The parasitic wasp genera Skiapus, Hellwigia, Nonnus, Chriodes, and Klutiana (Hymenoptera, Ichneumonidae): Recognition of the Nesomesochorinae stat. rev. and Nonninae stat. nov. and transfer of Skiapus and Hellwigia to the Ophioninae

The ichneumonid wasp genera Skiapus Morley, Hellwigia Gravenhorst, Nonnus Cresson, Chriodes Förster, and Klutiana Betrem are each formally removed from the Campopleginae on the basis of morphological, molecular, and simultaneous analyses using PAUP* and the optimization alignment program POY. Skiapus and Hellwigia are shown to form a monophyletic group with the Ophioninae to which they are transferred. Nonnus is treated as comprising the Nonninae stat. nov. and Chriodes and Klutiana (sometimes treated as a junior synonym of Chriodes) are treated as comprising the Nesomesochorinae stat. rev. The status of Nesomesochorinae and Nonninae is not fully resolved as they are not consistently recovered as separate groups, forming a single clade in some analyses. We keep them separate because of this uncertainty. Molecular synapomorphies within the D2–3 expansion region of the 28S rDNA gene show the utility of this gene region in determining subfamily‐level placement within the higher Ophioniformes.


Introduction
Much effort has recently been put into obtaining a wide range of ichneumonoids for DNA sequencing projects aimed at answering a range of evolutionary questions (Belshaw et al. 1998(Belshaw et al. , 2001Broad and Quicke 2000;Belshaw and Quicke 2002;Dowton et al. 2002). As a result, representatives of many genera whose systematic placement has hitherto been uncertain have been obtained, and in several cases it is now possible to offer a more definite conclusion about their relationships. Here we use morphological, molecular, and simultaneous analyses to explore the relationships of five genera currently included in the Campopleginae but which do not fit well there and might therefore belong to other groups.
The Campopleginae sensu lato is a very large, cosmopolitan ichneumonid subfamily and comprises approximately 70 valid genera and 1836 species (Yu and Horstmann 1997;Wahl 1999). Campoplegines are koinobiont endoparasitoids, nearly all attacking Lepidoptera larvae, though a few taxa use larvae of other groups as hosts, including tenthredinoid symphytans (Hymenoptera), Raphidioptera, Trichoptera and chrysomelid, curculionid and cerambycid beetles (Coleoptera). Although the Campopleginae is an important group of parasitoids from both economic and biological standpoints, there have been very few studies of relationships within the subfamily (Wahl 1991;Dbar 1993;Miah and Bhuyia 2001) and it has been considered to be one of the most taxonomically difficult groups in the Ichneumonidae (Townes 1970;Gauld and Mound 1982;Gauld 1984).
Although the overall limits of the Campopleginae have been relatively stable since Townes ' (1970) generic treatment, as with so many ichneumonoid subfamilies, morphological synapomorphies defining it are hardly apparent. Within the Campopleginae there are considerable disagreements about its classification at all levels (Townes 1970;Carlson 1979;Gauld 1984;Wahl 1991). Two aberrant tribes, the Hellwigiini and Nesomesochorini recognized by Townes et al. (1961Townes et al. ( , 1965 are morphologically distinct from the rest of the Campopleginae and do not appear to fit well there. The hellwigiines have long been considered as a distinct group and Horstmann (1969) went as far as to raise them to subfamily status, the Hellwigiinae, comprising the two genera Hellwigia Gravenhorst and Protohellwigia Brues. Townes (1970), at almost the same time, continued to treat the Hellwigiini within the Campopleginae (5Porizontinae sensu Townes) as a tribe including the genera Hellwigia (with which he synonymized Protohellwigia Brues) and Skiapus Morley. Wahl (1991) considered the phylogenetic placement of Rhimphoctona Fö rster, a genus with a relatively long ovipositor that attacks wood-boring beetle larvae, with reference to the higher categories within the subfamily. The study included both larval and adult characters and he concluded that the existing tribes were unsound and instead recognized five, less formal, genus groups, although he stated that all were monophyletic. Three of these were monotypic (the Nemeritis Holmgren, Gonotypus Fö rster, and Menaka Gupta groups), the Bathyplectes group included a small number of genera (Bathyplectes Fö rster, Rhimphoctona, Pyracmon Holmgren, Nepiesta Fö rster, and Leptoperilissus Schmiedeknecht), while the Dusona Cameron group embraced all the remaining genera (including those previously in the tribes Hellwigiini and Nesomesochorini). Rhimphoctona represented a basal group which formed the sister clade to the rest of the subfamily. However, in Miah's (1998) preliminary morphological analyses of campoplegine genera, five genera, included in the Dusona group by Wahl (1991Wahl ( , 1999, namely Skiapus, Hellwigia, Nonnus, Chriodes, and Klutiana, were never recovered with the other members of the subfamily. The first two were associated consistently with the Ophioninae and the latter usually with the Anomaloninae. Molecular analyses have further suggested that neither the Nesomesochorini nor Hellwigia are actually campoplegines. In the preliminary molecular and simultaneous molecular and morphological analyses of Quicke et al. (2000), Nonnus was found not to cluster with the three other Campopleginae included, though its placement elsewhere was not stable. More recently, the molecular phylogeny presented by Belshaw and Quicke (2002) placed Hellwigia within the Ophioninae and Nonnus and Chriodes as the sister group of the Anomaloninae, largely in agreement with Miah's morphological trees.
Here we present additional 28S rDNA sequence data, including all the genera of Hellwigiini and Nesomesochorini together with a far denser sampling of campoplegine and related genera. These data are analysed separately and in combination with a morphological data set, based on the individual exemplar genera, using direct optimization to deal with the length variation in the gene fragment used. The results lead us to transfer Hellwigia and Skiapus to the Ophioninae and to recognize the Nesomesochorinae and Nonninae as subfamilies.

Selection of taxa
The Campopleginae belongs to the ''Ophioniformes'' group of subfamilies as proposed by Wahl (1991). This group currently comprises the Ophioninae, Ctenopelmatinae, Banchinae, Mesochorinae, Metopiinae, Campopleginae, Tatogastrinae, Cremastinae, Tersilochinae, and Anomaloninae, though molecular and morphological data additionally indicate that the Lycorininae, Neorhacodinae, Oxytorinae, Phrudinae, Stilbopinae, and Tryphoninae also belong in the same large clade D. L. J. Quicke et al., in preparation). Within this complex Gauld (1985) and Wahl (1991) suggested Ophioninae and Cremastinae, respectively, as the sister group of Campopleginae. The first hypothesis was supported by the most parsimonious tree (MPT) resulting from analyses of 28S rDNA by Belshaw et al. (1998) without any bootstrap support, and subsequently with high support by Belshaw and Quicke (2002). In the present analyses we therefore selected a number of genera of Ophioninae along with a smaller selection from each of the Anomaloninae, Banchinae, Cremastinae, Ctenopelmatinae, and Tersilochinae, in order to allow us to test the monophyly of Campopleginae sensu lato. The Orthopelmatiformes (comprising the genus Orthopelma Taschenberg) was selected as the outgroup because in previous molecular and simultaneous analyses it nearly always formed the sister group to the enlarged Ophioniformes group .
The genera of Campopleginae included in our analyses covered a wide range of the subfamily based on the cladistic analyses of Miah (1998). Our selection was also influenced by the five genus groups recognized by Wahl (1991), but we were unable to obtain material for sequencing of the Nemeritis and Menaka groups (both monotypic). The taxa sequenced are listed in Table I together with the EMBL/GenBank accession numbers of the sequences obtained. Morphological characters were scored as far as possible for the same taxa as sequenced, but we also examined a range of other species and scored characters as polymorphic where intrageneric variation was encountered. When larval and internal characters had not been scored for the same species, the data presented are from congeners; no assumptions were made beyond that. We coded taxa for 67 morphological characters (Table II); the matrix is included as the Appendix.

Molecular protocols
DNA was extracted from single mid-legs preserved in absolute ethanol using the ethanol precipitation method with final elution into 30 ml of water. Polymerase chain reactions Five parasitic wasp genera (PCRs) were carried out in a GeneAmp9600 thermal cycler in 20 ml reactions containing 1.0 ml of DNA extract, 10 pmol of primers (forward: 59 GCG AAC AAG TAC CGT GAG GG 39; reverse: 59 TAG TTC ACC ATC TTT CGG GTC 39), 10 nmol of dNTPs (Amersham Pharmacia Biotech: APB), 1.0U of Taq polymerase (Bioline), 2 ml of 106 reaction buffer (2.0 mM MgCl 2 ). PCR conditions were 94uC for 30 s, 50uC for 30 s, and 72uC for 60 s (35 cycles with an initial denaturation for 2 min and a final extension for 7 min). PCR products were purified using GFX gel band purification kit (APB) and sequenced directly using BigDye terminators.

Data analysis
The morphological and molecular data sets were analysed both separately and simultaneously. Morphological data were analysed with maximum parsimony using PAUP* (Swofford 1998) treating multistate characters as both unordered and with selected characters set as ordered (see below). Initially we searched a large area of tree space by using 10,000 random additions holding only a single tree in memory for branch swapping (TBR) each addition. The shortest trees found were then used as starting trees for further branch-swapping with maxtrees set at 50,000. Successive approximations weighting was carried out on most parsimonious trees using the maximum value of the retention index as the reweighting function (Gauthier et al. 2000). Analyses involving molecular data were carried out using direct optimization (Wheeler 1996;Gladstein and Wheeler 2001) implemented using the program POY (version 3.0.11a, 20 May 2003) on a supercomputer at the University of Helsinki. The 28S D2-D3 sequences were initially arranged by eye to recognize regions for which homology could be assigned with high confidence. Nineteen putatively homologous regions were then Five parasitic wasp genera Clypeus: (0) not extended beyond tentorial pits; (1) extended beyond tentorial pits (Sanborne 1986;Wahl 1991). 3 Clypeus and face: (0) separated by a groove or depression; (1) not separated by a groove (Gauld 1985;Wahl 1991). 4 Number of flagellomeres: (0) 40 or fewer; (1) more than 40. 5 Median flagellar segments: (0) longer than wide or quadrate; (1) at least 1.05 times wider than long. 6 Antenna length: (0) less than fore wing; (1) equal to or greater than fore wing. 7 Ocelli: (0) small, separated from eye by more than 0.5 times their diameter; (1) enlarged, nearly touching eyes. 8 Emargination of eyes adjacent to antennal sockets: (0) weak or absent; (1) moderate; (2) strong (Townes 1970;Miah 1998).

Morphological analysis
The MPTs obtained, either with all characters unordered ( Figure 1a) or with selected ones ordered ( Figure 1b) were essentially similar, though the strict consensus tree of the latter was somewhat less resolved. Neither recovered the Campopleginae as monophyletic, but instead it was paraphyletic with respect to the Anomaloninae, Banchinae, Ctenopelmatinae, and Ophioninae in all cases and also with respect to the Cremastinae in non-successive approximation trees. The Nesomesochorini (Chriodes, Klutiana, and Nonnus) was recovered as monophyletic in all analyses. Skiapus and Hellwigia were included in the large polytomy in both the unordered and ordered analyses (Figure 1a, b), but in the successive approximations trees (Figure 1c) were associated with a derived clade comprising the Ophioninae and Anomaloninae.

Molecular analysis
The trees obtained from the three different POY analyses (with different gap:substitution ratios) were very similar and strict consensus trees for each are given in Figure 2a-c. In all cases the Campopleginae excluding Chriodes, Klutiana, Nonnus, Skiapus, and Hellwigia was monophyletic whereas Ctenopelmatinae was paraphyletic at least with respect to the Campopleginae and in the case of the 2:1 and 4:1 also with respect to the Ophioninae. In all cases, Skiapus and Hellwigia were recovered monophyletic with, and usually separately inside, the Ophioninae. In all analyses Chriodes, Klutiana, and Nonnus were monophyletic with Anomaloninae and in 3:1 and 4:1 Nonnus was recovered inside the Anomaloninae as the sister group of Trichomma Wesmael.

Simultaneous analysis of molecular and morphological data
The trees obtained from the three simultaneous analyses (with different gap:substitution ratios) were very similar and are presented in Figure 3a-c. In all cases the Campopleginae excluding Chriodes, Klutiana, Nonnus, Skiapus and Hellwigia was recovered as monophyletic and formed a sister group to a monophyletic Ctenopelmatinae, and in turn these were the sister group to the Cremastinae. In all trees Skiapus formed a sister group to the Ophioninae plus Hellwigia, the latter being recovered in a derived position with Euryophion Cameron and Thyreodon Brullé. In the 2:1 tree (Figure 3a) Chriodes, Klutiana, and Nonnus form a monophyletic group which is the sister group of the Anomaloninae plus Ophioninae (including Skiapus and Hellwigia). With a 3:1 gap:substitution ratio Nonnus remained in that position while Chriodes and Klutiana appeared as the sister group of the Cremastinae, Ctenopelmatinae and the restricted Campopleginae (Figure 3b). With the highest gap:substitution ratio, Chriodes, Klutiana, and Nonnus were again recovered as monophyletic, but here forming a sister group to a clade comprising Ophioninae (including Skiapus and Hellwigia), Anomaloninae, the restricted Campopleginae, Ctenopelmatinae, and Cremastinae.

Discussion and conclusions
That the Campopleginae sensu lato were not recovered as monophyletic in the analysis of morphological data set, despite inclusion of several characters normally considered as synapomorphies for the subfamily (for example, the silvery setae of the face, lack of a distinct groove between the clypeus and face and, where known, the Y-shaped prelabial sclerite of the final instar larva) is not too surprising since the Ichneumonidae are well known to show high levels of morphological homoplasy. This may explain why many studies treat groups at subfamily level rather than including representative genera or large numbers of characters. The 28S rDNA sequence data, on the other hand, reveal several substitutions that appear to be synapomorphies for the Campopleginae with the exceptions of Skiapus, Hellwigia, Nonnus, Chriodes, and Klutiana. Some of these are highlighted in Figure 4. The sequences obtained for Skiapus and Hellwigia both possess the two obvious molecular synapomorphies characteristic of all members of the Ophioninae sequenced to date [for example, inserts in fragments 3 and 4 in Figure 4 (boxed)] and lack the synapomorphies of the Campopleginae [for example, substitutions in fragments 1 and 2 in Figure 4 (black circles)]. Thus it is not surprising that Skiapus and Hellwigia are recovered in a monophyletic clade with the Ophioninae in the POY trees. However, these two genera are not recovered together either in purely molecular or simultaneous analyses, and the DNA sequence fragments shown in Figure 4 suggest why. Within the Ophioninae a number of Enicospilus species possess a four-base insertion in the D2 28S rDNA (Figure 4 fragment 3) that is present (albeit without base homology) in Skiapus. Given the lack of base homology and the fact that this is just a two-base pair insertion within an already variable piece of DNA, it would be unwise to attribute too much to this. All Ophioninae (sensu Gauld, 1985) may possess a high number of rectal pads, whereas campoplegines either possess the putatively plesiomorphic number (six) or fewer (four or five) (Pampel 1914;Quicke et al. 1999). The number of ophionine genera examined (just three) is, however, inadequate to draw conclusions about whether the whole subfamily is characterized by the large number of rectal pads (a character state, incidentally, also shared by the Anomaloninae, Rhyssinae, and some Acaenitinae in the Ichneumonidae, and by the genus Five parasitic wasp genera Euurobracon Ashmead, in the Braconidae). Thus, while our data show convincingly that Hellwigia and Skiapus belong in the same monophyletic group as the Ophioninae, we remain cautious about whether one or both are derived within the Ophioninae or form a sister group to that subfamily.
Whereas the Anomaloninae are well characterized as a monophyletic group based on morphology (Gauld 1976(Gauld , 1997, the 28S D2-3 rDNA gene fragment shows no unique synapomorphies (see Figure 4), and no obvious individual substitutions indicate a clear relationship with Nonnus, Chriodes, and Klutiana, though these genera were recovered either monophyletically or as a grade as the sister group of the Anomaloninae in the purely morphological analyses. However, in the simultaneous analyses (Figure 3) they showed a trend with increasing gap:change values from being a monophyletic sister group of the Anomaloninae+Ophioninae (Figure 3a), through a polyphyletic state (Figure 3b) to again forming a monophyletic group, but this time as a sister group of the clade comprising all the included taxa except for Banchinae and Tersilochinae (Figure 3c).
Nonnus, Chriodes, and Klutiana share a putatively synapomorphic, medially strongly incurved comb on the inner margin of the hind tibia (Table II: character 52; Figure 6, cf. Figure 5) and, within the Ophioniformes, the clavate fore and mid tibiae of the females (Table II: character 49). Nevertheless, despite the inclusion of these characters, they were only recovered as forming a monophyletic group in two of the three simultaneous analyses, thus there is only a weak signal in the combined data set that would support recognition of a single subfamily including Nonnus, Chriodes, and Klutiana. We therefore are here proposing that these be placed in two subfamily-level taxa, the Nonninae for Nonnus and the Nesomesochorinae for Chriodes and Klutiana. The Nesomesochorinae was originally proposed by Ashmead (1905) with type genus Nesomesochorus Ashmead (a junior objective synonym of Chriodes), and the Nonnini was proposed by Townes et al. (1961).
The Nesomesochorinae and Nonninae can be keyed out together, as the Nonnini, in Townes (1970, p 144) and are recognized by the reduced number of labial palp segments, in addition to the modified hind tibial comb (Table II: character 52). Chriodes and Klutiana have eyes that converge ventrally so that they are almost touching (Figures 7, 8), the claw is strongly pectinate but not to the apex (Figure 9), and the propodeum has an almost complete set of carinae ( Figure 10). Nonnus has no especially distinctive morphological features, but they are large wasps with a long ovipositor and white-banded antennae (see Townes 1970, Figure 140). Despite the lack of ''ophionoid facies'', the Palaearctic genus Hellwigia is superficially very like other ophionines, except that it lacks the spurious vein in the fore wing. Its fore wing veins are characteristically sinuous and vein 2m-cu is more or less interstitial (see Townes 1970, Figure 142). Skiapus is a highly characteristic genus of medium-sized ichneumonids from Africa. The mandibles are highly aberrant ( Figure 11) which are twisted and out-curved, the occipital carina is deeply indented medio-dorsally (Figure 12), the propodeum is very steep posteriorly with two complete transverse carinae located close to its anterior margin (Figure 13), the hind legs are especially long with very large coxae and all legs are strongly spinose (Figure 14). The hind coxa has a tooth ventrally ( Figure 15) and the claws are strongly pectinate, though not to the apex and not sinuous as in other ophionines ( Figure 16).
Finally, this study emphasizes the value of considering both molecular and morphological data in phylogeny reconstruction since both can provide convincing synapomorphies for groups. In the present example, only molecular data provide convincing evidence for monophyly of the Campopleginae (excluding Hellwigia, Skiapus, Nonnus, Chriodes, and Klutiana), while most support for the Anomaloninae comes from the morphological data with no molecular synapomorphies in the D2-3 28S rDNA region.