Mitochondrial DNA Variation Among Populations of Megaselia sulphurizona Borgmeier (Diptera: Phoridae): Evidence for a Cryptic Species Complex. Marc. R. Duquette A Thesis Submitted to the Department of Biology California State University, Bakersfield In partial fulfillment of the Degree of Masters of Science Fall 2013 Copyright By Marc Roland Duquette 2013 Mitochondrial DNA Variation Among Populations of Megaselia sulphurizona Borgmeier (Diptera: Phoridae): Evidence for a Cryptic Species Complex. Marc. R. Duquette Department of Biology, California State University, Bakersfield Abstract With over 1,300 known species, the genus Megaselia Rondani is the largest phorid genus, largely the result of high cryptic diversity and a past tendency for researchers to lump species into the genus that defied convenient classification. For many of these species, little information is available beyond one or two morphological characters. The first molecular study of such a species, Megaselia sulphurizona Borgmeier, is presented here. New sequence data from two mitochondrial loci (cytochrome oxidase, COI; NADH 1 dehydrogenase, ND1) for a total of 792 bp were obtained from 22 M. sulphurizona specimens collected from four populations in California, plus 12 other phorid taxa using standard DNA extraction and PCR techniques. Neighbor-joining, maximum likelihood, and maximum parsimony analyses revealed M. sulphurizona to be a cryptic species complex, constituting three distinct species in the Riverside, Monrovia, and Bakersfield/Tehachapi areas. These findings suggest greater cryptic diversity within M. sulphurizona, and have broader implications for phorid biodiversity. Table of Contents MITOCHONDRIAL DNA VARIATION AMONG POPULATIONS OF MEGASELIA SULPHURIZONA BORGMEIER (DIPTERA: PHORIDAE): EVIDENCE FOR A CRYPTIC SPECIES COMPLEX. List of Figures ................................................................................................................................. 3 List of Tables .................................................................................................................................. 5 CHAPTER 1: INTRODUCTION ................................................................................................ 6 Purpose of Study ......................................................................................................................... 6 Phorid Characterization .............................................................................................................. 7 Taxonomy ................................................................................................................................... 8 Importance of Group ................................................................................................................... 9 Literature Cited ......................................................................................................................... 13 CHAPTER 2 ................................................................................................................................ 17 Megaselia .................................................................................................................................. 17 Materials & Methods ................................................................................................................ 19 Results ....................................................................................................................................... 20 Discussion ................................................................................................................................. 21 Literature Cited ......................................................................................................................... 36 CHAPTER 3 ................................................................................................................................ 42 Conclusions ............................................................................................................................... 42 Suggested Future Work............................................................................................................. 43 Acknowledgements ................................................................................................................... 43 Literature Cited ......................................................................................................................... 44 2 List of Figures Chapter 1 Figure 1 ........................................................................................................................................ 11 Closeup of enlarged pedicel of a male Lecanocerus compressiceps. Hatfield, M. J., used under Creative Commons License. Figure 2 ........................................................................................................................................ 11 Basic Phorid anatomy. Holzenthal, R. W., used with permission. Figure 3 ........................................................................................................................................ 12 A. Adult female Apocephalus borealis. B. A. borealis ovipositing on honey bee. C. Two final instar A. borealis larvae exiting dead host bee. From Core, A., C. Runckel, J. Ivers, C. Quock, T. Siapno, S. DeNault, B. Brown, J. DeRisi, C. D. Smith, and J. Hafernik. 2012. A new threat to honey bees, the parasitic phorid fly Apocephalus borealis. PLoS ONE 7:e29639 doi:10.1371/journal.pone.0029639.g002. Used under Creative Commons License. Chapter 2 Figure 1 ........................................................................................................................................ 25 Map of sites where Megaselia sulphurizona samples were gathered. Accessed online at https://mapsengine.google.com/map/edit?mid=zLVaWtZ-rc14.kBiTkqE_5CYc. Copyright Google Maps. Figure 2 ........................................................................................................................................ 33 Neighbor-joining tree of 34 mitochondrial CO1+ND1 sequences representing four Megaselia sulphurizona populations, eight additional Megaselia species, and another four phorid species that served as outgroup taxa. Numbers on the branches represent bootstrap values (%). Distances 3 were calculated using number of pairwise differences using substitution model GTR+G with Akaike information criterion (AIC). Figure 3 ........................................................................................................................................ 34 Maximum-likelihood tree based on p-distance of 34 mitochondrial CO1+ND1 sequences representing four Megaselia sulphurizona populations, eight additional Megaselia species, and another four phorid species that served as outgroup taxa. Numbers on the branches represent bootstrap values (%). Distances were calculated using number of pairwise differences using substitution model GTR+G with Akaike information criterion (AIC). Figure 4 ........................................................................................................................................ 35 Bootstrap consensus maximum-parsimony tree of all sample sequences obtained from Close- Neighbor-Interchange algorithm (search level 1, 100 initial random trees). Tree length = 745, consistency index (CI) = 0.49, retention index (RI) = 0.79. Numbers on the branches represent bootstrap values (%). 4 List of Tables Table 1. ......................................................................................................................................... 26 Phorid taxa used in study with collection sites Table 2 .......................................................................................................................................... 27 Nucleotide frequencies for combined mitochondrial COI and ND1 sequences for all phorid samples. Table 3 .......................................................................................................................................... 29 Pairwise estimates of evolutionary divergence between Megaselia sequences. The number of base differences per sequence from between sequences is shown. The analysis involved 30 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were 792 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. Table 4 .......................................................................................................................................... 32 Estimates of average evolutionary divergence over sequence pairs within Megaselia groups. Table 5 .......................................................................................................................................... 32 Estimates of mean evolutionary divergence (bp) between groups of Megaselia sulphurizona. Table 6 .......................................................................................................................................... 32 Pairwise species delimitation between Riverside (RI), Monrovia (MO), and Bakersfield/Tehachapi (BT) Megaselia sulphurizona populations. 5 CHAPTER 1: INTRODUCTION Purpose of Study Species classification is often difficult, particularly with cryptic taxa. For example, the Phoridae (Diptera), with ~3,700 described species and an estimated 30-50,000 extant species displaying a wide variety of lifestyles and morphologies, stand among the most diverse lineages of insects. Unfortunately, this family