BUILDING THE TREE OF LIFE: RECONSTRUCTING THE

EVOLUTION OF A RECENT AND MEGADIVERSE BRANCH

(: 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 (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 (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 (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes

3.1 Introduction 70

3.1.1 71

3.1.2 72

3.1.3 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

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 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 and the 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. The Muscoidea are paraphyletic with a monophyletic Oestroidea being nested within the muscoid grade. The monophyly of three (the Fanniidae,

Muscidae, and Scathophagidae) of the four recognized families is confirmed while the

Anthomyiidae is apparently paraphyletic.

The fourth chapter concentrates on the Oestroidea, but it also includes the data from the previous chapters. Overall, the relationships between 247 calyptrate species representing all three superfamilies are reconstructed using molecular data from both mitochondrial (12S, 16S, COI, Cytb) and nuclear genes (18S, 28S, Ef1a and CAD). The monophyly of the Calyptratae, the superfamilies Hippoboscoidea and Oestroidea and the paraphyly of the muscoid grade are confirmed. A first comprehensive family-level hypothesis for the Oestroidea is proposed and the positions of two enigmatic species, the McAlpine’s fly and Mysctacinobia zelandica are clarified.

x LIST OF TABLES

TABLE DESCRIPTION PAGE NO. NO. 1.1 Species list and GenBank accession numbers. 16

1.2 Primer sequences used for PCR amplification and sequencing 17 (1 primer for initial amplification; 2 primer for reamplification).

1.3 Testing competing phylogenetic hypotheses for 24 Hippoboscoidea using the Templeton test (z-test; *=difference between topologies is significant).

2.1 Taxa used in study and known larval breeding habits 45

2.2 Primers used in study 48

2.3 Summary of jackknife support, ILD and symmetric tree 55 distances for different partitions of the dataset from the various analyses using different weighting regimes for different alignment strategies and treatment of gaps as missing or information

3.1 List of taxa used in study with Genbank accession numbers 78 (gixxx = submitted to Genbank).

4.1 Species used in study with total length (bp) and genes 128 amplified.

4.2 Primers used in study 135

xi LIST OF FIGURES

FIGURE PAGE DESCRIPTION NO. NO.

1.1 Previously proposed phylogenetic relationships within the 13 Hippoboscoidea. OWS = Old World ; NWS – New World Streblidae

1.2 Figure 1. 2A: Maximum parsimony tree based on combined 22 sequence data from CAD, COI, 16s and 28s (numbers in first line are bootstrap support values and posterior probabilities; - = bootstrap support < 50; numbers in second line are PBS values for CAD/COI/16s/28s; 1= Olfersini; 2= Hippoboscinae; 3=Lipopteninae; 4=Ornithomyini). Fig. 1.2 B: Subtree illustrating the topological difference between most parsimonious tree s with and without Brachytarsina speiseri (numbers = bootstrap support). Fig. 1.2 C: Subtree illustrating the topological difference between the most parsimonious tree in 2A and and the Bayesian tree (numbers = posterior probabilities).

1.3 Key events in the evolution of the Hippoboscoidea. 31

2.1 Most parsimonious tree for all manual alignments and indel 52 treatments under optimal analysis conditions (tv=4). Support values are indicated on branches as Jackknife support. The set of numbers toward the bottom of the squares represent the [transition, transversion, gap] costs used for the particular analysis. Black fields in the sensitivity plot signify agreement, white disagreement, and grey neither (polytomy).

xii

FIGURE PAGE DESCRIPTION NO. NO.

2.2 Most parsimonious tree for direct optimization (in POY) under 53 optimal analysis conditions (tv=4). Support values are indicated on branches as Jackknife support. The set of numbers to the bottom of each of the squares represent the [transition, transversion, gap] costs used for the particular analysis. Black fields in the sensitivity plot signify agreement, white disagreement, and grey neither (polytomy).

2.3 Most pasimonious tree under equal weighting using a manual 54 alignment

2.4 Number of times a node is recovered out of the ten different 61 analysis conditions (nodal stability) under manual alignment (•) and direct optimization alignments () versus Jackknife support at the node.

2.5 Tracing of natural history evolution in the Scathophagidae. One of 65 the optimizations when phytophagy is coded separately as dicot and monocot (represented by symbols) is shown here. In this case, the Scathophagid ancestor was feeding on monocot plants. Taxa for which there is either species-specific information or at least statements about the have been included.

3.1 Strict consensus of three most parsimonious trees (indel=5th 86 character); above node jackknife support for indel=5th character state; below node indel=missing; nodes shared with ML tree indicated by.

3.2 Likelihood tree from ML analysis (Garli) indicating bootstrap 88 support, nodes shared with selected guide tree indicated by  and majority rule consensus of all ten guide trees indicated by.

xiii FIGURE PAGE DESCRIPTION NO. NO.

3.3 12s guide tree from Clustal X. 100

3.4 16s guide tree from Clustal X. 101

3.5 28s guide tree from Clustal X. 102

3.6 18s guide tree from Clustal X. 103

3.7 Guide tree from Clustal X used in alignment of ribosomal genes. 104

3.8 Strict consensus of 23 most parsimonious trees (indel=missing). 105

3.9 Majority rule consensus of trees from parsimony analysis of 106 datasets based on the ten different guide trees.

4.1 Two different hypotheses for the family relationships within the 113 Oestroidea based on morphology a) McAlpine (1989) b) Pape (1992).

4.2 Phylogeny of the Calyptratae based on morphology in McAlpine, 125 1989.

4.3 Strict consensus of fifteen most parsimonious trees (indel=5th 139 character); value above node jackknife support for indel=5th character state; below node indel=missing.

4.4 Likelihood tree from Maximum likelihood analysis using Garli 143

xiv LIST OF PUBLICATIONS

1. Kutty, S.N., Bernasconi, M.V., Sifner, F., Meier, R., 2007. Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha : Calyptratae). Cladistics 23, 64-83.

2. Kutty, S.N., Pape, T., Pont, A.C., Wiegmann, B.M., Meier, R. The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes. Molecular Phylogenetics and Evolution. 49, 639-652.

3. Petersen, F.T., Meier, R., Kutty, S.N., Wiegmann, B.M., 2007. The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers. Mol. Phylogenet. Evol. 45, 111-122.

4. Su, K.F.Y., Kutty, S.N., Meier, R., 2008. Morphology versus Molecules: The phylogenetic relationships of (Diptera: Cyclorrhapha) based on morphology and DNA sequence data from ten genes. Cladistics. 24, 902-916.

5. Tan, S.H.D., Ali, F., Kutty, S. N., Meier, R. The need for specifying species concepts: how many species of silvered langurs (Trachypithecus cristatus group) should be recognized? Mol. Phylogenet. Evol. 49, 688-689.

6. Puniamoorthy, N., Jeevanandam, J., Kutty, S.N., 2008. Give south Indian authors their true names. Nature 452, 530-530.

7. Lim, S.G, Hwang, W. S., Kutty, S. N., Meier, R. and Grootaert, P., 2009. Mitochondrial and nuclear markers support the monophyly of and suggest a rapid origin of the subfamilies (Diptera: ). Sys. Entomology. Accepted.

xv INTRODUCTION

The concept of “Tree–of-Life” is one of main elements of Charles Darwin’s theory of evolution according to which the evolutionary relationships of the earth’s biodiversity, including all living and extinct forms over the past 3.5 billion years, can be depicted by a tree-like diagram. Having a tree-of-life for all species is important for modern biology because it provides the framework for all comparative biology. It reveals how the diversity evolved as well as the historical basis for similarity and differences among organisms (AToL, 2004). Building this Tree of Life is one of the most complex scientific problems facing modern biology because the number of species that are described and yet to be discovered is vast and a large amount of data has already been published. This includes the morphology of recent and extinct species, developmental data, behavior, etc. for many of these species. All this information has to be collected and new analytic tools are needed to reconstruct the relationships based on these data. This is the main goal of the US National Science Foundation funded project ‘Assembling the Tree of Life’ which involves many international teams that concentrate on different subsets of taxa. Obtaining an accurate and universal

‘Tree of Life’ has enormous research potentials and benefits to society by improving human health, improving agriculture, tracing developmental changes, protecting invasive species form ecosystems and also understating disease out breaks and evolution of strains (AToL, 2004).

With an estimated 150,000 described species, the order Diptera (Class

Insecta, Phylum Arthropoda), or true is one of the most diverse branch on the

Tree of Life (FLYTREE, 2006). Diptera is also is one of the four “megadiverse” orders of . As many flies are of economic and medical importance and are also

1 model organisms for research, understanding the relationships between the different fly is important. This is why the FLYTREE project was funded as part of the

NSF sponsored ‘Assembling the Tree of Life’ initiative. FLYTREE is an international research collaboration designed to elaborate and discover the details of fly relationships and diversity with the ultimate goal of providing a newly resolved phylogeny for this major branch of the Tree of Life (FLYTREE 2006). The most effective way of transmitting new phylogenetic evidence is via a published data matrix analyzed in a quantitative framework (Yeates and Wiegmann, 1999) and the

FLYTREE project is an ambitious endeavor that will be carried out in a three-tier approach with each tier tackling different Diptera relationships at different levels.

Eventually these data will be combined to build a meta-analysis based tree with 2000 species. The Diptera comprise 10% of all described species and FLYTREE is thus a major part of the Tree of Life project. Within Diptera, one major is the

Calyptratae, the subject of my PhD project.

The Calyptratae comprises about 18,000 described species representing 12% of dipteran diversity which forms a sizeable part of the Diptera diversity. The commonly known members of this group are house flies, blowflies, flesh-flies, tsetse flies, warble flies, etc and many species have close associations with humans, livestock and agriculture. Despite its medical and economic importance, the relationships within the calyptrates are very poorly understood and most literature on the phylogeny of the group (Hennig, 1973; McAlpine, 1989; Pape, 1992; Nirmala, 2001; etc.) is often controversial. The Calyptratae are morphologically and biologically very diverse and have invaded a large variety of habitats. They show an exceptional range of natural history traits. However, despite all this diversity, the clade appears to be relatively young with the oldest confirmed fossil being only 40 million years old.

2

Although phylogenetic studies, mostly based on morphological data, have been carried out for a few families and subfamilies of the group, a phylogenetic tree for the

Calyptratae is not available. At the onset of my thesis, I assessed what is known based on the available data. The reconstruction of phylogenies of large groups like the Calyptratae can be approached by either using the supermatrix method or the supertree method (Sanderson et al., 1998). I combined all the available data for species under study into a single matrix and used the information from each character directly in the supermatrix analysis. The trees from the different published studies are recoded in a matrix format and then combined using the MRP (Matrix representation with parsimony) technique in the supertree approach. The trees obtained by both approaches were poorly resolved in conflict and it became clear that new data were needed. Since the data overlap of both genes and taxa in the available datasets was poor, adding more data into a supermatrix of taxa from across the calyptrate group was regarded as the most appropriate approach. This is the technique that I have used for this project.

OBJECTIVES

My PhD project aims to resolve phylogenetic relationships in the group Calyptratae using molecular data. The general objectives are to address: i. monophyly and relationships between superfamilies. ii. monophyly and relationships between the recognized families (Glossinidae,

Hippoboscidae, Streblidae, Nycteribiidae, Fanniidae, Musciidae,

Anthomyiidae, Scathophagidae, , Oestridae, ,

Rhinophoridae, Sarcophagidae, Mystaciinobiidae). iii. position of the enigmatic species referred to as “McAlpine’s fly”.

3 iv. evolution of life history strategies like host choice and larval breeding habits

within the group. v. the performance of various alignment and test various analyses techniques

using the calyptrate dataset.

I approached this large and ambitious PhD project by subdividing the Calyptratae into smaller taxa. Three of the thesis chapters are thus based on the currently recognized three superfamilies (Hippoboscoidea, Muscoidea and Oestroidea) and one chapter is a detailed phylogenetic study on the family Scathophagidae (Calyptrate: Muscoidea):

Chapter 1: The phylogeny and evolution of host choice in the Hippoboscoidea

(Diptera) as reconstructed using four molecular markers.

Chapter 2: Sensitivity analysis, molecular systematics, and natural history

evolution of Scathophagidae (Diptera:Calyptrate: Musocidea).

Chapter 3: The Muscoidea (Diptera:Calyptratae) are paraphyletic: Evidence from

four mitochondrial and four nuclear genes.

Chapter 4: Molecular phylogeny of the Calyptratae (Diptera:Cyclorrhapha) with

emphasis on the Oestroidea.

The sequence of the chapters in the dissertation reflects how the project evolved which explains why different analysis and alignment techniques were tested in the different sections. Alignment techniques used in this project are 1) Clustal

(Thompson et al., 1994), 2) Manual alignment based on Clustal alignments, 3) Direct optimization (Wheeler, 1996), and 4) user alignments based on user-defined guide trees (Kumar and Filipski, 2007). The data were analyzed using 1) Parsimony executed PAUP* (Swofford, 2002) and TNT (Goloboff et al., 2000) 2) Maximum

4 Likelihood as implemented in Garli (Zwickl, 2006) and 3) Bayesian likelihood

(Huelsenbeck and Ronquist 2003). The variation in alignments and analyses between chapters reflects progress in the field during the duration of the PhD. Some analysis techniques that yielded poor results were also not applied in subsequent analyses. Some analyses technique could also not be used in the final chapter because the dataset was too large for the available computational resources.

This dissertation is my original work but like any project; this study and all resulting publications are a result of a collaborative effort and to give due credit to all involved

“we” has been used throughout this dissertation.

5

CHAPTER 1

The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed

using four molecular markers

1.1 INTRODUCTION

Hippoboscoidea are highly specialized ectoparasitic flies with four recognized family- level taxa: Glossinidae, Hippoboscidae, Streblidae, and Nycteribiidae (Hennig, 1973;

McAlpine, 1989; but see Griffiths, 1972). The well-known Glossinidae (tsetse flies) are free-living and only come into close contact with their host during feeding. The other three families, Hippoboscidae, Nycteribiidae, and Streblidae, are all genuine ectoparasites (i.e. species with a trophic and a spatial association to a host) spending all or most of their adult life within the fur or among the feathers of their mammal and bird hosts. These families exhibit a large number of unique and striking morphological and physiological adaptations, most of which are specifically associated with their ectoparasitic lifestyle. One of the most remarkable of these is adenotrophic viviparity (Meier et al., 1999). The larvae develop individually, in the female oviduct, where they are fed by secretions from accessory glands. The fully mature 3rd instar is deposited either as a motile larva, which quickly pupates within its last larval skin (Glossinidae, Hippoboscidae), or as a more or less soft pre- puparium (Streblidae, Nycteribiidae). At the time of deposition, the weight of the larva can exceed the weight of the female (Hill, 1963).

Although the group has received considerable taxonomic attention, comparatively little is known about the relationships among the families. As a consequence, phylogenetic assessments of the evolution of host choice have not yet been possible and much of the literature on the subject is highly speculative. A recently published molecular systematic analysis by Dittmar et al. (2006) addressed some of these problems, but it focused largely on the relationships within Streblidae and

Nycteribiidae, and included only a few species from the remaining families. Here, we

7 present results from a complementary phylogenetic study that includes a broader taxon sample from the Hippoboscidae and Glossinidae and we explore the use of different genetic markers than those used in Dittmar et al. (2006). In addition to the mitochondrial 16S rDNA used by Dittmar et al (2006), we sequenced fragments of the nuclear genes 28S ribosomal DNA (28S rDNA), the carbamoyl-phosphate synthase (CPSase) domain of CAD (Moulton and Wiegmann, 2004), and the mitochondrial gene cytochrome oxidase I (COI), for 35 species. The goal of the current study was to test the monophyly of the Hippoboscoidea, test the monophyly of the four subordinate families, clarify the phylogenetic relationships among the families, and to use the resulting trees to reconstruct key events in the evolution of

Hippoboscoidea.

1.1.1 Family portraits: Biology and Systematics

Nycteribiidae are obligate ectoparasites of bats with highly specialized and reduced adult morphology. The wings are completely reduced, the thorax is dorsoventrally flattened, the legs are inserted dorsally, and the head is folded backwards resting on the thorax. The flies thus have a spider-like appearance and are regularly delivered to spider taxonomists for identification (N. Scharff, pers. comm.). The adults spend all of their life in the fur of the host, leaving the host only for brief periods in order to affix a puparium to the wall or ceiling of the bat roost. Numerous morphological synapomorphies have left little doubt that the Nycteribiidae are monophyletic

(Hennig, 1973). Three subfamilies are recognized: the Archinycteribiinae and

Cyclopodiinae (on Megachiroptera), and the Nycteribiinae (on Microchiroptera;

Hennig, 1973; Theodor, 1967). Morphological support for this subdivision comes mainly from the number of tergites on the female abdomen, the position of the thoracic sutures and setae, shape of tibiae, and overall chaetotaxy. However, these

8 characters are highly variable (Theodor, 1967) and currently the most consistent character is host choice, although behavioral features such as host use may be particularly prone to convergence (Blomberg et al., 2003). Furthermore, character polarity is unknown; i.e., it is unclear whether the Archinycteribiinae+Cyclopodiinae

(Megachiroptera) or the Nycteribiinae (Microchiroptera) are based on a plesiomorphic host association.

The Streblidae are also obligate bat-ectoparasites, but unlike the Nycteribiidae most species retain fully functional wings for at least part of their life. An exception is

Ascodipteron Adensamer 1896 in which females, after mating, embed themselves in the tissue of the host. Wings and legs are shed and the fly attains a sack- or flask like appearance while the males retain their wings throughout life. The morphology of the remaining Streblidae is also unusually variable. For example, some species are dorsoventrally flattened, while the Nycterophiliinae are laterally flattened. Because of these unique traits, finding support for streblid monophyly has been difficult

(McAlpine, 1989). Most autapomorphies proposed by McAlpine (1989) are invalid because they are based on wing morphology and thus inapplicable for the other family of bat flies (Nycteribiidae); i.e., it remains unclear whether these features are autapomorphic for Streblidae or Streblidae+Nycteribiidae. Similarly problematic are

McAlpine’s (1989) characters pertaining to thorax morphology because the nycteribiid thorax is so highly modified that homologies are difficult to establish.

McAlpine (1989) also listed the absence of spermathecae as a streblid synapomorphy although Wenzel & Peterson (1987) considered the spermathecae

“probably present”. This conflict may be due to the fact that several hippoboscoid families have unsclerotized spermathecae (Maa & Peterson, 1987; Peterson &

Wenzel, 1987; Wenzel & Peterson, 1987), thus making the feature very difficult to

9 identify in, for example, pinned specimens. Currently, the Streblidae is subdivided into five subfamily-level taxa (McAlpine, 1989, but see Hennig 1973). The

Nycteriboscinae and the endoparasitic Ascodipterinae are restricted to the Old World, while the Trichobiinae, the Nycterophiliinae, and the Streblinae are found only in the

New World.

The Hippoboscidae contains approximately 150 species that infest the plumage of various birds or the fur of mammals. However, whether the ancestral host species was a bird or a mammal remains unknown. Bequaert (1954) maintained that the hippoboscid ancestor was a bird parasite, although he admitted that there was little evidence to support this hypothesis. In contrast, in an earlier publication Hennig

(1965), assuming a sister group relationship between the Hippoboscidae and the

Glossinidae, asserted that the most likely ancestral host of the Hippoboscidae was a mammal. The Hippoboscidae are dorsoventrally flattened, and, in contrast to the

Nycteribiidae, the head is prognathous and broadly confluent with the thorax. Overall, these flies have a crab-like appearance, although they are generally called “louse flies”. Most species have fully developed and functional wings, but some are stenopterous and a few apterous. Despite the morphological variability, the monophyly of the Hippoboscidae has been almost universally accepted (Bequaert,

1954; Hennig, 1973; McAlpine, 1989). The family is subdivided into three subfamilies

(Maa & Peterson, 1987). The Lipopteninae are restricted to mammals while the

Ornithomyiinae and Hippoboscinae parasitize both mammals and birds. However, the sole member of the Hippoboscinae known to infest birds is Struthiobosca struthionis (Janson, 1889) which is only found on ostriches, the ecologically most mammal-like bird.

10 The fourth family of Hippoboscoidea, the Glossinidae, is not only the least speciose

(22 spp.), but it is also by far the most familiar due to its notoriety as the vector for

Trypanosoma parasites that cause sleeping sickness in humans and nagana in livestock (Krinsky, 2002). Morphologically it is the least modified hippoboscoid family.

The adults are free-living and only come in contact with the host during feeding, and thus, apart from their proboscis, the glossinids look like typical calyptrate flies and do not display any of the spectacular adaptations for a ectoparasitism that are so common in the Hippoboscidae, Nycteribiidae and Streblidae. While retaining many ancestral calyptrate features, the monophyly of the Glossinidae was accepted by

Hennig (1973) and supported by a number of synapomorphies in McAlpine

(McAlpine, 1989: e.g., arista with long plumules on dorsal surface, very elongated palpi and proboscis).

1.1.2 Hippoboscoidea Phylogenetics

There are relatively few issues in hippoboscoid phylogeny and classification that are not contentious. Most authors agree that the Nycteribiidae and the Hippoboscoidea are well-supported monophyletic groups. The latter is supported by two cladistic analyses using DNA sequence data (Nirmala et al., 2001; Dittmar et al., 2006) and several morphological synapomorphies including, adult mouthparts that are uniquely modified for hematophagy, and adenotrophic viviparity with the deposition of mature

3rd instar larvae (Hennig, 1973; McAlpine, 1989). Arriving at the modern concept of

Hippoboscoidea, however, was a slow process. Initially, the Hippoboscoidea only included the three core families that are today known as “Pupipara” (Hippoboscidae,

Nycteribiidae, Streblidae). The monophyly of this grouping was initially controversial

(e.g., Bequaert, 1954; Falcoz, 1926; Hendel, 1936; Jobling, 1929; Lameere, 1906;

Müggenburg, 1892; Muir, 1912), but is now defended by many authors (e.g., Griffiths,

11 1972; Hennig, 1973) and sometimes the Nycteribiidae and Streblidae are even considered included within the Hippoboscidae (Biosystematic World Database of

Diptera: http://www.sel.barc.usda.gov/diptera/names/BDWDabou.htm; Griffiths,

1972). The Hippoboscoidea in its present composition including the Glossinidae and

Pupipara was only proposed in 1971 by Hennig (as “Glossinoidea”) although Speiser had already pointed out similarities in 1908.

Controversial are most other Hippoboscoidea relationships. For example, its position within the dipteran clade Calyptratae is not certain (see Bequaert, 1954). The current consensus is that the superfamily is probably the sister group to all remaining

Calyptratae (Hennig, 1973; McAlpine, 1989; Nirmala et al., 2001), but previously the most common placement was close to or within the Muscidae (Bequaert, 1954; Muir,

1912), or even as the sister group of the Oestridae (Lameere, 1906; Pollock 1971;

1973; but see Griffiths, 1976). To begin to address the placement of Hippoboscoidea within the Calyptratae, we include six species from four other calyptrate families as outgroups.

Similarly unclear are the interfamiliar relationships in Hippoboscoidea which hinder the reconstruction of key events in the evolution of this group. Hennig (1973) proposed that genuine ectoparasitism evolved once and that the two families of bat parasites form a monophyletic group (Fig 1.1), while McAlpine (1989) favored a sister group relationship between Glossinidae+Hippoboscidae and Streblidae +

Nycteribiidae. The latter view was also supported by Nirmala et al.’s (2001) molecular data and Dittmar et al.’s (2006) maximum likelihood analysis with the exception that the Streblidae was paraphyletic (Fig. 1.1). Streblid paraphyly was also found in

12 Dittmar et al.’s (2006) parsimony and Bayesian likelihood analyses which otherwise yielded again very different trees for suprafamiliar relationships (Fig. 1.1).

Figure 1.1. Previously proposed phylogenetic relationships within the Hippoboscoidea. OWS = Old World Streblidae; NWS – New World Streblidae

13 1.2 MATERIALS AND METHODS

1.2.1. Taxon sampling, DNA extraction and sequencing

Thirty species representing seven out of 10 subfamilies of Hippoboscoidea are included in our analysis. Specimen data are given in Table 1.1 and voucher specimens are deposited in the collection of the North Carolina State University.

Owing to uncertainty with regard to the placement of the Hippoboscoidea within the

Calyptratae, we included six calyptrate outgroups from four families and the acalyptrate, Drosophila melanogaster Meigen 1830, for rooting the tree.

DNA was either extracted from single legs using the DNAeasy kit (Qiagen, Santa

Clara, CA) following the manufacturer’s protocol, except that elution volume was 30

µl, or in some cases we punctured the specimen and used a CTAB phenol/ chloroform extraction protocol keeping the exoskeleton of the specimen as a voucher. A 760bp fragment of the carbamolyphosphate synthetase (CPS) region of the CAD gene (also know as “rudimentary” in Diptera) was amplified and sequenced according to the protocol described by Moulton & Wiegmann (2004). Initial amplification was carried out with the primers 787F and 1124R (Table 1.2). For reamplification and subsequent sequencing the primers 806F and 1098R were used.

Approximately 2kb of the 28S rDNA were amplified in three overlapping pieces using the primers listed in Table 1.2 and the PCR conditions given in Collins & Wiegmann

(2002). Partial sequences for COI and 16S were obtained using the primers specified in Table 1.2 under standard conditions (Savage et al., 2004).

For sequence editing and contig construction we used Sequencher 4.2 (Gene Codes

Corporation Inc, Ann Arbor, Michigan, USA). COI and CAD were unambiguously

14 aligned by eye using MacClade 4.03 (Maddison & Maddison, 2001) and neither displayed stop codons when translated to amino acid sequence. The sequences for

16S and 28S were aligned in ClustalX (Thompson et al., 1997) using the following settings: Gap opening cost (10) and gap extension cost (0.20). We subsequently performed minor manual adjustments in MacClade 4.03 (Maddison & Maddison,

2001). Alignments are available on request, and GenBank accession numbers are given in Table 1.1. Leading and trailing gaps were treated as missing data, while internal gaps were scored as a 5th character state.

15 Table 1.1. Species list and GenBank accession numbers.

16 Table 1.2. Primer sequences used for PCR amplification and sequencing (1 primer for initial amplification; 2 primer for reamplification).

1.2.2 Phylogenetic Analysis

The parsimony analyses were performed with TNT (Goloboff et al., 2003) using traditional search, 1000 random addition replicates, and TBR branch swapping. Non- parametric bootstrap values (Felsenstein, 1985) were calculated with the following settings: traditional search, 20.000 bootstrap replicates with 100 random addition analyses per replicate. Partitioned Bremer Support values (Baker and DeSalle, 1997) were calculated for individual nodes using TreeRot v2b (Sorenson, 1999) and PAUP*

4.0b10 (Swofford, 2002). Due to problems with sequencing several genes for

Brachytarsina speiseri (Jobling, 1934), the dataset was analyzed both with this species included and excluded. We also tested the competing topological hypotheses for hippoboscoid relationships from figure 1.1 through constrained tree

17 searches and statistical testing (Templeton test: Templeton, 1983). We defined constraint trees in MacClade (Maddison & Maddison, 2001) and used these in

PAUP* (Swofford, 2002) for finding most parsimonious solutions under the specified constraints. All outgroup taxa were included as an unresolved polytomy. We then used the implementation of the Templeton test in PAUP* (z-test) for determining whether the competing topologies can be rejected based on our data.

We also analyzed our data using Bayesian analysis as implemented in MrBayes 3.1

(Huelsenbeck and Ronquist 2003). All Bayesian sequence analyses were initiated from random starting trees and utilized the GTR+I+G model which was favored by the Akaike Information Criterion (AIC) as implemented in MrModeltest version 2.2

(Nylander 2004). We partitioned the data set by gene and ran it for 1,500,000 generations. A tree was sampled every 300 generations, resulting in 5,000 trees of which 25% were discarded as burn-in. Three independently repeated analyses resulted in similar tree topologies and comparable clade probabilities and substitution model parameters.

Key events in the evolution of Hippoboscoidea were reconstructed by mapping the following characters onto the most parsimonious tree: (1) hematophagy

(presence/absence), (2) host choice (mammals/birds), (3) host specialization on bats

(presence/absence), (4) Adenotrophic viviparity (presence/absence), (5) Motility of larva (capable of burrowing/incapable of burrowing/pupariation within female), (6)

Shedding of wings after finding host (presence/absence), (7) Reduction of forewing size (presence/absence), (8) Complete loss of forewing (presence/absence). These characters were mapped onto the most parsimonious tree excluding Brachytarsina

18 speiseri using MacClade 4.03 (Maddison & Maddison, 2001) and ACCTRAN. The only multistate character was coded as non-additive.

1.3 RESULTS

The length of the aligned sequences is 760 bp for CAD, 2561 bp for 28S rDNA, 542 bp for 16S, and for COI 1264 bp. In CAD, we found three trinucleotide insertions.

One is shared between Trichobius joblingi Wenzel, 1966 and Paratrichobius longicrus (Ribeiro, 1907), whereas the other two are unique to Megistopoda aranea

(Coquillett, 1899) and Ornithomya avicularia (Linnaeus, 1758), respectively. One nine-nucleotide indel is unique to Drosophila melanogaster. The total dataset is 5127 bp in length of which 1837 sites are parsimony informative.

The analysis excluding Brachytarsina speiseri, resulted in a single parsimonious tree with a total length of 8884 steps, a retention index (RI) of 0.441 and a consistency index (CI) of 0.6 (Fig. 1.2 A). The interfamiliar relationships on this tree are identical to the proposal by Hennig (1973; Fig. 1.1). The monophyly of the Hippoboscoidea and its placement as sister group to the rest of the Calyptratae is strongly supported

(BP=90). The Glossinidae (BP=100), the Hippoboscidae (BP=94) and the bat flies

(BP=99) are all found to be monophyletic. Within the Glossinidae, two of the three currently recognized species groups (palpalis- and morsitans-species groups) are recovered as monophyletic with strong support (BP = 100 and 98 respectively).

Glossina brevipalpis Newstead, 1910 of the fusca species group emerges as sister group to all remaining Glossinidae.

19 Within the Hippoboscoidea, the Glossinidae are the sister group of the remaining families (BP=95). Within the Hippoboscidae, the Hippoboscinae (BP= 100) and

Lipopteninae (BP= 100) are supported as monophyletic. The Ornithomyinae is paraphyletic. However, both constituent tribes, the Ornithomyini (BP= 100) and the

Olfersiini (BP= 86) are monophyletic. At the genus level, Ornithomya and Lipoptena are both paraphyletic. The Nycteribiidae (BP= 100) and Streblidae (BP= 100) are both well-supported monophyletic lineages, and together form a clade (BP= 99). The nycteribiid subfamily Nycteribiinae is also monophyletic (BP=88).

The maximum parsimony analysis including Brachytarsina speiseri (Fig. 1.2B) yields a single most parsimonious tree with a tree length of 9008 steps, a consistency index

(CI) of 0.436, and a retention index (RI) of 0.569. The topology is nearly identical to that found in the analysis excluding Brachytarsina speiseri with the exception that within the bat fly clade Brachytarsina speiseri is placed as sister group to the

Nycteribiidae, thus rendering the Streblidae paraphyletic, albeit without bootstrap support (Fig. 1.2B). The Bayesian analysis excluding Brachytarsina speiseri resulted in a tree with an identical ingroup topology to the most parsimonious tree depicted in

Figure 1.2A. Bayesian and parsimony analyses differed only in the resolution of outgroup relationships Figure 1.2C.

Hennig’s hypothesis for the interfamiliar relationships of Hippoboscoidea is identical to the most parsimonious tree excluding Brachytarsina. However, when

Brachytarsina is included, the Streblidae are paraphyletic, which disagrees with a statement by Hennig (1973) on the intrafamiliar relationships of the family that implies that he considered it monophyletic. Making Streblidae monophyletic requires three additional steps and the two topologies are also not significantly different as

20 judged by the Templeton test (Table 1.3). All topologies obtained by using previous hypotheses as topological search constraints (Fig. 1.1) require a large number of extra steps. These topologies are also rejected by the Templeton tests (Table 1.3).

However, these test results have to be interpreted with care given that one of the test assumptions is that all sites evolve independently.

All life history characters had only one most parsimonious optimization. According to our most parsimonious tree, hematophagy evolved once and the hippoboscoid ancestor fed on mammal blood. Feeding on bats evolved once and feeding on birds twice. Adenotrophic viviparity involving the deposition of a single third-instar larva or puparium evolved in the hippoboscoid ancestor and the most parsimonious tree is compatible with the scenario that the deposited larva was initially motile (e.g.,

Glossinidae), then lost its ability to burrow in the soil (Hippoboscoidea), before starting to pupate already within the female (bat flies). Shedding of wings after finding a host evolved once and one species in this clade subsequently completely lost the forewings. Forewing loss is also observed in a second clade. Stenoptery (reduction of wing size) evolved twice.

21 Figure 1. 2.

Figure 1. 2A: Maximum parsimony tree based on combined sequence data from CAD, COI, 16s and 28s (numbers in first line are bootstrap support values and posterior probabilities; - = bootstrap support < 50; numbers in second line are PBS values for CAD/COI/16s/28s; 1= Olfersini; 2= Hippoboscinae; 3=Lipopteninae; 4=Ornithomyini).

Fig. 1.2 B: Subtree illustrating the topological difference between most parsimonious tree s with and without Brachytarsina speiseri (numbers = bootstrap support).

Fig. 1.2 C: Subtree illustrating the topological difference between the most parsimonious tree in 2A and and the Bayesian tree ( numbers = posterior probabilities).

22 DISCUSSION

The Hippoboscoidea include some of the most important and interesting flies from a medical, veterinary, and morphological point of view. Yet, many questions regarding the evolution of these flies remain unanswered because key issues in

Hippoboscoidea phylogenetics remained unresolved. One of the main obstacles with regard to trees based on morphology has been the large number of reductions due to extreme morphological adaptations associated with ectoparasitism. Determining primary homology was especially difficult for the highly modified features of the wings, head, thorax and legs that are commonly used in higher-level fly phylogenetics. Here, DNA sequences provide a particularly valuable source of phylogenetic information because molecular evolution in the standard genes used as phylogenetic markers is likely to be largely independent of the selection causing morphological adaptations to ectoparasitism. It is all the more surprising that the first two attempts at addressing interfamilial relationships in Hippoboscoidea using DNA sequences yielded conflicting results (Fig. 1.1; Nirmala et al., 2001; Dittmar et al.,

2006). Nirmala et al. (2001) did not recover either of the two topologies that had been proposed based on morphology. Instead they found weak support for a third indicating a sister group relationship between the Hippoboscidae and the

Nycteribiidae and a monophyletic group consisting of the Glossinidae+Streblidae.

Conversely, Dittmar et al. (2006) found some support for McAlpine’s (1989) hypothesis in parsimony and Bayesian trees by recovering

Glossinidae+Hippoboscidae, but the placement of this clade relative to the bat flies

0varied and depended on the analysis method.

23 Our newly collected data appear to have strong phylogenetic structure, as evidenced by high levels of branch support. These new data are also decisive in that alternative topologies can be rejected based on Templeton tests (Table 1.3). We are able to confirm the monophyly of the following key clades with high bootstrap support (BS):

Hippoboscoidea (BS: 90), Glossinidae (BS: 100), Nycteribiidae (BS: 100), and

Hippoboscidae (BS: 94). We can also corroborate Hennig’s (1973) hypothesis of a sistergroup relationship between Glossinidae and all remaining Hippoboscoidea

(“Pupipara” BS: 95), and of a monophyletic bat fly clade consisting of Nycteribiidae and Streblidae (BS: 99).

Table 1.3. Testing competing phylogenetic hypotheses for Hippoboscoidea using the Templeton test (z-test; *=difference between topologies is significant).

We believe that two factors are likely responsible for finding strong support for interfamiliar relationships in our analysis. The first is better taxonomic coverage for the Hippoboscidae and Glossinidae. Nirmala et al. (2001) used three hippoboscoid species in their attempt to resolve relationships within the Calyptratae, while Dittmar et al. (2006) concentrated on resolving the relationships within the bat fly families.

For this reason, they included only a single glossinid and few hippoboscid species.

24 Taxon sampling can have a major effect on clade recovery and support in phylogenetic inference (e.g., Hillis 1998) especially in groups as diverse and old as holometabolous insects or calyptrate flies. Our more thorough taxon sampling was designed to test the monophyly of major hippoboscoid clades through more accurate optimization of ancestral states. This is especially important in cases where individual taxa may be highly autapomorphic in morphology or evolutionary rates. The second factor is the choice of genes. We used four genes and carried out a partitioned

Bremer support (PBS) analysis in order to investigate their relative contributions to node support. As a summary of gene contribution to the total tree support, we

calculated the sum of all PBS values (ΣPBS) for each gene. Ranked in descending order of performance (e.g., overall influence on the parsimony tree topology), our

genes were CAD (ΣPBS=473.9), COI (ΣPBS=306.9), 28S rDNA (ΣPBS=64.75), and 16S

rDNA (ΣPBS=2.45). Only the worst-performing gene, 16S rDNA, had been used in previous attempts to resolve the phylogenetic relationships within the

Hippoboscoidea (Nirmala et al, 2001; Dittmar et al., 2006). Overall, we find that in our analysis the nuclear genes CAD and 28S rDNA provide most of the support at the

“basal” nodes while COI provides support at the tips.

It is noteworthy, however, that for some crucial nodes there is strong disagreement between the two nuclear genes. For example, the monophyly of the Hippoboscoidea and the monophyly of the Hippoboscidae are strongly contradicted by 28S rDNA, and

CAD supports an arrangement of outgroup taxa that is in strong disagreement with all previous hypotheses of calyptrate relationships. Nonetheless, the additional evidence provided by just the small segment of CAD used here suggests that additional CAD sequence from contiguous regions of the gene may be quite valuable for additional studies of hippoboscoid relationships. In contrast, the small fragment of

25 mitochondrial 16S rDNA contributes little or no information to our phylogenetic trees; it appears to us that this gene could be omitted from future studies of these taxa.

1.4.1 Classificatory implications

The monophyly of the Hippoboscoidea as a whole is supported by numerous morphological characters (Hennig, 1973; McAlpine, 1989) and has been confirmed in all previous molecular analyses (Nirmala et al., 2001; Dittmar et al., 2006) including the present study. It now seems established beyond doubt. However, the exact placement of the superfamily within the Calyptratae had not been firmly settled. In our analysis, the Hippoboscoidea are placed deeply nested within the Calyptratae

(Fig. 1.2A), which is incongruent with McAlpine’s (1989) hypothesis of a sister group relationship between Hippoboscoidea and the remaining calyptrate flies. However, our outgroup tree topology remains only poorly supported and the Bayesian analysis finds a similar placement of Hippoboscoidea as found by Nirmala et al. (2001) and

Dittmar et al. (2006) in their parsimony and Bayesian analyses.

The monophyly of the Hippoboscidae has traditionally not been questioned and is here recovered with strong support (BP=94). But our taxon sample for Hippoboscidae and Glossinidae also allows us to address classification issues within both families.

Within the Hippoboscidae, our results are largely congruent with the family-level classification proposed by Maa (1969). The mammal parasites in the Lipopteninae and Hippoboscinae are sister- groups and both monophyletic. However, Maa’s

(1969) Ornithomyinae consisting of Ornithomyini and Olfersiini is more problematic.

The tribes are monophyletic and well supported, but the subfamily as a whole is paraphyletic with the Olfersiini being strongly supported (BS: 99) as sister group to

Hippoboscinae+Lipopteninae. Within the Ornithomyini, our finding of paraphyly for

26 the genus Ornithomya is not unexpected as the genera Crataerina and Stenepteryx were described by Maa (1963: 96) as “… a highly specialized form of the polyxenous, widely distributed genus Ornithomya”, a view shared by Bequaert (1954: 69), who stated that “[t]he evolution of Crataerina from an Ornithomya-like (…) bird-fly is obvious”. All three genera should be synonymized under Ornithomya.

Synonymization could also repair the paraphyly of Lipoptena caused by the sheep ked, Melophagus ovinus (Linnaeus, 1758) which is deeply nested within Lipoptena.

However, we suspect that there would be considerable resistance to a name change because both genus names are well established and Melophagus, which is intimately associated with the sheep ked, has priority over Lipoptena.

In the two previous molecular analyses of Hippoboscoidea, the Glossinidae had only been represented by a single taxon. For this reason, the authors were unable to test the monophyly of the glossinids and their subgenera. With our larger sample of species, we find the Glossinidae to be monophyletic (BS= 100) as was suggested by

Hennig (1973), Griffiths (1972), and McAlpine (1989) based on morphology. In addition, we find support for the species-group subdivision originally proposed by

Newstead (1911) and later confirmed by studies on habitat preference (Jordan,

1974). The fusca species group is sister to the remaining Glossinidae as had been suggested by Newstead et al. (1924) and Bursell (1958). Our results therefore support these two proposed classifications over the alternatives proposed by

Machado (1959) and Pollock (1973).

Relationships within the bat fly clade have been particularly difficult to resolve.

Nirmala et al. (2001) did not recover it as monophyletic, while Dittmar et al. (2006) presented various results depending on which analytical method was used (see Fig.

27 1.1). Here, we find strong molecular support for the clade Streblidae+Nycteribiidae, a clade that had previously been suggested by numerous authors familiar with the morphology of these families (e.g., Hennig, 1973; McAlpine, 1989). As in other analyses for this clade based on morphological or molecular characters, the monophyly of Nycteribiidae is unproblematic and the main problem is the delimitation and monophyly of the Streblidae. Dittmar et al. (2006) demonstrated streblid paraphyly by showing that Old World Streblidae+Nycteribiidae form a monophyletic group regardless of which analysis technique they used to evaluate their data. Our analyses corroborate the latter view, although weakly, because our matrix contains only a single Old World streblid for which we were unable to collect the full sequence data (Fig. 1.2). Dittmar et al. (2006) speculated that the difficulties in getting a consistent resolution of the basal relationships in the bat flies are the result of early rapid radiation within those groups. However, a visual inspection of branch lengths within the bat fly clade for our full combined molecular dataset did not reveal noticeably shorter branches than those found within the Hippoboscidae, and thus we see little evidence within our taxon and gene sample for a unique and rapid diversification in bat flies.

1.4.2 Host-shifts and diversification in Hippoboscoidea

Evolutionary changes in host use for organisms with specialized feeding habits such as these obligate vertebrate ectoparasites can be driven by reciprocal co-evolution between host and parasite (parallel cladogenesis), host tracking -in which parasites colonize and become established on closely related hosts- or by less predictable host shifts that are unconstrained by host (Page, 2003; Labandeira, 2002). It is compelling to consider the potential for parallel cladogenesis in Hippoboscoidea.

However, the age of the split between mammals and birds (Blair & Hedges, 2005:

28 >310 MYA) greatly predates the earliest divergences found in extant hippoboscoid flies given that the earliest confirmed calyptrate fossil is only from the Oligocene

(Michelsen, 2000). Parallel cladogenesis may be found in specific groups of bat flies, but definitive tests await more detailed and thoroughly sampled phylogenetic studies of nycteribiids and streblids.

Strikingly, within Hippoboscoidea true ectoparasitism originated only once in the common ancestor of the Pupipara (Fig. 1.3). This specialization involved a change from a free-living, and blood feeding fly (e.g., Glossinidae) to a fly with an obligate and close association with a particular vertebrate host. This specialization could have contributed to the large observed difference in species diversity between the sister groups Glossinidae (22 spp.) and Pupipara (630 spp.); and, it has been postulated that specialization of feeding structures, host finding behavior, and population subdivision associated with parasitism spurs species diversification (e.g. Mitter et al.,

1988; Price, 1980), although this may not be a general trend in carnivorous parasitic insects (Wiegmann et al., 1993). Calyptratae includes multiple diverse lineages of flies with specialized feeding habits such as blood-feeding, parasitoidism, vertebrate endoparasitism and phytophagy. Better resolution of the phylogeny of Calyptratae, including a more exact placement for the Hippoboscoidea, will be necessary to fully gauge the possible affects of trophic specialization on diversification rates within this group.

When host-association is mapped onto our combined molecular phylogeny (Fig. 3), we find that mammal feeding is ancestral for the Hippoboscoidea. Based on this tree, feeding on birds has evolved at least twice; once in the Ornithomyini and once within the Olfersini while the ancestor of the Hippoboscidae was still feeding on the blood of

29 mammals (contra Bequaert, 1954). However, this scenario is likely an oversimplification because our taxon sample is not extensive enough to illustrate the complete story. For example, we lack data for the bird-feeding Struthiobosca which is currently placed in the otherwise fully mammal-feeding Hippoboscinae. Better sampling of hippoboscines would also be necessary to allow determination of whether the ancestral host of this subfamily was a bird or mammal.

A major success story in Hippoboscoidea evolution was the host shift to bats. When we optimize a host-association as a two-state character onto our tree (mammal vs. bird), we find that the shift to bats as hosts originated from species already feeding on mammals. The bat flies comprise approximately 500 species, a much larger number of species than their sister group, the Hippoboscidae with its approximately

150 species. Given that sister groups are of equal age, either the speciation rates in the former must have been higher or hippoboscid species experienced higher extinction rates. Our species sample is insufficient for studying the evolution of host specificity in Nycteribiidae and Streblidae, but more research in this area will be of central importance in understanding the major differences in species richness between the bat- and hippoboscid flies.

30

Figure 1. 3. Key events in the evolution of the Hippoboscoidea.

31

1.4.3 Morphological and life history evolution in Hippoboscoidea

A partial or complete reduction of wings is often encountered within the

Hippoboscoidea, and the tree topology in our analysis clearly indicates at least four complete or partial losses. Additional losses and cases of wing shedding will very likely be found once the taxon sampling is better for Streblidae. For example,

Ascodipteron also lacks wings, but is not included in the data set. Within our taxon sample, the first reduction is within the Hippoboscidae where the monophyletic clade consisting of Stenepteryx+Crataerina is characterized by wings of reduced size.

Indeed this is one of the reasons for the separation of these genera from the more

“generalized” species in the genus Ornithomya with their fully developed wings.

Secondly, in the subfamily Lipopteninae the genus Melophagus is characterized by the complete loss of wings and halteres. The other species in the Lipopteninae emerge from the puparium with fully developed wings and use flight to find an appropriate host. They then shed their wings close to the base and spend the remainder of their life on the host. The third reduction of wings is found in the

Nycteribiidae, which, however, retain their halteres. Finally, the streblid Megistopoda aranea has straplike wings (i.e., is stenopterous), although, according to our cladogram, its ancestor was fully winged.

Loss of wings presumably allows easier and unhindered movement on the host and is common among ectoparasitic insects (Andersen, 1997). With at least four independent losses of wings within the superfamily, the Hippoboscoidea is no exception to this general pattern. However, while beneficial to an ectoparasite that needs to stay firmly attached while feeding and/or move about quickly on the host, the loss or reduction of wings must also severely impede dispersal and host finding.

32 This problem may explain why wing loss is common in bat flies (>50% of all species).

Bats are often gregarious and continue to use the same roosts for many generations which makes it relatively easy for bat flies to place their offspring in the direct vicinity of future hosts. A similarly conducive host ecology may also explain the complete loss of wings in the sheep ked, Melophagus ovinus. The females glue puparia to the fleece of the sheep and the frequent contact between host guarantees that the offspring will have access to a wide variety of hosts.

One of the most fascinating traits of all Hippoboscoidea is their ability to give birth to fully developed 3rd instar larvae. In the Glossinidae, the deposited larva is fully motile and actively burrows into the soil for pupariation. If the larva is deposited onto a suitable substrate, the larva can bury itself and start pupariation within 15 minutes. If deposited in an unsuitable location, it can delay the process by up to six hours

(Finlayson, 1967). In the Hippoboscidae, the deposited larvae are almost completely immobile, incapable of burrowing, and in most cases begin pupariation within the female (Bequaert, 1952). In the bat flies the immobilization is even more pronounced with deposited larvae being completely immobile. They are glued to the ceiling or wall of the bat roost by the female, after which they immediately pupariate (“pupiparity”).

Our phylogenetic hypothesis is compatible with a gradual loss of motility. The formation of the puparium inside the female or immediately after deposition would be a synapomorphy for Pupipara, while gluing the puparium to the walls of bat roosts would be a synapomorphy of the Streblidae+Nycteribiidae.

33 1.5 CONCLUSIONS

Our sequence data provide convincing phylogenetic evidence for the monophyly of

Hippoboscoidea, Glossinidae, Hippoboscidae, Nycteribiidae, Hippoboscinae,

Lipopteninae, Ornithomyini, Olfersini, Nycteribiinae, Trichobiinae, the Glossina palpalis-, and the Glossina morsitans species-groups within Glossinidae. The data also provide weak evidence for a paraphyletic Streblidae. Additional detailed studies based on a larger taxon sample within hippoboscoid families are needed to more fully investigate the evolution of specialization and host use in the families of

Hippoboscidae as well as its specific phylogenetic position within the calyptrate

Diptera. Nucleotide data should continue to provide an important independent phylogenetic data source for evolutionary investigations of these uniquely specialized flies.

34

CHAPTER 2

Sensitivity analysis, molecular systematics, and

natural history evolution of Scathophagidae

(Diptera: Cyclorrhapha: Calyptratae)

2.1 INTRODUCTION

The Cyclorrhapha contain about 60,000 described species and exhibit a remarkable diversity in breeding habits ranging from saprophagy over phytophagy to parasitism and predation. In order to understand how this diversity has evolved, studying the life history evolution across Cyclorrhapha is important. However, given the size of the

Cyclorrhapha, it may be advisable to first study the life history evolution of a small subclade with an unusual diversity in larval habits. A good choice for such a subclade is the Scathophagidae. One of the most striking features across this relatively small family of 250 described species is the extreme diversity in biology. Scathophagids breed in different types of dung or other decaying organic matter like rotting seaweed

(Gorodkov, 1986; Vockeroth, 1989), mine in leaves, bore in culms and feed on immature flowering heads, or on seed capsules and ovules. Larvae of a few species are also predators of small invertebrates or caddis fly egg masses; i.e., although commonly known as ‘dung flies’, only a few species in the genus actually breed in dung. In contrast to the larvae, adult scathophagids appear all to be predators of other invertebrates.

Scathophagid systematics is in a state of chaos at multiple levels. The

Scathophagidae along with the families Fanniidae, Muscidae and Anthomyiidae comprise the superfamily Muscoidea within the Calyptratae (McAlpine, 1989). This superfamily has been regarded by some authors as a group of convenience

(Michelsen, 1991; Bernasconi et al., 2000b) while others considered it monophyletic

(McAlpine, 1989). The position of the Scathophagidae within Muscoidea (if monophyletic) is similarly contentious. It is sometimes regarded as the sistergroup to the Anthomyiidae (Bernasconi et al, 2000b) or at other times as the sistergroup of all

36 remaining Muscoidea (McAlpine & Wood, 1989). Yet other authors treat the

Scathophagidae as a subclade of subfamily rank of either the Muscidae or

Anthomyiidae thus assuming a close relationship of Scathophagidae to either of these families (Hackman, 1956; Vockeroth, 1956, 1965, 1989). To make matters worse, there is not even convincing evidence for the monophyly of the

Scathophagidae and the same applies for its two currently recognized constituent subfamilies (Scathophaginae, Delininae: Gorodkov, 1986; Vockeroth, 1989).

Morphological characters have been successfully used for developing identification keys and creating an overall satisfactory genus-level taxonomy. However, as is evident from our account on scathophagid phylogenetics, morphological characters have been less successful for determining the position of Scathophagidae within

Calyptratae or reconstructing the relationships within the family (Bernasconi et al,

2000a, b, 2001).

In 2000, Bernasconi et al (2000a) addressed these phylogenetic problems by carrying out analyses based on COI sequences for 61 species representing 22 genera of the Scathophagidae. The most parsimonious tree provided moderate support for many genera, but the higher-level clades only had weak or no bootstrap support. Here, we present the results of a cladistic analysis of the Scathophagidae with additional DNA sequences from the mitochondrial genes 12S rRNA, 16S rRNA,

Cytochrome oxidase subunit I, Cytochrome b and the nuclear genes 28S rRNA,

Elongation factor 1-alpha (Ef1a) and RNA polymerase II (Pol II). In this analysis, we have also added two scathophagid species and include 11 outgroups representing the remaining muscoid families Fanniidae, Muscidae and Anthomyiidae in order to be able to rigorously test the monophyly of Scathophagidae and to identify its closest relative.

37

Phylogenetic research on Scathophagidae is particularly timely because the scathophagids are widely used in behavioral ecology. For many years, this only applied to (Scathophaginae) which was frequently used as a model organism in behavioral studies (e.g., Parker, 1970; Hosken, 1999). However, recently scathophagid research has become more comparative and now includes species from across the entire family although prior work on the phylogenetic relationships only yielded weakly supported hypotheses. For example, a recent study investigating the co-evolution of male and female reproductive characters utilized the tree based on COI, although many branches have little support (Minder et al., 2005).

The modes of feeding associated with the larvae of the Cyclorrhapha and

Scathophagidae can broadly be divided into phytophagy, saprophagy, parasitism, and predation. In general, decaying organic matter is considered the primitive larval medium for Cyclorrhapha from which more specialized forms of feeding like phytophagy and predation have evolved (Ferrar, 1987). The shift to phytophagy is seen as an evolutionary hurdle that, once overcome, can lead to rapid radiation and diversification (Mitter et al., 1988). Here, we identify the ancestral feeding habit of the

Scathophagidae and reconstruct the origins of saprophagy, predation, and phytophagy. Such tracing requires a robust and well-supported phylogenetic tree for the Scathophagidae that we propose here based on multiple genes.

2.1.1 Comparing alignment techniques through a sensitivity analysis

Character matrices consisting of molecular data can be analyzed using many different alignment strategies and weighting regimes. Depending on the strategy and weighting regimes that are used for the analysis, the reconstructed phylogeny will

38 differ (Wheeler, 1995; Wheeler and Hayashi, 1998). Here, we use a sensitivity analysis for identifying optimal analysis conditions for our dataset. Our study follows

Laamanen et al., (2005) in that we use the same transition, transversion and gap costs matrices and use multiple criteria for choosing optimal analysis conditions

(topological congruence, character incongruence, node support). Laamanen et al.

(2005) had found that character incongruence, tree support, and topological congruence favored similar analysis conditions.

However, in the current analysis we go beyond Laamanen et al. (2005) and other studies comparing alignment algorithms (Terry and Whiting, 2005) in that we compare three different alignment strategies: ClustalX (default parameters), Direct

Optimization (POY), and “manual” alignment. We believe that such comparison of different alignment strategies is particularly important because it is well established that they profoundly influence the outcome of cladistic analyses (e.g., Morrison &

Ellis, 1997). Yet, currently most systematists are either firm practitioners of manual alignment or strong believers in the superiority of optimization alignment; i.e., few analyses systematically compare the results obtained under the two most popular techniques, although such comparative data may help in choosing between the available methods and provide critical information for interpreting trees based on data obtained using different alignment strategies. Lastly, here we test Kluge & Grant’s

(2005) contention that “node stability” sensu Giribet (2003) and node support are largely synonymous concepts by studying the correlation between both measures for the nodes of our trees.

39

2.2 MATERIALS AND METHODS

2.2.1 Taxa and DNA extractions

Sixty-three species of Scathophagidae are included in the analysis. Whole flies were frozen and pulverized in liquid nitrogen before the DNA extraction was performed using the QIAamp tissue kit (QIAGEN) according to manufacturer’s instructions. DNA was eluted in 400 µl of distilled water or buffer. Compared to Bernasconi et al.

(2000a), we have added 13 taxa (Table 2.1) representing the other muscoid families

Fanniidae, Muscidae and Anthomyiidae. DNA extractions for the outgroup species and two scathophagid species were performed using phenol-chloroform extraction.

The specimens were lysed in CTAB buffer, 20ηL Proteinase K was added and the samples were incubated overnight at 55°C. DNA was extracted using phenol: chloroform: isoamyl alcohol (25:24:1) mix and precipitated with 100% ethanol. After washing the DNA pellets in 70% ethanol, they were dissolved in 50-100 µl of water.

2.2.2 DNA amplification and sequencing

Standard PCR amplifications were carried out with Bioline Taq and Takara Ex-Taq using approximately 1-5 µl of the DNA extractions to amplify the five different gene regions of interest. The genes sequenced for the Scathophagidae taxa are 12S, 16S,

28S, Ef1a and Pol II. Amplification of COI sequences for species lacking information in Bernasconi et al (2000a) was also carried out. The primers used in this study are given in Table 2.2. All genes were sequenced for the outgroups. The PCR cycles consisted of an initial denaturation step at 95°C for 7 minutes, followed by 95°C for

1.5 minutes, annealing at temperatures ranging from 44-50°C and extension at 72°C for 1.5 minutes. A final extension at 72°C for about 5 minutes was also added. The

40 amplified gene products were purified using Bioline Quick-Clean solution following the manufacturer’s protocol. Cycle sequencing reactions was performed on the purified products using BigDye Terminator v3.1. The products were then prepared for direct sequencing by removing the dye-terminator using 5µl of Agencourt CleanSEQ solution. The sequences were edited and assembled in Sequencher 4.0. The protein encoding genes COI, Cytb, Ef1a and Pol II were aligned in the same program and yielded gap-free alignments.

The ribosomal gene sequences for 12S, 16S and 28S were treated using three different techniques. First, an alignment was obtained using Clustal X 1.8 (Higgins &

Sharp, 1988) with the gap opening and extension cost set at the default (15:6.66).

Second, this Clustal alignment was manually re-aligned in MacClade (Maddison and

Maddison, 2000). This adjustment was taxon-blind; i.e., the taxa names were not usd during the manual optimization of the alignment. Third, direct optimization (Wheeler et al., 2003) was used as implemented in POY (Wheeler, 1996; Wheeler et al., 2003; documentation by De Laet and Wheeler, 2003) The indel cost was varied from 1-10

(see Fig. 2.2). For two reasons we refrained from differentiating between gap opening and gap extension costs (“affine gap costs”: Watermann et al., 1976; Gotoh,

1982), although recent analyses suggest such treatment may improve congruence between gene partitions (Aagesen, 2005). Firstly, we were concerned about potential violations of metricity in costs sets consisting an opening, extension, and base change cost. Secondly, comparing the results of affine gap costs in POY with the other alignment techniques would be difficult given that different gap opening and extension costs cannot be easily implemented in cladistic analyses based on fixed alignments.

41

2.2.3 Tree search strategies

The dataset including fixed alignments was analyzed using parsimony as implemented in PAUP* 4.0 (Swofford, 2002; using batch files for carrying out the various procedures needed for the sensitivity analysis; see supplementary material).

For each weighting regime, we carried out a heuristic search using 100 random sequence additions replicates and TRB branch swapping. Node support was assessed using Jackknife values (250 replicates, 100 random addition sequences each) obtained at 36.80% deletion as recommended in Farris et al. (1996). The data was analyzed using five cost matrices that define different weighting regimes for state transformations. Transitions were downweighted by assigning higher weights to transversions (2, 4, 6, 8 and 10). Third positions of protein encoding genes were downweighted by applying the same higher weights to the first and second positions and the sequences for the ribosomal genes. The analysis was carried out with gaps coded as missing data as well as using the gaps as fifth character state. When coded as fifth character state, gaps were given at most half the weights of the transversions to avoid violations of triangular inequalities (Wheeler, 1993).

The dataset was also analyzed using parsimony and direct optimization as implemented in POY (Wheeler et al., 2003; documentation by De Laet and Wheeler,

2003). The protein-encoding genes were entered as prealigned data. The sequences for 28S rDNA were divided into three fragments using hyper-conservative stem regions as break points while the relatively short 12S and 16S fragments remained whole. The sequences were analyzed using POY in a parallel computing mode on two clusters at Singapore’s National Grid (“Melon”: 4 nodes with 4 Xeon CPUs;

“Hydra3”: 10 nodes with 4 Itanium2 CPUs) using the following string of commands:

42 - parallel -jobspernode 2 -onan -onannum 2 –dpm -norandomizeoutgroup -maxtrees

10 -holdmaxtrees 100 -fitchtrees -seed -1 -slop 5 -checkslop 10 -dpm –multirandom

-replicates 10 -treefuse -fuselimit 10 -fusemingroup 5 –fusemaxtrees 100. For the preferred analysis condition, we increased the number of replicates to 100 (same tree length was found as in the 10 replicate analysis). The analysis was carried out using the same cost matrices that were used for the fixed alignments. Node support was assessed using the Jackboot option in POY (-parallel -jackboot -jobspernode 4 - onan -onannum 4 -dpm -norandomize outgroup -maxtrees 10 -holdmaxtrees 1000 - fitchtrees -seed -1 -slop 5 -checkslop 10 -dpm -multirandom -replicates 100). The number of replicates was increased to 250 for the preferred analysis condition.

2.2.4 Sensitivity analysis

Separate sensitivity analyses (Wheeler, 1995) were carried out for the three alignment strategies and two ways to partition the data: (1) protein-encoding genes versus ribosomal genes; (2) all genes versus each other. Branch support was assessed by summing the jack knife support values above 50 (Laamanen et al.,

2005). Character incongruence for the partitions was assessed using the ILD

(Incongruence Length Difference) as implemented in Laamanen et al., 2005 (Wheeler and Hayashi, 1998). We opted against RILD (Rescaled Incongruence Length

Difference) and MRI (The Meta-Retention Index) because we had previously found that RILD and ILD were highly correlated (Laamanen et al. 2005) and MRI remains unpublished (but see Aagesen et al., 2005). Topological congruence was determined for each partition by counting the number of nodes on the strict and semi-strict consensus trees (Laamanen et al., 2005). For the partitioning scheme using protein encoding genes versus ribosomal genes, the symmetric tree distance metric (Penny

43 and Hendy, 1985) and the corrected symmetric tree distance metric were computed

(Laamanen et al., 2005).

2.2.5 Natural history

Data for the breeding and feeding habits of larvae for the different species of the

Scathophagidae (Table 2.1) were compiled from the literature and personal observations of the third author. Natural history information was only available for a subset of the taxa used in this study and the tree was reduced by pruning all outgroups and terminals lacking natural history information. Larval breeding habits were mapped onto the most parsimonious tree (Fig. 2.5) using two different character definitions and “trace character” in MacClade. In the case of multiple optimizations, we inspected all equally parsimonious mappings. We first coded “phytophagy” as one character state in a multistate “natural history evolution” character (states: phytophagy, predation, saprophagy). Alternatively, we divided “phytophagy” into

“monocot phytophagy” and “dicot phytophagy.” This coding does not assume homology between feeding on monocots and dicots.

44

s t i b a h g n i d e e r b l a v r a l n w o n k d n a y d u t s n i d e s u a x a T

. 1 . 2 e l b a T

45

) d e u n i t n o c (

s t i b a h g n i d e e r b l a v r a l n w o n k d n a y d u t s n i d e s u a x a T

. 1 . 2 e l b a T

46

1 2. e bl a T

) d e u n i t n o c (

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47

Table 2.2. Primers used in study

48 2.3 RESULTS

Sequence data for the 78 taxa were compiled and concatenated in MacClade 4.0

(Maddison and Maddison, 2000). The ClustalX aligned dataset had 5060 characters whereas in the manually aligned dataset the number of characters is reduced to

5052. The jackknife support, ILD and symmetric tree distances for different partitions of the dataset from the various analyses using different weighting regimes for different alignment strategies and treatment of gaps as missing or information are summarized in Table 2.3.

2.3.1 Alignment techniques, indel treatment, character transformation weighting

The default Clustal alignment performs worst with regard to two optimality criteria: they have the highest character incongruence and lowest topological congruence regardless of whether indels are scored as missing values or 5th character states.

With regard to the third parameter, branch support, Clustal alignments outperform

POY, but not the manual alignment (however, it is unclear whether jackknife values based on fixed alignments can be directly compared to those from optimization alignment analyses). The manual alignment and direct optimization perform at similar levels. For the “protein vs. ribosomal genes” analysis, the lowest character incongruence is observed for POY while the manual alignment outperforms POY in the “all genes vs each other” sensitivity analysis. A similar pattern emerges with regard to topological congruence with both techniques yielding optimal values depending on which measure of topological congruence is used and how the dataset is partitioned.

49 Downweighting of third positions for protein-encoding genes increases character incongruence and lowers jackknife support as well as topological congruence for all three ways of aligning and two ways of partitioning the data. However, downweighting transitions improved tree support and lowered ILD values for all alignment techniques. The highest jackknife support was obtained for the weighting regime tv=4, when the analysis was carried out using gaps as information and the manually aligned dataset. The ILD values and symmetric tree distance between the protein and ribosomal partitions are lowest at tv=2 and tv=6 respectively but since the tree topologies for tv=2, 4 and 6 weightings are identical, we prefer the strict consensus tree of the two most parsimonious trees obtained at tv=4 (Fig. 2.1, 2.2).

This tree is used for our phylogenetic discussion and for tracing the natural history evolution. The tree topology of the favored POY treatment only differs with regard to outgroup arrangement; i.e., it is also very similar to the trees obtained based on manual alignment with transversion weighting. However, the equal weighting tree differs in several regards from the preferred tree (compare Fig. 2.1, 2.2 and Fig. 2.3).

All analyses of the dataset confirm the monophyly of the family Scathophagidae with the Anthomyiidae being its next closest relatives when the tree is rooted to Fannia armata. Based on our rooting and our very limited taxon sample, the Anthomyiidae are a paraphyletic group. However, the monophyly of the two subfamilies of

Scathophagidae is well supported and our phylogenetic hypothesis is thus consistent with the proposed subfamily classification of the Scathophagidae into the

Scathophaginae and Delininae (Gorodkov, 1986; Vockeroth, 1989) based on morphology. The data also confirm the monophyly of most genera including

Cordilura, , , Gimnomera, and and resolves the intergeneric relationships. The ancestral feeding habit in the Scathophagidae is

50 phytophagy from which saprophagy and predation have evolved. In general, we see that two shifts to saprophagy and one shift to predation has occurred. A second origin of predation is found in the otherwise saprophagous Scathophaga.

51 Fig. 2.1. Most parsimonious tree for all manual alignments and indel treatments under optimal analysis conditions (tv=4). Support values are indicated on branches as Jackknife support. The set of numbers toward the bottom of the squares represent the [transition, transversion, gap] costs used for the particular analysis. Black fields in the sensitivity plot signify agreement, white disagreement, and grey neither (polytomy).

52

Fig 2.2. Most parsimonious tree for direct optimization (in POY) under optimal analysis conditions (tv=4). Support values are indicated on branches as Jackknife support. The set of numbers to the bottom of each of the squares represent the [transition, transversion, gap] costs used for the particular analysis. Black fields in the sensitivity plot signify agreement, white disagreement, and grey neither (polytomy).

53

Fig 2.3. Most parsimonious tree under equal weighting using a manual alignment

54 s u o i r n a o i v t

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55 2.4 DISCUSSION

2.4.1 Sensitivity analyses and choice of alignment

Proposing a phylogenetic hypothesis for the Scathophagidae based on the results of our sensitivity analysis is at the same time difficult and relatively straightforward as the following discussion will reveal. We here use a number of different indicators to choose optimal analysis conditions and we find slight differences with regard to which weighting regime or alignment technique should be used. However, fortunately the tree(s) that are favored by all these indicators are either identical or very similar

(Fig. 1.2). Furthermore, we find an orderly pattern in the recommendations from the sensitivity analyses in that mostly similar weighting regimes yield optimal values (see also Terry and Whiting, 2005). This finding mirrors the results of a similar studies

(e.g., Laamanen et al., 2005; Aagesen, 2005; Aagesen et al., 2005) that indicated that sensitivity analyses can be based on several criteria (e.g., character incongruence, topological congruence, branch support) without obtaining wildly conflicting results.

We find that for the scathophagid data set, assigning higher weights to transversion than transitions is the preferred treatment. Such transversion weighting improves node support, character congruence, and topological congruence for all three different ways to align the ribosomal genes (ClustalX, Manual, POY). For our laboratory this is now the fourth sensitivity analysis in a row for which we find that transversion weighting is favored (Meier & Wiegmann, 2002; Damgaard et al., 2005;

Laamanen et al., 2005). We thus believe that this strategy may be of considerable importance for many data sets. For the scathophagid dataset, we find that several indicators favor a transversion weight from 1-10 with most preferring a weight of 2 or

56 4. Fortunately, the systematic conclusions are not affected since the tree topologies are identical for both weighting regimes. Unambiguous across all of our recent sensitivity analyses (Meier & Wiegmann, 2002; Damgaard et al., 2005; Laamanen et al., 2005) is the futility of downweighting third positions. Several authors had argued for such a treatment because third positions evolve at faster rates when compared to first and second positions (see references in Laamanen et al., 2005). In the present study, the downweighting of third positions never leads to any improvement in branch support, character congruence, or topological congruence and this conclusion holds for any of our three ways to align the data. This result adds to the mounting evidence that the downweighting of third position is generally not a useful technique (see references in Laamanen et al. 2005) and that third positions often contain important phylogenetic structure (e.g., Källersjö et al., 1999).

More controversial are our findings with regard to indel treatment and the preferred alignment technique. Giribet and Wheeler (1999) had strongly argued based on theory that (1) indels should always be coded as fifth character state and that (2) numerical alignments are always preferable over “manual” or “by eye” alignments.

With regard first issue, we find that for our Clustal alignment, coding indels as missing values decreases character incongruence and increases topological congruence and branch support; i.e., all optimality criteria indicate that indels should be treated as missing data. For the manual alignment, the results are more ambiguous. Depending on what measure is used and how the data are partitioned, character incongruence and topological congruence either improve or worsen while jackknife support is highest for the analysis coding indels as missing data. One could argue that these results may be due to problems with implementing the same indel costs during alignment and cladistic analysis, but unfortunately this cannot be tested

57 because the default alignment parameters of Clustal use affine gap costs and manual alignments do not utilize a fixed cost matrix. What is more important from empirical point of view is that our result indicates that at least for some datasets and alignments treating gaps as missing data may be appropriate. In any case, we find that for our Clustal-aligned dataset one can either honor the call for using indels as character states or one can optimize character incongruence and topological congruence. Both goals are not simultaneously attainable and we would argue that then the choice of the indel-coding technique becomes a matter of value judgment. It appears to us that overall character incongruence and topological congruence are more important than the theoretical arguments in favor of coding indels as fifth character states; i.e., if one were to use an unmodified Clustal alignment, one should code the gaps as missing values.

With regard to the second issue, the preference of numerical alignments over manual ones, we have to disagree with Giribet and Wheeler (1999), because our sensitivity analyses clearly reveal that the manual alignment is outperforming the Clustal alignment with regard to character incongruence, topological congruence, and branch support. We thus do not see any evidence that the “numerical” alignment produced by Clustal is a viable alternative to manual alignment for those users who would like to use a fixed alignment for their cladistic analysis (see also Laamanen et al., 2005). Our results thus lend some support to the wide-spread practice of using manual alignments. Note also, that the results based on such alignments are similar to those based on morphological character matrices in that they are perfectly repeatable at the cladistic analysis stage. For this reason alone, we find it difficult to see why a technique that is acceptable for morphological data should be rejected for

DNA sequence data. Of course, we agree with Wheeler (2003) that it would in

58 principle be preferable to use a technique that automates alignment and tree search.

But this does not answer the question of what one should do if automatization sacrifices phylogenetic accuracy as measured by partition congruence.

But is there an automated technique that performs as well as manual alignments?

We find that the results based on our manual alignment and direct optimization are indeed very similar with regard to character incongruence, topological congruence, and preferred tree. The main difference lies in branch support where the manual alignment greatly outperforms direct optimization. Unfortunately, it remains unclear whether jackknife values across the different techniques can be directly compared because the jackknifing of a fixed alignment employs different sampling techniques than jackknifing during optimization alignment. We thus urge further study in this area because more information on this issue will be important when comparing support values from different studies. Could it be that the jackknife values from POY are generally lower than those from analyses using fixed values? Only additional studies exploring different alignment techniques can resolve this issue. Fortunately, in the case of the Scathophagidae, the trees favored by POY and the manual alignment are topologically identical with regard to ingroup relationships; i.e., the choice of analysis technique does not influence our systematic conclusions. The only difference concerns the Muscidae which are not monophyletic on the POY trees although muscid monophyly is well supported based on morphological characters; i.e., the tree based on the manual alignments is here favored. This is similar to Meier and

Wiegmann’s analysis (2002) of coelopid relationships (Diptera). Here, only the analysis using a manual alignment of 16S rDNA recovered the monophyly of

Coelopidae and the authors favored the trees obtained from the manual alignments given the strength of the morphological evidence.

59

2.4.2 Node support and Node stability

One remarkable phenomenon displayed by our dataset is that the tree topology supported by equal weighting (Fig. 2.3) differs in several important regards from the topology of our favored tree while the latter is very stable with regard to different transversion and 3rd position weights. For example, the sister group relationship between and Nanna clades is not supported on the equally weighted trees and the clade consisting of the genera Hydromyza, Spaziphora, ,

Pogonota, Trichopalpus, and Okeniella which is a sister group to the

Scathophaga clade on the equally weighted tree moves to the base of the

Scathophaginae on the preferred tree. It is in discussing this phenomenon that

Giribet’s (2003) concept of node stability proves its utility. Many nodes on our scathophagid tree are extremely stable (Fig. 2.1,2.2) in that they are supported by most or all weighting matrices utilized in our sensitivity analyses and are furthermore insensitive to alignment techniques. Node stability sensu Giribet (2003) has been criticized by Kluge and Grant (2005) who argued that it is epistemologically unsound and questioned that node stability and node support are fundamentally different concepts. However, as pointed out by Giribet (2003), it is not uncommon to find nodes that are very stable across many analysis conditions, but have relatively low support as measured by jackknifing or bootstrapping. Our tree furnishes additional examples such as the Gimnomera+Scathophaga clade that is recovered under almost all analysis conditions although the jackknife support is usually less than 50%.

Overall, we find that a plot of nodal support versus nodal stability across the entire tree ( 2.4) reveals that the two measures are only mediocre predictors of each other.

Interestingly, node stability is higher for direct optimization while we pointed out earlier that node support is higher for a fixed alignment.

60

Fig 2.4. Number of times a node is recovered out of the ten different analysis conditions (nodal stability) under manual alignment (•) and direct optimization alignments () versus Jackknife support at the node

.

2.4.3 Systematic conclusions

We had earlier remarked that scathophagid systematics is in state of chaos at multiple levels. Some of this chaos can be resolved based on the results of our study.

Commenting on the monophyly of the Muscoidea and the position of Scathophagidae would require sampling non-muscoid calyptrate and acalyptrate outgroups while our study only included muscoid taxa. However, regardless of which branch is used for rooting, certain hypotheses are never supported. For example, a clade consisting of

Muscidae+Scathophagidae is non-monophyletic under all possible roots while other proposals such as a sistergroup relationship between Scathophagidae and the remaining Muscoidea and a clade composed of Anthomyiidae+Scathophagidae are viable options. The latter was favored in a preliminary analysis in which we used the acalyptrate Curtonotum helvum as a non-muscoid outgroup. Unfortunately, the data

61 for Curtonotum was too incomplete to allow for a formal inclusion in this study, but we consider it likely that Anthomyiidae+Scathophagidae will form a clade.

More definite answers can be provided with regard to the monophyly of

Scathophagidae and its two constituent subfamilies. All three taxa are well-supported as monophyletic. Equally well supported are a number of important genera such as

Cordilura, Nanna, Norellia, Gimnomera, Hydromyza and Spaziphora. Some of the remaining genera are more problematic, but in most cases the issues can be resolved by eliminating small or monotypic genera. For example, based on our data,

Pogonota (7 spp in genus) is paraphyletic with respect to Okeniella (3 spp in genus) which should probably be synonymized with the former. Chaetosa (2 spp.) is similarly paraphyletic with respect to Trichopalpus (5 spp.; Hackman, 1956; Vockeroth, pers. comm; Bernasconi et al., 2000a) and the former should probably be synonymized with the latter. However, formal synonymization should await a study of all species.

The relationships within the relatively speciose genus Cordilura are not well supported and relatively labile to variation in analysis conditions, but its monophyly is supported as long as the monotypic Phrosia is synonymized with Cordilura. The position of Phrosia has been controversial at the subfamily-level. Gorodkov (1986) had placed it in the Delininae, a position that had been questioned by other authors

(Püchel, pers. comm) who considered it part of the Scathophaginae based on morphological similarities of the adults to Cordilura. The latter position found some support in Bernasconi et al. (2000a), but is now very well supported by our new data set (jackknife=100). Both Cordilura and Phrosia have phytophagous larvae living in plants in wet habitats with the former apparently being restricted to Carex while

Phrosia is known to feed on Liliaceae.

62

The sister group of Cordilura is a clade containing Nanna, ,

Neorthacheta dissimilis, and Orthacheta cornuta. Again, there has been some taxonomic disagreement, especially with regard to the placement of Cleigastra apicalis in the scathophagid subfamilies (Collin, 1958; Kloet & Hincks, 1976;

Gorodkov, 1986; Nelson, 1988). Bernasconi et al. (2000a) favored a placement in the

Scathophaginae (Bernasconi et al., 2000a) and our analysis again suggests the same. Norellia and Gimnomera are both monophyletic with high jackknife support.

Based on the number of bristle rows on the tibia of the first leg, the genus Norellia has been divided into two subgenera (Sack, 1937; Šifner, 1995; Gorodkov, 1986):

Norellia, Norelliosoma. The only species in the subgenus Norellisoma (N. tipularia) is placed as the sisterspecies of all Norellia so that this subdivision is consistent with our phylogenetic hypothesis.

The monophyly of Scathophaga has been the subject to some speculation. We find that monophyly can only be restored if the monotypic Ceratinostoma is synonymized with Scathophaga while the proposed subgenus classification of Scathophaga is consistent with our tree (Cuny, 1983; Vockeroth, pers. comm). The subgenus

Scathophaga is monophyletic and consists on our tree of S. analis, S. inquinata, S. lutaria, S. cineraria, S. suilla, S. taeniopa, S. incola, S. furcata, S. tropicalis, and S. stercoraria. Also monophyletic is the subgenus Coniosternum (S. obscura, S. tinctinervis) which had previously been treated as a separate genus (Bernasconi et al., 2000a, 2001) and was thought to be closely related to Scathophaga (Hackman,

1956). A third group within Scathophaga consist of S. calida and S. litorea and

Ceratinostoma. The larvae of the latter all develop in decaying brown algae.

63 2.4.4 Natural history evolution

The Cyclorrhapha exploit a large diversity of habitats and breeding substrates with the ancestral larval breeding habit widely being thought to be saprophagy (Ferrar,

1987). From here, other breeding habits such as phytophagy, parasitism, and predation are thought to have evolved. However, our tree for Scathophagidae suggests a very different scenario for this family. The ancestral feeding mode is phytophagy. The larvae of the closest relative the Anthomyiidae, breed in different substrates including decaying organic matter, dung, flowering plants, ferns and fungi

(Ferrar, 1987). Determining the ground plan condition for this family is unfortunately not possible given that the phylogenetic relationships within Anthomyiidae are poorly understood and we only include few species. It thus remains unclear whether the phytophagy in Scathophagidae is homologous to the phytophagy known from many species of Anthomyiidae. However, contrary to scenarios proposed in the literature, saprophagy in the form of breeding in dung or other decaying organic matter is not the ancestral mode and has instead evolved twice from phytophagy. Predation has evolved two times independently, once from phytophagy and once from saprophagy

(Fig. 2.5).

64

Fig. 2.5. Tracing of natural history evolution in the Scathophagidae. One of the optimizations when phytophagy is coded separately as dicot and monocot (represented by symbols) is shown here. In this case, the Scathophagid ancestor was feeding on monocot plants. Taxa for which there is either species-specific information or at least statements about the genus have been included.

65 The first shift to saprophagy involves the genus Scathophaga. Most species are coprophagous while others have shifted to other decaying organic matter. Most notably, S. calida, S. litorea, and Ceratinostoma have become specialists for decaying brown algae stranded on beaches, a breeding substrate that is popular with many cyclorrhaphous flies that are otherwise predominantly known from dung

(Moeller, 1965; e.g., Sepsidae: Orygma; : Thoracochaeta). The second shift to saprophagy from phytophagy involves Cleigastra apicalis and can illustrate how saprophagy can evolve from phytophagy. The larvae of Cleigastra apicalis are found in galls made by Lipara (Choropidae) or tunnels made by lepidopterous larvae on Rumex and Phragmites. However, the Cleigastra larvae are not phytophagous or predacious as previously thought (see Chandler and Stubbs,

1969). Instead they feed on the frass of caterpillars in these tunnels (Groth, 1969); i.e., the saprophagous Cleigastra larvae are still intimately associated with other phytophagous insects although it is no longer directly feeding on plant tissue.

A few species of scathophagids are not only predatory as adults, but also have predatory larvae. Scathophaga obscura larvae feeds on the egg masses of caddis flies (Berte & Wallace, 1987) and according to our phylogenetic hypotheses evolved this predatory behavior from saprophagous ancestors. The same unusual substrate is used by Acanthocnema larvae which are found feeding on egg masses of caddis flies in swift streams (Hilton, 1981; Suwa, 1986; Anderson, 1997). Given the independent evolutionary origin, it is not surprising that Nelson (1992) found that the mode of feeding and mouth hook morphology differs significantly between

Acanthocnema and Scathophaga. Acanthocnema belongs to the second predatory clade in the Scathophagidae which also comprises Spaziphora, Chaetosa, and

Trichopalpus. This predatory clade has larvae living on lake shores and in sewage

66 where they feed on small invertebrates (Graham, 1939; Irwin, 1978). The larvae of the sistergroup, Hydromyza, mines in leaves or tunnels in submerged petioles of

Nymphaceae (Nuphar and Nymphaea) which suggests that predation evolved from a phytophagous ancestor with aquatic or semiaquatic larvae.

Most phytophagous species of Scathophagidae are only known from one host species, but these very narrow host-ranges could be due to the generally sparse information on the breeding habits of scathophagids. Unfortunately, the optimization of life history characters does not unambiguously resolve whether the scathophagid ancestor was feeding on monocots or dicots and whether it deposited its eggs on or into the plant host. The former is known from the Delininae which feed on

Orchidaceae, Liliaceae, and Commelinaceae while the Scathophaginae insert their eggs into the plant material and utilize a range of monocot and dicot hosts.

Depending on character coding, phytophagy itself only evolved once or several times. When phytophagy is a priori considered homologous and thus treated as a single character state, it is found at the base of the scathophagid tree and has been lost three times. However, a more complex picture emerges when a non-additive phytophagy presence/absence character is used and different character states are assigned to phytophagy on monocots and phytophagy on dicots. Fourteen equally parsimonious optimizations suggest a wide range of scenarios ranging from three origins of phytophagy with one host shift between monocots and dicots to a single origin of phytophagy and three host shifts. However, the relationship between host and the phytophagous scathophagid species is not as chaotic as this may suggest.

Some genera are entirely restricted to particular families of plants; i.e., it appears that over short time periods scathophagids rarely undergo dramatic host shifts. For example, Hydromyza are only known from water lilies (Nymphaeaceae), Cordilura

67 only known from Carex (Cyperaceae), Nanna only known from Gramineae, and

Gimnomera feeds on the seeds and ovules of Scrophulariaceae. An exception is

Norellisoma which appears to utilise a wide range of hosts (Liliaceae, Rosaceae,

Polygonaceae) and Norellia (Amaryllidaceae: Narcissus L. and Leucojum L.).

2.5 CONCLUSIONS

Our study manages to clarify many important issues surrounding the systematics of

Scathophagidae. It also provides a first insight into the relationships within the

Muscoidea and is a start for more extensive work on Calyptratae involving the remaining families in this group. We also demonstrate that simplistic ideas about the evolutionary relationships between saprophagy, phytophagy, and predation will have to be revised. In Scathophagidae, phytophagy is an important launching platform for becoming saprophagous and predacious. We are confident that the ongoing work on the Assembling the Tree of Life for Diptera will soon provide new data for testing long-standing hypotheses about natural history evolution in Diptera and other insects.

68

CHAPTER 3

The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial

and four nuclear genes

3.1 INTRODUCTION

With approximately 7,000 species in four families, the Muscoidea constitute a very significant portion of the described dipteran diversity (ca. 5%). Many of the muscoids are familiar flies that we encounter on a daily basis. For example, the most speciose family, the Muscidae, includes the (Musca domestica) and the stable fly

(Stomoxys calcitrans). The best known scathophagid is the yellow dung fly

(Scathophaga stercoraria), which is widely used as a model organism in behavioral biology, and some species of Anthomyiidae are important agricultural pests as larvae with the best-known examples being the onion fly (Delia antiqua) and the cabbage root fly (Delia radicum). The best-known species from the relatively small family

Fanniidae is the lesser housefly (Fannia canicularis), and some Fannia species play an important role in forensic entomology.

The Muscoidea are one of the three superfamilies in the Calyptratae, but due to the lack of a unique autapomorphy that would support the monophyly of Muscoidea, the taxon has often been considered a group of convenience or a potentially paraphyletic residual. For example, Michelsen (1991) characterized the Muscoidea as “the calyptratae less the Hippoboscoidea and Oestroidea”. However, the non-monophyly of the Muscoidea is far from universally accepted. For example, the Muscoidea are considered monophyletic by McAlpine (1989), who argued for the monophyly based on a combination of morphological character states such as the male anus being situated above the cerci, the male sternite ten forming bacilliform sclerites, and the female abdominal spiracle seven being located on tergite six (Hennig, 1973;

McAlpine, 1989). However, as some authors have pointed out, these character states may be plesiomorphic with respect to the Oestroidea (Michelsen, 1991). With regard

70 to the position of Muscoidea within Calyptratae, McAlpine (1989) proposed a sister group relationship between Muscoidea and Oestroidea based on the reduction of the male sternite 6, the female abdominal segments 6 and 7 being modified for oviposition, strongly developed vibrissae, a close connection between surstyli and cerci and a female hypoproct with lingulae.

Despite the Muscoidea being speciose and receiving considerable attention from applied entomologists, the phylogenetic relationships within the superfamily and the position of the Muscoidea within the Calyptratae have rarely been addressed. The constituent families of the Muscoidea are generally considered monophyletic but the phylogenetic relationships between these families are far from understood and additional research based on molecular, morphological and other data is necessary before this significant portion of Diptera diversity can be reliably placed on the tree of life (Bernasconi et al., 2000b).

3.1.1 Fanniidae

The smallest family in the Muscoidea is the Fanniidae with about 335 described species in four genera that are mostly found in the Holarctic and Neotropical regions.

As larvae almost all species feed on a wide variety of decaying organic matter and a few can cause human myiasis. The monophyly of this family has been supported by morphological character states such as the shape of the apical part of the subcosta

that curves evenly towards the costa and a strongly curved vein A2. Fanniid larvae are furthermore characterized by lateral fleshy projections. While the monophyly of the Fanniidae may seem strongly corroborated, the phylogenetic relationships within the family are still poorly understood. The monotypic Australofannia Pont is currently considered the sister group to the remaining members of the family (Pont, 1977)

71 because it retains the ejaculatory apodeme that apparently has been lost in all other

Fanniidae.

3.1.2 Muscidae

There are approximately 5000 described muscid species in some 170 genera and the family is amply represented in all biogeographical regions. The larvae are usually saprophagous while adults can be saprophagous, predacious, hematophagous, or feeders on nectar and pollen. Many muscids are vectors of disease. Presumably because of the large number of species, genera, and subfamilies, many different and often conflicting classifications and phylogenetic hypotheses have been proposed for this group (Malloch, 1934; Séguy, 1937; Roback, 1951; Hennig, 1955–1964, 1965;

Couri and Pont, 2000; Carvalho and Couri, 2002; Couri and Carvalho, 2003; Savage et al., 2004). Muscid monophyly is generally considered as uncontroversial, although it is supported by only a few morphological character states. These include the loss of both the female abdominal spiracles 6-7 and the male accessory glands (Séguy,

1937; Roback, 1951; Hennig, 1965, 1973; McAlpine, 1989; Michelsen, 1991;

Carvalho and Couri, 2002). Species of the Palaearctic Achanthiptera Lioy and the

Neotropical Cariocamyia Snyder have been stated to have independently re-acquired spiracle 6 (Carvalho et al., 2005), and the absence of male accessory glands, which has been confirmed for only a minority of the species, is shared with the

Scathophagidae. Muscid monophyly was recently corroborated using molecular data

(Schuehli et al., 2007). Currently, eight subfamilies are recognized (Achanthipterinae,

Atherigoninae, Azeliinae, Cyrtoneurininae, Coenosiinae, , Mydaeinae and

Phaoniinae), but the subfamilies and tribes in this family have undergone many classificatory changes and various hypotheses of relationship have been proposed

72 (Couri and Pont, 2000; Couri and Carvalho, 2003; Savage and Wheeler, 2004; Nihei and Carvalho, 2007; Schuehli et al., 2007).

3.1.3 Anthomyiidae

The Anthomyiidae are with approximately 2000 described species in some 50 genera most diverse in the Holarctic region. These flies are mostly found in wooded and moist habitats, and they may be very abundant in subarctic and mountainous areas.

The best known anthomyiid species are the onion fly and cabbage root fly that are major agricultural pests because their larvae are phytophagous as root/shoot miners on many economically important crops. As adults, anthomyiids feed on different types of rotting media like dung and decaying plant material, on nectar in flowers, or they are predacious on small insects. The most common larval breeding habits include phytophagy and saprophagy on decaying plant matter, but the family also includes several mycophagous species. Larvae of certain species are known to be internal parasites of grasshoppers (Acridomyia spp.), others are kleptoparasites in solitary bees (Leucophora spp.), and Coenosopsia spp. are dung breeders. The oldest confirmed fossil of a calyptrate fly belongs to the Anthomyiidae (Michelsen,

2000). It comes from Baltic amber, which has been dated as 40my old. Griffiths

(1972:144) stated that “The limits of the Anthomyiidae require clarification since no autapomorphous conditions can be put forward to demonstrate that the family, as presently delimited, is a probable monophyletic group”. Also Hennig (1973) noted the absence of derived ground plan features. However, other authors (Hennig, 1976;

Michelsen, 1991, 1996) have regarded the Anthomyiidae as monophyletic and supported the by many morphological character states. The main ones are the presence on hind tarsomere 1 of a strong ventro-basal seta, hair-like setulae subapically on the underside of scutellum, a surstylus with a sclerotised connection

73 to the cercus, a surstylus that is distally biramous (but often secondarily simplified), and fused male cerci. The Anthomyiidae were previously classified as a subfamily of a Muscidae sensu lato (even including the Fanniidae) and were further split into two or more subfamilies. Huckett (1965) considered the genera Fucellia Robineau-

Desvoidy and Circia Malloch [= Alliopsis Schnabl & Dziedzicki] a separate subfamily

(Fucelliinae) while the remaining anthomyiids were in his subfamily Anthomyiinae, which was divided into two tribes, viz. the Anthomyiini and Myopinini. This classification has since been completely abandoned, and the family now stands without any subfamilies (e.g. Suwa & Darvas, 1998) or is tentatively subdivided into four major subgroups: the Phaonantho Albuquerque genus-group and the subfamilies

Myopininae, Pegomyinae and Anthomyiinae (Michelsen 2000). A controversial issue is whether the New World genera Coenosopsia and Phaonanthe Albuquerque together constitute the extant sister group of the remaining family (Michelsen, 1991,

2000), or whether these two genera are not sistergroups and neither is a basal anthomyiid taxon (Nihei & Carvalho, 2004).

3.1.4 Scathophagidae

The Scathophagidae are another relatively small muscoid family with about 400 described species. This family exhibits an unusually varied natural history ranging from saprophagy over phytophagy to predation: some species breed in different types of dung or other decaying organic matter such as rotting seaweed, others mine in leaves, bore in culms and/or feed on immature flower heads or seed capsules and ovules. Larvae of a few species are also known to be predators of small invertebrates or caddis fly egg masses. The monophyly of the Scathophagidae has found no

(Griffiths, 1972) or only little (Hennig 1973) support from morphological characters, but two recent molecular studies have brought considerable progress with the result

74 that phylogenetic relationships in this family are now comparatively well understood

(Bernasconi et al., 2000a; Kutty et al., 2007). Kutty et al. (2007) reconstructed the relationships in the Scathophagidae from 63 species (representing 22 genera) based on the mitochondrial genes 12S rRNA, 16S rRNA, COI, Cytb and the nuclear genes

28S rRNA, Ef1a and RNA polymerase II (Pol II). The monophyly of the

Scathophagidae was corroborated with strong support. The two recognized subfamilies of the Scathophagidae, Scathophaginae and Delininae, emerged as monophyletic sister groups. The monophyly of most genera, including Cordilura,

Nanna, Norellia, Gimnomera, Hydromyza and Spaziphora, was also confirmed in the analysis.

3.1.5 Interfamilial relationships

The phylogenetic relationships between the families of Muscoidea are controversial.

This is well illustrated by the different hypotheses that exist for the position of the

Fanniidae within the Muscoidea. A sister group relationship has been suggested between the Fanniidae and Muscidae (Hennig 1965, 1973) and the Fanniidae were also considered a subfamily of the Muscidae (Huckett & Vockeroth, 1987).

Alternatively, the Fanniidae were proposed to be the sister group of the remaining

Muscoidea (Pont, 1977). The other families have similarly generated conflicting hypotheses. Based on morphological characters, the Muscidae and Anthomyiidae have been proposed as sister groups (Michelsen, 1991), whereas the

Scathophagidae have been regarded as the sister group to the Anthomyiidae on the basis of molecular data (Bernasconi et al., 2000a; Bernasconi et al., 2000b; Kutty et al., 2007). McAlpine (1989) concluded, based on several allegedly autapomorphic character states, that a taxon composed of Anthomyiidae, Muscidae and Fanniidae is

75 monophyletic, which suggested that the Scathophagidae are the sister group of the remaining Muscoidea.

Much taxonomic and systematic research on the various taxa within the Muscoidea has been carried out, but these studies mostly addressed issues at the species and genus levels. Comparatively few studies also address relationships across families and even fewer studies explicitly targeted the interfamilial relationships within the

Muscoidea. Exceptions include McAlpine (1989) and Hennig (1973), who both utilized morphological characters but nevertheless obtained conflicting results.

Therefore, it appears timely to use a different source of data; i.e. DNA sequences. A small-scale phylogenetic study using the genes Cytochrome oxidase subunit I and II was carried out by Bernasconi et al. (2000b), but the authors had to conclude that

“the exact relationships among the Muscoidea still remain unclear” and they stressed the need for further research. In our study, we test the monophyly of Muscoidea and address the position of the superfamily and its constituent families within the

Calyptratae. In particular, we address the relationship between the Muscoidea and the Oestroidea and the phylogenetic relationship between the four families of

Muscoidea. To this end we use DNA sequence data from eight genes (12S, 16S,

COI, Cytb, 18S, 28S, Ef1a and CAD) and 127 species from all four muscoid families, ten species from three families of Oestroidea, two species of Hippoboscoidea and seven outgroups from the . This study of the Muscoidea is our third contribution to a better understanding of calyptrate relationships. Previous studies addressed the intrafamilial relationships of the Scathophagidae (Kutty et al., 2007) and the Hippoboscoidea (Petersen et al., 2007) respectively.

76 3.2 MATERIALS AND METHODS

3.2.1 Taxa and DNA extraction

The Muscoidea are here represented by 127 exemplar species from the four constituent families (Table 3.1). With regard to the remaining two calyptrate superfamilies, we included two species from the Hippoboscoidea (Glossinidae and

Hippoboscidae respectively), while the Oestroidea are represented by ten species from the four major families (Calliphoridae, , Sarcophagidae and

Tachinidae). The probable calliphorid non-monophyly as shown by Rognes (1997) has not been an issue of the present study and will be addressed in a forthcoming paper. As outgroups we included seven acalyptrate species representing four different superfamilies: (Hemeromyia anthracina), Lauxanoidea

( sp.), (Lopa convexa, Gluma nitida) and

(Ceratitis capitata, Bactrocera dorsalis, Bactrocera oleae). Most of the DNA extractions utilized a CTAB extraction protocol as described in Kutty et al. (2007).

DNA extractions for some species were also carried out according to manufacturer’s instructions using the QIAamp tissue kit (QIAGEN, Santa Clara, CA).

3.2.2 DNA amplification

Standard PCR amplifications were carried out using either Takara Ex-Taq or Bioline

Taq on 1-5 μl of template DNA. Nine different gene regions were amplified which included the mitochondrial genes 12S ribosomal RNA, 16S ribosomal RNA,

Cytochrome oxidase I (in two parts), Cytochrome b, and the nuclear genes 18S ribosomal RNA, 28S ribosomal RNA, Elongation factor 1-alpha and a fragment of the carbamolyphosphate synthetase (CPS) region of the CAD gene. Due to the variable quality of the extracted DNA and the large phylogenetic distances between the

77 Table 3.1. List of taxa used in study with Genbank accession numbers.

78 Table 3.1. (contd.) List of taxa used in study with Genbank accession numbers.

79

Table 3.1. (contd.) List of taxa used in study with Genbank accession numbers.

80 exemplar species, not all genes successfully amplified. Furthermore, due to the conservative nature of the 18S sequences, we only amplified this gene for 20 species representing the major taxa in the analysis (see Table 3.1). The PCR cycles for all amplifications except CAD consisted of an initial denaturation step at 95°C for 7 minutes, followed by 95°C for 1.5 minutes, annealing at temperatures ranging from

44-50°C and extension at 72°C for 1.5 minutes. A final extension at 72°C for about 5 minutes was also added. The amplified gene products were purified using Bioline

Sure-Clean solution following the manufacturer’s protocol. For CAD, the PCR protocol described by Moulton and Wiegmann (2004) was used for the amplification, and gel extraction was carried out on the amplified product using QIAquick Gel extraction kit following the manufacturere’s protocol. Cycle sequencing was performed on the purified products using BigDye Terminator v3.1 and direct sequencing was carried out on an ABI 3100 genetic analyser (Perkin Elmer). The sequences were edited and assembled in Sequencher 4.0 (Gene Codes Corp., Ann

Arbor, MI).

3.2.3 Alignments

The protein encoding genes COI, Cytb, Ef1a and CAD were aligned based on amino acid translations in AlignmentHelper (McClellan, 2004), which uses ClustalW

(Thompson et al., 1994) for the amino acid alignment. The nucleotide alignments were indel-free for most genes except for CAD where codon insertions were found in two muscid species. Due to the large size of the data set, aligning the ribosomal genes was challenging. We rejected a manual alignment because it would yield non- repeatable results, while a visual inspection of the default alignments in ClustalX revealed unconvincing homology hypotheses. Given that the quality of alignments is heavily dependent on the guide trees used during the alignment (Kumar and Filipski,

81 2007), we inspected the default guide trees. These guide trees were in conflict with regard to many of the well supported monophyletic groups. For example, in the 12S guide tree we find that the muscids, scathophagids and anthomyiids scattered all across the tree and the same pattern was seen in the other ribosomal gene guide trees (see Fig 3.3). We thus decided to improve the guide tree using the following steps. We determined a preliminary alignment with ClustalX 2.0 (Thompson et al.,

1997) for the ribosomal genes 12S, 16S, 18S and 28S (gap opening and extension cost: 15:6.66). We then identified the indel-free fragments of the ribosomal genes that are likely to correspond to stem regions of the rRNAs and then concatenated the indel-free rDNA sequences with the aligned sequences for the protein-encoding genes. We then estimated a guide tree from this dataset by analyzing it with parsimony (TNT v2.0: Goloboff et al., 2000: new technology search at level 50, initial addseqs=9, find minimum tree length 5 times). This analysis yielded ten most parsimonious trees. Each of these topologies was then used as guide trees for aligning the full-length DNA sequence data for the ribosomal genes in ClustalX. We thus obtained ten different alignments. Using tree length as and optimality criterion, the alignment that yielded the shortest tree was used in all subsequent analyses, but the trees based on the remaining alignments were very similar (see Fig. 3.9).

3.2.4 Tree search strategies

The aligned Muscoidea dataset had 146 taxa and 7,202 characters. The dataset was analyzed using both maximum parsimony (MP) and maximum likelihood (ML). The tree was rooted using the acalyptrate Hemeromyia anthracina (), although any other acalyptrate family could have been used as outgroup given that we currently do not have a viable hypothesis as to which acalyptrate taxon may be the sister group to the Calyptratae. Maximum parsimony analyses were carried out in

82 TNT v2.0 (Goloboff et al., 2000: new technology search at level 50, initial addseqs=9, find minimum tree length 5 times), with indels coded once as missing data and once as fifth character states. Node support was assessed by jackknife resampling percentiles (250 replicates, same search options as above) obtained at 36% deletion as recommended by Farris et al. (1996). For the likelihood analyses, we used

MrModeltest version 2.2 (Nylander, 2004) for identifying the best fit model (GTR+I+V) based on the Akaike Information Criterion (AIC). The likelihood analyses were conducted with Garli v0.951 (Zwickl, 2006). Three independent runs were carried out and node support was assessed using a non-parametric bootstrap with 250 replicates using the automated stopping criterion set at 10,000 generations for each replicate.

3.3 RESULTS

After the alignment and concatenation of the eight genes 12S, 16S, COI, Cytb, 18S,

28S, Ef1a and CAD, 2,437 sites of the 7,202 base pairs were parsimony informative.

The parsimony analysis with indels coded as a fifth character state yielded three most parsimonious trees with a tree length of 24,267, while the analysis with indels coded as missing data yielded 23 most parsimonious tress with a tree length of

23,201. Parsimony analysis coding indels as missing data (see Fig. 3.8) and as a fifth character resulted in trees with identical family-level relationships and sharing approximately 85% of the nodes, which suggests that indel coding has only a minor influence on the tree topology (Fig 3.1). Since the parsimony analyses for indels coded as missing data and as a fifth character state respectively result in topologies that are congruent for higher level relationships, other indel coding methods like simple indel coding (SIC) and modified complex indel coding (MCIC; Simmons et al.,

83 2007; Simmons and Ochoterena, 2000) were not tested. The maximum likelihood tree is shown in Fig 3.2. The overall tree topologies of the MP and ML trees are also very similar and recover the same family-level relationships for the Muscoidea.

However, there is a conflict with regard to the Hippoboscoidea, which are polyphyletic on the ML tree as the Glossinidae emerge as the sister group of a clade consisting of the non-fanniid Muscoidea plus the Oestroidea. There are also other topological differences within the muscoid and oestroid families (compare Figs 3.1 &

3.2).

The Calyptratae are corroborated as monophyletic, with modest support in the maximum parsimony analysis and high support in the maximum likelihood analysis.

On the most parsimonious tree, the Hippoboscoidea are monophyletic and placed as the sister group to the remaining calyptrates, although with very modest support. The

Muscoidea are the only calyptrate superfamily that is paraphyletic and this paraphyly is found in all the different analyses regardless of indel coding and the use of maximum parsimony or maximum likelihood. The monophyly of the Oestroidea is well supported and this superfamily is nested within a paraphyletic Muscoidea. The

Fanniidae are the sister group to the remaining Muscoidea plus the Oestroidea. The

Muscidae are monophyletic and sister group to a clade composed of Anthomyiidae,

Scathophagidae and Oestroidea. The Anthomyiidae +Scathophagidae form a moderately supported clade and the Oestroidea are well supported as the sister group to this. In all analyses, we find a paraphyletic Anthomyiidae.

Most genera of the Musicdae are monophyletic: Coenosia, Helina, Hebecnema,

Limnophora, Mydaea, Mesembrina, Morellia, Musca, , Phaonia, Spilogona and . Only Hydrotaea does not emerge as monophyletic in the maximum

84 parsimony analysis, but the resampling analysis does not provide a conclusive result.

The maximum likelihood analysis has a highly corroborated monophyletic Hydrotaea.

At the subfamily level, we only recover a monophyletic Coenosiinae, while the remaining subfamilies are either para- or polyphyletic on the most parsimonious tree

(Mydaeinae, Phaoniinae, Azeliinae and Muscinae). However, on the maximum likelihood tree, the Muscinae are also monophyletic. The Scathophagidae are monophyletic as are most genera including Nanna, Gimnomera, Norellia and

Cordilura. The subfamily Delininae is monophyletic but nested within the

Scathophaginae. In all analyses, the Anthomyiidae are paraphyletic. The genera

Botanophila, Hylemya and Pegoplata are monophyletic, while Lasiomma is paraphyletic. The two subfamilies Anthomyiinae and Pegomyinae are para- or polyphyletic on the MP tree while the subfamily Anthomyiinae is monophyletic on the

ML tree. The Myopininae are monophyletic on both the ML and MP tree. Within the oestroids, the Sarcophagidae are monophyletic with high node support. Tachina ferox Panzer and the Sarcophagidae are sister groups. The calliphorid exemplars form a paraphyletic grade, with maculata being the sister group to

Eurychaeta palpalis.

85 Fig 3.1. Strict consensus of three most parsimonious trees (indel=5th character); above node jackknife support for indel=5th character state; below node indel=missing; nodes shared with ML tree indicated by.

86 Fig 3.1. (contd). Strict consensus of three most parsimonious trees (indel=5th character); above node jackknife support for indel=5th character state; below node indel=missing; nodes shared with ML tree indicated by.

87 Fig 3.2. Likelihood tree from ML analysis (Garli) indicating bootstrap support, nodes shared with selected guide tree indicated by  and majority rule consensus of all ten guide trees indicated by.

88 Fig 3.2. (contd.) Likelihood tree from ML analysis (Garli) indicating bootstrap support, nodes shared with selected guide tree indicated by  and majority rule consensus of all ten guide trees indicated by.

89 3.4 DISCUSSION

The Muscoidea are of economic importance, as many of these flies are pests on both agricultural crops and livestock, and similarly they are of medical importance as some species are vectors of diseases. Muscoid flies are also among the most common insects and many species live in close association with humans. However, the relationships among the main clades of the Muscoidea have remained poorly understood, and past analyses yielded very conflicting hypotheses. Even the taxonomic composition of the Muscoidea within Diptera has been controversial, and as mentioned by McAlpine (1989): ‘The name Muscoidea has probably been used in a wider variety of senses than any other suprageneric name in Diptera’ (p. 1496).

The usages range from encompassing all of (Coquillett, 1901) to being a subgroup of the Schizophora (Griffiths, 1972), to being a subgroup of the

Calyptratae (Roback, 1951; Hennig, 1973; McAlpine 1989).

However, the most commonly used concept of Muscoidea is that of Hennig (1973) and McAlpine (1989), who classified the Anthomyiidae, Fanniidae, Scathophagidae and Muscidae in the superfamily Muscoidea. In the absence of convincing evidence to the contrary, Hennig (1973) used as a working hypothesis that the Muscoidea are monophyletic, but that their interfamilial relationships and the relationships to the other calyptrate superfamilies (Oestroidea, Hippoboscoidea) are unknown. Michelsen

(1999), however, explicitly acknowledged the lack of support for muscoid monophyly by defining the Muscoidea as ‘the calyptratae less the Hippoboscoidea and

Oestroidea’. Based on our data we are able to test many of these hypotheses.

90 3.4.1 Comparison of tree hypotheses

The tree topologies from the three different analyses, MP with indels treated as a fifth character state, MP with indels treated as missing data and ML, are largely congruent. Most higher-level relationships are uncontroversial regardless of which indel treatment or the analysis strategy is used. The calyptrate monophyly is well supported on all trees. Another well supported relationship is the position of the monophyletic Oestroidea, which are always placed as the sister group to the clade

Anthomyiidae+Scathophagidae. The interfamilial relationship (Fanniidae + (Muscidae

+ (Anthomyiidae + Scathophagidae + Oestroidea))) is also recovered irrespective of the analytical method. Of the approximately 80% nodes shared between the MP and

ML trees, many relationships at the subfamily level are identical, including the monophyly of the subfamilies Delininae (Scathophagidae) and Coenosiinae

(Muscidae) and the sister group relationship between the Phaoniinae+Mydaeinae clade and Coenosiinae in the Muscidae. The terminal nodes are generally supported by high jackknife values on the MP tree and bootstrap values on the ML tree.

However, the node support for the higher level relationships is generally lower, which is similar to the findings of many recent phylogenetic analyses of higher-level phylogenetic relationships in Diptera (e.g. Tephritoidea: Han and Ro, 2005;

Empidoidea: Moulton and Wiegmann, 2007; : Holston et al., 2007;

Opomyzoidea: Scheffer et al., 2007). In the Muscoidea analysis the node support for many higher level relationships is similarly low, despite the use of large amounts of data and congruence between the tree topologies obtained using different analysis methods such as parsimony, Bayesian and maximum likelihood. The only major conflict between our MP and ML trees is the monophyly and position of

Hippoboscoidea. Regardless of indel codings, it is monophyletic on the MPTs, which is in agreement with the currently accepted hypothesis (Hennig, 1973; McAlpine,

91 1989; Nirmala et al., 2001; Dittmar et al., 2006; Petersen et al., 2007). However, the

Hippoboscoidea are not monophyletic on the ML tree.

3.4.2 Calyptrate monophyly

The monophyly of the Calyptratae is well supported by a large number of morphological characters but molecular data have consistently suggested that the calyptrates may be paraphyletic (Vossbrinck and Friedman, 1989; Bernasconi et al.,

2000b) with some acalyptrates being nested within them. We believe that this is due to very sparse taxon sampling in the earlier molecular analyses, because in our study calyptrate monophyly is consistently supported despite more rigorous testing via the inclusion of acalyptrate outgroups from four different superfamilies. All remaining molecular studies only included 1-2 outgroups.

3.4.3 Superfamily monophyly and the relationships between the calyptrate superfamilies

Among the three calyptrate superfamilies, the monophyly and phylogeny of the

Hippoboscoidea has been well studied and supported using both morphological and molecular data (Hennig, 1973; McAlpine, 1989; Nirmala et al., 2001; Dittmar et al.,

2006; Petersen et al., 2007). Due to insufficient gene overlap with the Petersen et al.

(2007) study, our dataset included only two representative species from this superfamily, but they form a monophyletic group in the parsimony analyses. The monophyly of and relationships among the remaining two superfamilies, Muscoidea and Oestroidea, has been more open to discussion. Previous studies suggested a sister group relationship between Hippoboscoidea and the remaining calyptrate flies

(McAlpine, 1989), whereas in Petersen et al. (2007) the Hippoboscoidea were deeply nested within the Calyptratae, although outgroup sampling was sparse and the

92 support for this hypothesis was very low. Based on our most parsimonious tree, we find that the Hippoboscoidea are the sister group of the Calyptratae, and that the

Oestroidea are monophyletic. However, the Muscoidea are likely paraphyletic with regard to the Oestroidea. This confirms Michelsen’s proposal, but is in conflict with

McAlpine’s (1989) hypothesis of monophyly. However, it is important to remember that McAlpine assessed all synapomorphies relative to the (hypothetical) groundplan of the Schizophora; i.e. all the characters proposed as supporting Muscoidea monophyly could have been plesiomorphic relative to the groundplan of the

Calyptratae. Our study is not the first to suggest that the Muscoidea are a paraphyletic grade (Michelsen, 1991; Bernasconi et al., 2000b; Nirmala et al., 2001), but our study is based on a much larger gene and taxon sample than previous analyses, and the most probable phylogenetic position of all muscoid families is indicated.

Once a group with a well-established name is shown to be paraphyletic, a new classification and/or new names have to be proposed. For example, Yeates and

Wiegmann (1999) proposed the informal names “lower Diptera” for and

“lower Cyclorrhapha” for instead of proposing new ranks and new names for subgroups within the non-Brachyceran Diptera and non-Schizophoran Cyclorrhapha.

We are in favour of this approach that was also adopted in a recent review of Diptera classification (Yeates et al., 2008) and propose that a better way of referring to the paraphyletic Muscoidea will be as the “muscoid grade”. An alternative would be a new superfamily-level classification that would either require that the Oestroidea are subsumed in the Muscoidea or that separate superfamilies are recognized for the

Fanniidae, Muscidae and Anthomyiidae+Scathophagidae respectively. We consider

93 the latter as an unnecessary inflation in the number of superfamilies, and at least two would contain a single family only and thereby be redundant.

3.4.4 Interfamilial relationships within the muscoid grade

The Fanniidae are placed as the sister group to a clade consisting of the Oestroidea plus the remaining families of the muscoid grade on the most parsimonious tree (Fig.

3.1). This position had been suggested based on molecular characters by

Bernasconi et al. (2000b), but was in conflict with the more traditional views, which placed the family either as the sister group of the Muscidae (Hennig, 1973) or as the sister group of Anthomyiidae+Muscidae (McAlpine, 1989). On the Bayesian tree, the

Fanniidae are in a similar position, but surprisingly Glossina pallidipes Austen is the sister group of Muscoidea+Oestroidea. Given the strong morphological support for a monophyletic Hippoboscoidea, we believe that the overall evidence supports the most parsimonious topology with Fanniidae being sister group to

Muscoidea+Oestroidea. In any case, Fanniidae are never the sister group of

Muscidae or Anthomyiidae as had been previously suggested.

With regard to the Muscidae, various authors have corroborated the monophyly of this family using both morphological and molecular data. This monophyly is further corroborated here. However, our analysis is the first to address the relative position of Muscidae within the calyptrates based on a large data set. In our analysis, the family is the sister group of Oestroidea +Scathophagidae+ Anthomyiidae. We also consistently find that Anthomyiidae+Scathophagidae form a monophyletic group. This relationship was suggested by Roback (1951), who included the Scathophagidae (as

Scopeumatinae) as a subfamily of the Anthomyiidae. This was based on vein

A1+CuA2 reaching the wing margin (probably a symplesiomorphy), and on the larval

94 morphology of Scathophaga stercoraria, which was stated to be “distinctly anthomyoid in all its characteristics” (p. 333). However, in spite of this, Roback also noted that “on the basis of the male genitalia, and the presence of the three sternopleurals the Anthomyiinae can be considered more advanced than the

Scopeumatinae, and closer to the remainder of the Muscoidea and the

Sarcophagoidea” (p. 334). No other author has to our knowledge proposed morphological data supporting the clade Anthomyiidae+Scathophagidae, but it is consistently supported by molecular data (Bernasconi et al., 2000b; Kutty et al.,

2007). The finding that the Oestroidea are the sister group to the clade

Anthomyiidae+Scathophagidae is a new result. Previously, it had been thought that the Oestroidea are the sister group to a monophyletic Muscoidea (Hennig, 1973;

McAlpine, 1989), or even that the Oestroidea were paraphyletic with regard to the

Muscoidea (Roback, 1951).

3.4.5 Family monophyly and relationships within families

The Fanniidae represented by two Fannia species are monophyletic and this is consistent with morphological studies on this family. The Muscidae are monophyletic but the support for this is not very high, which may be due to a relatively small taxon sample for this very large family. Although our species-level sample is small, we do include representatives of most subfamilies so that the test for monophyly is overall quite rigorous. The monophyly of the subfamily Coenosiinae is corroborated. On both the ML tree and the MP tree, Spilogona and Villeneuvia are sister groups and closely related to the Coenosia species, although Spilogona and Villeneuvia are generally considered more closely related to the clade Limnophora+Lispe (Hennig, 1965). The other subfamilies Azeliinae, Muscinae, Mydaeinae and Phaoniinae are not monophyletic in the MP tree, although the monophyly of the clade

95 Mydaeinae+Phaoniinae is well supported and with the genera Helina, Phaonia,

Mydaea and Hebecnema being monophyletic. The subfamily Coenosiinae and

Phaoniinae are also closely related as suggested by Scheuli et. al, (2007) and the

Mydaeinae+Phaoniinae+Coesnosiinae clade has moderate support. However, it is puzzling that the azeliine Muscina is sister group to Phaoniinae

+Mydaeinae+Coenosiinae with moderate support instead of being placed on the

Azeliinae+Muscinae branch. Similarly, the muscine genera Polietes and Mesembrina are suprisingly nested within the Azeliinae. The genera Thricops and Drymeia from the subfamily Azeliinae are monophyletic and so are the muscine genera Musca and

Morellia. The tribe Stomoxyini within the Muscinae is here represented by Stomoxys calcitrans and Haematobosca stimulans is monophyletic and the sistergroup relationship of these two species was also suggested in the molecular phylogeny of the Muscidae by Scheuli et. al, (2007) and Carvalho (1989). However, it is clear from some of the unexpected findings that a more extensive taxon sample for the

Muscidae will be needed in order to resolve tribal and subfamily level relationships.

In our analysis, the Anthomyiidae are paraphyletic but we find that none of the nodes that render this family paraphyletic have jackknife support on the MP tree (>50) or bootstrap support on the ML tree (>50). Of the three subfamilies recognised by

Michelsen (2000), only the Myopininae are monophyletic on both the ML and MP tree. The genera Botanophila, Hylemya and Pegoplata are monophyletic and only

Lasiomma is paraphyletic (remaining genera being represented by only a single exemplar species). It should be noted that the Anthomyiidae are the most poorly sampled muscoid family in our dataset and that additional species are needed for a more rigorous test of anthomyiid monophyly. In particular, exemplar species from the genera Coenosopsia and Phaonanthe should be included.

96

The Scathophagidae and most of its genera are monophyletic. However, the relationships between some of the genera and species differ from the phylogenetic hypothesis in Kutty et al. (2007). The most significant difference is a change in the position of the subfamily Delininae which is here nested within the Scathophaginae as opposed to being their sister group. However, it must be recalled that in Kutty et al. (2007) a sensitivity analysis had been conducted that revealed that the downweighting of transitions was favoured, while in this study all character changes are equally weighted because, due to the large size of the current data set, such a sensitivity analysis would have been computationally prohibitive. Most scathophagid genera are monophyletic as proposed by Kutty et al. (2007), but some relationships between the genera differ on the ML and MP trees. However, these branches have low node support in the MP and ML analyses.

Within the Oestroidea, the clade Sarcophagidae+Tachinidae is monophyletic as suggested by Pape (1992) and Rognes (1997) – although these authors presented different morphological evidence corroborating this sister group relationship. Our results indicate non-monophyly of Calliphoridae as the single exemplar species of the

Rhinophoridae emerges as the sister group of Eurychaeta palpalis (Calliphoridae:

Helicoboscinae), i.e. as nested within the calliphorids. This, however, is in conflict with the analyses of Rognes (1997) and Pape and Arnaud (2001), who found the

Rhinophoridae to be the sister group of either the clade Sarcophagidae+Tachinidae or of the Rhiniinae (a former blow fly subfamily, not included in this study and subsequently raised to family rank by Evenhuis et al. [2008]). Blow fly non- monophyly as argued by Rognes (1997) and Pape and Arnaud (2001) is caused by the Rhiniinae falling outside and the Oestridae falling inside the traditional

97 Calliphoridae, but this has not been tested in the present study. The position of

Oestroidea within the Calyptratae and the relationships between their constituent families are currently under study based on a much larger set of exemplar species and will be the focus of a future publication.

3.4.6 Alignments of the ribosomal genes

The use of ribosomal genes for the reconstruction of phylogenetic relationships has its advantages and disadvantages (Hillis and Dixon, 1991; Simon et al., 1994;

Caterino et al., 2000). Ribosomal genes vary in length across species of different ages and the genes can thus provide valuable information for many phylogenetic questions. Furthermore, due to the large number of copies, the genes can often be amplified for degraded templates. However, one of the major difficulties with ribosomal data is their alignment, i.e. establishing primary homologies between nucleotide sites from distantly related taxa. Some of the most commonly used techniques include alignments based on secondary structure of the respective ribosomal genes (Gutell, 1994; Hickson et al., 1996; De Rijk et al., 1999; Van de

Peer et al., 1999), progressive alignment with a guide tree as implemented in alignment programs like Clustal (Thompson et al., 1994), Muscle (Edgar, 2004),

Malign (with the evaluation of multiple guide trees: Wheeler and Gladstein, 1994), and optimization alignment (Wheeler, 1996). Given that alignment is critical for the results obtained in a phylogenetic analysis (Morrison and Ellis, 1997; Morrison,

2006), there are numerous other techniques that have been suggested in the literature apart from the methods mentioned above (Wheeler, 1995, 1996; Giribet and Wheeler, 1999; Simmons and Ochoterena, 2000; Hickson et al., 2000; Wheeler,

2003; Danforth et al., 2005; Benavides et al., 2007; Kumar and Filipski, 2007;

Simmons et al., 2007). In studies utilizing progressive alignment programs, the

98 authors frequently manually re-adjust the numerical alignments and/or exclude some parts of the alignment after inspection. However, manual re-alignments and data deletion have been criticized for being subjective. Many authors thus prefer numerical techniques (Giribet and Wheeler, 1999).

The alignments for the ribosomal genes in this study were based on an approach that is still rarely used although this technique has recently been promoted by Benavides et al. (2007) for the alignment of nuclear introns which faces similar issues related to length differences and variability. They used the conserved regions of the introns and nuclear coding genes to generate a guide tree for the alignment of the the complete intron sequences. Here, we similarly use a user-defined ‘guide tree’ estimated based on conserved sequences instead of relying on the Clustal’s default guide trees. This user-defined guide tree is then used for aligning the full-length ribosomal fragments

(including the variable regions) while Clustal’s guide trees are calculated from a distance matrix based on dissimilarity scores between sequence pairs. However, such dissimilarity scores are unlikely to reflect true evolutionary distances and the guide trees may thus be misleading. We believe that some of the guide trees generated by ClustalX for our ribosomal genes 12S, 16S, 18S and 28S fall into this category (see Fig 3.3-3.9). On these guide trees, we would expect the species of, for example, the Muscidae to cluster. Instead, they are scattered all over the guide tree

(e.g. see 12S guide tree). To reduce this source of error that can have serious effects on the downstream phylogenetic inferences (Kumar and Filipski, 2007), we thus generated a guide tree to improve alignments for the ribosomal genes in our dataset.

The guide tree used in our alignments was not based on subjective opinions about calyptrate relationships. Instead, we use the phylogenetic signal from those sequences that code for protein-encoding genes and those parts of the ribosomal

99

Fig 3.3. 12s guide tree from Clustal X

100

Fig 3.4 16s guide tree from Clustal X .

101

Fig 3.5 28s guide tree from Clustal X

102

Fig 3.6 18s guide tree from Clustal X

103

Fig 3.7 Guide tree from Clustal X used in alignment of ribosomal genes.

104

Fig 3.8 Strict consensus of 23 most parsimonious trees (indel=missing).

105

Fig 3.9 Majority rule consensus of trees from parsimony analysis of datasets based on the ten different guide trees.

106 genes that likely code for the stem-regions of rRNAs. The method that we use is thus numerical, repeatable, and computationally tractable. It also does not require the deletion of sequence data.

A main concern may be that the guide tree may have undue influence on the downstream phylogenetic results; i.e. that the trees based on all data merely reflect the relationships on the guide tree. We therefore compared the guide tree to our phylogenetic hypotheses based on MP and ML. We find that the two trees have a surprisingly small number of shared nodes. About 60% of the nodes are shared between the MP tree and the guide tree while only 50% nodes are the same between the guide tree and the ML tree (see Fig 3.2.). On examining the ten guide trees we find that the three taxa, the two Hippoboscoidea species and the Tachina are placed within the Muscidae (see Fig 3.7). This placement is in stark conflict with the well established monophyly of Muscidae, Tachinidae, and Oestroidea. However, this conflict disappears on the trees that are based on the guide-tree assisted alignment of all data (see Fig. 3.9). All three species are now placed in positions that are consistent with well supported higher-level hypotheses for calyptrates. It appears as if in this case the phylogenetic signal from the hypervariable regions of the ribosomal genes is valuable in that it placed the taxa in positions (on the most parsimonious tree) that are in agreement with previously suggested hypotheses. Alignment techniques based on user-defined guide trees have been accused of circularity

(Benavides et al., 2007), but not only are the guide tree and phylogenetic trees not identical, they also have several conflicting nodes. Furthermore, one could also argue that a strong influence of the guide tree on the downstream phylogenetic results is wanted given that it is based on the signal of the data. Using a user-defined guide tree based on those data that can be aligned with little ambiguity thus appears

107 to be an interesting option for those systematists who would like to avoid subjective manual alignments and data deletions and yet deal with large data sets that are difficult to analyze using techniques such as optimization alignment.

3.5 CONCLUSIONS

Our study provides a large amount of novel molecular evidence for the phylogenetic relationships of the Calyptratae, and in particular for the paraphyly of, and phylogenetic relationships within, what we suggest to call the muscoid grade, i.e., the

Fanniidae, Muscidae, Anthomyiidae and Scathophagidae. The position of this grade within the Calyptratae and the relationships of its constituent families to the remaining calyptrate superfamilies, viz. Hippoboscoidea and Oestroidea, are resolved. The relationships between the four muscoid families are established and the monophyly of the Fanniidae, Muscidae and Scathophagidae is further corroborated while no molecular support was obtained for anthomyiid monophyly.

108

CHAPTER 4

Molecular phylogeny of the Calyptratae (Diptera:

Cyclorrhapha) with emphasis on the superfamily

Oestroidea

4.1 INTRODUCTION

The Calyptratae (Diptera: ) is with 18,000 described species one of the largest and most diverse groups in the Diptera representing approximately 12% of all flies. The best known members of this group are the , blowflies, flesh-flies, tsetse flies, and warble flies. One of the most remarkable features of this group is the wide variety of life histories strategies that calyptrates utilize which can be broadly categorized as saprophagy on dead and decaying organic matter, parasitism and predation on other and isopods, phytophagy on both dicots and monocots plnats and haematophagy on livestock and humans. Despite the large morphological and biological diversity, calyptrates appear to be a rather recent group. The oldest known calyptrate fossil is an anthomyiid species from early Coenozoic Baltic amber

(Michelsen, 2000) which dates back to Eocene period (about 40 mya) and this diverse group of insects must have undergone rapid radiation. In order to understand the evolution of this radiation it is very crucial to have a well-supported and robust phylogenetic tree for the group.

Numerous phylogenetic studies have been carried out within families, subfamilies, and genera and many used morphological data (Bernasconi et al., 2000a;

Bernasconi et al., 2000b; Carvalho and Couri, 2002; Dittmar et al., 2006; Gleeson et al., 2000; McAlpine, 1989; Michelsen, 1991; Nihei and de Carvalho, 2004, 2007;

Pape, 2001; Rognes, 1997; Savage and Wheeler, 2004; Savage et al., 2004;

Schuehli et al., 2007; Stevens, 2003; Stireman, 2002; Verves, 1989) However, a phylogenetic tree based on a numerical analysis which establishes relationships within the Calyptratae: between the three superfamilies (Hippoboscoidea, Oestroidea and muscoidea grade), constituent families and between subfamilies is not available.

110 One of the reasons for this is the lack of oestroid coverage in all calyptrate analyses to date.

We address two main topics in this chapter. One of the goals of this study is resolving and understanding the relationships within the Oestroidea with good taxon coverage from the constituent families. This also will include the placement of enigmatic calyptrate taxa such as Mystacinobia and the McAlpine’s fly. However, placing these taxa and understanding Oestroidea relationships cannot be carried out in isolation and requires the inclusion of taxa and data from the other non-oestroid families. Hence, we have included all species data from the earlier studies on the

Hippoboscoidea and muscoidea grade, as better taxon sampling will enable us not only to address resolve relationships within the Oestroidea superfamily and within the

Calyptratae group but also help in testing existing hypotheses (including the results presented in previous sections) about calyptrate relationships.

4.1.1 Oestroidea phylogenetics

The monophyly of Oestroidea is not contentious and autapomorphies supporting this clade include a vertical row of bristles present on the meron, a patch of setulae on the anatergite ventral to the lower calypter, wing vein M deflected forwards to join C before the wing apex and phallus with cuticular denticles on ventral surface of distal section (Griffiths, 1982; McAlpine, 1989; Pape, 1992). The Oestroidea has changed ranks from a family (Griffiths 1972) to family group (Griffiths 1982) and is currently accepted as a superfamily (Hennig, 1958; Pape, 1986). In the literature, this superfamily has also been referred to as the Calliphoroidea (Hennig, 1958),

Tachinidae family group (Pape, 1992), and Tachinoidea (Pape, 1986; Rohdendorf,

1977). The Oestroidea of McAlpine (1989) comprise the six families Calliphoridae

111 Sarcophagidae, Rhinophoridae, Tachinidae, Oestridae and the Mystacinobiidae. The monophyly status of families like the Calliphoridae and placement of families (e.g.

Mystacinobia zelandica) and relationships between the families and are still controversial.

4.1.2 Oestroidea family relationships

Few molecular studies have been carried out with Oestroidea species but no clear conclusion could be drawn as they were based on very few calyptrate taxa and were single or double gene studies (e.g. Nirmala et al., 2001). The family level relationships in the Oestroidea have been addressed by few authors and are mostly based on morphological characters. The relationships between all the oestroid families were first addressed by McAlpine (1989) who proposed

(Calliphoridae+Mystacinobiidae)+ Sarcophagidae as sistergroup to Oestridae +

(Rhinophoridae+Tachinidae). (Pape, 1992) studied the phylogenetic relationships of the Tachinidae-family group (in which the Mystacinobiidae was not included) and recovered a conflicting hypothesis: Rhinophoridae + (Oestridae+Calliphoridae) and

(Tachinidae+Sarcophagidae). One if the most complicated aspects of the Oestroidea phylogeny is the fact that the Calliphoridae that traditionally had been regarded as monophyletic is now believed to be paraphyletic (McAlpine, 1989; Rognes, 1997).

This requires that all studies addressing oestroid relationships have to consider the calliphorid subfamilies as terminals. Earlier studies were not aware of this complication and these studies are thus difficult to evaluate.

Griffiths (1982) considered the Calliphoridae as the sistergroup of the remaining

Oestroidea while the Oestridae was the sistergroup to the rest of the Oestroidea in

(Hennig, 1976). Pape and Arnaud (2001) suggested that the calliphorid subfamily

112 Rhiniinae was the sistergroup to the family Rhinophoridae and Rognes (1997) suggested a position of the Oestridae close to the calliphorid subfamilies

Helicoboscinae and Bengaliinae. The clades (Oestridae+Tachinidae) (Roback,

1951), (Rhinophoridae+Sarcophagidae) (Rohdendorf, 1967), (Rhinophoridae +

Calliphoridae) (Tschorsnig, 1985a) and (Rhinophoridae +Tachinidae) (Wood, 1987) have also been variously suggested.

Fig 4.1. Two different hypotheses for the family relationships within the Oestroidea based on morphology a) McAlpine (1989) b) Pape (1992).

4.1.3 Oestroidea family portraits

Calliphoridae

This family consists of approximately 1,000 species and is represented in all biogeographical regions. The breeding habits of the larvae range from carrion breeding to attacks on necrotic tissues and wounds of living vertebrates which can lead to myiasis. Some species included in the Calliphoridae are the sheep blowflies

(Lucilia cuprina) and screw-worm fly (Chrysomya bezziana) that cause primary myiasis in livestock and humans and are major veterinary and medical pests. One of the most expensive and time consuming eradication programs was carried out for the

113 screw-worm fly which was responsible for the infestation of about more 2.7 million animals in Africa where large scale myiasis due to the Cochliomyia hominivorax species was documented. However, the inaccessibility of some areas in Africa and the need for constant vigilance still pose a threat of large scale infestations of livestock in this region.

The larvae of Auchmeromyia senegalensis are blood suckers on humans. Many genera are parasitoids on earthworms and snails (Bellardia, Onesia, Amenia,

Pollenia) while larvae from the genera Protocalliphora and Trypocalliphora are specialized for blood sucking on nestling birds. Other highly specialized breeding habits include breeding in pitcher plants (Nepenthomyia and Wilhelmina) and associations with nests of termites and ants (Stomorhina lunata and Villeneuviella).

However, it is not clear how and from which state the different life history strategies have evolved. This question can only be addressed once the phylogenetic relationships of the calliphorids are better known.

The recent literature suggests that the calliphorids may be paraphyletic, although the literature also includes authors who have supported calliphorid monophyly (Lehrer,

1970; McAlpine, 1989; Rognes, 1986, 1991; Stevens, 2003). The validity of this group has been questioned by Griffiths (1982), Hennig (1973) and Rognes (1997) and Pape (1992) further concluded that the Calliphoridae was paraphyletic based on different sets of morphological data. This family has also undergone many changes in the number, names, and classification of subfamilies (James, 1970; James, 1977;

Kurahashi, 1989; Pont, 1980; Schumann, 1986; Shewell, 1987). The earliest subfamily classification was by (Lehrer, 1970, 1972) but his system was not based on phylogenetic arguments (Rognes, 1991). (Hennig, 1973) was the first to use a

114 phylogenetic approach and he recognized five subfamilies: Calliphorinae (including

Polleniinae), Chrysomyinae, Rhiniinae, Ameninae, and Mesembrinellinae. The genus

Eurychaeta (=Helicobosca) was transferred from the family Sarcophagidae to the separate subfamily Helicoboscinae in the Calliphoridae (Rognes, 1986) and Rognes

(1993) revised this subfamily. In Rognes (1991), the paraphyletic Calliphoridae were subdivided into the subfamilies; Calliphorinae, Chrysomyinae, Luciliinae,

Melanomyinae, Polleniinae, Auchmeromyiinae, Bengaliinae, Mesembrinellinae,

Phumosiinae, Rhiniinae, and Toxotarsinae. The monophyly of the Ameniinae and

Mesembrinellinae were also confirmed by Crosskey (1965) and Guimarães, (1977) respectively. The Helicobosinae subfamily was placed as sister group to the rest of the calliphorid subfamilies in the analysis of the Oestroidea by Pape (1992) and the

Bengaliinae was sistergroup to the remaining non helicoboscinae calliphorids. The subfamilies Chrysomyinae, Rhiniinae and Toxotarsinae form a clade in this analysis.

Tachinidae

The Tachinidae is with about 10,000 described species one of the largest families in the Diptera. Tachinids are found in all terrestrial environments including forests, grasslands, deserts and mountain regions. The adults are morphologically very diverse in size and color while all larvae with known natural history are internal parasitoids of other arthropods. Particularly commonly parasitized are the larval stage of Lepidoptera and other major groups of phytophagous insects. For this reason, tachinids are used in biological control programs of crop and forest pests.

The tachinids also have evolved a wide variety of mechanisms by which they attack these hosts (O'Hara, 1985) and the oviposition strategies include minute eggs that are ingested and hatched in the host’s gut and planidial larvae that actively seek out the victim. The Tachinidae are a recent andactively radiating group of flies (Crosskey,

115 1976). They are considered to be monophyletic based on morphological characters which includes a well developed subscutellum in the adult, reduced mandibles and the fusion of the labrum to the rest of the cephaloskeleton in the first instar larvae

(Pape, 1992; Rognes, 1986; Tschorsnig and Richter, 1998; Wood, 1987).

Four subfamilies are currently recognized: the Tachininae, Exoristinae, Phasiinae, and Dexiinae (Herting, 1984 ; Herting et al., 1993; O’Hara and Wood, 2004;

Tschorsnig and Richter, 1998; Wood, 1987). Among these subfamilies, only the

Dexiinae is supported as monophyletic based on derived features of the male genitalia (Richter, 1992; Tschorsnig, 1985b; Wood, 1987). The subfamily Exoristinae was considered to be monophyletic by (Tschorsnig, 1985b) and (Stireman, 2002) supported its monophyly using molecular data from the nuclear genes 28S and

Elongation factor-1-alpha. The monophyly of Tachininae and Phasiinae is still contentious and many different relationship hypotheses for the subfamilies have been based mainly on egg morphology and the evolution of ovolarviparity in the family. (Herting, 1966, 1983) suggested a sistergroup relationship between the

Phasiinae+Exoristinae and Tachininae+Dexiinae while (Richter, 1992) proposed

Phasiinae+ (Exoristinae + (Tachininae + Dexiinae)) and (Shima, 1989) a sister group relationship between Phasiinae + Dexiinae and Exoristinae + Tachininae.

Rhinophoridae

The Rhinophoridae is a small family found in all geographical regions. All known

Rhinophoridae larvae are parasitoids of woodlice. The eggs are deposited in environments where woodlice congregate; i.e, the eggs are not attached to the potential hosts (Thompson, 1934). The first and second instars feed on the host blood while the third instar attack the tissue and organs of the woodlouse. The

116 monophyly of this family has been based on morphological characters from the first instar larva (Pape, 1992) but there are no convincing characters from the adults to support the monophyly of the Rhinophoridae (Wood, 1987). Rognes (1997) pointed out the possibility of the Rhinophoridae might not be a monophyletic group and some taxa (e.g. Rhinophora lepida) might have to be excluded in order to retain the monophyly of the family. The genus Bezzimyia was included in the Rhinophoridae by

(Pape and Arnaud, 2001) after having been transferred between the Rhinophoridae and Tachinidae. The Axiniidae (axe flies), formerly occasionally treated as a separate family, has also been considered close relatives and also a part of the family

Rhinophoridae (Colless, 1994; Colless and McAlpine, 1991; Crosskey, 1977; Pape,

1998b).

Oestridae

The Oestridae is an economically important family with approximately 150 species and are again found in all regions of the world and across various habitats. These larvae of all oestroids are parasites of mammals including domesticated livestock.

Heavy infestations can be so harmful to the animal that they can cause significant economic losses. The Oestridae do not feed in their adult stage and their entire nutrition is from larval feeding. Bot flies of subfamily Oestrinae are larviporous and the the 1st instar larvae (Cephenemyi sp.) are squirted into the nasal cavities of various mammals including domesticated livestock where they complete larval development. The Hypodermatinae species (Hypoderma sp.) are oviparous and the eggs are attached to body hairs and the 1st instar larval tunnels into the skin and live in ‘warbles’ under the skin surface of the host (domesticated animals). The species

Dermatobia hominis, from the subfamily Cuterebrinae is the only bot fly that attacks humans. This fly attaches eggs to the body of vectors, which includes mosquitoes,

117 and other haematophagous flies. The eggs hatch in response to the heat of the host and the larvae then enters the host through the bit or other wounds. The larvae develop inside the subcutaneous layers, and after approximately eight weeks, they drop out to pupate for at least a week, typically in the soil. The larvae of species from the Gasterophilinae are mainly found in the digestive tracts of herbivorous animals. These flies lay eggs in the fall on the face and around the lips and noses of horses, donkeys, and other equines and when the eggs hatch, the larvae proceed to either the nasal passages or the stomach of the host animal. Larvae that grow in the stomach are passed in the feces of the host, and they burrow into the soil to pupate and those that grow in the nasal passages drop out of the nose and pupate in the ground.

The monophyly of the Oestridae is well established by many authors (Pape, 1992;

Wood, 1986; Wood, 1987) on the basis of many convincing morphological characters that include first instar larvae with bands of thorn-like spines encircling, sternite 5 of the males with posterior margin simple or with shallow emargination and tergite 6 fused to syntergosternite 7 + 8. There are currently four recognized subfamilies in the

Oestridae: the Cuterebrinae, Gasterophilinae, Hypodermatinae and Oestrinae (Pape,

1992; Wood, 1986) which are biologically so distinct clades (Pape, 2001) that these subfamilies were previously given family status by various authors (Grunin, 1965,

1966, 1969; Papavero, 1977; Pont, 1980; Soos and Minar, 1986a, b, c). The subfamilial relationship (Cuterebrinae (Gasterophilinae (Oestrinae+

Hypodermatinae))) has been supported by morphology (Pape, 2001) while the following tree (Cuterebrinae (Oestrinae (Gasterophilinae + Hypodermatinae))) was supported by molecular sequences from cytochrome oxidase I gene (Otranto et al.,

2003).

118

Mystacinobiidae

This family includes only one species, Mystacinobia zelandica, known only from roosts of short-tailed bats (Mystacina tuberculata) from New Zealand. The adults are apterous, have reduced eyes, and cling on to the fur of the bat using specialized claws. However, in contrast to the streblids and nycteribiids, Mystacinobia only uses the bat for transport and not for feeding on its blood. The larvae and adults feed on the bat’s guano. Due to highly modified morphology of the flies, the position of

Mystacinobiidae in Diptera remains contentious. Holloway (1976) described the genus and placed it in the acalyptrate Schizophora under the Drosophiloidea, but

Griffiths (1982) placed Mystacinobia as a genus in the Calliphoridae by and

Kurahashi (1989) erected a subfamily Mystacinobiinae within the Calliphoridae.

Mystacinobia was placed in its own family within the Oestroidea by (McAlpine, 1989), a position that was confirmed by Rognes (1997). Gleeson et al. (2000) used mitochondrial 16S ribosomal DNA sequences to support the position of the

Mystacinobiidae as the sistergroup to the calliphorid clade within the Oestroidea.

Sarcophagidae

The Sarcophagidae, a large family with worldwide distribution, are commonly known as flesh flies because some species cause frequent myiases in livestock and man, but the biology of these flies is much more varied. While some species (genus

Wohlfahrtia) feed in mammalian tissues others have larvae (Senotainia tricuspis) that are internal parasites of honeybees. Larvae of species like the Dexosarcophaga termitaria and Brachicoma devia have been isolated from the nests of social insects like the termites and wasps. Some sarcophagids like the species Blasoxipha have also been used as biological control agents as they parasitize grasshoppers and

119 locusts and a few species from this genus have larvae that breed in pitcher plants where they feed on insects drowned in the pitcher.

Based on morphology, the Sarcophagidae is a very well corroborated monophyletic group (Pape, 1992) and three subfamilies are recognized: the Miltogramminae,

Paramacronychiinae, and Sarcophaginae. The majority of the species in the subfamily Miltogramminae are kleptoparasites in nests on eusocial Hymenoptera

(Pape, 1996; Spofford and Kurczewski, 1989). The generic classification and monophyly of Miltogramminae has been based non-larval characters (Pape, 1996;

Rohdendorf, 1967; Verves, 1989; Verves, 1994) though the interesting biology of this subfamily seems to have spurred a diversification in the first instar larva morphology

(Szpila and Pape, 2005a, b). The first instars are readily available because all

Sacrophagidae are ovoviviparous (Meier et al., 1999) and females tend to larviposit in captivity. The Paramacronychiinae and Sarcophaginae breed mostly in invertebrate and vertebrate carrion but there is a tendency within the

Paramacronychiinae to becoming facultative parasitoids on insects and other invertebrates. The larvae of the Paramacronychiinae and Sarcophaginae also commonly breed in animal carcasses, dead fish, decaying shellfish, snails and

Orthoptera. A sistergroup relationship between these subfamilies was suggested

(Pape, 1992, 1998a) and the Miltogramminae is considered to be the sistergroup of the (Paramacronychiinae + Sarcophaginae) clade (Pape, 1998a).

4.1.4 Is the McAlpine’s fly a calyptrate?

The so-called McAlpine’s fly was discovered in the 1970s in South East Australia.

The flies are found in woodlands and the common breeding medium dung where the larvae and adults are saprophagous (Ferrar, 1979). This fly possesses all calyptrate

120 autapomorphies, but it exhibits “generalized” calyptrate morphology and there are no obvious synapomorphies with currently recognized families that would allow for a placement into an existing calyptrate family (Michelsen & Pape in prep.). Based on the male genital characters, this fly was also considered closely related to the

Calliphoridae (Ferrar, 1979). However, McAlpine’s fly is thought to have affinities with the muscoid families Anthomyiidae and the Muscidae.

4.1.5 Calyptratae phylogenetics

The monophyletic Cyclorrhapha (Diptera) is traditionally divided into two groups,

‘Aschiza’ and ‘Schizophora’ based on the absence or presence, respectively, of a ptilinal fissure. While the ‘Aschiza’ are likely to be paraphyletic (Collins and

Wiegmann, 2002; Cumming et al., 1995; Griffiths, 1972; Griffiths, 1990; Hennig,

1973; Wada, 1991; Zatwarnicki, 1996), the Schizophora are well supported as monophyletic. It is traditionally divided into the ‘Acalyptrates’, again a group that is likely to be paraphyletic and the Calyptratae. Though the Calyptratae were traditionally supported as monophyletic based on the large lower calypter, now many additional characters like presence of a dorsolaterally placed cleft or seam in the antennal pedicel, presence of lower fronto-orbital bristles, development of prestomal teeth and the presence of abdominal spiracles 2–5 in the tergites have been employed as support of calyptratae monophyly (Griffiths, 1972; Hackman and

Väisänen, 1985; Hennig, 1973; McAlpine, 1989; Michelsen, 1991). Other morphological characters that support the monophyly of the Calyptratae include thorax with complete transverse line (’suture’) arising in front of the anterior notal wing processes and wing vein Costa with alternating slender and robust setae. The calyptrate monophyly appears overall very well supported, but most of the characters mentioned above are not unique to Calyptratae and are also found in some

121 acalyptrate families. Given that the acalyptrates are paraphyletic some of the characters are likely synapomorphies of some acalyptrates (super)family+

Calyptratae.

Calyptrate monophyly was also suggested in analyses utilizing molecular data

(Nirmala et al., 2001; Vossbrinck and Friedman, 1989). In Nirmala et al. (2001) two ribosomal gene regions 18S and 16S were used to reconstruct the relationships of the calyptrates. But this analysis included only sixteen species representing twelve families and could not make conclusions on relationships either between the superfamilies or families. This widely accepted classification of the Calyptratae is based on morphology and distinct biological characteristics but it remains untested with molecular data.

The monophyly of Hippoboscoidea has also been to be a monophyletic superfamily based on morphological characters (Griffiths, 1972; Hennig, 1973) supported by molecular data (Dittmar et al., 2006; Nirmala et al., 2001; Petersen et al., 2007). The

Hippoboscoidea are are ectoparasites on mammals, birds or bats and are well supported as a monophyletic superfamily based on molecular data and morphological characters such as adenotrophic viviparity (Meier et al., 1999) and wing characteristics (Petersen et al., 2007). Four families are included in this superfamily; the Glossinidae (free-living), Hippoboscidae (birds and mammals),

Streblidae (bats), and Nycteribiidae (bats) (Hennig, 1973; McAlpine, 1989) and the phylogenetic relationships largely follow host specialization. The monophyly of three familes: Hippoboscidae, Glossinidae and the Nycteribiidae is not in dispute and find support from both morphological characters and molecular data (Bequaert, 1952;

Dittmar et al., 2006; Griffiths, 1972; Hennig, 1973; Maa and Peterson, 1987;

122 McAlpine, 1989; Nirmala et al., 2001; Petersen et al., 2007) while the Streblidae were found to be paraphyletic by (Dittmar et al., 2006) and (Petersen et al., 2007). The two hippoboscoid families feeding on bats are considered sistergroups by various authors

(Hennig, 1973; McAlpine, 1989; Petersen et al., 2007) though this clade was not recovered in all analyses based on the molecular data in (Nirmala et al., 2001) and

(Dittmar et al., 2006) .

The Muscoidea was long considered a monophyletic group by McAlpine (1989) and

Hennig (1973) based on morphological support from characters such as the male anus being situated above the cerci, the male sternite ten forming bacilliform sclerites, and the female abdominal spiracle seven being located on tergite six.

However, some authors suspected that the Muscoidea is a paraphyletic grade

(Michelsen, 1991) and recent analysis based on molecular data confirmed the paraphyly. Monophyly has been established for three of the families: Fanniidae

(Pont, 1977), Muscidae (Carvalho and Couri, 2002; McAlpine, 1989; Michelsen,

1991; Roback, 1951; Schuehli et al., 2007; Séguy, 1937) and Scathophagidae

(Bernasconi et al., 2000a; Hennig, 1973; Kutty et al., 2007; McAlpine, 1989). Support came from morphological as well as molecular analyses. Though the systematics of the Fannidae still requires attention, the montypic species Australofannia is considered the sister group species to the other Fannidae (Pont, 1977). Eight subfamilies are recognized in the Muscidae (Achanthipterinae, Atherigoninae,

Azeliinae, Cyrtoneurininae, Coenosiinae, Muscinae, Mydaeinae and Phaoniinae), but the muscid subfamilies and tribes have undergone many classificatory changes and stability has yet to be achieved (Couri and Carvalho, 2003; Couri and Pont, 2000;

Nihei and de Carvalho, 2007; Savage et al., 2004; Schuehli et al., 2007). The family

Anthomyiidae has been discussed as both a monophyletic (Michelsen, 1991;

123 Michelsen, 1996) and paraphyletic (Griffiths, 1972) group and the subfamily-level classifications is similarly contentious. The two subfamilies of the Scathophagidae: the Scathophaginae and the Delininae are monophyletic in the most recent cladistic analyses based on DNA sequence data (Bernasconi et al., 2000a; Kutty et al., 2007).

Within the musoid grade, the Fannidae is considered to be the sistergroup to the other families and the Anthomyiidae and Scathophagidae are closely related to each other. However, a recent analysis revealed that the superfamily Oestroidea is nested within the muscoid grade (Kutty et al., submitted).

From the literature review, it becomes obvious that much more work is needed to resolve the phylogenetic relationships of the Oestroidea and also the calyptrates because of the presence of paraphyletic families and superfamilies. The phylogeny of the calyptrate group as a whole, was suggested by McAlpine (1989) in the Manual of

Nearctic Diptera and this manual remains the only source of comprehensive information on the Calyptrate phylogeny. But the characters and character coding for these phylogenies were based on synapomorphies related to the ground state plan of the Schizophora rather than to the sister group. Griffiths (1972) calyptrate study used manual tree reconstruction methods as opposed the various sophisticated tree reconstruction and analysis methods available today.

124 Fig 4.2. Phylogeny of the Calyptratae based on morphology in McAlpine, 1989.

Because of the diversity and large number of the different Calyptrate species, finding morphological characters and properly coding them across the Calyptratae is complicated and this group is thus an ideal case where molecular data can play an important role for reconstructing its relationships. The use of molecular data is appealing because a large number of characters can be used for this young radiation. The diversification of Diptera lineages occurred in the Mesozoic era

(Yeates and Wiegmann, 1999) and a time scale for brachyceran fly evolution estimated the divergence time of the calyptrate representative at 29–80 MYA

(Wiegmann et al., 2003). However, since this group radiated relatively fast during a short period of time, deducing the phylogeny of the calyptrates has its share of complications. Both mitochondrial and nuclear genes were thus here selected for amplification in order to recover the relationships between the higher-level taxa and the terminals. Mitochondrial genes have the advantage because of the ease of

125 amplification and faster evolutionary rates (Simmons et al., 2007), while nuclear genes can be more effective at resolving deeper nodes and higher-level relationships

(Lin and Danforth, 2004).

In this study, molecular data from eight genes; four mitochondrial genes 12S, 16S,

COI (two fragments), Cytb, and four nuclear genes 18S, 28S, Ef1a and CAD were used to reconstruct the phylogeny of the Calyptratae with 247 species representing four families of the Hippoboscoidea, four families of the muscoidea grade and six families of the Oestroidea. The results have implications for the systematics of many families (e.g. the Calliphoridae) in the group. This study of the Calyptratae encompasses data from the previous studies on the Scathophagidae (Kutty et al.,

2007), Hippoboscoidea (Petersen et al., 2007) and muscoidea grade (Kutty et al. submitted) along with a more extensive oestroid taxon sampling to investigate interfamily relationships within the Oestroidea.

4.2 MATERIALS AND METHODS

4.2.1 Taxa

The three superfamilies of Calyptratae are here represented by 247 species (Table

4.1). The four families of the Hippobocoidea are represented by 30 species and the muscoidea grade with three fanniids, 46 muscids, 17 anthomyiids and 64 scathophagids. The six families of the Oestroidea are represented by 91 species; one species form the Oestridae, four species from the Rhinophoridae, 48 species from the Sarcophogidae, nine species from the Tachinidae, 22 species from the

Calliphoridae. We also included the enigmatic calyptrates Mystacinobia zelandica and McAlpine’s fly. As outgroups we included 11 non-calyptrate species representing

126 four different superfamilies from the acalyptrate grade: Carnoidea (Hemeromyia anthracina), Lauxanoidea (Celyphidae sp.), Sciomyzoidea (Lopa convexa, Gluma nitida, Orygma luctuosum, Helcomyza mirabilis, buccata, Icaridion debile) and Tephritoidea (Ceratitis capitata, Bactrocera dorsalis, Bactrocera oleae).

4.2.2 DNA extraction

DNA extractions used two main protocols: CTAB extraction and the DNeasy Blood and Tissue Kit. The CTAB DNA extraction protocol was followed as described in

(Kutty et al., 2007). The DNA was precipitated with 100% ethanol, washed with 70% ethanol and then dissolved in 50-100 μl of water depending on the size of the pellet.

DNA extractions on some whole flies were also performed using the DNeasy

(QIAGEN, Santa Clara, CA) according to manufacturer’s instructions. DNA was eluted in 50-70 μl of distilled water.

127 Table 4.1 Species used in study with total length (bp) and genes amplified.

128 Table 4.1 (contd.) Species used in study with total length (bp) and genes amplified.

129 Table 4.1 (contd.) Species used in study with total length (bp) and genes amplified.

130 Table 4.1 (contd.) Species used in study with total length (bp) and genes amplified.

131 Table 4.1 (contd.) Species used in study with total length (bp) and genes amplified.

132 Table 4.1 (contd.) Species used in study with total length (bp) and genes amplified.

133 4.2.3 DNA amplification and sequencing

PCR reactions used 2.5µl of reaction buffer, 2µl dNTPs, 0.5-1.0 µl MgCl, 0.1µl of either Takara Ex-Taq or Bioline Taq. About 1-5 μl of template DNA was used for amplifications in 25µl reaction volume. Nine different gene regions were amplified.

The mitochondrial genes sequenced are 12S ribosomal gene, 16S ribosomal gene,

Cytochrome oxidase I (COI), Cytochrome b (Cytb) and nuclear genes are 18S ribosomal gene, 28S ribosomal gene, Elongation factor 1 alpha (Ef1a) and a fragment of the carbamolyphosphate synthetase (CPS) region of the CAD gene. The primers used in this study are given in Table 4.2. The PCR programs for all all gene amplifications except the CAD gene consisted of an initial denaturation step at 95°C for 7 minutes, followed by 95°C for 1.5 minutes, annealing at temperatures ranging from 44-50°C and extension at 72°C for 15 minutes. A final extension at 72°C for about 5 minutes was also added. For CAD, the amplification protocol described by

(Moulton and Wiegmann, 2004) was used for the amplification, and gel extraction was carried out on the amplified product using the gel extraction kit QIAquick

(QIAGEN, Santa Clara, CA) following manufacturer’s protocol. The amplified gene products were purified using Bioline’s Quick-Clean or Sure-Clean solution following the manufacturer’s protocol. Cycle sequencing reactions were performed on the purified products using BigDye Terminator v3.1. The products were then prepared for direct sequencing by removing the dye-terminator using the Agencourt CleanSEQ solution following manufacture’s protocol. The sequences were edited and assembled in Sequencher 4.0 (Gene Codes Corporation Inc, Ann Arbor, Michigan,

USA).

134

Table 4.2. Primers used in study

4.2.4 Alignments

The protein encoding genes COI (two gene fragments), Cytb, Ef1a and CAD were aligned using AlignmentHelper which uses ClustalW (Thompson et al., 1994) for aligning the nucleotides based on amino acid translations of the sequence

(McClellan, 2004). The protein coding genes, except for CAD4, were gap-free after alignment. The alignments of the ribosomal genes followed a different strategy. The

135 alignment of the ribosomal genes in Clustal usually does not yield satisfactory results. This is probably partially due to the use of a neighbor joining guide tree. In this study, we thus chose to use a more reasonable guide tree. The ribosomal genes were initially aligned in ClustalX using default parameters (gap opening: gap extension=15:6.66). The conserved regions of the ribosomal genes along with the protein encoding genes were then concatenated into a data set that we called the

“guide dataset.” This dataset was then analyzed in TNT 1.1 (New technology search at level 50, initial addseqs=9, find minimum tree length 5 times) and resulted in 13 trees. Each of these 13 tree topologies were then used as guide trees for the alignment of the four ribosomal genes 12s, 16s, 28S and 18S (all sequence data including the loops). The sets of aligned ribosomal genes were then concatenated with the protein encoding genes resulting in 13 datasets. The different datasets were analyzed in TNT 1.1 (New technology search at level 50, initial addseqs=9, find minimum tree length 5 times) and we used tree length as an optimization criterion for choosing among the data sets.

4.2.5 Taxa selection strategy for analysis

We have excluded certain species from our initial species list for this study. Gene amplifications for some species were unsuccessful and species with relatively less data were excluded. Certain other species especially the Oestridae subfamilies were excluded because of their erratic behavior in the analysis and their placement on the trees (affinity to Hippoboscoidea species). This exclusion was not carried out subjectively but by measuring the leaf stability using the program Phyutility (Smith and Dunn, 2008) on a jackknife tree (parsimony analysis) comprising of all the calyptrate species for which amplifications were carried out. Taxa with leaf stability values less than a threshold of 75 were excluded from the analysis.

136

4.2.6 Tree search strategies

The dataset was analyzed using both maximum parsimony (MP) and maximum likelihood (ML). The rooting of the trees utilized the acalyptrate Hemeromyia anthracina (Carnidae), although any other acalyptrate family could have been used as outgroup given that we currently do not have a viable hypothesis as to which acalyptrate taxon may be the sister group of the Calyptratae. Maximum parsimony analyses were carried out in TNT v2.0 (New technology search at level 50, initial addseqs=9, find minimum tree length 5 times) (Goloboff, 2000), with indels coded once as missing data and once as fifth character states. Node support was assessed by jackknife resampling percentiles (250 replicates, same search options as above) at 36% deletion as recommended by (Farris et al., 1996). The likelihood analyses were conducted with Garli v0.951 (Zwickl, 2006) with three independent runs carried out for 50,000 generations. The bootstraps for the ML analysis cold not be presented because of the constrain of computational time taken for a dataset of this size. The same concerns are present when the Bayesian analysis has to be carried out. The analysis takes an exceptionally large amount of time to complete the specified number of generations, and still the independent runs do not to attain stationarity and similar likelihood values. This problem has been encountered by other large scale analysis (Hackett et al., 2008), where time taken for the Bayesian analysis was extremely long and the different runs did not attain stationarity.

137 4.3 RESULTS

The aligned Calyptratae dataset had 248 taxa and 7314 characters. The parsimony analysis with indels treated as the 5th character state yielded 15 trees with a tree length of 43,878 (Fig 4.3). The superfamily relationships are similar in all 15 trees although there is some conflict within the Oestroidea. The three independent runs on

Garli (Zwickl, 2006) resulted in the same topologies and the topology of the

Maximum Likelihood tree (Fig 4.4) share many nodes with the MPTs. The parsimony analysis of the dataset with indels coded as missing data yielded 28 trees with a tree length of 42235. The higher-level relationships between the two tree topologies are the same and terminal nodes are shared (Fig 4.3). Since the maximum parsimony analysis recover similar tree topologies for indel=missing and indel=5th character state, the results and discussion will concentrate on the ML tree and MPT where indels are coded as 5th character state. On both the MPT and ML tree, the

Calyptratae monophyly is well supported. The trees rooted at Hemeromyia anthracina has all other non-calyptrate species forming a clade on the ML tree whereas on the MPT the Tephritoidea are the sistergroup to the calyptrates. On both tree topologies, the superfamily Oestroidea is a well supported monophyletic group and nested within the muscoid grade.

Within the Oestroidea, the family Sarcophagidae is moderately supported.

McAlpine’s fly and the Mystacinobia zelandica are sistergroups and this clade is placed as the sistergroup to the Sarcophagidae on all trees. This clade combined with the sarcophagid clade is the sistergoup to the rest of the oestroid species

(calliphorids, tachinids, rhinophorids and oestrids) and both the ML and MPT support this relationship.

138 Fig 4.3 Strict consensus of fifteen most parsimonious trees (indel=5th character); above node jackknife support for indel=5th character state; below node indel=missing.

139 Fig 4.3 (contd.) Strict consensus of fifteen most parsimonious trees (indel=5th character) value above node jackknife support for indel=5th character state; below node indel=missing

.

140 Fig 4.3 (contd.) Strict consensus of fifteen most parsimonious trees (indel=5th character); value above node jackknife support for indel=5th character state; below node indel=missing.

141 The Calliphoridae (including some subfamilies) is paraphyletic on the likelihood and parsimony trees. The calliphorid species are spread across the Rhinophoridae

+Tachinidae+Oestridae clade and the sistergroup to this clade is the monophyletic subfamily Rhiniinae. On the ML tree, the Tachinidae are monophyletic along with the two subfamilies Tachininae and Phasiinae. The Rhinophoridae is also recovered as a monophyletic group on the ML tree. On the parsimony tree however, the families

Tachinidae and Rhinophoridae are paraphyletic.

The three subfamilies of the Sarcophagidae; the Miltogramminae,

Paramacronychiinae and the Sarcophagiinae are monophyletic with the

Paramacronychiinae being sistergroups to the Miltogramiinae + Sarcophaginae clade on the MPT. On the ML tree, the subfamily Sarcophaginae shares a sistergroup relationship to the Paramacronychiinae+Miltogramminae clade. The monophyly of the subfamily Sarcophaginae is very well supported, but the relationships between the three subfamilies are not stable. The genera Sarcophaga, Helicobia and Peckia are monophyletic while Blaesoxipha and Tricharaea are paraphyletic. Wohlfahrtia and Sarcophila belonging to the subfamily Paramacronychiinae are monophyletic and are sistergroups.

Of the two subfamilies of the Tachinidae in the dataset, the Tachininae are monophyletic and the genus Gymnosoma from the subfamily Phasiinae is monophyletic on the MPT. These two subfamilies share a sistergroup relationship and the genera Tachina and Gymnosoma are monophyletic. The oestrid representative Cuterebra baeri is closely related to the tachinids more specifically to the genus Gymnosoma. The family Rhinophoridae is paraphyletic and the Stevenia species are sistergroups.

142 Fig 4.4 Likelihood tree from Maximum likelihood analysis using Garli.

143 Fig 4.4 (contd.) Likelihood tree from Maximum likelihood analysis using Garli.

144 Fig 4.4 (contd.) Likelihood tree from Maximum likelihood analysis using Garli.

145 The family Calliphoridae and some of its subfamilies are paraphyletic and the relationships are the same on both the MPT and ML tree. The subfamilies Luciliinae,

Rhiniinae and Polleniinae are monophyletic. The subfamily Mesembrinellinae is closely related to the Tachinidae whereas the Polleniinae and the Helicoboscinae species Eurychaeta palpalis is closely related to the Rhinophoridae. The

Calliphorinae and Chrysomiinae are paraphyletic and the Melanomyinae and

Bengaliinae are closely related to these subfamilies respectively. The genera

Chrysomya, Lucilia, Hypopygiopsis of the Calliphoridae are monophyletic.

Within the Calyptratae, the Hippoboscoidea are the sistergroup to the rest of the calyptrates. Within the Hippoboscoidea, the Glossinidae form a strongly supported monophyletic group and is the sister group to the other families on both the most parsimonious tree and likelihood tree. On both the ML and MPT, the Nycteribiidae and Streblidae species form a clade, but only the Nycteribiidae are monophyletic

(see Petersen et al., Dittmar et al). The family Hippoboscidae is paraphyletic on the

MPT and only the subfamilies Lipopteninae and Ornithomyini are monophyletic. On the ML tree, the Hippoboscidae and its four constituent subfamilies: the Olfersini,

Hippoboscinae, Lipopteninae, Ornithomyini are monophyletic.

The Muscoidea are a paraphyletic grade both the MP and ML analysis and the three species of Fannia form a well supported monophyletic Fannidae and this family is placed as the sistergroup to the rest of the muscoids and Oestroidea. The Muscidae are monophyletic with high support and is the sister group of the clade consisting of the Scathophagidae, Anthoymiidae and Oestroidea. The muscid subfamily

Coenosiinae is monophyletic while the other subfamilies the Mydaeinae, Phaoniinae,

Azeliinae and Muscinae are either para- or polyphyletic. The monophyletic genera in

146 the Muscidae include Coenosia, Helina, Hebecnema, Hydrotaea, Limnophora,

Mydaea, Mesembrina, Morellia, Musca, Muscina, Phaonia, Spilogona and Thricops.

The Anthomyiidae+Scathophagidae clade is well supported on both the ML and

MPT, but on the MPT the families appear to be paraphyletic with the anthomyiid species Mycophaga testacea and scathophagid Hydromyza clade being sistergroups. However, this paraphyly is not supported on the jackknife tree. On the

ML tree, the Scathophagidae is monophyletic and the sister group to this family is the paraphyletic Anthomyiidae. Within the Anthomyiidae on the MPT, the genera

Botanophila, Pegoplata and Eutrichota are monophyletic while the Lasiomma and

Hylemya are paraphyletic. The subfamily Delininae of the Scathophagdiae is monophyletic and the genera Nanna, Gimnomera, Norellia, Chaetosa, Pogonata,

Spaziphora, Hydromyza and Cordilura of the subfamily Scathophaginae are monophyletic.

4.4 DISCUSSION

Many of the hypotheses based on morphological studies in the calyptrates are here corroborated and the same time novel hypothesis with regard to the placement of some species can be proposed based on our molecular dataset. The trees from both the MP (indel=5th character state) and ML analysis share the same overall topology.

The main differences are seen only within the Oestroidea and the relationships within this family have low jackknife support and are unstable. Despite these differences, many of the important relationships within the group could be resolved.

147 4.4.1 Oestroidea monophyly

Oestroidea monophyly has not been doubted and traditionally had good support from morphological characters. Here, we can add support from DNA sequence characters.

However, we revise the composition of Oestroidea by demonstrating that McAlpine’s fly is nested within the superfamily. Furthermore, we demonstrate that the monophyletic Oestroidea are nested within the muscoid grade. The relationships recovered within the Oestridae differ from all previous hypotheses, including

McAlpine’s (1989) and Pape’s (1992) phylogenetic reconstruction for the Tachinidae family-group. The support for the interfamilial relationships is relatively low despite the large amount of genetic data. One of the reasons for this could be the difficulty in efficiently analyzing this dataset under more stringent conditions due to its size. We also find that Sarcophagidae with the relatively richer species sample appears to be well resolved (with node support) and a better taxon sampling could also improve the stability of relationships.

4.4.2 Placement of the McAlpine’s fly and M. zelandica in the Oestroidea

The results of this analysis are very significant in that it corroborates the position of

Mystacinobia zelandica within the Oestroid and also placed the McAlpine's fly as an oestroid. These two rare species are rarely studied and this project is the first to place these taxa in the calyptrate dataset. Particularly, novel is our suggestion that

=McAlpine’s fly is the sister species to Mystacinobia. Previously, McAlpine’s fly had only been placed within Calyptratae and its relationships were poorly understood.

Our placement as the sistergroup to Mystacinobia was initially surprising given that previous authors have considered this species to be either closely related or placed within the ‘calliphoridae’. However, there is now also morphological evidence for its

148 membership in the Oestroidea (Pape, pers. comm.) and this species will need to be placed into a new family within this superfamily. Given that the two species are morphologically very distinct, the McAlpine’s fly should get its own family and will become the first new family of Calyptratae in the last 30 years.

The position of M..zelandica within the Oestroidea had previously suggested.

However, Gleeson et al. (2000) had suggested that this species is closely related to the calliphorid grade. However, only data from one gene was available and the taxon sample in the study was relatively small. Here, we demonstrate oestroid membership based on a rich taxon sample and a large amount of gene data. This sistergroup relationship between two interesting flies restricted only to SE Australia and bat caves in New Zealand respectively is also highly stable.

4.4.3 Oestroidea family relationships

The Sarcophagidae and all three subfamilies the Miltogramminae,

Paramacronychiinae and Sarcophaginae are monophyletic. This monophyly of this family and subfamilies has support from morphological characters (Pape, 1992,

1996). Based on DNA sequence data, the relationships between the subfamilies are not stable. The Paramacronychiinae are either sistergroup to the Sarcophaginae

(MP), as suggested by morphology (Pape, 1992, 1998a), or the Miltogramminae

(ML). The relationships within the Miltogramminae and Paramacronychiinae are identical on both the MP and Ml tree but several higher-level hypotheses have poor support. Sarcophaga is supported on both the ML and MP trees with the New World species Sarcophaga arizonica and Sarcophaga triplasia being sistergroups. The genus Peckia is monophyletic on the MP tree but not recovered as monophyletic on the ML tree. Some species relationships that are supported by morphology (Pape,

149 pers. comm.) are recovered on the MP tree; this includes the sistergroup relationships between Tricharaea occidua and Sarcofahrtiopsis cuneata and the close relationships between Blaesoxipha plinthopyga and Spirobolomyia flavipalpis.

The other genera recovered as monophyletic on both trees include Wohlfahrtia,

Sarcophila, and Helicobia.

A sister group relationship between the Tachinidae and Sarcophagidae as suggested by (Pape, 1992) and (Rognes, 1997) is not obtained on either the MP or ML tree and the Rhinophoridae +Sarcophagidae clade hypothesized by (Rohdendorf, 1967) is similarly not recovered. On all tree topologies, the Sarcophagidae is the sistergroup to the Mystacinobiidae and McAlpine’s fly, a relationship not suggested before in literature.

Tachinid monophyly is only recovered on the likelihood tree. The species in our taxon sample represent two subfamilies (Tachininae, Phasiinae). Both are monophyletic on the ML tree. This is not the case on the MPT. Here, the oestrid species Cuterebra baeri and the calliphorid Mesembrinella quadrilineata are nested within the tachinid clade. We believe that this may be due to our poor taxon sample for Tachinidae.

Dipteran families that are actively radiating and contain large numbers of species require a better taxon sample in order to successfully resolve the relationships. We have seen this effect before for other calyptrate families like the Anthomyiidae,

Sarcophagidae, and Muscidae, and we believe that the Tachinidae that are one of the largest dipteran families and that are actively radiating group (Crosskey, 1976) would require a more extensive taxon sample.

150 The Rhinophoridae are close relatives of the Tachinidae though calliphorid species from three different subfamilies are present in this clade. The families Oestridae,

Rhinophoridae and Tachinidae have been considered close relatives by Roback

(1951), Wood (1987) and McAlpine (1989) and a similar position is observed on our tree topologies although the calliphorid subfamilies Polleniinae, Mesembrinellinae and Helicoboscinae are also included in this clade. Rhinophorid monophyly is recovered on the likelihood tree. The possibility that the Rhinophoridae might not be a monophyletic group and species like that Rhinophora lepida might have to be excluded to retain monophyly was suggested by (Rognes, 1997). However,

Rhinophora lepida is on all tree topologies placed with the other rhinophorids, albeit with low support. Though the removal of this species would result in a well supported rhinophorid clade, a strong affiliation of the Rhinophora to this clade cannot be ignored and we do not suggest the exclusion of this species from the Rhinophoridae.

The family Calliphoridae is a paraphyletic as had been suggested by many authors based on morphological characters (Griffiths, 1982; Hennig, 1973; Rognes, 1997).

However, we find that several subfamilies are similarly paraphyletic. This includes the

Calliphorinae and Chrysomyiinae. The position of the species Eurychaeta palpalis is within the non-sarcophagid clade and this is corroborated by morphology data

(Rognes, 1986, 1993) though it is not placed as the sistergroup to the other calliphorid species as suggested Pape (1992). In our study, this species appears to be more closely related to the rhinophorid species. The Rhiniinae were traditionally considered the sistergroup to the Rhinophoridae (Pape and Arnaud, 2001). However, we find that it is placed as the sistergroup to the clade comprising of the rhinophorids, tachinids, oestrids and calliphorid species. The Mesembrinella quadrilineata is either

151 in the Tachinidae or as sister group to the calliphorid Polleniinae depending on the analysis technique.

Given that morphology and DNA sequence data clearly document calliphorid paraphyly, we suggest that one should now refer to this assemblage of species as the calliphorid grade within the Oestroidea. Eventually this grade will have to be split, but since the morphological and DNA sequence data disagree on some key relationships, new taxa and names can only be proposed in the future.

4.4.4 Higher-level systematics of the Calyptratae

This dataset combines calyptrate species form the earlier studies (Chapter 1,2&3).

The better taxon sampling would enable a more rigorous test of the monophyly of the

Calyptratae and the relationships at the superfamily and family level and hence we revisit the earlier questions of monophyly/paraphyly and relationships of superfamilies and relationships between families in the Hippoboscoidea and muscoidea grade. The presence of ten non-calyptrate species has confirmed the monophyly of the Calyptrate with very strong support. The sistergroup of the calyptrates cannot still be determined as the relationships between the outgroups vary with different analysis techniques and it remain unclear which taxon should be used for rooting. Therefore, no conclusion with regard to the sistergroup of the

Calyptratae can be suggested.

The Hippoboscoidea is placed as the sistergroup to the rest of the calyptrates and is monophyletic. The family level relationships in our analysis differ from the results of the analysis in (Petersen et al., 2007) mainly with respect to the family

Hippoboscidae which is paraphyletic in our analysis. We believe the main difference

152 is that we could not use all of Petersen et al.’s data because a different set of genes was analyzed. The muscoid families again form a paraphyletic grade with the

Oestroidea being placed as the sistergroup to the Anthomyiidae and Scathophagidae clade. The interfamilial relationships between these families are stable and overall in agreement with the muscoidea grade study in chapter three. This includes the paraphyly of Anthomyiidae. However, Scathophagidae monophyly is not recovered in all analyses. The Fanniidae is again the sistergroup to a clade composed of the muscoid grade and Oestroidea.

The next interesting and obvious step in this study isto trace the diverse natural history across this group. With this calyptrate phylogeny and information on the larval breeding habits, the origin and evolution of the many different life strategies of calyptrate species can be explored.

The dataset for Calyptratae that was here assembled was due to its large size difficult to assemble. We faced numerous taxon sampling, amplification, and computational problems. The gene regions amplified in this study were selected based on the availability of conserved Diptera primers. In some cases, these primers had to be modified in order to improve amplification success. However, in a moderate number of cases, amplifications of all selected genes for a species were not be possible due to different reasons such as primer mismatch, presence of inhibiting substances, poor DNA quality, etc; i.e., certain species in this dataset have a fairly large amount of missing data which is a concern. However, simulation and empirical studies have shown that there is little relationship between the completeness of a taxon and its impact on an analysis (Wiens, 2003).

153 The phylogeny of such a diverse and large group require extensive taxon sampling in order to achieve phylogenetic accuracy (Zwickl and Hillis, 2002). Though the dataset includes exemplars from all families and almost all subfamilies, there is still a need for sampling additional taxa for some families within the Oestroidea especially the

Oestridae, Tachinidae, and the calliphorid grade. Without this sampling, the interfamilial relationships cannot be resolved with good node support. However, we also see some less satisfactory results and oddities from increased taxon sampling.

One such case is scathophagid paraphyly (MP analysis) and the other is hippoboscid paraphyly where increase in taxon sampling in the dataset has effects on groups that were highly stable prior to addition of more species. The paraphyly of the

Hippoboscidae could be attributed to the low gene overlap and the scathophagid paraphyly could be due to the difficulty to get accurate results given the data set size and this may also explain the differences between the analyses. The computational challenges of analyzing such a large data set is another issue of concern (Sanderson and Driskell, 2003). The analysis of a large dataset such as the calyptrate dataset required a large amount of computational time and resources. Though the maximum parsimony analysis (and jackknife resampling) was carried out successfully, the running time of a ML bootstrap analysis would have been excessively long and could not be completed.

However, it must be noted that regardless of these shortcomings, our dataset is still the largest ever assembled for the Calyptratae. Our dataset has the number of taxa and characters and our results will hopefully have a major impact on further studies of calyptrate relationships.

154 4.5 CONCLUSIONS

This study resolves some of key issues in Oestroidea relationships including providing evidence for the paraphyly of the calliphorid grade. A family-level hypothesis for the Oestroidea is proposed and the position of two enigmatic species, the McAlpine’s fly and Mysctacinobia zelandica within the Oestroidea is clarified. Our study also provides molecular evidence for the monophyly of the Calyptratae, the superfamilies Hippoboscoidea and Oestroidea. The paraphyly of the muscoid grade is also confirmed.

155 OVERALL CONCLUSIONS

This study has addressed many key issues in calyptrate phylogeny, an important branch of the Diptera tree of life. It provides the very first extensive molecular phylogenetic tree for the Calyptratae which includes representatives from almost all recognized families. While most of the systematic conclusions are in corroboration with previous morphological studies, there are some unexpected and interesting findings. The superfamily and family relationships have been addressed with a taxon sampling of 247 species representing majority of the families. The inclusion of 11 outgroup species from different families have confirmed and supported the monophyly of the Calyptratae beyond doubt. The placement of the three superfamilies the Hippoboscoidea, muscoidea grade and Oestroidea are resolved.

The monophyly of the Hippoboscoidea is well supported by molecular data. The interfamilial relationships within this superfamily are addressed and convincing phylogenetic evidence for the monophyly of Hippoboscoidea, Glossinidae,

Hippoboscidae, Nycteribiidae, Hippoboscinae, Lipopteninae, Ornithomyini, Olfersini, and the Nycteribiinae is provided by our data. The data also provide weak evidence for a paraphyletic Streblidae. The evolution of specialization and diversification of host shifts, morphological and life history in the families of Hippoboscidae are also studied. A single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates and at least two host shifts from mammals to birds have occurred. The hippoboscoid ancestor also evolved adenotrophic viviparity. Morphological characteristics such as wings have been lost in this superfamily and never regained.

The gradual reduction in the motility of the deposited final instar larvae from active

156 burrowing in the soil to true pupiparity where adult females glue the puparium within the confines of the bat roosts is compatible with this phylogenetic hypothesis.

At the superfamily level, the ‘Muscoidea’ is a paraphyletic group based molecular evidence from this study. The muscoid paraphyly is due to the placement of the superfamily Oestroidea within this grade. This paraphyly has been suspected based on morphological characters and this now finds further support from genetic data.

The muscoid families Fanniidae, Muscidae and Scathophagidae are monophyletic with the exception of the Anthomyiidae which are always recovered as a paraphyletic group. The Fanniidae is placed at the ‘base’ of the musocid grade and the

Anthomyiidae and Scathophagidae are sistergroups. The subfamily relationships within the Muscidae and Scathophagidae have also been addressed.

The systematics of the family Scathophagidae based on molecular data has been addressed in detail in this study. The phylogenetic relationships of 66

Scathophagidae species representing 22 genera and the two subfamilies are reconstructed. The scathophagid phylogenetic tree was used to trace its larval natural history evolution traits such as phytophagy, parasitism, and predation as this family mirrors the diversity of habitats and breeding substrates of the Cyclorrhapha.

Though the ancestral larval breeding habit widely being thought to be saprophagy in the form of breeding in dung or other decaying organic matter, this is not the case for the Scathophagidae. Instead, phytophagy is the ancestral larval breeding habit and saprophagy has evolved twice from phytophagy whereas predation has evolved two times independently, once from phytophagy and once from saprophagy. This dataset was also used to test various alignment (ClustalX, Manual, POY) and analysis strategies. Assigning higher weights to transversion than transitions was the

157 preferred treatment for this dataset and such transversion weighting improves node support, character congruence, and topological congruence for all three different ways to align the ribosomal genes.

The Oestroidea is a strongly supported monophyletic group. The interfamilial relationships in this superfamily are addressed and the results have implications in the systematics of this superfamily. This study supports the paraphyly of the family

Calliphoridae which has been suggested in literature. Two interesting species, the

Mystacinobia zelandica and McAlpine’s fly share a sistergroup relationship and are placed within this superfamily.

This study can be further extended to include more species from the different families especially the Muscidae and Tachinidae which are large families within the

Calyptratae. Also with the availability of a calyptrate tree, the origin and evolution of various different interesting traits of this group including natural history, morphological characteristics and behaviour can be further studied and understood.

.

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