... 17 References …………………………………………………………........... 18 CHAPTER 2: An Illustrated Identification Key to the Genera of Ulidiidae (Diptera: Tephritoidea) of the United States and Canada Abstract …………………………………………………………………... 34 Introduction ……………………………………………………...……….. 34 Materials and Methods ………………………………………………….... 36 Specimen Acquisition / Taxon Sampling ……………………….... 36 Specimen Imaging ………………………………………………... 37 Updates to the Key ……………………………………………….. 37 Dichotomous Key ………………….……………....…………………….. 38 Acknowledgments ………………………………………………………... 39 References ………………………………………………………………... 40 REFERENCES ………………………………………………………………………….. 44 APPENDICES …………………………………………………………………………... 53 Appendix A: Identification Key ……....……………....………...………... 54 iv LIST OF TABLES CHAPTER 1 Table 1. Taxa sampled …………………………………………………………… 27 Table 2. Genbank sequences ……………………………………………………... 28 CHAPTER 2 Table 1. Taxa of the United States and Canada ………………………………….. 43 v LIST OF FIGURES CHAPTER 1 Figure 1. Amino Acid Maximum Likelihood Phylogenetic Tree ………………... 29 Figure 2. Nucleotide Maximum Likelihood Phylogenetic Tree …………………. 30 Figure 3. COI-only Maximum Likelihood Phylogenetic Tree …………………... 31 Figure 4. COI+COII Maximum Likelihood Phylogenetic Tree ……....………..... 32 Figure 5. Nucleotide Bayesian Analysis Phylogenetic Tree ……………………... 33 vi CHAPTER 1: Molecular Phylogeny of the Picture-Winged Flies (Diptera: Tephritoidea: Ulidiidae) Using Anchored Hybrid Enrichment ABSTRACT The monophyly of the family Ulidiidae and its position relative to other members superfamily Tephritoidea (Insecta: Diptera) have both been established with strong molecular and morphological support. Relationships within the family, however, have received relatively little attention, especially in molecular phylogenetic analyses. Using both anchored hybrid enrichment (AHE) datasets and alignments of the mitochondrial genes cytochrome oxidase subunit 1 (COI) and II (COII), I performed analyses using a maximum likelihood model to investigate the phylogeny of the “picture-winged flies” (Ulidiidae). The inferred phylogenetic trees offer strong support for the monophyly of the family, its division into two subfamilies, and the monophyly of several of its tribes, while deviating from contemporary taxonomic classification in several significant ways (in e.g. the placement of Myennidini with the tribes of Ulidiinae, rather than Otitinae). Further research with an expanded set of taxa is needed to better resolve the constituents and placement of tribes and the monophyly or lack thereof of large, potentially paraphyletic, or enigmatic genera. INTRODUCTION Ulidiidae Macquart, 1835, the “picture winged flies,” is a family of acalyptrate true flies belonging to the superfamily Tephritoidea. It is a relatively small family, consisting of approximately 800 species distributed across two subfamilies (Diaz-Fleischer et al. 2000, Kameneva and Korneyev 2006). Its locus of diversity is in the “New World,” with the greatest 1 representation in the Neotropical bioregion (Kameneva et al., 2017). With its close relatives within Tephritoidea it shares their characteristic telescoping ovipositor (Marshall 2012); propensity towards showy courtship displays, often using the distinctively patterned wings that grant the family its common name (Sivinski 2000); and status as pestiferous on several crops significant to agribusiness, e.g. on corn in the American South (Goyal et al., 2010, Goyal et al., 2011, Goyal et al., 2012). Historically, consideration of the family as monophyletic has been questioned on the basis of the relative