Terms used for adult morphology follow Heraty et al. (2018). Terms used for larval morphology follow Heraty and Darling (1984), Darling and Miller (1991), and Darling (1992).

Specimens were extracted using the DNeasy blood and tissue kit manufactured by Qiagen (Valencia, CA, USA) with 1 μL RNAse A added after incubation. Two gene regions were sequenced. The ribosomal gene 28S D2 used the following primers and thermocycler protocol: D2F (CGG GTT GCT TGA GAG TGC AGC) and D2Ra (CTC CTT GGT CCG TGT TTC); initial denaturization: 94 °C 3 min; (denaturization: 94 °C 1 min; annealing: 55 °C 1 min; extension: 72 °C 1 min) ×34; final extension: 75 °C 7 min. The mitochondrial gene COI-barcoding (COI-BC) used the following primers and thermocycler protocol: LCO1490 (GGT CAA CAA ATC ATA AAG ATA TTG G) and HCO2198 (TAA ACT TCA GGG TGA CCA AAA AAT CA); initial denaturization: 93 °C 3 min; (denaturization: 93 °C 15 sec; annealing: 46 °C 45 sec; extension: 68 °C 45 sec) ×34; final extension: 68 °C 7 min. PCR products were purified with DNA Clean & Concentrator™ -5 kits by Zymo Research (Irvine, CA, USA). PCR product concentrations were measured using Nanodrop 2000c (Thermo Scientific™). Each gene was PCR amplified individually and Sanger sequenced using both primers. Samples for Sanger sequencing were sent to Retrogen Inc. (San Diego, CA, USA) for sequencing on an Applied Biosystems 3730xl DNA Analyzer. Chromatograms were inspected for base calling errors and edited in Mesquite v.3.31 (Maddison and Maddison 2017b) using Chromaseq v.1.2 (Maddison and Maddison 2017a).

We attempted to identify (or verify) specimens of parasitoid and host by comparing sequences with the online databases NCBI BLAST (Johnson et al. 2008) and BOLD (Ratnasingham and Hebert 2007) (Suppl. material 1: Tables S1, S2).

We tested the placement of Eutrichosomatinae within the PLC by first using the planidial morphology data matrix developed by Heraty and Darling (1984) and later modified by Darling (1992). Within the PLC, the subfamilies of Perilampidae are paraphyletic in recent molecular studies (Munro et al. 2011; Heraty et al. 2013), and we treat Chrysolampinae, Perilampinae, and Philomidinae as separate terminal taxa and equivalent to Eucharitidae. We refined these previous character sets to include only informative and unambiguous characters at the family or subfamily level. Parsimony analyses were performed in PAUP* v4.0a (Swofford 2002) with default settings. We provide a detailed list of the characters, including character states, polarity inferences, and modifications from earlier studies (Heraty and Darling 1984; Darling 1992), with justifications for inclusion or exclusion of those characters.

Character 1. Egg shape: 0 = ovoid; 1 = stalked. Stalked eggs were regarded by Heraty and Darling (1984) as a synapomorphy for Eucharitidae, however at least three genera within Eucharitidae (Indosema, Orasemorpha, and Timioderus) lack egg stalks (Heraty 1994), and Chrysolampinae (Darling and Miller 1991), Philomidinae (Heraty et al. 2019), and Eutrichosomatinae all have egg stalks. The polarity cannot be inferred because the presence of egg stalks seems to be widespread throughout Chalcidoidea (e.g. Aphelinus, Leucospis, Tetrastichus, Torymus, and many more (Parker 1924)). We excluded this character from the analysis.

Character 2. Egg sculpture: 0 = smooth; 1 = ridged. Smooth eggs were inferred to be plesiomorphic by Heraty and Darling (1984), with ridged eggs only appearing in Perilampinae. This character is an autapomorphy for Perilampinae, thus excluded.

Character 3. Sclerotization of terga (=tergites): 0 = absent; 1 = present. Heraty and Darling (1984) treated this character as a combination of sclerotization and distinctiveness of terga, which was present in all of our ingroup taxa. Darling (1992) made this into two distinct characters: character 3 was sclerotization and character 3’ was the shape of the terga (0 = completely encircling body; 1 = incomplete ventrad). All ingroup taxa were coded as sclerotized, and all ingroup taxa except Chrysolampinae and Eutrichosomatinae were coded as incomplete ventrad. The interpretation that Chrysolampinae and Eutrichosomatinae are more sclerotized than outgroup taxa with the terga completely encircling the body seems to be questionable, so we did not include the sclerotization character (3) but only used the shape character (3’), which is treated here as character 20.

Character 4. Setal pattern of tergum III (TIII): 0 = ventral setae absent; 1 = present. Heraty and Darling (1984) treated this character as a loss of the ventral setae in Chrysolampinae and Oraseminae (Eucharitidae). The ventral pair of setae are difficult to define by position and are more accurately coded as the third pair of setae on TIII that are located ventrolaterally. Having the seta located on the ventrolateral margin is an autapomorphy of Eucharitidae, although it is not found in all taxa (Heraty and Barber 1990). However, these ventrolateral setae do appear to be present in both Chrysolampinae (Darling and Miller 1991) and Eutrichosomatinae, rendering this character uninformative for this analysis, thus it was excluded.

Character 5. Distribution of dorsal setae: 0 = absent; 1 = setae present on TI–III, V, VII, IX, XI; 2 = setae present on TI–III, V, VII, IX; 3 = setae present on TI–III, V. Heraty and Darling (1984) and Darling (1992) treated the absence of setae as the plesiomorphic condition, which is not easily justified given the widespread presence of setae in chalcidoid first-instar larvae (e.g. Leucospis, Torymus, Eupelmus, Eurytoma (Parker 1924)). For this analysis, the only informative dorsal seta is on TX. We excluded character 5, but added the presence of a seta on TX as character 21.

Character 6. Dorsal fusion of terga I and II (TI and TII): 0 = absent; 1 = present. Heraty and Darling (1984) treated this as a synapomorphy of Eucharitidae, however Oraseminae lack this fusion, and Monacon and Krombeinius (Perilampinae) have the terga fused (Darling 1995; Darling and Roberts 1999). This character is uninformative for this analysis and therefore excluded.

Character 7. Ventral spines: 0 = absent; 1 = present. Heraty and Darling (1984) noted that ventral spines are present in many ectoparasitic chalcidoid larvae. Spines of any kind are absent in Eucharitidae, and because Perilampidae are polymorphic, they interpreted the absence of spines as a synapomorphy of these two families. Darling (1992) combined character 7 (spines) with character 8 (tubercles) using the states: 0 = absent; 1 = tubercles present; 2 = spicules (spines) present. This is problematic because multiple terms have been applied to these spines (e.g. the ventral spines in Chrysolampus have been referred to as tubercles, pustules, and spicules without any clear distinction (Darling and Miller 1991)). Additionally, Chrysolampinae and Philomidinae were coded as having spicules (but not tubercles), and Perilampinae are coded as having tubercles (but not spicules) despite contradicting descriptions (cf. Darling and Miller 1991). We treat spines as a separate character from tubercles. Spines, represented as hook-like structures on the ventral region of the body segments and located between the tergal margins (cf. figs 17, 34, 37 in Heraty and Darling 1984), are a feature found only in some Perilampinae. They are uninformative for this analysis and excluded.

Character 8. Lateral tubercles: 0 = absent; 1 = present. Eutrichosomatinae have a series of tubercles across the ventral region of body segments II–XII (Fig. 1B, D, F; tbs). Heraty and Darling (1984) treated this as an autapomorphy of Chrysolampinae based on the description by Askew (1980). This character was further explored and illustrated on two species of Chrysolampus by Darling and Miller (1991), occurring on body segments II–XII. A similar patch of tubercles was found on the ventral region of body segment I in Philomidinae (Darling 1992). We excluded this character because of the difficulty assessing the homology of the various types of ventral and lateral protuberances.

Figure 1. 

Eutrichosoma mirabile immature stages. A Eggs of Eutrichosoma mirabile (small, stalked) laid on top of the eggs of their weevil host (large, unstalked) within a seed pod of Vachellia constricta; inset: Eutrichosoma mirabile egg B SEM ventrolateral habitus image of a planidium of Eutrichosoma mirabile C planidium attached to host weevil larva D setal map of a Eutrichosoma mirabile planidium, modified from an illustration by Darling & Miller (1991) E head capsule of planidium, dorsal and ventral views F planidium TIII–IV, ventral tubercles G head, anterolateral view, showing the labial structure H TXIII with cerci, dorsolateral view. Abbreviations: ant = antenna, cer = cerci, cs = cranial spine, lp = labial palp, man = mandible, plst = pleurostomal seta, prl = prelabium, psb = pleurostomal bridge, psl = postlabium, set = seta, spi = spiracle, spn = spine, tbs = tubercles, I–XIII = terga numbered from anterior to posterior.

Character 9. Spiracles: 0 = spiracles on TII, IV, V, VI; 1 = spiracles on TII; 2 = absent. Heraty and Darling (1984) noted that the plesiomorphic condition in Chalcidoidea is having pairs of spiracles on TII, IV, V, and VI, which is the state found in Eutrichosomatinae and several outgroup taxa (Parker 1924). Perilampinae and Philomidinae have lost all spiracles except on TII, and Eucharitidae and Chrysolampinae lack spiracles entirely.

Character 10. Tergopleural line: 0 = absent; 1 = present. This longitudinal line of thin, unpigmented cuticle going through the lateral sides of TII–IX is found in most Eucharitidae. It is absent in most Oraseminae, although present in Orasemorpha (Heraty 2000). In Eucharitinae, it is absent only in Pseudochalcura (Heraty and Barber 1990). It is present in Gollumiellinae, which is sister to Oraseminae + Eucharitinae (Heraty 2004), hence it is interpreted as a synapomorphy of Eucharitidae. It is uninformative for this analysis and was excluded.

Character 11. Caudal cerci: 0 = present as undifferentiated setae; 1 = absent; 2 = present as differentiated (longer and/or stouter) setae. These structures are defined as setae arising on the dorsum of TXII (Heraty and Darling 1984), which are larger than the setae on other body segments and often prolonged as stout spines or long hairs. If this character is treated as a synapomorphy of Eucharitidae and Perilampidae, the definition is not adequate because several groups have short setae (e.g. Gollumiella longipetiolata Hedqvist (Heraty 2004), Hydrorhoa stevensoni (Risbec) (Heraty 2002), Australosema valgius (Walker) and Orasemorpha didentata (Girault) (Heraty 2000)) and some have other setae on the body as long as the setae on TXII (e.g. Perilampus chrysopae Crawford (Clancy 1946), Steffanolampus salicetum (Steffan) (Darling 1999)). Eutrichosomatinae and Philomidinae both have short socketed setae on TXII and TXIII that are located more laterally (Eutrichosomatinae; Fig. 1H) or ventrally (Philomidinae). These setae appear to be homologous with cerci but without the enlargement and proposed specialized locomotion functions associated with Perilampinae and Eucharitidae (e.g. providing support to hold the body in an upright posture); therefore, we created another character state (state 0) for these taxa. Only Chrysolampinae has entirely lost setae on TXII. The position of the cerci on Eucharitidae appears to be between TXII and TXIII, which is a synapomorphy for the family but uninformative for this analysis.

Character 12. Caudal pad: 0 = absent; 1 = present. The last segment (TXIII) is membranous and expanded in Eucharitidae and Perilampinae to adhere to surfaces (Heraty and Darling 1984). However, the morphology does not appear to be different from other taxa in the PLC. We also think that polarity (absent ancestrally) cannot be assumed for this character because multiple other chalcidoid taxa have differentiated posterior segments with “suckers” or other ornamentations (e.g. Leucospis (Parker 1924)). This was excluded from the analysis.

Character 13. Antenna: 0 = present; 1 = reduced; 2 = absent. The short, conical, papilliform antenna (state 0) is considered plesiomorphic (Heraty and Darling 1984). These are present in Eutrichosomatinae and Chrysolampinae and are widespread across Chalcidoidea (e.g. Leucospis, Torymus, Eupelmus, Eurytoma, and more (Parker 1924)). Philomidinae have low, broad swellings that may indicate reduced antennae (Darling 1992).

Character 14. Cranial setae: 0 = present; 1 = absent. It is noted by Darling (1992) that the distinction and homology between cranial setae and cranial spines is not clear, which resulted in a combination of characters 14 and 15 with the states: 0 = 3–4 pairs; 1 = reduced number. Philomidinae was the only taxon coded as having complete cranial setae, however, all Perilampinae have 3 pairs of setae on the cranium. Because of the difficulty assessing polarity and homology for this character, it has been excluded.

Character 15. Cranial spines: 0 = present; 1 = absent. Some species of Perilampus have stout, recurved spines (Heraty and Darling 1984), which may be derived setae. This character is uninformative and excluded from this analysis.

Character 16. Prelabium: 0 = membranous; 1 = with sclerotized marginal rim. The prelabium is a depressed area with a sclerotized marginal rim and labial palpi on the margins (Heraty and Darling 1984). Presence of the labial sclerite was considered a synapomorphy for Eucharitidae and Perilampidae and is most easily identified when the palpi appear within or on the marginal rim. In Philomidinae, the labial palpi are not associated with a sclerite, which is thought to be the plesiomorphic condition (Darling 1992). This structure is extremely minute and difficult to score for most slide-mounted larvae. It is excluded from this analysis because the structure could not be verified in many of the taxa, although it is clearly present in Eutrichosoma (Fig. 1B, D, E, G; prl).

Character 17. Postlabium: 0 = non-eversible; 1 = enlarged and eversible. In Eucharitidae and Perilampinae, the postlabium is an eversible membranous sac surrounding the prelabium (Heraty and Darling 1984). This may be the same in Philomidinae (Darling 1992). Observations of specimens with everted postlabia are the only accurate way to code this character, and we could not observe this in Eutrichosomatinae, therefore it was excluded.

Character 18. Labial plates: 0 = absent; 1 = present. These are two sclerites found posterior to the prelabium in Eucharitidae (absent in Oraseminae). This character is uninformative for this analysis and excluded.

Character 19. Pleurostomal setae: 0 = present; 1 = spine-like; 2 = fused spines. These are setae lateral to the mouth, and they are present in most chalcidoid taxa (Heraty and Darling 1984). These setae do not appear to be modified in Eutrichosomatinae, Chrysolampinae, Philomidinae, or most Perilampinae, despite being previously coded as spine-like (Darling 1992). In Eucharitidae this has been coded as fused spines, but what was observed was not a socketed seta but instead a pointed projection of the cranial cuticle. This character was excluded from the analysis.

Character 20. Shape of terga: 0 = completely encircling body; 1 = incomplete ventrad. This is character 3’ from Darling (1992). Terga encircling the body is seen in all other known chalcidoid first-instar larvae (Parker 1924) and is considered plesiomorphic for the PLC. Philomidinae, Perilampinae, and Eucharitidae all have incomplete terga. One exception is in the genus Monacon (Perilampinae), where the posterior terga (VII–XI) are ventrally fused, while the anterior terga are not (Darling and Roberts 1999). This could be treated as a third character state for a polymorphic coding of Perilampinae, but it would not have an impact on the relationships in this analysis.

Character 21 (new). Seta on tergum X: 0 = present; 1 = absent. Setae present on all terga is treated here as the plesiomorphic state for the PLC based on the presence in many other chalcidoid taxa. Most taxa in the PLC have lost setae on TX, with the exception of Eutrichosomatinae, which maintains the ground plan configuration. We chose to focus on TX because the presence of setae on other terga are either present in all taxa (TI–III, V), only lost in Eucharitidae (TVII, IX), or require interpretation of the dorsal/lateral/ventral homology of the multiple setae present (TIV, VI, VIII).

Character 22 (new). Seta on tergum XI: 0 = present; 1 = absent. Loss of setae on TXI is a synapomorphy of Eucharitidae and Perilampinae.

Character 23 (new). Behavior: 0 = not ectoparasitic koinobiont; 1 = ectoparasitic koinobiont. All members of the PLC are ectoparasitic koinobionts. There are several examples of planidia residing internally (e.g. Perilampus spp.) or transdermally (e.g. Orasema spp.) within a host, but they always emerge to an external position to continue development through later instars; therefore, we do not consider these taxa endoparasitic sensu Heraty and Murray (2013). No other chalcidoids (where behavior is known) are ectoparasitic koinobionts because of the unique challenge of reattaching to the host after each molt, which requires increased mobility and host-recognition.

Our dataset was reduced to seven characters (9, 11, 13, 20–23) for the parsimony analysis. We chose to discuss all of the characters previously used because they can be valuable for future phylogenetic analyses at different levels (e.g. the genera of Eucharitidae). The major limitation for this analysis was finding informative characters with minimal ambiguity in the interpretation of their homology, which can be difficult for groups with simple morphology and large evolutionary gaps between sampled taxa. The most parsimonious tree was 11 steps and included only one homoplastic character (Fig. 2). We recovered the same topology as Heraty and Darling (1984) and Darling (1992) with the addition of Eutrichosomatinae, which was placed as the most plesiomorphic member of the PLC.

Figure 2. 

Most parsimonious tree from larval morphology, PAUP* analysis. Character state changes on branches are indicated by black bars (synapomorphies) and white bars (homoplasies). Character state matrix and tree statistics included. Pseudocatolaccus asphondyliae is shown as an example of a generic hymenopteriform larva with morphology that fits a hypothetical ancestor to the PLC. Illustrations of Pseudocatolaccus asphondyliae modified from Parker (1924); Chrysolampus sisymbrii modified from Darling and Miller (1991); Aperilampus varians modified from Darling (1992); Moncacon robertsi modified from Darling and Roberts (1999); Orasema delicatula modified from Burks et al. (2015).

The inclusion of the new species of Eutrichosoma has modified the generic diagnosis (Bouček 1974). Margin of clypeus without incision; advanced occipital ridge directly posterior to the ocelli present or absent; the anterior transverse carina on the pronotal collar present or absent; mesoscutal midlobe completely separating the axillae medially, posteriorly reaching the mesoscutellum; fore wing without marginal fringe; postmarginal vein on the fore wing present, rudimentary (difficult to distinguish), or absent. It differs from Peckianus by the iridescent blue or green coloration of the body, posterior end of the mesoscutal midlobe broadly separating the axillae (by 0.18–0.23× the length of the mesoscutal midlobe; 0.08–0.09× in Peckianus), marginal vein on fore wing short (0.1–0.13× the length of the fore wing; 0.2–0.21× in Peckianus). It differs from Collessina by having a moderately setose body (setae sometimes spatulate), head and mesosoma more coarsely reticulate, antenna with two anelli, scape not reaching median ocellus, notauli and scutoscutellar sulcus distinct, marginal vein on fore wing with a continuous width (not increasing distally).