Abstract Wallace, Charles Reid. A

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.

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.

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

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

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

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

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 absence of synapomorphies that strongly unite its two subfamilies to the exclusion of the closely related and morphologically similar Tephritidae (Galinskaya et al., 2014,

Han and Ro 2016). However, a succession of contemporary analyses, both morphological

(Kameneva and Korneyev 2006) and molecular (Wiegmann et al., 2011, Galinskaya et al., 2014,

Han and Ro 2016), has supported its status as a singular monophyletic clade. Contemporary taxonomic consensus further divides the family into two subfamilies, Otitinae and Ulidiinae, consisting each of four subgroups: in Otitinae, three tribes (Cephaliini, Myennidini, Otitini) and a collection of incertae sedis genera speculated to be closely related to the Cephaliini; in Ulidiinae, four tribes (Lipsanini, Pterocallini, Seiopterini, Ulidiini) (Kameneva and Korneyev 2006).

Multiple analyses (Han et al. 2002, Han and Ro 2005, Han and Ro 2016) have supported the monophyly of Tephritoidea, which consists, in addition to Ulidiidae, of the families

Tephritidae, Lonchaeidae, Pallopteridae, Circumphallidae, Piophilidae, Eurygnathomyiidae,

Richardiidae, Platystomatidae, Ctenostylidae, Pyrgotidae, and which is split into two monophyletic groups (Han and Ro 2016). Ulidiidae is well established as a member of the

“higher Tephritoidea” (Kameneva and Korneyev 2010, Marshall 2012) or “Tephritidae group”

(Han and Ro 2016), accompanied by Pyrgotidae, Tephritidae, Richardiidae, Platystomatidae, and

Ctenostylidae, and in which it occupies a relatively basal position, at the second most proximal position to the point of division from the “Piophilidae group” (Han and Ro 2016).

Although both the monophyly of Ulidiidae and its placement within Tephritoidea are now well established, investigation of the family’s internal phylogeny has been lacking; proportionally little molecular phylogenetic work has been conducted on this small family.

Although Ulidiidae has been incorporated into numerous phylogenetic analyses of Tephritoidea and higher-level Diptera, few have included a broad sample of the family, with most limited to one or two taxa (Galinskaya et al. 2014, Han et al. 2002, Han and Ro 2005, Wiegmann et al.

2011). To date, only two relatively significant investigations of the molecular phylogeny of the family have been published: the inclusion of fifteen species distributed across both subfamilies and all but one tribe (Ulidiinae: Pterocallini) by Han and Ro (2016), and another by Galinskaya et al. (2014), which limited its genetic sampling to the barcording region (658 bp) of the cytochrome oxidase subunit 1 (COI) gene, and to taxa of the Palaearctic region. No study so far has included a broad cross-section of the family with taxa drawn primarily from the Neotropical bioregion, in which its diversity is concentrated; nor has any previous study investigated the family with the large datasets of phylogenomic study.

Phylogenomics, enabled by Next Generation sequencing technology and new bioinformatics analysis pipelines, is revolutionizing systematic practice and rapidly expanding its evidence base (Kjer et al. 2016, Trautwein et al. 2012). Many groups of organisms historically considered difficult or almost intractable through traditional phylogenetic analysis are now being examined through extensive genetic sampling (Allen et al., 2017, Branstetter et al., 2016,

Breinholt et al., 2017, Haddad et al., 2017). The robust support and resolution of phylogenomic trees is providing increased opportunities to resolve key questions regarding monophyly and

relationships (Misof et al., 2014, Dietrich et al., 2017), molecular evolution and phylogenetic information content (Léveillé-Bourret et al., 2016; Bossert et al., 2017); morphological homology and character transformations (Winterton et al., 2017), and tests of ecological, behavioral, and biogeographic hypotheses (Blaimer et al., 2016a). While genome and transcriptome sequencing strategies provide enormous numbers of gene loci (in the thousands), these methods remain expensive and time consuming, require fresh and abundant DNA or RNA, and are difficult to sample effectively for a standard sized species- or genus-level study.

Sequence capture methods that target specific genes to obtain hundreds of potentially informative orthologous markers have proven to be an effective strategy to obtain large numbers of specific loci without “deep” sequencing (Faircloth et al., 2012, Lemmon and Lemmon 2013).

Anchored hybrid enrichment (AHE) (Lemmon et al, 2012; Prum et al., 2015), is a gene capture method that relies on highly conserved gene regions used as “baits” or anchors to capture specific loci and then sequence into adjacent exonic and non-coding genomic regions. This method has proven effective in providing robust phylogenetic data at multiple levels and across diverse taxa, including Diptera (Young et al., 2016, Pauli et al., in press). These data provide a source of genomic information without requiring comprehensive sequencing and genomic scaffolding. Standard DNA extraction methods work well with AHE, allowing specimen sampling from field caught material, pinned dried specimens from museum collections, malaise trap sampled stored in ethanol, and archived DNA samples in laboratory freezers (Blaimer et al.,

2016b).

Ulidiidae is understudied as pertains to its natural history, which for the majority of its species remains unknown. The family, in contrast to its close relative Tephritidae, the “true fruit flies,” is mostly saprophagous. The natural history of Ulidiidae is best known for its larvae,

which have been documented in dung, rotting plant material, and living plant tissue, in which they have been documented as primary, secondary, and tertiary invaders (Arnett 2000, Bjerke et al. 1992, Brunel and Rull 2010, Ferrar 1987, Goyal et al., 2010, Goyal et al., 2011, Goyal et al.,

2012, Kameneva and Korneyev 2010, Marshall 2012, Steyskal 1987). This occasional shift from the presumably ancestral state of saprophagy to phytophagy is commonly noted as one of the family’s most noteworthy features (Ferrar 1987, Korneyev 2000, Marshall 2012), and these instances of true phytophagy are taxonomically isolated and distributed apparently without bias across the family (Table 1). Assuming the legitimacy of contemporary classification as a proxy for evolutionary relationships, this indicates that phytophagy is a trend within Ulidiidae that has arisen independently multiple times; further resolution of the family’s internal relationships would provide a scaffolding on which to organize further investigation of the family’s diverse ecologies, and to better contextualize this repeated pattern.

In this study, I aimed to expand the so-far limited molecular phylogenetic analysis of the sub-familial relationships of the Ulidiidae. Based on large-scale phylogenomic data acquired through AHE I have constructed trees based on Maximum Likelihood using nucleotide and amino acid data. Additional trees using COI and COII sequences were produced, to expand taxon sampling to taxa otherwise unavailable, thereby enabling closer comparison against previous studies, particularly Galinskaya et al. (2014), which heavily sampled the Ulidiinae tribe Ulidiini.

METHODS AND MATERIALS

Taxon Sampling

Taxon sampling was performed to maximize taxonomic diversity given available resources, with the dual goals of absolute diversity (number of individual genera and species)

and maximal coverage of subfamilial, suprageneric categorizations according to the most current classification, as articulated by Kameneva and Korneyev (2006). Most specimens were acquired from malaise trap samples collected the Neotropical bioregion, primarily Brazil, Peru, Bolivia, and Colombia. Additional specimens were provided from the Nearctic and Palearctic regions.

In total, 49 species from 31 genera were included, representing each subfamily and six of the seven tribes (no specimens from Ulidiini were acquired), as well as one representative (Idana marginata Say, 1830) of the incertae sedis group of genera of the subfamily Otitinae. A full list of ulidiid taxa obtained as specimens for DNA extraction is available in Table 1.

Given the establishment of Tephritidae as a close relative to Ulidiidae, several genera of

Tephritidae were chosen to act as outgroups: Anastrepha (Anastrepha striata Schiner, 1968;

Anastrepha leptozona Hendel, 1914), Aenigmatomyia (Aenigmatomyia nsp. shannon,

Aenigmatomyia unipuncta Malloch 1933), Tomoplagia (Tomoplagia sp. 1, Tomoplagia sp. 2), and Omomyia sp.

Additional sequences were obtained from Genbank through the nucleotide database of the National Center for Biotechnology Information (NCBI), for taxa identified to genus whose

COII or COII gene sequences are available (Table 2).

From Genbank, sequences of the mitochondrial CO1 gene were successfully obtained for

45 species across 20 genera distributed across both subfamilies and including at least one generic representative of each tribe (except Ulidiinae: Pterocallini), as established by Kameneva and

Korneyev (2006). Additionally, sequences of the mitochondrial COII gene were successfully obtained for 14 of those 45 species. This acquisition expanded taxonomic coverage by seven genera unrepresented in our AHE dataset, including all genera of the Ulidiinae tribe Ulidiini

(Physiphora, Timia, Ulidia).

DNA Extraction

All specimens except those of Idana marginata and Tetropismenus hirtus Loew, 1876 were from malaise trap samples and kept in 100% ethanol and frozen prior to DNA extraction.

Pinned specimens for Idana marginata and Tetropismenus hirtus were obtained, and kept in

100% ethanol overnight to prepare for extraction.

DNA was extracted from specimens using the DNEasy Tissue Extraction Kit (Qiagen,

Hilden, Germany) and sequenced on Illumina HiSeq at the North Carolina State University

Genetic Sciences Library.

Anchored Hybrid Enrichment

For AHE an existing Diptera-specific probe set was used that targets 559 gene loci from across the fly genome (Young et al., 2016). The probes were constructed through collaborative research by the Wiegmann lab in 2014-15 and are based on comparative genomic and transcriptomic analysis of 14 fly species sampled across the order. Probes are tiled to provide high coverage representation of a conserved target sequence across the 559 regions. Multiple ulidiid genomic samples (specimens or species) were obtained in a single sequencing run.

To conduct AHE, the laboratory protocols used in Lemon and Lemon (2012) and Young et al (2016) were used. For each DNA extract, I carried out genome sonication, probe x genomic

DNA hybridization, capture-loci library construction, and indexed multiplex Illumina single-read sequencing. These experiments were carried out in the Wiegmann laboratory at NCSU and

Illumina sequencing was provided by the Genomics services facility of the NCSU Genome

Sequencing Laboratory (GSL).

DNA was sonicated to a fragment size of 200–800 bp using the Covaris E220 Focused- ultrasonicator with Covaris micro-TUBES. Libraries were constructed following a protocol originally derived from Meyer & Kircher (2010), but with library fragments being size-selected using SPRI select beads (Beckman-Coulter Inc., Brea, CA, U.S.A.) at a 0.9°— ratio of bead to sample volume. Following addition of 8 bp indices, libraries were pooled in groups of 16 and enriched using an Agilent Custom SureSelect XT kit (ELID 3005721, Wilmington, Delaware).

Following enrichment, library pools were combined into single sequencing pool and sequenced on 1 PE150 Illumina 2500 lane.

Orthology Assessment and Informatics Pipeline

AHE hybridization provides high-coverage sequence harvests of target regions using

NextGen sequencing techonology. This results in large datasets comprised of hundreds to thousands of individual loci, as well as non-target mitochondrial genes for each species. To manage and analyze these data I used bioinformatic methods for comparative genomic analysis contained in the 1Kite Bioinformatics Pipeline (Misof et al., 2014) installed on the NCSU

Bionformatics Research Center (BRC) Computational Cluster. The data are stored and managed in an NCSU-based data repository and will be deposited in NCBI and DRYAD public databases at the time of publication.

The analysis of AHE data involves editing and filtering individual specimen-based indexed sequence, assembly of loci (Trinity software; Grabherr et al., 2011, Zhao et al., 2011), orthology search and BLAST analysis (Orthograph software; M. Petersen) to confirm gene and locus identity, removal of short, ambiguous, or non-dipteran sequences, multiple sequence alignment (MAFFT software; Katoh et al. 2005) alignment quality assessment (Alicut and

Aliscore software; Kück 2009), phylogenetic data concatenation and assessment (FasConCat software; Kück et al. 2010).

Phylogenetic analysis

Phylogenetic trees are constructed using the model-based maximum likelihood methods in parallelized-RAxML software (Stamatakis et al. 2012) installed on the NCSU BRC cluster or performed remotely on the CIPRES Science Gateway phylogenetic analysis web portal.

Analyses are carried out on aligned nucleotide data and translated amino acids for concatenated datasets of multiple aligned genes. Bayesian phylogenetic analyses were also performed for nucleotide data sets using MrBayes 3.2.6 (Ronquist et al. 2012). To evaluate issues of gene-tree discordance or individual gene signal using coalescent models (Degnan and Rosenberg 2009), I use the individual gene consensus methods provided in the Astral software package (Mirarab et al. 2014).

RESULTS

Alignments

Using RaxML for maximum likelihood (ML) analysis, I produced two trees: one of an amino acid dataset, and one of a nucleotide data set. The amino acid (AA) dataset consisted of 57 sampled taxa, and included information from 445 loci and 115,102 amino acids. The tree figures represent results from 500 bootstrap iterations. The nucleotide (NT) dataset consisted of 59 sampled taxa, and included information from 812 loci and 480,975 nucleotides total. The tree figures represent results from 500 bootstrap iterations.

Previously Un-Sequenced Taxa

Our dataset expanded the taxonomic breadth of extracted taxa in several significant ways, most notably the inclusion of members of the Ulidiinae tribe Pterocallini. No previous molecular phylogenetic treatment of the family Ulidiidae has included any members of Pterocallini, an exclusively Neotropical tribe, whereas our dataset included 11 of its 25 genera (Kameneva and

Korneyev 2010). Of those genera, two were proportionally very well represented: Pterocerina, represented by six species; and Xanthacrona, with inclusion of four of its five described species

(Kameneva and Korneyev 2010, Steyskal 1966). Additionally, we sampled included Idana marginata, a representative of one of the incertae sedis Otitinae genera, none of which have previously been sequenced or included in molecular phylogenetic analysis

Phylogenetic Analysis

Five phylogenetic analyses were run: two ML trees using the AHE dataset, limited to amino acid (Figure 1) and nucleotides (Figure 2); two ML trees using only mitochondrial gene sequences, COI (Figure 3) and an alignment of COI and COII (Figure 4); and one Bayesian analysis using nucleotide data from the AHE datasets (Figure 6).

Of the five, the Bayesian analysis (Figure 6) never reached convergence, ending with a standard deviation of split frequencies of 0.14. It is included for comparison against the other trees produced, most notably as pertains to the taxa Pterocerina trifasciata Hendel, 1909, Idana marginata, and Seioptera sp. Of these, both Pterocerina trifasciata and Idana marginata exhibited disproportionately long branch lengths in both ML AHE trees (Figures 1, 2) relative to other taxa. In the Bayesian tree, these two and Seioptera sp. form a clade with poor support (0.5 posterior probability), sister to (Cephaliini + Otitini).

There was absolute consensus across all trees to support the monophyly of both Ulidiidae and Tephritidae with near-absolute support.

Both trees constructed using only mitochondrial gene sequences offered overall poor support of higher-level intrafamilial relationships (Figures 3, 4). Both the COI and COI+COII trees returned 100% bootstrap support for the monophyly of the family, but no higher than 30% for any suprageneric relationships outside of the Cephaliini, the only subfamilial suprageneric clade returned with near uniformly high support across all analyses.

There is near complete agreement between the AA and NT AHE trees, regarding placement and constituency of clades. However, bootstrap support is overall ‘higher’ for suprageneric clades in the former than the latter. The major divergence between the two is in the placement of the potentially problematic Pterocerina trifasciata and Idana marginata.

The mitochondrial gene trees, in agreement with previous molecular studies, indicated

Ulidiinae as a paraphyletic assemblage of genera basal in comparison to the apparently monophyletic Otitinae; bootstrap support for this result, however, was nearly absent in both the

COI and COI+COII trees. The Maximum Likelihood analyses indicated a basal division of

Ulidiidae into two monophyletic sister groups which broadly circumscribe the two subfamilies as hypothesized based on morphological analysis (Kameneva and Korneyev 2006).

Six out of the seven defined tribes within Ulidiidae were represented in our analysis. Of those, five were represented by more than one taxon (Ulidiinae: Seiopterini represented by only one of its three constituent genera, Seioptera), and of those, Otitini was moderately well supported and the other three were strongly supported as monophyletic in each of the two ML trees constructed using the AHE dataset. With the exception of Pterocerina trifasciata (whose placement is variable between trees) and the apparently rogue Rhyparella decempunctata

Hendel, 1909 (placed with the Otitini in both; the only genus/species to appear in the “wrong” group), only Pterocallini (Ulidiinae) appears as a clade paraphyletic with respect to the monophyletic Myennidini and Lipsanini; Cephaliini and Otitini, together with Seioptera sp., constitute the other of the two sister clades.

In both ML AHE trees, Xanthacrona and Pseudopterocalla scutellata Schiner, 1868 form a monophyletic clade sister to a clade formed by Lipsanini, Myennidini, and the remaining

Pterocallini, although in the AA tree Xanthacrona is recovered as monophyletic, whereas in the

NT tree it is paraphyletic with respect to Pseudopterocalla scutellata (where it is shown as sister to Xanthacrona tripustulata Enderlein, 1921, although the relationships of that group are otherwise identical between the two trees).

Most other genera appear monophyletic (Tritoxa, Otites, Megalaemyia) or nearly monophyletic (all Tetanops species returned as closely related, but paraphyletic with respect to

Herina gyrans), with strong support. Paragorgopis (two species) and Pterocerina (seven species), together with Apterocerina argentea Hendel, 1914 also form a monophyletic clade together. Euxesta, represented by three species, are polyphyletic; two species are placed as sisters with 100% support, separated by another species nested within a clade with other members of

Lipsanini: Eumetopiella, Chaetopsis, Euphara, and Axiologina.

DISCUSSION

The results of this study differ significantly in several ways from both the results of previously phylogenetic analyses of the family, and of the accepted taxonomic convention, as articulated by Kameneva and Korneyev (2006) following the definition of a new tribe,

Myennidini.

The greatest point of commonality between all five analyses is the strong support of the family Ulidiidae as a monophyletic group. Further, no members of the Nearctic Cephaliini were included by Galinskaya et al. (2014), but were represented by Delphinia picta Fabricius, 1781 and Tritoxa flexa Wiedemann, 1830 in Han and Ro (2016). The latter study found these to be sister taxa and our analysis confirms this placement with strong bootstrap support.

In both Galinskaya et al. (2014) and Han and Ro (2016), the Ulidiinae are a paraphyletic assemblage relative to the Otitinae, with the latter emerging from within a grade of distinct, but difficult to resolve, tribal lineages. However, the representation of Ulidiinae in both is substantially more limited than in our analysis, especially in Galinskaya et al. (2014), in which the Ulidiinae are represented by the three genera assigned to the tribe Ulidiini, the only tribe of which our dataset has no representatives. Mitochondrial gene trees using COI and COII affirmed that finding, but with no significant bootstrap support for any phylogenetic resolution. Using the substantially large AHE dataset, our analysis shows a basal division of Ulidiidae into two reciprocally monophyletic sister groups.

The largest disruption to the contemporary classification of the family and its subfamilial groups is the placement of Myennidini (Otitinae) alongside Pterocallini and Lipsanini

(Ulidiinae). Myennidini as a tribe was first defined by Kameneva and Korneyev in 2006, in a paper in which genera previously assigned to either the Ulidiini or Pterocallini were redefined as a grouping together, and moved from Ulidiinae to Otitinae. Our new analysis supports the monophyly of Myennidini as a tribe, but not the movement from one subfamily to the other, suggesting that it is more closely related to other members of Ulidiinae.

A further point of complication is the placement of Seiopterini. Seiopterini is considered a tribe of Ulidiinae, comprised of three genera of Holarctic distribution: Seioptera,

Homalocephala, and Pseudoseioptera. In both this and Han and Ro (2016), Seiopterini is represented by a single member of a single genus (in ours: Seioptera, placed sister to Cephaliini and Otitini together, both belonging to Otitinae; in Han and Ro, Pseudoseioptera is placed sister to the Myennidini). The most extensive inclusion of Seiopterini was in Galinskaya et al. (2014), in which it was represented by both Seioptera vibrans and several species of Homalocephala, and in which the two were widely separated, though the placement of either genus was poorly supported. The monophyly of this tribe is well supported by morphological data (Kameneva and

Korneyev 1994, Korneyev 1999, Kameneva and Korneyev 2006). Further molecular systematic research with more extensive sampling is necessary to better establish the relationships between its three constituent genera, and their position relative to the rest of the family.

There remain many issues facing the resolution of intra-familial relationships of the

Ulidiidae which are beyond the scope of this study’s sampling, particularly as pertain to groups of noted phylogenetic ambiguity such as the incertae sedis genera within Otitinae and the larger and less specifically defined genera, particularly Euxesta. Kameneva and Korneyev (2006) note that Euxesta is defined less by synapomorphy than by absence of the definitive traits of other genera, and that it is heterogeneous and likely not monophyletic; insofar as the genus is represented in this analysis, this is tentatively supported by the separation of Euxesta sp. 1 from the clade formed by Euxesta nitidiventris Loew, 1873 and Euxesta sp. 2; the reliability of this result, however, is limited by the proportional underrepresentation of the genus, which is one of the family’s most speciose and diverse.

Similarly, the resolution of the placement of members of incertae sedis within Otitinae, and more specifically whether they are closer to Otitini or to Cephaliini, is largely beyond the scope of this analysis. The one incertae sedis member of our analysis, Idana marginata, was

placed differently between AHE trees, and even when placed within the majority-Otitinae clade, was closer to members of Otitini than to Cephaliini (Figure 2). In the Bayesian analysis, Idana marginata formed a moderately supported clade with Pterocerina trifasciata, which suggests grouping by long-branch attraction and the potential of a confounding effect in other analyses; the placement of either taxon may be regarded with skepticism, and further analyses (especially a new run of the Bayesian analysis) may benefit from their removal.

The relatively high and low support for AHE and mitochondrial gene analyses, respectively, in this paper serves as a demonstration of the relative predictive power of phylogenomic analysis and larger datasets. The support values, both bootstrap and posterior probabilities, were higher for AHE vs. mitochondrial gene trees, especially in the tree constructed exclusively using sequences of the barcoding region of the COI gene, which was already shown by Galinskaya et al. (2014) to offer poor resolution of relationships above the level of genus.

The Ulidiidae, being a majority saprophagous family with an established trend towards phytophagy and numerous intermediaries along a spectrum between a purely saprophagous or specialized phytophagous trophic habit, is ripe for character mapping analysis to investigate the evolution of larval ecology within the family. Broader hypotheses regarding the placement of

Ulidiidae within, and the evolution of, Tephritoidea indicate it should be an ancestrally saprophytic family (Brunel and Rull 2010), supported by the majority of what is so far known of ulidiid natural history (Diaz-Fleischer et al. 2000, Ferrar 1987, Kameneva and Korneyev 2006,

Kameneva and Korneyev 2010, Marshall 2012, Sivinski 2000). However, information on the biology of most species and general remains unavailable or poorly understood, and is biased towards taxa with a Nearctic distribution (primarily members of Otitinae, see Table 1) and

towards pestiferous species of economic importance (Brunel and Rull 2010). Furthermore, those taxa represented in our analyses include multiple congenerics of species whose biology has been documented, but not the species themselves. For example, several species of Euxesta and

Chaetopsis are documented as primary invaders of corn (Zea mays) plants in the American southeast into Central and South America. Our analysis lacks those species (e.g. Euxesta stigmatias Loew, 1868; Chaetopsis massyla Walker, 1849; Chaetopsis major Wulp, 1899), but includes at least one member of each genus, and offers tentative support for the suggestion that the large and heterogeneous Euxesta may not be monophyletic (Kameneva and Korneyev 2006) and therefore calls into question whether taxa assumed closely related to an individual species can act in character mapping analysis as a proxy for that species. Both increased documentation of the biology of ulidiids and more expansive taxonomic sampling are required to offer a more robust and nuanced reconstruction of the evolution of larval ecology within Ulidiidae.

Our results strongly support the monophyly of Ulidiidae, and to a somewhat lesser extent its further separation into two subfamilies; where it departs from consensus is in the relegation of tribes between subfamilies, particularly in the nesting of the Otitinae tribe Myennidini as a monophyletic clade within a clade formed by a Pterocallini, which itself is rendered paraphyletic by the aforementioned Myennidini and by Lipsanini. To offer a more reliable and extensive resolution of intra-familial relationships, further studies should expand their taxonomic sampling, with a strong representation of both Neotropical and Holarctic species, such that all tribes may be adequately and proportionally represented.

ACKNOWLEDGMENTS

This study was made possible by Dr. Allen Norrbom at the USDA/USNM through his donation of the specimens from which most of our extracted genomic material was derived.

Additional thanks to Dr. Keith Bayless and the Wiegmann lab for additional taxa, Dr. Matthew

Bertone and Dr. Valery Korneyev for assistance with the identification of taxa, and Brian Cassel for training and assistance with laboratory work to carry out the AHE experiments and with the bioinformatics analysis of NextGen sequence data. This study was supported in part through

NCSU Insect Museum Curatorial Research and Teaching Assistantships from the Department of

Entomology and Plant Pathology and National Science Foundation Dimensions of Biodiversity

Project DEB – to Brian M. Wiegmann.

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Table 1: All taxa of Ulidiidae extracted in lab, including information on larval ecology where known. For taxa identified only to genus, generic (vs specific) authorship included. Sources referenced: (1) Allen and Foote 1967, (2) Bjerke et al. 1992, (3) Brunel and Rull 2010, (4) Goyal et al. 2010, (5) Goyal et al. 2017, (6) Kameneva and Korneyev 2010, (7) Mahrt and Blickenstaff 1979, (8) Manis 1941, (9) Marshall 2012, (10) McAlpine 1951, (11) Melhus and Harris 1949, (12) Steyskal 1963, (13) Steyskal 1973, (14) Teskey 1976, (15) Valley, Novak and Foote 1969

Table 2: Sequences acquired through NCBI Genbank, with COI and COII accession numbers, organized by family, subfamily, and tribe.

TEPHRITIDAE

Figure 1: Phylogenetic tree using Maximum Likelihood model, of amino acids from AHE dataset. Bootstrap values at nodes. Ulidiid groups delineated by color; from top: Pterocallini, Otitini, Lipsanini, Myennidini, Seiopterini, Otitini, Cephaliini.

TEPHRITIDAE

Figure 2: Phylogenetic tree using Maximum Likelihood model, of nucleotides from AHE dataset. Bootstrap values at nodes. Ulidiid groups delineated by color; from top: Pterocallini, Lipsanini, Myennidini, Seiopterini, Otitini, Cephaliini.

Figure 3: Phylogenetic tree using Maximum Likelihood Model, using only COI barcoding sequence. Bootstrap values at nodes.

Figure 4: Phylogenetic tree using Maximum Likelihood model, using alignment of COI barcoding sequence and COII. Bootstrap values at nodes.

TEPHRITIDAE

Figure 5: Phylogenetic tree using Bayesian analysis, of nucleotides of AHE dataset. Posterior probability values at nodes. Ulidiid groups delineated by color; from top: Lipsanini, Pterocallini, Myennidini, Seiopterini, Otitini, and Cephaliini.

CHAPTER 2: An Illustrated Identification Key to the Genera of Ulidiidae (Diptera:

Tephritoidea) of the United States and Canada

ABSTRACT

Ulidiidae, the “picture-winged flies,” are a family of true flies belonging to the superfamily Tephritoidea. One hundred and twenty-seven of the family’s approximately 800 species, and 40 of its 128 genera, have been documented north of Mexico in the United States and Canada. Although the family is primarily saprophagous and commonly found in association with feces or decaying plant tissue, several species are known to be pestiferous on crops significant to agribusiness, primarily in the American Southeast. Here the taxonomic key provided in the “Otitidae” chapter of the Manual of Nearctic Diptera (Steyskal 1987) is updated with color photographs, including notes on taxonomy and distribution of species.

INTRODUCTION

The “picture-winged flies” (Ulidiidae) are one of the larger families of the Diptera superfamily

Tephritoidea, numbering approximately 800 species total, 127 of which are found in the United

States or Canada (Diaz-Fleischer et al. 2000, Steyskal 1987). They are a family of acalyptrate flies, generally small to medium, and broadly recognizable by the distinctively patterned wings that grant the family its common name, though this trait is not exclusive to them.

Currently, Ulidiidae is divided into two subfamilies: Ulidiinae Macquart, 1835, and

Otitinae Aldrich, 1932, each with three tribes (and an additional incertae sedis group of genera in the latter) (Kameneva and Korneyev 2006). These subfamilies are unequally distributed geographically; Otitinae is primarily Holarctic, and represents the slight majority of species

documented north of Mexico, whereas Ulidiinae is primarily concentrated in the Neotropical region of Central and South America (Galinskaya et al. 2014, Arnett 2000, Kameneva and

Korneyev 2006).

Unlike its close relative, Tephritidae, the ulidiids are primarily a saprophagous group

(Arnett 2000, Ferrar 1987). The greater number of picture winged flies can be found in association with feces or, more typically, rotting vegetables and fruits, where they commonly oviposit. Some publications (e.g. Arnett 2000) present the family as exclusively associated with plants, but, although there is a clear preference throughout the family for plant tissue, dead or alive, it is not exclusive (Ferrar 1987, Marshall 2012, Sivinski 2000). There is, however, a repeated trend within the Ulidiidae of true and/or opportunistic phytophagy, in which larvae act as primary, secondary, or tertiary invaders on living plant tissue (Goyal et al. 2012, Kameneva and Korneyev 2010). This is particularly well documented in those species that act as pests on plants such as sugar beet, onions, or corn (Bjerke et al. 1992, Chittenden 1927, Ferrar 1987,

Goyal et al. 2010, Goyal et al. 2011, Goyal et al. 2012).

Like the Tephritidae, adult ulidiids commonly use their distinctive wings to engage in often complex mating rituals. Male members of Callopistromyia raise their dappled wings at a

90 degree angle over their abdomen and “strut,” while others such as Delphinia picta Fabricius,

1791 make a “rowing” motion while walking over leaves (Sivinski 2000, Marshall 2012). Even more “extreme” examples of novel reproductive strategies can be found in species outside of a

Nearctic distribution, as in the stalk-eyed Plagiocephalus Wiedemann, 1830, or the post- copulatory ejaculate expulsion of Euxesta bilimeki Hendel, 1909 females (Brunel and Rull 2010,

Luis Rodriguez-Enriquez et al. 2013).

Unfortunately, the biology of many ulidiids, even in the relatively well-documented

North American taxa, remains unknown or understudied. One potential barrier is the near absence of taxonomic tools. There is so far no comprehensive illustrated guide to the taxa of ulidiids of any biogeographical region, and many described species and genera remain to be photographed or otherwise imaged, or else unavailable to a broad audience. This update to the key will fill some of this void by providing new images of many more taxa than have previously been available.

METHODS AND MATERIALS

Specimen Acquisition / Taxon Sampling

Specimens and data records were accessed from insect collections at three institutions: the Insect Museum in the Department of Entomology and Plant Pathology at North Carolina

State University (NCSU), the Florida State Collection of Arthropods (FSCA) at the University of

Florida at Gainesville, and the Diptera Collection in the National Museum of Natural History

(NMNH) of the Smithsonian Institution in Washington, DC.

Location data for taxa represented in the United States and Canada was based primarily on Steyskal’s (1987) descriptions, and supplemented with documentation of occurrences in published literature (Arnett 2000, Marshall 2012), the Catalog of Life (Roskov et al. 2017), the website Systema Dipterorum (Pape and Thompson 2017), the Global Biodiversity Information

Facility (GBIF.org), and collection information from the three institutions listed above. All genera, species, and documented distributions are listed in Table 1.

In total, Ulidiidae is represented in the United States and Canada by 40 genera and 124 currently valid species. Members of all recognized subfamilial, suprageneric categories (Otitinae:

Cephaliini, Myennidini, Otitini, and incertae sedis; Ulidiinae: Lipsanini, Pterocallini, Seiopterini,

Ulidiini), as of the most recent comprehensive taxonomic consideration of the family (Kameneva and Korneyev 2006), are represented by at least one genus, with a bias towards the primarily

Holarctic subfamily Otitinae.

Additional Notes

In Arnett (2000), Pseudotephritina Malloch, 1931 is treated as a sub-genus of

Pseudotephritis Johnson, 1902. Herein, however, I agree with Steyskal (1987) and Kameneva and Korneyev (2006), in treating it as a fully valid genus.

One additional genus, Prionella Robineau-Desvoidy, 1830, is indicated as appearing in the United States in Arnett (2000), and on Systema Diptorum (diptera.org). However, it is excluded from this analysis due to the ambiguity of its true distribution (Pape and Thompson

2017) and the apparent absence of any specimens held in North American collections.

Specimen Imaging

With the exception of Hiatus fulvipes Cresson, 1942 (type: Academy of Natural Sciences in Philadelphia; image: Dr. Jason Weintraub) and Texasa chaetifrons Steyskal, 1961 (type:

United States Natural Museum, image: Dr. Allen Norrbom), all images in this key were taken by the author. Each final image was produced through focus stacking. Initial photographs were taken using a Canon EOS 6D camera with the Canon MP-E 65mm 1-5x macro lens, at variable magnification and aperture depending on specimen, mounted on a Cognisys StackShot standard macro rail. Images were stacked using the Zerene Stacker system. Minor edits, to remove blemishes and to correct for color and clarity, were performed using Adobe Photoshop CS6.

For each trait used diagnostically in the key, an image was attempted; however, several traits were not visible from the available specimens, and are thus not illustrated photographically in this update to the key.

Updates to the Key

This key is a photographically illustrated key using the text of the “Otitidae” chapter of the

Manual of Nearctic Diptera by George Steyskal (1987). Most couplets did not require contemporary revision, however several were edited for greater clarity or to correct false generalizations:

 Couplet #6: “R2+3 distinctly sinuate” changed to “R2+3 usually sinuate,” as at least one

species of Tritoxa (Tritoxa cuneata Loew, 1873) possesses an R2+3 that is not.

 Couplet #20: References to the face being “convex,” “straight,” or “concave” were

removed. These statements were not necessary to differentiate taxa with this couplet, and

are believed to be a potential source of confusion.

 Couplet #22: Reference to the dm-cu of wing edited to say “biangulate or sinuate” in

reference to Oedopa Loew, to avoid confusion regarding Oedopa ascriptiva Hendel,

1909 whose dm-cu vein is not noticeably biangulate.

 Couplet #27: The description of 27’ was modified to “body often metallic, sometimes

reddish” to avoid confusion between Zacompsia Coquillet and nonmetallic, reddish

species of Euxesta (e.g. Euxesta rubida Curran, 1935).

 Couplet #29: “R2+3 sinuate” changed to “R2+3 distinctly sinuate,” to avoid confusion with

Idana marginata Say, 1830, whose R2+3 is somewhat, but not significantly, sinuate.

 Couplet #30: Reference to banded condition of tibiae removed; 30’ states “tibiae usually

distinctly banded,” but this is contradicted by couplet #32, which clarifies that neither

Pseudotephritina nor Xanthacrona possess distinctly banded tibiae, i.e. two of the four

relevant genera lack the previously specified characteristic.

 Couplet #35: For efficiency, references to scutellar bristles and the proepisternal bristles

were removed. These characters lacked resolute power of differentiation between the two

halves of the couplet, and were unnecessary, as the characteristics of the head are

sufficient.

 Couplet #41: This couplet has been removed. Tetanops myopaeformis Roder, 1881 is

acknowledged as a member of Tetanops, and therefore does not necessitate an additional

couplet. A key to species of Tetanops in North America can be found in Harriot (1942).

ACKNOWLEDGMENTS

I would like to thank Dr. Gary Steck at the Florida State Collection of Arthropods for facilitating access to the FSCA in Gainesville, and to Dr. Torsten Dikow and Dr. Allen Norrbom for facilitating access to the NMNH collection at the Smithsonian. Thanks to Dr. Allen Norrbom for photographing Texasa chaetifrons, whose type is held in the NMNH collection, and to Dr.

Jason Weintraub for photographing Hiatus fulvipes, whose type is held in the National Academy of Sciences in Philadelphia. Additionally, thanks to those involved with the Insect Museum and

Insect Systematics program at North Carolina State University: especially, Bob Blinn, the collection manager; Dr. Matthew Bertone, and my adviser, Dr. Brian Wiegmann. And thanks to the late Dr. George C. Steyskal, for all his exceptional scholarship and foundational work in fly taxonomy.

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