Received: 26 March 2019 | Revised: 25 August 2019 | Accepted: 26 August 2019 DOI: 10.1111/zsc.12390

ORIGINAL ARTICLE

Molecular phylogeny places the enigmatic subfamily Masoninae within the , not the

Donald L. J. Quicke1,2 | Andrew D. Austin3 | Erinn P. Fagan‐Jeffries3 | Paul D. N. Hebert4 | Buntika A. Butcher1,2

1Integrative Ecology Laboratory, Department of Biology, Faculty of Abstract Science, Chulalongkorn University, The Masoninae (type Masona van Achterberg) is a widespread but seldom col- Bangkok, Thailand lected group of morphologically aberrant tiny (body length 2 mm or less) that 2 Center of Excellence in Entomology: have always been considered as a subfamily of Braconidae, albeit on little support- Biology, Diversity of and Mites, Chulalongkorn University, Bangkok, ing evidence. Discovery of a fully winged female of Masona from remote Australia Thailand has enabled a reassessment of its relationships. This specimen yielded sequence data 3 Australian Centre for Evolutionary Biology for the nuclear 28S and 18S rDNA genes, the barcoding fragment of mitochondrial and Biodiversity, School of Biological Sciences, The University of Adelaide, cytochrome oxidase subunit 1 and the mitochondrial 16S rDNA gene. These were Adelaide, SA, Australia analysed separately and together, along with representatives of Braconidae and 4Centre for Biodiversity Genomics, Ichneumonidae. Maximum likelihood analyses of all four genes separately and com- Biodiversity Institute of Ontario, University bined concur that Masona is nested within the Ichneumonidae and, therefore, is not of Guelph, Guelph, ON, Canada a member of Braconidae. This is also supported by the position of the sternaulus low Correspondence on mesopleuron, absence of fore wing vein (RS + M)a (though the great reduction Buntika A. Butcher, Integrative Ecology in wing venation makes further interpretation problematic) and the articulated junc- Laboratory, Department of Biology, Faculty of Science, Chulalongkorn University, tion of metasomal tergites 2 and 3. On balance, molecular analyses place Masona Phayathai Road, Pathumwan, Bangkok, basally among the ophioniformes lineage of Ichneumonidae. Formal description of BKK 10330, Thailand. Masona timpaynei Email: [email protected] the Australian as Quicke sp. n. and a revised diagnosis of Masoninae are provided in S1 together with illustration of the holotype. Funding information Australian Biological Resources Study, KEYWORDS Grant/Award Number: RF214‐16 and Braconidae, Ichneumonidae, Masona, Masoninae, molecular phylogenetic analysis CT215‐08; Ratchadaphiseksomphot Fund; Chulalongkorn University, Grant/Award Number: CU‐GR_62_06_23_03

1 | INTRODUCTION afforded family‐level status largely because of aberrant biol- ogy or conspicuous character states that are now interpreted The currently comprises two extant fami- as autapomorphies, notably the extinct Eoichneumoninae lies, the Braconidae and Ichneumonidae, which collectively and the extant Aphidiinae, Apozyginae and Hybrizontinae comprise more than 44,000 described extant species (Quicke, (=Paxylommatinae) (Quicke, 2015). Apart from the extinct 2015; Yu, van Achterberg, & Horstmann, 2012) and un- grade taxon Eoichneumoninae and the rare Chilean Apozyginae doubtedly many times more. The extinct Praeichneumonidae which has yet to be sequenced, molecular data now firmly are also treated tentatively as members of the superfamily. place all of these among the extant ichneumonids or braconids. Several other taxa now placed in one or other of the two ex- The genus Masona was erected for a small group of highly tant families of Ichneumonoidea have at various times been aberrant wasps with prognathous heads in the

Zoologica Scripta. 2019;00:1–8. wileyonlinelibrary.com/journal/zsc © 2019 Royal Swedish Academy of Sciences | 1 2 | QUICKE et al. females and with body lengths between 1.1 and 1.8 mm. The n., as well as a large number of principally ichneumonid four described extant Masona species are known only from species. The diverse historical molecular phylogenetic stud- Australia (Queensland) and south‐east United States (Georgia ies from which most of the analysed DNA sequence data and Florida, including the Key Islands), but an undescribed originated mean that very few braconid and ichneumonid East African species was also noted by van Achterberg taxa are represented in GenBank by all the gene fragments (1995) and one of us (DQ) has recently been made aware of employed. As relatively little molecular phylogenetic re- its occurrence also in Asia (Cambodia) and South America search has been carried out on the Ichneumonidae apart (Brazil; Quicke, Chaul & Butcher, 2019). Two fossils of from detailed studies of a few subfamilies, we generated Masoninae from the New World have been described from new sequence data, particularly for the 16S rDNA gene, Miocene Dominican amber, one in the extant genus Masona for a taxonomically dispersed set of taxa. We have treated (van Achterberg, 2001) and one in the probably extinct genus genera as the basic taxonomic unit for this study, and thus Anoblepsis Engel & Bennett, 2008. in the analysed matrix, some genera are represented by a Van Achterberg (1995) named the genus Masona after combination of genes from multiple species. Details of Bill [W.R.M.] Mason (1911–1981) who was going to de- included sequences and GenBank accessions numbers are scribe it as a new family of because of its pe- provided in Appendix S2. culiar morphology and in particular its large gular sclerite. In describing Masoninae as a new subfamily of Braconidae, van Achterberg (1995) noted as the only putative synapomorphies 2.2 | Sequence generation and alignment the ‘united second and third metasomal tergites and the re- Sequencing the COI barcoding gene region involved duction of veins of fore wing’. Its wide [disjunct] geographic polymerase chain reaction (PCR) amplification, follow- distribution further led van Achterberg to suggest that it was ing standard protocols for DNA extraction, PCR and se- most likely an archaic group. The subfamily Masoninae it- quencing (Ivanova, deWaard, & Hebert, 2006; de Waard, self was originally described to include a second tribe, the Ivanova, Hajibabaei, & Hebert, 2008). PCR was performed Mannokeraiini, but this has since been shown to belong to the using the C_LepFolF/C_LepFolR primers. The COI frag- braconid subfamily Euphorinae (Belshaw & Quicke, 2002; ment for the taxa investigated was largely length invariant Sharanowski et al., 2011; Stigenberg et al., 2015). except for a two‐codon insertion in the sequence for the Up until now, all known female Masona species are apter- ichneumonid Alomya (Alomyinae), the position of which ous, but males are winged with very reduced venation (van was determined by reference to the translated amino acid Achterberg, 1995: Figures 1 and 2); the fore wing of Masona sequence. prognatha van Achterberg, 1995, having only weakly pig- The length variable 28S rDNA fragments were aligned mented indications of the pterostigma and veins M + CU, CU, following the braconid secondary structure model of 1‐M and 1‐1A, and the hind wing completely lacking veins. Gillespie, Yoder, and Wharton (2005); the 16S fragments Here, we present DNA sequence data for four gene frag- were aligned in accordance with the secondary structure ments together with a range of braconids and ichneumonids models of Buckley, Simon, Flook, and Misof (2000) and to re‐assess the relationships of the Masoninae. Additionally, Wu et al. (2014), while the 18S sequences were largely we describe new morphological characters pertinent to the length‐conserved. For all three ribosomal genes, length relationships of Masoninae from re‐examination of para- variable non‐homologous regions were excluded. The raw types of the Australian Masona similis van Achterberg, 1995 sequence files with secondary structure interpretation and (Figure 1a–e), a new Australian species described here as the analysed files are provided in Appendices S4 and S5, Masona timpaynei Quicke sp. n. (Appendices S1 and S2), respectively. and from Masona popeye Quicke & Chaul (Figure 1f). As a result of both molecular analyses and morphological con- siderations, we show that Masoninae are not members of the 2.3 | Phylogenetic analyses Braconidae but are actually ichneumonids, probably derived Sequences were analysed using maximum likelihood (ML) near the base of the informal ‘ophioniformes’ clade. with the programme RAxML (v.8) (Stamatakis, 2014), using a GTR + G rate model. To estimate support for nodes, we conducted a combined ML search and rapid bootstrap using 2 | MATERIALS AND METHODS the “‐f a” option and 100 runs. The combined data were parti- tioned in three different ways for separate analyses: (a) eight 2.1 | Taxon sampling partitions comprising three codon positions for COI, pairing Approximately half of our molecular data are derived from and unpairing bases for 16S and 28S bases separately, and publicly available sequences on GenBank. In addition, new 18S as a single partition because of the very few informa- sequence data were generated for M. timpaynei Quicke sp. tive positions; (b) four partitions with each gene fragment QUICKE et al. | 3 FIGURE 1 Masona morphology. (a) (c) (a–e) Masona similis van Achterberg, 1995, paratypes from Australia; (f) Masona popeye Quicke & Chaul, 2019 from Brazil. (a) Female habitus (near) lateral view. (b) Female habitus, dorsal view. (c) Male habitus, lateral view. (d) Face. (e, f) Metasomal tergites 1–3 showing abrupt end of lateral marginal thickening of tergum of tergite 2 and lateral articulation with tergite 3 (b)

(d)

(f) (e)

treated as a single partition; and (c) all data combined as a microscopy of the slide‐mounted, Australian winged fe- single partition. Trees were visualized using FigTree (1.4.3) male Masona was carried out using an IX83 microscope (Rambaut, 2016). (Olympus Company). Images of M. similis were generated using a Visionary Digital BK+ imaging system with a Canon EOS 7D 18 megapixel camera, and Zerene Stacker, Zerene 2.4 | Morphology Systems LLC, PMAX software and cropped and resized in New data are presented for the fully winged M. timpaynei Adobe Photoshop CS6 (Adobe Systems Inc.). Quicke sp. n. (see Appendix S1) from Western Australia, Terminology follows van Achterberg (1988) except for Millstream National Park, 17 June 2014, collected by Neil wing venation nomenclature, which follows Sharkey and Brougham, that was collected as part of the Global Malaise Wharton (1997); see also figure 2.2 in Quicke (2015) for trap Barcode Initiative, and for a new apterous species from comparison of wing venation naming systems. Brazil (Quicke et al., 2019). New DNA sequence data are The body parts of the new species from Australia are presented for the new Australian species. Phase contrast mounted in Canada balsam on two cavity microscope slides, 4 | QUICKE et al.

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Astiphr us r H B Ox u Ichneute Corsoncu cr Anomalon Op Co Hercusphon s Tr A y a mi sipoli le hysipolisn y thal Atel Tr D R s Rh s o omma Si a iadromusc Hor Rh n rmiu e aco imp oph Ho Cylloceri ntru eute Br h o us color Pimp Hyposooctonarus er n_bi Mason Di Prao a s adegm ter Histerom sosto I a a l Du a Me sch a Urosigalphus son nocero azus a Campoa oni leti Maxfischeria Yam s Eub ght s Nemeri s ata tonu tis tonia Odon rote Wrou Venturi a Wrough toc s Microc n Helcon olon Helco Meteoride a Stilb ops Masona Phaeogenes Apech this Di xetastes adrom us Euceros Hybrizon_buccatus E a No Lissonot a tosemus eleboe s Plat Deleboea Brachy D nchu ylab us mon Microl cyrt us Ba Co s mpsoph Ichneu eptes ero Jopp or s Xoride oc a us e P Ischn Ad Ic Tr helt olytrib s s e hneumo ogus Op Clasi u Polytriblo g Diaparsis ax Odontocolon G nat ylabus a hrud eli hu n lat s s a P s a s P li Zagryphus Dipl es H x List L rid s emit eogenes abe o Laben ea hu ya Ge s azon X ot e ha Lyme Cestrus na Gr s Ces Atel l P tus r lom Ly Tamaulipec es e s Campoleti ogna rsiloc Tamaulipic e x A a Li eut di T y s t meontru Agriotypus o t i is st Hyposot o s centruhousr Cl Adelp s e Diadegma th o it r hyt n han ar r e ogn t le es a a P u e e i a Mi s s si s Phrudu P om b St Co l u Diap n s Al s c a a delognathu Dusona a Ophion s Coll it i hi Brachro thus o u A De ato hyss u lom a tele s m o l l ep m R e Me Eu n us o cm eoxorid p ap mi Anomalon o r yr r plaz iacritus t N t ocer a A e i acru es m y r c Tr D i ia H D p g c en lich er yr P e as o pe Cyll o c t oc achyz s u Metopius D s h t X Yamata s Hy yl

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FIGURE 2 Maximum likelihood trees recovered for each of the four gene fragments showing recovered positions of Masona with respect to a range of braconids and ichneumonids one with the disarticulated head and mesosoma with one 3 | RESULTS fore wing attached, the other with the metasoma and one leg. These are deposited in the Australian National 3.1 | Molecular phylogenetics Collection, Canberra. DNA extracted from the Australian M. timpaynei sp. n. DNA Masona timpaynei sp. n. Quicke D.L.J. extract was successfully sequenced for the barcode gene ZooBank registration: urn:lsid:zoobank.org:pub: (564 BPs), 28S D2 + D3 (674 BPs), 16S (397 BPs) and 18S 832989BC‐03E8‐43DC‐BB31‐552F6683F6F3. QUICKE et al. | 5 TABLE 1 Results of BLAST searching Masona sequences on GenBank and accessions numbers of best matching sequences (GenBank accessed 9.ii.2019; full details and accessions numbers are given in Appendix S6)

CO1 16S 28S 18S Winged female Masona 88% to various unidentified 84% to cryptine and 93% to tersilochine, stilbopine and 98% to various and a diplazontine claseine genera ctenopelmatine genera cryptine genera Previously published Masona 84% to various Aphidiinae — 91% to a predicted (Formicidae) — spp (COI: JF963525; 28S: sequence, and to a rhyssaline AJ302914) braconid

(155 BPs). The GC contents of these were 26.1%, 60.5%, the closest matching ichneumonids represented a diversity of 20.9% and 53.5%, respectively. DNA sequences for the cur- subfamilies including members of the informal brachycyrti- rent and previously published Masona species were BLAST formes, ichneumoniformes and basal ophioniformes. searched (https​://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_ Individual ML analyses of the four genes all recovered the TYPE=Blast​Search) on 8 February 2019. The top matches Ichneumonidae separated from the Braconidae with 100% for each of the four gene fragments are summarized in Table bootstrap support (Figure 2). The 16S and COI sequences both 1 (see Appendix S5 for full details). The highest similarity recover Masona within a monophyletic ophioniformes clade, in matches (98%) were obtained for the highly conserved 18S the former case rather close to the base. The 28S tree recovered rDNA gene. All other sequences gave far lower scores rang- the ophioniformes as a grade taxon from within which were de- ing from 84% to 93%. For the newly generated barcode se- rived all other ichneumonid groupings, but Masona associated quence, the top 100 nearest matches were all members of the most closely to the basal ophioniformes subfamilies Ichneumonidae, ranging in sequence identity from 87% to and Stilbopinae. The 18S tree recovered Masona within the 88%; the top 100 nearest matches for the 28S sequence were Ichneumonidae together with Xoridinae and an . also all members of the Ichneumonidae, ranging in sequence In combined analyses of the four genes (Figure 3), identity from 90% to 93%, and the best matches for 16S and the ophioniformes were recovered as a basal ichneu- 18S similarly were members of the Ichneumonidae. However, monid grade rendered paraphyletic by the Xoridinae (eight

8 partitions 4 partitions 1 partition

Megastylus Trogus Diadromus Diacritus Platylabus Phaeogenes Allomacrus Notosemus Notosemus Orthocentrus Diadromus Platylabus Coleocentrus Phaeogenes Compsophorus Phaenolobus Compsophorus Joppa Yamatarotes Joppa Ichneumon

s Collyria Ichneumon Trogus Pimpla Adelognathus Adelognathus Brachyzapus Lymeon Ateleute Rhyssa Cestrus Tamaulipeca Dolichomitus Listrognathus Cestrus Apechthis Microleptes Lymeon Tromatobia Polytribax Listrognathus Delomerista Alomya Microleptes Neoxorides Tamaulipeca Polytribax Pimpliforme Xanthopimpla Ateleute Alomya Perithous Hemiteles Hemiteles Hyperacmus Gelis Gelis Cylloceria Agriotypus Agriotypus Diplazon Brachycyrtus Adelphion Adelphion Adelphion Brachycyrtus Brachycyrtus Euceros Clasis Brachycyrtiformes Euceros Clasis Euceros Clasis Dolichomitus Phaenolobus Diadromus Rhyssa Yamatarotes Phaeogenes Brachyzapus Xanthopimpla Platylabus Apechthis Apechthis Notosemus Tromatobia Pimpla Compsophorus Delomerista Dolichomitus Joppa Yamatarotes Rhyssa Trogus Phaenolobus Brachyzapus Ichneumon Collyria Tromatobia Adelognathus Pimpla Delomerista Ateleute Diacritus Diacritus Tamaulipeca Megastylus Megastylus Alomya Allomacrus Allomacrus Cestrus Orthocentrus Collyria e Lymeon Xanthopimpla Orthocentrus Listrognathus Perithous Coleocentrus Polytribax Coleocentrus Neoxorides Microleptes Neoxorides

Ichneumoniformes Perithous Gelis Diplazon Diplazon Hemiteles Hyperacmus Cylloceria Agriotypus Cylloceria Hyperacmus Grotea Labena Tersilochus Labeniformes Labena Grotea Diaparsis Tersilochus Tersilochus Stethantyx Diaparsis Diaparsis Lycorina Stethantyx Stethantyx Ischnoceros Lycorina Lycorina Odontocolon Odontocolon Xorides Xorides Xoridiformes Ischnoceros Ischnoceros Labena Xorides Odontocolon Grotea Masona Orthopelma Diadegma

Ichneumonida Deleboea Dusona Hyposoter Lissonota Campoletis Dusona Exetastes Diadegma Campoletis Scopesis Hyposoter Venturia Stilbops Rhimphoctona Sinophorus Banchus * Nemeritis Rhimphoctona Diadegma Sinophorus Nemeritis Hyposoter Venturia Rhynchophion Campoletis Ophion Ophion Dusona Rhynchophion Corsoncus Rhimphoctona 100% Metopius Ophionellus Nemeritis Corsoncus Anomalon Venturia bootstrap Ophionellus Trathala Sinophorus Anomalon Pristomerus

s Corsoncus Trathala Hybrizon Ophionellus Pristomerus Physotarsus Anomalon Hybrizon Metopius Pristomerus Physotarsus Hadrodactylus Trathala Hadrodactylus Alexeter Hybrizon Alexeter Euryproctus Physotarsus Euryproctus Opheltes Ophion Pion Absyrtus Rhynchophion Absyrtus Pion Metopius Opheltes 100% Tatogaster

Ophioniforme Pion Tatogaster Deleboea Hadrodactylus Deleboea bootstrap Lissonota Alexeter Lissonota Exetastes Oxytorus Exetastes Banchus Euryproctus Banchus Stilbops Opheltes Scopesis Scopesis Absyrtus Stilbops Neorhacodes Tatogaster Neorhacodes Phytodietus Mesochorus Phytodietus Masona Astiphromma Masona Mesochorus Phrudus Tryphon Astiphromma Tryphon Hercus Phrudus 100% Hercus Oxytorus Zagryphus Zagryphus Acrotomus Oxytorus * bootstrap Neorhacodes Zagryphus * Acrotomus * Phytodietus Astiphromma * Hercus Acrotomus Mesochorus Tryphon Orthopelma Phrudus Orthopelma Hormius Urosigalphus Rhysipolis Bracon Stantonia Bracon Rhysipolis Meteoridea Histeromerus Hormius e Ascogaster Histeromerus Lysiphlebus Lysiphlebus Praon Ichneutes Toxoneuron Praon Mesostoa Euphorinae_gen_sp Mesostoa Maxfischeria Leiophron Maxfischeria Microctonus Euphorinae_gen_sp Ascogaster Toxoneuron Rhysipolis Leiophron Ichneutes Hormius Microctonus Urosigalphus Bracon Stantonia Histeromerus Ascogaster Toxoneuron Meteoridea Lysiphlebus Ichneutes Leiophron Praon Meteoridea Euphorinae_gen_sp Mesostoa Urosigalphus Braconida Microctonus Maxfischeria Stantonia Eubazus Eubazus Eubazus Wroughtonia Wroughtonia Wroughtonia Helcon Helcon Helcon

FIGURE 3 Maximum likelihood trees based on four concatenated gene fragments showing recovered positions of Masona (red arrow and asterisk) with three different treatments of DNA partitions 6 | QUICKE et al. partition analysis) with Orthopelma basal, by Xoridinae and Sexually dimorphic wing development is a common trait in Orthopelma (four partition analysis) or by Xoridinae plus smaller‐bodied ichneumonoids (Quicke, 2015). In com- Labeninae (with Orthopelma basal). In all analyses, Masona mon with females of the other species of Masona, it lacks was recovered within this ophioniformes grade. ocelli. However, the pedicellus is small relative to the sca- pus in other females but large (up to 80% length) in males (Figure 1d; also figure 753 in van Achterberg, 1995). Thus, 3.2 | Morphological characters our female M. timpaynei sp. n. is male‐like in this respect Previously described Masona species are strongly sexu- too. A similarly large pedicellus occurs in some ophioniform ally dimorphic with apterous prognathous females and fully Ichneumonidae such as in Neorhacodinae (see figure 113c in winged, more orthognathous males (Figure 1a,b cf 1c). Broad, Shaw, & Fitton, 2018) and many (see Females lack ocelli (Figure 1b), but ocelli are present in males figure 152 in Broad et al., 2018). (Figure 1d). In both sexes, the face is short and rather feature- Robust legs (Appendix S1 Figure A) are probably adap- less and the mandibles strongly twisted and sickle‐shaped. tive to the wasps having to push bodily through some sub- No groove separates metasomal tergites 2 and 3 dorsally, strate to reach hosts, being found in the Ichneumonidae, and, in most specimens, these tergites form a more‐or‐less notably in many Metopiinae, Orthocentrinae and continuous curve in profile. However, close examination of Hyperacmus Holmgren, 1858. The shape of the ovipositor M. similis (Figure 1e) and M. popeye (Figure 1f) shows that dorsal valve, narrowed, apically almost needle‐like and with in both, the two tergites are flexibly connected, that is a nar- an elongate notch (Appendix S6 Figure G), strongly sug- row lateral margin of the second tergite is more strongly scle- gests that Masona are endoparasitoids (Belshaw, Grafen, & rotized than the third, and posteriorly is modified forming Quicke, 2003), and is reminiscent of the ovipositor of some points of articulation with the third tergite. and Metopiinae. Given the morphological The first winged female Masona species is described and assessment above, a revised diagnosis of Masoninae is pro- illustrated as new in online supporting materials (Appendix vided in Appendix S1. S1) despite being fragmented and slide‐mounted because of its systematic importance. It is not only winged but was col- lected in a Malaise trap rather than in a ground‐layer trap, 4 | DISCUSSION suggesting it is well capable of flying and possibly attacks hosts in a different niche than its apterous congeners. It is su- The new molecular and morphological data presented here perficially similar to the males of the two other species of the show that Masoninae are members of the Ichneumonidae genus for which males are known (M. similis and M. progna- rather than the Braconidae. In the original description of the tha van Achterberg, 1995) (Figure 1c). Its head is only moder- subfamily, van Achterberg (1995) wrote ‘I include the genus ately elongate and not very prognathous. Wing venation was [Masona] in the Braconidae because it shares its synapomor- hardly visible in the intact specimen with normal transmitted phies such as the united second and third tergites and the or incident light (Appendix S1 ), but after slide‐mounting, reduction of veins of fore wing’. Many braconids have far phase contrast microscopy revealed a more extensive pattern more reduced venation than most ichneumonids (Matthews, of fore wing venation (Appendices S1 Figure D and S6 Figure 1974), a trend that is probably associated with their generally A) comprising M + CU, 1M, 1CU, 2CU, r‐rs, m‐cu and a smaller body size, but the bulk of Braconidae has as com- combined longitudinal RS + M that divides. Veins (RS + M) plete a wing venation as ichneumonids. However, the more a and 2m‐cu are absent. The arrangement is almost identical important feature would seem to be the connection between to that of the ichneumonid genus Neorhacodes Hedicke, 1922 the second and third metasomal tergites which in Braconidae (Neorhacodinae; Appendix S6 Figure B). No venation was (excepting Aphidiinae) are immovably fused, whereas in the observable in the hind wing. In addition, the pedicellus of the Ichneumonidae, they are flexibly joined (Sharkey & Wahl, new Australian species is very large, approximately 0.8 × the 1992). In Masona, there is no groove separating them, and length of the scapus, a condition also shown by hormiine in many specimens, these tergites appear superficially to be braconids and neorhacodine ichneumonids. connected in a braconid‐like way. However, they are often an- The lower part of the mesopleuron of most species pos- gled with respect to one another and closer inspection reveals sesses a longitudinal groove that we interpret as a sternaulus that the third tergite is articulated laterally with the second rather than a precoxal sulcus because of its more ventral loca- (Figure 1e,f). The second and third tergites are also conver- tion (Appendix S1 Figures A,E). gently fused in various derived lineages of Ichneumonidae The Australian specimen is assigned here to Masona de- (Sharkey & Wahl, 1992). The other three morphological apo- spite a few differences from the females of all the other known morphies of the Braconidae mentioned by Sharkey & Wahl species. Notably, it is fully winged, with a head shape typical are all characters of the hind wing which are not discernible of that of males (known only for M. similis and M. prognatha). in any known Masoninae. QUICKE et al. | 7 Masona species are variable with regard to the devel- This study highlights the need to generate robust molec- opment of a lateral groove on the mesopleuron. Having ular data sets for highly modified, morphologically reduced taken it that Masona is a braconid, van Achterberg (1995, Hymenoptera, to test hypotheses on their phylogenetic place- 2001) referred to this structure as the precoxal sulcus (see ment. In many cases, these taxa are rarely collected and so Appendix S6 Figure D) whereas had it been described in the such studies are invariably opportunistic. Ichneumonidae the groove would have been referred to as a sternaulus. For a long time, it was broadly assumed that these ACKNOWLEDGEMENTS were homologous although the groove in the Ichneumonidae is located more ventrally. However, the occurrence of We are very grateful to Somrudee Attachit (Mahidol both grooves in the opiine braconid Sternaulopius Fischer University) for her assistance and expertise in phase contrast (Wharton, 2006) (Appendix S6E) shows that they are differ- microscopy. Júlio Cezar Mário Chaul (Universidade Federal ent structures. Importantly, the groove in Masona is situated de Viçosa, Brazil) kindly allowed us to use a photograph far ventrally (Appendices S1E and S6C), suggesting it is of Masona popeye from Brazil. Andrew Bennett kindly al- probably a sternaulus rather than a precoxal sulcus. lowed us to use DNA extracts from various specimens to generate additional sequence data. We also wish to thank Gavin R. Broad and an anonymous referee for their helpful 4.1 | Molecular data comments, and Mike Sharkey for discussion of wing vena- The first published molecular data for a masonine were tion nomenclature. This work was supported by the Senior a 28S rDNA gene fragment used by Belshaw and Quicke Postdoctoral Fellowship under Ratchadaphiseksomphot (2002) in a study of ancestral biological state reconstruc- Fund, Graduate School, Chulalongkorn University to tions in the Ichneumonoidea. The aberrant nature of the se- DLJQ and Ratchadaphiseksomphot Fund, Chulalongkorn quence was highlighted in that paper with the statement ‘The University (CU‐GR_62_06_23_03) to BAB. Involvement only other taxa for which placement was uncertain with re- in this project by ADA and EFJ was supported by the spect to these rootings were the Xoridinae, Labeninae, and Australian Biological Resources Study grants RF214‐16 Hybrizon Fallén, 1813 in the Ichneumonidae and Masona and CT215‐08, and PDNH was funded by a NSERC in the Braconidae’. Depending upon tree reconstruction Discovery grant. method and parameters, the Masona sequence associated with either the braconid sigalphoid subfamily complex ORCID OR amongst the outgroups used comprising the , and Dryinidae, which, being aculeate hymenop- Donald L. J. Quicke https://orcid. terans were generally thought to be the likeliest sister group org/0000-0003-4471-6775 of the Ichneumonoidea at that time. Quicke et al. (2012) Andrew D. Austin https://orcid. published a barcode for another Masona species from org/0000-0002-9602-2276 Australia (GenBank: JF963525, sample ID BCLDQ1128, Erinn P. Fagan‐Jeffries https://orcid. BOLD BIN BOLD: AAI1711) and in the published tree it org/0000-0002-3322-6255 was recovered as sister group to the Aphidiinae though on Paul D. N. Hebert a long branch. https://orcid. Our molecular trees (Figures 2 and 3) failed to recover org/0000-0002-3081-6700 several groups as monophyletic which have usually been Buntika A. Butcher https://orcid. recovered in other investigations and make morphological org/0000-0002-0541-0709 sense. Notably, in several cases, the Ichneumonidae were rooted among the ophioniformes, whereas the latter were recovered monophyletic by Quicke et al. (2009) who anal- REFERENCES ysed an elided 28S rDNA data set combined with morphol- Belshaw, R., Grafen, A., & Quicke, D. L. J. (2003). Inferring life ogy. Indeed, Quicke et al.’s (2019) analyses of 28S sequence history from ovipositor morphology in parasitoid wasps using data alone from 1,001 ichneumonid species often recovered phylogenetic regression and discriminant analysis. 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