A combined morphological and molecular phylogenetic analysis was performed to evaluate the subfamily relationships of the parasitoid wasp family Ichneumonidae (Hymenoptera). Data were obtained by coding 135 morphological and 6 biological characters for 131 exemplar species of ichneumonids and 3 species of Braconidae (the latter as outgroups). The species of ichneumonids represent all of the 42 currently recognized subfamilies. In addition, molecular sequence data (cytochrome oxidase I “DNA barcoding” region, the D2 region of 28S rDNA and part of the F2 copy of elongation factor 1-alpha) were obtained from specimens of the same species that were coded for morphology (1309 base pairs total). The data were analyzed using parsimony and Bayesian analyses. The parsimony analysis using all data recovered previously recognized informal subfamily groupings (Pimpliformes, Ophioniformes, Ichneumoniformes), although the relationships of these three groups to each other differed from previous studies and some of the subfamily relationships within these groupings had not previously been suggested. Specifically, Ophioniformes was the sister group to (Ichneumoniformes + Pimplformes), and Labeninae was placed near Ichneumoniformes, not as sister group to all Ichneumonidae except Xoridinae. The parsimony analysis using only morphological characters was poorly resolved and did not recover any of the three informal subfamily groupings and very few of the relationships were similar to the total-evidence parsimony analysis. The molecular-only parsimony analysis and both Bayesian analyses (total-evidence and molecular-only) recovered Pimpliformes, a restricted Ichneumoniformes grouping and many of the subfamily groupings recovered in the total-evidence parsimony analysis. A comparison and discussion of the results obtained by each phylogenetic method and different data sets is provided. It is concluded that the molecular characters produced results that were relatively consistent with traditional, non-phylogenetic concepts of relationships between the ichneumonid subfamilies, whereas the morphological characters did not (at least not by themselves). The inclusion of both molecular and morphological characters using parsimony produced a topology that was the closest to the traditional subfamily relationships. The method of analysis did not greatly affect the overall topology for the molecular-only analyses, but there were differences between Bayesian and parsimony results for the total-evidence analyses (especially near the root of the tree). The Bayesian results did not seem to be altered very much by the inclusion of morphological characters, unlike in the parsimony analysis. In summary, the following groups were supported in multiple analyses regardless of the characters used or method of tree-building: Pimpliformes, higher Ophioniformes, higher Pimpliformes, (Claseinae + Pedunculinae), (Banchinae + Stilbopinae), Campopleginae, Cremastinae, Diplazontinae, Ichneumoninae (including Alomya), Labeninae, Ophioninae, Poemeniinae, Rhyssinae, and Tersilochinae sensu stricto. Conversely, Ctenopelmatinae and Tryphoninae were never recovered without inclusion of other taxa. Based on the hypothesis of relationships obtained by the total-evidence parsimony analysis, the following formal taxonomic changes are proposed: Alomyinae Förster (= Alomya Panzer and Megalomya Uchida) is once again synonymized with Ichneumoninae and is now considered a tribe (Alomyini rev. stat.); and Notostilbops Townes is transferred from Stilbopinae to Banchinae, tribe Atrophini.

Ichneumonidae, phylogeny, parsimony, Bayesian, classification, taxonomy

The catalogue of Yu et al. (2016) listed 25,285 described species of ichneumonids in 1601 genera. In terms of the subfamily classification within Ichneumonidae, there is general consensus for the taxonomic limits of most subfamilies; however, some authors disagree on a small minority (see Table 1). Townes (1969) established the modern subfamily classification recognizing 25 subfamilies. Morphological studies by several authors between 1969 and 2002 increased this number gradually to 37 (Wahl 1990, 1993; Gauld 1991; Porter 1998; Gauld et al. 2002a). The latter study recognized 37 subfamilies including the Pedunculinae, but not the Claseinae (both proposed by Porter 1998). Thus the total number recognized by morphology-based studies alone is 38 (see column 1 of Table 1).

From 1998–2009, several studies used single gene molecular evidence (the D2–D3 region of 28S ribosomal DNA) to examine ichneumonid subfamily relationships, either combined with morphological characters (Quicke et al. 2005; Quicke et al. 2009) or using molecular characters alone (Belshaw et al. 1998; Laurenne et al. 2006). It was not until Quicke et al. (2005) that formal changes to the subfamily classification were proposed based on studies using molecular data. This study proposed two additional subfamilies: Nesomesochorinae and Nonninae for three genera previously included in Campopleginae. Further, Laurenne et al. (2006) proposed the resurrection of Alomyinae (included in Ichneumoninae in previous classifications) (Wahl and Mason 1995). Subsequently, Quicke et al. (2009) evaluated sequences of 28S D2–D3 ribosomal DNA from 1001 species of Ichneumonidae and proposed the resurrection of one subfamily and the synonymy of three others (see column 2 of Table 1) (for a total of 39 subfamilies). The state of knowledge in ichneumonid systematics was summarized by Quicke (2015) and Broad et al. (2018).

More recently, Broad (2016) reversed one of the synonymies proposed by Quicke et al. (2009) by once again recognizing Neorhacodinae. Santos (2017), using both morphology and multi-gene sequence data, studied the relationships within Cryptinae and raised Phygadeuontinae (from the tribe Phygadeuontini) and Ateleutinae (from the tribe Cryptini, subtribe Ateleutina). In summary, 42 ichneumonid subfamilies were recognized prior to this study. Since Santos (2017), two other studies have been published which discuss ichneumonid subfamily relationships but neither of these proposed changes to the subfamily classification. Broad et al. (2018) provided an updated handbook to the ichneumonids of Britain and Ireland including discussion of all subfamilies, and Klopfstein et al. (2019) used transcriptomes and target DNA enrichment to examine the relationships of Pimplinae and related subfamilies.

In terms of the relationships of the subfamilies, prior to about 1990, relationships could only be inferred by the their relative arrangement in Henry Townes’s Genera of Ichneumonidae (Townes 1969, 1970a, 1970b, 1971), a classification that was largely based on the supporting evidence (especially larval) in the character outline of Townes (1969) (pp. 29–35). As Townes (1969) wrote (p. 29): “Examination of the larval characters gave final proof of the basic faults in the old system and helped in the formulation of a new one”. Since 1990, phylogenetic hypotheses have been proposed for several groupings of subfamilies within Ichneumonidae. The informal name Pimpliformes was proposed for five subfamilies putatively related to Pimplinae (Wahl, 1990), and this group was further divided into eight subfamilies by Gauld (1991) through establishment of Diacritinae, Poemeniinae and Rhyssinae. Further, Wahl (1991) proposed the name Ophioniformes for eight subfamilies and Wahl (1993a) hypothesized that the two large subfamilies Cryptinae and Ichneumoninae were related and proposed the informal name Ichneumoniformes to comprise these two subfamilies along with Brachycyrtinae. The studies by Quicke and colleagues have for the most part, upheld these three groupings. Their studies also suggested placement for other subfamilies, for example, Quicke et al. (2009) placed an additional 8 subfamilies within the Ophioniformes (for a total of 16), 3 additional subfamilies in Ichneumoniformes (for a total of 5), and 1 additional subfamily (Collyriinae) in Pimpliformes (for a total of 9). Still, the study of Quicke et al. (2009) had two subfamilies with uncertain affinity (Eucerotinae and Microleptinae). Their study included the most comprehensive taxon sampling of any phylogenetic study of Ichneumonidae, including species from all subfamilies, but the analysis included only one gene and morphology was coded mostly at the subfamily and tribal levels (88 terminal taxa). More recent studies (Santos 2017 which sampled heavily within Ichneumoniformes and Klopfstein et al. 2019 that focused on Pimpliformes) included much more molecular data, but with a more limited taxonomic scope. The overall purpose of this study is to examine the relationships of the subfamilies of Ichneumonidae based on phylogenetic analyses using both morphological and molecular sequence data. The study is of interest because it includes the largest morphological data set for Ichneumonidae that has been coded for individual exemplar species. Novel morphological characters are introduced, especially for larvae, and the nuclear gene elongation factor 1-alpha is sequenced across the entire family for the first time. The results will be compared to previous hypotheses of relationships (e.g., Wahl and Gauld 1998, Quicke et al. 2009, Santos 2017, Klopfstein et al. 2019) and will provide another hypothesis of relationships against which future studies can be compared.

For the purposes of this study, the following definitions for the subfamily groupings are used:

1) Ophioniformes includes the following 18 subfamilies: Anomaloninae, Banchinae, Campopleginae, Cremastinae, Ctenopelmatinae, Hybrizontinae, Lycorininae, Mesochorinae, Metopiinae, Oxytorinae, Neorhacodinae, Nesomesochorinae, Ophioninae, Sisyrostolinae, Stilbopinae, Tatogastrinae, Tersilochinae and Tryphoninae. This concept is the same as that of Quicke et al. (2009)–the 16 subfamilies listed under Ophioniformes in their table 4 plus Neorhacodinae resurrected by Broad (2016) and Tatogastrinae which was presumably accidentally omitted from their table. Note that this group includes the eight subfamilies included by Wahl (1991) when the name was proposed. Gauld et al. (1997) included nine subfamilies: the eight of Wahl (1991) and Tersilochinae. Gauld et al. (1997) also coined the name Tryphoniformes to comprise Adelognathinae, Eucerotinae, Tryphoninae and Townesioninae (the latter now synonymized with Banchinae). Adelognathinae and Eucerotinae are now believed more closely related to Ichneumoniformes (Quicke et al. 2009, Santos 2017) and Tryphoninae is thought to belong to Ophioniformes (Quicke et al. 2009), as sister group to all other subfamilies, therefore we do not use the term Tryphoniformes. Within Ophioniformes is a group of closely related subfamilies that Quicke et al. (2009) called the “higher Ophioniformes” which we define as Anomaloninae, Campopleginae, Cremastinae, Nesomesochorinae and Ophioninae.

2) Pimpliformes includes the following nine subfamilies: Acaenitinae, Cylloceriinae, Diacritinae, Diplazontinae, Orthocentrinae, Pimplinae, Poemeniinae, Rhyssinae and in addition, Collyriinae. This definition corresponds to the concept of Quicke et al. (2009), which was the same as Wahl and Gauld (1998), except for the inclusion of Collyriinae in Quicke et al. (2009). The higher Pimpliformes is comprised of Pimplinae, Poemeniinae and Rhyssinae (Quicke et al. 2009). Note that the term “higher Pimpliformes” was used previously by Gauld (1991) for a group comprised of Diplazontinae, Orthocentrinae and Microleptinae, but this is not the definition used in the current study. Gauld (1991) believed that Rhyssinae was sister subfamily to all others in Pimpliformes, followed by a polytomy of Pimplinae, Poemeniinae and Acaenitinae leading to the koinobiont, endoparasitoid fly parasitoids (Cylloceriinae and his “higher Pimpliformes”). The sister group relationship of Rhyssinae to the rest of Pimpliformes was not upheld by the studies of Wahl and Gauld (1998) and Quicke et al. (2009), but the grouping of Rhyssinae, Poemeniinae and Pimplinae was strongly supported in both studies.

3) Ichneumoniformes is considered in two senses: a) in the strict sense (Ichneumoniformes sensu stricto) as originally proposed by Wahl (1993a) and including only Brachycyrtinae, Cryptinae (including Ateleutinae and Phygadeuontinae) and Ichneumoninae (including Alomyinae), and b) in the broad sense (Ichneumoniformes sensu lato) comprised of Ichneumoniformes s.s. plus seven related subfamilies which will be further described and discussed in the Results and discussion section.

In addition, Quicke et al. (2009) defined the following higher groups:

4) Xoridiformes comprised of Xoridinae

5) Labeniformes comprised of Labeninae

6) Orthopelmatiformes comprised of Orthopelmatinae

7) Brachycyrtiformes comprised of Brachycyrtinae, Claseinae and Pedunculinae.

See the Results section on Support/ relationships of subfamilies (below) for discussion on the support/ placement of these groups in the current study.