Proc. Natl. Acad. Sci. USA Vol. 91, pp. 9911-9915, October 1994 Evolution Molecular phylogeny of the insect order Hymenoptera: Apocritan relationships (Symphyta/molecular systematlcs/mItochondral DNA/16S ribosomal RNA) MARK DOWTON AND ANDREW D. AUSTIN Department of Crop Protection, Waite Campus, University of Adelaide, Glen Osmond, S.A. 5064, Australia Communicated by Charles D. Michener, June 7, 1994 (receivedfor review March 5, 1994) ABSTRACT Phylogenetic relationships among the major monomorpha (the Ichneumonidae and Braconidae; refs. groups of hymenopteran insects were investigated by using 8-12), most remaining apocritan relationships are conten- comparative sequence information from the mitochondrial 16S tious (6) (Fig. 1 Right). More generally, any phylogeny ofthe rRNA gene. The placement of the ectoparasitic Stephanldae as apocritan wasps that is based on morphology suffers from the sister group to the remaining Apocrita confirmed ectopar- problems associated with reductional synapomorphies be- asitism as the ground plan biology for the Apocrita. Endopar- cause of the extremely small size of many members of the asitism evolved at least eight times within the Apocrita, and the Proctotrupomorpha and Ceraphronoidea (=1 mm; refs. 8 and consequent associaton with polydnaviruses and virus-like par- 11). ticles evolved at least three times. The Evaniomorpha were Molecular systematic studies have thus far been unable to consistently placed as basal to the remaining Apocrita but were provide a robust systematic framework for the Hymenoptera. not resolved as monophyletic. The Gasteruptildae were re- One study (13, 14) using the 16S rRNA gene reemphasized solved as the sister group to the Evanlidae, but the relationship the sister-group relationship between the Ichneumonidae and between the Trigonalyoidea and the Evanioidea was unclear. the Braconidae, but the omission ofmost ofthe major groups The Proctotrupomorpha (sensu Rasnitsyn) was resolved by precluded the placement of the Ichneumonoidea within an topology-dependent permutation tall probability (T-PTP) test- apocritan phylogeny. A survey of a wider range of hy- ing as monophyletic, with strong evidence for a sister group menopteran groups used 18S rRNA sequences (15, 16). relationship between the Platygastroldea and the Chalcldoldea. However, the level of variation was too low to resolve any Strong evidence was found for the monophyly of the Ichneu- relationships. In the present study, we investigate the evo- monomorpha (Ichneumonidae + Braconidae) and the sister- lution of the parasitoid life-style in the Hymenoptera using group relationship between-the Aculeata (Vespomorpha) and molecular sequence datafrom the 16S rRNA gene; we survey the Ichneumonomorpha. 10 of the 14 superfamilies of the Apocrita and 4 of the 7 superfamilies ofthe Symphyta (sawflies) in an attempt to lay The Hymenoptera (ants, bees, and wasps) comprise one of the foundations for a phylogenetic understanding ofthe entire the largest (115,000 described species) (1) and most biologi- order.* cally diverse group ofinsects (2) containing both eusocial and parasitic groups. Apart from the termites, the order Hy- MATERIALS AND METHODS menoptera contains most other eusocial insects, while the DNA Amplification and Sequendng. Sequences were ob- parasitic Hymenoptera are the most important group of tained either from the literature or generated in our labora- biological control agents for insect and weed pests (3). tory from the following 14 superfamilies: Tenthredinoidea Although our understanding of the evolution of these com- [Perga condei (Benson), Phylacteophaga froggatti (Riek), plex behaviors depends upon a robust phylogeny (4-6), and Tenthredinidae indet. (14)]; Siricoidea [Tremex columba historical relationships among the Hymenoptera are cur- (L.) (14)]; Cephoidea [Hartigia trimaculata (Say)]; Orus- rently poorly understood. Molecular systematic studies are soidea [Orussus terminalis (Newman)]; Stephanoidea likely to provide important evidence to resolve these histories [Schlettererius cinctipes (Cresson)]; Ichneumonoidea [Cote- (4). For example, evidence that advanced eusociality in bees sia glomerata (L.), Cotesia rubecula (Marshall), Cotesia has multiple origins was provided recently after comparison flavipes Cameron (14), Digonogastra kimballi (Kirkland) ofmitochondrial 16S rRNA gene sequences (5). However, no (14), Alabagrus stigma (Brulle) (14), Ichneumon promis- comparable molecular systematic study has been performed sorius (Erichson), Venturia canescens (Gravenhorst), and on the parasitic Hymenoptera to evaluate the current hy- Xanthopimpla stemmator (Thunberg) (14)]; Vespoidea [Myr- pothesis that endoparasitoids and their association with sym- mecia forficata (F.) and Polistes versicolor (Olivier) (14)]; biotic polydnaviruses arose multiple times from ectoparasitic Apoidea [Apis mellifera (L.) (17)]; Platygastroidea [Scelio ancestors (6, 7). fulgidus (Crawford) and Trissolcus basalis (Wollaston)]; The first fully resolved phylogenetic hypothesis concern- Chalcidoidea [Aphytis melinus (De Bach), Encarsiaformosa ing the Hymenoptera was advanced by Rasnitsyn (8). Within (Gahan), and Pteromalus puparum (L.)]; Proctotrupoidea the suborder Apocrita (which contains all of the parasitic [Ropronia garmani (Ashmead) and Vanhornia eunemidarum Hymenoptera apart from the ectoparasitic Orussidae), he (Crawford)]; Cynipoidea [Ibalia leucospoides (Hochen- proposed four major lineages; the Ichneumonomorpha, the warth)]; Trigonalyoidea [Poecilogonalos costalis (Cresson) Vespomorpha (Aculeata), the Proctotrupomorpha, and the and Orthogonalys pulchella (Cresson)]; and Evanioidea [Eu- Evaniomorpha, with sister-group relationships between the foenus sp., Gasteruption sp., and Evania sp.]. Dipteran former and latter pairs (Fig. 1 Left). Although there is strong outgroups were Drosophila yakuba (Burla) (18) and Aedes morphological evidence for the monophyly of the Ichneu- Abbreviation: T-PTP, topology-dependent permutation tail proba- The publication costs ofthis article were defrayed in part by page charge bility. payment. This article must therefore be hereby marked "advertisement" *The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. U06953-U06975). 9911 Downloaded by guest on September 30, 2021 9912 Evolution: Dowton and Austin Proc. Natl. Acad. Sci. USA 91 (1994) Orussomorpha Orussomorpha Ichneumonidae Stephanidae Ichneumonomorpha Braconidae Ichneumonidae Vespomorpha (Aculeata) Ichneumonomorpha Braconidae Cynipoidea Proctotrupoidea Aculeata Chalcidoidea Proctotrupomorpha Cynipoidea Platygastroidea Proctotrupoidea Mymarommatidaej Proctotrupomorpha Chalcidoidea Stephanidae Megalyridae Scelionoidea_ Trigonalyidae Trigonalyidae Megaspilidae Evaniomorpha Megalyridae Ceraphronidae Evaniomorpha Ceraphronoidea Gasteruptiidae Evaniidae Evanioidea I FIG. 1. Cladogram of the Hymenoptera as proposed by Rasnitsyn (8) as it appears in ref. 6 (Left) and as revised in the review by Whitfield (6) (Right). (Reproduced from ref. 6 with permission.) aegypti (L.) (19). Genomic DNA was extracted from frozen using CLUSTAL V (26), based on the secondary structure (-800C) or 100% ethanol-stored specimens as described (20). model of the Drosophila yakuba 16S gene (27). This strategy Those specimens that were held in 100%o ethanol were has been used to align other 16S sequences of varying length washed three times for 15 min in 10 mM Tris HCl, pH 8/100 (28). Gaps were coded as single characters, irrespective of mM NaCl/1 mM MgCl2 before extraction (21). A portion of length, as described (29). Briefly, gaps were coded as missing the 16S gene was then amplified with primers as described data, and an additional "gap" data matrix was constructed. (22) but edited according to the honeybee sequence (17). The Where overlapping gaps occurred, each size gap was as- primers used were: 16SWa (5'-CGTCGATTTGAACT- signed a particular character state. Phylogenetic analysis was CAAATC-3'; anneals to nucleotides 1146-1165 of the hon- performed by maximum parsimony using PAUP version 3.1.1 eybee 16S rRNA gene) and 16SWb (5'-CACCTGTTTAT- (29), with transitions and transversions weighted equally. CAAAAACAT-3'; anneals to nucleotides 616-635). Where The number of informative characters was 386. Both most amplifications were unsuccessful under a variety of condi- parsimonious as well as near-most parsimonious trees were tions, one of the following primers was used in place of examined to investigate the cladistic structure of the se- 16SWa: 16S.Sh (5'-AGATTTTAAAAGTCGAACAG-3'; an- quence data (30). Analyses were also performed with tran- neals to nucleotides 1121-1140) or 16Wa.1 (5'-GACT- sitions weighted relative to transversions at 1:2, 1:3, 1:4, and TACGTCGATTTGAACT-3'; anneals to nucleotides 1152- 1:5. One-hundred thousand trees were drawn at random from 1171). The PCRproduct was thus in the range of480-550 base the set of all possible trees, and the number of trees was pairs. PCR reactions were optimized as described (23). plotted as a function of the number of steps in the tree. This Reactions were carried out in 67 mM Tris HCl buffer, pH plot showed a left-skewed distribution (g, = -0.53), indicat- 8.8/16.6 mM (NH4)2SO4/0.2 mg of gelatin per ml/0.45% ing that the data contains a strong phylogenetic signal (P < Triton X-100 containing also 2-4 mM MgCl2, 25 juM dNTPs, 0.01; ref. 31). The degree of support for various nodes was 0.05-0.20 ,uM