Australian Lasioglossum + Homalictus Form a Monophyletic ... · form a monophyletic group. Bootstrap support for the Australian clade ranged from 73% to 77%, depending on the method

Syst. Biol. 50(2):268–283, 2001

Australian Lasioglossum + Homalictus Form a Monophyletic Group:Resolving the “Australian Enigma”

BRYAN N. DANFORTH AND SHUQING JI

Department of Entomology, Comstock Hall, Cornell University, Ithaca, New York 14853-0901 , USA;E-mail: [email protected]

Abstract.—The bee genus Lasioglossum includes >1,000 species of bees distributed on all continentsexcept Antarctica. Lasioglossum is a major component of the bee fauna in the Holarctic, Ethiopian,and Asian regions and is an important group for investigating the evolution of social behavior inbees. Given its cosmopolitan distribution, the historical biogeography of the genus is of considerableinterest. We reconstructed phylogenetic relationships among the subgenera and species within La-sioglossum s.s., using DNA sequence data from a slowly evolving nuclear gene, elongation factor-1®.The entire data set includes >1,604 aligned nucleotide sites (including three exons plus two introns)for 89 species (17 outgroups plus 72 ingroups). Parsimony and maximum likelihood analyses providestrong evidence that the primarily Indoaustralian subgenera (Homalictus, Chilalictus, Parasphecodes)form a monophyletic group. Bootstrap support for the Australian clade ranged from 73% to 77%,depending on the method of analysis. Monophyly of the Australian Lasioglossum suggests that a sin-gle colonization event (by way of Southeast Asia and New Guinea) gave rise to a lineage of >350native Indoaustralian bees. We discuss the implications of Australian monophyly for resolving the“Australian enigma”—the similarity in social behavior among the Australian halictine bees relativeto that of Holarctic groups. [Biogeography; elongation factor-1®; maximum likelihood; phylogeny;social evolution.]

The Australian bee fauna is remarkable inmany ways. More than half of the species ofAustralian bees belong to one family (Colleti-dae), which is commonly considered to bethe most plesiomorphic of bees (Alexanderand Michener, 1995). In addition, major fam-ilies in other parts of the world are absent inAustralia (Andrenidae and Melittidae), andAustralia is surprisingly depauperate in par-asitic bees (Wcislo, 1988). Although the en-demic Australian Colletidae (Euryglossinae)and Stenotritidae and the predominantlyAustralian Paracolletini and Hylaeinae mayrepresent groups that became isolated onAustralia during the breakup of Gondwanain the Mesozoic, many other groups ofbees have clearly colonized Australia fromthe north by way of Southeast Asia andNew Guinea (Michener, 1979a). Numerousindependent colonization events have oc-curred within the bee family Halictidae asshown by the several distantly related generanow occupying parts of Australia: NomioidesSchenck (Nomioidinae, 1 sp.; Yeates andExley, 1986), Lipotriches Gerstaecker (D No-mia Latreille; Nomiinae, »70 spp.; Cardale,1993; Michener, 2000), Sphecodes Latreille(Halictinae, 2 sp.; Cardale, 1993), Pachyhal-ictus Cockerell (Halictinae, 2 spp.; Walker,1993, 1996), Lasioglossum Curtis (Halictinae,many species; Michener, 1965; Walker, 1995);

and Homalictus Cockerell (Halictinae, manyspecies; Walker, 1986, 1997). By far thelargest groups of Indoaustralian halictidbees are in the related genera Lasioglossumand Homalictus, which together account fornearly 350 species of Australian halictinebees.

The genus Lasioglossum includes >1,000species worldwide with numerous subgen-era and species groups recognized. The sub-generic groupings within Lasioglossum aretreated by some authors as separate genera(see Krombein et al., 1979; Moure and Hurd,1987) because they comprise such large anddiverse taxa. Others treat Lasioglossum as agenus consisting of many subgenera (Ebmer,1987; Michener, 2000). For the purposes ofthis paper, we will refer to the genus La-sioglossum and its numerous subgenera asfollows L. (Chilalictus) for the Lasioglossumsubgenus Chilalictus, as L. (Paraphecodes) forthe Lasioglossum subgenus Parasphecodes , andso on for all subgenera listed in Table 1.

Michener (2000) divided the subgeneraof Lasioglossum into two groups: the Hemi-halictus series, which includes all subgen-era with a weakened �rst r-m cross vein infemales, and the Lasioglossum series, whichincludes all subgenera with a completelysclerotized �rst r-m cross vein (Table 1).Six of the eight subgenera within the

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2001 DANFORTH AND JI—RESOLVING THE “AUSTRALIAN ENIGMA” 269

TABLE 1. Classi�cation of the subgenera of Lasioglossum (modi�ed slightly from Michener, 2000).

Subgenus (no. species) Distribution

Lasioglossum seriesCtenonomia Cameron (>100) Paleotropical (mostly Southeast Asia)Lasioglossum Curtis s.s. (>150)a Holarctic and MesoamericanAustralictus Michener (10) Australia (widespread)Callalictus Michener (8) Australia (VIC, SA, NSW, QLD)Chilalictus Michener (134) Australia (widespread) and New Caledonia (1 sp.)Glossalictus Michener (1) Australia (WA)Parasphecodes Smith (92) Australia (widespread) and New GuineaPseudochilalictus Michener (1) Australia (NSW and QLD)Homalictus Cockerell (94)b Indoaustralia (widespread)Echthralictus Perkins and Cheesman (2)b,c Samoa

Hemihalictus seriesAcanthalictus Cockerell (1) SiberiaAustrevylaeus Michener (9) Australia and New ZealandDialictus Robertson (>300)a Nearctic/NeotropicalEvylaeus Robertson (>100)a Holarctic/NeotropicalHemihalictus Cockerell (1) NearcticParadialictus Pauly (1) Africa (Zaire)Paralictus Robertson (3)c NearcticSellalictus Pauly (11) Africa (Zaire to Cape Prov.)Sphecodogastra Ashmead (8) NearcticSudila Cameron (6) Sri Lanka and Malaysia

aIndicates subgenera with both solitary and eusocial species.bPreviously not considered as part of Lasioglossum.cIndicates socially parasitic subgenera.

Lasioglossum series consist predominantly orexclusively of endemic Australian species:Australictus, Callalictus, Chilalictus, Glos-salictus, Parasphecodes and Pseudochilalictus(Table 1).

Although Homalictus (plus the cleptopar-asitic derivative, Echthralictus Perkins andCheesman, found in Samoa [Perkins andCheesman, 1928; Michener, 1965, 1978]) hasnot recently been considered a subgenus ofLasioglossum (Michener, 2000), both morpho-logical characters (Lasioglossum sensu lato andHomalictus share weakened 2r-m and 2m-cucross veins in females (see Fig. 1 in Danforth[1999]) and molecular data presented belowindicate that Homalictus arises from withinLasioglossum. We have therefore chosen to re-fer to Homalictus as a subgenus of Lasioglos-sum throughout this paper. Homalictus hasits center of diversity in Australia, but manyspecies occur in New Guinea (Pauly, 1986;Michener, 1980a), as far north as Sri Lankaand Southeast Asia, and southward and east-ward to Indonesia (Pauly, 1980), the Philip-pines (Cockerell, 1919; Michener, 1980b), andthe islands of Fiji (Michener, 1979b) andSamoa. However all the available evidencesuggests that Homalictus has its origins inAustralia (Michener, 1979a).

Members of Indoaustralian Lasioglossumand Homalictus are distinct both behav-iorally and, to a lesser extent, morpholog-ically, from their Holarctic relatives in thegenus Lasioglossum. First, like most Aus-tralian bees, they primarily visit plants inthe family Myrtaceae (such as Melaleuca andEucalyptus; Walker, 1986; Michener, 1965)for pollen and nectar. Other importantsources of pollen and nectar include plantsin the families Mimosaceae (such as Aca-cia), Proteaceae (such as Banksia), and Papil-ionaceae (Bernhardt and Walker, 1984, 1985;Bernhardt, 1987; Walker, 1986), and to a lesserextent, Amaranthaceae, Asteraceae, Lami-aceae, Dilleniaceae, Frankeniaceae, Good-eniaceae, Haemodoraceae, Haloragaceae,Myoporaceae, Portulacaceae, Rutaceae, Sola-naceae, Sterculiaceae, and Xanthorrhoeaceae(T. Houston, pers. comm.). Australian hal-ictines are generally considered narrowlypolylectic, in that most species restrict pollenforaging to Myrtaceae but will visit a di-verse array of genera depending on local-ity. Nevertheless, several species are clearlyoligolectic. Lasioglossum (Chilalictus) mega-cephalum is restricted to Goodeniaceae, L.(Chilalictus) frankenia is oligolectic on Franke-nia (Frankeniaceae), and numerous species

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270 SYSTEMATIC BIOLOGY VOL. 50

FIGURE 1. Strict consensus tree based on analysis of unweighted nucleotide data; exons plus introns with indelmutations coded as described in Danforth et al. (1999) (1,540 nucleotide positions; 534 parsimony-informativecharacters; CI D 0.3946, RI D 0.7541, length D 2,477). Outgroups included Halictus (Seladonia) confusus, Halictus(Halictus) farinosus, H. (H.) rubicundus, H. (H.) ligatus, H. (H.) poeyi, Agapostemon kohliellus, A. sericeus, A. tyleri,A. virescens, Pseudagapostemon brasilensis, Mexalictus arizonensis, Sphecodes minor, Sphecodes ranunculi, Augochloropsismetallica, Megalopta genalis, Augochlora pura, and Neocorynura discolor.

of Chilalictus are oligolectic on Wahlenber-gia (Campanulaceae) (Walker, 1995). Onlytwo subgenera of Holarctic Lasioglossum,Hemihalictus (Daly, 1961) and Sphecodogas-

tra (McGinley, 2000), are known to beoligolectic.

More importantly, the Australian Lasioglos-sum and Homalictus have a unique array

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of behavioral attributes that distinguishesthem from Lasioglossum in other parts of theworld (Michener, 1960; Knerer and Schwarz,1976, 1978). All species studied to date ex-hibit either solitary or communal nestingbehavior (Michener, 1960, 1974), such thatmultiple females share a nest but do not co-operate in cell provisioning or show repro-ductive division of labor. Michener (1960)conducted a broad survey of species in Homa-lictus, Chilalictus, and Parasphecodes by dis-secting females collected on �owers. Forvirtually all species examined, 100% of fe-males are fertilized. Such results providestrong, though indirect, evidence that thesespecies are not eusocial. More detailed stud-ies involving nest excavations and dissec-tions of foraging and resident females havebeen conducted on additional species, in-cluding L. (Chilalictus) lanarium (Knerer andSchwarz, 1978), L. (Chilalictus) cognatum (asL. [Chilalictus] inclinans, Knerer and Schwarz,1978), L. (Chilalictus) platycephalum (as L.[Chilalictus] mesembryanthemiellum ; Knererand Schwarz, 1978; McConnell-Garner andKukuk, 1997), L. (Chilalictus) leai (as Hal-ictus leai; Cardale and Turner, 1966), andL. (Chilalictus) hemichalceum (Rayment, 1955;Houston, 1970; Kukuk and Schwarz, 1987,1988; Kukuk and Crozier, 1990; Kukuk, 1992;Kukuk and Sage, 1994; Ward and Kukuk,1998). In all cases, nests contained multiple,reproductively active females, and in somecases there was evidence of overlap in gen-erations. Nests may be huge in some species,such as Homalictus urbanus, which has up to160 females per nest (T. Houston unpubl. ob-servation, cited in Walker, 1986).

Among the more remarkable aspects ofcommunal Australian Lasioglossum is thatthey defend their nests by plugging thenest entrance with the metasoma (Rayment,1935; Michener, 1960; P. Kukuk, pers. comm.;B.N.D., pers. unpubl.) while showing low ag-gression towards conspeci�cs (Kukuk andCrozier, 1990; Kukuk, 1992; Kukuk and Sage,1994). Molecular genetic studies indicate thatnestmates in communal Chilalictus are unre-lated (Kukuk and Sage, 1994), as one wouldexpect for communal (as opposed to euso-cial) species. Communal nesting is rare inthe Holarctic groups of Lasioglossum. Speciesin the nominate subgenus, Lasioglossum s.s.,have been observed by numerous authorsto be solitary, whereas most species inthe Hemihalictus series are primitively eu-

social (e.g., numerous species of Dialictusand Evylaeus; see Michener [1990], Packer[1993], Wcislo [1997], and Yanega [1997] forreviews).

At least one species of Australian Lasioglos-sum (L. [Chilalictus] hemichalceum) shows dis-crete male dimorphism whereas male pos-itive head allometry is widespread in thesubgenus Chilalictus (Walker, 1995). Large-headed males in L. (Chilalictus) hemichalceumhave been interpreted as guards (Houston,1970; Kukuk and Schwarz, 1988).

Nest architecture in the Australian La-sioglossum and Homalictus is also distinctfrom that of their Holarctic relatives. AllAustralian Lasioglossum and Homalictus con-struct cells either in series (e.g., in Homalic-tus and some Chilalictus) or in clusters (someChilalictus) (Knerer and Schwarz, 1976). Ho-larctic Lasioglossum typically construct ses-sile cells off of a central nest tunnel, as in L.(Evylaeus) marginatum, L. (Evylaeus) malachu-rum, and many species of L. (Dialictus), al-though some species (L. [Evylaeus] duplex)construct cells in clusters (Michener, 1974).

This unique suite of social attributespresent in the Australian Lasioglossum andHomalictus was referred to by Knerer andSchwarz (1976) as the “Australian enigma.”They presumed that the social behaviorof the Australian halictines was conver-gently evolved, perhaps in response toheavy ant predation on ground-nestingbees, or in response to mutillid wasp at-tack (Rayment citations in Michener, 1960).The classi�cation of Australian halictinebees would not have suggested a com-mon ancestral origin for Australian La-sioglossum and Homalictus, because Homal-ictus was considered a distinct genus, andeven the Australian subgenera of Lasioglos-sum are not obviously monophyletic asbased on morphology (Michener, 1965). Inaddition, the Australian subgenera of La-sioglossum exhibit substantial morphologi-cal diversity. Within Chilalictus alone aresmall, metallic greenish species that super-�cially resemble North American Dialictus(in fact, they were classi�ed as such [us-ing the synonymous name Chloralictus] priorto Michener’s [1965] study), small blackspecies similar to Northern Hemisphere Evy-laeus, and large species with metasomalhair bands and imbricate mesosomal sculp-turing that resemble Northern HemisphereLasioglossum s.s.

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We sought to test the hypothesis thatthe Australian subgenera of Lasioglossumplus Homalictus form a monophyletic groupby analyzing a large nucleotide data setfor a diverse array of species within La-sioglossum and Homalictus plus outgroups. Ifthe Indoaustralian Lasioglossum C Homalic-tus form a monophyletic group, we wouldconclude the unique social attributes of theAustralian halictine bees are derived from acommon ancestor that also had those traits,rather than through convergent evolution insocial behavior. Likewise, monophyly wouldsuggest a single colonization of Australia inthe distant past, rather than multiple recentcolonizations.

We chose a nuclear, protein-coding gene,elongation factor-1® (EF-1®), for this study.EF-1® encodes an enzyme involved in theGTP-dependent binding of charged tRNAsto the acceptor site of the ribosome duringtranslation (Maroni, 1993). Previous cladisticanalyses of EF-1® sequence data have foundthat this gene provides useful phylogeneticinformation across a wide range of diver-gence times (Friedlander et al., 1992, 1994).Within insects, EF-1® has been shown to re-cover higher-level relationships in the mothsubfamily Heliothinae (Cho et al., 1995),the moth superfamily Noctuoidea (Mitchellet al., 1997), and the bee genus Halictus(Danforth et al., 1999).

MATERIALS AND METHODS

Bees for this study were collected byB.N.D. or generously provided by colleagues(see Acknowledgments). Specimens used forsequencing were primarily preserved in 95%ethanol, but recently collected pinned spec-imens (<5 years old) and frozen specimenswere also used. The outgroup and ingrouptaxa included in this study, locality data,specimen voucher numbers, and GenBankaccession numbers are listed in Table 2.

DNA extractions followed standard pro-tocols detailed in Danforth (1999). Two setsof polymerase chain reaction (PCR) prod-ucts were used to generate the data set. Ini-tially, primers were designed based ona com-parison of published Drosophila (Hovemannet al., 1988), Apis (Walldorf and Hovemann,1990), and moth (Cho et al., 1995) sequences.Primers that initially ampli�ed at least somehalictid species included For1-deg, For3, andCho10 (all primer sequences are listed in

Danforth et al., 1999). On the basis of theinitial comparisons of the F1 and F2 copiesof EF-1® in halictid bees, we developed anew, F2-speci�c reverse primer (F2-Rev1). Forthe downstream (30) end of EF-1® we usedprimers For3/Cho10. These primers amplifyboth EF-1® copies; however, the presence ofan »200–250-bp intron in the F2 copy allowsthese PCR products to be separated on low-melting-point agarose gels. Only the F2 copywas included in the present analysis.

PCR ampli�cations were carried outby fol-lowing standard protocols (Palumbi, 1996),with the following cycle conditions: at 94±C,1 min for denaturation; at 50–56±C, 1 min forannealing; at 72±C, 1 min to 1 min 20 secfor extension. Before sequencing, the PCRproducts were either gel-puri�ed in low-melting-point agarose gels (FMC, Rockland,ME) overnight at 4±C or puri�ed directly byusing the Promega (Madison, WI) WizardPCR Preps DNA Puri�cation kit.

For manual sequencing we used 33P-labeled dideoxy chain termination reac-tions (Thermo Sequenase radiolabeled ter-minator cycle sequencing kit; Amersham,Cleveland, OH) and standard 8% polyacry-lamide gel electrophoresis, as indicated in theAmersham product manual.

Automated sequencing of PCR productswas performed with an ABI 377 automatedsequencer available through the Cornell Au-tomated Sequencing Facility. Overall, we se-quenced EF-1® F2 in 89 species, three ofwhich were represented by more than one lo-cality (giving a total of 92 OTUs). The regionanalyzed below corresponds to positions 196to 1266 in the coding region of the insectEF-1® gene (Danforth and Ji, 1998), meaningour data set spans 77% of the 1386 bp codingregion (Walldorf and Hovemann, 1990). As inour previous report (Danforth and Ji, 1998),we found two introns within the region ana-lyzed (at locations 753/754 and 1029/1030).

Taxon Sampling

Although it was not possible to obtain rep-resentatives of all Lasioglossum subgenera,this study includes species from all the ma-jor subgenera. Of the 18 widely recognizedsubgeneric groupings (5 of which are mono-typic), we included at least 1 member of 9 ofthese groups and have sampled extensivelywithin the 3 major North American andEuropean subgenera Dialictus, Evylaeus,

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TABLE 2. Taxa included in this study, collecting localities, specimen voucher codes, and GenBank accessionnumbers.

Voucher GenBankSpecies Locality codea accession

Outgroup taxaAugochlora pura (Say) Ithaca, New York, USA Aupu333 AF140314Augochloropsis metallica (Fabricius) Ithaca, New York, USA Aume334 AF140315Megalopta genalis Meade-Waldo Smithsonian Tropical Res. Station, Mgge247 AF140316

Republic of PanamaNeocorynura discolor (Smith) Colombia Ncdi249 AF140317Agapostemon kohliellus (Vachal) Dominican Republic Agko12 AF140318Agapostemon sericeus (Forster) Ithaca, New York, USA Agse162 AF140319Agapostemon tyleri (Cockerell) Portal, Arizona, USA Agty230 AF140320Agapostemon virescens (Fabricius) Ithaca, NY, USA Agvr161 AF140321Pseudagapostemon brasiliensis Cure Minas Gerais, Brazil Psbr347 AF140323Halictus (Halictus) farinosus Smith Logan, Utah, USA Hafa25 AF140332Halictus (Halictus) ligatus Say Rock Hill, South Carolina, USA Hali(c) AF140300Halictus (Halictus) poeyi Lepeletier Rock Hill, South Carolina, USA Hapo(d) AF140303Halictus (Halictus) rubicundus (Christ) Missoula, Montana, USA Haru32 AF140335Halictus (Seladonia) confusus Smith Junius Ponds, New York, USA Haco301 AF140304Mexalictus arizonensis Eickwort Miller Canyon, Arizona, USA Mxaz97 AF140322Sphecodes minor Robertson Sydney, Nova Scotia, Canada Spmi21 AF140324Sphecodes ranunculi Robertson Ithaca, New York, USA Spra337 AF140325

Ingroup taxaL. (Chilalictus) convexum (Smith) Cobboboonee S.F., Victoria, Australia Chcv156 AF264790L. (Chilalictus) conspicuum (Smith) Cobboboonee S.F., Victoria, Australia Chcs155 AF264789L. (Chilalictus) cognatum (Smith) Cobboboonee S.F., Victoria, Australia Chcg317 AF264788L. (Chilalictus) erythrurum (Cockerell) 6 km E. SA/WA border, S. Australia Chey308 AF264791L. (Chilalictus) �orale (Smith) 6 km E. SA/WA border, S. Australia Ch�320 AF264792L. (Chilalictus) lanarium (Smith) Cobboboonee S.F., Victoria, Australia Chla316 AF264793L. (Chilalictus) mediopolitum (Cockerell) 6 km E. SA/WA border, S. Australia Chmd291 AF264794L. (Chilalictus) mirandum (Cockerell) Bluff Knoll, Stirling Range NP, Chmi319 AF264795

W. Australia, AustraliaL. (Chilalictus) parasphecodum (Walker) 6 km E. SA/WA border, S. Australia Chps318 AF26496L. (Chilalictus) supralucens (Cockerell) Bluff Knoll, Stirling Range NP, Chsu295 AF26497

W. Australia, AustraliaL. (Dialictus) cressonii (Robertson) Ontario, Canada Dicr66 AF264801L. (“Dialictus”) �gueresi Wcislo Republic of Panama Di�341 AF264802L. (Dialictus) gundlachii (Baker) Puerto Rico Digu48 AF264803L. (Dialictus) hyalinum (Crawford) Mt. Lemmon, Arizona, USA Diha277 AF264804L. (Dialictus) imitatum (Smith) Ithaca, New York, USA Diim27 AF264805L. (Dialictus) parvum (Cresson) Puerto Rico Dipa7 AF264806L. (Dialictus) pilosum (Smith) Junius Ponds, New York, USA Dipi71 AF264807L. (Dialictus) rohweri (Ellis) Junius Ponds, New York, USA Dirh79 AF264808L. (Dialictus) tegulare (Robertson) Junius Ponds, New York, USA Ditg81 AF264809L. (Dialictus) umbripenne (Ellis) Republic of Panama Dium322 AF264810L. (Dialictus) vierecki (Crawford) Junius Ponds, New York, USA Divi67 AF264811L. (Dialictus) zephyrum (Smith) Junius Ponds, New York, USA Dizp74 AF264812L. (Evylaeus) albipes (Fabricius) Les Eyzies, Dordogne, France (social) Eval99 AF264814L. (Evylaeus) albipes (Fabricius) Longemer and Col de la Schlucht, Vosges, Eval104 AF264813

France (solitary)L. (Evylaeus) apristum (Vachal) Mt. Sanbe, Shimane Prefecture, Japan Evap145 AF264815L. (Evylaeus) boreale Svensson Inuvik, NWT, Canada Evbo262 AF264816L. (Evylaeus) calceatum (Scopoli) Les Eyzies, Dordogne, France Evca105 AF264817L. (Evylaeus) cinctipes (Provancher) Ithaca, New York, USA Evci311 AF264818L. (Evylaeus) comagenense Knerer and Atwood Sydney, Nova Scotia, Canada Evco255 AF264819L. (Evylaeus) duplex (Dalla Torre) Sendai, Miyagi Prefecture, Japan Evdu142 AF264820L. (Evylaeus) fulvicorne (Kirby) Ventoux, Vaucluse, France Evfu310 AF264821L. (Evylaeus) gattaca Danforth and Wcislo Chiriqui Province, Republic of Panama Evsp324 AF264834L. (Evylaeus) laticeps (Schenck) Les Eyzies, Dordogne, France Evla117 AF264822L. (Evylaeus) lineare (Schenck) Pont-Saint-Vincent, Meurthe et Moselle, Evli137 AF264823

FranceL. (Evylaeus) marginatum (Brulle) Les Eyzies, Dordogne, France Evmg108 AF264825L. (Evylaeus) malachurum (Kirby) Les Eyzies, Dordogne, France Evml111 AF264826L. (Evylaeus) mediterraneum (Bluthgen) Les Eyzies, Dordogne, France Evme289 AF264824

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TABLE 2. Continued

Voucher GenBankSpecies Locality codea accession

L. (Evylaeus) morio (Fabricius) Les Eyzies, Dordogne, France Evmo148 AF264827L. (Evylaeus) nigripes (Lepeletier) Beaumont du Ventoux, Vaucluse, France Evng129 AF264828L. (Evylaeus) pauxillum (Schenck) Vienna, Austria Evpa131 AF264829L. (Evylaeus) pectorale (Smith) Florida, USA Evpe10 AF264830L. (Evylaeus) politum (Schenck) Les Eyzies, Dordogne, France Evpo122 AF264831L. (Evylaeus) puncticolle (Morawitz) Les Eyzies, Dordogne, France Evpu128 AF264832L. (Evylaeus) quebecense (Crawford) No locality data Evqu325 AF264833L. (Evylaeus) subtropicum Sakagami Iriomote Is., Okinawa Prefecture, Evsu139 AF264835

JapanL. (Evylaeus) truncatum (Robertson) Ithaca, New York, USA Evtr312 AF264836L. (Evylaeus) villosulum (Kirby) Les Eyzies, Dordogne, France Evvi125 AF264837L. (Hemihalictus) lustrans (Cockerell) Bastrop, Texas, USA Helu186 AF264838L. (Homalictus) megastigmus (Cockerell) Bluff Knoll, Stirling Range NP, Homg360 AF264839

W. Australia, AustraliaL. (Homalictus) punctatus (Smith) Adelaide, S. Australia, Australia Hopu245 AF264840L. (Lasioglossum) albocinctum Lucas France Laab315 AF338386L. (Lasioglossum) callizonium (Perez) Berja, Almeria Prov., Spain Laca380 AF264841L. (Lasioglossum) coriaceum (Smith) No locality data Laco15 AF264842L. (Lasioglossum) desertum (Smith) Rose Canyon Lake, Arizona, USA Lade251 AF264843L. (Lasioglossum) discum (Smith) France Ladi313 AF264850L. (Lasioglossum) fuscipenne (Smith) Michigan, USA Lafu65 AF264844L. (Lasioglossum) laevigatum (Kirby) Les Eyzies, Dordogne, France Lala23 AF264845L. (Lasioglossum) lativentre (Schenck) Les Eyzies, Dordogne, France Lalt120 AF264848L. (Lasioglossum) leucozonium (Schrank) Les Eyzies, Dordogne, France Lale133 AF264846L. (Lasioglossum) leucozonium (Schrank) Ithaca vicinity, New York, USA Lale170 AF264847L. (Lasioglossum) majus (Nylander) France Lamj314 AF264849L. (Lasioglossum) pavonotum (Cockerell) Point Reyes Natl. Sea Shore, California, Lapa339 AF264851

USAL. (Lasioglossum) sexnotatum (Kirby) Morigny-Champigny, Essonne, France Lasx136 AF264853L. (Lasioglossum) sisymbrii (Cockerell) Chiricahua Mts., Arizona, USA Lasi253 AF264852L. (Lasioglossum) titusi (Crawford) Twentynine Palms, California, USA Lati167 AF264854L. (Lasioglossum) zonulum (Smith) Ithaca, New York, USA Lazo284 AF264855L. (Paralictus) asteris Mitchell Ithaca, New York, USA Paas30 AF264856L. (Parasphecodes) hybodinum (Cockl.) 6 km E. SA/WA border, S. Australia, Pahy299 AF264857

AustraliaL. (Parasphecodes) olgae (Rayment) Cobboboonee S.F., Victoria, Australia Ctsp153 AF264798L. (Parasphecodes) olgae (Rayment) S. Australia, Australia Ctsp397 AF264800L. (Parasphecodes) sp. Cobboboonee S.F., Victoria, Australia Pasp160 AF264858L. (Sphecodogastra) noctivagum Linsley Monahans Sand Hills, Texas, USA Stno258 AF264859

and MacSwainL. (Sphecodogastra) oenotherae (Stevens) Ithaca, New York, USA Stoe54 AF264860L. (Sudila) alphenum (Cameron) Hakgala Botanical Garden, NE District, Sual390 AF264861

Sri LankaL. (Subgen. Nov. N.) NDA(1)-A Cobboboonee S.F., Victoria, Australia Ctsp297 AF264799

aVoucher specimens and DNA extractions are housed in the Cornell University Insect Collection.

and Lasioglossum s.s., as well as 2 majorAustralian subgenera, Chilalictus (Walker,1995) and Parasphecodes. For the subgenusEvylaeus we included representatives of sev-eral species groups (as de�ned by Ebmer,1995, 1997). Among the acarinate Evylaeus(as de�ned in Ebmer, 1997) we included rep-resentatives of the morio, brevicorne, lucidu-lum/tarsatum, politum, and puncticolle speciesgroups. Among the carinate Evylaeus (as de-�ned in Ebmer, 1995) we included represen-

tatives of the calceatum, fulvicorne/fratellum ,interruptum, laticeps, malachurum, margina-tum, and pauxillum species groups. The onlylarge species groups missing from our dataset are the marginellum, punctatissimum , andtrincinctum groups. In this paper we focus onthe relationships among the Lasioglossum se-ries of subgenera. A later paper (Danforth,in prep.) will focus on the subgeneric andspecies–group relationships within the Hemi-halictus series.

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Parsimony Analysis

Phylogenetic analyses of nucleotide andamino acid sequences were performed witha beta test version of PAUP¤ (PAUP version4.0b2; Swofford, 1999). For equal weightsparsimony analyses we used heuristicsearch with TBR branch swapping, randomaddition sequence for taxa, and 50 replicatesper search. Bootstrap analysis (Felsenstein,1985) was used to evaluate branch supporton parsimony trees. Bootstrap values werecalculated based on 100 replicates with 10random sequence additions per replicate andmaxtrees set at 200.

Because our data set includes noncod-ing intron sequences, we inferred inser-tion/deletion mutations in the two includedintrons. However, the intron regions couldbe aligned with little dif�culty and the gapswere generally short (1 to 4 bp). When ana-lyzing the introns we used gap-coding meth-ods developed by Herve Sauquet and de-scribed in Danforth et al. (1999). This assignsto individual indel mutations (of whateverlength) a weight equal to a single nucleotidesubstitution while at the same time retain-ing information onsequence variation withinindels. We report below only the analysesbased on this gap-coding method, but othermethods (coding gaps as missing data andas a �fth state) gave similar results. Align-ments for both the original and the recodeddata sets are available from B.N.D.

Maximum Likelihood Analysis

For maximum likelihood (ML) analyseswe initially used the equal weights par-simony trees obtained based on the gap-coded matrix to estimate the log likelihoodof each tree under 20 distinct models of se-quence evolution (Frati et al., 1997; Huelsen-beck and Crandall, 1997; Sullivan andSwofford 1997). The four basic models wereJukes–Cantor (JC), Kimura two-parameter(K2P), Hasegawa–Kishino–Yano (HKY) andthe General Time Reversible (GTR) model(Swofford et al., 1996). Within each model wehad �ve methods of accounting for rate het-erogeneity: no rate heterogeneity, gamma-distributed rates (G), proportion of invariantsites (I), gamma C invariant sites (I C G), andsite-speci�c rates (SSR; where each codon po-sition plus introns were assigned a differentrate). Using SSR was appropriate in this case,because rate catagories could be identi�ed a

priori and because clear differences in ratesamong sites were apparent (see below).

Once likelihoods were calculated basedon equal weights parsimony trees, we per-formed branch swapping, using appropri-ate ML models with a series of increasinglyexhaustive branch swapping algorithms, inthe following order: NNI, SPR(1), SPR(2),TBR(1), and TBR(2). Before each round ofbranch swapping, the ML parameters werereestimated based on the trees currentlyin memory and applied to the next roundof branch swapping. The parameter esti-mates resulting from this search algorithmare discussed below. In all cases our branchswapping algorithms converged on thesame tree irrespective of the model selected(see below).

For the ML analyses we excluded the fol-lowing taxa to reduce search time: L. (Paras-phecodes) olgae (Ctsp153), L. (Lasioglossum)albocinctum, L. (Lasioglossum) leucozonium(Lale133), L. (Dialictus) imitatum, L. (Evylaeus)albipes (Eval104), L. (Evylaeus) comagenense,L. (Evylaeus) duplex, and L. (Sphecodogastra)oenotherae. These sequences were all verysimilar to other sequences in the data set,either because they represented additionalspecimens of the same species, or becausethey were closely related to another speciesin the data set.

RESULTS

Alignment

The 92 sequences were aligned by usingMegAlign in the Lasergene software pack-age (DNASTAR Inc., Madison, WI). Apis mel-lifera (Walldorf and Hovemann, 1990) wasincluded as a reference to determine the read-ing frame of the sequences. The region ana-lyzed consists of two introns and three exons,as judged by comparison with the Apis cod-ing sequence. Intron/exon junctions wereuniversally AG/GT or AG/GA motifs.

Together, the three exons represent 1,074bp of aligned sequence with no inser-tion/deletion (indel) mutations observed.Intron 1 (positions 559–844) includes 286aligned nucleotide sites (with 11 gap-codedcharacters), and intron 2 (positions 1121–1364) includes 244 aligned nucleotides in all(with 11 gap-coded characters). The entiredata set includes 1,604 aligned nucleotidesites plus 22 numerical characters represent-ing gap-coded variations. For the purposes

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of the analysis below we deleted two regions.First, we deleted an A/T-rich insertion (posi-tions 597–659) in intron 1 that was impossibleto align and was present in only 21 species(this proved to be a synapomorphic inser-tion; see below). Second, we deleted a 9-bpregion (positions 1542–1550) in exon 3 thatwas subject to compression on manual se-quencing gels.

Base Composition

The overall base composition and the basecomposition broken down by character par-tition is shown in Table 3. Overall, the basecomposition was only slightly A/T-biased(55%). The A/T bias was most signi�cant inintrons, where A and T accounted for 65% ofthe nucleotides. Heterogeneity in the propor-tion of bases among taxa was not signi�cant(chi-square test; Table 3).

Phylogenetic Analysis

In all analyses presented below we in-cluded 17 outgroup taxa in the following hal-ictine genera: Halictus Latreille, AgapostemonGuerin-Meneville, Pseudagapostemon Schrot-tky, Sphecodes Latreille, Mexalictus Eickwort,Augochlora Smith, Augochloropsis Cockerell,Megalopta Smith, and Neocorynura Schrottky(Table 2).

Equal weights parsimony analyses.—Figure 1shows a strict consensus tree of the 336equally parsimonious trees obtained basedon an analysis of the entire data set (exons Cintrons). Two major clades within Lasioglos-sum are evident, supporting Michener’s di-vision of the genus into the Hemihalictus andLasioglossum series (Michener, 2000: Table 1).The subgenera of the Lasioglossum series con-tained three major clades. The �rst, the basalbranch, includes species of Lasioglossum s.s.from Europe and North America, includingL. (L.) laevigatum, L. (L.) lativentre, L. (L.) sexno-tatum (European species) plus L. (L.) pavono-tum, L. (L.) fuscipenne, L. (L.) desertum, L. (L.)

TABLE 3. Base composition of EF-1® sequence data.

A C G T P-valuea

Exon 26.5 24.7 24.2 24.5 1.0nt1 28.7 18.2 38.3 14.8 1.0nt2 30.2 26.1 16.2 27.5 1.0nt3 20.7 29.8 18.1 31.3 1.0

Intron 29.5 16.0 19.0 35.5 1.0Overall 27.4 22.2 22.7 27.6 1.0

aProbability of rejecting the null hypothesis of homogeneityamong taxa in base composition.

coriaceum, L. (L.) sisymbrii, and L. (L.) titusi (allNorth American species). Most of the speciesincluded in this group have a weakly sculp-tured propodeal dorsal area that is long inrelation to the metanotum. The branch sec-ond includes L. (L.) leucozonium and L. (L.)zonulum (both of which occur in North Amer-ica and Europe) and the exclusively Palaearc-tic species, L. (L.) discum, L. (L.) callizonium,L. (L.) majus, and L. (L.) albocinctum. Thesespecies (plus L. [L.] aegyptiellum and L. [L.]subopacum) are referred to as the Lasioglos-sum leucozonium species group (see Packer,1998). The leucozonium group is united by atleast four morphological characters (Packer,1998), including (1) a patch of erect setaeon the male S6 (Packer’s character 63), (2)a �attened apical gonostylus (Packer’s char-acter 76), (3) ventral retrorse lobes of thegonostylus lacking (Packer’s character 78),and (4) relatively short and coarsely sculp-tured propodeal dorsal area in females. Sisterto the leucozonium group is the third branch,a lineage of Indoaustralian subgenera andspecies, including Parasphecodes, Homalictus,Chilalictus, and Australian species tentativelyplaced in a new subgenus (L : [Subgen.Nov. N.] NDA(1)-A; K. Walker, pers. comm.).This group will be referred to below as the“Australian clade” (Fig. 1).

Within the Hemihalictus series, relation-ships among species are reasonably wellresolved. Our EF-1® data set recovers amonophyletic subgenus Dialictus, places thesubgenera Hemihalictus and Sudila in the“acarinate Evylaeus,” and recovers mono-phyly of the Evylaeus calceatum group. Re-lationships within the Hemihalictus seriesimply that Evylaeus is paraphyletic with re-spect to several other subgenera includedin this study (including Dialictus, Hemihal-ictus, Sudila, Sphecodogastra, and Paralictus).According to the equal weights parsimonyanalysis, neither the carinate nor the acari-nate Evylaeus are monophyletic (Fig. 1).

Clades that are well supported by boot-strap values include Lasioglossum s.l. (97%),the Lasioglossum series of subgenera(95%), the Hemihalictus series of subgenera(100%), the Lasioglossum leucozonium group(100%), the leucozonium group C Australianclade (100%), and the Australian clade(76%) (Fig. 2). Interestingly, the A/T-richinsertion in intron 1 (positions 597–659)has proved to be a unique and unreversedsynapomorphy of the leucozonium group

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FIGURE 2. 50% bootstrap consensus tree based on analysis of unweighted nucleotide data; exons plus intronswith indel mutations coded as described in Danforth et al. (1999). Outgroups as in Figure 1.

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plus the Australian clade, providing strongsupport for the monophyly of this group.Bootstrap support for the Australian cladevaried from 73% to 77%, depending on howgaps were treated in the parsimony analysis.Six characters, all third position transitions,support Australian monophyly.

The EF-1® data provide strong supportfor Australian monophyly and for the inclu-sion of Homalictus within Lasioglossum (seeabove). Relationships within the Hemihalic-tus series are well resolved, and many higher-level groupings are clearly recovered by theEF-1® data, including monophyly of Dialic-tus, close relationship between Dialictus andthe acarinate Evylaeus, and clear resolutionwithin the carinate Evylaeus.

Inclusion of introns in the parsimony anal-ysis is crucial to reconstructing relationshipswithin Lasioglossum. Although exons accountfor roughly twice the number of nucleotidesites sequenced, they account for only halfof the parsimony-informative sites (Table 4).Virtually all of the variation in exons (86.4%)is in third position silent sites. As a result,the total number of parsimony-informativeamino acid changes was very small (Table 4).

Maximum likelihood analyses.—We ap-plied ML to our data for two reasons.First, rate heterogeneity among sites issubstantial. Coding sequences alone (exons)exhibit large differences among �rst, second,and third positions (with third positionsevolving an order of magnitude fasterthan second positions). The inclusion ofnoncoding introns provides an additionalsource of rate heterogeneity in that theintrons evolve considerably faster than doexons overall. Second, there is clear evidenceof transition/transversion bias. Depend-ing on the model of sequence evolutionselected, transitions occur at a rate 3.67 to4.12 times that of transversions, indicatingthat transformations of character stateswithin positions are not all equally probable.

TABLE 4. Composition of introns and exons.

Parsimony- Parsimony-Total Const. uninformative informative

Exons 1,074 731 63 280nt1 358 318 14 26nt2 358 335 11 12nt3 358 78 38 242

Intronsa 489 168 60 261Amino acids 358 314 19 25

aBased on gap-coded data set.

FIGURE 3. ¡ln likelihoods based on the equalweights parsimony trees for 20 models of sequenceevolution. Likelihoods improved slightly with branchswapping, as described in Results. SSR, site-speci�crates for introns and �rst, second, and third positions.The arrow indicates the model used for tree searching.

As expected, the log likelihood val-ues increased with increasingly complexmodels (Fig. 3). Allowing for variable transi-tion/transversion ratios and accounting forrate heterogeneity among sites improved thelikelihood scores considerably; however, in-cluding empirical base frequencies (HKY) asopposed to equal base frequencies (K2P) didnot improve the likelihood score as judgedby the likelihood ratio test (¡2 ln 3 D ¡9.42,df D 3, not signi�cant; Huelsenbeck andCrandall, 1997). We chose to use the K2Pmodel with SSR because this was the sim-plest model that substantially improved thelikelihood scores, and because this modelused shorter search times on the Power MacG3 computer for the ML analysis.

Branch swapping led to only slight in-creases in ¡ln likelihood (from 15370.82 to15361.75), indicating that the parsimony treescome very close to the tree topologies esti-mated under ML. In an analysis of the en-tire data set (exons and introns) we obtainedone tree (¡ln likelihood D 15361.75; Fig. 4).We also performed branch swapping undermore complex models (e.g., HKY C SSR andGTR C SSR). In either case we obtained thesame �nal tree topology as obtained withthe simpler model (K2P C SSR). Estimatesof the relative rate of substitution indicatedthird positions evolve at roughly the samerate as introns, and both introns and third po-sitions evolve roughly an order of magnitudefaster than either �rst or second positions:introns, 1.64; nucteotide1, 0.17; nucteotide2,0.06; nucteotide3, 1.93 (based on the K2P CSSR model).

The tree topology obtained by using like-lihood (Fig. 4) recovers many of the samehigher nodes as the consensus of equally

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FIGURE 4. Maximum likelihood analysis based on the K2P C SSR model. ¡Ln likelihood D 15361.75. Branchlengths shown are proportional to character changes.

parsimonious trees (Fig. 1) and the 50%bootstrap consensus tree (Fig. 2). Using ML,we recovered monophyletic Hemihalictusand Lasioglossum series, a monophyletic leu-cozonium group, a monophyletic Australianclade, and a sister group relationshipbetween the leucozonium group and theAustralian clade.

DISCUSSION

Phylogenetic Results

Although we were unable to include repre-sentatives of all the Australian subgenera, wethink it likely that the Australian subgeneranot included (Callalictus, Pseudochilalictus,and Australictus) are closely related to those

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that were included in our analysis. Species ofAustralictus and Callalictus are similar mor-phologically to species of Parasphecodes. Therelationship of Pseudochilalictus (a monotypicsubgenus) to the other subgenera is not clear,but Pseudochilalictus may be closely related toParasphecodes (possibly rendering Parasphe-codes paraphyletic; K. Walker, pers. comm.)

The results presented above providestrong and unambiguous support for mono-phyly of Homalictus plus the Australian La-sioglossum, irrespective of the data partitionsanalyzed, the methods used for coding gaps,or the methods of analysis (parsimony vs.likelihood). This hypothesis is novel, but itis not incompatible with any morphologicalcharacters.

Biogeographic Implications

The sister group relationship implied bythese data between the Lasioglossum leucozo-nium group and the Australian clade makessense biogeographically. The subgenus La-sioglossum is widespread across the Palaearc-tic from Western Europe to Japan and south-ward to Southeast Asia. The leucozoniumgroup is also widespread across the Palearc-tic region. The genus Lasioglossum (like Hal-ictus, a closely related genus) is primarily aNorthern Hemisphere group. The Australianclade represents the only major radiation ofLasioglossum in the Southern Hemisphere.

The presence of species of Homalictus out-side of Australia has probably resulted fromdispersal from Australia, rather than the re-verse, as suggested by Michener (1979a),given that the majority of species are Aus-tralian endemics.

The results presented here for Australianhalictine bees parallel the results for birdhigher-level relationships as determined byDNA–DNA hybridization studies (Sibleyand Ahlquist, 1985, 1990; Sibley et al., 1988).The major lineage of passerine birds of theworld (the oscines, Suborder Passeres) iscomposed of two large, monophyletic, sis-ter clades: the Parvorder Corvida and theParvorder Passerida. These two lineages areestimated to have diverged in the Eoceneor Oligocene, according to molecular clockestimates from DNA–DNA hybridization(Sibley and Ahlquist, 1990). The three ma-jor superfamilies within the Corvida includethe Menuroidea (31 spp.), the Meliphagoidea(276 spp.), and the Corvoidea (794 spp.).

Relationships implied by the DNA hy-bridization studies place the Meliphagoideaand Corvoidea as sister groups (Sibley,et al., 1988). Within the Parvorder Passeridaare three recognized superfamilies: Mus-cicapoidea (610 spp.), Sylvioidea (1,195spp.), and Passeroidea (1,651 spp.), and theSylvioidea and Passeroidea are sister groups.Considering the zoogeographic distributionsof these groups, virtually all of the fam-ilies within the Parvorder Corvida clearlyare endemic to Australia or share a com-mon ancestor that was originally Austro-Papuan. Two of the corvid superfamiliesare exclusively Australian (Menuroidea andMeliphagoidea), and the majority of familieswithin the Corvoidea are Austro-Papuan en-demics. Derived members of the ParvorderCorvida have dispersed from Australia toother parts of the world, including Eurasia,North America, and South America. Groupsthat have dispersed from Australia or thathave been derived from Australian ances-tors include the Families Irenidae, Laniidae,Vireonidae, and three subfamilies withinthe family Corvidae (Corvinae, Aegithini-nae, and Malaconotinae).

Although representatives of the ParvorderPasserida are found in Australia, theseare recent colonists from groups with ori-gins in Eurasia and Africa (Sibley andAhlquist, 1990). Because of the distinctionbetween the two Parvorders, Sibley andAhlquist distinguished between the “oldendemics” (including Australian membersof the Parvorder Corvida) and the “newendemics” (including the Australian mem-bers of the Parvorder Passerida). Of the 700species of Austro-Papuan passerines, 400(57%) are “old endemics.” The recognitionof Australian endemism in the Corvida re-solved many problems in bird phylogeny be-cause convergent evolution among membersof the two Parvorders had in many cases ob-scured the true phylogenetic af�nities.

Our results for halictine bees parallel thoseof Sibley and Ahlquist for birds. A majorradiation within Australia has given riseto an endemic fauna (400 species in thepasserine birds and >350 species of halic-tine bees) that shows convergent featureswith relatives from other parts of the world.As with Australian passerines, halictine beesthat originated in Australia have given riseto descendants now present in neighbor-ing regions, including Sri Lanka, Southeast

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Asia, New Guinea, the Philippines (Homa-lictus), and Samoa (Echthralictus). That theAustralian Lasioglossum C Homalictus forma monophyletic group helps resolve manyquestions in halictid bee social evolutionand biogeography. Other major Australianradiations include the marsupial mammals(Archer, 1981) and the plant family Myr-taceae (Beadle, 1981).

Evidence of Australian monophyly amongLasioglossum subgenera also helps resolvethe “Australian enigma” posed by Knererand Schwarz (1976). Similarity among theAustralian Lasioglossum in �ower associa-tions, nest architecture, and sociality (withmost Australian Lasioglossum being commu-nal rather than eusocial) probably re�ectscommon ancestry rather than convergentevolution. Ecological factors such as mutil-lid parasitism and ant predation may havefavored communal associations among nest-mates in the early Australian colonists.

ACKNOWLEDGMENTS

We are grateful to the following collaborators for pro-viding specimens for this and future studies of halic-tid relationships: John Ascher, Manfred Ayasse, Con-nal D. Eardley, Michael Engel, Terry Griswold, Pene-lope F. Kukuk, Pat Lincoln, Yasuo Maeta, RyouichiMiyanaga, Robert Minckley, John L. Neff, Beth Nor-den, Alain Pauly, Robert Paxton, Tai Roulston, Jerome G.Rozen, Jr., Michael Schwarz, Roy R. Snelling, William T.Wcislo, and Douglas Yanega. We are particularly grate-ful to Stephan Reyes, Laurence Packer, Cecile Plateaux-Quenu, Kenneth Walker, and Andreas W. Ebmer for pro-viding identi�cations and crucial taxa and for their in-terest in this work. The following people commented onearly drafts of this paper: John Ascher, Karl Magnacca,and Charles Michener, and two reviewers provided nu-merous suggestions that substantially improved the pa-per. This project was supported by a National ScienceFoundation Research Grant in Systematic Biology (DEB-9508647) with travel funds provided by the Cornell In-ternational Agriculture Program.

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Received 9 November 1999; accepted 29 March 1999Associate Editor: R. Leschen

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Australian Lasioglossum + Homalictus Form a Monophyletic ... · form a monophyletic group. Bootstrap support for the Australian clade ranged from 73% to 77%, depending on the method