Higher-Level Phylogeny of the Therevidae (Diptera: Insecta) Based

Molecular Phylogenetics and EvolutionVol. 15, No. 3, June, pp. 440–451, 2000doi:10.1006/mpev.1999.0771, available online at http://www.idealibrary.com on

on 28S Ribosomal and Elongation Factor-1a Gene SequencesLonglong Yang,* Brian M. Wiegmann,* ,1 David K. Yeates,† and Michael E. Irwin‡

*Department of Entomology, Box 7613, North Carolina State University, Raleigh, North Carolina 27695; †Department of Zoology andEntomology, The University of Queensland, Brisbane, Queensland 4072, Australia; and ‡Department of Natural Resources and

Environmental Sciences, University of Illinois at Urbana–Champaign, Urbana, Illinois 61820

Received June 8, 1999; revised December 30, 1999

gram that brings a wealth of new evidence to bear on

Therevidae (stilleto flies) are a little-known family of

asiloid brachyceran Diptera (Insecta). Separate andcombined phylogenetic analyses of 1200 bases of the28S ribosomal DNA and 1100 bases of elongation fac-tor-1a were used to infer phylogenetic relationshipswithin the family. The position of the enigmatic taxonApsilocephala Krober is evaluated in light of the mo-lecular evidence. In all analyses, molecular datastrongly support the monophyly of Therevidae, ex-cluding Apsilocephala, and the division of Therevidaeinto two main clades corresponding to a previous clas-sification of the family into the subfamilies Phycinaeand Therevinae. Despite strong support for some rela-tionships within these groups, relationships at thebase of the two main clades are weakly supported.Short branch lengths for Australasian clades at thebase of the Therevinae may represent a rapid radia-tion of therevids in Australia. © 2000 Academic Press

Key Words: Therevidae; stiletto fly; elongation fac-tor-1a; 28S ribosomal DNA; phylogeny; Diptera.

INTRODUCTION

Therevidae (Diptera: Asiloidea), stiletto flies, are amedium-sized family of lower brachyceran Dipterawith more than 800 described species and in excess of4000 species estimated in recent surveys (Yeates, 1994;Yeates and Wiegmann, 1999). Therevidae occur in allbiogeographic regions, but species diversity and abun-dance are highest in arid and semiarid regions, wherethe larvae thrive as predators of soil arthropods (Irwinand Lyneborg, 1981b). Therevidae are most oftenplaced in the dipteran superfamily Asiloidea alongwith the families Asilidae, Apioceridae, Mydidae,Scenopinidae, and Bombyliidae (Hennig, 1973; Wood-ley, 1989; Yeates, 1994). Very little is known abouttherevid biology and evolution; however, the family isthe focus of a comprehensive systematic research pro-

1 To whom correspondence should be addressed. Fax: (919) 515-7746. E-mail: bwiegman@unity.ncsu.edu.

1055-7903/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

440

therevid diversity (Irwin and Kampmeier, 1997). Thisstudy, part of that program, is the first to apply nucle-otide sequence data to infer higher-level phylogeneticrelationships within the family.

Few studies have addressed classification and rela-tionships of Therevidae below the family level. Irwinand Lyneborg (1981a) separated the family into twosubfamilies, Phycinae and Therevinae, based primar-ily on the male and female terminalia (Irwin, 1976).Several groups of genera were also identified accordingto their similar geographic distributions (Irwin andLyneborg, 1981b), but no formal classification specify-ing tribes for the majority of genera has been proposed.Recent collections in understudied regions such asAustralia, Patagonia, and southern Africa havebrought to light a large number of new genera andspecies (Irwin and Yeates, 1998; Winterton et al.,1999). This newly discovered diversity, together withcurrent efforts at curation and databasing of preservedmuseum specimens, has made it increasingly neces-sary to obtain a phylogeny-based classification forTherevidae above the genus level.

The monophyly of the family Therevidae has neverbeen firmly established by morphological evidence(Woodley, 1989; Yeates, 1994). This may be due to thedifficulty in placing several enigmatic groups that havebeen previously allied with Therevidae. In particular,Apsilocephala longistyla Krober, which was describedin the Therevidae by Krober (1914), has charactersthat have also been interpreted as indicating place-ment in Bombyliidae (Irwin and Lyneborg, 1981a,b) orEremoneura (5 Empidoidea 1 Cyclorrhapha) (Nagatomiet al., 1991b). The systematic position of Apsilocephalahas been enigmatic since Irwin (1976) removed the genusfrom Therevidae based on characters of the female repro-ductive system. Nagatomi et al. (1991a) erected a newfamily, Apsilocephalidae, for Apsilocephala, ClesthentiaWhite, and Clesthentiella Nagatomi et al., and suggesteda possible relationship to Eremoneura based on theirinterpretation of male genitalic structures as homologs of

the surstyli of higher Diptera. This placement was ques-

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closely to Irwin and Lyneborg’s (1981a) division of the

441RELATIONSHIPS OF THEREVIDAE

tioned by Sinclair et al. (1994), who retained Apsilo-ephala as incertae sedis in Therevidae. Yeates’ (1994)orphological analysis also supported the separation ofpsilocephala from Therevidae as a separate asiloid lin-age sister group to all Therevidae, Apioceridae, and Asi-idae. Even more recently, Nagatomi and Yang (1998)ynonymized Apsilocephalidae with the Rhagionempidi-ae, which had previously included only a few fossil taxa,ased primarily on similarities in wing venation. In gen-ral, the morphological evidence for any clear placementf Apsilocephala, Clesthentia, and Clesthentiella is eitherimited, conflicting, or controversial (Sinclair et al., 1994;agatomi and Yang, 1998). We are currently compiling

omprehensive morphological data sets to reexaminehese hypotheses through combined analysis of molecu-ar and morphological variation.

The family Scenopinidae (window flies) is generallyegarded as the sister group to Therevidae, based onhe secondary division of larval abdominal segmentsWoodley, 1989; Yeates and Irwin, 1992). However, thelacement of Scenopinidae 1 Therevidae among thether asiloid families is not yet resolved (Yeates, 1994;eates and Wiegmann, 1999).Family-level divergences among lower brachyceraniptera are likely to be as old as the Jurassic (150–200ya), based on known compression fossils (Hennig, 1981;venhuis, 1994). Clade ages within the family are moreifficult to predict, but therevid diversity is well repre-ented in Baltic amber (Eocene/Oligocene 25–50 mya)Evenhuis, 1994; Grimaldi and Cumming, 1999).

In this study, nucleotide sequences from the 28Sibosomal (28S rDNA) and elongation factor-1a

(EF-1a) genes were applied to reconstruct relation-ships among the major lineages of Therevidae. Both ofthese genes have been widely used in insect molecularsystematics. Ribosomal DNAs are among the mostcommonly used genes in phylogenetics (Hillis andDixon, 1991), with various regions of the ribosomalrepeat unit having been applied to almost all levels offly diversity (e.g., Whiting et al., 1997; Friedrich andTautz, 1997a,b; Pawlowski et al., 1996; Pelandakis etal., 1991; Porter and Collins, 1996). EF-1a, a protein-coding gene, has been applied within both families andsubfamilies in insects (Cho et al., 1995; Mitchell et al.,1997; Danforth and Ji, 1998). This gene, though highlyconserved in amino acid sequence, has the unexpectedproperty of retaining useful phylogenetic signal in itsthird positions for Tertiary (65 mya) and younger di-vergences (Cho et al., 1995; Mitchell et al., 1997).

Sequences from 28S rDNA and EF-1a were analyzedseparately and in combination to reconstruct relation-ships among therevid genera. Our nucleotide evidencedemonstrates that (1) the Therevidae are a monophyleticgroup, but this group excludes the controversial genusApsilocephala; (2) the earliest therevid divergences di-vide the family into two major clades corresponding

family into Phycinae and Therevinae; and (3) a large,diverse radiation of Australasian taxa is placed at thebase of the Therevinae, but the current molecular dataare insufficient to specify conclusively relationshipswithin and among lineages of Therevinae.

MATERIALS AND METHODS

Taxon Sampling

A total of 34 genera of Therevidae (including Apsilo-cephala) were included in the study, representing abroad geographic sampling of known taxa (Table 1).Three genera of the putative therevid sister groupScenopinidae were sampled as outgroups. An addi-tional outgroup species from the genus HeterotropusLoew (family Bombyliidae) was also sequenced. Speci-mens were collected directly into 95% ethanol andstored at 280°C for preservation of nucleic acids.

Laboratory Methods

Total genomic nucleic acids were extracted using theguanidinium isothiocyanate homogenization protocolof Chirgwin et al. (1979) (after Cho et al., 1995). A1.2-kb fragment near the 39 end of the 28S rRNA genewas amplified in two overlapping segments using prim-ers from Hamby et al. (1988) modified to match thepublished sequence for the dipteran Drosophila mela-nogaster Meigen (Hancock et al., 1988) (Table 2). Prim-ers rc28H and 28K were used in combination toamplify a 750-bp fragment (5982–6739 in D. melano-gaster), and primers rc28Q and 28Z amplify an over-lapping 850-bp fragment (6385–7220 in D. melano-gaster). Internal primers 28Q and 28X were used incycle sequencing reactions for some templates (Table2). Amplification of 28S was carried out using standardthree-step PCR with 50°C annealing temperature and30 cycles.

To amplify the elongation factor-1a gene, primersEF4 and EF5 were developed to specifically amplifyapproximately 1.1 kb (Table 2). Both standard PCRfrom DNA templates and RT-PCR of mRNA tran-scripts according to protocols in Kawasaki (1990) wereused to amplify EF-1a. Internal primers 46, 47, and 51were also used in sequencing reactions for these tem-plates (Table 2).

Sequences were obtained by dye terminator cyclesequencing using the ABI Taq FS enzyme (PE AppliedBiosystems, Foster City, CA), gel fractionated, andbase-called on the ABI PRISM 377 DNA sequencer (PEApplied Biosystems) of the North Carolina State Uni-versity DNA Sequencing Facility. Opposite strandswere confirmed for all templates. ABI trace files wereedited and contigs assembled using the program GAP4in the STADEN software package (Staden, 1996) on aSUN SPARC 5 workstation.

TABLE 1

442 YANG ET AL.

Nucleotide Alignment and Phylogenetic AnalysisAlignments were constructed manually using the

multiple alignment editor of the Genetic Data Environ-ment (GDE 2.2) (Smith et al., 1994). Highly conserved

Taxa Sampled in The

Taxon

So

MEI No.

BombyliidaeHeterotropus sp.

ScenopinidaeProratinae

Alloxytropus sp. MEI 050694Scenopininae

Stenomphrale teutankhameni MEI 029578Brevitrichia sp.

ApsilocephalidaeApsilocephala sp. MEI 075533

TherevidaePhycinae

Henicomyia hubbardi MEI 027097Ataenogera sp.Phycus niger MEI 089483Phycus niger MEI 089478Stenogephyra sp. MEI 089474Actorthia micans MEI 029581Ruppellia basalis MEI 089480Orthactia penicillata MEI 089470Efflatouniella sp. MEI 050689Neotabuda sp. MEI 089473Pherocera albihalteralis MEI 027102

TherevinaeAcraspisa sp. MEI 089456Agapophytus sp. MEI 053710Ectinorhynchus sp.Entesia sp. MEI 106357Nanexila manni MEI 089188Nanexila vittata MEI 089466Taenogerella elizabethae MEI 053719Bonjeania clamosis MEI 053734Neodialineura striatithorax MEI 053713Neodialineura striatithorax (2) MEI 053714Parapsilocephala sp. MEI 053716Parapsilocephala sp. 1 MEI 089467Parapsilocephala bicolor MEI 089462Anabarhynchus tristis MEI 053735Megalinga bolbocera MEI 090265Brachylinga sp. MEI 090258Lysilinga aurantiaca MEI 089531Penniverpa sp. MEI 090271Chromolepida pruinosa MEI 090268Cyclotelus pictipennis MEI 089430Ozodiceromyia sp. MEI 089435Acrosathe sp. MEI 106328Hoplosathe frauenfeldi MEI 050690Nebritus pellucidus MEI 103266Pandivirilia sp. MEI 089518Tabudamima melanophleba MEI 106327Thereva nobilitata

Note. Source numbers refer to M. E. Irwin collection numbers corrand Irwin, 1998).

regions of the 28S rDNA gene were unambiguouslyalignable. Positions within the 28S divergent domainsfor which alternative ad hoc placement of indels couldbias the phylogenetic outcome were excluded from the

idae and Outgroups

e GenBank Accession No.

Distribution 28S EF-1a

S. Africa AF147823 AF148082

Israel AF147826 AF148084

Israel AF147824 AF148085Guatemala AF147825 AF148086

New Mexico AF147827 AF148083

Arizona AF147828Guatemala AF147829 AF148089S. Africa AF147830 —Namibia — AF148090S. Africa AF147831 AF148088Israel AF147832 AF148092Namibia AF147833 AF148094S. Africa AF147834 AF148093Israel AF147835 AF148091S. Africa — AF148095Texas AF147836 AF148087

Australia AF147837 AF148107Australia AF147840 AF148106Australia — AF148098Chile — AF148099Australia AF147841 —Australia — AF148096Australia AF147842 AF148097Australia AF147843 AF148102Australia AF147844 —Australia — AF148100Australia AF147845 —Australia — AF148101Australia AF147846 AF148105Australia AF147847 AF148108Guatemala AF147848 AF148110Guatemala AF147849 AF148114Australia AF147850 AF148113Guatemala AF147851 AF148109Guatemala AF124487 AF124488Illinois AF147852 AF148112Illinois AF147853 AF148111California — AF148115Israel AF147854 AF148118Baja, CA — AF148120Colorado AF147855 AF148116California — AF148117England AF147856 AF148121

onding to a collection/specimen database (MANDALA) (Kampmeier

rev

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data for all 38 genera were analyzed. In the combined

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443RELATIONSHIPS OF THEREVIDAE

analysis. These excluded sites were concentrated injust three A-T rich regions, each less than 15–20 nu-cleotides in length. Alignment gaps within aligned re-gions were treated as missing data. Alignment ofEF-1a was unambiguous within the coding region frag-

ent sequenced. Aligned sequences for both genes,nnotated to indicate positions used in the correspond-ng phylogenetic data set, are obtainable on the web-ite http://www2.ncsu.edu/unity/users/b/bwiegman/ublic_html/align.html or on request from the senioruthor.Phylogenetic trees were constructed using equallyeighted parsimony methods in PAUP*4.0d64 (Swof-

ord, 1999). Heuristic searches were performed using00 replicate random addition searches with tree bi-ection–reconnection (TBR) branch swapping. Bremerupport values (Bremer, 1988, 1994) were calculatedy heuristic search (TBR) with 10 replicates of randomdditions using the “constraint” and “enforce converse”ptions in PAUP*4.0d64. Bootstrap support valuesFelsenstein, 1985) were obtained from 200 replicateesampled data sets and heuristic searches (TBR). Theartition homogeneity test option in PAUP* was usedo calculate IMF values (Mickevich and Farris, 1981;

Farris et al., 1995; Johnson and Soltis, 1998) with 100replicates to test for homogeneity of signal in the twosequence data sets.

Because a few taxa were sampled for only one of thetwo genes (Table 1), a data set with all taxa was firstanalyzed separately for each gene. Next, only the 31genera with both 28S and EF-1a gene sequences werencluded in a combined data set. Finally, combined

Sequences of 28S and EF-1a Primers Usedin PCR and Sequencing

Gene Primer Sequence

8S rc28H 59-CTACTATCCAGCGAAACC-(6000)28k 59-CTTCGATGTCGGCTCTTG-(6582)rc28Q 59-GGACATTGCCAGGTAGGGAGTT-(6406)28Q 59-AACTCCCTACCTGGCAAT-(6389)rc28X 59-CGCCTCTAAGGTCGTATCCG-(6930)28Z 59-GCAAAGGATAAGCTTCAGTGG-(7200)

F-1a rc40 59-GTCGTSATYGGWCACGTMGATTCYGG-(2118)46 59-TGAGGAAATCAARAAGGAAG-(2582)rc47 59-GGAACAGTACCYGTGGGTCG-(2859)51 59-CATGTTGTCRTGCCATCC-(2645)54 59-CCGTTCCAGTGGTTSCGGCG-(3424)rcEF4 59-GARGGTGGTATYACMATTGA-(2283)EF5 59-CTCATATCACGTACAGCRAARGG-(3329)

Note. Degenerate positions are identified by the IUB single-lettercode for nucleotide bases: R 5 A/G; Y 5 C/T; M 5 A/C; W 5 A/T; S 5

/G; K 5 G/T; rc, reverse complement, which indicates that primerinds to the sense strand of the DNA. Numbers in brackets at the 39nd of each primer refer to nucleotide position relative to the Dro-ophila melanogaster sequence.

analysis, those genera lacking either of the two genesequences were coded as missing data. In a few cases,sequences that we obtained for the two genes weresampled from separate individuals of the same species(Phycus niger, Neodialineura striatithorax; Table 1) orfrom different species of the same genus (Nanexilamanni, N. vittata, Parapsilocephala sp., Parapsilo-cephala sp. 1; Table 1) and then combined as a singletaxon for genus-level phylogenetic analysis. In the lat-ter case, generic identification was unambiguous foreach species contributing to a “combined-species”taxon. Finally, partitioned Bremer support values(Baker and DeSalle, 1997) were calculated using theprogram TreeRot v.2 (Sorenson, 1999) to examine therelative support for the combined topology from each ofthe two gene partitions.

RESULTS

Aligned Data Sets and Nucleotide Variation

Of the approximately 1270 bp amplified from ther-evid 28S rDNA, 1162 bp were included as an alignablesequence for phylogenetic analysis. Of these sites, 237bp were variable (21%) and 138 bp phylogeneticallyinformative (14%). Uncorrected pairwise sequence di-vergence ranged from 0.26 to 6.90% between therevidgenera and from 4.93 to 8.98% between outgroup taxaand therevid genera. A few lineages show an increasedrate of 28S rDNA evolution within Therevidae, espe-cially Pherocera Cole, which is 5.17–6.90% divergentfrom other therevid genera. In general, pairwise diver-gence increases with depth in the phylogenetic hierar-chy, as is expected under homogeneous rates. Averagebase frequencies for this fragment are A 5 30.70%, C 517.30%, G 5 22.51%, and T 5 29.49%, and a x2 test forbase composition showed no significant deviation fromthese proportions among taxa (x2 5 6.77, df 5 96,P 5 1.0).

For EF-1a, 993 bp of the 1064-bp amplified fragmentwere alignable, with 366 bp variable (37%) and 299 bp(32%) phylogenetically informative. Amplification ofEF-1a for three therevid genera (Orthactia Krober,Efflatouniella Krober, and Megalinga Irwin &Lyneborg) and two outgroups (Heterotropus Loew, Al-oxytropus Bezzi) from standard genomic PCR revealed53- to 79-bp intron interrupting the coding sequence

53 bp downstream from primer EF4 [position 2736 in. melanogaster (Hovemann et al., 1988)]. We found novidence for multiple copies of EF-1a in the Therevidae

(cf. Danforth and Ji, 1998).EF-1a showed a higher level of variability than 28S

for these taxa. Uncorrected pairwise divergence rangedfrom 2 to 17% between therevid genera and from 10 to18% between outgroups and therevid genera. Like 28S,these values increase with increasing phylogenetic

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444 YANG ET AL.

depth of comparison. Mean base frequency for EF-1aare A 5 29.24%, C 5 20.86%, G 5 21.52%, and T 528.36%. A x2 test for base frequency heterogeneityamong taxa showed that base composition proportionswere different across taxa (x2 5 135.57, df 5 111,

5 0.0565). This result was due largely to the highercentage of A and T observed in Pherocera andtenogephyra (33.6 and 31.8%, respectively), a sharp

ncrease above the means for other taxa. Excludingherocera and Stenogephyra from the data equalizedroportions across taxa (x2 5 75.91, df 5 105, P 5.99).

ubstitutional Saturation

Expected transition:transversion ratios have beensed as a gauge of substitutional saturation, the pointt which an excess of superimposed substitutions areikely to confound phylogeny estimates (Swofford et al.,996; Naylor and Brown, 1998; Yang, 1998). 28S

FIG. 1. Phylogenetic relationships based on analysis of unweiconsensus of 32 MP trees; tree length 5 573, CI 5 0.555, RI 5 0.566nd that below each branch is the decay index. (b) One of the 32CCTRAN optimization.

DNA, total EF-1a, and third positions of EF-1a allshowed no evidence of saturation, as measured by aconstant linear increase in substitution type with in-creasing phylogenetic depth of pairwise comparisons(not shown).

Phylogenetic Analysis of 28S Ribosomal DNA

Parsimony analysis of the 1162-bp 28S data setyields 32 most-parsimonious trees (MPTs) (length 5573, CI 5 0.56, RI 5 0.56; Figs. 1a and 1b). A strictconsensus tree of the 32 MPTs is presented in Fig. 1a.These trees show strong support for the monophyly ofTherevidae, exclusive of the genus Apsilocephala. Theontroversial genus Apsilocephala unexpectedly joinshe tree within the outgroups, with the scenopinidenus Alloxytropus (Scenopinidae; Proratinae) as a sis-er group to Therevidae. Within Therevidae, the basalivision of the family comprises two large clades, aphycine” clade and a “therevine” clade (Fig. 1a). Sup-

ed 28S rDNA gene sequences of 33 taxa in 32 genera. (a) Stricte number above each branch is the bootstrap value (200 replicates)trees. Branch lengths indicate assigned character changes under

ght. ThMP

sd

hE

445RELATIONSHIPS OF THEREVIDAE

port for groupings within these two clades is generallylow, except for a strongly supported (98% bootstrap)clade grouping Penniverpa and nine higher therevinegenera. Parsimonious assignment of character changes(ACCTRAN) on the MPTs reveals somewhat higherlevels of variation within the phycine group (Steno-gephyra, Pherocera) (Fig. 1b). Assigned branch lengthsare shorter and taxa are more homogeneous across thetherevine genera (Fig. 1b).

Phylogenetic Analysis of Elongation Factor-1a

Parsimony analysis of 993 aligned sites from EF-1ayields 21 equally parsimonious trees (length 5 1746;CI 5 0.32; RI 5 0.52; Figs. 2a and 2b). The EF-1aresults largely agree with the general pattern of rela-tionships inferred from the 28S gene. Moderate to high(.50%) bootstrap values support the sister group rela-tionship of Scenopinidae 1 Therevidae. The strict con-ensus confirms the monophyly of the family Therevi-ae, again excluding Apsilocephala, and the

FIG. 1—

monophyly of the two main branches. Like 28S, EF-1astrongly supports a clade consisting of Penniverpa 1

igher therevines. The most-parsimonious trees forF-1a and 28S differ in the relative placement of the

mostly australasian taxa at the base of the “therevine”clade. Both genes show limited character support forrelationships in this area of therevid phylogeny.

Within the two subfamilies, some placements differfrom those suggested by 28S. Bootstrap support forgenus-level groupings is generally greater for EF-1athan for the 28S rDNA. A major difference betweenresults for the two genes is the position of Apsilo-cephala. The EF-1a data place Apsilocephala outsidethe Therevidae 1 Scenopinidae.

Combined Analysis of 28S rDNA and EF-1a

Among the 38 genera sampled, only 31 were se-quenced for both 28S and EF-1a. The combined datafor these taxa comprise 2155 sites, of which 1162 are28S and 993 are EF-1a. Calculation of the incongru-

ntinued

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446 YANG ET AL.

ence length difference (ILD; Mickevich and Farris,1981; Farris et al., 1995; Johnson and Soltis, 1998) bythe partition homogeneity test in PAUP* showed nosignificant heterogeneity in these data (P 5 0.240).

Parsimony analysis of the combined 28S and EF-1adata yields three minimum-length trees (length 52150, CI 5 0.40, RI 5 0.51). The strict consensus (Fig.3a) and higher bootstrap values both strongly supportmonophyly of the family Therevidae, monophyly of thetwo main branches found in the individual gene anal-yses, and monophyly for the sister group relationshipof Scenopinidae and Therevidae. Similar to the EF-1atrees, Apsilocephala is placed basal to the Scenopini-

ae 1 Therevidae. Bootstrap values for many genus-evel groupings were amplified in the combined result.

FIG. 2. Phylogenetic relationships based on analysis of unweighof 21 MP trees; tree length 5 1746, CI 5 0.321, RI 5 0.518. The numelow each branch is the decay index. (b) One of the 21 MP trees. Bptimization.

Support for relationships at the base of the therevineclade remain low in the combined analysis (Fig. 3a).

Addition of genera for which only one of the twogenes was sequenced [Henicomyia (28S only);Neotabuda Krober, Ectinorhynchus Macquart, Entesia

ldroyd, Nebritus Coquillett, Acrosathe Irwin &yneborg, and Tabudamima Irwin & Lyneborg (EF-1a

only)] to the analysis yields 14 minimum-length trees(length 5 2337, CI 5 0.38, RI 5 0.52), with strictconsensus shown in Fig. 3b. These trees have Henico-myia placed either as the basal therevid lineage or asthe basal-most branch of the phycine clade, with higherbootstrap support (51%, not shown). This full-taxon-setcombined analysis is topologically concordant with the31-taxon combined analysis, but lacks resolution due

EF-1a gene sequences of 38 taxa in 37 genera. (a) Strict consensusr above each branch is the bootstrap value (200 replicates) and thatnch lengths indicate assigned character changes under ACCTRAN

tedbera

b

M

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447RELATIONSHIPS OF THEREVIDAE

to low support for the placement of Henicomyia andasal therevines.

DISCUSSION

olecular Phylogenetic Evidence for Relationships ofTherevidae

Both nuclear gene fragments sequenced in our studyppear to contain useful phylogenetic information fornferring relationships within the Therevidae andmong its closest relatives. The utility of these twoenes for asiloid phylogenetics is supported by the gen-ral agreement of tree topologies found in separatenalyses of the data. Despite some differing arrange-ents among terminals in the results of the separate

nalyses, there are no large differences in the higher-evel placement of taxa. Previous studies employing

FIG. 2—

hese genes (28S rDNA: Whiting et al., 1997; Friedrichnd Tautz, 1997b; Wiegmann et al., 2000; EF-1a: Cho

et al., 1995; Mitchell et al., 1997; Danforth and Ji,1998), as well as levels of pairwise nucleotide variationobserved here, suggest that 28S rDNA should be mostinformative among the oldest divergences, possibly asold as the late Jurassic in Therevidae and other asiloidfamilies (Hennig, 1981; Grimaldi and Cumming, 1999).EF-1a seems most informative below the family level,corresponding to Tertiary-aged and younger diver-gences (Mitchell et al., 1997; Friedlander et al., 1998).Synapomorphies from the mostly third position varia-tion of EF-1a are more evenly distributed throughoutthe tree, whereas 28S variation is greatest at the fam-ily level and among taxa in the subfamily Phycinae.This differential support for portions of the tree is alsoreflected in partitioned Bremer support values for the

ntinued

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448 YANG ET AL.

two genes calculated on the combined topologies of tailed analyses of the enigmatic adult morphology of

ma

449RELATIONSHIPS OF THEREVIDAE

Figs. 3a and 3b.We consider our results from combining data from

both genes (Fig. 3) to be the best hypothesis of therevidrelationships currently available. Following others(Kluge, 1989; Baker and DeSalle, 1997; Siddall, 1997),we favor the “total-evidence” tree because broad-based,quantitative, phylogenetic hypotheses have never beenpreviously proposed for the Therevidae and becauseboth genes are somewhat limited in their individualsupport for therevid relationships. While each genecontributes homoplasy to the combined analysis, thetwo data sets show significant character congruenceaccording to the partition homogeneity test, result inlargely concordant topologies when analyzed sepa-rately, and show no apparent source of character bias(e.g., saturated transitions, base composition bias) thatwould lead us to reject one gene in favor of the other.

Our data support the first detailed empirical hypoth-esis of phylogenetic relationships for the family Ther-evidae. The 28S rDNA, EF-1a, and combined analysesof these genes all show strong support for the mono-phyly of Therevidae, division of the family into twomain branches corresponding to Irwin and Lyneborg’s(1981a) Phycinae and Therevinae, and a higher ther-evine clade which includes the genera Penniverpa,Megalinga, Thereva, Hoplosathe, Pandivirilia,Brachylinga, Lysilinga, Chromolepida, Cyclotelus, andOzodiceromyia. The combined analyses differ only inthe level of resolution available among basal clades ofthe two subfamilies and among the more derived ther-evine lineages. These differences are attributable toconflicting signal for the position of Henicomyia at thebase of the Phycinae and lack of resolution or conflictarising from low character variation among Therevi-nae (Fig. 1b).

The molecular data presented here exclude Apsilo-cephala from Therevidae but cannot rule out a place-ment within the Scenopinidae or basal to Scenopini-dae 1 Therevidae. The monophyly of Therevidaeexclusive of Apsilocephala is well supported in bothindividual and combined analyses. Constraining the32-taxon combined data analysis to include Apsilo-cephala within the Therevidae requires 13 additionalsteps in parsimony analyses. Our result is consistentwith the elevation of this taxon to family-level statusby Nagatomi et al. (1991a,b), but our findings alsoquestion the monophyly of Scenopinidae and suggest apossible subordinate position for the apsilocephalidswithin Scenopinidae. We are currently preparing de-

FIG. 3. Phylogenetic relationships based on combined 28S and EFost-parsimonious trees; tree length 5 2150, CI 5 0.395, RI 5 0.512

nd that below each branch is the partitioned Bremer support valuerDNA and EF-1a gene sequences for all 38 genera showing the stricbranch is the bootstrap value (200 replicates).

these flies together with a large survey of Therevidaeand Scenopinidae for a subsequent paper. Combinedmorphological and molecular data sets may be neces-sary to significantly improve our understanding of thelimits of the Therevidae. Of particular importance, forexample, would be the discovery of the still unknownlarvae of Apsilocephala and Clesthenthia to determinewhether they have the secondary abdominal segmen-tation synapomorphy of Scenopinidae 1 Therevidae.Ultimately, a more exact placement for Apsilocephali-dae will require both increased taxon and charactersampling from the Asiloidea and other basalBrachycera.

Within the Phycinae, both genes support a close as-sociation of the genera Ruppellia, Actorthia, and Or-thactia; EF-1a also groups Neotabuda with these gen-era. Therevinae are a much more diverse clade, withAustralasian taxa at the base. Node support is gener-ally weak among these basal lineages. The combineddata (Fig. 3) place Nanexila as the sister to all othertherevines and then group the other Australasian gen-era in two main clades. These groupings are consistentwith preliminary analyses of morphological evidence(Winterton et al., 1999). Combined analysis also groupsAnabarhynchus, Penniverpa, and nine “higher-ther-evine” genera (Fig. 3). Classification of Therevidae intoadditional subfamily-, tribe-, and genus-level group-ings will require additional molecular data, as well asemerging detailed morphological analyses (Irwin andYeates, 1998; Winterton et al., 1999).

Radiation of Australasian Therevidae

All three molecular phylogenetic analyses showweak support for relationships among the Australa-sian taxa at the base of the Therevinae (Figs. 1–3).Clades containing short branch lengths within largerphylogenetic trees are often interpreted as having un-dergone rapid radiation, with short intervals of sharedancestry leaving few synapomorphies. This inference isconsistent with an hypothesis of rapid radiation ofTherevidae within Australia. Branch lengths inferredfrom both genes sequenced in this study imply a rapidradiation among these lineages. Australian thereviddiversity is greater than that found in all other biogeo-graphic regions in species, genera, and morphologicaldivergence.

Based on the postulated age of the family and thatthe distal clades of therevines have radiated exten-sively in the Holarctic, the basal therevine radiation

gene sequences of 32 taxa in 31 genera. (a) Strict consensus of threehe number above each branch is the bootstrap value (200 replicates)F-1a/28S rDNA). (b) Phylogenetic relationships from combined 28Sonsensus of 14 equally parsimonious trees. The number above each

-1a. T(E

t c

probably dates from Pangean times (before 160 mya; Bremer, K. (1988). The limits of amino acid sequence data in angio-sperm phylogenetic reconstruction. Evolution 42: 795–803.

H

H

H

HH

H

I

I

I

450 YANG ET AL.

Veevers, 1991). It is likely that the Australasian ther-evine lineages were restricted to Australasia by ecolog-ical barriers in Gondwana. Few palaeoenvironmentalreconstructions have been proposed for the supercon-tinent, but the interior is likely to have been arid, muchlike the centers of the large continents today (Ander-son, 1981). At the time of the early radiation of ther-evines, Australia occupied the eastern margin of Gond-wana and the vegetation was dominated by moisttemperate forests (Barlow, 1981). Areas retaining thisenvironment today may harbor further basal lineagesof therevines and will be the focus of future surveys.Radiations at lower taxonomic levels in Australia mayhave occurred relatively recently in response to theincreasing aridity of the Tertiary period (Barlow,1981), and this scenario is concordant with the largenumbers of congeneric species found during taxonomicrevisionary research on Australian therevids (e.g.,Winterton et al., 2000). The limited support that wefound for groupings within the Australian fauna sug-gests that additional information from genes and mor-phology will likely prove necessary to better delimit thetaxonomic breadth, geographic range, and temporalscale of the diversification of Australian therevids.

ACKNOWLEDGMENTS

We are grateful to E. Schlinger and F. C. Thompson for supportand assistance with this project. We thank L. L. Deitz and R. M. Roe,Department of Entomology, North Carolina State University and M.Purugganan, Department of Genetics, North Carolina State Univer-sity for comments on an earlier draft of the manuscript. We alsothank Brian Cassel, Jason Cryan, and Gail Kampmeier for invalu-able technical assistance. Our special thanks go to all the membersof the therevid PEET team, especially Shaun Winterton, DonaldWebb, Stephen Gaimari, Mark Metz, Martin Hauser, and KevinHolston for providing specimens, taxonomic information, advice, andsupport. The North Carolina State University Automated DNA Se-quencing Facility provided sequencing for this project; we gratefullyacknowledge the advice, technical expertise, and efficiency offered byDr. Arthur Johnson and the NCSU DNA Sequencing Facility tech-nical staff. This project was supported by funds from the NationalScience Foundation (DEB-9521925), the Schlinger Foundation, theNorth Carolina State University College of Agriculture and LifeSciences, and the North Carolina Agricultural Research Service(NCARS).

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