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Home , Aculeata, Ampulicidae, Apoidea, Bembicinae, Bembicini, Crabronidae

Zoologica Scripta

Identifying the sister group to the : a molecular phylogeny of with an emphasis on the superfamily

ANDREW H. DEBEVEC,SOPHIE CARDINAL &BRYAN N. DANFORTH

Submitted: 16 January 2012 Debevec, AH., Cardinal, S & Danforth, BN. (2012). Identifying the sister group to the Accepted: 20 April 2012 bees: a molecular phylogeny of Aculeata with an emphasis on the superfamily Apoidea. doi:10.1111/j.1463-6409.2012.00549.x —Zoologica Scripta, 41, 527–535. The hymenopteran superfamily Apoidea includes the bees (Anthophila) as well as four predatory families (Heterogynaidae, , and ) collec- tively referred to as the ‘‘sphecoid’’ or ‘‘apoid’’ . The most widely cited studies sug- gest that bees are sister to the wasp family Crabronidae, but alternative hypotheses have been proposed based on both morphological and molecular data. We combined DNA sequence data from previously published studies and newly generated data for four nuclear genes (28S, long-wavelength rhodopsin, elongation factor-1a and wingless) to identify the likely sister group to the bees. Analysis of our four-gene data set by maximum likelihood and Bayesian methods indicates that bees most likely arise from within a paraphyletic Crabronidae. Possible sister groups to the bees include , or Philanthinae + Pemphredoninae. We used Bayesian methods to explore the robustness of our results. Bayes Factor tests strongly rejected the hypotheses of crabronid as well as placement of Heterogynaidae within Crabronidae. Our results were also stable to alternative rootings of the bees. These findings provide additional support for the hypothe- sis that bees arise from within Crabronidae, rather than being sister to Crabronidae, thus altering our understanding of ancestry and evolutionary history. Corresponding author: Andrew H. Debevec, 320 Morrill Hall, 505 S. Goodwin Ave, Urbana, IL 61801, USA. E-mail: [email protected] and Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL Sophie Cardinal, Canadian National Collection of , Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada. E-mail: [email protected] Bryan N. Danforth Department of Entomology, Cornell University, Ithaca, NY, USA. E-mail: [email protected]

Introduction evolution that remains unclear is identification of the sister Bees (Anthophila) are currently one of the most important group to the bees within the ‘‘spheciform’’ (or ‘‘apoid’’) lineages of pollinators on earth. Bees appear to have wasps. Correctly identifying the sister group to the bees arisen near, or shortly after, the first appearance of has important implications for understanding early bee flowering plants in the fossil record, approximately 140– evolution as well as estimating the antiquity of bees based 110 myBP (Grimaldi 1999; Grimaldi & Engel 2005; on the apoid fossil record. Michener 2007). Bees are thought to have played an Bees and spheciform wasps have long been regarded as important role in the diversification of the angiosperms in forming a monophyletic group (Comstock 1924). Modern, the early to mid- (Grimaldi 1999). A number of character-based, phylogenetic studies (Lomholdt 1982; recent molecular and combined molecular and morpholog- Alexander 1992; Prentice 1998; Melo 1999; Ohl & Bleid- ical studies have provided a solid basis for understanding orn 2006; Lohrmann et al. 2008) have corroborated this relationships at the subfamily and the family level within hypothesis and establish unambiguously that bees arose bees (Danforth et al. 2006a,b; Almeida & Danforth 2009; from within a paraphyletic group of predatory wasps Michez et al. 2009; Cardinal et al. 2010; Brady et al. 2011; variously referred to as the ‘‘Sphecidae’’ (Bohart & Litman et al. 2011). However, one important aspect of bee Menke 1976), the ‘‘sphecoid wasps’’ (Melo 1999) or the

ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 527 Phylogeny of Aculeata d A. H. Debevec et al.

‘‘’’ (Michener 2007). The monophyletic the bees was Pemphredoninae (‘‘aphid wasps’’), based on group including the bees and the wasp families Heterogy- similarities in nesting , as well as other features. naidae, Ampulicidae, Sphecidae and Crabronidae is Recent molecular studies based on limited sam- referred to as Apoidea (Prentice 1998; Melo 1999; Mich- pling have provided additional support for the hypothesis ener 2007). Monophyly of Apoidea is supported by a num- that Crabronidae is not monophyletic and that bees may ber of morphological characters, including (i) the rounded arise from within Crabronidae. Ohl & Bleidorn (2006) pronotal lobe well separated from the tegula, (ii) ventral analysed apoid wasp and bee relationships based on a sin- extension of the pronotum to encircle or nearly encircle gle, nuclear gene, long-wavelength rhodopsin. Outgroups the thorax behind the front coxa and (iii) enlargement of included (Chrysididae) and the metapostnotum (propodeal triangle) (Brothers 1975, (Pompilidae, , and ), and the 1976; Michener 2007). ingroup included 10 of apoid wasps (including all While bees clearly belong within the Apoidea based four recognized families) and eight species of bees (includ- both on morphological (Lomholdt 1982; Alexander 1992; ing six of the seven extant bee families). In both ML and Prentice 1998; Melo 1999 [but see Lanham 1980]) and on Bayesian analyses, bees were nested within a paraphyletic molecular (Ohl & Bleidorn 2006; Lohrmann et al. 2008; Crabronidae (including Heterogynaidae). However, the Pilgrim et al. 2008) data, the exact placement of bees is limited number of crabronid exemplars makes it difficult controversial. Lomholdt (1982), Prentice (1998) and Melo to determine the exact sister group to the bees. (1999) obtained morphological support for bees + - Lohrmann et al. 2008 expanded on the Ohl & Bleidorn ronidae, and this hypothesis has been the most widely (2006) data set by adding more taxa and an additional cited in the apoid wasp (and bee) literature (e.g. Michener mitochondrial gene (COI). Their results support 2007; Fig. 1). Monophyly of Crabronidae is viewed as of Crabronidae (including Heterogynaidae) and suggest a strongly supported by a number of authors (Lomholdt sister group relationship between bees and subfamily Phil- 1982; Melo 1999; Michener 2007) because all Crabronidae anthinae (as suggested by Alexander 1992; see below). possess double-salivary openings in the larvae, which is Unfortunately, their study did not include Pemphredoni- shared neither with bees nor with the related apoid wasp nae, which has also been hypothesized to be the sister families (Michener 2007). Three additional characters sup- group to the bees (Malyshev 1968). Pilgrim et al. (2008), porting crabronid monophyly were listed by Melo (1999; based on the analysis of four nuclear genes elongation fac- p. 37) based on his analysis. However, a number of stud- tor 1-a, F2 copy [EF-1a], long-wavelength rhodopsin ies, including those based on morphology (Alexander [opsin], wingless and 28S) and extensive taxon sampling 1992) and molecular data (Ohl & Bleidorn 2006), have across Vespoidea, but limited sampling within Apoidea, suggested that Crabronidae may not be monophyletic and obtained varying results depending on the data set and that bees may have arisen from within Crabronidae. Like- method of analysis: (i) bees sister to Crabronidae (molecu- wise, Malyshev (1968) proposed that the sister group to lar data analysed by parsimony), (ii) bees sister to Crabron-

A Alexander 1992 B Prentice 1998; Melo 1999 Ampulicidae Ampulicidae

Sphecidae Heterogynaidae

Heterogynaidae Sphecidae

Crabronidae Crabronidae

Anthophila Anthophila

C Melo 1999 D Ohl and Bleidorn 2006 Ampulicidae Sphecidae (incl. Ampulicidae)

Heterogynaidae

Sphecidae Heterogynaidae

Crabronidae Philanthinae Fig. 1 —A–D, Previous phylogenies of Anthophila Anthophila Apoidea.

528 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 A. H. Debevec et al. d Phylogeny of Aculeata idae + Sphecidae (combined molecular and morphological Dolichurinae (Ampulicidae; 69 spp.), Dinetinae (Crabroni- data analysed by both parsimony and Bayesian methods) dae; 12 spp.), Eremiaspheciinae (Crabronidae; 18 spp.) and (iii) bees nested within a paraphyletic Crabronidae and Mellinae (Crabronidae; 18 spp.) (Pulawski 2010). All (molecular data analysed by Bayesian methods). It is difficult families and subfamilies of bees were represented in the to draw conclusions regarding apoid relationships from the data set. Pilgrim et al. (2008) study because their data set did not include sufficient numbers of apoid wasps (or bees) to confi- Sequence data dently establish the sister group to the bees, nor did they We obtained sequence data from four different fragments include Ampulicidae and Heterogynaidae, two of the four including ribosomal 28S (1200 bp) and three protein- recognized families of apoid wasps. In summary, no previ- coding nuclear genes: opsin (600 bp), EF-1a F2 copy ous studies utilizing molecular or morphological and molec- (1100 bp) and wingless (500 bp). Most sequences were ular data have had adequate taxonomic sampling to firmly obtained from GenBank, and newly generated sequences establish the placement of bees within Apoidea, or relation- were produced following standard protocols described in ships between the major apoid wasp lineages. Danforth et al. (2006b). PCR conditions and primer pairs We analysed relationships among the major lineages of used are listed in Table S4. Aculeata (including Chrysidoidea, used as outgroups, Ves- poidea and Apoidea) using a comprehensive sample of taxa Alignment and the same four nuclear genes used by Pilgrim et al. 28S ribosomal RNA was aligned through comparison with (2008): EF-1a (F2 copy), opsin, wingless and 28S. We a secondary structure model for the , Apis mellif- combined data from previous studies of Aculeata (Pilgrim era (Gillespie et al. 2006). Scripts written by the first et al. 2008), Apoidea (Ohl & Bleidorn 2006), (Brady author were used to verify that the number of non-canoni- et al. 2006) and bees (Danforth et al. 2006b; Cardinal et al. cal base pairings obtained was as expected. Regions of 2010), as well as new sequences obtained for this study. ambiguous alignment and regions of expansion and con- Our new taxa were mostly from the apoid wasp families traction were discarded. The protein-coding genes (EF- Ampulicidae, Heterogynaidae, Sphecidae and Crabronidae. 1a, opsin and wingless) were aligned using the default Because some previous studies had obtained evidence of settings in MUSCLE v.3.8 (Edgar 2004) and adjusted by crabronid paraphyly with respect to the bees, we included eye to maintain indels in units of three base pairs. Introns as many crabronid subfamilies and tribes as possible to were discarded from EF-1a and opsin; wingless had no fully resolve the placement of bees with respect to the introns. The alignment used is publically available on crabronid tribes and subfamilies. Our goal was to identify TreeBase (http://purl.org/phylo/treebase/phylows/study/ the sister group to the bees, but our data set also allows us TB2:S12543). to evaluate relationships within Apoidea (e.g. placement of Ampulicidae and Heterogynaidae) and the likely vespoid Phylogenetic analyses sister group to Apoidea as a whole. In all analyses, the protein-coding data were partitioned by codon position, with 28S having its own partition (four Materials and methods partitions in total). On the basis of the results of model Taxon sampling testing performed in JModelTest v.0.1.1 (Guindon & All taxa from the Pilgrim et al. (2008), Danforth et al. Gascuel 2003; Posada 2008), a GTR+I+G model was (2006a,b) and Ohl & Bleidorn (2006) studies were applied to each partition. included, as well as some taxa from Cardinal et al. (2010) A maximum likelihood analysis was performed using and Brady et al. (2006) (Table S1). One taxon, Hedychridi- RAxML v.7.0.3 (Stamatakis 2006a). 3000 rapid bootstrap um sp. (Chrysididae), from Pilgrim et al. (2008) was replicates were performed using GTR+CAT, which excluded as it had an extremely long branch and was approximates a GTR+I+G model (Stamatakis 2006b). impossible to confidently align with other sequences Bayesian analyses were performed using Parallel Mr (Table S2). An additional 19 species of previously unse- Bayes v. 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & quenced apoid wasps were also included (Table S3). In Huelsenbeck 2003; Altekar et al. 2004). Four runs of total, the data set includes five Chrysidoidea (as out- 10 million generations using four chains each were per- groups), 88 Vespoidea, 50 apoid wasps and 88 bees. All formed. Convergence was verified using Tracer v. 1.5 four families of apoid wasps (Heterogynaidae, Ampulici- (Rambaut & Drummond 2007), and burnin was assessed dae, Sphecidae and Crabronidae), 11 of the 15 subfamilies based on when stationarity was reached for each run. Log- and 18 of the 35 tribes are represented in the data set Combiner v. 1.6.1 and TreeAnnotator v. 1.6.1, part of the (Pulawski 2010). The four subfamilies not included were BEAST package (Drummond & Rambaut 2007), were

ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 529 Phylogeny of Aculeata d A. H. Debevec et al. used to combine the runs and summarize the results using is sister to a monophyletic group consisting of Sphecidae a maximum credibility tree. All of the Bayesian anal- s.s., Crabronidae and Anthophila, whereas in the ML tree, yses were performed on the Cornell Biological Services it is nested within Crabronidae. In both the Bayesian and Unit BioHPC computing cluster from Cornell University the ML trees, Crabronidae is paraphyletic. Many of these (http://cbsu.tc.cornell.edu/). nodes are weakly supported, but the BF test comparing the To further explore the level of support found in our unconstrained analysis to the analysis in which Crabroni- data set for (i) the paraphyly of Crabronidae, (ii) the phy- dae was constrained to be monophyletic strongly supports logenetic placement of Heterogynaidae and (iii) the sister a paraphyletic Crabronidae with a BF score of 59.28. We group to bees, three different constrained analyses were also did a BF test in which we included Heterogynaidae run. In the first constrained analysis, Crabronidae was within Crabronidae and once again found strong support forced to be monophyletic. In the second, Crabroni- for a paraphyletic Crabronidae with a BF score of 101.88. dae + Heterogynaidae was forced to be monophyletic. In Crabroninae, one of the crabronid subfamilies, formed the the third analysis, we constrained the family-level relation- monophyletic sister group to Sphecidae s.s. (PP = 1, ships within bees to be congruent with those found in B = 59). is also recovered as paraphyletic, with Danforth et al. (2006b). We then compared the log har- tribe recovered as sister to (PP = 0.98, monic mean (as calculated by the sump command in B = 56). Pemphredoninae + Philanthinae (PP = 0.74, MrBayes) of each constrained analysis to that of the B = 36) is recovered as the sister group to Anthophila unconstrained analysis using a Bayes Factor (BF) test (Kass (PP = 1, B = 63). However, because the family-level rela- & Raftery 1995), where the BF test statistic was calculated tionships within bees differ from those previously sug- as being twice the difference between the harmonic mean gested based on larger data sets, we also ran an analysis in of the posterior sample of likelihoods from the uncon- which the bee topology was constrained to match that of strained and constrained analyses. A BF test statistic of 10 Danforth et al. (2006b): ( + (( + Megachi- or higher has been suggested as a cut-off point for indicat- lidae) + ( + ( + ( + Collet- ing significant support for one model over another (Kass idae))))). The BF score comparing this constrained analysis & Raftery 1995). to the unconstrained one did not strongly support one topology over the other (BF score of )5.24). The sister Results group to bees, however, did change when the bees were The tree with the highest likelihood score from the maxi- constrained to the Danforth et al. (2006b) topology. In this mum likelihood analysis was mostly congruent with the analysis, we recovered Philanthinae as sister to bees Bayesian maximum clade credibility tree (Fig. 2). The level (PP = 0.41) as did Alexander (1992) and Prentice (1998) in of support varied throughout the tree with some nodes some of their morphological analyses. being highly supported by both posterior probability (PP) and bootstrap (B) values, and others having low support. Discussion Nodes with high bootstrap values always had high PP val- Vespoid paraphyly ues, whereas some nodes with high PP values had low Our results largely corroborate the results of Pilgrim et al. bootstrap support. Our results support the conclusions of (2008) in supporting a paraphyletic Vespoidea, with a Pilgrim et al. (2008) regarding the paraphyly of Vespoidea. monophyletic Apoidea arising from within Vespoidea. Our We recover four independent vespoid lineages. The first data set provides significantly expanded taxon sampling for clade consists of and (PP = 1, Apoidea and Formicidae and provides stronger support for B = 91). The second clade, which contains most of the monophyly of certain groups, including the clade consist- non- Vespoidea, is weakly supported (PP = 0.75, ing of Formicidae, Scoliidae, s.s. and B = 55), while the third clade, which consists of , Apoidea. The congruence between our results and the is highly supported (PP = 1, B = 99). The fourth clade, Pilgrim et al. (2008) results would support the hypothesis composed of the families Bradynobaenidae sensu stricto (s.s.) that paraphyly of Vespoidea is not an artefact of limited or and Scoliidae (PP = 0.98, B = 76), is the sister group to biased taxon sampling. These results also provide addi- Apoidea (PP = 1, B = 94). tional support for the superfamily classification proposed Our results also support a monophyletic group of Form- by Pilgrim et al. (2008). icidae, Scoliidae, Bradynobaenidae sensu stricto (Bradynoba- eninae and Apterogyninae) and Apoidea (PP = 1, B = 78). The sister group to Apoidea Apoidea is found to be monophyletic (PP = 1, B = 98) with Previous morphological studies of aculeate wasp and bee Ampulicidae being recovered as sister to the rest of Apoi- relationships (Brothers 1975, 1999; Brothers & Carpenter dea (PP = 1, B = 63). In the Bayesian tree, Heterogynaidae 1993) have all presented trees in which Apoidea and

530 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 A. H. Debevec et al. d Phylogeny of Aculeata

1 0.74 100 [2] 1 46 0.37 100 Plumariidae [2] - Chrysididae [1] 1 1 100 Vespidae [5] 91 1 100 Rhopalosomatidae [2] 1 0.84 99 Chyphotinae (Bradynobaenidae) [5] 71 1 99 Typhoctinae (Bradynobaenidae) [3] 0.76 63 Methochinae (Tiphiidae) [1] 1 1 0.96 75 Myzininae (Tiphiidae) [2] 76 1 43 0.65 100 Thynninae (Tiphiidae) [4] 0.98 - Anthoboscinae in part (Tiphiidae) [1] 0.74 0.75 66 - 25 46 0.94 42 Anthoboscinae* (Tiphiidae) [1] 24 Diamminae (Tiphiidae) [1] 1 0.70 100 Pompilidae [9] 61 1 0.75 0.99 100 Mutillidae (excluding Myrmosinae) [7] 55 45 1 1 100 [3] 70 1 100 Myrmosinae (Mutillidae) [2] 1 1 100 Tiphiinae (Tiphiidae) [3] 100 1 0.75 0.97 54 100 Brachycistidinae (Tiphiidae) [3] 48 [1] 1 99 Formicidae [25] Bradynobaenidae s.s. 1 1 100 Bradynobaeninae [2] 100 1 1 0.98 100 Apterogyninae [2] 78 76 0.86 Proscoliinae (Scoliidae) [1] 78 1 100 Scoliinae (Scoliidae) [5] 1 1 94 100 Ampulicidae [2] 1 100 Heterogynaidae [2] 1 0.97 [3] 98 1 73 97 1 1 100 Chloriontinae [3] 1 100 1 Apoidea 100 Sphecini () [4] - 1 1 1 97 0.99 96 Prionychini (Sphecinae) [2] 59 79 1 100 [4] 1 0.33 100 Crabroninae [9] 63 1 61 (Bembicinae) [9] Bayes 1 0.58 0.98 100 Nyssonini (Bembicinae) [2] ML 19 - 56 Astatinae [1] 23 1 Heterogynaidae [2] 0.61 100 0.96 Chrysidoidea 9 0.74 52 Pemphredoninae [2] 36 1 97 Philanthinae [7] Vespoidea 1 1 63 - 79 Melittidae s.l. [15] 39 1 Ampulicidae 100 Andrenidae [9] 1 1 s.s. 100 100 Andrenidae [9] Sphecidae 1 0.88 1 100 [25] Heterogynaidae Anthophila - 1 100 Stenotritidae [1] 99 1 0.88 100 Halictidae [12] Crabronidae 65 1 1 100 [5] 95 1 Anthophila 94 Apidae [21] 0.07

Fig. 2 Maximum clade credibility tree recovered tree from Bayesian analysis with alternative relationships based on maximum likelihood analysis of the combined molecular data set collapsed at the family or subfamily level. Numbers above nodes represent posterior probabilities from Bayesian analysis; numbers below nodes represent maximum likelihood bootstrap values (from 3000 pseudoreplicates). See Figs S1 and S2 for full results. *Anthoboscinae (Tiphiidae) was recovered as paraphyletic in the Bayesian analysis but monophyletic in the ML analysis. The position marked with an asterisk is where one taxon was recovered in the Bayesian analysis, but where both taxa were placed in the ML analysis.

Vespoidea are each recovered as reciprocally monophyletic 1999), eight morphological characters (chars. 40, 48, 56, sister groups. In the most recent analysis of aculeate 73, 107, 187, 194) were found to support vespoid mono- family-level relationships based on morphology (Brothers phyly, but none of these eight characters was a unique and

ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 531 Phylogeny of Aculeata d A. H. Debevec et al. unreversed synapomorphy. One character that is widely suggests that the many characters previously considered cited as synapomorphic for Vespoidea is the reduction in plesiomorphic are in fact derived characters, as previously the prepectus, although the exact nature of the reduction suggested based on molecular evidence (Ohl & Bleidorn varies within Vespoidea (e.g. Brothers & Carpenter 1993; 2006). see Pilgrim et al. 2008). Previous morphological studies of One of our most significant and well-supported results aculeate relationships (e.g. Brothers 1975, 1999; Brothers is that Crabronidae is not monophyletic and that bees & Carpenter 1993) have generally assumed vespoid mono- arise from within Crabronidae. Our Bayes Factor tests, in phyly, in spite of the fact that there is limited morphologi- which we constrained Crabronidae to be monophyletic cal support for the group (Gauld & Bolton 1988; (both with and without Heterogynaidae), were highly sig- Ronquist 1999; Pilgrim et al. 2008). nificant, indicating that our data are significantly incon- While a recent molecular study of Aculeata (Pilgrim gruent with the hypothesis of crabronid monophyly. In et al. 2008) obtained results suggesting that Apoidea arises addition, the placement of bees sister to Pemphredoni- from within Vespoidea, the study failed to provide a clear nae + Philanthinae is supported by a posterior probability indication of the likely sister group to Apoidea. Pilgrim of 1.0 and ML bootstrap of 63. et al. (2008) obtained variable placement of Apoidea The hypothesis that bees arise from within Crabronidae depending on their methods of analysis (parsimony, maxi- is not a new idea. In 1992, Byron Alexander published a mum likelihood and Bayesian) and whether they included remarkably prescient paper on apoid phylogeny that fore- morphological data in the analysis. Using Bayesian meth- shadowed many of the conclusions that we have obtained. ods on the molecular data alone, they obtained results His analysis of 86 larval and adult morphological charac- similar to those presented earlier: Apoidea sister to Scolii- ters revealed that the family Crabronidae is paraphyletic dae + Bradynobaenidae s.s. However, inclusion of morpho- with respect to the bees. In other words, the double-sali- logical data yielded different results (Apoidea as sister to vary openings in the larvae of Crabronidae, while viewed Scoliidae, Bradynobaenidae s.s., Formicidae, Vespidae and as strongly supporting crabronid monophyly by some Rhopalosomatidae). Based on our data set, the clade con- authors (Prentice 1998; Melo 1999; Michener 2007), may taining Apoidea, Scoliidae and Bradynobaenidae is well be homoplasious when analysed along with other morpho- supported (PP = 1; B = 94; Fig. 2). Future phylogenetic logical characters. Alexander analysed his data set in a studies of Apoidea may consider including representatives variety of ways (see his Table 4). While all analyses sug- of Scoliidae and Bradynobaenidae s.s. as outgroups for any gested Crabronidae is paraphyletic with respect to the analysis. These results may also provide information for bees, the analyses differ widely in the likely sister group to fossil-calibrated dating studies as the earliest appearance of the bees. The following possibilities were obtained by Scoliidae and ⁄ or Bradynobaenidae s.s. may help estimate Alexander: (i) bees sister to Philanthinae (Analyses 1, 5, 6), the age of the Apoidea. (ii) bees arising from within a paraphyletic Philanthinae (Analysis 2), (iii) bees sister to Laphyragonini (Ere- Relationships between the families of Apoidea miaspheciinae) (Analyses 3, 7) and (iv) bees sister to Xeno- Previous studies of apoid relationships based on morpho- sphecini (Mellininae) (Analyses 4, 8). The analysis that logical (Alexander 1992; Prentice 1998; Melo 1999) and comes closest to the one we have obtained is his Analysis molecular data (Ohl & Bleidorn 2006) have provided a 5 in which bees are sister to Philanthinae, with Pemphre- variety of alternative topologies (Fig. 1). While the mono- doninae (including and Pemphredonini) closely phyly of Apoidea is not in doubt, relationships between related to this group, but paraphyletic. the component wasp families (Ampulicidae, Heterognynai- In Michael Prentice’s unpublished PhD thesis (1998; dae, Sphecidae and Crabronidae) and bees have never been verified by Hanson & Menke 2006), many of the same clearly resolved. Placement of Heterogynaidae is particu- relationships as presented in Alexander were recovered. In larly problematic (reviewed in Ohl & Bleidorn 2006), as is fact, a clade of Philanthinae and Anthophila was suggested the monophyly of Crabonidae and the exact placement of in a majority of the trees presented. A close association of bees. Our results strongly support the placement of Ampu- Anthophila with Eremiaspheciini (Eremiaspheciinae) was licidae as sister to the remaining Apoidea (as in Melo also recovered often, as well as an association of Antho- 1999), but our results are not particularly clear about the phila, Eremiaspheciini, Pemphredoninae and Philanthinae. placement of Heterogynaidae. Our Bayesian results place As there is currently no molecular data available for Ere- Heterogynaidae as sister to the monophyletic group miaspheciinae, a subfamily consisting of two tribes, each including Crabronidae, Sphecidae and bees, while our ML with one , and only 18 described species, this could results place Heterogynaidae within a paraphyletic not be investigated in the current study. Likewise, no Crabronidae (Fig. 2). The placement of Heterogynaidae molecular data for subfamily Mellininae, which contains

532 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 5, September 2012, pp 527–535 A. H. Debevec et al. d Phylogeny of Aculeata tribe Xenosphecini and was recovered in close association molecular evidence, including the Bayes Factor test with bees by Alexander (1992), are available. This subfam- rejecting crabronid monophyly, as increasingly strong sup- ily contains only two tribes, each with one genus, and only port for the hypothesis of crabronid paraphyly with 18 described species. respect to the bees. The most likely sister group(s) to the In several analyses performed by Prentice (1998), a rela- bees are Pemphredoninae, Philanthinae, or Pemphredoni- tionship of Pemphredoninae, Philanthinae and bees was nae + Philanthinae (Fig. 3). suggested. However, this result was not discussed. Alexan- der (1992) also recovered this relationship, but in only a Acknowledgements few analyses and with a paraphyletic Pemphredoninae. We are very grateful to Dr Wojciech Pulawski for his The low support given to the monophyletic group of assistance with apoid wasp identifications and for making Philanthinae and Pemphredoninae recovered in this study his catalogue of the apoid wasps available. Michael Ohl (PP = 0.74; B = 36) suggests an unresolved clade of Pem- generously provided specimens of nocticola for phredoninae, Philanthinae and Anthophila. One morpho- our analysis and Susanne Schulmeister kindly provided logical character supporting the close affiliation of specimens of compressa. Part of this work was car- Anthophila and Pemphredoninae is the presence of a sin- ried out by using the resources of the Computational Biol- gle mid-tibial spur in both groups (Danforth & Poinar ogy Service Unit from Cornell University, which is 2011). Most other apoid wasps have two mid-tibial spurs partially funded by Microsoft Corporation. The project (Bohart & Menke 1976), whereas all bees and Pemphre- was partially supported by the Hughes Scholars Under- doninae have a single mid-tibial spur (Michener 2007). graduate Research Program (Fellowship to AHD) as well There is significant morphological support for a sister as a National Science Foundation grant (DEB-0814544) to group relationship between Philanthinae and Anthophila. BND and SC (with one REU supplement that directly Prentice (1998) specifically addressed the question of a supported AHD). clade consisting of Anthophila and Philanthinae and sug- gested seven morphological synapomorphies: (i) position of antennal socket that is very well separated from the ep- References istomal sulcus; (ii) great expansion of the cardinal discal Alexander, B. A. (1992). An exploratory analysis of cladistic cavity; (iii) presence of a broad prearticular portion of the relationships within the superfamily Apoidea, with special reference to sphecid wasps (). 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Rambaut, A. & Drummond, A. J. (2007). Tracer v1.4. Available long-branched taxon. In this region (D3-2 to D3-3¢), a via http://beast.bio.ed.ac.uk/Tracer. large deletion has occurred, reducing the length of from Ronquist, F. (1999). Phylogeny of the Hymenoptera (Insecta): the 97 bp in other Aculeata to just 37 bp. While the sequences state of the art. Zoologica Scripta, 28, 3–11. are definitely correct according to BLAST searches, they Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: bayesian phylogenetic inference under mixed models. Bioinformatics, 19, have several abnormalities, including not just this loss but 1572–1574. also extreme distance on the tree. In fact, the exemplar Stamatakis, A. (2006a). RAxML-VI-HPC: maximum likelihood- included from the same family, Parnopes grandior, also based phylogenetic analyses with thousands of taxa and mixed appears long-branched on the tree, although it is more models. Bioinformatics, 22, 2688–2690. similar to other aculeates than the Hedychridium sp. Stamatakis, A. (2006b). Phylogenetic models of rate heterogeneity: sequences. a high performance computing perspective. Proceedings of the Table S3 Voucher and locality information for newly 20th IEEE International Parallel & Distributed Processing Symposium, Conferenced occurred in Rhodos, Greece in sequenced taxa in this study. April 2006. Paper available online (http://www.hicomb.org/ Table S4 Primers used for PCR amplification and HiCOMB2008/papers/HICOMB2006-05.pdf). sequencing in this study. Conditions were as follows: (1) Ward, P. S. & Brady, S. G. (2003). Phylogeny and biogeography 28S: initial denaturation of 45 s at 94 C; 35 cycles of of the ant subfamily Myrmeciinae (Hymenoptera: Formicidae). 1 min at 94 C, 1 min at 58 C, 1 min at 72 C; final , 17, 361–386. extension of 5 min at 72 C; (2) Opsin: initial denaturation of 5 min at 94 C; 35 cycles of 1 min at 94 C, 1 min at Supporting Information 54 C, 1 min at 72 C; final extension of 5 min at 72 C; Additional Supporting Information may be found in the (3) EF1-a: initial denaturation of 5 min at 94 C; 35 cycles online version of this article: of 1 min at 94 C, 1 min at 54 C, 1 min 30 s at 72 C; Fig. S1 Maximum clade credibility tree recovered from final extension of 5 min at 72 C; (4) wingless: initial dena- Bayesian analysis. Numbers above nodes represent poster- turation of 5 min at 94 C; 35 cycles of 45 s at 94 C, ior probabilities. 45 s at 58 C, 45 s at 72 C; final extension of 5 min at Fig. S2 Recovered tree with highest likelihood score 72 C. from ML analysis. Numbers above nodes represent boot- Please note: Wiley-Blackwell are not responsible for the strap values (from 3000 pseudoreplicates). content or functionality of any supporting materials sup- Table S1 Voucher numbers for all taxa used in this plied by the authors. Any queries (other than missing study, and GenBank accession numbers. material) should be directed to the corresponding author Table S2 This region of 28S ribosomal DNA is indica- for the article. tive of the larger problem of Hedychridium sp. being a

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