Systematic Entomology (2015), 40, 570–591 DOI: 10.1111/Syen.12122

Systematic Entomology (2015), 40, 570–591 DOI: 10.1111/syen.12122

Phylogeny of the parasitic wasp subfamily Euphorinae (Braconidae) and evolution of its host preferences

JULIA STIGENBERG1, CHARLES ANDREW BORING2 and FREDRIK RONQUIST3

1Entomology Section, Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden, 2Department of Recent Invertebrates, Sam Noble Museum of Natural History, Norman, OK, U.S.A. and 3Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Stockholm, Sweden

Abstract. The braconid subfamily Euphorinae is a large, cosmopolitan group of endoparasitoid wasps. The majority of species attack adult hosts, a strategy that is rare among parasitic wasps, but there are also many species that attack nymphs and larval stages. Euphorine hosts may belong to a variety of insect orders (Coleoptera, Hemiptera, Hymenoptera, Neuroptera, Psocoptera, Orthoptera and Lepidoptera) although most euphorine tribes are confined to Coleoptera. Here we investigate the phylogenetic relationships of the Euphorinae based on molecular data (3 kb of nucleotide data from four markers: 18S, 28S, CAD and COI) and propose a higher-level classification based upon the resulting phylogeny. We also infer the evolution of host associations and discuss the diversification of the Euphorinae. Results from both Bayesian inference and maximum-likelihood analysis show that the subfamily, as previously circumscribed, is paraphyletic. We propose that the subfamily be expanded to include the tribes Meteorini and Planitorini (Mannokeraia + Planitorus), so that it corresponds to a clade that is strongly supported as monophyletic in our analyses. Based on our results, a revised higher classification of the Euphorinae is proposed, in which 52 extant genera and 14 tribes are recognized. We reinstate the genus Microctonus belonging to the tribe Perilitini, and synonymize Ussuraridelus with Holdawayella, Sinuatophorus with Eucosmophorus. Furthermore, we propose the following tribal rearrangements: Spathicopis and Stenothremma are transferred to Perilitini; Tuberidelus, Eucosmopho- rus and Plynops to Cosmophorini; Ecclitura to Dinocampini; Chrysopophthorus, Holdawayella and Wesmaelia to Helorimorphini; Proclithroporus and Heia to Towne- silitini. The monotypic tribe Cryptoxilonini is synonymized with Cosmophorini. The genera Pygostolus and Litostolus are placed in a separate tribe, Pygostolini, previously recognized as a subtribe among the Centistini. Parsimony-based ancestral state reconstructions suggest that the ancestor of Euphorinae was a parasitoid of lepidopteran larvae, and that a host shift to larval Coleoptera occurred only in one clade of the Meteorini, some members of which secondarily shifted back to larval lepidopteran hosts. In the remainder of the subfamily, there was an initial shift from larval to adult coleopterans, followed by subsequent shifts to adults or larvae of Hemiptera, Hymenoptera, Neuroptera, Orthoptera and Psocoptera.

Introduction nymphal and larval stages, especially when they co-occur with adults in the same habitat (Obrycki et al., 1985; Shaw, 1985). Parasitoid wasps rarely attack adult hosts, but this is the predom- The diversity of euphorine hosts is exceptional, including rep- inant mode of parasitism among the Euphorinae (Hymenoptera: resentatives from seven different orders of insects: Coleoptera, Braconidae). Target hosts of the Euphorinae also include Hemiptera, Hymenoptera, Neuroptera, Orthoptera, Psocoptera and Lepidoptera (Shaw, 1988; Chen & van Achterberg, 1997). Correspondence: Julia Stigenberg, Swedish Museum of Natural A robust phylogeny of the Euphorinae is most desirable in order History, P.O. Box 50007, SE-10405 Stockholm, Sweden. E-mail: [email protected]

570 © 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Phylogeny of the Euphorinae 571 to understand transitions in host associations. This would bring based on data from four gene regions (18S, CAD, 28S and COI), insight into the origin of adult parasitism and further transitions and the dataset includes representatives from 42 of the 49 cur- in host associations within this group. rently recognized extant genera (Yu & van Achterberg, 2011). Shaw (1985) presented a tribal classification for 37 euphorine Based on the results, we propose a revised higher-level classifi- genera following a phylogenetic analysis of 68 external morpho- cation of the Euphorinae. We also map host preferences onto the logical characters. In his PhD dissertation, McDonald (2001) euphorine phylogeny and discuss the evolutionary implications. re-evaluated the morphological characters and the cladistic methods used by Shaw and proposed a new phylogeny. Although the results had important implications for euphorine classifica- Materials and methods tion, the dissertation was never published and the work went largely unnoticed. Since 1985, 23 additional genera or subgen- Taxon sampling era have been described and placed in the Euphorinae. As the known diversity has expanded, a need has arisen for a reclassifi- A total of 217 taxa were sampled out of which 16 species cation of the Euphorinae genera. Since Shaw’s revision, most of represented outgroups (Table S1). Two additional taxa (Asia- the new insights into the phylogenetic relationships of Euphori- centistes sp. and Tainiterma sp.) were initially considered part nae have come from molecular analyses of higher-level rela- of the ingroup, but were shown in the analysis to belong to tionships in the Ichneumonoidea or Braconidae (Dowton et al., the outgroup. The taxon sampling of the ingroup comprised 1998; Belshaw et al., 2000; Belshaw & Quicke, 2002; Dowton representatives from 52 genera, including exemplars of sev- et al., 2002; Li et al., 2003; Shi et al., 2005; Pitz et al., 2007; Sha- eral subgenera (Table S1). All tribes within the Euphorinae ranowski et al., 2011). The results from these studies have shown were represented. Several included genera have never before dramatically different results with respect to tribal and inter- been incorporated in a molecular analysis: Allurus, Asiacentistes generic relationships of the Euphorinae. This is not surprising (see discussion regarding placement), Betelgeuse, Bracteodes, because the scope of those studies included only a few of the 49 Centistina, Cryptoxilos, Heia, Litostolus, Mama, Myiocephalus, recognized, extant genera of Euphorinae. The most recent phy- Napo, Plynops, Proclithrophorus, Rilipertus, Eucosmophorus, logeny of the Braconidae by Sharanowski et al. (2011) showed Spathicopis, Stenothremma, Syntretellus, Tainiterma (see dis- ≥ high support [posterior probability (PP) 0.95] for the mono- cussion regarding placement), Townesilitus, Tuberidelus and phyly of the euphoroid complex in all their concatenated anal- Holdawayella. yses. The euphoroid complex (sensu Sharanowski et al., 2011) The inclusion of Cenocoelini, Ecnomios, Mannokeraia and includes, in addition to Euphorinae, also the subfamilies Ceno- Planitorus in our analysis was based on the results of Shara- coelinae, Ecnomiinae, Neoneurinae and Meteorinae, the latter nowski et al. (2011). The Meteorini are heavily represented in two of which are sometimes treated as tribes within the Euphori- our dataset due to the availability of material and sequences nae. This study includes additional representatives to further test (Stigenberg & Ronquist, 2011), as well as to the ambiguous the monophyly of the euphorid complex. placement of Meteorini in previous analyses (Dowton et al., The uncertainty concerning phylogenetic relationships is 1998; Belshaw et al., 2000; Belshaw & Quicke, 2002; Dowton reflected in disagreement with respect to the classification. The et al., 2002; Shi et al., 2005; Pitz et al., 2007; Sharanowski et al., lineage including Meteorus and Zele is recognized by many 2011), either at the base of Braconidae or at other various posi- authors as a separate subfamily, the Meteorinae (Shaw, 1985, tions within Braconidae. 1988, 2004; Maeto, 1990; Shaw & Huddleston, 1991; Hanson Outgroups included 11 braconid and 5 ichneumonid taxa. Due & Gauld, 1995; Wharton et al., 1997; Zitani et al., 1998; Li to the placements of Asiacentistes and Tainiterma in preliminary et al., 2003), but many others treat it among the Euphorinae as analyses we included several possible sister taxa to stabilize their the tribe Meteorini (Huddleston & Short, 1978; van Achterberg, placement: Cenocoelius (three spp.; Cenocoelinae), Blacus (two 1984; Chen & van Achterberg, 1997; Belokobylskij, 2000b; Sti- spp.) and Diospilus (Brachistinae), Wroughtonia (Helconinae) genberg & Ronquist, 2011). Both classifications may well be and Homolobus (Homolobinae). We also included several dif- consistent with the true phylogeny; there is little argument that ferent ichneumonids as well as several cyclostome braconids as the Meteorus + Zele lineage either forms the sister-group to the a more functional outgroup to help root the tree. Euphorinae or a basal clade inside the Euphorinae. If the former is true, then the decision on the subfamily or tribal status of the lineage is simply subjective, reflecting the author’s views of how the morphological and biological differences between these Terminology and Image capture groups should be reflected in the classification. There are also groups of taxa that have been classified as Terminology used for morphological characters follow Whar- genera or subgenera by various authors, representing cases of ton et al., 1997. Images were taken using an InfinityX digital ‘splitting’ or ‘lumping’. For example, different authors consider camera mounted on an Olympus SZX12 stereo microscope. the genus Leiophron to include some or all of the taxa Eupho- The DeltaPix InSight software package was used for imaging riana, Euphoriella, Euphorus and Peristenus as subgenera. and stacking. Voucher images of nearly all sequenced ingroup Here, we present the first molecular analysis of the taxa are available from the online image repository Morphbank higher-level relationships among euphorines. The analysis is (http://www.morphbank.net).

Molecular methods Molecular data

DNA sequences were generated for four genes: partial 28S A total of 3043 bp of nucleotide data were generated from domain 2 (rDNA), partial 18S (rDNA), partial CAD and the 5′ the four studied markers. Sequences were assembled and edited region of mitochondrial COI (Table S2). These genes have been with Invitrogen Vector NTI software v9, the Staden package (Staden, 1998) and Geneious Pro 5.6.5. The COI and CAD used in previous studies of braconid phylogenetics at various sequences were aligned in Mega5 (Tamura et al., 2011) after levels (Belshaw et al., 2000; Belshaw & Quicke, 2002; Dowton translating the nucleotides into amino acids. The rDNA (28S et al., 2002; Zaldivar-Riverón et al., 2006; Sharanowski et al., and 18S) sequences were aligned separately with the MAFFT 2011; Stigenberg & Ronquist, 2011). The specific part of the L-INS-I alignment algorithm (Katoh et al., 2002). CAD gene used here was primarily developed as a phylogenetic The uncertain species identifications of the GenBank records marker for the insect order Trichoptera by Johanson & Malm made it impossible in most cases to reliably match them with (2010). sequences from other genes of the same taxon. For instance, Specimens were extracted, amplified and sequenced either we found three COI sequences labelled as Elasmosoma in Gen- at the Molecular Systematics Laboratory, Swedish Museum Bank, one 28S sequence and one 18S sequence. With the infor- of Natural History or at the Department of Entomology mation provided in GenBank, we could not assign the sequences and the Advanced Genetic Technologies Center, Univer- to the same individual, not even to the same species. GenBank sity of Kentucky. DNA was extracted from single dry or sequences were therefore mostly used in the single-gene ethanol-preserved specimens using three different methods, analyses; we have assigned them the prefix GB_XXX when following the Qiagen protocol from the DNeasy Tissue Kit presenting them in the phylogenetic trees and in Table S1. (Qiagen, Valencia, CA, U.S.A.), using Chelex InstaGene Matrix (Bio-Rad, Hercules, CA, U.S.A.) or by automated isolation of DNA using GeneMole robot and MoleStrips, Phylogenetic analyses with an elution volume of 100 μL. DNA fragments were amplified using Ready-To-Go PCR beads (Amersham Phar- Phylogenetic analyses were performed with Bayesian macia Biotech, Amersham, U.K.). Product yield, specificity inference (BI) (MrBayes v3.2.2, Ronquist et al., 2012) and of amplification and contamination were investigated using maximum-likelihood (ML) [RAxML Web-Servers, Stamatakis agarose gel electrophoresis. PCR products were purified et al., 2008 (http://embnet.vital-it.ch/raxml-bb/index.php)] using EXOFAP (EXO1 and FastAP). Gene regions were methods. All BI analyses were performed through the Cipres sequenced with the same primers, using the BigDye Ter- web portal for phylogenetic analysis (Miller et al., 2010) minator v3.1 Cycle Sequencing Kit (Applied Biosystems, (http://www.phylo.org). All trees were rooted between ich- Waltham, MA, U.S.A.). Sequencing reactions were puri- neumonids and braconids, with braconids always recovered as fied using the DyeEx 96 kit (Qiagen) and cycle sequencing monophyletic. reactions were run on an ABI Prism 3100 Genetic Ana- lyzer (Applied Biosystems). Gene regions were sequenced in both directions. All steps were performed according to BI the manufacturer’s instructions. Primer sequences and poly- merase chain reaction protocols are presented in Tables S2 We first performed single-gene analyses to evaluate the signal from the individual genes. The COI and CAD alignments were and S3. each divided into two character sets based on codon positions Voseq 1.7.3 (Peña & Malm, 2012) database was used for stor- (1 + 2 vs 3) in these analyses, whereas the 28S and 18S datasets ing voucher and DNA sequence data. For some sampled taxa, were not partitioned. For the concatenated combined-data anal- we were not able to obtain sequences from all four molecular yses, the data were divided into six partitions (28S, 18S, COI markers used in the study. Therefore, we combined sequences first and second, COI third, CAD first and second, and CAD from two or more specimens representing the taxon (Table S1). third). Based on the test of rate heterogeneity in mega5 (Tamura Some sequences used in our analyses were downloaded from et al., 2011), a Mixed+G model was selected for the 18S and GenBank (Table S1). All sequences obtained as part of this 28S datasets, and for the COI and CAD datasets a Mixed+G+I study were deposited in GenBank under accession numbers: model was used in both single-gene and combined analyses. KJ591053–KJ591543 and KJ608350. The Mixed substitution rate model uses reversible-jump Vouchers are deposited in the Hymenoptera Institute Collec- MCMC to integrate over all reversible 4 × 4 nucleotide substitution of the University of Kentucky or in the entomology col- tion models (Huelsenbeck et al., 2004). The parameters ‘pinvar’, lection of the Swedish Museum of Natural History. Specimens ‘revmat’, ‘shape’ and ‘statefreq’ were unlinked between parti- with DNA voucher prefixes BJS###, JS### and AB### were tions. Each Bayesian single-gene analysis ran for a minimum of identified by C. A. Boring and extracted by B. Sharanowski, J. 10 000 000 generations, sampling every 1000 generations. The Sherman and C. A. Boring, respectively. Specimens with DNA concatenated dataset was run for 30 000 000 generations. Two voucher prefixes DNA_### and JS10_###### were extracted by independent runs were performed during each analysis, with J. Stigenberg (Table S1). four chains per run. The ‘burn-in’ value was set to 25% of the

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 573 sampled trees. The average standard deviation of split frequen- in a poorly resolved tree (not shown) but including all sites cies (ASDSF) was below 0.015 for all runs. and dividing each of these genes into two site partitions, codon positions 1 + 2 vs 3, improved the signal considerably. Thus, the latter approach was used in all BI analyses involving COI ML and CAD. Results of the phylogenetic analyses are presented in Figs 1, 2 and S1–S4. Conflict between the ML and BI trees only ML analyses used RAxML (Stamatakis et al., 2008) and were involved weakly supported nodes. run for the concatenated dataset. The concatenated analysis with all four genes was run using a GTR+G model. The data were divided into six distinct data partitions and bootstrapped (100 Single-gene analyses replicates) to assess ML support of individual groups. In the separate analyses of the genes 18S, 28S and COI, Host associations many clades were weakly supported and often a basal polytomy was recovered in the majority-rule consensus, although most Host association data were based mostly on the information tribes were still well supported (Figures S1–S3). The CAD tree from the database Taxapad (Yu & van Achterberg, 2011). When was considerably more resolved, and even some of the deeper terminal taxa were polymorphic (had several hosts), we tried nodes were highly supported (Figure S4). The 18S analysis to determine the ground-plan state by comparing each taxon produced a largely unresolved polytomy. The separate analyses with its closest relatives. For instance, Leiophron (Euphorus) of the genes 28S, COI and CAD suggest different placements fulvipes Curtis and L.(E.) similis Curtis have host records of for the Meteorini: either as a derived euphorine tribe (COI, both adult Coleoptera and Psocoptera (Yu & van Achterberg, CAD) or as a basal tribe (28S). Both the COI and CAD analyses 2011) but these are the only species of the subgenus Euphorus produced a paraphyletic Euphorinae (sensu Shaw, 1985; Yu & with host records for adult Coleoptera. Related Leiophron van Achterberg, 2011) with some outgroup taxa nested among species are all associated with Psocoptera. Therefore, the the ingroup taxa. ground-plan hosts are likely to be Psocoptera, and this was the host state we used here. Leiophron (Leiophron) spp. have host records of Hemiptera and Psocoptera, though L.(L.) psocivora Concatenated analyses Tobias is the only species with a host record for Psocoptera (Yu & van Achterberg, 2011). Therefore, Hemiptera are the Both the ML and the BI analyses of the concatenated data likely ground-plan hosts of this subgenus. In Peristenus,43 recovered a fairly well supported and well-resolved topology species are recorded as having Hemiptera hosts, whereas only (Figs 1, 2). There were only minor differences between the ML three species attack adult Coleoptera (Yu & van Achterberg, and BI trees. The well-supported clades that emerged from the 2011). All three species of Peristenus with host records of adult single-gene analyses (Figures S1–S4) were almost all recovered Coleoptera also have host records of Hemiptera (P.accincta also in the concatenated analyses (Figs 1, 2). Haliday, P.orchesiae Curtis, P.pallipes Curtis) (Yu & van The analyses of the concatenated data recovered Euphori- Achterberg, 2011). Therefore, the ground-plan hosts of all nae including Meteorini as monophyletic with high support Peristenus species are likely to be Hemiptera. [PP 1.0; bootstrap proportion (BP) in the ML analysis 0.70], Reconstruction of host preferences of ancestral lineages was with Cenocoelinae as its sister group (PP 1.0 and BP 0.96 for based on Fitch parsimony mapping of host data (Li et al., 2008) Cenocoelinae + Euphorinae). Surprisingly, one of the ingroup onto the consensus tree resulting from our analyses of the taxa, Asiacentistes, ended up as the sister group of Cenocoeli- combined data. The results of the phylogenetic analyses were nae + Euphorinae in the strongly supported lineage (PP 1.0, BP imported into Mesquite v2.75 (Maddison & Maddison, 2009) 0.96) that may be referred to as the ‘euphoroid’ clade. Asiacen- for these reconstructions. tistes is currently placed in the tribe Centistini of the Euphorinae, where it apparently does not belong. Within the Euphorinae (including Meteorini), most of the Results existing tribes can be approximately matched to strongly supported lineages in our analysis. However, to obtain a natural Data matrix higher-level classification of the Euphorinae, it is obvious that many taxa need to be moved among tribes. More than half of the The concatenated dataset contained 193 specimens. Single- tribes in the summarized classification of Yu & van Achterberg gene analyses included 165 taxa for 18S, 198 taxa for 28S, 171 (2011) were found to be para- or polyphyletic as currently taxa for COI and 85 taxa for CAD (Table S3). The concatenated circumscribed. For instance, the tribe Centistini sensu Yu & data matrix of the four genes included 3043 characters. The gene van Achterberg (2011) is problematic due to four genera that with the largest number of parsimony-informative (PI) sites was are misplaced there: Asiacentistes, Pygostolus, Litostolus and COI, closely followed by CAD. 18S had the least number of PI Spathicopis. The genus Asiacentistes apparently does not belong sites (Table S4). Excluding the third position from the separate to the Euphorinae, as mentioned above. Pygostolus forms a COI and CAD datasets in the single-gene BI analyses resulted separate lineage together with Litostolus within the Euphorinae,

1 Outgroup: Ichneumonidae 98 1 1 100 100 1 1 Outgroup: Braconidae 100 85 1 1 76 100

0.48 51 0.99 90

1 88

1 98

1 1 91 100 Meteorini 1 75

1 100

1 76 1 0.92 100 Ecnomiini 16 1 100 Dinocampini

1 100 Planitorini 0.99 1 49 100 Neoneurini 0.93 37 0.71 1 63 0.95 100 26 Centistini

0.99 57 Pygostolini 0.69 12

0.79 1 94 100 0.59 Cosmophorini 0.71 51 6

1 100 Myiocephalini 1 92 1 1 56 99 1 41

0.94 Syntretini 22

0.89 2 1 1 100 94 1 Helorimorphini 58 0.38 44 1 100 1 99 1 Euphorini 99

1 0.83 99 7 1 0.6 100 41

0.98 70 Townesilitini

1 88 0.28 16 0.99 1 32 68

1 100 1 89 0.97 36

1 100 1 Perilitini 100

1 92

0.49 33 0.5

Fig. 1. Cladogram from maximum-likelihood (ML) concatenated analysis showing results from both ML and Bayesian inference (BI) combined analysis (18S, 28S, CAD and COI). Support values from ML and BI analyses are plotted on to the ML topology. Values above branches are posterior probabilities (PP), values below branches are bootstrap support. Only deeper-level node values are shown. The scale bar represents expected number of substitutions per site.

(A)

Fig. 2. (A, B) Phylogeny retrieved from a Bayesian phylogenetic analysis of the concatenated molecular data (18S, 28S, CAD and COI). Numbers on branches are posterior probabilities (PP), the PP value 1 is indicated with an asterix (*), values > PP 0.95 at genus-level is indicated with a plus (+). The scale bar represents expected number of substitutions per site.

(B) * JS10_00274_Litostolus_sp. * JS10_00201_Pygostolus_sticticus JS10_00202_Pygostolus_sticticus Pygostolini 0.7 * JS10_00500_Pygostolus_falcatus * JS10_00193_Pygostolus_falcatus JS10_00204_Pygostolus_falcatus * JS10_00517_Ropalophorus_clavicornis JS10_00533_Ropalophorus_clavicornis AB088_Cosmophorus_(Eucosmophorus)_sp. * * JS144_Cosmophorus_sp. * * 1_Cosmo_Cosmophorus_ Cosmophorini 0.74 JS10_00210_Cosmophorus_klugi 0.6 AB096_Tuberidelus_flavicephalus * AB067_Plynops_hansoni * JS10_00531_Cryptoxilos_sp. * JS040_Plynops_sp1 JS10_00514_Myiocephalus_boops * JS10_00164_Myiocephalus_boops Myiocephalini From tree A JS10_00169_Myiocephalus_boops * JS10_00145_Syntretus_zuijleni JS10_00015_Bracteodes_sp. * AB084_Syntretus_sp5 * 0.97 JS10_00521_Syntretus_(Exosyntretus)_elabsus * JS10_00305_Syntretellus_morpho_species_9 * JS10_00321_Syntretellus_morpho_species_1 * JS10_00306_Syntretellus_morpho_species_6 JS10_00307_Syntretellus_morpho_species_7 JS10_00135_Syntretus_fuscivalvis JS10_00156_Syntretus_zuijleni Syntretini JS10_00528_Syntretus_komorovi 0.94 JS10_00137_Syntretus_falcifer JS10_00152_Syntretus_sp. 0.86 JS10_00147_Syntretus_taegeri 0.98 JS10_00134_Syntretus_idalius 0.92 0.85 * JS10_00138_Syntretus_idalius JS10_00150_Syntretus_idalius JS10_00153_Syntretus_idalius JS10_00159a_Syntretus_idalius * JS10_00519_Chrysopophthorus_hungaricus * AB021_Chrysopophthorus_sp1 * JS115_Chrysopophthorus_sp2 * JS10_00529_Wesmaelia_aff._decurta Helorimorphini * JS122_Wesmaelia_sp2 JS10_00520_Holdawayella_minuta * 0.99 JS10_00265_Aridelus_sp. 0.99 JS10_00507_Aridelus_egregius JS10_00508_Aridelus_rufotestaceus AB020_Peristenus_sp3 * AB016_Peristenus_sp1 0.59 AB023_Peristenus_sp4 * JS10_00515_Leiophron_(Euphoriana)_dispar JS10_00499_Mama_mariae * JS10_00242_Leiophron_sp. Euphorini 0.85 JS129_Leiophron_(Euphoriella)sp7 + JS10_00247_Leiophron_pallidistigma 0.86 JS10_00244_Leiophron_fulvipes JS068_Leiophron_sp1 JS120_Leiophron_(Euphoriella)sp6 AB089_Gen_nov. JS10_00040_Streblocera_(Lecythodella)_sp. JS10_00495_Streblocera_(Eutanycerus)_major * JS10_00497_Streblocera_(Streblocera)_carinata * JS10_00217_Streblocera_longiscapa JS10_00496_Streblocera_(Asiastreblocera)_dayuensis 0.62 + JS10_00513_Streblocera_(Villocera)_villosa + JS10_00498_Streblocera_(Cosmophoridia)_flaviceps + JS10_00530_Gen_nov. JS10_00518_Heia_robustipes 01_Yves_Gen_nov. Townesilitini 0.98 02_Yves_Gen_nov. * JS119_Marshiella_bobella * AB092_Proclithrophorus_sp. * JS10_00523_Proclithrphorus_mandibularis JS10_00218_Townesilitus_sp. * JS10_00223_Townesilitus_sp. 0.59 JS10_00224_Townesilitus_sp. JS10_00512_Townesilitus_deceptor * JS10_00222_Townesilitus_sp. 0.58 * JS10_00221_Townesilitus_sp. JS10_00219_Townesilitus_sp. JS074_Townesilitus_sp. JS10_00216_Perilitus_sp. * JS10_00213_Perilitus_sp. * JS10_00215_Perilitus_sp. * AB102_Microctonus_sp4 03_Yves_Miroctonus_sp JS10_00509_Perilitus_falciger JS10_00525_Rilipertus_intricatus 0.97 AB079_Microctonus_sp7 AB085_Microctonus_sp8 0.59 0.55 JS10_00283_Microctonus_sp. * 0.67 AB100_Microctonus_sp3 JS10_00501_Spathicopis_flavocephala Perilitini * JS10_00172_Spathicopis_flavocephala * JS10_00176_Spathicopis_flavocephala 0.92 JS10_00472_Spathicopis_flavocephala AB097_Orionis_eximius AB101_Orionis_sp1 * * JS10_00511_Perilitus_coxator * JS10_00526_Perilitus_coxator JS10_00510_Perilitus_flavifacies AB099_Perilitus_sp4 * JS10_00266_Gen_nov. JS10_00273_Gen_nov. JS124_Perilitus_sp1 0.05 0.80 JS10_00437_Stenothremma_sp. 0.87 JS10_00398_Stenothremma_sp.

Fig. 2. Continued.

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 577 outside any currently recognized tribe, and Spathicopis is nested conclusion, then, our analysis indicates that the Euphorinae were inside a clade that otherwise corresponds to the Perilitini sensu originally parasitoids of Coleoptera larvae. Yu & van Achterberg (2011). The tribe Dinocampini sensu Yu Within the rest of the Euphorinae, the mapping of the host & van Achterberg (2011) is polyphyletic due to Ropalophorus, associations onto the phylogeny implies two origins of parasitiz- which is nested within taxa belonging to the tribe Cosmophorini. ing Hymenoptera (in Neoneurini and Syntretini), and one origin Three genera of the tribe Euphorini sensu Yu & van Achterberg of parasitizing Hemiptera (in the common ancestor of Helori- (2011) (Chrysopophthorus, Stenothremma and Wesmaelia) morphini and Euphorini). Some members of the latter clade have are nested inside the Helorimorphini and Perilitini. The tribe subsequently shifted from Hemiptera to other hosts. In the Helo- Perilitini sensu Yu & van Achterberg (2011) includes four gen- rimorphini, the genus Chrysopophthorus has come to parasitize era (Ecclitura, Marshiella, Streblocera, Tuberidelus)thatare Chrysopidae (Neuroptera), and among the Euphorini, certain more closely related to taxa placed in the tribes Dinocampini, species of the genus Leiophron have shifted to parasitizing Pso- Townesilitini and Cosmophorini than to other taxa placed in the cidae (Psocoptera). Host associations of the tribes Planitorini, Perilitini. Ecnomiini and Tainitermini are still unknown. The relationships Based on the results of our phylogenetic analyses and morpho- of Planitorini and Ecnomiini are so uncertain that it is difficult logical study of additional taxa that could not be sequenced for to infer the ancestral host associations in these tribes. this study (Table S5), we propose a new tribal classification of the Euphorinae (Figs 1, 2 and Appendix). The new classification recognizes 14 tribes, all of which were included in our phyloge- Discussion netic analyses. In our analyses of the concatenated data all of the 14 studied tribes were strongly supported as monophyletic in the Placement of Euphorinae within Braconidae BI analysis (PP 1.0). In the ML analysis, all but the tribe Pygos- tolini (BP 0.57) were highly supported (BP >94) (Figs 1, 2). Very few studies address the position of the Euphorinae with The concatenated analyses did not resolve many of the rela- respect to the other subfamilies of Braconidae. The molecular tionships among tribes (as circumscribed here). For instance, phylogenetic analyses by Sharanowski et al. (2011) provided Meteorini was recovered as the sister group of the remain- strong support for a euphoroid clade consisting of (following Euphorinae with moderate support (PP 0.88, BP 0.21 for ing their classification) Cenocoelinae, Neoneurinae, Ecnomi- ‘core Euphorinae’, i.e. Euphorinae excluding Meteorini). Only inae, Meteorinae and Euphorinae among the noncyclostome two sister-group relationships between tribes were strongly braconids, with Cenocoelinae being the sister group to the supported: Townesilitini + Perilitini (PP 1.0, BP 0.73) and rest of the subfamilies. This agrees well with our results that Syntretini + Myiocephalini (PP 1.0, BP 93). The relationships indicated a sister-group relationship between the Cenocoelinae between Planitorini + (Neoneurini + Centistini) is also sup- and Euphorinae as circumscribed here (including Neoneurinae, ported, although with moderately strong support (PP 0.90, BP Ecnomiinae and Meteorinae). 0.65). The deeper intertribal nodes in the core Euphorinae were Our analyses indicated that neither Asiacentistes nor not as well supported. The relationships of several clades remain Tainiterma belong in the Euphorinae. This is remarkable uncertain, including the positions of Ecnomiini, Pygostolini, given previous taxonomic treatments of these taxa. The type and Helorimorphini + Euphorini. The whole clade containing of Asiacentistes, Centistes (Centistes) alekseevi Belokobylskij, (Helorimorphini + Euphorini) + (Townesilitini + Perilitini) is was originally placed in Centistes but Belokobylskij (1995) later moderately strongly supported (PP 0.80, BP 0.73). Two other considered it better placed in its own genus, even though he con- sister-group relationships between tribes are suggested, but with sidered it closely related to Centistes and Centistoides. He later low support values: Ecnomiini + Dinocampini (PP 0.75, BP not reaffirmed its placement in the Centistini (Belokobylskij, 2004). supported) and Helorimorphini + Euphorini (PP 0.76, BP 42). However, there is some evidence indicating that Centistini, as circumscribed by Belokobylskij, is not a monophyletic group. Members of the tribe Centistini are morphologically quite Evolution of host preferences similar to members of the tribe Brachistini of the Brachistinae (Sharanowski et al., 2011). The Centistini are placed in the When host preferences are mapped onto the tree from the Euphorinae mainly because of the curved forewing vein RS combined molecular analysis using the Fitch algorithm, it is (radial vein), which is considered a defining characteristic of the fairly clear that the Euphorinae were originally parasitoids of Euphorinae (Wharton et al., 1997). Interestingly, some members Coleoptera (Fig. 3). The sister group of the Euphorinae, Ceno- of the Brachistinae also have a similarly bent RS. A key charac- coelinae, are confined to coleopteran hosts. Meteorini, the sister ter separating members of Brachistini from Centistini is the vein group of the remaining Euphorinae, attack both Coleoptera and M + CU, which is sclerotized in the former but not in the latter. Lepidoptera hosts, and the Euphorinae excluding Meteorini are Interestingly, Asiacentistes and Centistoides differ from other predominantly coleopteran parasitoids. Both the Cenocoelinae Centistini in having a sclerotized M + CU, like the Brachistini, and Meteorini are completely specialized on immature hosts indicating that they may be misplaced in the Centistini or (larvae), which is the overwhelmingly predominant biology in even in the Euphorinae. Our analyses indicated that Asiacen- the Braconidae as a whole. This clearly suggests that larval tistes may be the sister group to Cenocoelinae + Euphorinae parasitism represents the ancestral state of the Euphorinae. In (Figs 1, 2). If so, it might have to be placed in a subfamily of

Ichneumonidae

Braconidae

Asiacentistes sp. Cenocoelius sp.

Meteorus sp.

Zele sp. Meteorini

Meteorus sp. Lepidoptera Coleoptera Ecnomios sp. Ecnomiini Dinocampus coccinellae Napo townsendi Centistina sp. Betelgeuse sp. Dinocampini Ecclitura primoris Planitorus sp. Mannokeraia sp. Planitorini Elasmosoma michaeli Neoneurus sp. Neoneurini Allurus muricatus Centistes ater Syrrhizus sp. Centistes sp. Centistini Syrrhizus sp. Centistes sp.

Litostolus sp. Pygostolus falcatus Pygostolini Ropalophorus clavicornis Tuberidelus flavicephalus Plynops hansoni Cryptoxilos sp. Cosmophorini C. (Eucosmophorus) sp. Cosmophorus sp. Myiocephalus boops Syntretus sp. Myiocephalini Bracteodes sp. Syntretus sp. Syntretellus sp. Syntretini Syntretus sp.

Chrysopophthorus sp. Wesmaelia sp. Helorimorphini Holdawayella sp. Aridelus sp. Peristenus sp. Leiophron dispar Mama mariae Euphorini Leiophron sp.

Gen nov. Marshiella bobella Proclithrophorus sp.

Townesilitus sp. Townesilitini

Host preferences Streblocera sp.

Coleoptera larva Gen nov. Heia robustipes Lepidoptera larva Gen nov. Coleoptera Perilitus sp. Hymenoptera Microctonus sp. Perilitus falciger Psocoptera Rilipertus intricatus Hemiptera Microctonus sp. Perilitus sp. Neuroptera Gen nov. Perilitini Perilitus sp. Unknown Stenothremma sp. Spathicopis flavocephala Orionis sp. 0.5 Perilitus coxator

Fig. 3. Host preferences plotted on the ML tree (see Fig. 1) using the Fitch algorithm and ML in Mesquite. The diagram illustrates the binary state proportional likelihood of host reconstruction (Coleoptera 0.9 vs Lepidoptera 0.1). Polymorphic terminal taxa were scored for the likely ground-plan state (see text). Host records from Yu & van Achterberg (2011); Stigenberg & Ronquist (2011) and Stigenberg & Shaw (2013).

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 579 its own. However, further study of the phylogenetic position of morphologically. The Helorimorphini stand out by having Asiacentistes, Centistoides and possibly other taxa belonging unusual features such as a long ovipositor, a long and slender to the Brachistini and Centistini is clearly needed before such a petiole, and aberrant surface sculpture, whereas the Euphorini new subfamily can be properly circumscribed. have a short ovipositor, a short and sturdy petiole and surface The type of Tainiterma pachytarsis van Achterberg & Shaw sculpture similar to other euphorines. was placed among the Euphorinae in a monotypic tribe, the Our analyses also supported a sister-group relationship Tainitermini (van Achterberg & Shaw, 2001). The Tainitermini between Townesilitini and Perilitini. This clade was strongly resemble the tribe Centistini in characters such as the wing supported both in the concatenated analysis (PP 1.0, BP 0.73) venation, carination of the propodeum, the size of the laterope andinthe28S analysis (PP 0.1). They are fairly similar mor- and the widened ovipositor sheaths (van Achterberg & Shaw, phologically, both in terms of wing venation and other attributes 2001). Tainiterma pachytarsis was unexpectedly returned out- such as ovipositor and petiolar structure, which is reflected by side of the Euphorinae, as sister to Diospilus (Brachistinae). their intertwined taxonomic history (see discussion under each This species was included in this study with data from 28S tribe). They are also similar in that they both attack Coleoptera. only; we hoped that including this taxon, with limited data, The sister-group relationship between Planitorini + would resolve its placement or at least give indication for (Neoneurini + Centistini) was also fairly strongly supported in sister-group relationships. It remains possible that T. pachytarsis the concatenated analyses (PP 0.90; BP 0.65). Morphologically, belongs among the Helconinae, but its placement may also these three tribes are very different; especially the Planitorini be an artefact from limited data. Here, we treat the Tainiter- stand out with its strongly protuberant face. mini as unplaced but we are confident in excluding it from Our results did not resolve the position of many tribes. This the Euphorinae. may be caused by insufficient taxon sampling, misleading signal Our analyses presented here, as well as those in Stigenberg & from individual genes, or rapid diversification in the early Ronquist (2011), support the long-standing hypothesis that the evolution of the group, as has been speculated for similar cases Meteorini form the sister-group of the remaining Euphorinae. occurring elsewhere in the Braconidae and in the Platygastroidea However, the support is not overwhelming, so future analyses (Whitfield et al., 2002; Murphy et al., 2007; Li et al., 2008). may well show that the Meteorini are more deeply nested inside We were hoping that increasing the density of taxon sampling the Euphorinae. Because the Euphorinae including Meteorini would help to resolve these relationships. However, compared have been consistently and strongly supported as a monophyletic to the analyses presented in Stigenberg (2013), we have added clade both in our analyses and in previous studies, and because it 61 new taxa without getting much better resolution at the deep is possible to characterize this clade morphologically, we prefer level in the euphorine phylogeny. Hopefully, an increase in the to include the Meteorini as a tribe in the Euphorinae rather than number of genes sampled will help resolve these relationships treating it as a separate subfamily. in the future. Undoubtedly, even richer taxon sampling will also contribute.

Euphorine relationships

Perhaps the most important outcome of the present study is Evolution of host associations in Euphorinae that it identifies a set of major lineages that largely correspond to the tribes recognized by Yu & van Achterberg (2011) as strongly The original ideas about the evolution of host utilisation in supported monophyletic groups, while also demonstrating that euphorines go back to Tobias (1966). In the ‘Chrysomelidae- a number of taxa are currently misplaced. The new tribal clas- hypothesis’, as denoted by Shaw (1985), Tobias stated that adult sification proposed by us (Appendix) is based on these strongly parasitism in Euphorinae originally evolved from parasitism supported lineages. Many of the tribes recognized here are rela- of chrysomelid larvae. Chrysomelidae parasitism is rather rare tively homogeneous with respect to their morphology and biol- among the Braconidae (Shaw, 2004), but is found in the tribes ogy, although the identification of potential morphological and Perilitini, Centistini, Microctonini and Townesilitini (Shaw, biological synapomorphies will require a more detailed analysis 1988), which are scattered in the Euphorinae phylogeny. There- of the available evidence. fore, Tobias suggested that chrysomelids are likely to be the In addition to supporting a sister-group relationship between ancestral hosts of the Euphorinae, which must have shifted from the Meteorini and the core Euphorinae, our analyses pro- attacking larvae, as other braconids do, to attacking adults early vide some evidence concerning relationships among tribes in in their evolution. Our current results show that the earliest the Euphorinae. The clade Helorimorphini + Euphorini was euphorines were larval parasitoids, and that the shift to adult strongly supported in the separate analysis of the CAD gene parasitism did not occur until later in their radiation, when the (PP 0.99) and in the concatenated analysis (PP 0.76, BP core Euphorinae originated (Figs 1–3). 0.42). This relationship makes sense considering the similari- Our results support the study of Stigenberg & Ronquist (2011) ties between these tribes in host associations, as both special- with respect to the likely ancestral host of the Meteorini, namely ize on hemimetabolan hosts (Psocoptera, Hemiptera), with the lepidopteran larvae. The Meteorini include larval parasitoids of exception of Chrysopophthorus of the tribe Helorimorphini that both Coleoptera and Lepidoptera hosts, and it is obvious that parasitizes Neuroptera. However, the tribes are quite distant there have been multiple shifts between hosts of the two orders.

However, whether the ancestral host was a coleopteran or a lep- Centistini Capek idopteran is not immediately obvious from the mapping of host (Figs 6–8) preferences onto the Meteorini tree alone; signal from outgroups Shaw (1985) included Allurus, Centistes, Pygostolus and is essential in resolving this issue. The larger phylogenetic Spathicopis within Centistini based on his phylogenetic analysis context considered here suggests that the Meteorini evolved of morphological data. van Achterberg (1977, 1985, 1992) from parasitoids of Coleoptera larvae, even though they must added Litostolus and Centistoides and Belokobylskij (1995) have shifted to lepidopteran hosts before the radiation of the added Asiacentistes (Fig. 4). crown group given the predominance of lepidopteran hosts In the phylogenetic analyses by Belshaw & Quicke (2002) among the basal lineages (Stigenberg & Shaw, 2013). and Li et al. (2003), Pygostolus and Centistes ended up in dif- Overall, the conclusions on phylogenetic relationships pre- ferent clades, suggesting that Pygostolus was misplaced in the sented by Tobias (1965, 1966), based on his knowledge of mor- Centistini. This result is confirmed by our analyses. Specif- phology and biology, are remarkably accurate when it comes to ically, our results show that Pygostolus and Litostolus form the larger picture. For example, he included both Neoneurini and a monophyletic lineage of their own, which we recognize as Meteorini at a basal position in the Euphorinae (Tobias, 1966). a separate tribe, the Pygostolini. Our phylogenetic analyses Shaw was the first to use a computerized algorithm approach also show that Spathicopis was incorrectly placed in the Cen- to untangle Euphorinae phylogeny (Shaw, 1985). His analysis tistini. It is deeply nested inside the Perilitini, to which it is was based on 68 morphological characters, and the results dif- transferred here. fered considerably from the ideas presented by Tobias. To a large Our analyses placed Asiacentistes as sister to Cenocoeli- extent, the conflicting results are due to different approaches nae + Euphorinae. This is surprising given the current place- to the weighting of morphological characters. Shaw’s method ment of Asiacentistes in Centistini. However, several morpho- is based on computerized analysis of equally weighted mor- logical traits of Asiacentistes suggest that it may be misplaced phological characters, whereas Tobias assigns more importance in the Centistini, such as the sclerotized forewing vein M + CU, to certain characters, and constructs his trees intuitively. Both the lack of a malar suture and the eyes lacking setae, all of hypotheses have their merits but share a great deal of conflict which are unusual Centistini characters. In fact, these charac- with our current results (Table S1). ters are common in the Helconinae, possibly indicating that Asiacentistes may not belong to Euphorinae, even though it Taxonomy does have some characteristic euphorine features, such as the shape of the petiole. Because of the clear molecular signal in Proposed higher-level classification our study, we exclude Asiacentistes from the Centistini and the Euphorinae. Based on the phylogenetic analyses, we propose a new Specimens of Centistoides were not included in our molecular higher-level classification of the Euphorinae (Appendix) that analysis, although holotype and additional specimens were recognizes 14 tribes: Centistini, Cosmophorini, Dinocampini, examined later and compared to core Centistini (Allurus and Ecnomiini, Euphorini, Helorimorphini, Meteorini, Myio- Centistes). Our tentative conclusion is that Centistoides should cephalini, Neoneurini, Perilitini, Planitorini, Pygostolini, be excluded from the Centistini and the Euphorinae, and that it Syntretini and Townesilitini. The Pygostolini were described may be related phylogenetically to Asiacentistes but we refrain by Belokobylskij (2000a) as a subtribe among the Centistini, from removing it formally from Euphorinae until more evidence but we recognize it as a separate tribe for the first time. All is available. the other tribes have figured in earlier classifications ofthe Euphorinae, but many of them are circumscribed differently The two remaining genera that were originally placed in here than they have been in the past. The tribe Tainitermini, the Centistini – Allurus and Centistes – are characterized by previously included in the subfamily, is excluded from the immobile cerci and a reduced ventral proctigeral sclerite, both Euphorinae here based on our results. potential synapomorphies defining these genera as a mono- Because our phylogenetic analyses indicate that Asiacentistes phyletic group. Here, we restrict the Centistini to that clade does not belong to the Euphorinae, it is tentatively excluded (but see discussion on Centistoides above). Although the mono- from the subfamily. However, as detailed above, further study phyly of the Centistini, circumscribed in this way, was well sup- of the phylogenetic position of Asiacentistes, and also that of ported in all of our analyses, the different subgenera of Centistes Centistoides (no molecular data available at this point), would recognized earlier (van Achterberg, 1985; Belokobylskij, 1992, be extremely valuable. 2000d) were not. Our analyses indicate that both the subgen- The probable placements of genera not included in the present era Syrrhizus and Ancylocentrus are paraphyletic with respect study are discussed below under each tribal heading (see to Centistes s.s. Furthermore, Centistes s.l. appears to be para- also Table S5). The conclusions presented here are based on phyletic with respect to Allurus. However, our study included morphological comparison of the missing taxa to taxa included a relatively small sample of Centistes representatives, and we in this study. In some cases, type specimens of the missing taxa refrain from proposing changes to the current generic and sub- were unavailable, and either nontype specimens or the original generic classification of the tribe until a more comprehensive descriptions were used as the basis for our conclusions on likely analysis can be completed. Meanwhile, it may be advantageous phylogenetic relationships (see Table S5 for details). to follow Shaw (1985) in not recognizing subgenera in Centistes.

Centistini

Cosmophorini

Dinocampini

Ecnomiini Euphorini

Helorimorphini Myiocephalini

Meteorini

Neoneurini

Figs. 4–51. 4, Illustrations of the forewing venation of Asiacentistes;5,Cenocoelius; 6–51, 46 Euphorinae genera. 15, 19 and 44, Redrawn from their original illustrations (De Saeger, 1946; Shaw, 1996, 2012). References for following figures; 8 (van Achterberg, 1992); 20, 41 (van Achterberg, 1995); 38, 51 (Chen & van Achterberg, 1997); 10 (van Achterberg & Quicke, 2000); 9, 14, 16, 43 (Wharton et al., 1997); 21 (Park & van Achterberg, 2004); 24, 39 (Belokobylskij 2000).

Figs. 4–51. Continued.

The Centistini, as circumscribed here, parasitize adult Eucosmphorus) within the Cosmophorini. Here we redefine Coleoptera of the families Chrysomelidae, Curculionidae and Cosmophorini to also include Ropalophorus (transferred from Staphylinidae. Dinocampini), Cryptoxilos and Plynops (transferred from Euphorini), and Tuberidelus (transferred from Perilitini). Diagnosis. Maxillary palpi with six segments, labial palpi Cosmophorus, Cryptoxilos, Plynops, Ropalophorus and Eucos- three segments, eyes with short but sparse setae; first meta- mophorus are all parasitoids of adult scolytine beetles in the somal tergite (petiole) sessile, short and with laterope but Curculionidae (Coleoptera). McDonald’s cladistic analysis lacking dorsope; cerci immobile and the ventral proctigeral (2001) grouped Cryptoxilos, Plynops and Cosmophorus based sclerite reduced; ovipositor broad and curved; tarsal claws on several characters, such as the reduction/loss of wing veins. simple or apically split (bifurcate); marginal cell of forewing Ropalophorus was placed as sister to Betelgeuse in McDonalds large, longer than stigma, vein r present, vein 2M desclero- analyses, that is, outside the Cosmophorini. In fact, the genus tized, presence of vein (RS + M)b, vein m-cu distinctly shorter Ropalophorus is quite different from the other Cosmophorini, than vein 2RS. with its clavate antennae (simple in other genera), complete wing veins (reduced in other genera), and sessile petiole (stalked Cosmophorini Muesebeck & Walkley in other genera). Previously, it has been placed in three different (Figs 9–14) tribes (Dinocampini, Perilitini and Euphorini). It was the study Van Achterberg & Quicke (2000) included only Cosmophorus by Li et al. (2003), which first indicated a relationship with and Sinuatophorus (here recognized as a junior synonym of Cosmophorus (BP >0.97 for Cosmophorus + Ropalophorus

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 583 in all four of their analyses). The close relationship between Diagnosis. Maxillary palpi with 3–5 segments, labial palpi Cosmophorus and Eucosmophorus (sister groups in our analy- with two segments, eyes with setae; first metasomal tergite ses) is clearly supported by morphological characters, such as petiolate, ovipositor long and slender; tarsal claws simple; the shape and structure of the petiole, the nervature of the wings marginal cell of forewing variable in shape, vein r about half and the shape of the head, and hence we retain Eucosmophorus the length of vein m-cu, vein 2M sclerotized, vein (RS + M)b at as subgenus to Cosmophorus as suggested by Belokobylskij. least as long as vein r, vein m-cu shorter than vein 2RS. The genera Tuberidelus, Cryptoxilos and Plynops appeared as a monophyletic clade in our analyses. Morphologically, the close Ecnomiini van Achterberg relationship among these genera is evidenced by the appearance (Figs 20, 21) of the wing – such as the small marginal cell and the loss Mason (1979) described Ecnomios from Papua New Guinea. of nervature – but also by the slightly rugose, striated dorsal He placed the genus within the Orgilinae. Park & van Achter- structure of the petiole. We were unable to obtain sequences berg (1994) redefined it as its own subfamily Ecnomiinae. from the Cosmophorus subgenera Regiphorus van Achterberg, Belshaw & Quicke (2002) and Sharanowski et al. (2011) showed so we cannot comment on its status. that Ecnomiinae should be included among the Euphorinae. Two genera are currently recognized among the Ecnomiini: Ecnomios and Korecnomios. We were only able to include spec- Diagnosis. Maxillary palpi with 4–5 segments, labial palpi imens from Ecnomios in our molecular analysis, but the results with 1–2 segments, eyes with short setae, propodeum and first clearly show that they belong within the Euphorinae, even if the metasomal tergite varying widely in shape, first metasomal ter- exact placement of Ecnomiini is not well resolved. According to gite parallel-sided or somewhat widened posteriorly or narrowed Quicke (personal comment) Korecnomios should not be placed apically, ovipositor broad; tarsal claws simple; marginal cell of among the Euphorinae but somewhere within the Helconinae, forewing variable in shape regarding sclerotization and size; rendering the tribe Ecnomiini paraphyletic. Unfortunately we small, equal to or smaller than stigma, vein r present or absent, were unable to investigate this further and until this has been vein 2M sclerotized, vein (RS + M)b not present, length of vein properly investigated, we tentatively keep Korecnomios within m-cu and vein 2RS variable. the Ecnomiini. Host records are unknown.

Dinocampini Shaw Diagnosis. Maxillary palpi with five segments, labial palpi (Figs 15–19) with three segments, eyes bare; first metasomal tergite sessile, Shaw (1985) proposed this tribe for the genera Centistina, ovipositor slender and short, hardly protruding beyond meta- Dinocampus and Ropalophorus, united by the following mor- soma; tarsal claws simple; marginal cell of forewing variable, phological characters: ocular setae present, antennal scape 3× vein r long, almost as long as vein m-cu, sclerotization of vein longer than wide and labial palpus reduced to two segments. 2M variable, vein (RS + M)bshorterthanveinr,veinm-cu Later, Shaw (1988, 2002, 2012) included the genera Betelgeuse slightly shorter than vein 2RS. and Napo. Our molecular analysis confirmed that all of the above genera belong in the Dinocampini except Ropalophorus that Euphorini Förster we transferred to the Cosmophorini (see that tribe). Our anal- (Figs 22–24) ysis also showed that the genus Ecclitura should be included In order to avoid confusion, it seems essential to preface this within the Dinocampini. The placement of Ecclitura has been section by mentioning that the taxa Euphoriana, Euphoriella, discussed in several publications. Ecclitura was formerly placed Euphorus and Peristenus have all at some point, in a vari- within the Perilitini by Tobias (1986) and further discussed by ety of combinations, been synonymized with or treated as Belokobylskij (2000, 2013). In the phylogenetic study by Shaw subgenera of Leiophron. Euphoriana wasdesignatedasa (1985), however, Ecclitura was suggested as a sister-group of new genus by Gahan (1913), and then synonymized with Streblocera, thus placing it in the Microctonini sensu Shaw Leiophron by Loan (1974). Chen & van Achterberg (1997) (1985). Our analyses are the first to place the genus in the redefined Leiophron and recognized Euphoriana as a dis- Dinocampini. tinct subgenus. Belokobylskij (2000a) revised Leiophron and The Dinocampini are defined by having a long antennal transferred a number of species to Leiophron (Euphoriana). scape and labial palpus with only two segments, petiole with- Euphoriella was originally described by Ashmead (1900). Chen out dorsope except for the genus Centistina. Among the & van Achterberg (1997) treated Euphoriella as a subgenus of Centistina there are some species (e.g. C. zitaniae) with a Leiophron. well-developed dorsope, despite the fact that the dorsope is Euphorus was originally described by Nees von Esenbeck missing from all other known members of the tribe. It is (1834). Muesebeck (1936) synonymized Peristenus with Eupho- unclear at this point whether this represents a secondary rus and later synonymized Euphorus with Leiophron (Peris- reappearance of the dorsope, or that the dorsope has been tenus was later resurrected) (Muesebeck, 1958). Chen & van reduced or lost multiple times within the tribe. Host records for Achterberg (1997) resurrected Euphorus, and finally Belokobyl- Dinocampini are adult Coleoptera of the family Coccinellidae skij (2000a) again treated Euphorus as a subgenus of Leiophron. (Shaw, 2012). Peristenus was described by Förster (1862). It was treated as

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 584 J. Stigenberg et al. a subgenus of Leiophron by Tobias (1986), shortly thereafter redefine the tribe to also include the genera Chrysopophthorus reinstated as a separate genus by Shaw (1987), and then treated and Wesmaelia, both placed in Euphorini by Yu & van Achter- again as a subgenus of Leiophron by Papp (1992). Chen & berg (2011). In Shaw’s (1985) analysis, Aridelus came out as van Achterberg (1997) later resurrected Peristenus, and finally sister group to Wesmaelia and Chrysopophthorus,justasitdidin Belokobylskij (2000a) again treated Peristenus as a subgenus of McDonald’s (2001) reanalysis of Shaw’s paper. In our molecular Leiophron. Thus, the inclusiveness of the taxon Leiophron,in phylogeny, however, it is Chrysopophthorus that comes out as particular, can vary depending upon the author’s treatment of sister to Aridelus + Wesmaelia. the genus. Shaw’s (1985) and McDonald’s (2001) analyses placed Hold- Shaw (1985, 1996) included the genera Aridelus, Chrysopoph- awayella in the Euphorini. However, this is likely due to erro- thorus, Cryptoxilos, Euphoriella, Holdawayella, Leiophron, neous character coding. The critical character in Shaw’s and in Peristenus, Stenothremma and Wesmaelia within Euphorini. McDonald’s analyses concerns the median frontal carina. It is Belokobylskij added Mama and transferred Aridelus to Helo- either: (0) absent; (1) ending near middle of frons at least 3 ocel- rimorphini (Belokobylskij, 2000a), and later treated Peristenus lus widths below median ocellus; or (2) extending to median as a subgenus of Leiophron (Belokobylskij, 2000c). ocellus. In Aridelus this character is coded as (2), whereas in Here, we redefine Euphorini to exclude all but Leiophron, Holdawayella the character was noted as absent (state 0). How- Peristenus and Mama among the taxa included in the molecular ever, the specimen we studied (Holdawayella minuta, synonym analysis. The monophyly of this clade is strongly supported by of Ussuraridelus minutus, determined by Belokobylskij) has our results. The classification of Euphoriana, Euphoriella and a frontal carina extending all the way to the median ocellus Euphorus either as separate genera or subgenera of Leiophron and should be coded as (2). The holotype of Holdawayella remains unsettled; they are all treated here as subgenera of tingiphaga, which is the type species of Holdawayella,hasa Leiophron. faint median carina, but it clearly extends almost to the ocelli, Specimens of Euphorini used in this study were identified to so it also has state 2 of this character. In addition to this error, the lowest level possible in order to examine potential mono- there is also an error in the number of segments in the maxil- phyletic subgroups within the tribe. The identification was lary palp. Both Shaw and McDonald coded Holdawayella as based on the key and diagnostic characters presented in Chen & having five-segmented maxillary palpi but the description of van Achterberg (1997), but these were sometimes problematic. Holdawayella tingiphaga by Loan (1967) states six-segmented In particular, their couplet 22 included qualifiers and exceptions palpi. The specimen of Holdawayella minuta we studied also has that prevented unambiguous identification of some specimens 6-segmented palpi. When McDonald’s morphological matrix is included in our analyses. It was particularly difficult to dis- re-run with these corrections using MrBayes 3.2.2. (Ronquist tinguish Euphorus spp. and Leiophron (Leiophron) spp., and et al., 2012), Holdawayella turns up as sister to Aridelus.We one specimen (labelled as Leiophron (Euphorus/Leiophron)) tested coding the median frontal carina character both as state 2 exhibited characteristics of both groups (e.g. forewing vein and state 1 in Holdawayella; in both cases, Holdawayella ends (RS + M)a present, hindwing vein cu-a absent, occipital carina up as sister to Aridelus. It is only with state 0 that Holdawayella interrupted dorsally). ends up within the Euphorini. Unfortunately, our analysis did not produce any answers con- The biology of Holdawayella is more consistent with a cerning the validity of the subgenera and better sampling is placement in the Helorimorphini than in the Euphorini. Hold- needed to untangle this question. There are many host associ- awayella overwinters as an immature larva in the host, like ation references concerning the Euphorini, such as Coleoptera other Helorimorphini (data available for Wesmaelia), but unlike (four families), Hemiptera (four families) and Psocoptera (six the Euphorini (data available for Leiophron, Peristenus and families). However, the reliable host records are confined to Euphoriella), which overwinter as adults in the cocoon (Loan, Chrysomelidae, Miridae and Psocidae. 1967). The reduced wing nervature of Holdawayella is similar to that of many Euphorini, but this is clearly a trait that is asso- Diagnosis. Maxillary palpi with five segments, labial palpi ciated with small body size (Tobias & Belokobylskij, 1981) and with 2–3 segments, eyes bare; first metasomal tergite petiolate, that has evolved many times within the Euphorinae. ovipositor slender and short, hardly protruding beyond meta- The members of Helorimorphini parasitize both Neuroptera soma; tarsal claws simple; marginal cell of forewing equal or and Hemiptera. Chrysopophthorus parasitize the genera smaller than stigma, seldom large, vein 3RSb strongly bent; Chrysopa and Mallada (Chrysopidae) whilst the reminder vein r short or absent, vein 2M desclerotized, vein (RS + M)b of the Helorimorphini parasitize on Nabidae and Pentatomidae, not present, length of vein m-cu (if present) shorter than length as well as other families among the Hemiptera. Wesmaelia of vein 2RS. parasitize on the genus Nabis, Holdawayella on Corythucha and Aridelus on several families, such as Pentatomidae and Acanthosomatidae (Yu & van Achterberg, 2011). Helorimorphini Schmiedeknecht (Figs 25–28) Yu et al. (2011) included only the genera Aridelus (Helorimor- Diagnosis. Maxillary palpi with six segments, labial palpi pha is a junior synonym) and Ussuraridelus (here recognized as 3–4 segments, eye setosity variable, presence of a setal comb on a junior synonym of Holdawayella) in Helorimorphini. Here we the inner apex of the hind tibia; first metasomal tergite petiolate,

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 585 ovipositor short or 0.2–0.3× forewing, claws simple or with length of vein m-cu and vein 2RS almost equal in length, lacking pectinate basal lobe; marginal cell of forewing variable, vein vein RS + M. r long, equal to vein m-cu, vein 2M distinctly curved, vein (RS + M)b present, length of vein m-cu and vein 2RS variable. Neoneurini Bengtsson (Figs 32, 33) Meteorini Cresson The Neoneurini display some rather unusual morphological (Figs 30, 31) characters (discussed below). They have been placed in vari- The Meteorini include the genera Meteorus and Zele.This ous systematic positions in the Braconidae. Early authors placed group has long been recognized as a monophyletic clade at the them in Braconinae, Agathidinae or Microgastrinae, but these base of the euphorine tree. Both Marshall (1897) and Tobias arrangements were discussed and refuted by Bengtsson (1918), (1966) placed Meteorus as sister to the Euphorinae and Shaw who placed them in their own subfamily Neoneurinae. Tobias (1985) rooted his Euphorinae tree with Meteorus.However, (1966) placed the group within the Euphorinae based on ‘mor- other authors have recognized the clade as a separate subfam- phological and biological peculiarities’, but this placement was ily and questioned the close relationship to other euphorines not accepted by later workers (van Achterberg, 1984; Shaw, (Förster, 1862; Shaw, 1985; Maeto, 1990; Zitani, 2003). The 1985). Today, the inclusion as a tribe among the Euphorinae is first cladistic analysis of the placement of the clade that included well accepted (Gómez Durán & van Achterberg, 2011; Shara- appropriate outgroups was presented in the dissertation by nowski et al., 2011). Belokobylskij (2000a) includes the genera McDonald (2001); it showed that the meteorines are basal Elasmosoma, Euneoneurus, Kollasmosoma, Neoneurus, Pare- euphorines. This is confirmed by our results. lasmosoma and Sinoneoneurus within the Neoneurini. Elas- In our analyses Meteorus was rendered paraphyletic by Zele, mosoma and Neoneurus were the only genera included in our which is consistent with the analyses of Belshaw & Quicke molecular analysis, but our morphological study of additional (2002) and Stigenberg & Ronquist (2011). This is probably material supports Belokobylskij’s circumscription of the tribe. best solved by dividing the genus Meteorus into two genera. Our analyses of the concatenated data indicated a sister-group We refrain from proposing such a change here because further relationship between Neoneurini and Planitorini (PP 94%) but analysis is needed before all three Meteorini genera resulting they are morphologically strikingly dissimilar. The wing vena- from such an action can be clearly defined by morphological tion of the Neoneurini is very unusual: the radial cell (marginal characters. cell) in the forewing is divided in two, a supplementary cell that according to Tobias (1966) is not formed by the radial vein (RS) Diagnosis. Maxillary palpi with six segments, labial palpi ‘but by a darkened straight fold of the wing’. Tobias (1966) with three segments, eyes with setae; first metasomal tergite points to the similarity to the wing venation of ants, and pro- petiolate, ovipositor length variable; tarsal claws variable, sim- poses that this is due to a special kind of mimicry, also seen ple or lobed; marginal cell of forewing larger than stigma, vein amongst Hybrizontinae (Ichneumonidae) and Polyaulon Förster 3RSb straight; vein r variable in length, vein 2M sclerotized, vein 1869 (Ichneumonidae, Cryptinae). The Neoneurini parasitize (RS + M)bvariableinlength,lengthofveinm-cuinrelationto adult worker ants (Formicidae). vein 2RS variable. Diagnosis. Maxillary palpi with 2–3 segments, labial palpi Myiocephalini Chen & van Achterberg with 1–3 segments, eyes bare; first metasomal tergite sessile, (Fig. 29) ovipositor short, bent, variable in broadness; tarsal claws slen- The genus Myiocephalus Marshall (recognized as Loxo- der; wing venation largely obsolete except for a strong thick cephalus Förster) was placed in its own tribe Loxocephalini by costa, and vein r present. Shaw (1985). Foissner & van Achterberg (1997) pointed out that the generic name Loxocephalus Förster was preoccupied, and Perilitini Förster that the later name Myiocephalus Marshall must be used instead. (Figs 34–39) The tribal name Myiocephalini was first used by Chen & van Belokobylskij (2000a) included Dinocampus, Ropalopho- Myiocephalus Achterberg (1997). The genus is associated with rus, Marshiella, Perilitus, Rilipertus and Streblocera within Formica ant nests of the genus but has never been reared (Don- Perilitini, and treated Microctonus and Townesilitus as sub- isthorpe, 1927). This tribe is sister to the Syntretini and there genera of Perilitus. In the cladistics analysis by Shaw (1987) are some unique morphological characters supporting this place- both Orionis and Stenothremma were placed among the ment, like the bulging eyes and smooth mesosoma. Euphorini according to the following arrangement; Orionis + Stenothremma + (Chrysopophthorus + Wesmaelia) + Aridelus. Diagnosis. Maxillary palpi with five segments, labial palpi Here we redefine Perilitini to include Microctonus, Orio- with three segments, eyes bulging; first metasomal tergite nis, Perilitus, Rilipertus, Spathicopis and Stenothremma.The slender with deep laterope, ovipositor length about 0.8× as long excluded genera were moved as follows: Marshiella (Townesil- as first tergite; claws simple; marginal cell of forewing variable, itini), Streblocera (Townesilitini) and Tuberidelus (here placed vein r about half the length of m-cu, vein (RS + M)b not present, within Cosmophorini).

The genus Perilitus came out as paraphyletic in our analysis, et al. (2011). The monophyly of Planitorini was well supported as suspected by Shaw (1985). The European Perilitus speci- in all of our analyses. mens were placed in a sister-group relationship with Microc- tonus, whilst the specimens from other parts of the world Diagnosis. Maxillary palpi with 5 segments, labial palpi with (Africa/Madagascar/French Guiana) formed the sister group of a 4 segments, eyes with sparse setae; first metasomal tergite clade comprising Orionis, Spathicopis and Stenothremma.This variable, petiolate to subsessile, ovipositor length variable; indicates that some revision of the generic classification of the tarsal claws robust and lobed; marginal cell of forewing almost Perilitini is needed, but this will have to be done in the context reaching apex of wing, vein 3RSb straight; vein r short, vein of an analysis including more representatives of the tribe. Some (RS + M)b absent, second submarginal cell present, length of authors have treated Microctonus as a subgenus of Perilitus,but vein m-cu equal to length of vein 2RS. this does not solve the problem and we suggest that Microctonus be maintained as a separate genus. Further study may indicate that the best solution is to restrict Perilitus to European species, Pygostolini Belokobylskij retain Microctonus as a separate genus, and create a new genus (Figs 42, 43) for the nonEuropean members of Perilitus. Haliday first described the genus Pygostolus in Haliday, The Perilitini parasitize adult Coleoptera, and are recorded 1833. Belokobylskij (2004) included the genus in Centistini but from several families. We consider the records from Ceramby- placed it in a separate subtribe Pygostolina that also included cidae and Chrysomelidae as the most reliable ones. Blackith Spathicopis. Our results show that Pygostolus forms an isolated (1967) reported rearing specimens of Perilitus morabinarum euphorine lineage together with the genus Litostolus and we Blackith from wild-caught adult Orthoptera in the family propose a separate tribe for them, Pygostolini. Spathicopis is Eumastacidae. However, the phylogenetic placement of this not closely related to Pygostolus; it belongs to the Perilitini, to euphorine species needs to be confirmed. The illustrations by which it is transferred here. Pygostolini parasitize Coleoptera in Blackith (1967) suggest that the species may well belong to the the family Curculionidae. Dinocampini or Euphorini rather than the Perilitini. The host association with Orthoptera makes us believe that the species is Diagnosis. Maxillary palpi with 5 segments, labial palpi likely to belong to the Helorimorphini + Euphorini, where there with 3 segments, eyes bare; first metasomal tergite variable, have been several major host shifts to other insect orders such subsessile to sessile, with deep dorsope and laterope; ovipositor as Hemiptera, Psocoptera and Neuroptera. length variable, straight to bent, blade-like; tarsal claws simple The biology of the genus Stenothremma is unknown; deter- but robust, marginal cell of forewing almost reaching apex mining the host of this genus as well as the seemingly para- of wing, vein 3RSb straight, vein m-cu slightly shorter than phyletic Perilitus could help provide important insights into the vein 2RS. evolution of host associations within this tribe. The nonEuro- pean Perilitus species are in dire need of review. Syntretini Shaw (Figs 44–46) Diagnosis. Maxillary palpi with 5–6 segments, labial palpi Shaw (1985) included Bracteodes, Falcosyntretus, Syn- with three segments, eyes sparsely setose or bare; first meta- tretellus, Syntretomorpha, Syntretoriana,andSyntretus within somal tergite petiolate or subsessile, ovipositor length variable; Syntretini. Belokobylskij (1993, 1998, 2000a) added Sculp- marginal cell of forewing variable, 3RSb strongly bent and size tosyntretus and Exosyntretus, and recognized Parasyntretus as as stigma to straight and to apical part of wing, vein r long, vein a subgenus of Syntretus. van Achterberg & Haeselbarth (2003) (RS + M)b absent, length of vein m-cu and vein 2RS equal or synonymized Exosyntretus, Falcosyntretus and Parasyntre- 2RS slightly longer, vein RS + M present in all but Microctonus tus with Syntretus, provisionally recognized Exosyntretus as a Spathicopis and . subgenus of Syntretus, and suggested synonymizing Sculptosyn- tretus with Syntretus. We included representatives of the genera Planitorini van Achterberg Bracteodes, Syntretellus, Parasyntretus, Syntretus and subgen- (Figs 40, 41) era Falcosyntretus in our analyses. The inferred relationships The Planitorini include the genera Planitorus and Mannok- emerging from our analysis only support the synonymization eraia. These genera are endemic to Australia, and have unknown of Falcosyntretus with Syntretus. Syntretellus is well defined life histories. One species of Mannokeraia has been described, as a monophyletic clade and sister to Parasyntretus. These two and it has apterous females. Specimens of additional species are in turn sister to Bracteodes, well separated from the rest of have been collected and included in this study. These species, yet Syntretus. to be described, show strong sexual dimorphism apart from both The genera Sculptosyntretus, Syntretomorpha and Syntretori- sexes being winged. Planitorus and Mannokeraia were origi- ana (sensu Belokobylskij, 1993; Shaw, 1985) are monotypic and nally described as members of Betylobraconinae and Masoni- have traditionally been included amongst Syntretini. Because nae, respectively (van Achterberg, 1995). The first evidence for the Syntretini are defined by numerous, easily recognized mor- including Mannokeraia and Planitorus amongst the Euphorinae phological synapomorphies, the inclusion of these genera in was presented by Belshaw & Quicke (2002) and Sharanowski the tribe is not in question. However, there is a possibility that

© 2015 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society. 40, 570–591 Phylogeny of the Euphorinae 587 some of these monotypic genera should be synonymized with Figure S2. Phylogeny retrieved from a Bayesian phylo- Syntretus because they appear to be exceptional species of Syn- genetic analysis of the 28S molecular data. Numbers on tretus with striking autapomorphies. The Syntretini parasitize branches are posterior probabilities (PP), the PP value 1 is Hymenoptera in the family Apidae. indicated with an asterix (*). Only deeper-level node values are shown. The scale bar represents the expected number of Diagnosis. Maxillari palpi 5–6 segmented, labial palpi 3 substitutions per site. segmented, eyes with setae; first metasomal tergite petiolated, Figure S3. Phylogeny retrieved from a Bayesian phylo- ovipositor length variable, straight to bent; tarsal claws bifur- genetic analysis of the COI molecular data. Numbers on cate; marginal cell of forewing of same length as stigma, vein branches are posterior probabilities (PP), the PP value 1 is 3RSb bent; vein r long, slightly longer than vein 1CUb, vein RS + M absent, second submarginal cell absent, length of vein indicated with an asterix (*). Only deeper-level node values m-cu variable to length of vein 2RS. are shown. The scale bar represents expected number of substitutions per site. Townesilitini Shaw Figure S4. Phylogeny retrieved from a Bayesian phyloge- (Figs 47–51) netic analysis of the CAD molecular data. Numbers on Shaw (1985) included the genera Townesilitus and Marshiella branches are posterior probabilities (PP), the PP value 1 is in the tribe Townesilitini. In contrast, Belokobylskij (2000a) indicated with an asterix (*). Only deeper-level node values treated Townesilitus as a subgenus of Perilitus. We reinstate are shown. The scale bar represents expected number of sub- the tribe Townesilitini with the following genera included: stitutions per site. Marshiella, Townesilitus, Streblocera (transferred from Microc- Table S1. Exemplars used for this study including amplified tonini) and Proclithrophorus (placed in Microctonini by Shaw (1985) and later placed in its own tribe Proclithrophorini by genes, GenBank ID numbers and either references to original Tobias (1986)). publication or collection (Coll), determinator (Det), country More sampling is needed to untangle the boundaries and of origin (Country). The classification of Euphorinae used affiliations of the genus Streblocera with respect to other here is based on the new tribal arrangement proposed in members of the tribe. The genus Streblocera is paraphyletic the current paper. Country codes are based on ISO 3166 with respect to Heia and to the specimen marked as ‘Gen. international standards. Determinator prefixes: CvA, van nov’ in our analyses, which indicates that its circumscription Achterberg; AB, C. A. Boring; JS, J. Stigenberg; SB, S. may have to be changed. The genus Streblocera is currently Belokobylskij, SS, S. Shaw. divided into six subgenera. All are represented in our molecular Table S2. Primer sequences. analysis but there are too few representatives to allow any definite conclusions on their status and on the relationships Table S3. PCR conditions. among them. The Townesilitini parasitize Coleoptera of the Table S4. List of genes with number (N) of taxa, alignment family Chrysomelidae. length (L), and total number (N) of parsimony informative characters for each gene. Diagnosis. Maxillari palpi 5–6 segmented, labial palpi 3 Table S5. Genera and subgenera that were not available segmented, ventral rim of clypeus with granulated sculpture, for the molecular characterization of the Euphorinae but eyes bare; first metasomal tergite petiolate; marginal cell of forewing of same length as stigma or shorter, vein 3RSb bent, that were studied morphologically either from specimens or vein r at least half the length of m-cu, vein RS + Mabsent, original descriptions. The tribal classification indicated here second submarginal cell absent, vein m-cu equal in length to or is the one proposed in the current paper. The tribal placement shorter than vein 2RS. of the taxa is the same in Yu & van Achterberg (2011).

Supporting Information Acknowledgements

Additional Supporting Information may be found in the online We are grateful to Seraina Klopfstein, Tobias Malm and Markus version of this article under the DOI reference: Englund for their help with methods, discussions and comments 10.1111/syen.12122 on the manuscript. We also thank Rasa Bukontaite for invaluable assistance with lab work, and Johannes Bergsten for insightful Figure S1. Phylogeny retrieved from a Bayesian phylo- comments on the manuscript. We are also grateful to Lars genetic analysis of the 18S molecular data. Numbers on Vilhelmsen, Sergey Belokobylskij, Kees van Achterberg, Mark branches are posterior probabilities (PP), the PP value 1 is Shaw, Scott Shaw, Mike Sharkey, Barbara Sharanowski, Donald indicated with an asterix (*). Only deeper-level node values Quicke, Caroline Boudreault, José Fernandez-Triana and Yves are shown. The scale bar represents expected number of sub- Braet for help with material and comments. Kevin Pitz kindly stitutions per site. allowed us to use his photograph of the Cenocoelius forewing

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Appendix I. Myiocephalini, Chen & van Achterberg ∘ Proposed classification of the extant Euphorinae including Myiocephalus, Marshall* 14 tribes, 52 genera and 17 subgenera (including nominate subgenera). Synonyms are not listed. An asterix (*) after a taxon Meteorini,Cresson ∘ indicates that it was included in our molecular analyses and a Meteorus, Haliday* ∘ circle (∘) indicates if a specimen was studied morphologically. Zele, Curtis* Genera in bold have been transferred since the summary by Yu & van Achterberg (2011). Neoneurini, Bengtsson Elasmosoma, Ruthe*∘ Centistini, Capek Euneoneurus, Tobias & Yuldashev∘ Allurus,Förster*∘ Kollasmosoma, van Achterberg & Argaman∘

Neoneurus, Haliday*∘ Syntretellus,deSaeger*∘ Parelasmosoma, Tobias & Yuldashev∘ Syntretomorpha,Papp Sinoneoneurus, He, Chen & van Achterberg Syntretoriana, Parrot Syntretus,Förster*∘ Perilitini,Förster Subgenus Syntretus Orionis Shaw*∘ Subgenus Falcosyntretus, Tobias*∘ Perilitus, Nees*∘ Subgenus Exosyntretus, Belokobylskij Microctonus,Wesmael*∘ Subgenus Parasyntretus, Belokobylskij*∘ Stenothremma,Shaw*∘ Spathicopis, van Achterberg*∘ Townesilitini,Shaw Rilipertus, Haeselbarth*∘ Townesilitus, Haeselbarth & Loan*∘ Marshiella,Shaw*∘ Planitorini, van Achterberg Streblocera, Westwood Planitorus, van Achterberg*∘ Subgenus Streblocera*∘ Mannokeraia, van Achterberg*∘ Subgenus Lecythodella, Enderlein ∘ Subgenus Asiastreblocera Belokobylskij*∘ Pygostolini, Belokobylskij Subgenus Cosmophoridia Hedqvist*∘ Pygostolus, Haliday*∘ Subgenus Eutanycerus Förster*∘ Litostolus, van Achterberg*∘ Subgenus Villocera Papp*∘ Proclithrophorus, Tobias & Belokobylskij*∘ Syntretini,Shaw Heia, Chen & van Achterberg*∘ Bracteodes,deSaeger*∘ Sculptosyntretus, Belokobylskij