ABSTRACT
TRAUTWEIN, MICHELLE DENEE. Multi-gene Phylogenetics to Resolve Key Areas of the Fly Tree of Life. (Under the direction of Brian M. Wiegmann.)
FLYTREE (an NSF Assembling the Tree of Life project) is a large collaborative project aimed at reconstructing relationships among major lineages of Diptera. Previous morphological and molecular work, along with preliminary analyses of phylogenomic and total evidence data sets from FLYTREE, have provided evidence that while much of the fly tree of life can be confidently resolved, some regions remain challenging to decipher. Flies are a species-rich lineage of insects that originated more than 240 MYA in the Mesozoic. Ancient radiations, particularly if they occurred rapidly, can be difficult to resolve, even with large amounts of data. Phylogenetic inference can be misled by both systematic and stochastic error. The reliance on rigorous data exploration to decipher phylogenetic signal from noise can be crucial to the accurate recovery of evolutionary relationships. This study utilizes multiple nuclear genes and data exploration to address three persistently problematic regions of dipteran evolution. The first chapter evaluates the relationships of the lower brachyceran superfamily Asiloidea, the putative sister-group to Eremoneura (Cyclorrhapha +
Empidoidea). CAD + 28S support traditional asiloid clades and recover multiple hypotheses for the sister group to higher flies, primarily due to the indeterminate placement of the family Bombyliidae (bee flies) and the enigmatic genus Hilarimorpha. The genus Apystomyia is strongly supported as sister to
Cyclorrhapha. Taxon stability and the effects of additional genes are explored.
The second chapter addresses the phylogenetics of the subfamilies of
Bombyliidae by analyzing CAD + 28S alone and with morphology. The monophyly of 8 of 15 subfamilies are confirmed along with the polyphyly of
Bombyliinae. A hypothesis for the interrelationships of bee fly subfamilies is presented. Topological incongruence and the effect of the removal of conflict- inducing taxa are explored. The third chapter relies on six-nuclear genes to identify the sister-group of Diptera by resolving the phylogeny of Holometabola.
Traditional supraordinal groupings are confirmed. Mecoptera+Siphonaptera are sister to Diptera. Strepsiptera, previously hypothesized as the closest relative of
Diptera, is confidently placed as the sister-group to Coleoptera. A thorough exploration to rule out the effects of long-branch attraction is presented. Multi-gene Phylogenetics to Resolve Key Areas in the Fly Tree of Life
by Michelle Denee Trautwein
A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fufillment of the requirements for the Degree of Doctor of Philosophy
Entomology
Raleigh, North Carolina
2009
APPROVED BY:
! ! BIOGRAPHY
Michelle Trautwein was born in Philadelphia, Pennsylvania in 1976 and relocated to Austin, Texas six months later. She began her studies at the University of
Texas in 1994 as an art major. Three years later, after an entomology class and a summer catching frogs in Costa Rica, she changed her major to science and graduated in 1999 with a BS in Biology. Before focusing on insects, she assisted with research on pigeon guillemots in Alaska, prairie dogs in Utah, and bottlenose dolphins in Florida. An internship at the Smithsonian studying Diptera brought her back to insects and reintroduced her to flies. Michelle began her graduate studies at North Carolina State University in Raleigh in 2003. Her work and interests are focused on the evolutionary relationships of flies and phylogenomics in general.
! ""! ! ! ACKNOWLEDGMENTS
Thank you to my professors and mentors who introduced me to
entomology (flies in particular), evolution, and phylogenetics: Riley
Nelson, Wayne Mathis, Amnon Friedberg, Lewis Deitz and Brian
Wiegmann. Thank you for your time, your wisdom and your friendship.
Additional thanks go to the other students and post-docs in the Wiegmann
lab who have supported my work and made my years at NC State
memorable.
! """! ! ! TABLE OF CONTENTS
Page
LIST OF TABLES…………………………………………………………………….….v
LIST OF FIGURES ……………………………………………………………………..vi
Chapter 1: A multi-gene phylogeny of the superfamily Asiloidea (Insecta:
Diptera): exploring the effects of taxon sampling and the resolving power of additional genes…….………………………………………………………………1
Abstract.………………………………………………………………………….2
Introduction….…………………………………………………………………...3
Methods and Materials….………………………………………………………7
Results ………………………………………………………………………….14
Discussion………………………………………………………………………20
Conclusions ..………………………………………………………………...... 28
Acknowledgments ……………………….………………………………...... 29
References ……………………………………………………………………..30
Chapter 2: The evolutionary relationships of bee fly subfamilies: short branches, long branches and topological incongruence …………………….52
Abstract…………………………………………………………………………53
Introduction……………………………………………………………………..54
Materials and Methods………………………………………………………..55
Results and Discussion……………………………………………………….61
Conclusions …………………………………………………………………...70
! "#! ! ! Acknowledgments ..……………………….………………………………...... 72
References ……………………………………………………………………..73
Chapter 3: Identifying the sister-group to Diptera: a multi-gene phylogeny of the holometabolous insects …………………………………………………….92
Abstract ………………………………………………………….……………..93
Introduction …………………………………………………….………………94
Results and Discussion ………………………………………….…………...97
Materials and Methods ..….………………………………………………….105
Acknowledgments ..……………………….………………………………....109
References ……………………………………………………………………110
! #! ! ! LIST OF TABLES
Chapter 1
Table 1. Sampled taxa ……………………………………………………….38
Table 2. Clade recovery results with varying methods of analysis,
treatment of data and taxon inclusion …………………………..40
Table 3. Leaf stability values of all taxa, stable taxa and reduced taxa…41
Table 4. Testing of a priori hypotheses in parsimony ……………..………43
Chapter 2
Table 1. Sampled taxa ………………………….…………………..……….80
Chapter 3
Table 1. Taxa, genes and genbank numbers for Holometabola and out-
groups………………………………………………………….….118
Table 2. Clade recovery results from ML analyses with varied taxon and
character inclusion used to counter LBA ………………………121
! #"! ! ! LIST OF FIGURES
Chapter 1
Figure 1. Strict consensus of 6 maximum parsimony trees of 28S+CAD
(bp 1+2). All taxa included …………………………………..…….45
Figure 2. Bayesian tree of 28S+CAD (bp 1+2). All taxa included ………46
Figure 3. Maximum- likelihood tree of 28S+CAD (bp 1+2). All taxa
included ……………………………………………………….……..47
Figure 4. Maximum parsimony analysis of 28S, CAD, TPI, and CO1 …..48
Figure 5. Comparison of MP bootstrap consensus tree including all taxa
and stable taxa only ……………………………………………….. 49
Figure 6. MP, ML, and BI congruent topology of reduced taxa …………..50
Figure 7. Four-cluster likelihood mapping image testing the support for a
monophyletic Asiloidea ………………………………………...... 51
Chapter 2
Figure 1. Strict consensus of 7 maximum parsimony trees of 28S+CAD
(bp 1+2). All taxa included ………………………………………85
Figure 2. Maximum- likelihood tree of 28S+CAD (bp 1+2). All taxa
included …………………………………………………………….86
Figure 3. Bayesian tree of 28S+CAD (bp 1+2). All taxa included ……….87
! #""! ! ! Figure 4. Consensus network showing conflict between MP, ML, and BI
topologies including all taxa ……………………………………...88
Figure 5. Consensus network revealing largely congruent topologies from
MP, ML and BI when conflict-inducing taxa are removed. …….89
Figure 6. Bayesian tree of 28S+CAD (bp 1+2) with 5 conflict-inducing taxa
removed …………………………………………………………….90
Figure 7. Total evidence tree. Bayesian analysis of 28S+CAD (bp 1+2)
plus155 morphological characters . .……………………………..91
Chapter 3
Figure 1. The phylogeny of the holometabolous insects based on 6
nuclear protein-coding genes …………………………………….122
Figure 2. The congruent ML and BI topology with branch lengths and
support values ……………………………………………….…….123
Figure 3a-b. Likelihood-mapping images showing the strength of our
phylogenetic signal and the conflicting data supporting the
placement of Strepsiptera ………………………………….…….124
Figure 4. Neighbor-Nets showing conflicting splits with all taxa included
and with Strepsiptera excluded ……………………………...…..126
! #"""! ! !
Chapter 1:
A multi-gene phylogeny of the superfamily Asiloidea (Insecta: Diptera):
exploring the effects of taxon sampling and the resolving power of additional
genes
! $! ! !
Abstract
Asiloidea are a group of nine lower brachyceran fly families made up of generally large-sized flower visitors, parasitoids and aerial predators of other insects.
Traditionally, the Asiloidea has been viewed as a monophyletic assemblage and the closest relative to the large, successful dipteran radiation Eremonuera
(Cyclorrhapha+Empidoidea). The evidence for asiloid monphyly is limited, and previous morphological and molecular studies demonstrate that this region of fly evolution is marked by very few characters delimiting the relationships between the presumed families of Asiloidea and Eremoneura. Adding to the phylogenetic complexity are the enigmatic ‘asiloid’ genera Hilarimorpha and Apystomyia, currently united in the family Hilarimorphidae, that retain morphological characters of both asiloids and higher flies. In this study we use the nuclear protein-coding gene CAD and 28S rDNA to test the monophyly of the Asiloidea and resolve its relationship to the Eremoneura. To this end, we also explore the effects of taxon sampling on support values and topological stability, the resolving power of additional genes, and hypothesis-testing using flour-cluster likelihood mapping.
! %! ! ! Introduction
! Brachyceran flies are a large Mesozoic radiation of approximately 100,000 described species that includes the great majority of species diversity within the insect order Diptera (Blagoderov et al., 2007; Yeates et al., 2007). More than
80% of brachyceran species, including the well-known flies, Musca domestica
(house fly) and Drosophila melanogaster (vinegar fly), occur within Eremoneura
(Cyclorrhapha + Empidoidea), or higher flies. Eremoneura are among the best supported of all dipteran clades (Yeates and Wiegmann, 1999), with 13 morphological synapomorphies and the strong support of molecular evidence
(Wiegmann et al., 2003). Divergence time estimates suggest that the origin and diversification of the Eremoneura began approximately 170 MYA and expanded with the origin and radiation of angiosperm plants (Wiegmann et al., 2003). The lower brachyceran superfamily Asiloidea, with 12,000 species, has been hypothesized to be the possible sister group to the Eremoneura (Hennig, 1973;
Woodley, 1989). Because asiloid monophyly is not well established, however, it remains unclear whether Eremoneura and Asiloidea share a most recent common ancestor, or whether the Eremoneura orginated from within the
Asiloidea with closest relatives in one or more of the asiloid family-level lineages.
Asiloid flies are generally large, showy flower visitors as adults and almost exclusively substrate-dwelling predators as larvae. Two of the largest families are significant exceptions to this rule: the larvae of Bombyliidae or bee flies, are insect parasitoids, and Asilidae, also known as robber flies, prey on insects as
! &! ! ! adults. Asiloid flies are distributed worldwide, with their greatest diversity occurring in arid, sandy areas.
There are nine families included in Asiloidea: Asilidae, Mydidae,
Bombyliidae, Scenopinidae, Apsilocephalidae, Apioceridae, Hilarimorphidae,
Therevidae, and Evocoidae. The interrelationships of these families are largely unconfirmed, and the evidence that they all share a recent common ancestor is tenuous. Morphologically, the families from which larvae are known are joined only by a larval spiracle in the penultimate abdominal segment (Woodley, 1989;
Yeates, 1994). Even this single unifying character is subject to homoplasy, appearing in Nemestrinoidea, Xylophagomorpha, and Vermileonidae, and may be an adaptation to terrestrial or parasitic habitats (Sinclair et al., 1994).
Molecular evidence from 28S ribosomal DNA in a broader study of brachyeran phylogeny does not support a monophyletic Asiloidea (Wiegmann et al., 2003).
In recent years, there have been several significant studies of asiloid families that have addressed some long-standing phylogenetic questions.
Molecular and morphological data have shown support for a therevoid clade including Therevidae, Scenopinidae, Apsilocephalidae, as well as, the newly described monotypic family Evocoidae (Yeates et al., 2003). A proposed sister- group relationship between Mydidae and Apioceridae is supported by the presence of multiple rectal papillae and shared wing venation features (Woodley,
1989), and molecular support from 28S ribosomal DNA (Irwin and Wiegmann,
2001). The monophyly of Asilidae, the robber flies, is supported by ribosomal
! '! ! ! DNA and morphology (Bybee et al., 2004; Dikow, 2009), although their position relative to the rest of Asiloidea is as yet unknown (Grimaldi, 2005). In contrast,
Bombyliidae are a heterogeneous assemblage with weak morphological support for monophyly (Yeates, 1994; 2002). They have been most recently hypothesized as sister group to (Woodley, 1989; Yeates, 2002) or paraphyletic with (Sinclair et al., 1994) all other Asiloidea.
Several enigmatic genera are also included in Asiloidea and these have contributed to the complexity of determining the superfamily’s monophyly and to difficulties in deciphering closest relatives of Eremoneura (Yeates and
Wiegmann, 1999; Yeates, 2002). Hilarimorpha and Apystomyia are little known asiloid flies that exhibit morphological characters shared with both the Asiloidea and the Eremoneura, and thus appear to be transitional species (Wiegmann et al., 1993). Both of these genera have previously been placed in Bombyliidae
(Hennig, 1973; Woodley, 1989), Therevidae (Sinclair et al., 1994), or their own separate families (Webb, 1974; Nagatomi and Liu, 1994); but the most current morphological evidence joins Apystomyia and Hilarimorpha together in a single family, Hilarimorphidae (Yeates, 1994; 2002). Hilarimorphidae has been considered the sister-group to Bombyliidae (Yeates, 1994) or sister to the entire
Eremoneura (Yeates, 2002; Grimaldi and Engel, 2005), however both hypotheses lack convincing morphological support.
Compounding the difficulty of determining Apystomyia’s phylogenetic placement is the fact that these exceedingly rare flies have been difficult, if not
! (! ! ! impossible, to acquire. First described by Melander in 1950, only 12 specimens were known in museum collections and many were damaged over time. Despite multiple collection attempts, Apystomyia was not captured again until 2005.
These new specimens have provided us with the opportunity to bring molecular data to bear on the hypothesis of Apystomyia’s inclusion in Asiloidea.
The most recent quantitative study that addressed the phylogeny of
Asiloidea in part was based solely on morphological characters (Yeates, 2002).
No previous phylogenetic work has focused primarily on the superfamily or a broad sampling of its taxa. In our molecular study of Asiloidea, we examine the use of the large, nuclear protein-encoding gene CAD and 28S ribosomal DNA to resolve further the relationships among the asiloid Diptera, to test the monophyly of the superfamily, and to determine the sister-group to Eremoneura. Both CAD and 28S have been shown to exhibit considerable phylogenetic signal for inferring Mesozoic-aged divergences amongst the Diptera (Moulton and
Wiegmann, 2004; 2007; Winterton et al., 2007). In addition, we examine the effects of taxon sampling on support values and topological stability and further test the placement of the anomalous genera Apystomyia and Hilarimorpha with additional sequence from 28S, the mitochondrial gene CO1 and the nuclear protein-coding gene TPI (Hardy, 2007) from a subsample of taxa. We explore our hypotheses through four-cluster likelihood mapping.
! )! ! ! Materials and Methods
Taxa Sampled
A total of 50 species representing 49 genera of orthorrhaphous
Brachycera, Asiloidea and Eremoneura were sampled for nucleotide sequencing.
All 9 families of Asiloidea are represented, including the newly described monotypic family Evocoidae, and the enigmatic genera Hilarimorpha and
Apystomyia. Five taxa representing families from diverse non-asiloid lower brachyceran lineages were sampled as outgroups (Table 1).
To test further the placement of Apystomyia and Hilarimorpha, an 8 taxa subsample representing Asiloidea, Eremoneura and lower Brachycera were sampled to seek corroboration from additional genes. The 8 taxon subsample was primarily drawn from within the 50 taxon set.
DNA Extraction, Amplification and Sequencing
Genomic DNA was extracted using the DNeasy DNA extraction kit
(QIAGEN Inc., Valencia, CA). The standard protocol was altered by extending the length of time the specimen was in proteinase K solution to two days, in order to effectively break down chitin but to avoid grinding the specimen. Final elution was reduced to 30 µl to avoid diluting the DNA solution.
Sequence data were collected from all 50 taxa for two nuclear genes, the protein-coding gene CAD (carbamoyl phosphate synthetase-aspartate transcarbamoylase-dihydroorotase) and 28S ribosomal DNA. For CAD,
! *! ! ! approximately 4000 bp from the carbomoylphosphate synthase (CPS) domain of the gene were amplified and sequenced using degenerate primers designed by
Moulton and Wiegmann (2004). To amplify approximately 1000 bp from the 3’ end of 28S rDNA, we used published Diptera primers (Yang, 2000). For 8 of the 50 taxa, 2000 additional base pairs of 28S rDNA were sequenced along with approximately 1400 bp of the mitochondrial gene cytochrome c oxidase subunit 1
(CO1) and 500 bp of the nuclear protein coding gene triose phosphate isomerase
(TPI) using primers developed by Hardy (2007) and Junwook Kim. PCR parameters varied for CAD, 28S CO1 and TPI. PCR products were extracted from agarose gels and purified with the Qiaquick Gel Extraction kit (Qiagen,
Santa Clara, CA). Big Dye Sequencing kits (Applied Biosystems, Foster City,
CA) were used for sequencing reactions and sequencing was completed at the
North Carolina State University Genome Sequencing Laboratory (GSL).
Sequences were contiged and edited using Sequencher 4.1 (Gene Codes Corp.,
Ann Arbor, MI).
Alignment was carried out manually using Se-Al 2.0 (Rambaut, 2002).
CAD, TPI and CO1 were aligned according to the amino acid translation. Introns in CAD, hypervariable regions of 28S and other positions of ambiguous alignment were removed from the data set. To detect existing base compositional bias, a chi-square test for homogeneity of base frequencies across taxa was performed for each gene and each codon position of CAD
! +! ! ! independently using Paup* 4.0b10 (Swofford, 2002). The test was repeated for
TPI, CO1 and the additional 28S sequence.
Phylogeny Estimation
Maximum parsimony analyses were carried out using Paup* 4.0b10
(Swofford, 2002). Heuristic search with TBR branch swapping and 100 random addition replicates were completed to find the shortest trees. Node support was obtained by acquiring bootstrap values from heuristic searches of 500 re- sampled data sets with 50 random addition replicates. Analyses including all taxa were conducted for the CAD and 28S rDNA as independent partitions, as well as with combined data sets that included all nucleotides equally weighted.
To overcome misleading phylogenetic signal due to potential saturation in the 3rd positions of CAD, analyses of the concatenated data set were also done with the
3rd positions of CAD removed and with CAD translated to amino acids.
To further test the placement of Hilarimorpha and Apystomyia, parsimony analyses of the larger 28S rDNA fragment, CAD, COI and TPI were conducted for the 8 taxon set. Analyses were carried out with all sequence data combined, and with the 3rd positions of CAD, CO1 and TPI excluded or included, and translated to amino acids.
In order to proceed with a Bayesian analysis, an appropriate model of nucleotide evolution, in this case GTR + I + G, was chosen by using Mr.
Modeltest (Nylander, 2005) to assess the adequacy of substitution models based
! ,! ! ! on the Akaike Information Coefficient (AIC) and nested likelihood ratio tests.
The choice of a model remained the same when genes were analyzed independently or as a concatenated data set. Using Mr.Bayes (Huelsenbeck and
Ronquist, 2001), Bayesian analyses were conducted for 20,000,000 generations, trees were sampled every 1000, and the first 25% (5000 trees) were discarded as burn-in. Analysis was completed with all taxa included and the third positions of CAD removed.
Maximum likelihood analyses were performed on the combined 28S and
CAD data set using Garli (Zwickl, 2006). A GTR+I+G model was implemented.
GARLI analyses were completed with all taxa included and the third positions of
CAD removed.
Stability Testing and Variations on Taxon Sampling
Because taxon choice had a strong effect on our preliminary estimates of tree topology, we sought to identify unstable or ‘rogue’ taxa by quantifying the instability of each taxon in parsimony analyses. The removal of ‘rogue’ taxa has been shown to increase resolution and support values in phylogenies
(Sanderson and Shaffer, 2002). Leaf stability indices were generated using
RadCon (Thorley and Page, 2000). RadCon calculates leaf stability for an individual taxon by analyzing bootstrap trees and determining the average support for the relationships of 3-taxon statements, or triplets, that include a particular taxon. We have relied on the ‘difference’ measurement, defined as the
! $-! ! ! difference between the bootstrap values of the two best-supported triplets that include a particular taxon. Using Paup* 4.0b10 (Swofford, 2002), a bootstrap analysis including all taxa and excluding the 3rd positions of CAD, was completed with 500 replicates and 50 random addition sequences. The trees were imported into RadCon (Thorley and Page, 2000) and the stability of each leaf in the set of rooted bootstrap trees was then calculated. The taxa with instability ratings lower than the overall average were deleted from the data set, resulting in a taxon set we refer to as ‘stable’, the parsimony analysis was then repeated, and new bootstrap values were obtained. The stability of each leaf of the pruned tree was then calculated.
The removal of unstable taxa involves deleting some taxa that are critical to our tests of specific phylogenetic hypotheses. Also, the removal of unstable taxa does not address the instability introduced by choice among possible outgroup taxa. Therefore, we compiled a ‘reduced’ data set that includes only two outgroups, a nemestrinid (Hirmoneura sp.), one of the most closely related outgroups to the asiloid flies, and a xylophagid (Heterostomus sp.). This reduced data set includes all families of Asiloidea. By experimenting with alternative samples of included taxa in preliminary analyses, we found that choice among sampled bombyliids, the especially varied Mythicomyiinae, had a strong effect on the placement of other taxa under all methods of analysis; consequently, our
‘Reduced’ data set includes only 5 bombyliids and a single representative of
! $$! ! ! Mythicomyiinae. The stability of the ‘reduced’ data set was calculated using the above mentioned RadCon method.
Maximium likelihood and Bayesian analyses of the ‘stable’ and ‘reduced’ data set were also completed.
Hypothesis testing in parsimony
To test the statistical significance of alternative topologies under parsimony, Templeton (Wilcoxon signed-ranks) tests and Kishino-Hasegawa tests were performed using Paup* 4.0b10 (Swofford, 2002). Hypothesis testing was done using the ‘reduced’ taxon set and with the 3rd positions of CAD removed. Due to its ambiguous placement, Hilarimorpha was removed from analyses that did not specifically address its placement, such as in tests regarding asiloid monophyly. Trees were estimated with topological constraints representing a priori hypotheses and were then compared to the most parsimonious tree.
Four-cluster likelihood mapping analysis
In order to visualize the strength of phylogenetic signal in our data set and to test for support for alternative hypotheses of relationships amongst asiloid flies, four-cluster likelihood mapping analyses were completed using the program
Tree Puzzle (Schmidt et al., 2002; Strimmer and Von Haeseler, 1997). This quartet-puzzling method allows the user to partition taxa into any four clusters,
! $%! ! ! resulting in three possible tree topologies, and returns a visual summary of the phylogenetic information supporting each of the three topologies. The relative frequencies of the likelihoods for each topology are plotted in an equilateral triangle with each point of the triangle representing a different topology. Support for alternate hypothesis regarding the monophyly of Asiloidea and the placement of Apystomyia and Hilarimorpha were examined using the ‘reduced’ taxon set
(Table 3), with the additional inclusion of the family Evocoidae in order to provide representation for all putative asiloid families. We tested asiloid monophyly by comparing support for alternate placements of Bombyliidae, the remaining asiloid flies, Eremonura, and two lower brachyceran outgroups. Alternative placements for Apystomyia were evaluated by comparing support for placing Apystomyia as closest relative to: all asiloid flies, Eremonura, and two lower brachyceran outgroups. The position of Hilarimorpha in Asiloidea was similarly tested.
Monophyly of the Hilarimorphidae, including Apystomyia, was tested by comparing support for the alternate placements of Hilarimorpha, Apystomyia, the asiloid flies, including Bombyliidae, and Eremoneura. We used exact parameter estimates and a neighbor-joining tree + quartet sampling. A GTR+I+G model of substitution was implemented with the rates of substitution input from estimations made in Paup* (Swofford, 2002). The gamma distribution alpha and the percentage of invariant sites were estimated from the data set.
! $&! ! ! Results
The final concatenated sequence alignment of all 50 taxa for CAD and
28S includes 4772 base pairs (28S=970, CAD=3801). A chi-square test for base composition homogeneity revealed significant heterogeneity among taxa for the full combined gene data set (p <0.0001). To identify the source of base composition heterogeneity within the data, each codon position of CAD, and the
28S gene were tested as independent partitions. Only the 3rd codon positions of
CAD show significant heterogeneity. The 3rd positions of CAD exhibit A/T bias
(70% average) in most taxa and G/T richness in other groups, specifically in the mythicomiine bombyliids (57%) and in some members of the Cyclorrhapha, primarily Paraplatypeza (85%). Exclusion of 3rd positions of CAD restores homogeneity of base pair composition for the concatenated data set.
To test the placement of Hilarimorpha and Apystomyia, we evaluated an additional data set limited to eight taxa sampled from across the higher-level taxonomic diversity of our study. This taxon-limited data set includes 8674 base pairs (28S =2917, CO1=1490, TPI= 486, CAD=3801). A chi-square test for base composition homogeneity across taxa shows significant heterogeneity among sites across taxa (p <0.0001). Tested independently, the 3rd positions of CAD and CO1 reject the null hypothesis of homogeneity. The exclusion of the 3rds positions of these two genes restores homogeneity of base pair composition.
! $'! ! ! Phylogenetic analyses including all taxa
Table 2 summarizes all topologies obtained in altenative phylogenetic analyses. Parsimony analysis of 28S and CAD nucleotides 1+2 (3505 total characters, 2148 constant, 469 parsimony uninformative, 866 parsimony informative) yields six trees of length 5748, the strict consensus of which is shown in Fig. 1. All asiloid and eremoneuran families are monophyletic with the exception of Mydidae, which is unresolved in respect to Asilidae and the
Therevoid clade. Asiloidea, excluding Bombyliidae, and Eremoneura are also monophyletic, but the relationship between them is unresolved. Amongst the asiloid families, two higher-level clades are recovered: a therevoid clade, including Therevidae, Apsilocephalidae and Scenopinidae, and a second clade including Asilidae. Bootstrap support and resolution is generally low for most nodes of the tree.
Other higher-level phylogenetic studies using CAD have shown that parsimony analyses that include the 3rd positions can result in the spurious phylogenetic placement of taxa, likely due to saturation or base composition bias
(Moulton and Wiegmann, 2004; Bertone et al., 2008). An analysis of the combined data set including the 3rd positions resulted in a tree with erroneous placements of outgroups amongst Cyclorrhaphans and bombyliids. Analyses using CAD + 28S, with CAD translated to amino acids recovered all of the families found in the n1+n2 tree along with the Eremoneura, however, the relationships between these groupings were entirely unresolved.
! $(! ! ! In all parsimony analyses that achieve resolution, the genus Apystomyia is placed as a basal cyclorraphan. Hilarimorpha lacks a stable placement and appears in various analyses as unplaced, the sister group to either Asiloidea,
Eremoneura, Asiloidea+Eremoneura, or sister to Bombyliidae. In general, our parsimony trees are sensitive to taxon sampling and outgroup selection.
A Bayesian analysis of all taxa with the 3rd positions of CAD removed differs from the corresponding parsimony analysis in that the Asiloidea is monophyletic, though lowly supported (58%) (Fig. 2). The relationship between the Eremonura, the Asiloidea and a clade including Evocoa and Hilarimorpha is unresolved. The monophyly of Therevidae, Asilidae, Eremoneura, Cyclorrhapha and Empidoidea are supported by 100 pp. The sister-group relationship between
Asilidae and Mydidae plus Apioceridae is supported by 100 pp. The sister-group relationship between Apystomyia and Cyclorrhapha is also supported by 100 pp.
A maximum likelihood analysis of all taxa with 3rd positions of CAD excluded differs from the parsimony and Bayesian trees in that bombyliid subfamily Mythicomyiinae are the sister-group to Eremoneura (Fig. 3). Similar to the parsimony and Bayesian tree, Asiloidea, excluding Bombyliidae, is made up of a monophyletic therevoid clade, including the new family Evocoidae, and a clade of asilids, mydids and apiocerids. The ML analyses also place Apystomyia as sister-group to Cyclorrhapha. Hilarimorpha is the sister-group to the asiloids, excluding the bee flies, plus Eremonura (including Mythicomyiinae).
! $)! ! ! A parsimony analysis of 28S and CAD, TPI and CO1 nucleotides 1+2
(6754 total characters, 5327 constant, 686 parsimony informative, 741 parsimony uninformative) results in a single tree of length 2632 (Fig. 4). Asiloidea and the
Eremoneura (83 bp) are monophyletic. Apystomyia is the sister-group to the
Cyclorrhapha (67 bp). Hilarimorpha is the sister-group to Asiloidea +
Eremoneura. When analyzed as amino acids, the bootstrap support for the sister-group relationship between Apystomyia and Cyclorrhapha is 95.
Stability testing and variations in taxon sampling
Leaf stability values are shown in Table 3. The average value of the difference between the bootstrap values of the two best-supported triplets containing a particular taxon ( = ‘difference’) is 0.5646. Out of 51 taxa, 16 taxa were deemed unstable due to difference values below 0.5646. The unstable taxa included all 3 members of the Scenopinidae, 5 bombyliids, including the 4
Mythicomyiinae and Heterotropus, Hilarimorpha, a mydid, an apiocerid and outgroups belonging to Nemestrinoidea. Removing unstable taxa and recalculating the parsimony bootstrap values, increases the average leaf stability to 0.8186.
Removing unstable taxa also dramatically increases the resolution of the bootstrap consensus tree, resulting in higher support for clades across the tree, including many that had not previously been supported. In contrast to the conflicting results retrieved when all taxa are included, the removal of unstable
! $*! ! ! taxa produces largely congruent results from parsimony, Bayesian, and likelihood analyses. The ‘stable’-taxon tree exhibits a monophyletic Bombyliidae (61 bp, 92 pp), Asiloidea excluding Bombyliidae (78 bp, 100pp) and Eremoneura (93 bp,
100pp). Parsimony and model-based methods of analysis of the stable-taxon data shows no support for a higher-level relationship between Bombyliidae and all remaining asiloid flies plus Eremoneura. Fig. 5 compares the resolution of the bootstrap trees of analyses including all taxa and stable taxa only.
The removal of unstable taxa, although useful for increasing support values, also removed many taxa that are critical to our phylogenetic hypotheses.
Furthermore, the removal of unstable taxa did not account for the topological effects due to outgroup selection. By adding several phylogenetically important taxa back into our analyses, removing several bombyliids and reducing the number of outgroups, we found a taxon set, referred to as ‘reduced’ that maintained a high average leaf stability (0.7024) and returned concordant results from parsimony, Bayesian and maximum likelihood analyses. The ‘reduced’ taxon tree (Fig. 6), like the ‘stable’ taxon tree, results in Bombyliidae as the sister group to the remaining asiloid flies + Eremoneura (in MP and ML, yet unresolved in BI). The trees resulting from different analysis methods conflict only in their placement of Hilarimorpha. Bootstrap and posterior probabilities are not as high for the reduced data set as they are for the stable data set, but they are higher than those resulting from the inclusion of all taxa.
! $+! ! ! Under all methods of analysis, no variation of taxon sampling returns high support for the relationship between Eremoneura, Bombyliidae and the remaining
Asiloidea.
Hypothesis testing
Under parsimony, Templeton and Kishino-Hasegawa tests of alternative a priori hypotheses of asiloid relationships using the ‘reduced’ taxon set, fail to reject alternate placements of Bombyliidae (as part of a monophyletic Asiloidea, or as sister-group to Eremoneura) and Hilarimorpha. Nevertheless, the same tests significantly reject topologies that include Apystomyia in Asiloidea and grouping of Hilarimorpha+Apystomyia (Hilarimorphidae) as sister to Eremoneura
(Table 4).
Four-cluster likelihood analysis
A four-cluster likelihood analysis testing asiloid monophyly shows a high percentage of non-phylogenetic signal present in the data set regarding this question (17.8%, 10-15% is considered high). Support for the relationship between asiloids (excluding bombyliids), Eremoneura, and bombyliids is divided, though the highest percentage of data points fall in favor of a monophyletic
Asiloidea (33.4%) (Fig. 7). Apystomyia is strongly supported as the closest relative of the higher flies (90.4%). When the placement of Hilarimorpha is tested between its affinity to a monophyletic Asiloidea, Eremoneura, or the outgroups, it
! $,! ! ! groups with the outgoups (50.2%, and 18.9% and 21.4% respectively). However, when the outgroups are removed and Apystomyia is included in order to test for support for the family Hilarimorphidae (Apystomyia + Hilarimorpha),
Hilarimorpha clusters with Asiloidea (81.8%) with much greater frequency that
Apystomyia (14.8%). Though these mixed results fail to resolve the placement of
Hilarimorpha, they do demonstrate the lack of support for the family
Hilarimorphidae.
Discussion
The more ancient and rapid a divergence, the more difficult it is to recover a well-resolved phylogenetic history with limited amounts of data (Rokas et al.,
2005a; Whitfield and Lockhart, 2007; Whitfield and Kjer, 2008). Asiloidea likely originated close to 200 million years ago (Wiegmann et al., 2003), and the current systematic status of the relationship between Asiloidea, Bombyliidae
(considered a member of Asiloidea), and Eremoneura exhibit some of the characteristics of a rapid radiation: namely, a topology lacking resolution or support, and short internal branches. Independent data sets, morphological and molecular, show agreement on which internal branches are short on our tree estimates, suggesting that rapid diversification is a key feature of early asiloid fly evolution (Poe and Chubb, 2004; Whitfield and Lockhart, 2007). Morphology alone has been unable to confirm the monophyly of Asiloidea or its relationship to the rest of the large majority of Diptera due to a dearth of characters uniting the
! %-! ! ! group (Yeates, 2002; Woodley, 1989) and the ambiguous placement of a collection of several asiloid-like genera (Yeates and Wiegmann, 1999). In a previous molecular analysis of Brachycera using 28S ribosomal DNA, the asiloid flies appear in a mostly unsupported arrangement in an otherwise well-supported topology (Wiegmann et al., 2003).
The results of our analyses echo previous morphological and molecular studies by presenting an unsubstantiated picture of the relationship between asiloid flies and Eremoneura. Methods of analysis, along with variations in outgroup selection and taxon sampling, yield conflicting topologies regarding a monophyletic Asiloidea. The incongruence in our results amongst higher-level groupings lies primarily with the placement of Bombyliidae, which appears either as sister group to the remaining asiloid flies, as proposed by Woodley (1989), or as the sister group to Eremoneura. When all asiloid families are represented in our analyses, these higher-level relationships are never accompanied by bootstrap support above 50 or resolution in BI, and hypothesis testing shows no significant difference between competing topologies; therefore a strongly supported estimate of relationships between asiloid flies and Eremoneura remains elusive.
Taxon sampling, the inclusion of unstable or ‘rogue’ taxa, as well as outgroup selection have a strong effect on our tree topologies, their stability and support values, a common finding that has been addressed in other phylogenetic studies (Leconitre et al., 1993; Sanderson and Shaffer, 2002; Rokas, 2005a). By
! %$! ! ! eliminating taxa from our data set that were deemed unstable though a leaf stability analysis, overall node support increased and results from different methods of analysis converged on similar topologies. By reintroducing key taxa while maintaining increased resolution, support values and stability, we find our best current estimates of asiloid relationships (Fig. 6). Though several analyses find resolution for the relationship between Bombyliidae, the remaining Asiloidea and Eremoneura, these results are not supported and are unstable in respect to taxon sampling and method of analysis. Lecointre et al. (1993) define a robust clade as ‘a node with a BP that is high and does not significantly vary whatever the species sample used to represent the corresponding group.’ The relationship between the asiloid flies (excluding the Bombyliidae) and the Eremoneura cannot be considered robust based on our current data.
The amount and type of sequence data we acquired has previously provided sufficient phylogenetic signal to resolve dipteran relationships in other analyses (Moulton and Wiegmann, 2004; 2007; Bertone et al., 2008; Winterton et al., 2007; Scheffer et al., 2007). Moreover, a four-cluster likelihood mapping analysis addressing the question of asiloid monophyly indicates that a large percentage of our data (17.8%) (Fig. 7) consists of nonphylogenetic information
(greater than 10-15% is considered inadequate for tree reconstruction) (Thorley and Page, 2000). Similarly, Rokas et al. (2005a) used as evidence of a rapid radiation in Metazoa, the observation that based on the same sampled genes, relationships among major metazoan lineages remained unresolved and
! %%! ! ! unsupported, while its sister kingdom, Fungi exhibited a well resolved and well supported tree. A similar comparison can be made of Asiloidea and the closely related superfamily Empidoidea. The actual sister group of Asiloidea is unknown, but their close relationship to Eremoneura (Empidoidea +
Cyclorrhapha) has been established along with evidence that they may have diverged at a similar time (Wiegmann et al., 2003). A phylogenetic analysis of the major empidoid lineages based on a large fraction of the CPSase region of CAD resulted in a highly supported, well resolved tree for basal lineages (Moulton and
Wiegmann, 2007). Yet, contemporaneus, deep diverging relationships in
Asiloidea are not resolved with high support using CAD alone, or together in combination with 28S rDNA.
Increasing the number of characters in a data set is often cited as the best means to increase resolution and node support (Rosenberg and Kumar, 2001;
Rokas, 2005b); a parsimony analysis that included 2 additional protein coding genes, CO1 and TPI, and an additional 2000 base pairs of 28S rDNA for a subsampling of taxa, did not improve the bootstrap support for the Asiloidea or its relationship to Eremoneura. Furthermore, the resulting topology, though lacking bootstrap support, yields a monophyletic Asiloidea as the sister group to
Eremoneura, which stands in conflict with the topology that our previous analyses of fewer genes and more taxa had converged upon (Fig. 4).
Though the broader issue of the higher-level relationships between asiloid flies and Eremoneura has not been clearly resolved by our data, they have
! %&! ! ! provided corroborating evidence for some of the relationships amongst asiloid families. Our results nevertheless suggest that Asiloidea could be defined in a more conservative, yet more reliably monophyletic arrangement than the currently accepted definition of the clade, by excluding the large, diverse familily
Bombyliidae. Hennig (1973) referred to this group as Asiliformia, but did not identify any morphological synapomorphies (Sinclair et al.,1994). This more conservative composition of Asiloidea is robust to analysis conditions and data sets and is consistently comprised of two monophyletic clades: the therevoid clade and a clade consisting of the Mydidae, Apioceridae and Asilidae.
In accord with other morphological and molecular hypotheses (Yeates et al., 2003), the majority of our analyses confirm the existence of a “therevoid” clade that includes Scenopinidae, Evocoidae and Apsilocephalidae + Therevidae as sister-groups. The instability of Scenopinidae in our data set prevents the
Therevoid clade from being accompanied by high support values; however, analyses of the ‘stable’ taxonset, that exclude the Scenopinidae, show
Apsilocephalidae + Therevidae supported by 100 pp and 94 bp. This result, along with the morphological and molecular evidence used by Yeates (2002) and
Yeates et al. (2003), contradicts the suggestion of Nagatomi et al. (1991) of a putative relationship between Apsilocephalidae and Eremoneura. The recently described monotypic family Evocoidae was placed in the therevoid clade in model-based analyses, as expected based on a previous study using morphological data and sequence from 28S rDNA (Yeates et al., 2003).
! %'! ! ! The clade joining Asilidae, Mydidae and Apioceridae, recovered in all of our analyses, is supported by 100 pp in all Bayesian analyses. This clade was previously proposed based on the potential synapomorphies of adults with a sunken vertex and overall larval similarity (Woodley, 1989), but the relationship of
Asilidae to the rest of Asiloidea has remained contentious (Yeates, 2002). The
Asilidae are consistently monophyletic in all methods of analysis of all taxon sets, in agreement with morphological and molecular hypotheses (Bybee et al., 2004;
Dikow, 2009). The relationship between Mydidae and Apioceridae, though also supported by 100 pp in all Bayesian analyses, lacks definitive resolution. Mydids are most often found to be paraphyletic with respect to apiocerids (Irwin and
Wiegmann, 2001).
The most surprising and strongly supported finding in our study is the placement of Apystomyia, the small, rare asiloid-like fly that has been collected only 3 or 4 times in southern California. The name Apystomyia, appropriate for this fly, in Greek means ‘a fly of which nothing is known’ (Melander, 1950). Since its discovery, the fly’s taxonomic placement has been controversial due to its unique morphology, including features that are common to both asiloid and eremoneuran flies. Apystomyia retains the synapomorphic male genitalia of the
Asiloidea, however its epandrium, reduced wing venation, and overall vestiture, with bristle-like setae, suggest affinity to the early cyclorrhaphan lineages.
Melander initially described Apystomyia as a member of the bombyliid subfamily
Heterotropinae, a catch-all grouping of hard-to-place flies. Melander’s reasoning
! %(! ! ! for including Apystomyia in Heterotropinae reflects the ambiguity of its classification. He states, “This enigmatic little fly does not seem to be related to any other genus, and its assignment to the Heterotropinae is made because it does not conform to any other subfamily or family.” Over time, Apystomyia has been treated as an anomalous bombyliid (Hull, 1973; Woodley, 1989; Nagatomi et al., 1991) and as a therevid (Sinclair et al., 1994). In 1994, Yeates’ quantitative morphological analysis of Bombyliidae placed Apystomyia in the previously monogeneric family Hilarimorphidae as the sister group to
Bombyliidae. His more recent morphological analysis of Brachycera, however, resulted in the family Hilarimorphidae, including Apystomyia, being placed as the sister group to Eremoneura (Yeates, 2002).
Our results do not reflect a close relationship between Hilarimorpha and
Apystomyia, and remove Apystomyia even further from its traditional placement in Asiloidea. Our molecular data unambiguously place Apystomyia as the sister group to Cyclorrhapha. This surprising result appears both stable and strongly supported among all treatments of data and methods of analysis. Parsimony and
Bayesian analyses show high support for this new placement of Apystomyia. A leaf stability test shows that under parsimony, Apystomyia is among the most stable of all taxa included in the data set and appears to be unaffected by outgroup selection or the inclusion of other ‘rogue taxa’. Alternative hypotheses testing in parsimony rejects the placement of Apystomyia in Bombyliidae or elsewhere in Asiloidea. A four-cluster maximum likelihood analysis shows that
! %)! ! ! our data contain phylogenetic signal that favor the relationship between
Apystomyia and Cyclorrhapha (90.4%) while rejecting its close affinity with
Asiloidea (5.5%). The implications of this unexpected finding are that
Apystomyia appears to be a relict or transitional lineage that has retained morphological similarity to the most recent common ancestor of both Asiloidea and Eremoneura, and is sister to the large successful Cyclorrhaphan lineage, the major synapomorphies of which it lacks. This pattern of unique, depauperate, difficult to place lineages recovered as sister to major radiations is a recurring phylogenetic pattern for modern studies of many major clades of living organisms: flowering plants (Soltis et al., 1999; Davies et al., 2004), Lepidoptera
(Wiegmann et al., 2000), Coleoptera (Maddison et al., 1999), Diptera (Bertone et al., 2008). As of yet, there are no convincing morphological synapomorphies uniting Apystomyia and the Cyclorrhapha, thus, amongst dipterists, this placement of Apystomyia will undoubtedly be subject to detailed examination further.
Like Apystomyia, Hilarimorpha’s placement within Asiloidea has varied, being considered a therevid (Sinclair et al., 1994) an empidid (Nagatomi et al.,
1991), or more frequently a bombyliid (Woodley, 1989; Webb, 1974), or even a close relative to bombyliids (Nagatomi and Liu, 1994; Yeates, 1994). Our data failed to resolve the placement of Hilarimorpha. In all parsimony, Bayesian and likelihood trees, the location of Hilarimorpha lacks support and varies from tree to tree. In a leaf stability analysis, Hilarimorpha ranks as the most unstable taxon in
! %*! ! ! the data set. Testing of a priori hypotheses does not reject the inclusion of
Hilarimorpha in Asiloidea or as the sister group to Eremoneura. A four-cluster likelihood mapping analysis shows that our data contains more phylogenetic signal supporting the hypothesis of a sister group relationship between Asiloidea and Hilarimorpha (81.8%) than of a sister group relationship of Hilarimorpha and
Eremoneura (0.6%) or Apystomyia (14.8%). Due to the ambiguity of our results, we suggest that Hilarimorpha continue to be considered incertae sedis amongst
Heterodactyla (Asilodea+Eremoneura).
Conclusions
Our results, in concordance with previous molecular and morphological work, show that relationships in this region of the dipteran tree of life continue to defy attempts at resolution using standard phylogenetic methods. The low support values for clades on our trees lead us to seek heuristic estimates of topological stability and congruence across analysis methods and treatments of data to arrive at a current best estimate of asiloid relationships. If the relationships between Asiloidea, Bombyliidae and Eremonura reflect an ancient, rapid radiation, as evidence currently indicates, our efforts to recover highly supported evolutionary relationships amongst them will likely continue to be a major challenge for dipteran systematics.
! %+! ! ! Acknowledgments
We are grateful to D.K. Yeates, M.E. Irwin and N.I. Evenhuis for the provision and identification of specimens. Additionall thanks go to Brian Cassel for assistance in the collection of molecular data. This project was supported by US
National Science Foundation (NSF) Assembling the Tree of Life (ATOL) grant
EF-03394 to BMW and DKY.
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Table 1. Sampled taxa
Taxon Genus species Outgroups TABANAMORPHA Pelecorhyncus Pelecorhynchidae personatus Vermelionidae Leptynoma sp. XYLOPHAGAMORPHA Xylophagidae Heterostomus sp. STRATIOMYOMOPRPHA Stratiomyidae Actina sp. Pantophthalmidae Pantopthalmus sp. NEMESTRINOIDEA Nemestrinidae Hirmoneura sp. Ingroup ASILOIDEA Asilidae Diogmites Dasypogon diadem Machimus Ommatius Apioceridae Apiocera haruspex Mydidae Tongamyia Scenopinidae Prorates sp. Scenopinus sp. Stenomphrale sp. Therevidae Ectinorhynchus sp. Effatouniella sp. Lysilinga sp. Bombyliidae Heterotropus sp. Sericosoma sp. Geminaria sp. Antonia sp. Pantarbes sp. Neosardus sp. Neosardus sp. Lordotus sp. Aphoebantus sp. Bombylius major Pteraulax sp. Epacmus sp. Tomomyza sp. Mythicomyiinae Cyrtosiini Genus sp.
! &,! ! ! Table 1. Continued
Mnemomyia sp. Glabellula sp. Mythicomyia sp. Hilarimorphidae Hilarimorpha sp. Apystomyia sp. EMPIDOIDEA Empididae Empis sp. Empididae Hilara sp. Dolichopodidae Dolichopus sp. Atelestidae Atelestus pulicarus Atelestidae Mehyperus sudeticus CYCLORRHAPHA Lonchopteridae Lonchoptera fusca Pipunculidae Pipunculus houghi Syrphidae Rhingia sp. Platypezidae Paraplatypeza atra
! '-! ! !
Table 2. Clade recovery with under MP, ML and BI with varying taxon and character inclusion
! "#! Table 3. Leaf stability values generated from MP bootstrap trees for all taxa, stable taxa only and our reduced taxa data set.
All taxa Stable taxa Reduced taxa Leaf Difference Leaf Difference Leaf Difference Hilarimorpha 0.3306 Mydidae 0.7617 Stenomphrale 0.5291 Stenomphrale 0.4076 Sericosoma 0.7884 Scenopinus 0.5291 Scenopinus 0.4076 Actina 0.7904 Hirmoneura 0.5372 Ogcodes 0.4139 Bombylius 0.7923 Mythicomyia 0.5373 Mesophysa 0.4268 Diogmites 0.7988 Hilarimorpha 0.5617 Heterotropus 0.4278 Dasypogon 0.7988 Prorates 0.6172 Evocoa 0.4329 Pantopthalmus 0.7994 Heterostomus 0.6287 Mnemomyia 0.4866 Apsilocephala 0.8032 Apiocera 0.6425 Cyrtosiini 0.4866 Efflatouniella 0.8037 Tongomyia 0.6552 Mythicomyia 0.4873 Ectinorhyncus 0.8038 Mydidae 0.6598 Glabellula 0.4873 Laxotela 0.8038 Apsilocephala 0.7079 Leptynoma 0.4876 Tomomyza 0.8047 Bombylius 0.7101 Hirmoneura 0.5146 Pteralaux 0.8050 Lordotus 0.7109 Tongomyia 0.5485 Ommatius 0.8050 Neosardus 0.7123 Prorates 0.5552 Machimus 0.8050 Epacmus 0.7128 Apiocera 0.5616 Pantarbes 0.8060 Diogmites 0.7215 Average 0.5646 Neosardus 0.8154 Dasypogon 0.7215 Mydidae 0.5728 Neosardus 0.8154 Efflatouniella 0.7271 Dolichopus 0.5772 Epacmus 0.8175 Ectinorhyncus 0.7297 Apystomyia 0.5793 Aphoebantus 0.8175 Laxotela 0.7297 Atelestus 0.5797 Lordotus 0.8196 Ommatius 0.7347 Meghypherus 0.5797 Amphicosmus 0.8196 Machimus 0.7347 Empis 0.5797 Geminaria 0.8196 Dolichopus 0.7846 Hilara 0.5797 Heterostomus 0.8325 Apystomyia 0.7860 Lonchoptera 0.5830 Dolichopus 0.8484 Lonchoptera 0.7930 Paraplatypeza 0.5832 Lonchoptera 0.8495 Paraplatypeza 0.7934 Pipunculus 0.5835 Paraplatypeza 0.8500 Atelestus 0.7948 Rhingia 0.5835 Apystomyia 0.8501 Meghypherus 0.7948 Diogmites 0.5849 Rhingia 0.8506 Empis 0.7950 Dasypogon 0.5849 Pipunculus 0.8507 Hilara 0.7950 Apsilocephala 0.5963 Empis 0.8517 Rhingia 0.7952 Ommatius 0.5988 Hilara 0.8517 Pipunculus 0.7953 Machimus 0.5988 Atelestus 0.8518 Average 0.7024 Efflatouniella 0.6011 Meghypherus 0.8518 Ectinorhyncus 0.6015 Average 0.8186