Phylogenetic Reassignment of Basal Cyclostome Braconid Parasitoid Wasps (Hymenoptera) with Description of a New, Enigmatic Afrotropical Tribe with a Highly

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Zoological Journal of the Linnean Society, 2020, 190, 1002–1019. With 6 figures.

Phylogenetic reassignment of basal cyclostome braconid parasitoid wasps (Hymenoptera) with description of a new, enigmatic Afrotropical tribe with a highly

anomalous 28S D2 secondary structure Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020

DONALD L. J. QUICKE1,2, SERGEY A. BELOKOBYLSKIJ3,4, YVES BRAET5,6, CORNELIS VAN ACHTERBERG7, PAUL D. N. HEBERT8, SEAN W. J. PROSSER8, ANDREW D. AUSTIN9, ERINN P. FAGAN-JEFFRIES9, DARREN F. WARD10, MARK R. SHAW11 and BUNTIKA A. BUTCHER1,2*

1Integrative Ecology Laboratory, Department of Biology, Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, BKK 10330, Thailand 2Center of Excellence in Entomology: Bee Biology, Diversity of Insects and Mites, Chulalongkorn University, Phayathai Road, Pathumwan, BKK 10330, Thailand 3Zoological Institute, Russian Academy of Sciences, St Petersburg, Russia 4Museum and Institute of Zoology, Polish Academy of Sciences, Warsaw, Poland 5Royal Belgian Institute of Natural Sciences, O.D. Phylogeny and Taxonomy, Entomology, Vautier street 29, B-1000 Brussels, Belgium 6Unité d’Entomologie fonctionnelle et évolutive, Gembloux Agro-Bio Tech, Université de Liège, Passage des Déportés 2, B-5030 Gembloux, Belgium 7Department of Terrestrial Zoology, Naturalis Biodiversity Center, Postbus 9517, 2300 RA, Leiden, The Netherlands 8Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada N1G 2W1 9Australian Centre for Evolutionary Biology and Biodiversity, School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005, Australia 10New Zealand Arthropod Collection, Landcare Research, Auckland, New Zealand 11National Museums of Scotland, Chambers Street, Edinburgh EH1 1JF, UK

Received 22 May 2019; revised 17 December 2019; accepted for publication 17 March 2020

A new tribe of braconid wasps provisionally included in the Rhyssalinae, Laibaleini trib. nov., type genus Laibalea gen. nov. (type species Laibalea enigmatica sp. nov.), from Kenya and the Central African Republic, is described. A molecular dataset, with emphasis on basally derived taxa based on four gene fragments (28S D2–D3 expansion region, COI barcode, elongation factor 1-alpha and 16S ribosomal DNA), was analysed both alone and in combination with a morphological dataset. Molecular phylogenetic placement of the new species into an existing subfamily is complicated by the extreme sequence divergence of the three sequences obtained for Laibalea. In both the combined sequence analysis and the combined DNA plus morphological tree, Laibalea is recovered as a sister group to the Rhyssalinae plus all non-cyclostome lineage braconids excluding Mesostoinae, Maxfischeriinae and Aphidiinae. A consensus of morphological characters and molecular analyses suggests inclusion of Laibalea either in the otherwise principally Holarctic subfamily Rhyssalinae or perhap more basally, in the principally Gondwanan Mesostoinae s.l., although we cannot exclude the possibility that it might represent a separate basal lineage. We place Laibalea in its own tribe,

*Corresponding author. E-mail: [email protected] [Version of record, published online 18 May 2020; http://zoobank.org/ urn:lsid:zoobank. org:pub:23206719-96D4-45BD-A36D-2D9E7BB9E88B]

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, 190, 1002–1019 1002 NEW BASAL CYCLOSTOME BRACONID FROM AFRICA 1003 provisionally included in Rhyssalinae. The DNA sequence data are presented for several genera for the first time. Avga, the type genus of Avgini, is shown not to belong to Mesostoinae s.l. or Hormiinae, but its exact relationships remain uncertain. The generic compositions of Rhyssalinae and Mesostoinae s.l. are revised. Anachyra, Apoavga, Neptihormius, Neoavga and Opiopterus are shown to belong to Mesostoinae s.s. A key to the tribes of Rhyssalinae is provided.

ADDITIONAL KEYWORDS: 16S rDNA – 28S rDNA – Avgini – cyclostomes – DNA barcodes – elongation factor 1-alpha – Mesostoinae – Rhyssalinae.

INTRODUCTION In this study, we describe these samples as a new Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 Afrotropical genus and use a combination of molecular The cyclostome braconids are a large monophyletic and morphological data to assess its relationships and group of parasitic wasps that include many idiobiont to examine the generic composition of several other ectoparasitoids of the larvae of various other cyclostome braconid subfamilies and tribes that have holometabolous insects, notably Lepidoptera and been contentious and were essentially based on weak Coleoptera, but with at least two transitions to koinobiont and largely informal morphological assessments. endoparasitoidism of Lepidoptera and Diptera (Shaw & Huddleston, 1991; Quicke, 2015). Along with a basal clade comprising the aphid-parasitizing Aphidiinae, the Maxfischeriinae, whose biology is unknown, MATERIAL AND METHODS and the biologically and morphologically diverse Taxon sampling Mesostoinae (Quicke et al., 2019a), they form the sister group to an entirely koinobiont endoparasitoid lineage Our dataset included virtually all available sequences comprising all of the remaining braconid subfamilies for the subfamilies Mesostoinae and Rhyssalinae, (Quicke, 2015). The cyclostomes include five relatively together with multiple representatives from each well-defined and species-rich subfamilies (Alysiinae, of eight other subfamilies (Aphidiinae, Doryctinae, Braconinae, Doryctinae, Opiinae and Rogadinae) and Exothecinae, Hormiinae, Maxfischeriinae, a number of typically smaller-bodied genera that Pambolinae, Rhysipolinae and Rogadinae) biased have variously been included in ≤ 15 subfamilies. towards taxa that previous studies have indicated Many of these subfamilies and genera are hard to as being placed basally in their respective groups. define morphologically and, although considerable Three non-cyclostome braconids were included as advances have been made over recent years, no robust outgroups: Helcon tardator Nees, 1812 (Helconinae) classification or phylogenetic hypothesis exists for representing the ‘helconoid’ complex (Sharanowski them as yet. et al., 2011), Microctonus aethiopoides Loan, 1975 Two specimens of an enigmatic cyclostome genus (Euphorinae) representing the ‘euphoroid’ complex and species were recently collected in Africa, one from (Sharanowski et al., 2011) and Urosigalphus Kakamega Forest, western Kenya and a second from sp. (Acampsohelconinae), which is unplaced the Central African Republic. Initial morphological but putatively retains the hindwing vein 2CU appraisal suggested it might belong to one of the plesiomorphically. Newly generated DNA sequences relatively basally derived subfamilies, Rhyssalinae are deposited in GenBank, and accession numbers or Mesostoinae, a view supported, in part, by the and provenances of all included taxa are given in the spiracles of metasomal tergites 2–6 being located Supporting Information (Table S1). Given that our in the laterotergites, an extremely large forewing dataset includes the first sequences for ten genera second submarginal cell, and the presence in one of (Anachyra van Achterberg, 1995, Avga Nixon, 1940, the forewings of a trace of vein 2m-cu. Vein 2m-cu is Chremyloides van Achterberg, 1995, Chremylomorpha absent in extant Braconidae (contra Ichneumonidae) Belokobylskij, 1986, Neoavga Belokobylskij, 1989, except for the Chilean genus Apozyx Mason, 1978 Neptihormius van Achterberg & Berry, 2004, (Apozyginae) and, occasionally, as an atavism in the Opiopterus Szépligeti, 1908, Pseudochremylus rhyssaline genus Histeromerus Wesmael, 1838 and in van Achterberg, 1995, Pseudohormius Tobias & a member of the Doryctinae (Tobias & Belokobylskij, Alexeev, 1973 and Spathiophaenodus Belokobylskij 1983). Alternatively, it might be related to Chremylus & Villemant, 2016), their relationships are also Haliday, 1833, because of its wing venation and discussed below. A previously published sequence metasomal structure. Chremylus is currently classified referred to as Anachyra (Zaldivar-Riverón et al., in the Pambolinae, which is phylogenetically distant to 2006) is the result of misidentification of a species of the Rhyssalinae (Quicke, 2015). Tebennotoma Enderlein, 1912.

Molecular methods and analyses aligned according to the secondary structure models Nearly all taxa analysed were represented by the of Buckley et al. (2000) and Wu et al. (2014). The barcoding region of the mitochondrial cytochrome c length-variable 28S sequences were aligned according oxidase subunit 1 (COI) gene. In addition, many to the secondary structure model of Gillespie et al. taxa were also represented by sequence data for (2005), but see the Results section for further details nuclear 28S ribosomal DNA (rDNA; D2+D3 expansion of the data alignment of the new specimens. The 28S region), and further subsets had sequence data for the rDNA and 16S rDNA bases were treated as either nuclear elongation factor 1-alpha (EF1α) gene and the pairing or non-pairing (Butcher et al., 2014; Quicke mitochondrial 16S rDNA gene. et al., 2016, 2019a), and the bases of COI and EF1α were partitioned into the three codon positions. We Most DNA sequencing was carried out according Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 to standard protocols (Ivanova et al., 2006; deWaard used PartitionFinder 2 v.2.1.1 (Lanfear et al., 2016) et al., 2008; Hebert et al., 2013). In the case of both on the CIPRES Science Gateway (Miller et al., 2010) the new specimens, DNA was extracted from a single with the concatenated molecular alignment using leg of a holotype. For 16S, we used the 16Saf forward the Akaike information criterion corrected for small primer of Rattan et al. (2006) and reverse primer sample size for best model correction, linked branch DQ16S-R (5′-DQ16S-R-3′), a modified version of the lengths and a greedy search (Lanfear et al., 2012) reverse primer of Whitfield (1997). For elongation using PhyML (Guindon et al., 2010). The suggested factor 1-alpha sequencing, we used the primer pair partitioning and model scheme comprised nine EF1af_DipHym and EF1ar_DipHym (Hovemann et al., partitions, with the 16S pairing and non-pairing not 1988; Belshaw & Quicke, 1997). We checked our new receiving support for separation (for model details, and published elongation factor sequences to confirm see Supporting Information, Table S2). Using these, that they represented the F2 copy by running separate the concatenated data were run on the RAxML-NG analyses of known EF1α homologues (e.g. from Heraty BlackBox webserver (Kozlov et al., 2019) with scaled et al., 2011; Klopfstein et al., 2011) and excluded a few branch lengths, otherwise using default parameters that were found to be homologous to the F1 copy. This and bootstrapping (100 replicates). was important because the two hymenopteran gene A dataset of morphological characters was compiled copies have evolved independently for far longer than for each of the terminals included in the molecular the age of Braconidae and will cluster separately in dataset (see Supporting Information, Table S3). It trees if a mix of copies is present. Primer sequences comprises 34 binary characters that have been found are available at http://v3.boldsystems.org/index.php/ widely to be informative between tribe- and subfamily- Public_Primer_PrimerSearch. level groups among cyclostome braconids. The Next-generation sequencing was used to obtain at combined molecular and morphological dataset was least partial DNA barcodes for several, predominantly analysed as above, but with the morphological data as Australian and eastern Palaearctic, cyclostome genera, a tenth partition using a BIN+G model. for which only old specimens were available. Briefly, Trees for all analyses were viewed in FigTree v.1.4.2 we used the method of Prosser et al. (2016), modified (http://tree.bio.ed.ac.uk/software/figtree/). for use on a Sequel SMRT sequencing platform (Pacific Biosciences). The resulting raw sequence data were processed into high-quality reads though the circular Morphology and terminology consensus sequence (CCS) algorithm (SMRTLink Terminology follows Van Achterberg (1979, 1988), v.4.0.0.190159) using a minimum pass of three and a except for the nomenclature of wing venation, which minimum predicted accuracy of 0.99. The CCS reads follows Quicke (2015), based largely on Sharkey & were then trimmed of primer and adapter sequences Wharton (1997); also see Quicke (2015: fig. 2.2) for using Cutadapt (DOI:10.14806/ej.17.1.200) and a comparison of wing venation naming systems. assembled (de novo) into full-length barcodes using a The specimen that was chosen to become the type custom R script (see Supporting Information, Appendix was imaged using a Leica M205 C courtesy of Leica S1). The final contig assemblies were validated and Microsystems [Histocenter (Thailand) Co. Ltd]; the uploaded to BOLD under Process IDs RHYMS001- paratype was imaged using a Leica IC 3D digital 17, RHYMS002-17, RHYMS003-17, RHYMS005-17, camera mounted on a Leica MZ16 microscope and RHYMS007-17, RHYMS008-17, RHYMS009-17 and using the Leica Application Suite imaging system RHYMS010-17. (Museum and Institute of Zoology, Warsaw, Poland). The raw sequence files with secondary structure The types of new species are lodged in the interpretation and the analysed files are provided in entomological collection of Tel Aviv University, Israel the Supporting Information (Appendices S2 and S3, (TAU; holotype) and Musee royal de l’Afrique centrale, respectively). The length-variable 16S sequences were Tervuren, Belgium (MRAC; paratype).

RESULTS AT:GC ratio (Table 3), with the most extreme (highest and lowest) base contents found in other taxa. Sequence data and 28S secondary structure In general, secondary structure alignment The new Kenyan specimen yielded sequences for three of cyclostome 28S D2 sequences is relatively genes (CO1, 641 bp; 16S, 433 bp; and 28S, 285 bp), but straightforward, although there is considerable failed to amplify for EF1α. All three of its recovered variation in length in some non-homologous regions. gene sequences were highly divergent from other known This was not the case for the new genus, which sequences. An initial BLAST search of the National displayed so many peculiarities that it was not Center for Biotechnology Information GenBank database possible even to identify all homologous loop and stem (March 2019) showed all gene fragments to be closest regions. To assist with interpretation, we submitted Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 to various cyclostome Braconidae and to some non- the sequences for each of the three main stems of cyclostome microgastroids. COI showed the highest the D2 region to the Web-based RNAfold programme similarity (82%) to members of Rhyssalinae, Doryctinae using the minimum energy criterion, both for the new and Microgastrinae and 81% to some Aphidiinae. The genus and for a range of other cyclostomes. The results maximum BLAST coverage for the 28S D2 region was obtained for other taxa were in excellent agreement only 63%, and that match had highest identity (80%) with the secondary structure given by Gillespie (2005) with unidentified doryctines, rogadines, rhyssalines, and those for Rhyssalus clavator Haliday, 1833 (Fig. 1). opiines and alysiines. BLAST searching the 16S sequence In contrast, 28S D2 from the new genus displayed showed highest identity (82–84%) to various opiines, many base changes and possessed stem regions that doryctines and a euphorine. These low similarities to showed no possibility of forming base-paired stems other sequences in databases suggest an isolated position. as in other taxa. Only some of these pairs could be The base composition of the 28S sequence of the recognized as homologous with high certainty (Fig. 1), new taxon was far more AT biased than other included such as a few sequence stretches of the third ‘arm’. braconids (exemplar values are given in Table 1), and this Interestingly, the 3i side spur and terminal loop region was the case both for the entire sequence and for those of the third arm (3g–3p), which is consistent in all alignable bases included in the phylogenetic analyses. other taxa, was absent in the new genus; the forward This anomalous base composition might explain why it (5′–3′) read of 21 bases showed virtually perfect base appeared on a long branch and could make its recovered homology with other cyclostomes, but formation of position unreliable. Its 16S sequences were relatively a stemmed loop seemed impossible (Fig. 1C). Out of A rich but T poor (Table 2), thus the low AT:GC ratio the 423 bases sequenced for the 28S gene of the new was largely attributable to the low T content. However, taxon, we were able to align only 271 with reasonable the range of variation in base composition for this gene confidence for inclusion in the phylogenetic analyses. was rather low overall. The COI sequence of the new In comparison, we were for example able to align 375 taxon was not extreme for any of the bases, nor for its bases for Dolopsidea Hincks, 1944.

Table 1. Summary base composition DNA statistics for 28S D2 expansion region of nuclear ribosomal DNA gene based on the alignable positions included in analyses, for selected cyclostome braconid wasp taxa

Taxon 28S D2 characteristics

AT:GC A (%) T (%) G (%) C (%)

Laibalea enigmatica 1.22† 24† 31† 24* 21* Mesostoa kerri (Mesostoinae s.s.) 1.08 22 30 26 22 Andesipolis sp. (Mesostoinae s.l.) 0.99 21 29 27 23 Maxfischeria tricolor (Maxfischeriinae) 0.97 21 28 27 24 Thoracoplites (Rhyssalinae) 0.84 18 28 29 25 Bolivar tuxtlae (Doryctinae) 0.92 17* 31† 29 23 Cedria sp. (Lysiterminae) 1.03 23 28 27 22 Pseudochremylus (Lysiterminae) – – – – – Pentatermus (? Lysiterminae) 0.91 21 27 29 24 Xenarcha abnormis (Exothecinae) 1.05 21 30 26 23 Stiropius sp. (Rogadinae) 1.02 21 30 26 23 Helcon tardator (Helconinae) 0.75* 17* 26* 31† 26†

*Lowest values. †Highest values.

Table 2. Summary base composition DNA statistics for 16S gene for selected cyclostome braconid wasp taxa

Taxon 16S characteristics

AT:GC A (%) T (%) G (%) C (%)

Laibalea enigmatica 4.2* 47† 34* 10† 9† Mesostoa kerri (Mesostoinae s.s.) 4.8 46 36 9 8 Andesipolis sp. (Mesostoinae s.l.) 5.0 46 38 9 8 Aspilodemon sp. (Mesostoinae s.l.) 6.1† 46 40† 7 7

Dolopsidea (Rhyssalinae) 4.2* 46 35 10† 9† Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 Heterospilus sp. (Doryctinae) 5.5 46 38 8 7 Cedria sp. (Lysiterminae) – – – – – Pseudochremylus (Lysiterminae) 5.1 46 37 8 8 Pambolus sp. (Pambolinae) 5.7 47† 38 6* 6* Stiropius sp. (Rogadinae) – – – – – Helcon tardator (Helconinae) 5.9 45* 40† 8 6*

*Lowest value. †Highest value.

Table 3. Summary base composition DNA statistics for COI gene for selected cyclostome braconid wasp taxa

Taxon COI characteristics

AT:GC A (%) T (%) G (%) C (%)

Laibalea enigmatica 2.7 28 44 17 10 Mesostoa kerri (Mesostoinae s.s.) 3.0 29 45 15 10 Andesipolis sp. (Mesostoinae s.l.) 2.3* 27* 43* 19† 11† Maxfischeria tricolor (Maxfischeriinae) 2.8 29 45 17 9 Thoracoplites (Rhyssalinae) 3.2† 31 46 14* 10 Bolivar tuxtlae (Doryctinae) 3.0 27* 48† 14* 11† Cedria sp. (Lysiterminae) 2.8 30 43* 16 10 Pseudochremylus (Lysiterminae) 3.0 30 45 16 9* Xenarcha sp. (Exothecinae) 2.7 30 44 16 11† Stiropius sp. (Rogadinae) 3.0 30 45 15 10 Helcon tardator (Helconinae) 3.0 34† 41 15 10

*Lowest value. †Highest value.

Phylogenetic results Rhyssalinae, which were recovered as a sister group In each of the separate single gene analyses, the new to the Aphidiinae within a paraphyletic Mesostoinae taxon was placed among basal cyclostomes (Fig. 2A–C). (Fig. 2C). With the 28S gene fragment, and with only those bases In the most likely tree, based on concatenated data from that were confidently homologizable included, it was the four gene fragments, the new genus was recovered recovered in Mesostoinae, closest to Mesostoa van as sister to Rhyssalinae plus all other cyclostomes Achterberg, 1985 (Fig. 2A). Within the tree obtained excluding Aphidiinae + Maxfischeriinae + Mesostoinae for the small 16S rDNA dataset, the new genus was s.l. (Fig. 3A). However, in repeated analyses using sister to Mesostoinae + Aphidiinae + Rhyssalinae the same parameters, the new taxon was placed (Fig. 2B), although the relationships among these sister to the Mesostoinae + Maxfischeriinae s.l. three subfamilies were reversed compared with the clade, indicating that the position was unstable, more frequently found (Aphidiinae + Mesostoinae which was confirmed by the low bootstrap support s.l.) + (Rhyssalinae + (other cyclostomes)) (e.g. on the relevant nodes of the tree (see Supporting Sharanowski et al., 2011). Analysis of the COI Information, Fig. S1). With the morphological dataset dataset recovered the new genus nested within the combined with the concatenated molecular dataset,

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Figure 1. Comparisons of minimum energy secondary structure alignments of Rhyssalus clavator (a typical representative cyclostome braconid) and Laibalea enigmatica, 28S D2 ribosomal RNA with stem region names, and with colour bars marking bases included in the final analysed alignment, green indicating positions corresponding to base-pairing stems in both taxa, red positions that are non-pairing in both taxa, and purple indicating bases that appear to be pairing in one but not the other. A, first arm. B, second arm. C, third arm.

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Figure 2. Maximum likelihood molecular phylogenetic trees based on separate genes. A, 28S. B, 16S. C, COI. the new genus was sister to Rhyssalinae plus all Given the long branch lengths in both cases, it is not other cyclostomes excluding Aphidiinae + Mesostoinae surprising that this relationship had low bootstrap s.l. (including Maxfischeriinae) (Fig. 3B). In both support. cases, the same basal cyclostome topology was Our resulting trees showed that some previous found, i.e. (Aphidiinae + Mesostoinae s.l.(including subfamilial placements, based solely on morphology, Maxfischeriinae)) + (Rhyssalinae + (other cyclostomes)). were probably wrong. In particular, our analyses

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Figure 3. Maximum likelihood molecular phylogenetic trees of selected cyclostome braconid wasps. A, concatenated analysis of COI, 16S, 28S and EF1α sequences. B, concatenated DNA plus morphological characters, showing placement of Laibalea enigmatica (red). showed that Avga (including Pseudobiosteres Hedwig, as an intermingled group but close to the Exothecinae 1961), the type genus of Avgini, is not a member of rather than more basally. However, several subfamilies Hormiinae, where it has usually been placed (Fig. 3; also were not recovered as monophyletic, notably see Supporting Information, Fig. S2), nor is it closely Rhyssalinae, Pambolinae and Dorytinae. related to Allobracon Gahan, 1915, Austrohormius Belokobylskij, 1989, Parachremylus Granger, 1949, Parahormius Nixon, 1940 or Proavga Belokobylskij, SYSTEMATICS 1989, all genera that have previously been included in Avgini (Van Achterberg, 1995) but are, in fact, widely Family Braconidae dispersed among the cylostome braconids. Furthermore, Subfamily Rhyssalinae Xenosternum Muesebeck, 1935, which was long regarded as a member of Exothecinae (e.g. Whitfield & Wharton, Tribe Laibaleini Quicke, Butcher & 1997), was recovered here with the sequenced Avga Belokobylskij trib. nov. species; a relationship proposed based on morphology lsid: urn:lsid:zoobank.org:act:4B8713FC-8A82- by Belokobylskij (1993, 2019). These are all small- 4262-BC25-5421D5EBB963 bodied wasps, and interpretations of relationships based on morphology are confounded by character losses, Type genus: Laibalea Quicke & Butcher, here reductions and homoplasy (Wharton, 1993). designated. Analysis of the morphological data alone recovered Laibalea near the base of a basal grade of Mesostoinae Tribal diagnosis: Occipital carina not fused and far genera (Fig. S3). Avga and Xenosternum were recovered removed from hypostomal carina and separately

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Figure 4. Laibalea enigmatica, holotype. A, habitus, dorsal view. B, head, facial view. C, head, dorsal view. D, head, oblique posterior view, showing wide separation of occipital (black arrow) and hypostomal (white arrow) carinae. E, F, left and right wings, respectively (E reversed to facilitate comparison). reaching base of mandible (Fig. 4D, arrows, Fig. (Fig. 6C). Precoxal sulcus absent. Vein 2CUb of forewing 5D). Cyclostome: hypoclypeal depression present interstitial (Fig. 4E). Vein 1m-cu short, much shorter and labrum concave and glabrous. Malar suture than 2RS (Fig. 4E, F). Vein RS+M long, a little shorter present (Fig. 5C). Maxillary palp with six segments; than 2RS. Vein 2a of forewing absent. Hindwing with fifth segment partially fused to sixth, approximately veins cu-a and m-cu present. Spiracles of tergites 1–6 half length of sixth. Propleuron with posterodorsal located in laterotergites, far removed from border flange. Mesoscutum with midpit posteriorly (Fig. 6C). with notum. Ovipositor without dorsal nodus; ventral Postpectal carina absent. Propodeum areolate valves with six sharp ventral serrations (Fig. 6E).

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Figure 5. Laibalea enigmatica, paratype. A, habitus, dorsal view. B, head, dorsal view. C, head, facial view. D, head, side view.

Laibalea Quicke & Butcher gen. nov. Type species: Laibalea enigmatica Quicke & Butcher, here designated. (Figs 4–6) Diagnosis: urn:lsid:zoobank.org:act:95D501C6-8ED8-4278-B931- D32557AEE903 Head - Terminal flagellomere virtually without apical spine. Flagellar segments each with a few elongate Etymology: Genus named for Laibale Friedman (Tel placoid sensilla that extend 80% of length of the Aviv University, Tel Aviv, Israel) in recognition of his flagellomere; internal aperture short, occupying 20% expertise in collecting specimens. Gender: feminine. of sensillum length. Scapus long, curved, flaring

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Figure 6. Laibalea enigmatica, holotype. A, mesosoma, metasoma and legs, lateral view. B, most of mesopleuron, propodeum and metasomal segments 1–4, lateral view with arrows indicating spiracles 2–4. C, back of head, mesosoma and first metasomal tergite, dorsal view. D, metasoma, dorsal view. E, distal part of ovipositor, lateral view. apically, ~3.0× longer than subglobose pedicellus. Eyes continuous with groove running along anterior border glabrous. Cyclostome (hypoclypeal depression present of eye, not reaching base of mandible. Ocelli small, and labrum concave and glabrous). Malar suture arranged in a triangle with base longer than sides.

Key to tribes of the subfamily Rhyssalinae 1. Vein RS+M of forewing long, a little shorter than 2RS; vein 1m-cu short (Fig. 4E). Veins 2CU, 2M (sometimes referred to as 2-SR+M being vertical) and 3M (the posterior border of the second submarginal cell) of forewing arising from virtually a single point (Fig. 4E). Clypeus high (Fig. 5C). Occipital carina not fused, remaining far removed from hypostomal carina and separately reaching base of mandible (Fig. 4D). Precoxal sulcus absent (Fig. 6A) �� Laibaleini Vein RS+M of forewing short, much shorter than 2RS; vein m-cu long. Veins 2CU, 2M (sometimes referred to as 2-SR+M being vertical) and 3M (the posterior border of the second submarginal cell) of forewing

arising from two different points. Clypeus low. Occipital carina ventrally fused with hypostomal carina and Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 together reaching base of mandible. Precoxal sulcus almost always present �� 2 2. Postgenal bridge of head wide. Propleuron without posterodorsal flange. Prepectal carina completely absent. Fore-tibia with cluster of numerous spines. Hind-basitarsus almost twice as long as second to fifth tarsal segment combined. First metasomal tergite without dorsope �� Histeromerini Postgenal bridge of head narrow or absent. Propleuron with posterodorsal flange. Prepectal carina present. Fore-tibia without or with a few spines. Hind-basitarsus always shorter than second to fifth tarsal segment combined. First metasomal tergite usually with distinct dorsope �� 3 3. Eyes distinctly setose. Antenna subclavate. Postpectal carina present. Tarsal claws with small basal lobe. Propodeum densely granulate. Dorsope of the first metasomal tergite indistinct. (Both sexes apterous) ...... Achaiabraconini Eyes glabrous or, rarely, indistinctly setose. Antenna filiform or setiform. Postpectal carina absent. Tarsal claws without distinct basal lobe. Propodeum areolate. Dorsope of first tergite present �� 4 4. Forewing marginal cell open apically. First subdiscal cell of forewing widely open. Vein cu-a of hindwing mainly or entirely absent; subbasal cell open. Hind-tibia of male usually distinctly claviform. Second and third metasomal tergite enlarged and more or less covering the following segments. Ovipositor sheath short, not protruding beyond apex of metasoma �� Acrisidini Forewing marginal cell closed apically. First subdiscal cell of forewing closed. Vein cu-a of hindwing present; subbasal cell closed. Hind-tibia of male usually not claviform (except many Rhyssalus). Second and third metasomal tergite not enlarged or weakly enlarged and, usually, almost not covering the following segments (except some genera). Ovipositor sheath distinctly protruding beyond apex of metasoma �� Rhyssalini

Antennal sockets situated close to each other. Head Vein 1cu-a postfurcal. Second subdiscal cell closed dorsally rather densely setose. Frons nearly flat, with distally; vein 2-1A+2cu-a curving to join vein 1CUb weak scrobes behind antennal sockets. shortly before origin of short (almost interstitial) vein m-cu. Veins 2CU, 2M (sometimes referred to as Mesosoma - Pronotum not produced into a 2-SR+M being vertical) and 3M (the posterior border conspicuous neck. Antescutal depression present, of the second submarginal cell) arising from virtually narrow. Mesoscutum rather overhanging pronotum, a single point. Vein 2-1A+2cu-a continuously tubular densely short-setose, with narrow, longitudinal midpit and without bulla. Both transverse anal veins (2A posteriorly; notauli present anteriorly, not meeting and a) absent; veins 1-1A and 2-1A running close to and posteriorly absent. Scutellar sulcus wide, rather posterior margin of wing. flat, bipartite, with single medial carina. Mesopleuron largely smooth, shiny and glabrous except densely Hind wing - Vein R1 long. Veins cu-a and m-cu present. setose anteriorly (Fig. 6A). Mesosternum setose. Prepectal carina absent laterally, present ventrally Legs - Femora of all legs large, swollen, ~5.0× longer but weak and largely hidden by large fore-coxae. than wide in lateral view. Apex of hind-tibia without Precoxal sulcus absent. Median area of metanotum comb of specialized setae. Hind-basitarsus long, weakly ridged medially but without a distinct carina distinctly tapering distally. Claws with small, rounded (Fig. 6C). Propodeum with midlongitudinal carina on basal lobes. anterior 0.25. Metasoma - First tergite with strongly developed Forewing - Second submarginal cell large, dorsal carinae that protrude anteriorly, forming a pair pentagonal. Vein 1rs-m present with single median of crests, and run separately to posterior margin of bulla. Vein (RS+M)a weakly curved, not bisinuous. tergum, defining a triangular area; dorsope moderately

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, 190, 1002–1019 1014 D. L. J. QUICKE ET AL. deep. Median tergites of segments 2–6 marginally less posteriorly than long, largely with irregular rugulose strongly sclerotized than laterotergites, but far from sculpture. Body mostly black; legs dark reddish brown, membranous, without sculpture. Suture between but forelegs paler. second and third metasomal tergites only indicated laterally; third tergite with single transverse, Variation: Paratype: Length of body 2.8 mm, of subposterior row of long setae. forewing 2.5 mm, of antenna 2.5 mm and of ovipositor (part exserted beyond apex of metasoma) 1.5 mm. Vein r-rs of forewing inclivous to pterostigma; Laibalea enigmatica Quicke & Butcher sp. nov. vein m-cu of forewing shortly present; hind-femur 3.3×

(Figs 4–6) longer than wide; first metasomal tergite 1.8× wider Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 posteriorly than long. urn:lsid:zoobank.org:pub:23206719-96D4-45BD- A36D-2D9E7BB9E88B Remarks Diagnosis: See genus diagnosis. The holotype has an abnormally developed head, being microphthalmic on the right-hand side Etymology: From Latin aenigma, a mystery, an (Fig. 4A–D; this is an occasional atavism among enigma, referring to the mysterious nature of this Braconidae and other Hymenoptera; e.g. Balazuc, species and its uncertain taxonomic placement. 1958; Bin, 1976; Bordera & Tormos, 1986). The other side of the head appears normal and agrees well Type material: HOLOTYPE: female, ‘KENYA, with that of the paratype from the Central African Kakamega Forest, 1525 m, 0°13′N, 34°52′E, 23 Republic (Fig. 5). The Kenyan specimen was selected September 2005, coll. L. Friedman’, collected by as the holotype because we obtained DNA sequence sweep-netting a roadside verge (L. Friedman, pers. data for it, whereas the paratype failed to yield comm.) (TAU). PARATYPE: female, ‘République sequenceable DNA. Centrafricaine, P.N. Ndoki, loc 1, 28–31 January 2012, forêt à Gilbertiodendron, Malaise trap, expedit. Sangha 2012, P. Moretto leg.’ (MRAC). Contents of Rhyssalinae as here understood Length of body 2.7 mm, of forewing 2.9 mm, of Achaiabraconini: Achaiabracon Belokobylskij, 2009. antenna 2.4 mm and of ovipositor (part exserted Acrisidini: Acrisis Foerster, 1863; Proacrisis beyond apex of metasoma) 1.0 mm. Tobias, 1983. Histeromerini: Histeromerus Wesmael, 1838. Rhyssalini (Oncophanini): Dolopsidea Hincks, Description 1944; Lysitermoides van Achterberg, 1995; Antenna with 21 flagellomeres. Median flagellomeres Oncophanes Foerster, 1863 (including subgenus submoniliform; 3.5× longer than wide. First flagellomere Koreophanes Belokobylskij, 2004); Pseudobathystomus 2.0× longer than wide, 1.1× longer than second and 1.2× Belokobylskij, 1986 (including subgenus longer than third, the latter being 2.5× longer than wide. Atlantobathystomus Belokobylskij & Koponen, 2004); Ocelli small; transverse diameter of posterior ocellus Rhyssalus Haliday, 1833; Thoracoplites Fischer, 1961; ~0.2× shortest distance between posterior ocellus and eye. Tobiason Belokobylskij, 2004. Vertex shiny, with dense setiferous punctures. Maxillary Laibaleini trib. nov.: Laibalea Quicke & Butcher gen. palps with segments 3 and 4 having long, anteriorly nov. directed setae. Mesosoma 1.5× longer than high (lateral view). Mesoscutum shiny except for small setiferous punctures. Notauli narrow, impressed, crenulated Contents of Mesostoinae s.l. as here on anterior 0.6 of mesoscutum, absent posteriorly. understood Forewing vein r-rs emerging marginally before middle Anachyra van Achterberg, 1995; Andesipolis of pterostigma, almost perpendicularly to pterostigma; Whitfield & Choi 2004; Apoavga van Achterberg, vein r-rs 0.65× 2RS. Pterostigma 5.5× longer than 1985; Aspilodemon Fischer, 1966; Austrohormius wide. Lengths of veins r-rs: 3RSa: 3RSb = 1.0: 1.5: 4.5. Belokobylskij, 1989; Doryctomorpha Ashmead, 1900; Hindwing vein M+Cu 0.9× 1-M. Base of hindwing evenly Hydrangeocola Brèthes, 1927; Mesostoa van Achterberg, setose. Lengths of fore-femur: tibia: tarsus = 1.0: 1.0: 1.0. 1985; Metaspathius Brues, 1922; Neptihormius van Lengths of hind-femur: tibia: tarsus = 1.15: 1.15: 1.0. Achterberg & Berry, 2004; Opiopterus Szépligeti, 1908; Length of hind-basitarsus 3.5× second tarsal segment. Praonopterus Tobias, 1988; Proavga Belokobylskij, First metasomal tergite strongly sclerotized, 1.5× wider 1989; Rhyssaloides Belokobylskij, 1999.

Morphology also suggests the inclusion of protrudes somewhat over the pronotum (Fig. 6A, C), Caenopachyella Szépligeti, 1908; Canberria a characteristic of most members of Mesostoinae s.l. Belokobylskij, 1991; Hormiitis van Achterberg, 1985; and, likewise, its second submarginal cell is large and Zeachremylus Belokobylskij, 2018. pentagonal. The forewing lacks veins 1a and 2a, the latter often being present in Rhyssalinae (although also in the mesostoine Andesipolis). Nevertheless, its membership of Mesostoinae is challenged by the DISCUSSION presence of hindwing vein m-cu. The presence of m-cu Morphologically, the new genus is difficult to place and, has generally been considered as plesiomorphic within although the available molecular data suggest that it Braconidae owing to the dogma that veins are more Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 nests among basal cyclostome braconids, the highly normally lost than gained. However, this vein is absent apomorphic nature of the sequences makes its correct in the extinct Praeichneumonidae, extant and extinct placement problematic. The basalmost members of Ichneumonidae, all known Cretaceous Braconidae cyclostome braconid wasps, based on both molecular and including some that are definitely cyclostomes morphological phylogenetic analysis, are Aphidiinae, (Basibuyuk et al., 1999; Ortega-Blanco et al., 2009, Maxfischeriinae and a highly heterogeneous set of taxa 2011; Perrichot et al., 2009; Belokobylskij, 2012; currently treated as Mesostoinae s.l. (Quicke, 2015; Engel, 2016; Engel & Wang, 2016; Engel et al., 2018), Quicke et al., 2019a). The Aphidiinae, despite being Aphidiinae, Maxfischeriinae (Boring et al., 2011) and most diverse in north temperate regions, are likely to in the extant Chilean genus Apozyx (Apozyginae). have originated in Gondwana (Belshaw et al., 2000). Furthermore, it is absent in all extant non-cyclostome The Maxfischeriinae are known only from Australia braconids, although it is well developed in one fossil (Boring et al., 2011), whereas the Mesostoinae s.l. are non-cyclostome braconid from the earliest Eocene currently restricted largely to Australia, New Zealand (Belokobylskij et al., 2010), potentially as a new or and South America (Quicke, 2015; Shimbori et al., re-expressed derived character because the probable 2017; Quicke et al., 2019a). It is possible that, given a closest relatives of this Eocene wasp and all other non- more robust phylogeny and expanded taxon sampling, cyclostomes lack the hindwing vein m-cu. the Mesostoinae s.l. will be split into two or more More problematic is the interpretation of the smaller monophyletic subfamilies in the future. The hindwing venation of Austrohormius. Belokobylskij ‘next’ clade, forming a sister group to the remaining (1989a) and Van Achterberg (1995) distinguish this cyclostomes, is Rhyssalinae, whose composition has genus on the basis of its complete lack of hindwing also been modified in recent years owing mainly to vein cu-a and, in contrast to other Mesostoinae s.l., molecular studies, but it is morphologically rather its possession of hindwing vein m-cu. In the more cohesive. interpretation of Quicke et al. (2019a–c) (and in the Laibalea shows most of the plesiomorphous present study), vein cu-a is present but has migrated character states that one might expect a ‘proto- far distally, thereby appearing to be vein m-cu. That braconid’ to possess, although it lacks forewing anal this is the case is supported by our observation of a veins. Therefore, its molecular affinities are consistent continuity of vein 1-1A and a distal vein cu-a in some with its morphology. It is also interesting that one Austrohormius and Metaspathius specimens (Quicke of the forewings has vein 2m-cu partly indicated, a et al., 2019c). In summary, a combination of the fossil character that occurs occasionally in Rhyssalinae record and phylogenetics suggests that the absence of (commonly in Histeromerus) and also in Neurocrassus hindwing vein m-cu in the Mesostoinae s.l. might be rarus (Belokobylskij, 1982) (Doryctinae) (Tobias & plesiomorphic. In that case, its presence in Laibalea Belokobylskij, 1983) as an atavism, whereas it is would suggest that it is derived higher up in the consistently present in the Neotropical Apozyginae cyclostome tree than our current analyses show and, (Mason, 1978). tentatively, could be included in the Rhyssalinae. Shimbori et al. (2017) and Quicke et al. (2019a) Strangely, no known ichneumonoid has both hindwing recently redefined the Mesostoinae s.l., although vein CU1b (= 2CU) and m-cu, which led Quicke et al. there is no single character or set of characters that (1999) to suggest that perhaps these are one and the are perfectly diagnostic. Laibalea agrees well with same, with the origin of the former having migrated morphological diagnoses by either Shimbori et al. distally, as is evidenced by a transition series in (2017) or Quicke et al. (2019a) of the heterogeneous set the labenine ichneumonid genus Apechoneura of genera comprising Mesostoinae s.l., including a weak Kriechbaumer, 1890. hypostomal carina with the occipital carina reaching to Laibalea also shares some morphological characters the base of the mandible, having a short pronotal collar, with Lysiterminae and with some genera near the notauli present only anteriorly and the mesoscutum Chremylus (Van Achterberg, 1995), including a with a midpit. The mesoscutum of the new genus also strongly reclivous forewing vein m-cu. Within this

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, 190, 1002–1019 1016 D. L. J. QUICKE ET AL. section of Braconidae, reduction of the vein 1-RS molecular methods. Specimen availability is limiting might be not the strongest derived character state, but for many groups, which means that internal anatomy, it is similar to the condition in the Australian genus such as the female venom apparatus (Zaldívar-Riverón Chremyloides. Both also have strong dorsal carinae of et al., 2004), internal ovipositor structure (Rahman the first metasomal segment, a long and near-vertical et al., 1998) and male genitalia (Tobias, 1967), are forewing vein r-rs and a reduced precoxal sulcus. Of essentially unknown for Mesostoinae s.l. and hardly course, several striking differences are also present, better known for Rhyssalinae and other problematic such as reduction of veins rs-m of the forewing and taxa. When a robust set of evolutionary relationships cu-a of the hindwing, the transverse clypeus and is finally obtained for a densely sampled set of basal the position of the metasomal spiracles (in the cyclostome taxa, there will undoubtedly be a need for Downloaded from https://academic.oup.com/zoolinnean/article/190/3/1002/5838741 by guest on 15 December 2020 laterotergite in Laibalea compared with the notum in considerable restructuring of the classification at the Lysiterminae). Here, we present the first molecular levels of both subfamily and tribe. data for Chremyloides and show that it forms a clade with two other Australian genera, Chremylomorpha and Pseudochremylus, and that these genera belong ACKNOWLEDGEMENTS to a derived clade near Cedria Wilkinson, 1934, far removed from Rhyssalinae, Mesostoinae and Laibalea. We thank Laibale Friedman and Netta Dorchin for The Cedriini (Cedria, Chremyloides, Chremylomorpha their help and hospitality in Israel, and the Israeli and Pseudochremylus) are recovered in a position Taxonomy Initiative for support for D.L.J.Q.’s visit to removed from Lysiterminae, in agreement with Israel. We thank the Animal Systematics Research Wharton (1993), who considered the metasomal Unit, Department of Biology, Faculty of Science, carapace to have evolved independently in the two Chulalongkorn University for allowing use of their groups (Tobias & Dudarenko, 1974). Cell^D imaging facility. We are also grateful to Philippe If we accept that Laibalea belongs to Rhyssalinae s.l., Moretto for the gift of specimens collected during the which it would key to in existing keys (e.g. Whitfield & Sangha mission 2012 (www.insectesdumonde.org) Wharton, 1997), then it is the second member of this organized by Philippe Annoyer in the Ndoki National group recorded from Africa; the other is Thoracoplites, Park (Central African Republic) and to all the Central which was originally placed in Opiinae and has now African Republic authorities who supported and been recorded from Madagascar, Kenya, Central helped the realization of this mission. Collection of African Republic (Simon van Noort and Robert material was caried out under the permit 019/UB/ Kula, pers. comm.), Côte d’Ivoire (Y.B., unpublished DSV2012. Florence Trus (Tervuren, Belgium) kindly observation) and the Republic of South African provided montage images of the paratype specimen (S.A.B., unpublished observation). In the present in IRSBN. Jayme Sones and Ramya Manjunath study, we also record the rhyssaline Proacrisis from at BOLD have been extraordinarily helpful with Madagascar and an undescribed mesostoine genus obtaining permissions and sequence submissions. from Thailand. Thus, the geographical distributions The Canada Research Chairs Program, the Natural of many of these small-bodied wasp groups is believed Sciences and Engineering Research Council, and to be considerably under-recorded. Finally, we provide the Canada First Research Excellence Fund to Food the first molecular data for of the placements of From Thought provided support to PDNH. This work three other rare genera: Afrotritermus Belokobylskij, was supported by the Senior Postdoctoral Fellowship 1995 is shown to belong to Lysiterminae, whereas under Ratchadaphiseksomphot Fund, Graduate Pachystigmus Hellén, 1927 belongs to Rhysipolinae, School, Chulalongkorn University to D.L.J.Q. and where it is related to Pseudavga Tobias, 1964 (cf. Ratchadaphiseksomphot Endowment Fund (CU- Shaw & Sims, 2015; Shaw, 2017). The position GR_62_06_23_03), Chulalongkorn University to of Aulosaphanes Belokobylskij, 1990 within B.A.B. The involvement of A.D.A. and E.P.F.-J. in the Lysiterminae (Acanthormiini) (Belokobylskij, 2004) is project was supported by funding from the Australian confirmed in both concatenated analyses (Fig. 3A, B). Biological Resources Study (RF214-16C and T215-08, It is clear that both gene coverage and taxon respectively); D.F.W. received funding from Ministry sampling are currently inadequate to resolve the for Business, Innovation, and Employment to Landcare relationships of the basal cyclostome braconids fully. Research within the Characterising New Zealand’s Nevertheless, considerable progress has been made in Land Biota Portfolio; S.A.B. was partly supported by recognizing the limitations of morphology as currently the Russian Foundation for Basic Research (project understood in the phylogenetic placement of many no. 19–04–00027) and the Russian State Research small cyclostome braconids. It is possible that there are Project no. АААА-А19-119020690101-6. no perfect diagnostic characters and that a phylogeny- Disclosure statement. No potential conflict of matching classification will be achieved only through interest was reported by the authors.

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