phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae)

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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 LEFEUVRE4 and SIMON VAN NOORT1,5 1

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

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. © 2016 The Linnean Society of London, Zoological Journal of the Linnean Society,2016, 178, 128–148 doi: 10.1111/zoj.12405

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

INTRODUCTION The Ophioninae (Hymenoptera: Ichneumonidae) are relatively large-bodied parasitoid wasps that utilize the larvae of various moths (Lepidoptera), and the majority of the species are typically nocturnal or cre-

*Corresponding author. E-mail: [email protected]

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puscular and are frequently collected at light. They are usually pale, moderate- to large-sized wasps with long slender antennae and enlarged ocelli (Fig. 1), a suite of characters that is known as the ‘ophionoid facies’ (Gauld & Huddleston, 1976) and which has arisen independently in several hymenopteran taxa (Quicke, 2015). Although a large number of specimens have been collected throughout the world during the past two centuries, reliable host data are

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE comparatively scarce, especially because of partial or erroneous identifications. The subfamily Ophioninae is represented by approximately 1000 species assigned to 32 extant genera. Remarkably, 80% of these species belong to one of the two major genera Ophion and Enicospilus (Yu, van Achterberg & Horstmann, 2012). The taxonomic classification of the species within the

A

B

C

Figure 1. (A) Afrophion nubilicarpus; (B) Enicospilus drakensbergi; (C) Thyreodon atriventris.

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Ophioninae is, however, complex. Earlier treatments by Cushman (1947) and later by Townes (1971) attempted to subdivide the subfamily into coherent genus-groups or tribes, by the presence or absence of alar sclerites, reduction of the membranous flange on the fore tibia, extension of the postpectal carina or even torsion of the mandibles, but all these characters appear to be evolutionarily plastic and prone to homoplasy (Gauld, 1985). With the increasing potential of computing tools, Ian Gauld attempted to reconstruct the life history of the Ophioninae, focusing 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; Fern andez-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 & Sperling, 2014; Schwarzfeld, Broad & Sperling, 2016), 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 Ophioninae 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 traditional assumption that many ichneumonid taxa are of temperate origin and diversified thereafter in the tropics (Quicke, 2012; Veijalainen et al., 2012). Molecular phylogenetic and morphological studies of the family also suggest that two morphologically distant 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 nevertheless 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 region of the nuclear ribosomal 28S genes and combined these data with a set of discrete

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morphological characters. It was anticipated that the CO1 gene will provide better resolution at the species level, while the more slowly evolving 28S may provide better results at the genus level (Klopfstein, Kropf & Quicke, 2010). Our integrated approach enabled us to (1) better define the evolutionary lineages within the subfamily, (2) clarify the evolutionary relationships between these major lineages and (3) provide a definitive argument for the assignment of the highly derived, palaeotropical genus Skiapus to Ophioninae.

MATERIAL AND METHODS M OLECULAR

DATASETS

A total of 74 species belonging to 24 genera of Ophioninae were included in the analyses, together with 13 outgroup species selected from the closely to more distantly related Anomaloninae, Banchinae, Campopleginae, Cremastinae and Hybrizontinae (Quicke et al., 1994, 2009). The localities, dates of collection and accession numbers of all sequenced individuals are summarized in Appendix 1. Most of the specimens were recently collected by the Iziko South African Museum in Cape Town (curator Simon van Noort) and the California Academy of Sciences in San Francisco (curator Brian Fischer). All specimens had been stored in 90–96% ethanol for varying periods, ranging from 1 to 15 years. Additional sequences were obtained from 15- to 25-year-old dried specimens stored in the Iziko South African Museum collections. All the Afrotropical specimens were identified to species using a recently developed Lucid matrix key (Rousse & van Noort, 2014). To obtain comprehensive taxonomic coverage, the final genetic data set was augmented with sequences available from the GenBank (Benson et al., 2013) and Bold Systems (Ratnasingham & Hebert, 2007) databases.

M ORPHOLOGICAL

CHARACTERS DATASET

A dataset of 62 morphological characters was constructed (Appendix 2). All characters were treated as unordered in the analyses. Each individual specimen was scored to include the range of polymorphisms represented in each species. The species for which no specimens were available in the collection were scored according to detailed descriptions (Gauld & Mitchell, 1977, 1978, 1981; Horstmann, 1981; Dasch, 1984; Gauld, 1985, 1988; Gauld et al., 1997; Gauld & Janzen, 2004; Quicke et al., 2005; Villemant, Yoshida & Quiles, 2012) and the morphometric index of the Taxapad database (Yu et al., 2012).

M OLECULAR

PROTOCOLS

For most specimens, DNA extraction was performed on entire specimens using a phenol/chloroform/isoamyl procedure (Sambrook, Fritsch & Maniatis, 1989). Chemical penetration of the specimen was facilitated by performing a ventro-longitudinal incision on the metasoma. Two molecular markers were then amplified and sequenced (Table 1): the mitochondrial cytochrome oxidase 1 (CO1) and the D2–D3 expansion of the nuclear ribosomal sequence 28S. Some of the older dried specimens had to be additionally amplified following a nested PCR protocol, using first a CO1 PCR-product diluted to 1:100 and then reamplified with purposely designed internal primers. All PCR products were subsequently purified using the QIAquick PCR purification kit (Qiagen) and cycle-sequenced using BigDye terminator chemistry (Applied Biosystems). The CO1 sequences were manually aligned, and the 28S D2–D3 sequences were aligned with MAFFT6 (Katoh & Toh 2008) using the G-INS-I algorithm, then manually optimized.

A NALYSED

MATRICES

Three molecular matrices were built based on the gene region sequenced: CO1, 28S and CO1 + 28S (the combined analyses utilized a trimmed data set where only individuals for which both sequences were available were included). Finally the CO1 + 28S data were analysed with (_morph) or without (_nomorph) morphological matrices. The topologies of the _morph and _nomorph trees were compared using a maximum agreement subtree congruence test (de Vienne, Giraud & Martin, 2007).

B AYESIAN

ANALYSIS

Phylogenetic analyses were done using MrBayes 3.2 (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 were set with a Γ-shaped range of distribution. The nucleotide substitution model settings were determined with jModeltest2 and Bayesian information criteria (Darriba et al., 2012). The morphological characters were treated as an undivided partition, the CO1 characters were subdivided into two partitions codons 1–2 and codon 3, and the 28S D2–D3 characters were subdivided into four partitions according to the secondary structure of this region: D2p, D2u, D3p and D3u (p = paired, u = unpaired; Gillespie, Yoder & Wharton, 2005; Butcher et al., 2014). All the ambiguous regions to align indicated as out by Gillespie et al., (2005; NHR and RAA) were excluded.

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Table 1. Primers and PCR protocols Primer

Sequence

Tm (°C)

PCR programme

C1-J-1718 C1-N-2329

50 -GGAGGATTTGGAAATTGATTAGTTCC-30 50 -ACTGTAAATATATGATGAGCTCA-30

54.2 49.5

28S-D2–D3 fwd 28S-D2–D3 rev

50 -GCGAACAAGTACCGTGAGGG-30 50 -TAGTTCACCATCTTT-30

61.4 34.1

CO1 nested fwd CO1 nested rev

50 -ATTGGGTCTCCTCCTCCTGAT-30 50 -GGAGTTCCCGATATAGCTTTTCCT-30

59.7 60.0

94 °C, 3 min 35 cycles (94 °C, 30 s; 50 °C, 30 s; 72 °C, 45 s) 72 °C, 7 min 94 °C, 2 min 35 cycles (96 °C, 15 s; 57 °C, 30 s; 72 °C, 30 s) 72 °C, 7 min 94 °C, 3 min 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 Monte Carlo Markov chains each for 5–12 million generations, until the average standard deviation reached below 0.01. The chains were sampled every 1000 generations, and 25% of the samples were discarded as burnin. The output run files were checked with Tracer 1.6 to ensure the stability of the eventual traces (all effective sample size ≫ 100), and the output summarized tree was displayed with FigTree 1.4 (both available in the BEAST package; Drummond et al., 2012) with posterior probability (pp) as values of node robustness. In the results below, the nodes with pp of 90% or more are considered as significantly supported.

RESULTS C O1

ANALYSES

The 584-bp CO1 matrix consisted of 11 outgroup species and 60 ophionine species (belonging to 17 genera). The selected substitution models proposed by jModeltest2 for codons 1–2 and codon 3 were GTR + I + Γ and HKY + Γ, respectively. The analysis ran for 5 million generations. Ophioninae (including Skiapus) are monophyletic and fully separated from the outgroups (Fig. 2, green). The internal topology of the subfamily is represented by a large polytomy. All Enicospilus species are recovered in a robust monophyletic clade (1.00 pp) with Lepiscelus distans embedded within it (Fig. 2, red). Ophion was recovered as polyphyletic and associated with Xylophion (0.96 pp) and Afrophion (0.69 pp). Overall, the relative placement of Enicospilus + Lepiscelus, the Ophion lineage and the remaining genera are not satisfactorily resolved here, with Dicamptus and Laticoleus not even recovered as monophyletic genera. Thyreodon is weakly associated with Stauropoctonus and Barytatocephalus with 0.60 pp.

28S

ANALYSES

The 675-bp 28S matrix included 63 ophionine species (24 genera). The selected substitution models proposed by jmodeltest2 for D2p, D2u, D3p and D3u were GTR + Γ, SYM + Γ, HKY + I and GTR + Γ, respectively. Ophioninae (including Skiapus) are recovered as monophyletic, separated from the outgroups (Fig. 3, green). As in the CO1 analyses, the internal topology is rather weakly resolved in the 28S analyses. The subfamily is recovered with 1.00 pp but with a large basal polytomy from which emerge two genusgroups: Ophion (Fig. 3, red) and Enicospilus (Fig. 3, blue), both separated from Thyreodon, Euryophion, Stauropoctonus, Eremotylus, Sicophion, Lepiscelus and Hellwigia. The genus Ophion is polyphyletic and only partly supported by 0.78 pp in a polyphyletic lineage mixed with Xylophion, Alophophion, Rhopalophion and Afrophion, the assemblage being linked with low support (0.60 pp) to Dictyonotus + Rhynchophion. The second major, but weakly supported lineage (0.69 pp) leads to a robust Laticoleus + Enicospilus clade (0.99 pp) non-significantly associated (0.83 pp) with Dicamptus and Hellwigiella. Skiapus is considered here as a strongly derived genus within the basal polytomy of the Enicospilus lineage, but with low support.

C O1 + 28S

ANALYSES

The combined matrix combined eight outgroup species and 53 ophionine species represented by 17 genera in a 1259-bp-long alignment. The CO1 + 28S_nomorph and CO1 + 28S_morph ran for 5 and 12 million generations, respectively. The two trees were significantly more congruent than expected by chance (Icong = 4. 3; P = 1.97 9 1028).

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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 analyses (Fig. 4). When excluding morphological data (Fig. 4A), Skiapus is recovered with support of 0.59 pp as sister taxon to the remainder of Ophioninae. With 0.90 pp, the remaining ophionine genera are subdivided into two Ophion + Thyreodon (Fig. 4A, purple and blue) and Enicospilus (Fig. 4A, red) genus-groups, including most genera but not Barytatocephalus. The first genus-group includes two strongly supported sister-clades: (0.94 pp)

Ophion + Xylophion + Rhopalophion + Afrophion; and (0.89 pp) Thyreodon + Rhynchophion + Dictyonotus + Stauropoctonus + Eremotylus. Euryophion is in turn significantly associated (0.90 pp) with these two clades. The Enicospilus genus group comprises a well-supported (1.00 pp) association between Enicospilus and Laticoleus, to which Dicamptus and Hellwigiella are associated with support of 0.91 pp. In CO1 + 28S_morph (Fig. 4B), the subfamily is 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). © 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

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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, Ophion and Enicospilus. The Eremotylus group comprising Eremotylus + Stauropoctonus is supported by 0.88 pp. Ophion is still recovered as polyphyletic and grouped (1.00 pp) with Afrophion, Xylophion and Rhopalophion. Thyreodon is grouped (0.99 pp) with Dictyonotus and Rhynchophion, this clade being moderately associated (0.86 pp) with Euryophion. The Ophion, Eremotylus and Thyreodon groups are in turn moderately associated (0.83 pp) in a single polytomic clade. Finally, the Enicospilus lineage is formed by a significantly supported (1.00 pp) clade of Enicospilus + Lepiscelus + Laticoleus, associated with a moderate probability (0.85 pp) to Dicamptus and Hellwigiella. Skiapus appears here as a highly derived genus weakly associated (0.53 pp) with the Enicospilus lineage.

DISCUSSION T AXONOMY All analyses support the monophyly of the Ophioninae, including Hellwigia and Skiapus. Skiapus thus

clearly does not belong to the Hybrizontinae to which it was ambiguously associated in former 28S analyses because of the aberrant structure of this gene in Hybrizon (Gillespie et al., 2005; Quicke et al., 2009). Historical higher taxon association of Skiapus reflected its puzzling evolutionary association: the genus was first reluctantly placed in Banchinae by its author (Morley, 1917), then transferred to Campopleginae as an anomalous tribe (Townes, 1970) before being recognized as belonging to the Ophioninae based on molecular data and the presence of a spurious vein on the fore wing (Quicke et al., 2005). However, its unusual habitus (nothing is known about its biology) led Quicke et al. (2005) to consider the possibility that the genus may be divergent enough to represent a new subfamily. Our present data support Skiapus as a strongly derived genus of Ophioninae, although its definitive placement within the subfamily is not fully resolved. Data from our analyses support the subdivision of Ophioninae into three clades (Table 2) which essentially match the three genus-groups defined in the morphological analysis of Gauld (1985). A fourth possible clade, including Eremotylus and Stauropoctonus,

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

Tribe Ophionini stat. rev.

Thyreodonini tribe nov.

Enicospilini stat. rev.

Incertae sedis

Morphological synapomorphies Ramellus present; 1 m-cu angled; mesopleural furrow extended; thyridia close to the margin of tergite 2 Laterotergite 2 pendant; propodeum with anterior transverse carina absent Spiracular sclerite partially to totally occluded

Genera included Ophion Xylophion Afrophion Rhopalophion Alophophion Sclerophion* Thyreodon Rhynchophion Dictyonotus

Dicamptus Enicospilus Laticoleus Hellwigiella Euryophion Stauropoctonus Eremotylus Janzophion Leptophion Pamophion Skiapus Hellwigia Agathophiona* Barytatocephalus Lepiscelus Ophiogastrella Orientospilus* Prethophion* Riekophion Sicophion* Simophion* Trophophion*

appears only in CO1 + 28S_morph with a non-significant probability and no support from the other analyses. We therefore do not discuss this clade any further. We consider the first three clades to represent tribes, because they are supported by both morphological and molecular apomorphies, and hence are relevant to formulate the evolutionary history of the Ophioninae. The clades are represented by the tribes Ophionini and Enicospilini stat. rev. whose names are resurrected and redefined, and the Thyreodonini tribe nov.

Distribution (specific diversity; Yu et al., 2012) Worldwide, mostly Holarctic (138) Australasian (2) South Africa (2) Afrotropical (3) Neotropical (7) Oriental (2) Mostly Neotropical (45) Mostly Neotropical (5) Southern Africa and eastern Asia (4) Afrotropical, Oriental and Australasian (32) Worldwide, mostly pantropical (699) Afrotropical (11) Mediterranean (1) Mostly Afrotropical (8) Worldwide except Nearctic Holarctic and Neotropical (17) Central America (2) Oriental and Australasian (30) Australia (1) Afrotropical and Korea (2) Palaearctic (3) Mexico (1) Eastern Europe to Caucasus (5) Afrotropical (1) Neotropical (6) India, Madagascar and South Africa (3) Neotropical (1) Australia (3) Neotropical (2) Central Asia and Central America (4) Western USA (1)

Comments Paraphyletic

Thyreodonini? Thyreodonini? Thyreodonini? Enicospilini? Enicospilini? Enicospilini? Skiapini? Skiapini?

Ophionini and Enicospilini sensu Townes (1971) were defined according to the length of the membranous flange on the front tibial spur, the flange being strongly reduced in the latter. The Ophionini were therefore defined on a plesiomorphic feature. Moreover, the flange reduction was later acknowledged as homoplastic (Gauld, 1977). Our analyses support homoplasy of this structure, the reduction of the flange occurring independently in Enicospilini, Thyreodonini and in Xylophion (Ophionini) in our cladogram. Since Townes’ classification was

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considered unsatisfactory because of the lack of phylogenetic support (Gauld, 1977), it is necessary for us to redefine his tribal concepts based on our data. Ophionini stat. rev We here morphologically define the tribe Ophionini by the angled 1 m-cu and the presence of a ramellus within the disco-submarginal cell (Fig. 5A, B). This feature is often considered as a plesiomorphic condition for ichneumonid wasps, because it appeared in the early fossil subfamilies of Ichneumonidae (Townes, 1973; Quicke, 2015). However, it evolved independently within many of the extant subfamilies, and in the ‘higher ophioniformes’ (i.e. Campopleginae, Cremastinae, Anomaloninae and Ophioninae). The basal character state is a rounded 1 m-cu without any ramellus. The reappearance of this feature in the Ophionini may thus be considered as a reversal. This hypothesis is further supported by the presence of an exceptionally long ramellus in Rhopalophion species, which is far longer than in any other extant ichneumonid taxon. Ophionini also exhibit a relatively well-developed mesopleural fovea. The plesiomorphic condition, in Ichneumonidae, is the presence of only a small pit positioned mid-posteriorly on the mesopleuron below the speculum (Gauld, 1985). In Ophionini, the pit is extended into a longitudinal or diagonal groove extending toward the anterior edge of the mesopleuron. Finally, the tribe is also defined by the close position of the thyridium to the anterior margin of the second metasomal tergite. The distance of separation between the thyridium and the anterior tergal margin is less than the thyridium length in all members of the Ophionini, whereas this distance is greater than the length of the thyridium in all other genera of the subfamily Ophioninae. As defined here, Ophionini includes the four genera Ophion, Xylophion, Rhopalophion and Afrophion. The tribe is consistently retrieved when 28S and combined DNA data are analysed. Support for the Ophionini clade is further strengthened by the addition of morphological data. The minor genera Alophophion and Sclerophion (seven and two species, respectively) most probably also belong to this tribe because they share the morphological apomorphies (Gauld, 1985), and for Alophophion this hypothesis is confirmed by the 28S marker. Ophion is by far the most speciose of these genera and contains 90% of the species in the tribe, and is most diverse in the Holarctic region. However, the delimitation of the genus itself is problematic. In all our analyses, Ophion is depicted as polyphyletic. Ophion minutus and the related unidentified sp4 are in particular well differentiated from the main Ophion clade, as was also emphasized by Schwarzfeld et al. (2016) in

their molecular analysis combining CO1 and 28S. The genus was considered to be probably paraphyletic by Gauld (1985) with respect to some of the minor genera (Agatophiona, Rhopalophion, Sclerophion, Afrophion, Xylophion, Alophophion) in his genus-group. Several studies attempted to clarify the internal phylogeny of the genus (Gauld, 1980; Schwarzfeld & Sperling, 2014; Schwarzfeld et al., 2016). The actual biodiversity has been furthermore grossly underestimated based on pure morphological taxonomy: a recent molecular revision of the Nearctic and European diversities shows there are probably 90– 121 actual species (Schwarzfeld & Sperling, 2015) instead of the about 50 expected by pure morphology (Gauld, 1985). To clarify the definition of Ophion and the sibling minor genera would, however, require a global revision of the tribe, which is beyond the scope of the present study. Thyreodonini tribe nov The genus-group circumscribed by Gauld (1985) based on Thyreodon (Thyreodon + Barytatocephalus + Euryophion + Rhynchophion + Dictyonotus) was defined by several morphological apomorphies: the long and straight, or barely curved hind tarsal claws; the absence of transverse propodeal carinae; and the rounded hind tibial spurs. Moreover, the genus-group is also characterized within Ophioninae by the reduction of the flagellar length and often reduced ocelli, characters associated with dry hot areas and/or diurnal habits in contrast to the vast majority of other ophionine species. Gauld (1985) also highlighted some morphological similarities between his Thyreodon and Enicospilus genus-groups. All these observations suggest that some of the morphological features characterizing Gauld’s Thyreodon genus-group are homoplastic as a result of ecological convergence. The associations provided by morphological assessment in the present analyses therefore have to be treated with caution as they could be determined by homoplasy. The molecular data demonstrate that the Thyreodon clade represents an evolutionary lineage distinct from the Ophion and Enicospilus clades, but delimitation of this lineage is made ambiguous by a lack of congruence between the molecular and morphological data. The CO1 marker (Fig. 2) clusters Thyreodon with Stauropoctonus and Barytatocephalus while 28S (Fig. 3) links it to Stauropoctonus only, both associations lacking significant support. In the combined CO1 + 28S_nomorph, Thyreodon is strongly associated with Dictyonotus, Rhynchophion, Eremotylus and Stauropoctonus (Fig. 4A), and Euryophion is depicted as basal to the Thyreodon + Ophion clade. Conversely, the addition

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A

B

C

D

E

F

G

H

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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 gauldetmitchellorum (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 Thyreodon + Dictyonotus + Rhynchophion from Stauropoctonus + Eremotylus and moderately groups it with Euryophion. Morphologically these four genera share

the total reduction of the anterior propodeal carina and the pendant laterotergite 2 (Fig. 5E–G). The definition of the tribe Thyreodonini tribe nov. to circumscribe the Thyreodon evolutionary lineage

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is thus somewhat arbitrary. The tribe is defined by the unambiguous inclusion of the three genera Thyreodon, Dictyonotus and Rhynchophion which are strongly correlated by both molecular and morphological synapomorphies (Fig. 4B). Euryophion might also belong to Thyreodonini because of its morphology, but the molecular data alone tend to consider it as the sister genus of the Ophionini +Thyreodonini clade. Thyreodon is a moderately diversified genus of 45, mostly Neotropical species, while Rhynchophion includes four Neotropical species, Dictyonotus four species in the Old World tropics and Euryophion eight species in the Old World tropics. The possible inclusion of Eremotylus and Stauropoctonus is supported by molecular data (Fig. 4A). Barytatocephalus is excluded as there are no synapomorphies, nor any ecological or geographical data to support its inclusion in the tribe. Enicospilini stat. rev The Enicospilus genus-group was defined by Gauld (1985) as a large clade including Enicospilus, Dicamptus, Laticoleus, Leptophion and the minor genera (containing 1–7 species) Ophiogastrella, Stauropoctonus, Lepiscelus, Pamophion, Orientospilus, Prethophion and Simophion. As a result of this large circumscription, he could only determine a single weak apomorphy: the loss of the laterotergite on metasomal tergite 1. Our molecular data do not fully support Gauld’s definition of this group, but the data do confirm the definition of a distinct lineage based on Enicospilus. The tribe Enicospilini stat. rev., circumscribed by the combined molecular markers, comprises Enicospilus, Laticoleus, Hellwigiella and Dicamptus. The clade is slightly less well supported when morphological data are included, probably because of the anomalous association with Skiapus. All four genera are morphologically characterized by the occlusion of the spiracular sclerite (Fig. 5C, D). Although this feature is also present in some other Ichneumonidae, it is unambiguously derived within the Ophioninae, because the plesiomorphic condition in ophioniforms is an exposed sclerite. Noticeably, this feature is also shared by genera that were more or less strongly related to them by the 28S sequence data: Pamophion, Leptophion and Janzophion. Thus, the tribe Enicospilini comprises four genera: the mostly pantropical and highly diversified genus Enicospilus encompassing 95% of its species, Laticoleus (Afrotropical), Dicamptus (Afrotropical and Indo-Australasian) and the monobasic Hellwigiella from the Mediterranean region. Pamophion, Leptophion and Janzophion may also be part of this tribe, but no CO1 data were available to confirm their affinities.

U NPLACED

GENERA

The placement of the monobasic genus Lepiscelus as a highly derived species embedded within Enicospilus (CO1 analysis, Fig. 2) is probably an artefact, as the CO1 sequence obtained for L. distans was only 299 bp. Moreover, the genus is, in part, characterized by features which may also be found with lesser prevalence in some Enicospilus species, i.e. the reduction of the occipital carina and the flanged apical expansion of the mid and hind trochantelli. Gauld (1985) pointed out the morphological similarities of L. distans with some Enicospilus species in dry environments. Conversely, the 28S analyses provided contradictory results, and additionally L. distans does not have the occluded spiracular sclerite possessed by the remainder of the tribe. Thus, no strong molecular or morphological support is available to definitely place this genus within Ophioninae. Recovered affinities of the genus Skiapus were highly variable across analyses, making placement of this genus within the subfamily ambiguous. The molecular data provided non-convergent results across markers as a result of the distinct DNA sequences of the genus. The CO1 marker weakly linked Skiapus to D. maxipol, which is itself a rather aberrant species of Dicamptus; the 28S sequence and the morphological characters placed the genus close to the Enicospilus lineage, but these placements are most probably anomalous because of long branch lengths. This is probably a consequence of its atypical 28S sequence and its very uncharacteristic morphology. Excluding morphological data, the combined markers placed Skiapus as a sister to the remaining Ophioninae.

P HYLOGENY

AND BIOGEOGRAPHY

The reconstruction of the evolutionary history of Ophioninae is complex for two main reasons. Firstly, the origin and subsequent expansion route of these lineages is difficult to ascertain because of their strong dispersal abilities. Ophioninae are good dispersers and colonizers because of their flight abilities and their relatively large host ranges, which has enabled them to reach and colonize remote oceanic islands (Quicke, 2015). As a result, their biogeographical history is likely to be driven by a succession of wide expansions and subsequent replacements by ecologically more successful taxa. This may explain why the lineages contain a contrasting mix of widespread and highly diversified genera (Enicospilus and Ophion, with c. 700 species in the Southern Hemisphere and 150 species in the Northern Hemisphere, respectively) and few

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A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE intermediate genera of 30–50 species (Thyreodon, Leptophion, Eremotylus, Alophophion and Dicamptus), along with numerous localized, monotypic or nearly so, and often highly derived genera (Xylophion, Rhynchophion, Dictyonotus, Skiapus, Sicophion, Janzophion, Hellwigia, etc.), which together represent 80% of the generic diversity, but less than 10% of the species (Yu et al. 2012). The second reason is the absence of reliable dating calibration data. Based on limited available fossil data, Gauld (1985) hypothesized that ‘possibly [the Ophioninae] radiated around the beginning of the Tertiary some 65–70 million years ago’. The family Ichneumonidae is proposed to have radiated around 140 Mya (Quicke, 2015), following the diversification of their lepidopteran major hosts during the Cretaceous era (Grimaldi & Engel, 2005). The following assumptions are therefore based on a first radiation dating, which cannot be certified at the moment. The lineage leading to Skiapus is tentatively considered as a basal divergence of Ophioninae as depicted by the combination of molecular data. Even though weakly supported, this ancient radiation hypothesis is supported by the vicariant distribution of Skiapus whose three described and seldom collected species occur in Africa and Korea. The extant species of Skiapus are probably remnants of an ancient, more diverse, lineage. As proposed by Quicke et al. (2005), Skiapus most probably forms a monophyletic clade with Hellwigia (result not confirmed here by our partial data for Hellwigia). This ancient radiation would thus form a transitional lineage between Ophioninae and the remainder of the Ichneumonidae, characterized by an intermediate wing venation. Ophioninae possibly diversified c. 65–70 Mya, when Eurasia and Africa were still connected to North America, while South America and Australia were separated by large oceans after Gondwana broke up 30 Myr before (Le Gall et al., 2010). According to our analyses, the main subfamily split into two major lineages, one leading to Enicospilus and another subsequently splitting into the Ophion and Thyreodon lineages. Gauld (1985) proposed Ophion and the related genera as the basal lineage of Ophioninae, based on his assertion that the fore wing venation configuration is plesiomorphic in these taxa. On the contrary, our molecular data and the current hypotheses concerning the phylogeny of the higher ophioniforms (Quicke et al., 2009) strongly suggest this might indeed be a character reversal. Hence the diversification of the tribe Ophionini took place after the appearance of the ancestors of the other tribes. These ancestral species probably evolved in Eurasia or Africa, concurrently with at least the Enicospilus lineage.

139

The Ophionini may have been the first lineage to diversify and expand, and putatively would have been previously far more widespread, as shown by the existence of the localized extant genera (Afrophion and Xylophion), which are phylogenetically but no longer geographically connected to the Holarctic genus Ophion. In the tropical areas the Ophionini may subsequently have been replaced by Enicospilus and related genera, but this latter lineage was far less successful in temperate areas where Enicospilini are currently weakly represented. Hence Ophion and Enicospilus became exclusively predominant in temperate and tropical areas, respectively. It also appears that Enicospilus colonized the Neotropical area after the break-up of Gondwana, because it is the only genus of Enicospilini present in this region and because the Neotropical species-group diversity of Enicospilus is far lower than in the Old World tropics (Gauld, 1988). Enicospilus might have reached the Neotropical region either via North America, or through temporary inter-continental connections (Coney, 1982) as was proposed for Campto€ksj€ typus (Ichneumonidae: Pimplinae) (S€ aa arvi, Gauld & Salo, 2004). Ophionini and Thyreodonini share a common ancestor: this is shown by the combined molecular analysis and also by the 28S analysis where some Thyreodonini genera are alternatively weakly linked to the Ophionini clade. Thyreodonini probably first diversified in the Old World tropics, where Dictyonotus is still present. This is also suggested by the Afrotropical and Oriental genus Euryophion, which is either part of Thyreodonini or the sister genus of the clade Thyreodonini + Ophionini. The colonization of North America possibly occurred thereafter, via the ancient northern connection with Eurasia before the eventual breakup of Laurasia or later via the Bering Strait. This is suggested by the putative association of Eremotylus and Stauropoctonus to Thyreodonini, supported by molecular data, but without morphological confirmation. These two genera have an intermediate distribution, especially Eremotylus whose diversity peaks in the Nearctic region. Following this hypothesis, Thyreodon arose and diversified with relative success in South America. The continent was connected to North America by the formation of the Panama Isthmus 13–15 Mya (Montes et al., 2015). The relative success of Thyreodon may be the result of colonization by this genus of a region that was not yet under the ecological hegemony of Ophion or Enicospilus. This global scenario hypothesis needs to be treated with caution because it is based on limited available data. The hypothesis relies on weak dating assumptions and moreover does not encompass many of the minor genera, which were not supported by any available molecular data, or if available the data

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proved to be insufficient to unambiguously link these genera to one or another lineage. Future assessments will need to take into account additional fossil and/or molecular data allowing for a robust molecular clock assessment.

ACKNOWLEDGEMENTS The completion of this work was greatly helped by the intervention of numerous contributors. We thank all of them find for providing specimens, pictures, DNA sequences and/or bibliographical references. Thanks to Terry Reynolds, Nyembezi Mgocheki and Pierre-Olivier Maquart (Iziko South African Museums), Dicky Yu (Canadian National Collections), Bernardo Santos (American Museum of Natural History), Jo€elle Sadeyen (Cirad), Gavin Broad (Natural History Museum), Franck M€ uller (Museum National d’Histoire Naturelle), Brian Fischer and Bob Zuparko (California Academy of Sciences), Andr es Fabi an Herrera Florez (University of Manitoba) and Jayme Sones (University of Guelph). Some sequencing was done for us by Sam Bolton, Katherine Powell and Will Nash. S.v.N. was funded by South African NRF (National Research Foundation) grants: GUN 2068865; GUN 61497; GUN 79004; GUN 79211; GUN 81139; GUN 86958. Part of the South African field work conducted by S.v.N. was funded by the National Science Foundation under PlatyPBI grant No. DEB-0614764 to N. F. Johnson and A. D. Austin. P.R. was funded by SABI (South African Biodiversity Initiative) NRF post-doctoral fellowship GUN 81609 and a Claude Leon Foundation post-doctoral fellowship. Cape Nature, the Eastern Cape Department of Environmental Affairs and the Northern Cape Department of Nature and Environmental Conservation provided collecting permits for South Africa. The Ugandan Wildlife Authority and UNCST provided permits to conduct research in Uganda. Field work in the Central African Republic was supported by WWF-US and WWF-CARPO. The Ministers of Water, Forests and the Environment and the High Commissioners for tertiary Education and Research of the Central African Republic granted permission to carry out the inventory survey and to export the specimens as part of the WWF-US CAR field expedition conducted in 2001.

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APPENDICES Appendix 1. Species used in the analyses. *Genbank accession numbers are CO1/28S Species Anomaloninae Anomalon sp. Agrypon sp. aff. nelsoni Agrypon varitarsum (Wesmael) Parania sp. Banchinae Apophua hispida (Benoit) Glypta fumiferanae Viereck Occia jereza Ugalde & Gauld Campopleginae Diadegma mollipla (Holmgren) Dusona sp. Venturia canescens (Gravenhorst) Campoletis sonorensis (Cameron) Cremastinae Eiphosoma sp. Hybrizontinae Hybrizon ghilarovi Tobias Ophioninae Afrophion hynnis (Gauld & Mitchell) Afrophion nubilicarpus (Tosquinet) Alophophion sp. Barytatocephalus mocsaryi (Brauns, 1895) Dicamptus maxipol Rousse & van Noort Dicamptus pulchellus (Morley) Dicamptus sp.1 Dicamptus sp.2 Dicamptus sp.3 Dictyonotus purpurascens (Smith) Enicospilus albiger (Kriechbaumer) Enicospilus antefurcalis (Sz epligeti) Enicospilus biimpressus (Brull e) Enicospilus capensis (Thunberg) Enicospilus divisus (Seyrig) Enicospilus dolosus (Tosquinet) Enicospilus equatus Gauld & Mitchell Enicospilus finalis Gauld & Mitchell Enicospilus grandiflavus Townes Enicospilus herero (Enderlein) Enicospilus justus (Seyrig) Enicospilus leucocotis (Tosquinet) Enicospilus mamatsus Gauld & Mitchell Enicospilus sp. nov. Enicospilus ramidulus (L.) Enicospilus rundiensis Bischoff Enicospilus senescens (Tosquinet) Enicospilus transvaalensis Cameron Enicospilus umbratus Gauld & Mitchell

Collection country

Accession no.*

Zimbabwe Costa Rica UK Madagascar

GenBank: GenBank: GenBank: GenBank:

Uganda Canada Costa Rica

Bold: OPHY005-15 GenBank: HQ107295/HQ025539 GenBank: JQ576059/EU378350

South Africa Central African Republic USA (culture) USA (culture)

GenBank: AJ888008/AJ508222.2 Bold: OPHY008-15 GenBank: DQ538851/DQ539001 GenBank: DQ538850/DQ539000

Bolivia

GenBank: JF963241/EU378426

Russia

GenBank: JF963427/EU378579

South Africa South Africa UK (Falklands) Turkey South Africa Gambia Madagascar Taiwan Taiwan Japan South Africa Madagascar Uganda South Africa Uganda Central African Republic Central African Republic Uganda South Africa South Africa Uganda South Africa Madagascar French Polynesia UK Uganda Uganda South Africa Madagascar

GenBank: JF962473/AY593084 Bold: OPHY001-15 GenBank: –/AY593085.1 GenBank: JF962965/AY593086 Bold: OPHY006-15 Bold: OPHY007-15 Bold: AAI6316 Bold: ASQIC064-09 Bold: ASQIC065-09 GenBank: –/EU378703 Bold: OPHY017-15 Bold: OPHY009-15 Bold: OPHY021-15 Bold: OPHY025-15 Bold: OPHY032-15 Bold: OPHY033-15 Bold: OPHY035-15 Bold: OPHY036-15 Bold: OPHY038-15 Bold: OPHY040-15 Bold: OPHY044-15 Bold: OPHY046-15 Bold: OPHY051-15 Bold: OPHY054-15 GenBank: JF963318/DQKP100 Bold: OPHY060-15 Bold: OPHY071-15 Bold: OPHY077-15 GenBank: JF963320/EU378709

JF962903/– JF792887/– –/AJ302927 –/EU378323

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Appendix 1. Continued

Species Eremotylus curvinervis (Kriechbaumer) Eremotylus sp.1 Eremotylus sp.2 Eremotylus sp.3 Eremotylus sp.4 Eremotylus sp.5 Euryophion ikuthanus (Kriechbaumer) Euryophion nigripennis Cameron Euryophion sp. Hellwigia obscura Gravenhorst Hellwigiella dichromoptera (Costa) Janzophion nebosus Gauld Laticoleus curvatus Delobel Laticoleus spilus Gauld & Mitchell Laticoleus unicolor (Sz epligeti) 1 Laticoleus unicolor (Sz epligeti) 2 Lepiscelus distans (Seyrig) Leptophion anici Gauld Ophiogastrella maculithorax Brues Ophion costatus Ratzeburg Ophion minutus Kriechbaumer Ophion obscuratus Fabricius Ophion scutellaris Thomson Ophion aff. scutellaris Ophion idoneus Viereck Ophion sp.1 Ophion sp.2 Ophion sp.3 Ophion sp.4 Ophion sp.5 Pamophion sorus Gauld Rhopalophion discinervus (Morley) Rhynchophion flammipennis (Ashmead) Riekophion emandibulator (Morley) Sicophion fenestralis Gauld Skiapus coalescens Morley Skiapus sp.1 Skiapus sp.2 Stauropoctonus bicarinatus (Cushman) Thyreodon atriventris (Cresson) 1 Thyreodon atriventris (Cresson) 2 Thyreodon laticinctus Cresson Thyreodon sp.1 Xylophion sevrapek Villemant

Collection country

Accession no.*

UK USA USA USA USA USA Tanzania South Africa Togo France Spain Costa Rica Madagascar South Africa South Africa Madagascar South Africa Australia Costa Rica UK UK UK UK UK Canada Madagascar Madagascar Taiwan UK UK Australia South Africa Costa Rica Australia Costa Rica Gambia Tanzania Togo Costa Rica Costa Rica Nicaragua Costa Rica Canada Vanuatu

Bold: BBHYA1627-12 Bold: BBHYG815-10 Bold: HYMBB144-09 Bold: HYMBB269-09 Bold: HYMBB283-09 Bold: OPHY080-15 GenBank: –/AJ302854 GenBank: –/AJ302858 GenBank: JF957051/EU378710 GenBank: –/EU378711 Bold: OPHY081-15 Bold: OPHY082-15 Bold: OPHY083-15 Bold: OPHY084-15 GenBank: –/AY593089 GenBank: –/EU378714 GenBank: JF963664/EU378716 GenBank: JF963665/EU378717 GenBank: FN662468/Z97889 GenBank: JF963667/EU378720 GenBank: KF594819/KF616305 GenBank: KF594578/KF616320 BOLD: AAI3373 BOLD: ASQIC068-09 BOLD: ASQIC071-09 BOLD: ASQIC109-09 BOLD: ASQIC112-09 GenBank: –/EU378722 Bold: OPHY085-15 GenBank: JF793291/AY593090 GenBank: –/EU378726 GenBank: –/EU378727 Bold: OPHY086-15 GenBank: JF963839/AY604252 Bold: AAJ5095 Bold: AAB7595 GenBank: JF793300/AJ302876 Bold: ABV8816

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Appendix 2. Morphological characters Head 01 02 03 04

Labial palps: (0) four-segmented; (1) three-segmented. Maxillary palps: (0) five-segmented; (1) four-segmented. Central segments of maxillary palps: (0) slender; (1) enlarged, globose. 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°. 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.

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE

145

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; (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.

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

Afrophion hynnis Afrophion nubilicarpus Agrypon varitarsum Apophua hispida Barytatocephalus mocsaryi Campoletis sonorensis Diadegma mollipla Dicamptus maxipol Dicamptus pulchellus Dicamptus sp Dictyonotus purpurascens Dusona sp. Eiphosoma sp. Enicospilus albiger Enicospilus antefurcalis Enicospilus biimpressus Enicospilus capensis Enicospilus divisus Enicospilus dolosus Enicospilus equatus Enicospilus finalis Enicospilus grandiflavus Enicospilus herero Enicospilus justus Enicospilus leucocotis Enicospilus mamatsus Enicospilus sp. Enicospilus ramidulus Enicospilus rundiensis Enicospilus senescens Enicospilus transvaalensis Enicospilus umbratus Eremotylus sp Euryophion sp Hellwigiella dichromoptera Laticoleus unicolor Lepiscelus distans Occia jereza Ophion costatus Ophion idoneus Ophion minutus Ophion obscuratus Ophion scutellaris

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 01 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 0 0 2 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 1 12 1 1 1 2 2 2 1 1 1 2 12 3 1 1 2 12 12 0 0 1 0 0 0 0 0 0 0 0

3 4 5

Appendix 3. Morphological matrix

0 0 0 0 0 1 0 0 0 0 0 01 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0

6 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0

0 0 0 0 01 0 0 0 01 ? 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

7 8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 01 0 0 1 1 0 0 1 0 1 1 0 0 01 0 0 2 0 1 0 0 0 0 0 0

9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 1 1 1 1 0 0 1 1 01 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 ? 0 1 0 ? 1 0 0 0 0 0

0 0 01 1 1 01 01 0 0 0 0 0 1 0 1 0 1 1 1 1 1 0 1 1 0 0 0 1 1 01 1 01 0 0 1 1 1 0 ? ? ? 0 ?

0 0 0 0 1 1 0 1 1 1 1 1 0 0 1 0 1 1 0 0 1 1 1 1 1 0 0 1 0 0 1 0 0 1 1 1 0 1 ? 1 ? 1 1

0 0 1 0 0 01 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 1 01 0 0 1 1 1 0 1 01 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1

01 01 0 1 ? 0 0 1 0 ? 0 0 ? 1 1 1 0 1 1 1 1 1 1 0 1 1 1 1 0 0 1 1 ? 0 0 1 0 0 ? ? ? ? ?

1 1 2 0 0 0 0 0 2 2 0 0 ? 2 2 12 1 2 2 2 2 0 1 2 01 01 2 12 1 2 2 2 ? 0 0 1 2 2 ? ? ? 1 ?

1 1 0 0 0 0 0 0 1 1 0 0 0 1 1 1 0 1 1 2 1 1 1 1 0 1 2 1 1 1 1 1 12 0 0 0 1 0 1 12 12 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 01 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 01 0 0 2 1 ? ? ? 0 ?

0 0 0 0 0 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 0 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 0 0 0 0

0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 01 0 0 0 0 1 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0

1 1 0 0 01 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 1 1 1

1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 01 1 0 1 1 1 1 1 1

0 0 0 1 ? 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 1 0 0 0 1 1 0 0 0 0 0 0 01 0 0 0 1 0 0 0 0 0

1 1 1 0 0 1 0 0 0 0 0 0 1 1 0 0 1 0 0 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 1 1 0 1 1 1 1 1

1 1 ? 0 ? ? ? 0 0 0 0 0 ? 1 01 01 0 1 1 01 01 0 0 01 0 01 0 1 1 1 1 0 ? 0 0 1 0 ? ? ? ? 0 ?

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

146 P. ROUSSE ET AL.

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

Afrophion hynnis Afrophion nubilicarpus Agrypon varitarsum Apophua hispida Barytatocephalus mocsaryi Campoletis sonorensis Diadegma mollipla Dicamptus maxipol Dicamptus pulchellus Dicamptus sp Dictyonotus purpurascens Dusona sp Eiphosoma sp Enicospilus albiger Enicospilus antefurcalis Enicospilus biimpressus Enicospilus capensis Enicospilus divisus Enicospilus dolosus Enicospilus equatus Enicospilus finalis Enicospilus grandiflavus Enicospilus herero

Ophion sp nr scutellaris Ophion sp Rhopalophion discinervus Rhynchophion flammipennis Skiapus coalescens Skiapus sp Stauropoctonus sp Thyreodon atriventris Thyreodon laticinctus Venturia canescens Xylophion sevrapek

.Appendix Continued3. Continued

0 0 0 01 01 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 1 1 0 0

0

0 0

0

1 1 2 0 0 0 0

0

0 0

0

1 1 3 0 0 0 0

0

0 0

0

3 4 5

0 0 0 0 0 0 0

0

0 0

0

6

0 0 0 0 0 0 0

0

0 0

0

0 0 0 0 0 0 0

0

0 0

0

7 8

3 3 0 0 0 0 0

0

0 0

0

9

1 1 0 0 0 0 0

0

0 0

0

0 0 ? 0 1 1 1

1

0 0

0

1 1 01 0 0 01 1

0

? 0

?

1 1 0 0 0 0 1

1

1 01

1

0 0 0 1 1 0 0

1

0 0

0

0 0 0 0 0 1 0

0

0 0

0

0 0 0 1 1 0 0

0

0 0

0

1 1 1 0 0 0 1

0

1 1

1

1 1 01 0 0 0 1

1

? 1

?

0 0 01 0 0 0 1

0

? 1

?

0 0 2 2 0 0 1

0

12 1

12

1 1 0 0 0 0 0

0

0 0

0

0 0 01 0 0 0 0

0

? 0

?

1 1 0 0 0 1 0

0

0 0

0

0 0 ? 0 0 0 0

0

0 0

0

0 0 0 0 0 0 0

1

0 0

0

0 0 0 0 0 0 0

0

0 0

0

0 0 1 01 01 0 1

1

1 1

1

0 0 0 1 1 0 1

1

1 1

1

1 1 0 0 0 0 0

?

0 1

0

0 0 0 1 1 1 0

0

1 0

1

0 0 ? 0 0 ? 0

?

? 0

?

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

0 0 0 0 01 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 0

0 0 1 0 0 0 0 0 1 0 0 0

0 0 0 0 0 2

1 0 2 0 2

1 0 2 2 2 2 2 2 2 2 2 2

1 1 2 2 2 2

1 1 2 0 2

1 0 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 1

0 0 1 0 1

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1

1 1 1 0 1

0 0 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 0

0 0 0 0 0

0 1 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 0

1 1 1 0 1

0 ? 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 1

0 0 0 0 1

1 2 1 1 1 1 1 1 1 1 1 1

01 1 1 1 1 1

0 0 2 2 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 0

0 0 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 1

1 1 0 0 1

0 0 2 2 2 2 2 2 2 2 2 2

0 0 2 2 2 2

2 2 0 0 2

0 0 2 2 2 2 2 2 2 2 2 2

0 0 2 2 2 0

2 1 0 0 1

0 0 1 1 1 1 1 1 0 1 1 1

0 0 0 1 1 0

1 0 0 0 0

0 0 0 1 1 01 1 0 0 1 0 0

0 0 0 0 0 0

0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1

0 0 0 1 1

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0

1 1 0 0 0

1 0 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 2

0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 0

0 0 0 1 1 1 1 1 1 1 1 1

0 0 01 0 ? 0

1 1 0 0 0

0 0 0 1 1 1 0 1 0 1 0 01

0 0 0 2 2 0

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0

0 ? 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 2

1 1 ? 1 0

2 02 1 1 1 1 1 1 1 1 1 1

1 2 1 1 1 0

0 0 2 0 0

0 0 2 2 2 2 2 2 2 2 2 2

0 0 1 1 1 2

0 0 0 0 2

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1

0 0 0 0 1

1 ? 0 0 0 1 0 0 1 1 0 0

? ? 1 1 1 ?

1 1 01 1 01

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

0

2

0

1

A REASSESSMENT OF THE PHYLOGENY OF OPHIONINAE

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148

147

Enicospilus justus Enicospilus leucocotis Enicospilus mamatsus Enicospilus sp Enicospilus ramidulus Enicospilus rundiensis Enicospilus senescens Enicospilus transvaalensis Enicospilus umbratus Eremotylus sp Euryophion sp Hellwigiella dichromoptera Laticoleus unicolor Lepiscelus distans Occia jereza Ophion costatus Ophion idoneus Ophion minutus Ophion obscuratus Ophion scutellaris Ophion sp nr scutellaris Ophion sp Rhopalophion discinervus Rhynchophion flammipennis Skiapus coalescens Skiapus sp Stauropoctonus sp Thyreodon atriventris Thyreodon laticinctus Venturia canescens Xylophion sevrapek

Appendix 3. Continued

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0

0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0 0 0 0 0 0

0 0

0

0 0 01 01 01 0 0

0 0 0 1 1 0 0

1

0 0

0 0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 2 2 0 0

2

0 01

0 2 2 0 0 0 0 0 0

0 2 2 0

0 1 1 0 0 0 0 0

2 2 2 2 2 0 2

2

01 0

2 2 2 01 01 0 2 01 01

2 ? 2 2

2 2 2 2 2 2 2 2

1 1 1 1 1 0 0

1

0 1

1 1 1 0 0 0 0 0 0

1 1 1 1

1 1 1 1 1 1 1 1

1 1 1 1 1 1 1

1

1 1

1 1 0 1 1 1 1 1 1

1 1 1 1

1 1 1 1 1 1 1 1

01 01 1 0 0 0 0

0

0 0

1 1 0 0 0 0 0 0 0

1 0 0 0

1 1 1 1 1 1 1 1

0 0 1 1 1 1 1

0

1 1

1 1 1 1 1 1 1 1 1

1 1 0 0

1 1 1 1 1 1 1 1

1 1 0 1 1 0 0

1

0 0

1 1 0 0 0 0 0 0 0

1 0 0 0

1 1 1 1 1 1 1 1

2 2 1 1 1 1 0

1

0 0

1 1 0 0 0 0 0 0 0

1 1 12 1

1 1 1 1 1 1 1 1

0 0 1 1 1 0 0

1

0 0

0 0 0 0 0 0 0 0 0

0 0 01 1

0 0 0 0 0 0 0 0

1 1 1 1 1 0 1

1

1 1

1 1 0 1 1 1 1 1 1

1 1 1 1

1 1 1 1 1 1 1 1

1 1 2 2 2 0 2

2

2 2

2 2 0 2 2 2 2 2 2

2 2 2 2

2 2 2 2 2 2 2 2

0 0 1 0 0 0 1

0

1 2

2 1 0 1 1 1 1 1 1

2 1 1 1

2 2 2 2 2 2 2 2

0 0 0 0 0 0 0

0

0 0

0 0 0 0 0 0 0 0 0

0 0 0 0

1 0 0 0 1 1 0 1

0 0 0 0 0 0 0

0

0 0

0 0 0 0 0 0 0 0 0

0 0 0 0

01 0 0 1 1 1 0 1

1 1 1 1 1 0 0

1

0 0

1 1 0 0 0 0 0 0 0

1 0 1 1

1 1 1 1 1 1 1 1

0 0 0 0 0 0 1

0

1 1

0 0 0 1 1 1 1 1 1

0 0 0 0

0 0 0 0 0 0 0 0

2 2 2 2 2 0 0

2

0 0

1 1 1 0 0 0 0 0 0

1 1 2 0

1 1 1 1 1 1 1 1

0 0 1 0 0 0 0

0

0 0

1 0 0 0 0 0 0 0 0

0 1 1 1

0 0 0 0 0 0 0 0

0 0 1 0 0 0 1

0

0 1

1 1 0 0 0 0 0 0 0

1 1 1 1

1 1 1 1 1 1 1 1

0 0 0 0 0 0 0

0

0 0

0 0 0 0 0 0 0 0 0

2 0 0 0

0 1 1 1 1 1 1 1

0 0 0 0 0 0 1

0

1 0

0 1 0 1 1 1 1 1 1

0 ? 1 1

0 0 0 0 0 0 0 0

1 1 01 ? ? 1 ?

01

? 1

0 0 ? ? ? ? 1 ? ?

1 01 12 ?

1 2 1 0 1 1 1 1

0 0 0 0 0 2 0

0

0 0

1 0 0 0 0 0 0 0 0

0 0 0 0

1 1 1 0 1 1 1 1

1 1 2 2 2 0 1

2

0 0

2 2 0 0 0 0 0 0 0

2 0 2 0

2 2 2 2 2 2 2 2

0 0 0 0 0 0 0

0

0 0

0 1 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 1 1 0 0

1

0 0

0 0 0 0 0 0 0 0 0

0 0 1 1

0 0 0 0 0 0 0 0

0 0 0 1 1 0 0

1

0 1

0 1 0 0 0 0 0 0 0

0 01 1 1

0 0 0 0 0 0 0 0

2 2 01 01 01 01 1

01

01 1

1 1 01 01 01 01 01 01 01

0 ? 0 01

1 0 0 1 1 1 1 1

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

148 P. ROUSSE ET AL.

© 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016, 178, 128–148