Phylogeny of the Subfamily Ophioninae (Hymenoptera: Ichneumonidae)

Zoological Journal of the Linnean Society, 2016, 178, 128–148. With 5 ﬁgures

A molecular and morphological reassessment of the phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae)

PASCAL ROUSSE1,2*, DONALD L. J. QUICKE3, CONRAD A. MATTHEE2, PIERRE Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 LEFEUVRE4 and SIMON VAN NOORT1,5

1Natural History Department, Iziko South African Museum, PO Box 61, Cape Town 8000, South Africa 2Department of Botany and Zoology, Evolutionary Genomics Group, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa 3Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, Thailand 4Cirad, UMR PVBMT, 7 Chemin Ligne Paradis, 97410 St Pierre, France 5Department of Biological Sciences, University of Cape Town, Private Bag, Rondebosch 7701, South Africa

Received 22 September 2015; revised 19 January 2016; accepted for publication 27 January 2016

The phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae) is investigated using molecular markers and morphological characters. We analysed the mitochondrial DNA CO1 and the nuclear 28S D2–D3 gene fragments for 74 species of Ophioninae from 25 out of the 32 recognized genera, which collectively represent 98% of described species diversity of the subfamily. Molecular markers were analysed separately and combined, with or without the adjunction of a matrix of 62 morphological characters using Bayesian inference. Our results reveal three distinct lineages, each including one of most speciose genera: Ophion, Enicospilus and Thyreodon. The comparison of the molecular data, and combined molecular plus morphological data led to the deﬁnition of the three tribes: Ophionini stat. rev. (Ophion + Alophophion + Rhopalophion + Xylophion + Afrophion); Enicospilini stat. rev. (Enicospilus + Laticoleus + Dicamptus + Hellwigiella); and Thyreodonini tribe nov. (Thyreodon + Dictyonotus + Rhynchophion). The possible association of other genera to one or another of these lineages is discussed. Ophion is a polyphyletic assemblage and requires a further revision to deﬁne the delimitation with close genera. The enigmatic Old World genus Skiapus is strongly supported as belonging to the Ophioninae, although its placement within the subfamily is ambiguous as a result of its derived genotype and phenotype. Finally, we propose a biogeographical scenario supported by this phylogeny and based on the limited available fossil data.

ADDITIONAL KEYWORDS: 28S D2–D3 – Bayesian analysis – CO1 – evolutionary history – evolutionary lineage – morphological characters – phylogenetics – systematics – taxonomy.

puscular and are frequently collected at light. They INTRODUCTION are usually pale, moderate- to large-sized wasps with The Ophioninae (Hymenoptera: Ichneumonidae) are long slender antennae and enlarged ocelli (Fig. 1), a relatively large-bodied parasitoid wasps that utilize suite of characters that is known as the ‘ophionoid the larvae of various moths (Lepidoptera), and the facies’ (Gauld & Huddleston, 1976) and which has majority of the species are typically nocturnal or cre- arisen independently in several hymenopteran taxa (Quicke, 2015). Although a large number of specimens have been collected throughout the world dur- *Corresponding author. E-mail: [email protected] ing the past two centuries, reliable host data are

128 © 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE 129 comparatively scarce, especially because of partial or Ophioninae is, however, complex. Earlier treatments erroneous identifications. by Cushman (1947) and later by Townes (1971) The subfamily Ophioninae is represented by attempted to subdivide the subfamily into coherent approximately 1000 species assigned to 32 extant genus-groups or tribes, by the presence or absence of genera. Remarkably, 80% of these species belong to alar sclerites, reduction of the membranous flange on one of the two major genera Ophion and Enicospilus the fore tibia, extension of the postpectal carina or (Yu, van Achterberg & Horstmann, 2012). The taxo- even torsion of the mandibles, but all these charac- nomic classification of the species within the ters appear to be evolutionarily plastic and prone to homoplasy (Gauld, 1985). With the increasing poten- tial of computing tools, Ian Gauld attempted to Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 A reconstruct the life history of the Ophioninae, focus- ing first on the Ophion genus-group (Gauld, 1980) then on the entire subfamily (Gauld, 1985). Although significant progress was made, it should be noted that many of the current taxonomic hypotheses are based on these revisions, which were based only on assessment of morphological characters (Gauld & Mitchell, 1977, 1978; Oosterbroek, 1978; Brock, 1982; Gauld & Carter, 1983; Gauld, 1988; Lee & Kim, 2002; Fernandez-Triana, 2005; Kim, Suh & Lee, 2009; Rousse & van Noort, 2014). More recently, the genus Ophion was investigated with more modern morphological methodologies (discrete and morphometrics) and/or molecular tools (Schwarzfeld & Sper- ling, 2014; Schwarzfeld, Broad & Sperling, 2016), B which highlighted the existence of numerous cryptic species and that the diversity of the family is probably severely underestimated. In the absence of a comprehensive phylogenetic tree for the Ophioninae the interpretation of the evolutionary history of the subfamily is fraught with conjecture. According to Gauld (1985) the Ophioni- nae radiated during the early Tertiary, with one primitive group in the northern hemisphere (represented by the genus Ophion) and a sister lineage from which all the other genera diverged. Recent taxonomic studies have cast some doubt on the tradi- tional assumption that many ichneumonid taxa are of temperate origin and diversified thereafter in the tropics (Quicke, 2012; Veijalainen et al., 2012). C Molecular phylogenetic and morphological studies of the family also suggest that two morphologically dis- tant genera Skiapus and Hellwigia, not considered by Gauld as part of this assemblage, and which had previously been classified within the Campopleginae, are also probably derived members of the Ophioninae (Quicke et al., 2005). The phylogenetic position of the most aberrant of these two genera, Skiapus, is never- theless still debated as it is also sometimes associated with another aberrant subfamily, the Hybrizontinae (Quicke et al., 2009). In an attempt to provide a more robust phylogeny for the Ophioninae, we sequenced the mitochondrial CO1 and the nuclear D2–D3 expansion Figure 1. (A) Afrophion nubilicarpus; (B) Enicospilus region of the nuclear ribosomal 28S genes and drakensbergi; (C) Thyreodon atriventris. combined these data with a set of discrete

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 130 P. ROUSSE ET AL. morphological characters. It was anticipated that MOLECULAR PROTOCOLS the CO1 gene will provide better resolution at the species level, while the more slowly evolving 28S For most specimens, DNA extraction was performed may provide better results at the genus level on entire specimens using a phenol/chloroform/isoa- (Klopfstein, Kropf & Quicke, 2010). Our integrated myl procedure (Sambrook, Fritsch & Maniatis, 1989). approach enabled us to (1) better define the evolu- Chemical penetration of the specimen was facilitated tionary lineages within the subfamily, (2) clarify by performing a ventro-longitudinal incision on the the evolutionary relationships between these major metasoma. Two molecular markers were then ampli- lineages and (3) provide a definitive argument for fied and sequenced (Table 1): the mitochondrial cyto- the assignment of the highly derived, palaeotropical chrome oxidase 1 (CO1) and the D2–D3 expansion of Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 genus Skiapus to Ophioninae. the nuclear ribosomal sequence 28S. Some of the older dried specimens had to be additionally amplified following a nested PCR protocol, using first a MATERIAL AND METHODS CO1 PCR-product diluted to 1:100 and then re- amplified with purposely designed internal primers. MOLECULAR DATASETS All PCR products were subsequently purified using A total of 74 species belonging to 24 genera of the QIAquick PCR purification kit (Qiagen) and Ophioninae were included in the analyses, together cycle-sequenced using BigDye terminator chemistry with 13 outgroup species selected from the closely to (Applied Biosystems). The CO1 sequences were man- more distantly related Anomaloninae, Banchinae, ually aligned, and the 28S D2–D3 sequences were Campopleginae, Cremastinae and Hybrizontinae aligned with MAFFT6 (Katoh & Toh 2008) using the (Quicke et al., 1994, 2009). The localities, dates of G-INS-I algorithm, then manually optimized. collection and accession numbers of all sequenced individuals are summarized in Appendix 1. Most of ANALYSED MATRICES the specimens were recently collected by the Iziko South African Museum in Cape Town (curator Simon Three molecular matrices were built based on the van Noort) and the California Academy of Sciences gene region sequenced: CO1, 28S and CO1 + 28S in San Francisco (curator Brian Fischer). All speci- (the combined analyses utilized a trimmed data set mens had been stored in 90–96% ethanol for varying where only individuals for which both sequences periods, ranging from 1 to 15 years. Additional were available were included). Finally the sequences were obtained from 15- to 25-year-old CO1 + 28S data were analysed with (_morph) or dried specimens stored in the Iziko South African without (_nomorph) morphological matrices. The Museum collections. All the Afrotropical specimens topologies of the _morph and _nomorph trees were were identified to species using a recently developed compared using a maximum agreement subtree con- Lucid matrix key (Rousse & van Noort, 2014). To gruence test (de Vienne, Giraud & Martin, 2007). obtain comprehensive taxonomic coverage, the final genetic data set was augmented with sequences BAYESIAN ANALYSIS available from the GenBank (Benson et al., 2013) and Bold Systems (Ratnasingham & Hebert, 2007) Phylogenetic analyses were done using MrBayes 3.2 databases. (Ronquist et al., 2012) with a standard non-clock tree for all partitions. All parameters except topology and branch lengths were unlinked across partitions. The MORPHOLOGICAL CHARACTERS DATASET morphological characters were set with a Γ-shaped A dataset of 62 morphological characters was con- range of distribution. The nucleotide substitution structed (Appendix 2). All characters were treated model settings were determined with jModeltest2 as unordered in the analyses. Each individual spec- and Bayesian information criteria (Darriba et al., imen was scored to include the range of polymor- 2012). The morphological characters were treated as phisms represented in each species. The species for an undivided partition, the CO1 characters were sub- which no specimens were available in the collection divided into two partitions codons 1–2 and codon 3, were scored according to detailed descriptions and the 28S D2–D3 characters were subdivided into (Gauld & Mitchell, 1977, 1978, 1981; Horstmann, four partitions according to the secondary structure 1981; Dasch, 1984; Gauld, 1985, 1988; Gauld et al., of this region: D2p, D2u, D3p and D3u (p = paired, 1997; Gauld & Janzen, 2004; Quicke et al., 2005; u = unpaired; Gillespie, Yoder & Wharton, 2005; Villemant, Yoshida & Quiles, 2012) and the mor- Butcher et al., 2014). All the ambiguous regions to phometric index of the Taxapad database (Yu align indicated as out by Gillespie et al., (2005; NHR et al., 2012). and RAA) were excluded.

Table 1. Primers and PCR protocols

Primer Sequence Tm (°C) PCR programme

C1-J-1718 50-GGAGGATTTGGAAATTGATTAGTTCC-30 54.2 94 °C, 3 min C1-N-2329 50-ACTGTAAATATATGATGAGCTCA-30 49.5 35 cycles (94 °C, 30 s; 50 °C, 30 s; 72 °C, 45 s) 72 °C, 7 min 28S-D2–D3 fwd 50-GCGAACAAGTACCGTGAGGG-30 61.4 94 °C, 2 min 28S-D2–D3 rev 50-TAGTTCACCATCTTT-30 34.1 35 cycles

(96 °C, 15 s; 57 °C, 30 s; 72 °C, 30 s) Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 72 °C, 7 min CO1 nested fwd 50-ATTGGGTCTCCTCCTCCTGAT-30 59.7 94 °C, 3 min CO1 nested rev 50-GGAGTTCCCGATATAGCTTTTCCT-30 60.0 35 cycles (94 °C, 30 s; 55 °C, 30 s; 72 °C, 45 s) 72 °C, 7 min

The analyses were run with two parallel runs of four 28S ANALYSES Monte Carlo Markov chains each for 5–12 million generations, until the average standard deviation reached The 675-bp 28S matrix included 63 ophionine species below 0.01. The chains were sampled every 1000 gener- (24 genera). The selected substitution models pro- ations, and 25% of the samples were discarded as burn- posed by jmodeltest2 for D2p, D2u, D3p and D3u in. The output run files were checked with Tracer 1.6 were GTR + Γ, SYM + Γ, HKY + I and GTR + Γ, to ensure the stability of the eventual traces (all effec- respectively. tive sample size ≫ 100), and the output summarized Ophioninae (including Skiapus) are recovered as tree was displayed with FigTree 1.4 (both available in monophyletic, separated from the outgroups (Fig. 3, the BEAST package; Drummond et al., 2012) with pos- green). As in the CO1 analyses, the internal topology terior probability (pp) as values of node robustness. In is rather weakly resolved in the 28S analyses. The the results below, the nodes with pp of 90% or more are subfamily is recovered with 1.00 pp but with a large considered as significantly supported. basal polytomy from which emerge two genus- groups: Ophion (Fig. 3, red) and Enicospilus (Fig. 3, blue), both separated from Thyreodon, Euryophion, RESULTS Stauropoctonus, Eremotylus, Sicophion, Lepiscelus and Hellwigia. The genus Ophion is polyphyletic and CO1 ANALYSES only partly supported by 0.78 pp in a polyphyletic The 584-bp CO1 matrix consisted of 11 outgroup spe- lineage mixed with Xylophion, Alophophion, Rhopa- cies and 60 ophionine species (belonging to 17 gen- lophion and Afrophion, the assemblage being linked era). The selected substitution models proposed by with low support (0.60 pp) to Dictyonotus + Rhyn- jModeltest2 for codons 1–2 and codon 3 were chophion. The second major, but weakly supported GTR + I + Γ and HKY + Γ, respectively. The analysis lineage (0.69 pp) leads to a robust Laticoleus + Eni- ran for 5 million generations. cospilus clade (0.99 pp) non-significantly associated Ophioninae (including Skiapus) are monophyletic (0.83 pp) with Dicamptus and Hellwigiella. Skiapus and fully separated from the outgroups (Fig. 2, green). is considered here as a strongly derived genus within The internal topology of the subfamily is represented the basal polytomy of the Enicospilus lineage, but by a large polytomy. All Enicospilus species are recov- with low support. ered in a robust monophyletic clade (1.00 pp) with Lepiscelus distans embedded within it (Fig. 2, red). CO1 + 28S ANALYSES Ophion was recovered as polyphyletic and associated with Xylophion (0.96 pp) and Afrophion (0.69 pp). The combined matrix combined eight outgroup Overall, the relative placement of Enicospilus + Lepis- species and 53 ophionine species represented by 17 celus,theOphion lineage and the remaining genera genera in a 1259-bp-long alignment. The CO1 + are not satisfactorily resolved here, with Dicamptus 28S_nomorph and CO1 + 28S_morph ran for 5 and 12 and Laticoleus not even recovered as monophyletic million generations, respectively. The two trees were genera. Thyreodon is weakly associated with Stau- significantly more congruent than expected by chance 28 ropoctonus and Barytatocephalus with 0.60 pp. (Icong =4.3;P = 1.97 9 10 ).

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Figure 2. CO1 phylogeny; highlighted are the genus-groups discussed in the text (9 scale bars are mean nucleotide substitution rates per site, nodes with posterior probability, pp > 0.90 are considered as signiﬁcant).

Ophioninae are recovered as monophyletic in both Ophion + Xylophion + Rhopalophion + Afrophion; analyses (Fig. 4). When excluding morphological data and (0.89 pp) Thyreodon + Rhynchophion + Dicty- (Fig. 4A), Skiapus is recovered with support of onotus + Stauropoctonus + Eremotylus. Euryophion 0.59 pp as sister taxon to the remainder of Ophioni- is in turn signiﬁcantly associated (0.90 pp) with nae. With 0.90 pp, the remaining ophionine genera these two clades. The Enicospilus genus group com- are subdivided into two Ophion + Thyreodon prises a well-supported (1.00 pp) association between (Fig. 4A, purple and blue) and Enicospilus (Fig. 4A, Enicospilus and Laticoleus, to which Dicamptus and red) genus-groups, including most genera but not Hellwigiella are associated with support of 0.91 pp. Barytatocephalus. The ﬁrst genus-group includes In CO1 + 28S_morph (Fig. 4B), the subfamily is two strongly supported sister-clades: (0.94 pp) mainly subdivided (0.92 pp) into four groups of

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Figure 3. 28S phylogeny; highlighted are the genus-groups discussed in the text (9 scale bars are mean nucleotide substitution rates per site, nodes with posterior probability, pp > 0.90 are considered as signiﬁcant).

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Figure 4. Combined CO1 + 28S trees, with (A) or without (B) the inclusion of morphological characters; highlighted are the genus-groups discussed in the text (9 scale bars are mean nucleotide substitution rates per site, nodes with posterior probability, pp > 0.90 are considered as significant). genera centred around Eremotylus, Thyreodon, clearly does not belong to the Hybrizontinae to which Ophion and Enicospilus. The Eremotylus group com- it was ambiguously associated in former 28S analy- prising Eremotylus + Stauropoctonus is supported by ses because of the aberrant structure of this gene in 0.88 pp. Ophion is still recovered as polyphyletic and Hybrizon (Gillespie et al., 2005; Quicke et al., 2009). grouped (1.00 pp) with Afrophion, Xylophion and Historical higher taxon association of Skiapus Rhopalophion. Thyreodon is grouped (0.99 pp) with reflected its puzzling evolutionary association: the Dictyonotus and Rhynchophion, this clade being genus was first reluctantly placed in Banchinae by moderately associated (0.86 pp) with Euryophion. its author (Morley, 1917), then transferred to Cam- The Ophion, Eremotylus and Thyreodon groups are popleginae as an anomalous tribe (Townes, 1970) in turn moderately associated (0.83 pp) in a single before being recognized as belonging to the Ophioni- polytomic clade. Finally, the Enicospilus lineage is nae based on molecular data and the presence of a formed by a significantly supported (1.00 pp) clade of spurious vein on the fore wing (Quicke et al., 2005). Enicospilus + Lepiscelus + Laticoleus, associated However, its unusual habitus (nothing is known with a moderate probability (0.85 pp) to Dicamptus about its biology) led Quicke et al. (2005) to consider and Hellwigiella. Skiapus appears here as a highly the possibility that the genus may be divergent derived genus weakly associated (0.53 pp) with the enough to represent a new subfamily. Our present Enicospilus lineage. data support Skiapus as a strongly derived genus of Ophioninae, although its definitive placement within the subfamily is not fully resolved. DISCUSSION Data from our analyses support the subdivision of Ophioninae into three clades (Table 2) which essen- TAXONOMY tially match the three genus-groups defined in the All analyses support the monophyly of the Ophioni- morphological analysis of Gauld (1985). A fourth pos- nae, including Hellwigia and Skiapus. Skiapus thus sible clade, including Eremotylus and Stauropoctonus,

Table 2. Proposed taxonomic reassessment of the subfamily Ophioninae according to the three tribes by molecular and morphological data (*no molecular data available)

Morphological Distribution (speciﬁc Tribe synapomorphies Genera included diversity; Yu et al., 2012) Comments

Ophionini stat. rev. Ramellus present; Ophion Worldwide, mostly Paraphyletic 1 m-cu angled; Holarctic (138) mesopleural furrow Xylophion Australasian (2) extended; thyridia Afrophion South Africa (2)

close to the margin Rhopalophion Afrotropical (3) Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 of tergite 2 Alophophion Neotropical (7) Sclerophion* Oriental (2) Thyreodonini tribe nov. Laterotergite 2 Thyreodon Mostly Neotropical (45) pendant; propodeum Rhynchophion Mostly Neotropical (5) with anterior Dictyonotus Southern Africa and transverse carina eastern Asia (4) absent Enicospilini stat. rev. Spiracular sclerite Dicamptus Afrotropical, Oriental partially to totally and Australasian (32) occluded Enicospilus Worldwide, mostly pantropical (699) Laticoleus Afrotropical (11) Hellwigiella Mediterranean (1) Incertae sedis Euryophion Mostly Afrotropical (8) Thyreodonini? Stauropoctonus Worldwide except Nearctic Thyreodonini? Eremotylus Holarctic and Neotropical (17) Thyreodonini? Janzophion Central America (2) Enicospilini? Leptophion Oriental and Australasian (30) Enicospilini? Pamophion Australia (1) Enicospilini? Skiapus Afrotropical and Korea (2) Skiapini? Hellwigia Palaearctic (3) Skiapini? Agathophiona* Mexico (1) Barytatocephalus Eastern Europe to Caucasus (5) Lepiscelus Afrotropical (1) Ophiogastrella Neotropical (6) Orientospilus* India, Madagascar and South Africa (3) Prethophion* Neotropical (1) Riekophion Australia (3) Sicophion* Neotropical (2) Simophion* Central Asia and Central America (4) Trophophion* Western USA (1) appears only in CO1 + 28S_morph with a non-sig- Ophionini and Enicospilini sensu Townes (1971) nificant probability and no support from the other were defined according to the length of the membra- analyses. We therefore do not discuss this clade any nous flange on the front tibial spur, the flange being further. We consider the first three clades to repre- strongly reduced in the latter. The Ophionini were sent tribes, because they are supported by both therefore defined on a plesiomorphic feature. More- morphological and molecular apomorphies, and over, the flange reduction was later acknowledged as hence are relevant to formulate the evolutionary homoplastic (Gauld, 1977). Our analyses support history of the Ophioninae. The clades are repre- homoplasy of this structure, the reduction of the sented by the tribes Ophionini and Enicospilini stat. flange occurring independently in Enicospilini, rev. whose names are resurrected and redefined, Thyreodonini and in Xylophion (Ophionini) in our and the Thyreodonini tribe nov. cladogram. Since Townes’ classification was

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 136 P. ROUSSE ET AL. considered unsatisfactory because of the lack of phy- their molecular analysis combining CO1 and 28S. logenetic support (Gauld, 1977), it is necessary for us The genus was considered to be probably para- to redefine his tribal concepts based on our data. phyletic by Gauld (1985) with respect to some of the minor genera (Agatophiona, Rhopalophion, Sclero- Ophionini stat. rev phion, Afrophion, Xylophion, Alophophion) in his We here morphologically define the tribe Ophionini genus-group. Several studies attempted to clarify the by the angled 1 m-cu and the presence of a ramellus internal phylogeny of the genus (Gauld, 1980; Sch- within the disco-submarginal cell (Fig. 5A, B). This warzfeld & Sperling, 2014; Schwarzfeld et al., 2016). feature is often considered as a plesiomorphic condi- The actual biodiversity has been furthermore grossly tion for ichneumonid wasps, because it appeared in underestimated based on pure morphological taxon- Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 the early fossil subfamilies of Ichneumonidae omy: a recent molecular revision of the Nearctic and (Townes, 1973; Quicke, 2015). However, it evolved European diversities shows there are probably 90– independently within many of the extant subfami- 121 actual species (Schwarzfeld & Sperling, 2015) lies, and in the ‘higher ophioniformes’ (i.e. Cam- instead of the about 50 expected by pure morphology popleginae, Cremastinae, Anomaloninae and (Gauld, 1985). To clarify the definition of Ophion and Ophioninae). The basal character state is a rounded the sibling minor genera would, however, require a 1 m-cu without any ramellus. The reappearance of global revision of the tribe, which is beyond the scope this feature in the Ophionini may thus be considered of the present study. as a reversal. This hypothesis is further supported by the presence of an exceptionally long ramellus in Thyreodonini tribe nov Rhopalophion species, which is far longer than in The genus-group circumscribed by Gauld (1985) any other extant ichneumonid taxon. based on Thyreodon (Thyreodon + Barytato- Ophionini also exhibit a relatively well-developed cephalus + Euryophion + Rhynchophion + Dictyono- mesopleural fovea. The plesiomorphic condition, in tus) was defined by several morphological Ichneumonidae, is the presence of only a small pit apomorphies: the long and straight, or barely positioned mid-posteriorly on the mesopleuron below curved hind tarsal claws; the absence of transverse the speculum (Gauld, 1985). In Ophionini, the pit is propodeal carinae; and the rounded hind tibial extended into a longitudinal or diagonal groove spurs. Moreover, the genus-group is also character- extending toward the anterior edge of the mesopleu- ized within Ophioninae by the reduction of the ron. Finally, the tribe is also defined by the close flagellar length and often reduced ocelli, characters position of the thyridium to the anterior margin of associated with dry hot areas and/or diurnal habits the second metasomal tergite. The distance of sepa- in contrast to the vast majority of other ophionine ration between the thyridium and the anterior tergal species. Gauld (1985) also highlighted some mor- margin is less than the thyridium length in all mem- phological similarities between his Thyreodon and bers of the Ophionini, whereas this distance is Enicospilus genus-groups. All these observations greater than the length of the thyridium in all other suggest that some of the morphological features genera of the subfamily Ophioninae. characterizing Gauld’s Thyreodon genus-group are As defined here, Ophionini includes the four gen- homoplastic as a result of ecological convergence. era Ophion, Xylophion, Rhopalophion and Afrophion. The associations provided by morphological assess- The tribe is consistently retrieved when 28S and ment in the present analyses therefore have to be combined DNA data are analysed. Support for the treated with caution as they could be determined Ophionini clade is further strengthened by the addi- by homoplasy. tion of morphological data. The minor genera Alopho- The molecular data demonstrate that the Thyre- phion and Sclerophion (seven and two species, odon clade represents an evolutionary lineage dis- respectively) most probably also belong to this tribe tinct from the Ophion and Enicospilus clades, but because they share the morphological apomorphies delimitation of this lineage is made ambiguous by a (Gauld, 1985), and for Alophophion this hypothesis is lack of congruence between the molecular and mor- confirmed by the 28S marker. Ophion is by far the phological data. The CO1 marker (Fig. 2) clusters most speciose of these genera and contains 90% of Thyreodon with Stauropoctonus and Barytato- the species in the tribe, and is most diverse in the cephalus while 28S (Fig. 3) links it to Stauropoc- Holarctic region. However, the delimitation of the tonus only, both associations lacking significant genus itself is problematic. In all our analyses, support. In the combined CO1 + 28S_nomorph, Ophion is depicted as polyphyletic. Ophion minutus Thyreodon is strongly associated with Dictyonotus, and the related unidentified sp4 are in particular Rhynchophion, Eremotylus and Stauropoctonus well differentiated from the main Ophion clade, as (Fig. 4A), and Euryophion is depicted as basal to the was also emphasized by Schwarzfeld et al. (2016) in Thyreodon + Ophion clade. Conversely, the addition

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Figure 5. Morphological synapomorphies of the three ophionine tribes. (A, B) details of the forewing of (A) Afrophion nubilicarpus and (B) Rhopalophion discinervus (both Ophionini) showing angled 1 m-cu and the short to long ramellus (arrow) in the disco-submarginal cell. (C, D) details of the postero-dorsal corner of pronotum showing the spiracular sclerite (arrow), occluded in (C) Enicospilus drakensbergi (Enicospilini) and fully exposed in (D) Afrophion nubilicarpus (Ophionini). (E, F) dorsal view of propodeum showing the basal transverse carina (arrows) in (F) Enicospilus gauldet- mitchellorum (Enicospilini), and totally reduced in (E) Dictyonotus nigrocyaneus (Thyreodonini). (G, H) lateral view of the ﬁrst metasomal tergites showing the latero-tergite 2, pendant below level of spiracle (arrow) in (G) Thyreodon atriventris (Thyreodonini) and folded in (H) Dicamptus maxipol (Enicospilini; pn, pronotum; ms, mesoscutum; mp, mesopleuron; t2, metasomal tergite 2). of morphological data (Fig. 4B) dissociates Thyre- the total reduction of the anterior propodeal carina odon + Dictyonotus + Rhynchophion from Stauropoc- and the pendant laterotergite 2 (Fig. 5E–G). tonus + Eremotylus and moderately groups it with The deﬁnition of the tribe Thyreodonini tribe nov. Euryophion. Morphologically these four genera share to circumscribe the Thyreodon evolutionary lineage

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 138 P. ROUSSE ET AL. is thus somewhat arbitrary. The tribe is defined by UNPLACED GENERA the unambiguous inclusion of the three genera Thyreodon, Dictyonotus and Rhynchophion which are The placement of the monobasic genus Lepiscelus as strongly correlated by both molecular and morpho- a highly derived species embedded within Eni- logical synapomorphies (Fig. 4B). Euryophion might cospilus (CO1 analysis, Fig. 2) is probably an arte- also belong to Thyreodonini because of its morphol- fact, as the CO1 sequence obtained for L. distans ogy, but the molecular data alone tend to consider it was only 299 bp. Moreover, the genus is, in part, as the sister genus of the Ophionini +Thyreodonini characterized by features which may also be found clade. Thyreodon is a moderately diversified genus of with lesser prevalence in some Enicospilus species, 45, mostly Neotropical species, while Rhynchophion i.e. the reduction of the occipital carina and the Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 includes four Neotropical species, Dictyonotus four flanged apical expansion of the mid and hind tro- species in the Old World tropics and Euryophion chantelli. Gauld (1985) pointed out the morphological eight species in the Old World tropics. The possible similarities of L. distans with some Enicospilus spe- inclusion of Eremotylus and Stauropoctonus is sup- cies in dry environments. Conversely, the 28S analy- ported by molecular data (Fig. 4A). Barytatocephalus ses provided contradictory results, and additionally is excluded as there are no synapomorphies, nor any L. distans does not have the occluded spiracular scle- ecological or geographical data to support its inclu- rite possessed by the remainder of the tribe. Thus, sion in the tribe. no strong molecular or morphological support is available to definitely place this genus within Enicospilini stat. rev Ophioninae. The Enicospilus genus-group was defined by Gauld Recovered affinities of the genus Skiapus were (1985) as a large clade including Enicospilus, highly variable across analyses, making placement of Dicamptus, Laticoleus, Leptophion and the minor this genus within the subfamily ambiguous. The genera (containing 1–7 species) Ophiogastrella, Stau- molecular data provided non-convergent results ropoctonus, Lepiscelus, Pamophion, Orientospilus, across markers as a result of the distinct DNA Prethophion and Simophion. As a result of this large sequences of the genus. The CO1 marker weakly circumscription, he could only determine a single linked Skiapus to D. maxipol, which is itself a rather weak apomorphy: the loss of the laterotergite on aberrant species of Dicamptus; the 28S sequence and metasomal tergite 1. Our molecular data do not fully the morphological characters placed the genus close support Gauld’s definition of this group, but the data to the Enicospilus lineage, but these placements are do confirm the definition of a distinct lineage based most probably anomalous because of long branch on Enicospilus. lengths. This is probably a consequence of its atypi- The tribe Enicospilini stat. rev., circumscribed by cal 28S sequence and its very uncharacteristic mor- the combined molecular markers, comprises Eni- phology. Excluding morphological data, the combined cospilus, Laticoleus, Hellwigiella and Dicamptus. markers placed Skiapus as a sister to the remaining The clade is slightly less well supported when mor- Ophioninae. phological data are included, probably because of the anomalous association with Skiapus. All four PHYLOGENY AND BIOGEOGRAPHY genera are morphologically characterized by the occlusion of the spiracular sclerite (Fig. 5C, D). The reconstruction of the evolutionary history of Although this feature is also present in some other Ophioninae is complex for two main reasons. Firstly, Ichneumonidae, it is unambiguously derived within the origin and subsequent expansion route of these the Ophioninae, because the plesiomorphic condition lineages is difficult to ascertain because of their in ophioniforms is an exposed sclerite. Noticeably, strong dispersal abilities. Ophioninae are good dis- this feature is also shared by genera that were persers and colonizers because of their flight abilities more or less strongly related to them by the 28S and their relatively large host ranges, which has sequence data: Pamophion, Leptophion and Jan- enabled them to reach and colonize remote oceanic zophion. Thus, the tribe Enicospilini comprises four islands (Quicke, 2015). As a result, their biogeo- genera: the mostly pantropical and highly diversi- graphical history is likely to be driven by a succes- fied genus Enicospilus encompassing 95% of its spe- sion of wide expansions and subsequent cies, Laticoleus (Afrotropical), Dicamptus replacements by ecologically more successful taxa. (Afrotropical and Indo-Australasian) and the This may explain why the lineages contain a con- monobasic Hellwigiella from the Mediterranean trasting mix of widespread and highly diversified region. Pamophion, Leptophion and Janzophion genera (Enicospilus and Ophion, with c. 700 species may also be part of this tribe, but no CO1 data in the Southern Hemisphere and 150 species in the were available to confirm their affinities. Northern Hemisphere, respectively) and few

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE 139 intermediate genera of 30–50 species (Thyreodon, The Ophionini may have been the first lineage to Leptophion, Eremotylus, Alophophion and Dicamp- diversify and expand, and putatively would have tus), along with numerous localized, monotypic or been previously far more widespread, as shown by nearly so, and often highly derived genera (Xylo- the existence of the localized extant genera (Afro- phion, Rhynchophion, Dictyonotus, Skiapus, Sico- phion and Xylophion), which are phylogenetically phion, Janzophion, Hellwigia, etc.), which together but no longer geographically connected to the Holarc- represent 80% of the generic diversity, but less than tic genus Ophion. In the tropical areas the Ophionini 10% of the species (Yu et al. 2012). may subsequently have been replaced by Enicospilus The second reason is the absence of reliable dating and related genera, but this latter lineage was far calibration data. Based on limited available fossil less successful in temperate areas where Enicospilini Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 data, Gauld (1985) hypothesized that ‘possibly [the are currently weakly represented. Hence Ophion and Ophioninae] radiated around the beginning of the Enicospilus became exclusively predominant in tem- Tertiary some 65–70 million years ago’. The family perate and tropical areas, respectively. It also Ichneumonidae is proposed to have radiated around appears that Enicospilus colonized the Neotropical 140 Mya (Quicke, 2015), following the diversification area after the break-up of Gondwana, because it is of their lepidopteran major hosts during the Creta- the only genus of Enicospilini present in this region ceous era (Grimaldi & Engel, 2005). The following and because the Neotropical species-group diversity assumptions are therefore based on a first radiation of Enicospilus is far lower than in the Old World dating, which cannot be certified at the moment. tropics (Gauld, 1988). Enicospilus might have The lineage leading to Skiapus is tentatively con- reached the Neotropical region either via North sidered as a basal divergence of Ophioninae as America, or through temporary inter-continental con- depicted by the combination of molecular data. Even nections (Coney, 1982) as was proposed for Campto- though weakly supported, this ancient radiation typus (Ichneumonidae: Pimplinae) (Sa€aksj€ arvi,€ Gauld hypothesis is supported by the vicariant distribution & Salo, 2004). of Skiapus whose three described and seldom col- Ophionini and Thyreodonini share a common ances- lected species occur in Africa and Korea. The extant tor: this is shown by the combined molecular analysis species of Skiapus are probably remnants of an and also by the 28S analysis where some Thyreodonini ancient, more diverse, lineage. As proposed by genera are alternatively weakly linked to the Ophion- Quicke et al. (2005), Skiapus most probably forms a ini clade. Thyreodonini probably first diversified in monophyletic clade with Hellwigia (result not con- the Old World tropics, where Dictyonotus is still pre- firmed here by our partial data for Hellwigia). This sent. This is also suggested by the Afrotropical and ancient radiation would thus form a transitional lin- Oriental genus Euryophion, which is either part of eage between Ophioninae and the remainder of the Thyreodonini or the sister genus of the clade Thyre- Ichneumonidae, characterized by an intermediate odonini + Ophionini. The colonization of North Amer- wing venation. ica possibly occurred thereafter, via the ancient Ophioninae possibly diversified c.65–70 Mya, northern connection with Eurasia before the eventual when Eurasia and Africa were still connected to breakup of Laurasia or later via the Bering Strait. North America, while South America and Australia This is suggested by the putative association of Ere- were separated by large oceans after Gondwana motylus and Stauropoctonus to Thyreodonini, sup- broke up 30 Myr before (Le Gall et al., 2010). Accord- ported by molecular data, but without morphological ing to our analyses, the main subfamily split into confirmation. These two genera have an intermediate two major lineages, one leading to Enicospilus and distribution, especially Eremotylus whose diversity another subsequently splitting into the Ophion and peaks in the Nearctic region. Following this hypothe- Thyreodon lineages. Gauld (1985) proposed Ophion sis, Thyreodon arose and diversified with relative suc- and the related genera as the basal lineage of cess in South America. The continent was connected Ophioninae, based on his assertion that the fore to North America by the formation of the Panama wing venation configuration is plesiomorphic in these Isthmus 13–15 Mya (Montes et al., 2015). The relative taxa. On the contrary, our molecular data and the success of Thyreodon may be the result of colonization current hypotheses concerning the phylogeny of the by this genus of a region that was not yet under the higher ophioniforms (Quicke et al., 2009) strongly ecological hegemony of Ophion or Enicospilus. suggest this might indeed be a character reversal. This global scenario hypothesis needs to be treated Hence the diversification of the tribe Ophionini took with caution because it is based on limited available place after the appearance of the ancestors of the data. The hypothesis relies on weak dating assump- other tribes. These ancestral species probably tions and moreover does not encompass many of the evolved in Eurasia or Africa, concurrently with at minor genera, which were not supported by any least the Enicospilus lineage. available molecular data, or if available the data

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148 140 P. ROUSSE ET AL. proved to be insufficient to unambiguously link these Butcher BA, Zaldivar-Riveron A, Van de Kamp T, Dos genera to one or another lineage. Future assess- Santos Rolo T, Baumbach T, Quicke DLJ. 2014. Exten- ments will need to take into account additional fossil sion of historical range of Betylobraconinae (Hymenoptera: and/or molecular data allowing for a robust Braconidae) into Palaearctic Region based on a Baltic molecular clock assessment. amber fossil, and description of a new species of Mesocen- trus Szepligeti from Papua New Guinea. Zootaxa 3869: 449–463. ACKNOWLEDGEMENTS Coney PJ. 1982. Plate tectonic constraints on the biogeogra- The completion of this work was greatly helped by phy of Middle America and the Caribbean region. Annals of the Missouri Botanical Garden 69: 432–443. the intervention of numerous contributors. We thank Cushman RA. 1947. 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APPENDICES Appendix 1. Species used in the analyses. *Genbank accession numbers are CO1/28S

Species Collection country Accession no.* Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 Anomaloninae Anomalon sp. Zimbabwe GenBank: JF962903/– Agrypon sp. aff. nelsoni Costa Rica GenBank: JF792887/– Agrypon varitarsum (Wesmael) UK GenBank: –/AJ302927 Parania sp. Madagascar GenBank: –/EU378323 Banchinae Apophua hispida (Benoit) Uganda Bold: OPHY005-15 Glypta fumiferanae Viereck Canada GenBank: HQ107295/HQ025539 Occia jereza Ugalde & Gauld Costa Rica GenBank: JQ576059/EU378350 Campopleginae Diadegma mollipla (Holmgren) South Africa GenBank: AJ888008/AJ508222.2 Dusona sp. Central African Republic Bold: OPHY008-15 Venturia canescens (Gravenhorst) USA (culture) GenBank: DQ538851/DQ539001 Campoletis sonorensis (Cameron) USA (culture) GenBank: DQ538850/DQ539000 Cremastinae Eiphosoma sp. Bolivia GenBank: JF963241/EU378426 Hybrizontinae Hybrizon ghilarovi Tobias Russia GenBank: JF963427/EU378579 Ophioninae Afrophion hynnis (Gauld & Mitchell) South Africa GenBank: JF962473/AY593084 Afrophion nubilicarpus (Tosquinet) South Africa Bold: OPHY001-15 Alophophion sp. UK (Falklands) GenBank: –/AY593085.1 Barytatocephalus mocsaryi (Brauns, 1895) Turkey GenBank: JF962965/AY593086 Dicamptus maxipol Rousse & van Noort South Africa Bold: OPHY006-15 Dicamptus pulchellus (Morley) Gambia Bold: OPHY007-15 Dicamptus sp.1 Madagascar Bold: AAI6316 Dicamptus sp.2 Taiwan Bold: ASQIC064-09 Dicamptus sp.3 Taiwan Bold: ASQIC065-09 Dictyonotus purpurascens (Smith) Japan GenBank: –/EU378703 Enicospilus albiger (Kriechbaumer) South Africa Bold: OPHY017-15 Enicospilus antefurcalis (Szepligeti) Madagascar Bold: OPHY009-15 Enicospilus biimpressus (Brulle) Uganda Bold: OPHY021-15 Enicospilus capensis (Thunberg) South Africa Bold: OPHY025-15 Enicospilus divisus (Seyrig) Uganda Bold: OPHY032-15 Enicospilus dolosus (Tosquinet) Central African Republic Bold: OPHY033-15 Enicospilus equatus Gauld & Mitchell Central African Republic Bold: OPHY035-15 Enicospilus ﬁnalis Gauld & Mitchell Uganda Bold: OPHY036-15 Enicospilus grandiﬂavus Townes South Africa Bold: OPHY038-15 Enicospilus herero (Enderlein) South Africa Bold: OPHY040-15 Enicospilus justus (Seyrig) Uganda Bold: OPHY044-15 Enicospilus leucocotis (Tosquinet) South Africa Bold: OPHY046-15 Enicospilus mamatsus Gauld & Mitchell Madagascar Bold: OPHY051-15 Enicospilus sp. nov. French Polynesia Bold: OPHY054-15 Enicospilus ramidulus (L.) UK GenBank: JF963318/DQKP100 Enicospilus rundiensis Bischoff Uganda Bold: OPHY060-15 Enicospilus senescens (Tosquinet) Uganda Bold: OPHY071-15 Enicospilus transvaalensis Cameron South Africa Bold: OPHY077-15 Enicospilus umbratus Gauld & Mitchell Madagascar GenBank: JF963320/EU378709

Appendix 1. Continued

Species Collection country Accession no.*

Eremotylus curvinervis (Kriechbaumer) UK - Eremotylus sp.1 USA Bold: BBHYA1627-12 Eremotylus sp.2 USA Bold: BBHYG815-10 Eremotylus sp.3 USA Bold: HYMBB144-09 Eremotylus sp.4 USA Bold: HYMBB269-09 Eremotylus sp.5 USA Bold: HYMBB283-09

Euryophion ikuthanus (Kriechbaumer) Tanzania - Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 Euryophion nigripennis Cameron South Africa Bold: OPHY080-15 Euryophion sp. Togo GenBank: –/AJ302854 Hellwigia obscura Gravenhorst France GenBank: –/AJ302858 Hellwigiella dichromoptera (Costa) Spain GenBank: JF957051/EU378710 Janzophion nebosus Gauld Costa Rica GenBank: –/EU378711 Laticoleus curvatus Delobel Madagascar Bold: OPHY081-15 Laticoleus spilus Gauld & Mitchell South Africa - Laticoleus unicolor (Szepligeti) 1 South Africa Bold: OPHY082-15 Laticoleus unicolor (Szepligeti) 2 Madagascar Bold: OPHY083-15 Lepiscelus distans (Seyrig) South Africa Bold: OPHY084-15 Leptophion anici Gauld Australia GenBank: –/AY593089 Ophiogastrella maculithorax Brues Costa Rica GenBank: –/EU378714 Ophion costatus Ratzeburg UK GenBank: JF963664/EU378716 Ophion minutus Kriechbaumer UK GenBank: JF963665/EU378717 Ophion obscuratus Fabricius UK GenBank: FN662468/Z97889 Ophion scutellaris Thomson UK GenBank: JF963667/EU378720 Ophion aff. scutellaris UK GenBank: KF594819/KF616305 Ophion idoneus Viereck Canada GenBank: KF594578/KF616320 Ophion sp.1 Madagascar BOLD: AAI3373 Ophion sp.2 Madagascar BOLD: ASQIC068-09 Ophion sp.3 Taiwan BOLD: ASQIC071-09 Ophion sp.4 UK BOLD: ASQIC109-09 Ophion sp.5 UK BOLD: ASQIC112-09 Pamophion sorus Gauld Australia GenBank: –/EU378722 Rhopalophion discinervus (Morley) South Africa Bold: OPHY085-15 Rhynchophion ﬂammipennis (Ashmead) Costa Rica GenBank: JF793291/AY593090 Riekophion emandibulator (Morley) Australia GenBank: –/EU378726 Sicophion fenestralis Gauld Costa Rica GenBank: –/EU378727 Skiapus coalescens Morley Gambia Bold: OPHY086-15 Skiapus sp.1 Tanzania GenBank: JF963839/AY604252 Skiapus sp.2 Togo - Stauropoctonus bicarinatus (Cushman) Costa Rica Bold: AAJ5095 Thyreodon atriventris (Cresson) 1 Costa Rica Bold: AAB7595 Thyreodon atriventris (Cresson) 2 Nicaragua - Thyreodon laticinctus Cresson Costa Rica GenBank: JF793300/AJ302876 Thyreodon sp.1 Canada - Xylophion sevrapek Villemant Vanuatu Bold: ABV8816

Appendix 2. Morphological characters

Head

01 Labial palps: (0) four-segmented; (1) three-segmented. 02 Maxillary palps: (0) ﬁve-segmented; (1) four-segmented. 03 Central segments of maxillary palps: (0) slender; (1) enlarged, globose. 04 Width of mandible: (0) not to slightly narrowed, apically at least 0.59 as wide as basally; (1) moderately tapered, apically 0.4–0.59 as wide as basally; (2) strongly tapered, apically less than 0.49 as wide as basally. 05 Torsion of mandibles: (0) not or hardly twisted, teeth in a plane which is less than 5° from the main mandible

plane; (1) moderately twisted, 5–25°; (2) strongly twisted, 25–50°; (3) very strongly twisted, more than 50°. Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 06 Ventral mandible flange: (0) absent or indistinct; (1) present, strong. 07 Basal swelling on mandible: (0) absent; (1) present. 08 Mid-longitudinal groove on mandible outer surface: (0) absent; (1) present. 09 Upper mandible tooth: (0) 1.0–1.59 longer than lower tooth; (1) more than 1.59 longer than lower tooth; (2) shorter than lower tooth: (3) mandible unidentate 10 Bending of mandibular teeth: (0) teeth not bent; (1) strongly bent downwards, teeth axis nearly perpendicular to mandible main axis. 11 Malar space: (0) less than 0.49 basal width of mandible; (1) at least 0.49 basal width of mandible. 12 Clypeus in profile: (0) flat; (1) convex. 13 Ventral margin of clypeus: (0) in-turned or not differentiated; (1) impressed or out-turned. 14 Median tooth on ventral margin of clypeus: (0) absent; (1) present. 15 Clypeus and face: (0) separated by a more or less distinct groove; (1) confluent. 16 Mid-longitudinal carina on frons: (0) absent or not expanded; (1) strongly raised between toruli. 17 Length of antenna: (0) shorter than fore wing; (1) equal or greater than fore wing length. 18 Relative length of first and second flagellomeres: (0) flagellomere 1 less than 1.69 longer than flagellomere 2; (1) flagellomere 1 at least 1.69 longer than flagellomere 2. 19 Elongation of 20th flagellomere: (0) less than 1.69 longer than wide; (1) 1.6–2.09 longer than wide; (2) more than 2.09 longer than wide. 20 Ocelli size: (0) not enlarged, median ocellus diameter less than 0.59 inter-ocular distance through median ocellus; (1) moderately enlarged, median ocellus diameter 0.5–0.79 inter-ocular distance through median ocellus; (2) strongly enlarged, median ocellus diameter more than 0.79 inter-ocular distance through median ocellus. 21 Strong depression between posterior ocelli and occipital carina: (0) absent; (1) present. 22 Occipital carina: (0) complete; (1) shortly interrupted mid-dorsally; (2) totally absent dorsally, laterally absent or vestigial. Mesosoma 23 Latero-ventral projecting flange of propleuron: (0) absent; (1) present, overlapping anterior margin of mesopleuron. 24 Spiracle of mesopleuron: (0) fully exposed; (1) partly to totally occluded by the expansion of the upper corner of pronotum. 25 Epicnemial carina: (0) reaching above level of ventral corner of pronotum; (1) shortened to absent above ventral corner of propleuron. 26 Postero-ventral tubercle on mesopleuron: (0) absent; (1) present. 27 Mesopleural fovea: (0) absent to distinct as an isolated pit; (1) present and extended into a longitudinal groove. 28 Postpectal carina (posterior transverse carina of mesosternum): (0) complete; (1) partially to totally obsolescent ventrally. 29 Submetapleural carina: (0) not distinctly broadened anteriorly; (1) enlarged into a broad flange anteriorly. 30 Notauli: (0) indistinct or vestigial; (1) distinct. 31 Elongation of scutellum: (0) less than 1.69 longer than basally wide; (1) at least 1.69 longer than basally wide. 32 Hind margin of metanotum: (0) unspecialized; (1) swollen backwards, reaching propodeal spiracle. 33 Base of propodeum: (0) not unusually swollen; (1) strongly swollen, spiracles in a deep anterior transverse trough (1). 34 Elongation of propodeal spiracle: (0) less than 49 longer than wide; (1) at least 49 longer than wide. 35 Anterior transverse carina of propodeum: (0) complete; (1) partially absent; (2) totally absent. 36 Posterior carina of propodeum: (0) complete; (1) partially absent; (2) totally absent. 37 Mid-longitudinal carina of propodeum: (0) present, strong to vestigial; (1) totally absent.

Appendix 2. Continued

Head

Metasoma 38 Spiracle of the first tergite: (0) at or anterior to middle; (1) distinctly posterior to middle. 39 Laterotergite of first tergite: (0) present; (1) vestigial to absent. 40 Elongation of second tergite: (0) less than 39 longer than apically high in profile; (1) more than 39 longer than apically high in profile. 41 Convex median area (umbo) on anterior margin of tergite 2: (0) present; (1) absent.

42 Position of thyridia: (0) close to anterior margin of tergite 2; (1) remote by more than their own length; Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 (2) thyridia absent. 43 Laterotergite of tergite 2: (0) indistinct, folded inside; (1) pendant. Fore wing 44 Vein 3Cu with adventitious vein along wing margin: (0) absent; (1) present. 45 Position of 2 m-cu: (0) distal or opposite to rs–m; (1) basal to rs–m. 46 Glabrous area in the discoido-submarginal cell: (0) absent; (1) reduced, not reaching beyond anterior third of Rs+2 m; (2) extending beyond. 47 Proximal sclerite of fore wing: (0) absent; (1) present. 48 Central sclerite of fore wing: (0) absent; (1) present. 49 1 m–cu: (0) angled; (1) more or less curved, without sharp angle. 50 Ramellus inside discoido-submarginal cell: (0) absent; (1) present. 51 Pterostigma: (0) triangular, apically abruptly narrowed; (1) elongate and narrow, evenly tapered toward apex; (2) linear 52 Anterior curve of Rs+2r, near pterostigma: (0) straight to curved; (1) distinctly angled. 53 Anterior thickness of Rs+2r: (0) not distinctly thickened; (1) thickened, Rs+2r anteriorly at least twice thicker than centrally. 54 Central shape of Rs+2r: (0) straight to about so; (1) slightly sinuate; (2) strongly sinuate or bowed. Hind wing 55 Vein Rs: (0) straight to barely curved; (1) distinctly curved. 56 Number of distal hamuli: (0) five and less; (1) six to nine; (2) ten and more. 57 Interception of Cu&cu-a: (0) at or above middle; (1) below middle. Legs 58 Membranous flange on fore tibial spur: (0) at least 0.39 length of spur; (1) less than 0.39 length of spur; (2) totally absent. 59 Apical edge of hind and mid trochantelli: (0) unspecialized; (1) expanded into a broad flange or sharp tooth. 60 Cross section of hind tibial spurs: (0) flattened; (1) cylindrical. 61 Shape of hind tarsal claws: (0) evenly curved, not unusually elongate; (1) straight and elongate. 62 Pectination of female outer claw: (0) pectinate, with more than 10 pectinae; (1) pectinate, with at most 10 pectinae; (2) not pectinate.

1 2 345 6 78 9 10111213141516171819202122232425262728293031 ROUSSE P.

Afrophion hynnis 0000000000000000 1011100000011011 Afrophion nubilicarpus 0000000000000000 1011100000011011 Agrypon varitarsum 0 000 0 00 0 00 0010 10 1 1 0 2 00 00 0 00 0 0 01 ? Apophua hispida 0 000 0 00 0 00 1 1 0 00 0 1 1 0 00 10 1 00 0 1 10 0 Barytatocephalus mocsaryi 0000001010011100001?00000000010?0 ? AL. ET

06TeLnenSceyo London, of Society Linnean The 2016 Diadegma mollipla 0 000 0 00 0 00 1010 01 0 0 0 0 00 11 0 00 0 0 00 ? Dicamptus maxipol 0 000 0 00 0 00 1 0 1 00 0 1 1 0 00 00 1 00 0 0 00 0 Dicamptus pulchellus 00000010100001000102100010000000 Dicamptus sp 0 000 0 00 ? 00 0 0 1 00 0 1 ? 2 10 00 1 ?0 0 0 00 0 Dictyonotus purpurascens 0 000 0 00 0 00 1 0 1 10 0 0 0 0 00 00 0 00 1 0 10 0 Dusona sp. 00000010000101010100000100000000 Eiphosoma sp. 0000010000011000001??00010000001 ? Enicospilus albiger 0 002 1 00 0 10 0 0 0 00 0 1 1 2 10 00 1 00 0 0 01 1 Enicospilus antefurcalis 0 0021200 1 00 0 1 1 00 0 1 1 2 10 00 1 00 0 0 00 01 Enicospilus biimpressus 0002100000000000 1112100101 00000001 Enicospilus capensis 0 002 1 00 1 10 0 1 1 00 0 1 0 1 00 00 1 00 0 0 01 0 Enicospilus divisus 0 002 1 00 0 00 0 1 1 00 0 1 1 2 10 00 1 00 0 0 00 1 Enicospilus dolosus 00022000010010000112100010000101 Enicospilus equatus 0 002 2 00 0 00 0 1 0 00 0 1 1 2 20 00 1 00 0 0 11 01

olgclJunlo h ina Society Linnean the of Journal Zoological Enicospilus ﬁnalis 0 002 2 00 0 00 0 1 1 00 0 1 1 2 10 00 1 00 0 0 00 01 Enicospilus grandiﬂavus 0 002 1 00 0 10 0 0 1 00 0 1 1 0 10 00 1 0100 0 11 0 Enicospilus herero 0 002 1 00 1 10 0 1 1 00 0 1 1 1 10 00 1 00 0 0 00 0 Enicospilus justus 0 002 1 00 0 00 0 1 1 00 0 1 0 2 10 00 1 00 0 0 00 01 Enicospilus leucocotis 0 002 2 00 0 00 1 0 1 00 0 1 10100 00 1 00 0 0 01 0 Enicospilus mamatsus 0 0021200 0 10 0 0 0 00 0 1 10110 00 1 00 0 0 10 01 Enicospilus sp. 0 002 3 00 0 00 0 0 0 00 0 1 1 2 20 00 1 10 0 0 10 0 Enicospilus ramidulus 0 002 1 00 1 10 0 1 1 00 0 1 11210 00 1 00 0 0 00 1 Enicospilus rundiensis 0 002 1 00 0 10 0 1 0 00 0 1 0 1 10 00 1 00 0 0 01 1 Enicospilus senescens 0 002 2 00 0 00 0010 00 0 1 0 2 10 00 1 00 0 0 00 1 Enicospilus transvaalensis 00021200000011000112100010000001 Enicospilus umbratus 0 0021200 0010 0010 00 0 1 1 2 10 00 1 00 0 0 00 0 Eremotylus sp 0001000000 ? 0 0 000 0 ? ?120 000 0? 0 ? 00 ? Euryophion sp 01 1 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 01 0 0 1 0 1 0 01 0 0 Hellwigiella dichromoptera 0 000 1 10 0 20 1 1 1 00 0 1 0 0 00 00 1 00 00100 0 Laticoleus unicolor 0 000 0 01 0 00 0 1 1 00 0 1 1 1 00 00 1 10 0 1 01 1 2016, , Lepiscelus distans 0002000010?10000102102000000010 Occia jereza 0 000 0 00 0 00 1 0 1 00 0 1 0 2 00 10 1 10 0 1 10 ? Ophion costatus 00000000000??0001??10 ?00001101 ? 178 Ophion idoneus 0000000000 0 ? 1 000 1 ? ?120 ?00 00 1 1 01 ?

128–148 , Ophion minutus 0000000000 0 ? ? 000 1 ? ?120 ?00 00 1 1 01 ? Ophion obscuratus 0 000 0 00 0 00 0 0 1 00 0 1 ? 1 10 00 0 00 1 1 01 0

Ophion scutellaris 00000000000?10001??10 ?00001101 ? Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 September 23 on guest by https://academic.oup.com/zoolinnean/article/178/1/128/2667458 from Downloaded © .AppendixContinued 3. Continued 06TeLnenSceyo London, of Society Linnean The 2016

1 2 345 6 78 9 10111213141516171819202122232425262728293031

Ophion sp nr 00000000000?10001??120 ?00 001101 ? scutellaris Ophion sp 00000000000?10001??120 ?00 001101 ? Rhopalophion 0 000 0 00 0 00 0 00100 0 1 1 1 10 00 0 00 1 1 10 0 discinervus Rhynchophion 000000000010110001000000 1011?0 ? ﬂammipennis Skiapus coalescens 00101100031011000110010100000100 Skiapus sp 00101100031011000110010100000100 Stauropoctonus sp 0 0 0 2 3 0 0 0 0 0 ? 01 0 0 0 0 1 01 01 2 0 01 0 ? 0 0 1 0 0 0 ? Thyreodon atriventris 001000000000010100020000 00011010 olgclJunlo h ina Society Linnean the of Journal Zoological Thyreodon laticinctus 001000000010010100000000 00011010 Venturia canescens 0 000 0 00 0 00 1010 01 0 0 0 0 00 01 0 00 0 0 01 ?

Xylophion sevrapek 000000000011100011110000 0011000 OPHIONINAE OF PHYLOGENY THE OF REASSESSMENT A

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

Afrophion hynnis 0001101 010 001221 00100 10010000 01 Afrophion nubilicarpus 0000101 010 001210 00100 10010000 01 Agrypon varitarsum 0002211 010 200000 00000 000 ?2000 001 Apophua hispida 0000000 000 200000 01000 00010000 01 Barytatocephalus 00102211011 001210 01010 00000200 101 mocsaryi Campoletis sonorensis 0000101 0000100000 01000 00001000 0? Diadegma mollipla 0000101 000 100000 01000 00002000 0? Dicamptus maxipol 0000211 111 101220 01010010011100 01 2016, , Dicamptus pulchellus 0000211 111 101221 01010 02011100 01 Dicamptus sp 0000211 111 101221 01010 ?2011100 01

178 Dictyonotus 1012211 001 111200 01021 00020201 1? purpurascens 128–148 , Dusona sp 0000111 000 100000 01010 00002000 01 Eiphosoma sp 0000001 01? 200000 01000 000 ?02000 0? Enicospilus albiger 0001211 111 101221 01010 00011200 00 Enicospilus antefurcalis 0000211 111 101221 11010 11011200 00 Enicospilus biimpressus 0000211 111 101221 11010 11011200 00 Enicospilus capensis 0000211 111 101221011010 11011200 01 Enicospilus divisus 0000211 111 101221 11010 10011200 00 Enicospilus dolosus 0000211 111 101221 01010 11011200 00 Enicospilus equatus 0001211 111 101220 01010 10011200 01 Enicospilus ﬁnalis 0000211 111 101221 11010 11011200 01 Enicospilus grandiﬂavus 0000211 111 101221 01010 10011200 00

Enicospilus herero 0000211 111 101221 01010 101011200 00 147 Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 September 23 on guest by https://academic.oup.com/zoolinnean/article/178/1/128/2667458 from Downloaded Appendix 3. Continued 148

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 .ROUSSE P.

Enicospilus justus 0000211 11110122101101010011200 01 Enicospilus leucocotis 0001211 1111012200101011021200 00 Enicospilus mamatsus 0001211 1111012200101011011200 00 Enicospilus sp 0000211 1111012201101011000200 01 Enicospilus ramidulus 0000211 1111012211101011011200 01 AL. ET

06TeLnenSceyo London, of Society Linnean The 2016 Enicospilus senescens 0000211 1111012200101011011200 01 Enicospilus 0000211 1111012211101011011200 01 transvaalensis Enicospilus umbratus 0000211 1111012200101012010200 00 Eremotylus sp 0002?11 0101012100001110?01000001? Euryophion sp 0002211 0001201121001021101120201 10 Hellwigiella 0000211 00011121001001101 ?0001 101 dichromoptera Laticoleus unicolor 0000211 1111012200101110001200 01 Lepiscelus distans 0002211 1111012100101010100210 11 Occia jereza 0002210 01000000000010000 ?0000 001 Ophion costatus 00000101010 00121000100001 ?0000 001 Ophion idoneus 00000101010 00121000100001 ?0000 001 Ophion minutus 0000001 01000121000100001 ?0000 001 Ophion obscuratus 0000201 0100012100010000110000 001 olgclJunlo h ina Society Linnean the of Journal Zoological Ophion scutellaris 00000101010 00121000100001 ?0000 001 Ophion sp nr 00000101010 00121000100001 ?0000 001 scutellaris Ophion sp 00000101010 00121000100001 ?0000 001 Rhopalophion 00001011010 0012200010010010000 11 discinervus Rhynchophion 0012211 00111120001020000010201 101 ﬂammipennis Skiapus coalescens 00002110101 2011000102000010100 02 Skiapus sp 00002110101 2011000102000010100 02 Stauropoctonus sp 01000211110 11121001021100010200 001 Thyreodon atriventris 01112211011 11120001020000 ?0201 101 Thyreodon laticinctus 01112211011 11120001020000 ?0201 101 Venturia canescens 0000001 0101000000000000012000 001 Xylophion sevrapek 0000201 01000121000100101 ?0100 01 2016, , 178

128–148 , Downloaded from https://academic.oup.com/zoolinnean/article/178/1/128/2667458 by guest on 23 September 2021 September 23 on guest by https://academic.oup.com/zoolinnean/article/178/1/128/2667458 from Downloaded