Phylogenomics of Ichneumonoidea (Hymenoptera) and Implications for Evolution of Mode of Parasitism and Viral Endogenization

bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.157719; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Title: Phylogenomics of Ichneumonoidea (Hymenoptera) and implications for evolution of mode of parasitism and viral endogenization

Authors:

Barbara J. Sharanowski1; Ryan D. Ridenbaugh1; Patrick K. Piekarski1,2; Gavin R. Broad3; Gaelen R. Burke4; Andrew R. Deans5; Alan R. Lemmon6; Emily C. Moriarty Lemmon7; Gloria J. Diehl1; James B. Whitfield8; Heather M. Hines5,9

Affiliations:

1Department of Biology, University of Central Florida, Orlando, FL 32816, USA

2Laboratory of Social Evolution and Behavior, The Rockefeller University, New York, NY 10065, USA

3Department of Life Sciences, the Natural History Museum, Cromwell Road, London SW7 5BD, UK

4Department of Entomology, University of Georgia, Athens, GA 30606, USA

5Department of Entomology, Pennsylvania State University, University Park, PA, 16802

6Department of Scientific Computing, Florida State University, Tallahassee, FL 32306, USA

7Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA

8Department of Entomology, University of Illinois, Urbana, IL 61801, USA

9Department of Biology, Pennsylvania State University, University Park, PA, 16802

Corresponding Author: Barbara Sharanowski: [email protected]

Keywords: Idiobiosis, Koinobiosis, Ectoparasitism, Endoparasitism, Polydnavirus, Codon Use Bias, Ancestral State Reconstruction, Parasitoid wasps, Braconidae, Ichneumonidae

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.157719; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract:

Ichneumonoidea is one of the most diverse lineages of animals on the planet with more than 48,000 described species and many more undescribed. Parasitoid wasps of this superfamily are beneficial insects that attack and kill other arthropods and are important for understanding diversification and the evolution of life history strategies related to parasitoidism. Further, some lineages of parasitoids within Ichneumonoidea have acquired endogenous virus elements (EVEs) that are permanently a part of the wasp’s genome and benefit the wasp through host immune disruption and behavioral control. Unfortunately, understanding the evolution of viral acquisition, parasitism strategies, diversification, and host immune disruption mechanisms, is deeply limited by the lack of a robust phylogenetic framework for Ichneumonoidea. Here we design probes targeting 541 genes across 91 taxa to test phylogenetic relationships, the evolution of parasitoid strategies, and the utility of probes to capture polydnavirus genes across a diverse array of taxa. Phylogenetic relationships among Ichneumonoidea were largely well resolved with most higher-level relationships maximally supported. We noted codon use biases between the outgroups, Braconidae, and Ichneumonidae and within Pimplinae, which were largely solved through analyses of amino acids rather than nucleotide data. These biases may impact phylogenetic reconstruction and caution for outgroup selection is recommended. Ancestral state reconstructions were variable for Braconidae across analyses, but consistent for reconstruction of idiobiosis/koinobiosis in Ichneumonidae. The data suggest many transitions between parasitoid life history traits across the whole superfamily. The two subfamilies within Ichneumonidae that have polydnaviruses are supported as distantly related, providing strong evidence for two independent acquisitions of ichnoviruses. Polydnavirus capture using our designed probes was only partially successful and suggests that more targeted approaches would be needed for this strategy to be effective for surveying taxa for these viral genes. In total, these data provide a robust framework for the evolution of Ichneumonoidea.

1 1. Introduction

2 Ichneumonoidea is one of 15 superfamilies of parasitoid Hymenoptera, wasps whose larvae feed on 3 other arthropods. As a result of an exceptional rate of species diversification, this lineage comprises 4 ~48,000 valid species, 33% of all Hymenoptera (145,000 spp.; (Huber, 2009)) and 3% of all known life 5 (Chapman, 2009; Yu et al., 2016). The true diversity may be many times higher when undescribed species 6 are considered. For example, estimates of known and unknown diversity in a single polydnavirus-carrying 7 subfamily (Microgastrinae) within the group might exceed the total described species for the superfamily 8 (Rodriguez et al., 2013). Ichneumonoidea has been challenging for higher level systematics because of its 9 rapid radiation and relatively high levels of morphological convergence (Quicke, 2015; Sharkey, 2007). 10 Further, the sheer size of the lineage and the relative dearth of taxonomic experts in this group has 11 presented many challenges for large phylogenetic studies. 12 The diversity of Ichneumonoidea may be due to the varied methods the wasps use to attack and 13 kill their hosts as the evolution of new mechanisms to exploit hosts may have opened up new niches, 14 enabling adaptive radiations (Price, 1980). Wasp species may be external (ectoparasitic) or internal 15 (endoparasitic) parasitoids, although the latter often leave the host to pupate (Austin and Dowton, 2000). 16 Some ichneumonoids are idiobionts, arresting the development of their host upon oviposition; whereas 17 others are koinobionts, allowing the host to continue development, often allowing the host to molt to 18 other life stages (Askew and Shaw, 1986). Further, ichneumonoids attack their arthropod hosts in all life 19 stages, from eggs to adults, and hosts in different habitats, from exposed to concealed (Quicke, 2015). 20 Understanding core phylogenetic relationships in Ichneumonoidea is pertinent to research across 21 multiple fields. Ichneumonoid parasitoid wasps are beneficial insects, providing natural management of 22 pests in crops and forests, and are estimated to provide >$17 billion in these ecosystem services (Pimentel 23 et al., 1997). Many ichneumonoids have been released in biological control programs against major 24 invasive pests, such as the Emerald ash borer (Bauer et al., 2008; Kula et al., 2010) and the Mediterranean 25 fruit fly (Vargas et al., 2001). Several ichneumonoid wasps are important models for understanding 26 parasitoid physiology (Ballesteros et al., 2017; Webb et al., 2006), interactions with host immune 27 response (Lovallo et al., 2002; Schmidt et al., 2001; Strand, 2012), development of novel transgenic 28 control methods for insect pests (Beckage and Gelman, 2004; Webb et al., 2017), and the process and 29 evolution of viral endogenization to produce beneficial viruses (Bézier et al., 2009; Burke et al., 2018a; 30 Pichon et al., 2015; Strand and Burke, 2012). Given their immense diversity, Ichneumonoidea also 31 present an exceptional opportunity to understand the factors that promote diversification, a critically 32 important question as we face increasing species extinctions with continued habitat destruction and global 33 climate change.

34 35 1.1 Phylogenetic history of Ichneumonoidea

36 Ichneumonoidea is robustly monophyletic (Sharanowski et al., 2011; Sharkey et al., 2010) and 37 currently comprises the monophyletic families: Ichneumonidae, Braconidae and Trachypetidae (Quicke et 38 al., 2020b; Sharkey, 2007). Previously in Braconidae, Trachypetidae was recently elevated to family 39 status (Quicke et al., 2020b), however the data supporting this taxonomic status is not robust, and as we 40 do not sample this lineage here, we do not discuss it further. The classification of the other two families 41 has also been in flux, with Braconidae having anywhere from 17 to 50 subfamilies and Ichneumonidae 42 with 24 to 44 subfamilies (Belshaw and Quicke, 2002; Bennett et al., 2019; Quicke et al., 2020a). 43 Braconidae, represented by over 21,000 valid species (Yu et al., 2016), 41 currently recognized 44 subfamilies (Sharanowski et al., 2011), and approximately 109 tribes, are highly valued for their use in 45 biocontrol of pestiferous hosts (Austin and Dowton, 2000; Peixoto et al., 2018; Zhang et al., 2017). A 46 molecular phylogeny of Braconidae using 7 gene regions was completed by Sharanowski et al. (2011). 47 Hypotheses of deep level relationships were well supported, allowing for a revised classification, but 48 relationships within one of the three major lineages (cyclostomes) remain largely unresolved 49 (Sharanowski et al., 2011; Zaldivar-Riverón et al., 2006), and will require greater taxon representation to 50 allow more comprehensive reclassification.

51 Although Ichneumonidae is currently larger in terms of number of described species than 52 Braconidae, the family is much less understood phylogenetically. Ichneumonidae is comprised of over 53 25,000 valid species, 38-43 subfamilies, and approximately 57 tribes (Bennett et al., 2019; Broad et al., 54 2018; Quicke et al., 2020a; Quicke, 2015; Quicke et al., 2009; Yu et al., 2012). Until recently, the three 55 molecular studies that examined higher-level relationships within Ichneumonidae (Belshaw et al., 1998; 56 Quicke et al., 2000; Quicke et al., 2009) were all based on one gene (28S rDNA), severely limiting 57 phylogenetic resolution. Phylogenetic and revisionary studies have been attempted for some lineages (e.g. 58 Bennett, 2001; Gauld et al., 2002; Gauld, 1985; Wahl and Gauld, 1998), with only a few based on 59 molecular data (Klopfstein et al., 2010; e.g. Laurenne et al., 2006; Matsumoto, 2016; Rousse et al., 2016; 60 Santos, 2017). There are three informal lineages in Ichneumonidae that have been recognized for many 61 years, namely Ichneumoniformes, Ophioniformes and Pimpliformes (Wahl, 1986, 1991; Wahl, 1993a; 62 Wahl and Gauld, 1998). These definitions were largely kept but redefined in 2009 with Quicke et al.’s 63 (2009) combined 28S and morphological analyses based on dense taxonomic sampling (1001 wasps). 64 Quicke et al. (2000) also added three additional informal groupings for single subfamily lineages 65 (Xoridiformes, Labeniformes, Orthopelmatiformes), the latter two with uncertain placement, even when

66 the taxonomic sampling was robust (Quicke et al., 2009). This classification, based on a single gene, was 67 largely followed in Quicke’s (2015) comprehensive and seminal book on Ichneumonoidea.

68 Several recent studies have been published with more comprehensive taxon, genetic, and 69 morphological sampling. Santos (2017) examined relationships among Cryptinae and higher-level 70 relationships within Ichneumoniformes using extensive taxon sampling, morphological characters, and 71 seven genes and revised the taxonomy for some lineages. Klopfstein et al. (2019) analyzed relationships 72 among the Pimpliformes using anchored hybrid enrichment with 93 genes and 723 genes from a reduced 73 taxon transcriptome dataset. They showed the evolutionary history of Pimpliformes was confounded by 74 rapid radiations across several lineages. Finally, Bennett et al. (2019) examined relationships among the 75 whole family using extensive morphological characters and three loci, and currently represents the most 76 detailed analysis and summary of ichneumonid relationships to date. Here we follow the revised higher- 77 level groupings for Ichneumonidae proposed by Bennett et al. (2019).

78 Given the immense diversity on both Braconidae and Ichneumonidae, continued research on their 79 phylogenetic relationships, evolution, and taxonomy are warranted given their importance in pest control 80 and interesting evolutionary history with endogenous viruses. Further, members of both families have 81 been underutilized in biological control due to numerous cryptic species (Derocles et al., 2016; Peixoto et 82 al., 2018) and limited taxonomic resources facilitating accurate identification of many groups, particularly 83 for species (Broad et al., 2018; Quicke, 2015; Veijalainen et al., 2012; Wahl, 1993b). 84 85 1.2 Evolution of viral symbiosis in Ichneumonoidea 86 Some ichneumonoids overcome host defenses through the use of viruses produced by genes that 87 are integrated within the wasp genome and modify host physiology and immunity (Kroemer and Webb, 88 2004; Lavine and Beckage, 1995; Strand and Burke, 2012). The acquisition of these endogenous virus 89 elements (EVEs) in Ichneumonoidea promotes reproductive success and may be a factor promoting 90 diversity (Burke et al., 2018a; Burke and Strand, 2014; Federici and Bigot, 2003; Gauthier et al., 2017; 91 Whitfield, 1990). These viruses in the family Polydnaviridae (PDV, genera Bracovirus, Ichnovirus) are 92 only known from a few clades across Ichneumonoidea. Within Ichneumonidae, viruses are known from 93 two subfamilies (Banchinae and Campopleginae) that are both members of the Ophioniformes (Quicke, 94 2015; Quicke et al., 2009). In both lineages, the viruses are ichnoviruses (PDVs) that have some 95 homologous genes, but whether this represents one or two independent acquisitions remains unclear 96 (Béliveau et al., 2015; Lapointe et al., 2007; Volkoff et al., 2012; Volkoff et al., 2010), largely due to a 97 lack of knowledge on Ichneumonid relationships and viral presence across the family. There is one known 98 case of an ichneumonid, Venturia canescens (Campopleginae), which does not produce an ichnovirus but

99 instead has an alphanudivirus-like EVE that no longer packages DNA but produces virus-like particles 100 (VLPs) (Pichon et al. 2015). Thus, for Ichneumonidae, there have been at least 2 or 3 independent 101 acquisitions of EVEs. 102 Within Braconidae, EVEs are best known within the diverse microgastroid complex, in which all 103 known viruses are bracoviruses (Polydnaviridae) that were likely derived from a common ancient (~100 104 mya) betanudivirus (Nudiviridae) (Bézier et al., 2009; Bézier et al., 2013; Murphy et al., 2008; Whitfield, 105 1997, 2002; Whitfield and O’Connor, 2012). The latest discovery of an independent and recent 106 acquisition of endogenous virus related to alphanudiviruses (FaENV - Nudiviridae) in a different lineage 107 of Braconidae (some species of Fopius (Opiinae)) suggests that viral endogenization may be more 108 common than previously thought (Burke et al., 2018a). Unlike bracoviruses and ichnoviruses, FaENV in 109 species of Fopius, like in Venturia, lost the ability to package DNA and instead produce VLPs. Thus, both 110 Ichneumonidae and Braconidae have at least two subfamilies that produce viruses (PDVs and VLPs), but 111 virion morphology, virus replication machinery, genome rearrangement and gene homology suggest these 112 endogenization events likely represent four or five independent acquisitions (Béliveau et al., 2015; Burke 113 et al., 2018a; Herniou et al., 2013; Thézé et al., 2011). 114 115 1.3 Importance of a stable phylogenetic framework for Ichneumonoidea 116 Unfortunately, understanding of viral acquisition and evolution, as well as parasitoid evolution, 117 host use, development, diversification, and host immune disruption, is severely limited by the lack of a 118 robust phylogenetic framework (Desjardins et al., 2008; Gauld, 1988; Lapointe et al., 2007; Pennacchio 119 and Strand, 2006; Whitfield and O’Connor, 2012). Outside of low genetic sampling of previous studies, 120 the difficulty of resolving clades in Ichneumonoidea is likely due in part to several rapid radiations 121 (Banks and Whitfield, 2006; Klopfstein et al., 2019; Whitfield and Kjer, 2008). Further, previous 122 phylogenetic analyses based on morphology (Belshaw et al., 2003; Dowton et al., 2002; Pitz et al., 2007; 123 Quicke et al., 2000; Sharkey and Wahl, 1992; van Achterberg and Quicke, 1992; Wahl and Gauld, 1998; 124 Wharton et al., 1992) have discovered high levels of convergence, thereby obfuscating true relationships 125 (Gauld and Mound, 1982; Quicke, 2015; Whitfield, 2002; Wild et al., 2013). Thus, deep genetic sampling 126 will likely be necessary to understand relationships in Ichneumonoidea. Several recent studies of other 127 insect groups that likely had rapid radiations have shown the power of a phylogenomics approach using 128 hundreds of genes (Branstetter et al., 2017; Peters et al., 2017; Piekarski et al., 2018), even if taxonomic 129 sampling was relatively low (Johnson et al., 2013; Misof et al., 2014; Savard et al., 2006; Simon et al., 130 2009). 131 In this study we resolve several deep phylogenetic relationships for Ichneumonoidea using 132 anchored hybrid enrichment (AHE) (Lemmon et al., 2012) and focusing more on the poorly resolved

133 ichneumonids. We sequenced whole genomes of 11 ichneumonoid wasps and utilize these genomes along 134 with previously sequenced Hymenopteran genomes to design a novel probe set targeted for 135 Ichneumonoidea, but capable of capturing genes across multiple lineages of Hymenoptera. We sequenced 136 479 genes for 80 ingroup exemplars, one of the largest molecular datasets for this immensely diverse 137 superfamily to date. These data provide a robust phylogenetic framework for Ichneumonoidea and 138 establish molecular tools for future sampling in the lineage. We reconstruct ancestral states for two traits 139 related to parasitism: idiobiosis versus koinobiosis, and ecto- versus endoparasitism. Further we examine 140 the utility of probes to capture polydnaviruses across a broad array of taxa. Our data help resolve previous 141 phylogenetic uncertainties and improve the ability to address interesting biological questions in this rapid 142 radiation. 143 144 2. METHODS

145 2.1 Genomic Sequencing and Probe Design

146 The goals for probe design for AHE (Lemmon et al., 2012) were to specifically target 147 ichneumonoids, but also to have broader applicability across all of Hymenoptera. Thus, we utilized 148 published Hymenoptera sequences and sequenced several new genomes to design the probe set. The 149 probe design was generated by optimizing across published genomes from the bees Apis mellifera 150 (Honeybee Genome Sequencing Consortium, 2006) and Bombus impatiens (Sadd et al., 2015), the 151 parasitoid wasp Nasonia vitripennis (Werren et al., 2010), seven ant species (Gadau et al., 2012), and two 152 braconid genomes from i5k (http://arthropodgenomes.org/wiki/i5K): (Microgastrinae: Microplitis 153 demolitor (Burke et al., 2018b) and Opiinae: Diachasma alleoleum (Tvedte et al., 2019)). To these we 154 added whole genome sequences from 11 additional ichneumonoid wasps, including four braconids and 155 seven ichneumonids that represent diverse lineages across the ichneumonoid phylogeny (Table 1). For 156 whole genome sequencing of these 11 individuals, DNA was extracted from whole specimens using a 157 DNeasy Blood and Tissue Kit with RNase treatment. DNA was only used if good quality scores and 158 DNA concentration were obtained from the Nanodrop, and gel electrophoresis showed minimal DNA 159 degradation. Indexed libraries with ~250 bp inserts were prepared for sequencing following Lemmon et 160 al. (2012), and sequenced at 100bp paired end to achieve ~15x genomic coverage on two HiSeq2500 161 sequencing lanes (79 Gb total output). Reads were submitted to the NCBI Sequence Read Archive 162 (FigShare 10.6084/m9.figshare.12497468).

163

164 Raw reads from newly sequenced taxa were combined with assembled genome data to design the 165 Hymenoptera probe set. In general, we followed locus selection procedures outlined in Hamilton et al. 166 (2016). Initial Hymenoptera probe design involved finding orthologs to an optimal set of 941 insect loci 167 established in Coleoptera (Haddad et al., 2018). Using the red flour beetle (Tribolium castaneum) 168 sequence as a reference, we scanned the assembled genomes of two divergent hymenopteran species 169 (Bombus impatiens and Nasonia vitripennis) for each of the Coleoptera anchored hybrid enrichment loci. 170 The best matching region in each of the two Hymenoptera genomes was identified and a 4000 bp region 171 containing each locus was extracted and aligned using MAFFT v7.023b (Katoh and Standley, 2013b) 172 with –genafpair and –maxiterate 1000 flags. We then scanned the 12 assembled and raw reads from 11 173 unassembled genomes outlined above for targeted anchor regions using Nasonia vitripennis sequences. 174 For the 12 assembled genomes, we isolated up to 4000 bp surrounding the region that best matched the 175 reference. For the unassembled genomes, reads were merged and trimmed following Rokyta et al. (2012), 176 and then mapped to Nasonia vitripennis. The consensus sequences from the resulting assemblies were 177 extended in flanking regions to produce up to a 4000 bp sequence for each species at each locus following 178 protocols outlines in Hamilton et al. (2016). The resulting loci from N. vitripennis and the 23 target 179 species were then aligned using MAFFT. These were visually inspected to confirm adequate alignment in 180 Geneious (Kearse et al., 2012), and then trimmed and masked to isolate well-aligned regions. To ensure 181 sufficient enrichment efficiency, we removed loci that contained fewer than 50% of the taxa, resulting in 182 528 anchor loci.

183 We also added a few custom selected genes into the probeset: five recognized venom proteins 184 (calreticulin, phenoloxidases, chitinase, trehalase, aspartylglucosaminidase); two genes (ACC and CAD) 185 used in a previous braconid phylogeny (Sharanowski et al., 2011); and five polydnavirus genes (see 186 below). For each of these, rather than use the genomes, probes were designed from a reference set of 187 previously published gene sequences.

188 Polydnaviruses were included to enable comparison of taxon incidence, number of origins, age of 189 virus endogenization events and evolution of polydnaviruses among the Ichneumonoidea while also 190 assessing the ability of the anchored enrichment approach to capture more rapidly evolving adaptive 191 genes. Furthermore, this capture could increase the numbers of these genes across taxa to enable an 192 improved framework for examining their evolution in future. The PDV genes included two markers 193 identified from bracoviruses (Bézier et al., 2009) from three species: HzNVorf128, a gene of viral origin 194 associated with replication, and p74 per os infectivity factor, a viral envelope gene. We also designed 195 probes for two ichnovirus replication or structural proteins that occur across three species of 196 Campopleginae, including EST data derived from Sharanowski et al. (2010): IVSP4 and U1 (Burke et al.,

197 2013; Lorenzi et al., 2019; Volkoff et al., 2012; Volkoff et al., 2010). These genes are part of the 198 replication machinery maintained within the host genome and were selected because they are more likely 199 to be single copy or involve just a few copies. In addition, we included sequences representing the 200 vankyrin gene family across all PDV lineages to test the ability to capture gene family representatives 201 (Kroemer and Webb, 2005). Unlike the other loci, vankyrins are packaged within PDVs and are likely not 202 of viral origin (Huguet et al., 2012).

203 Repetitive Kmer alignment regions among all loci were masked following Hamilton et al. (2016). To 204 build the final probe set we tiled 120 bp probes at 2X density across the 24 taxon-specific sequences for 205 each locus. The final probe set included 57,066 probes from 541 loci covering 212,392 total base pairs. 206 Loci included in the probe design consisted of a diverse array of genes, with no significant enrichment of 207 any particular class of genes based on Blast2GO (Conesa et al., 2005) analysis.

208 2.2 Taxon Sampling

209 To provide a deeper phylogenetic framework for systematic revision in Ichneumonoidea, we 210 focused our taxon sampling on the lesser-understood ichneumonids, but also included sampling of 211 braconids to enable comparison with a previously published 7-gene phylogeny on Braconidae 212 (Sharanowski et al., 2011). We utilized 59 Ichneumonidae species representing 25 subfamilies and 21 213 Braconidae species representing 17 subfamilies (Table 1). For Ichneumonidae, taxa were selected to 214 maximize the number of represented lineages and provide denser sampling for lineages that were 215 suspected to be paraphyletic based on previous studies (Quicke et al., 2009) or contain PDVs (Bigot et al., 216 2008; Strand and Burke, 2015). For Braconidae, taxa were selected to include representatives of major 217 lineages to assess how our results obtained with the anchored hybrid enrichment compare to a more 218 traditional molecular study completed by Sharanowski et al. (2011). Species level identifications were not 219 possible for the majority of taxa, as many genera require revisionary works or there are not adequate keys 220 or descriptions of species to ensure accuracy. Outgroups consisted of genomic data from published model 221 genomes that were included in our probe design (Table 1). We follow the higher-level groupings of 222 Sharanowski et al. (2011) for Braconidae and Bennett et al. (2019) for Ichneumonidae, except we keep 223 the grouping Labeniformes for members of Labeninae, and do not include this lineage within 224 Ichneumoniformes s.l. All specimens were deposited in the University of Central Florida Collection of 225 Arthropods (UCFC) except specimens from Sweden, which were deposited at Station Linné, Swedish 226 Malaise Trap Project (SMTP).

227 2.3 Hybrid Enrichment Genome Capture & Data Processing

228 The remaining sequence data were obtained at Florida State University’s Center for Anchored 229 Phylogenomics (www.anchoredphylogeny.com) using hybrid enrichment sequence capture with 230 SureSelect. For this approach, genomic DNA samples were extracted using the Qiagen DNEasy kit with 231 quality assessed using gel electrophoresis and a Nanodrop. Indexed libraries were prepared following a 232 protocol modified from Meyer & Kircher (2010) but modified to accommodate the Beckmann-Coulter 233 FXP liquid handling robot. Libraries were pooled and enriched using an Agilent Custom SureSelect kit 234 containing all 57,066 probes and were sequenced using PE 100bp PE sequencing on the Illumina HiSeq 235 2000.

236 The resulting paired Illumina anchored hybrid enrichment reads were demultiplexed with no 237 mismatches tolerated after quality filtering using the Illumina Casava high chastity filter. Read pairs were 238 then merged using a Bayesian approach that identifies the sequence overlap, removes adapter sequences, 239 and corrects sequencing errors in overlapping regions (Rokyta et al. 2012). The resulting reads were then 240 assembled using a quasi de novo assembler described by Hamilton et al (2016), with the following 241 references (Table 1): Megachile rotundata, Microplitis demolitor, Aleiodes sp. (PSUC_FEM 10030003), 242 Lissonota sp. (PSUCFEM_10030012), and Odontocolon sp. (PSUC_FEM 10030002). The effects of low- 243 level contamination and misindexing were avoided by filtering out assembly clusters with fewer than 100 244 reads. To establish orthology, homologous consensus sequences from each assembly cluster passing the 245 coverage filter were evaluated to compute a pairwise distance matrix that was used to identify orthosets 246 using a neighbor-joining approach (following Hamilton et al., 2016). After assembly and preliminary 247 trimming using the pipeline, 479 loci were retained.

248 2.4 Alignment and Dataset Filtering

249 Orthologous sequences were aligned using MAFFT (v7.023b, with –genafpair and –maxiterate 250 1000 flags (Katoh and Standley, 2013a). Alignments were manually inspected for errors after 251 autotrimming (mingoodsites=12, minpropsame=0.4 and 20 missing taxa allowed) following the approach 252 outlined in Hamilton et al. (2016). Alignments were then manually checked to ensure the proper reading 253 frame was retained after auto-trimming by using the gene maps of Apis mellifera, Nasonia vitripennis, 254 and Solenopsis invicta as a guide, and where necessary, non-coding regions were removed (7 genes 255 captured parts of introns and 3 genes captured parts of untranslated regions (UTRs).

256 2.5 Model testing and Phylogenetic Analyses

257 Each locus alignment was translated into a protein dataset and both protein and nucleotide 258 datasets were concatenated using SequenceMatrix 1.8 (Vaidya et al., 2011) and analyzed separately. We

259 analyzed the datasets using Maximum Likelihood with IQ-Tree v.1.6.10 (Nguyen et al., 2014) on either 260 the Stokes HPC at the University of Central Florida Advanced Research Computing Center or on the 261 CIPRES Science Gateway (Miller et al., 2009; Miller et al., 2010). Bayesian analyses were also 262 performed on the concatenated nucleotide dataset with the third codon removed using MrBayes 3.2.6 263 (Ronquist et al., 2012). The full nucleotide or protein dataset was not completed using MrBayes, as 264 analyses were taking longer than 3 months and often did not converge, even when constraints of known 265 monophyletic lineages were applied. For the Nucleotide Bayesian analysis with the third codon position 266 removed, we constrained monophyly for Braconidae, Ichneumonidae, Ants, and Bees. PartitionFinder 267 V.2.1.1 (Lanfear et al., 2012; Lanfear et al., 2016) was used to find the best-fit models of molecular 268 evolution and partitioning scheme using each locus as a priori subsets for the nucleotide dataset. For each 269 treatment, an hcluster search with branch lengths linked was performed and model selection was based on 270 the Bayesian Information Criterion (Schwarz, 1978). For the protein dataset, the best fit models of 271 molecular evolution and partitioning scheme was inferred using ModelFinder (Chernomor et al., 2016; 272 Kalyaanamoorthy et al., 2017) within IQ-Tree v.1.6.10, limited to the following models with the option 273 for invariant and gamma parameters: Blosum62, Dayhoff, JTT, LG, VT, and WAG. All alignment 274 datasets can be found on FigShare (10.6084/m9.figshare.12497468).

275 We also ran saturation, relative synonymous codon usage, and long branch exclusion tests and ran 276 the nucleotide dataset with the 3rd position removed using IQ-tree using the methods outlined above. 277 Nodal support in IQ-tree analyses were assessed with 1000 pseudo-replicates of both the ultrafast 278 bootstrap (Hoang et al., 2017) and Shimodaira-Hasegawa-like approximate Likelihood Ratio Test (SH- 279 aLRT) (Guindon et al., 2010). The Bayesian analysis was run with 45 million generations sampling every 280 1000 generations with a 25% burn-in. The analysis consisted of two independent runs with four chains 281 each. Convergence of the Bayesian analysis was assessed and confirmed using diagnostics in Tracer v.1.6 282 (Rambaut et al., 2013). To test for saturation, we calculated the number of pairwise differences and 283 Tajima Nei genetic distance for each codon position using MEGA v.7.0 (Kumar et al., 2016), and plotted 284 against each other using R (R Team, 2016) and the package “ggplot2”(Wickham, 2016). To test for codon 285 bias in the nucleotide dataset we performed a principal component analysis using relative synonymous 286 codon usage (RSCU) and amino acid composition, and performed a phylogenetic ANOVA using the 287 effective number of codons (ENC) in R with the following packages: “seqinr” (Charif and Lobry, 2007), 288 “phangorn” (Schliep, 2011), “geiger” (Harmon et al., 2008), “vhica” (Wallau et al., 2016), and “dplyr” 289 (Wickham et al., 2015). In addition to this we calculated GC content for each taxon and synonymous 290 skew on a scale of 0 to 1 for each of the six-fold degenerate amino acids (Rota-Stabelli et al., 2013) using 291 the formula introduced by Perna and Kocher (1995), and mapped these metrics onto the nucleotide

292 phylogeny using the R packages “ape” (Paradis et al., 2004) and “phytools” (Revell, 2012). As there were 293 differences in some relationships between the nucleotide and amino acid analyses, we tested stability of 294 the relationships recovered in the amino acid analyses by performing a series of taxon exclusion tests 295 (Bergsten, 2005). For each analysis, one or more taxa were removed and the analysis rerun in IQ-tree 296 following the methods outlined above. Choices of taxa to exclude were based on length of branches and 297 instability in phylogenetic position between the nucleotide and amino acid datasets.

298 Successfully captured PDV genes were aligned along with the available GenBank sequences used 299 to facilitate capture (FigShare 10.6084/m9.figshare.12497468) using Geneious with the native alignment 300 algorithm and default parameters. These alignments were run through jmodeltest2 (Darriba et al., 2012) to 301 assign models of evolution and were then run in MrBayes v.3.2.6 (10 M generations, 3 runs) using 302 CIPRES Science Gateway (Miller et al., 2010). All were confirmed to have converged with Tracer v. 1.6 303 (Rambaut et al., 2013). As no taxa were captured with U1, these analyses included three genes: six taxa 304 for Bracovirus HzNVorf128 at 1035 aligned bases and run under a GTR+G model, four taxa for 305 Bracovirus per os infectivity factor p74 run with 2144 bps under a GTR+I model, and ten sequences for 306 the Ichnovirus IVSP4, 1198 bp run under a GTR+I model.

307 2.6 Ancestral State Reconstructions

308 To examine evolutionary transitions of parasitoid biology, ancestral states were reconstructed for 309 two traits: ectoparasitism (0) versus endoparasitism (1); and idiobiosis (0) versus koinobiosis (1) 310 (Supplementary Table S1). Reconstructions were completed using the IQ-tree phylogeny of the amino 311 acid analysis and repeated with Labeninae removed (due to instability in this lineage’s phylogenetic 312 position; see results). To assess robustness of results across phylogenetic uncertainty, the analysis was 313 repeated with the IQ-tree phylogeny based on nucleotide data with the third position removed (nt-3out) 314 and repeated with Labeninae removed. Binary characters were analyzed using Maximum Likelihood 315 using Mesquite version 3.2 (Maddison and Maddison, 2019). Two models were tested for likelihood 316 reconstructions: the Markov k-state one parameter (Mk1), which has an equal rate for character gains and 317 losses, and the asymmetrical two-parameter Markov k-state (Assym2), which applies a separate rate. To 318 test whether the characters evolved in drastically different ways between Braconidae and Ichneumonidae, 319 we tested whether the same model was a better fit when only one family was included for analysis.

320 3. Results

321 3.1 Sequence Data

322 Selective enrichment was good, with 27.3% of reads on average (range 14.6–49.0%) mapped to 323 loci. Most of the loci were recovered across individuals, with an average of 478 of the 541 genes captured 324 per individual. This is nearly the same number of genes that were recovered from blasts of taxa sequenced 325 from whole genome data against references for each gene (n=480), suggesting that nearly all genes 326 present with close affinity to reference sequences were captured. Many of the loci had comprehensive or 327 nearly comprehensive coverage, with 206 of the loci having high read numbers (>100) mapped across all 328 35 taxa (1 taxon failed in the sequencing run) and 297 loci having high read mapping across at least 33 of 329 the 35 taxa. Average length per locus was 990 bps (Supplementary Fig. 1). After trimming only seven 330 genes had introns partially captured and three had portions of untranslated regions captured, which were 331 manually trimmed out of the dataset. The concatenated datasets had a total of 91 taxa and 479 genes with 332 lengths of 150,435 base pairs and 50,145 amino acids for the nucleotide and amino acid datasets, 333 respectively. The nucleotide dataset with the third position removed had 100,290 base pairs.

334 3.2 Evolutionary Relationships among Ichneumonoidea

335 Saturation was indicated in the third codon position of the concatenated dataset (Fig. S2), and 336 thus the maximum likelihood phylogeny with all data included (Fig. S3) is not discussed further given the 337 likelihood for systematic error. The maximum likelihood amino acid phylogeny is depicted in Fig. 1A-C. 338 Nodal supports for the nucleotide dataset with the 3rd codon position removed (nt-3out) for the maximum 339 likelihood (Fig. S4) and Bayesian (Fig. S5) phylogenies are also indicated on Fig. 1A-C where 340 relationships were identical. All analyses recovered a maximally supported and monophyletic 341 Ichneumonoidea, Braconidae, and Ichneumonidae (Fig 1a). Maximum support indicates a value of 100 342 for Shimodaira-Hasegawa approximate Likelihood Ratio Tests (aLRT), 100 for Ultra-Fast bootstraps 343 (UFb), and 1.0 for posterior probability (PP).

344 Within Braconidae, several higher-level lineages were also maximally supported across all 345 analyses, including the cyclostomes s.l., cyclostomes s.s., non-cyclostomes, the Aphidioid, Helconoid and 346 Microgastroid complexes, and the Alysioid subcomplex (Fig. 1B), similar to results reported in 347 Sharanowski et al. (2011). Relationships among all cyclostomes were also consistent with Sharanowski et 348 al. (2011), despite the low taxonomic sampling: the Aphidioid complex was sister to the cyclostomes s.s., 349 Rhyssalinae was recovered as sister to the remaining cyclostomes s.s., and Braconinae was recovered as 350 sister to the Alysioid complex with maximal support (Fig. 1B). The placements of Rogadinae and 351 Pambolinae were labile between the amino acid (Fig. 1B) and nt-3out analyses (Figs. S4-5); however, 352 support for Rogadinae as sister to Braconinae + the Alysioid complex increased when different outgroups 353 were used (Fig. S6, Table S2) and when Aphidiinae was removed (Fig. S7). Many relationships within

354 the non-cyclostomes were identical to results in Sharanowski et al. (2011). For example, Euphorinae was 355 consistently recovered as sister to the Helconoid complex (Fig.1b): Acampsohelconinae (Brachistinae 356 (Helconinae + Macrocentroid subcomplex)). One notable exception was the placement of Ichneutinae, 357 which was variably placed as sister to Agathidinae + the Microgastroid complex (Fig. 1B) or sister to 358 Agathidinae (Figs S4-5) with variable support. Most previous studies have recovered Ichneutinae as a 359 paraphyletic group sister to the Microgastroid complex s.s. (Belshaw et al., 2000; Belshaw and Quicke, 360 2002; Dowton et al., 2002; Quicke and van Achterberg, 1990; Sharanowski et al., 2011). When 361 Agathidinae was removed, Ichneutinae was recovered as sister to the Microgastroid complex (Fig. S8) 362 with very high support (≥ 99), possibly indicating that low taxonomic sampling or long branch attraction 363 may have been an issue for the unexpected placement of Ichneutinae.

364 Within Ichneumonidae, Xoridinae was always recovered as sister to all remaining ichneumonids 365 with strong support (>95, Fig. 1C), consistent with previous analyses (Klopfstein et al., 2019; Quicke et 366 al., 1999b) where Xoridinae was not set as the root (but see Bennett et al., 2019). The next basal lineage 367 was Labeninae in the protein analysis (Fig. 1C), but this placement had very low support. In the nt-3out 368 analyses, Labeninae was recovered as sister to Orthopelmatinae with relatively strong support (Figs. S4- 369 S5), and these taxa were sister to Ichneumoniformes + Pimpliformes. Other recent studies with large 370 taxon or gene sampling also had variable placement of Labeninae (Bennett et al., 2019; Klopfstein et al., 371 2019; Santos, 2017). These variable results may reflect the low taxonomic sampling, especially non- 372 sampling of potentially related subfamilies, such as Brachycyrtinae, which has been recovered as the 373 sister group to Labeninae (Bennett et al., 2019). In general, Labeninae was the most labile lineage across 374 analyses and when different taxa were excluded (Table S2, Figs. S6-S17). When Xoridinae was excluded, 375 the branch support for Labeninae as the most basal taxon is increased (Fig. S9). Further when Labeninae 376 was excluded (Fig. S10), the support for all remaining Ichneumonids (except Xoridinae) as a 377 monophyletic lineage was substantially increased (≥ 99).

378 Several higher-level relationships among Ichneumonidae were maximally supported, including 379 Ichneumoniformes, Pimpliformes, and Ophioniformes (Fig. 1C). Ophioniformes was recovered as sister 380 to all remaining ichneumonids (excluding Xoridinae and Labeninae) with the following relationships: 381 Pimpliformes (Orthopelmatinae + Ichneumoniformes). The amino acid analysis recovered 382 Orthopelmatinae as sister to the Ichneumoniformes, but with low support (Fig. 1C); however, in the 383 outgroup test (Fig. S6) and several taxon exclusion tests, support for this relationship increased (Table S2, 384 Figs. S6, S13-14, S17). Although Orthopelmatinae and Eucerotinae had very long branches, when 385 Eucerotinae was excluded (Fig. S13), Orthopelmatinae was recovered as sister to Ichneumoniformes s.l. 386 with increased support. Interestingly, when both Labeninae and Orthopelmatinae were excluded (Table

387 S2, Fig. S12), support for the major backbone relationships increased (≥ 99): Ophioniformes 388 (Ichneumoniformes + Pimpliformes).

389 Eucerotinae was always recovered as sister to a clade containing Adelognathinae, Cryptinae, and 390 Ichneumoninae with maximal support (Fig. 1C). Despite our low taxonomic sampling for this lineage, 391 these results support Eucerotinae within Ichneumoniformes s.l. (following Bennett et al., 2019). However, 392 our lack of members of several subfamilies within Ichneumoniformes s.l. (Brachycyrtinae, Claseinae, 393 Agriotypinae, Microleptinae, Pedunculinae, Ateleutinae, Phygadeuontinae) limits understanding of 394 phylogenetic relationships in this lineage. The representatives we included for Ichneumoninae and 395 Cryptinae were always recovered as monophyletic with maximum support, however, the placement of 396 Adelognathinae varied with respect to these subfamilies across the amino-acid (Fig. 1C) and Bayesian nt- 397 3out analysis (Fig. S5).

398 Similar to Klopfstein et al. (2019), relationships within Pimpliformes varied widely across the 399 amino acid (Fig. 1C) analysis and nt-3out analyses (Figs. S4-5). In the amino acid analysis, Acaenitinae 400 was recovered as sister to the remaining Pimpliformes with strong support (Fig. 1C), whereas 401 Diplazontinae + Orthocentrinae were recovered at the base of the radiation in the nucleotide analyses with 402 strong support (Figs S4-5). Interestingly, the relationships recovered in the amino acid analysis (Fig. 1C) 403 remained stable across all outgroup and taxon exclusion tests (Table S2, Figs. S6-17) and were largely 404 consistent with relationships proposed in a comprehensive morphological study by Wahl and Gauld 405 (1998). We investigated codon use biases and observed that Diplazontinae has more similar codon usage 406 to Ichneumoniformes s.s. than the rest of Pimpliformes (Fig. S18), and this may account for its more basal 407 position in the nt-3out analyses (Figs. S4-5). Like long branch attraction (Bergsten, 2005), false 408 homologies created through the codon use patterns may pull convergent taxa falsely together.

409 Consistent and well supported relationships within Pimpliformes included: Diplazontinae + 410 Orthocentrinae (consistent with Wahl, 1990; Wahl and Gauld, 1998); Collyriinae + Cyllocerinae; and 411 monophyly of the Ephialtini and Pimplini tribes of Pimplinae, consistent with the findings of Klopfstein 412 et al. (2019) (Fig. 1C). Pimplinae was recovered as paraphyletic when nucleotide data was analyzed, but 413 monophyletic when the amino acid data was analyzed (a similar issue was found in Klopfstein et al., 414 2019). There was also a distinct difference in codon usage between the Ephialtini and Pimplini clades of 415 Pimplinae (Fig. S19). Thus, the amino acid analysis probably more accurately reflects the true monophyly 416 of Pimplinae, which has been supported by some morphological studies (Gauld et al., 2002; Wahl and 417 Gauld, 1998). The lone specimen of Poemeniinae (Neoxorides sp.) was recovered as sister to the 418 Pimplinae, suggesting its close affiliation (Bennett et al., 2019; Quicke et al., 2009; Wahl and Gauld,

419 1998). The inclusion of Collyriinae in Pimpliformes supports previous studies (Bennett et al., 2019; 420 Klopfstein et al., 2019; Quicke et al., 2009; Sheng et al., 2012).

421 Within Ophioniformes, the following subfamilies (that had more than one exemplar) were 422 recovered as monophyletic across all analyses with maximum support (Fig. 1C): Tryphoninae, Banchinae, 423 Tersilochinae, Metopiinae, Anomaloninae, Cremastinae, and Campopleginae. Tryphoninae was 424 consistently recovered and maximally supported as the sister to all remaining Ophioniformes. Banchinae 425 was the next most basal lineage (consistent with Klopfstein et al., 2019; Quicke et al., 2009), but this 426 placement was poorly supported (Fig. 1C). However, this placement was largely consistent across the 427 outgroup and taxon exclusion analyses (Table S2), except when Mesochorinae was removed (Fig. S16). 428 Further, in the nt-3out analyses (Figs S4-5), Banchinae was recovered as sister to Tersilochinae with 429 strong support, and both were sister to the remaining Ophioniformes (except Tryphoninae) (Figs. S4-5). 430 The remaining Ophioniformes were consistently recovered in a maximally supported clade, including: 431 Mesochorinae, Oxytorinae, Ctenopelmatinae, Metopiinae, Cremastinae, Hybrizontinae, Anomaloninae, 432 Ophioninae, and Campopleginae. Of these, Mesochorinae was recovered as sister to the rest, but with 433 variable support (Fig. 1C). Ctenopelmatinae was unsurprisingly paraphyletic as was found previously 434 (Bennett et al., 2019; Quicke et al., 2009). Metopiinae was consistently recovered as sister to a maximally 435 supported clade that roughly corresponds to Quicke et al.’s (2009) “higher Ophioniformes,” including 436 Cremastinae, Hybrizontinae, Anomaloninae, Ophioninae, and Campopleginae. Interestingly, 437 Hybrizontinae (a group that has varied widely in placement across all previous analyses) was recovered as 438 sister to Cremastinae with strong but variable support across all analyses (Fig. 1C). When Hybrizontinae 439 was excluded (Fig. S15), Anomaloninae was recovered as sister to Cremastinae with relatively high 440 support (>93). Ophioninae was consistently recovered as the sister group to Campopleginae with maximal 441 support across all analyses.

442 3.3 Ancestral State Reconstructions

443 Ancestral biological states were reconstructed on phylogenies that varied in the data type they 444 were estimated from (amino acid or nt-3out) and taxon representation: either all taxa were included, all 445 taxa but Labeninae (due to its uncertain placement), and with or without outgroups included. Further, 446 each family was tested individually based on the amino acid analysis, with all other taxa removed. As our 447 sampling was not comprehensive, we only discuss ancestral state reconstructions for higher-level 448 relationships, including Ichneumonoidea, Braconidae and Ichneumonidae. Within Braconidae we also 449 discuss reconstructions for the ancestors of the Cyclostomes s.l., Cyclostomes s.s., and Non-cyclostomes, 450 and within Ichneumonidae, ancestors of Ichneumoniformes s.l., Pimpliformes, Ophioniformes, and

451 Ophioniformes excluding Tryphoninae (Fig. 2A and B). The full reconstructions for each trait and input 452 phylogeny can be found on FigShare (10.6084/m9.figshare.12497468).

453 The Assym2 model (separate parameters for gains and reversals) consistently outperformed the 454 Mk1 model (equal rates of transition between character states) for character 1 (idiobiont vs koinobiont) 455 when all Ichneumonoidea were analyzed together (Table 2). Under these analyses, the ancestors of 456 Ichneumonoidea, Ichneumonidae, and Braconidae were reconstructed as idiobionts, although this was 457 only consistently significant for Ichneumonidae (Fig. 2A, Table 3). Within Braconidae, the ancestors of 458 the Cyclostomes s.l. and s.s. were also recovered as idiobionts, but this was only consistently significant 459 for the latter. Unsurprisingly, the ancestor of the non-cyclostomes was significantly koinobiont as all 460 known members are koinobiont (Fig. 2A, Table 3). Within Ichneumonidae, the Ichneumoniformes s.l. and 461 Pimpliformes were ancestrally idiobiont, whereas Ophioniformes was koinobiont, and all reconstructions 462 were significant (Fig. 2A, Table 3). When the families were analyzed individually, the above results 463 remained consistent for Ichneumonidae, but not Braconidae as the Mk1 model was preferred for 464 Braconidae (Table 2). Under this model all major braconid nodes were reconstructed as koinobiont and 465 significant for all lineages except the cyclostomes s.s. (Fig. 2A, Table 3), suggesting this character is 466 sensitive to the data used to infer the ancestral states for Braconidae.

467 For character 2 (ecto- vs. endoparasitism), the ancestral states were very sensitive to the 468 phylogeny utilized in the analysis (Fig. 2B). The Mk1 model was supported for character 2 when 469 Labeninae and outgroups were both excluded, when the nt-3out phylogeny was used, or when the 470 Braconidae or Ichneumonidae were analyzed individually (Table 2). Under the Mk1 model, all major 471 nodes were reconstructed as endoparasitoid and this consistently significant for most reconstructions 472 except the Ichneumonoidea, Ichneumonidae, Ophioniformes, and cyclostomes s.s. (Fig. 2B, Table 3). For 473 Ichneumonidae, these reconstructions suggested that the Ichneumonidae, Ichneumoniformes s.l., and 474 Pimpliformes had idiobiont endoparasitoid ancestors. When the Assym2 model was most appropriate 475 (Table 2), all major braconid nodes were reconstructed as ectoparasitoid, except the non-cyclostomes, and 476 only the cyclostomes s.s. had significant reconstructions (Table 3). There was also inconsistency across 477 the Ichneumonidae, where under the Assym2 model, only the Pimpliformes and Ophioniformes without 478 Tryphoninae were recovered as endoparasitoid and these reconstructions were not significant (Fig. 2B, 479 Table 3). These data suggest that ancestral reconstruction, particularly of ecto- vs. endoparasitism, is 480 sensitive to the taxon representation and type of data used in the analysis.

481 3.4 Capture of Polydnavirus Genes

482 Capture of polydnavirus genes had limited success. The U1 ichnovirus replication gene was not 483 captured in any of the taxa in our dataset including the campoplegines most likely to harbor a copy of it. 484 The IVSP4 gene was represented in the probe set by two paralogs from Hyposoter didymator (IVSP4-1 485 and IVSP4-2) and one gene copy from Campoletis sonorensis (Fig. 3A). Glypta fumiferanae (Banchinae) 486 was not used as the probes were designed prior to the publication of this genome (Béliveau et al., 2015). 487 We were able to capture matches to both IVSP4-1 and IVSP4-2 in another Hyposoter species with 488 considerable divergence from the other Hyposoter (I9995), suggesting these genes may be rapidly 489 evolving. We were also able to capture several copies of this gene family from Campoletis: two 490 individuals of Campoletis (I2339, I10007, Table 1) matched very closely to the original Campoletis 491 sonorensis sequence and Campoletis sp. 1 (I10007) exhibited two additional paralogs (copy 2, 3) that 492 were more closely related to the other Campoletis sp. 1 paralog than to paralogs of Hyposoter (Fig. 3A). 493 Casinaria sp. showed affinities to a copy in Campoletis (copy 3). This demonstrates that this ichnovirus 494 replication gene is part of a gene family that likely has lineage specific diversification. Hyposoter, 495 Campoletis sp. 1 and 2, and Casinaria sp. 1 are recovered in a maximally supported lineage and represent 496 just one lineage of Campopleginae (Fig. 1C). Interestingly, these genes were not captured in species of 497 Dusona and Campoplex, suggesting either these genera do not have ichnoviruses, or the ichnovirus genes 498 are highly divergent from other lineages within Campopleginae. No IVSP4 family members were 499 captured in Lissonota despite being present in the genome (Burke et al., 2020), but this may be due to not 500 having a banchine sequence in the original alignment used to design the probes. Although in Hyposoter 501 both IVSP4 copies were captured, it is interesting that not all copies were captured in each case, which 502 again could be a complication of this approach for capturing gene copies successfully or for indication of 503 potential gains and losses of ancestrally attained gene copies.

504 In the bracovirus replication genes, HzNVorf128 was represented by a Chelonus (Cheloninae) and 505 a Cotesia (Microgastrinae) species in the original probe design and was effectively captured and located 506 for all taxa in our sampling thought to include bracoviruses (Fig. 3B). The relationships inferred from 507 HzNVorf128 were congruent with the expected phylogenetic relationships (Fig. 1B), suggesting the 508 expected co-phylogeny of the replication machinery with the taxa they harbor (Fig. 3B). Also, there was 509 no indication of gene duplications in this gene, making it a good candidate to evaluate further in PDV 510 analysis. The per os infectivity factor p74, however, was captured/identified in the genomes of the two 511 closely related microgastroids to the probe sequences, with Schoenlandella (Cardiochilinae) not captured 512 and the gene not found in the Phanerotoma genomic sequence (Fig. 3C). It likely is evolving more 513 rapidly, thus eluding successful identification beyond close relatives. We included the vankyrin genes to 514 test if we could delineate members of a gene family using hybrid capture and because they are present in

515 each of the lineages that have PDVs (bracoviruses and ichnoviruses). Some individuals exhibited an 516 overabundance of vankyrins in the capture, e.g., 2% of the Australoglypta (Banchinae) sequence was 517 comprised solely of vankyrins, and other individuals had very low and likely non-specific capture. Given 518 the complexity of these results and establishing paralogy among divergent gene family members, we 519 excluded vankyrins from further analysis.

520

521 4. Discussion

522 4.1 Utility of Probe Set for Phylogenetics

523 The designed 541 probes set worked very well for capturing genes across Ichneumonoidea, with 524 an average of 478 genes captured per individual and 491 genes used in the final dataset analyzed here. 525 The probes were designed from alignments across taxa within the Holometabola and thus genes were 526 largely conserved. Further, the vast majority of genes captured only coding regions after trimming, 527 making them appropriate genes for examining macroevolutionary relationships, such as for 528 Ichneumonoidea, which has been estimated to have diverged in either the late Jurassic or early Triassic 529 (Peters et al., 2017; Zhang et al., 2015). These probes have also been successfully used to capture loci for 530 examining phylogenetic relationships and the evolution of eusociality in Vespidae (Piekarski et al., 2018), 531 indicating they have wide applicability across different hymenopteran taxa.

532 4.2 Evolutionary Relationships among Ichneumonoidea

533 Sampling across the superfamily was sparse relative to the total number of described species 534 within Ichneumonoidea (> 44,000, Yu et al., 2012), however, the inferred phylogenies were largely 535 stable. This was true across different analytical methods and datasets and when taxa with suspected long 536 branches or labile placement were excluded. Numerous maximally supported higher-level relationships 537 were inferred using the 491 gene dataset, providing a robust higher-level evolutionary picture for 538 Ichneumonoidea.

539 Although the taxon sampling for Braconidae was very low, the core higher-level relationships 540 recovered from traditional molecular phylogenetic approaches with a handful of genes (Sharanowski et 541 al., 2011) are largely recapitulated using a phylogenomics approach with denser genetic sampling. 542 Interestingly, regions of the Braconidae phylogeny with low support were similar to regions of low 543 support from Sharanowski et al. (2011), a symptom typical of rapid radiations (Giarla and Esselstyn, 544 2015; Xi et al., 2012), including in Braconidae (Banks and Whitfield, 2006; Zaldivar-Riverón et al.,

545 2008). Thus, a combined dense taxonomic and genetic sampling approach will be necessary to fully 546 understand the phylogenetic history of this extremely diverse family of parasitoid wasps.

547 The relationships of Ichneumonidae were also largely stable and highly supported with a few 548 exceptions. All analyses and datasets recovered Xoridinae as the sister to all remaining Ichneumonids, an 549 important finding since some previous analyses have used Xoridinae to root the Ichneumonid phylogeny 550 (Quicke et al., 2000; Quicke et al., 2009) and its placement impacts the understanding of the evolution of 551 parasitism (Belshaw and Quicke, 2002; Gauld, 1988). Subfamilies with previously ambiguous placement 552 include Orthopelmatinae and Eucerotinae (Quicke et al., 2000; Quicke et al., 2009). In our analyses, 553 Orthopelmatinae was recovered as sister to Ichneumoniformes s.l. with low support in the amino acid 554 analysis. However, support for this relationship increased when different outgroups were used or when 555 specific taxa were individually excluded (e.g. Eucerotinae, Adelognathinae). This relationship was not 556 recovered in the nt-3out relationship because of the placement of Labeninae, but still suggested an affinity 557 with Ichneumoniformes s.l. Orthopelmatinae has been considered an aberrant group, sometimes placed in 558 its own Orthopelmatiformes group (Quicke et al., 2000; Quicke et al., 2009), of which the placement has 559 been unstable across numerous previous analyses, either as sister to Ophioniformes (Quicke et al., 2000; 560 Quicke et al., 2009), sister to Pimpliformes (Quicke et al., 2000, morphological analysis), as the second 561 most basal lineage next to Xoridinae (under parsimony, Bennett et al., 2019), or within the Ophioniformes 562 (Quicke et al., 2009). Whether or not Orthopelmatinae is sister to Ichneumoniformes s.l. will require 563 denser taxonomic sampling, specifically across more basal lineages.

564 Eucerotinae has also been variably treated due to its unique (within Ichneumonoidea) larval 565 biology and stalked eggs (see Bennett et al., 2019 for detailed review), but has more recently been 566 associated with taxa not sampled here, including Agriotypinae, Claseinae, Pedunculinae, Brachycyrtinae, 567 and/or Labeninae, along with Ichneumoniformes (Bennett et al., 2019; Gauld and Wahl, 2002; Quicke et 568 al., 2009). Here, Eucerotinae was consistently recovered as sister to Ichneumoniformes with maximum 569 support, consistent with recent studies with more extensive taxonomic sampling (Bennett et al., 2019; 570 Santos, 2017). Further our data supports the assertion of Bennett et al. (2019) that Eucerotinae should be 571 placed within the Ichneumoniformes (Bennett et al.’s Ichneumoniformes s.l.).

572 While monophyletic, the placement of Labeninae varied widely across analyses and taxon 573 exclusion schemes, and this impacted the support values of a handful of deeper nodes within 574 Ichneumonidae. When labile taxa were removed or different outgroups were used, support values tended 575 to increase across the backbone of the ichneumonid phylogeny. We recovered Labeninae as sister to the 576 rest of the Ichneumonidae, except Xoridinae, similar to some analyses in other previous studies

577 (Klopfstein et al., 2019; Quicke et al., 2009; Santos, 2017). The placement of Labeninae has varied 578 widely across these and other studies (Bennett et al., 2019; Quicke et al., 2000) and its true placement 579 represents an important future challenge for understanding ichneumonid evolution.

580 Quicke et al. (2009) separated Ophioniformes into the ‘higher Ophioniformes’ (Campopleginae, 581 Cremastinae, Anomaloninae, Ophioninae, Mesochorinae, Nesomesochorinae, and Tatogastrinae, the last 582 two not sampled here) and a ‘basal grade’ of other subfamilies (Tryphoninae, Banchinae, Tersilochinae, 583 Oxytorinae, Ctenopelmatinae, Metopiinae, Neorhacodinae, Sisyrostolinae, and Stilbopinae, the last three 584 not sampled here). Alternatively, Bennett et al. (2019) defined the ‘higher Ophioniformes’ as including 585 only Anomaloninae, Campopleginae, Cremastinae, Ophioninae, and Nesomesochorinae, but did not state 586 why. Perhaps the smaller set of taxa relates to support for this grouping across some of their analyses, or 587 maybe due to the somewhat contradictory statement of the groups’ membership in the discussion of 588 Quicke et al. (2009): “The probable monophyly of the “higher” ophioniformes (Anomaloninae, 589 Campopleginae, Cremastinae, Nesomesochorinae, Ophioninae, but not Tersilochinae nor Tatogastrinae) 590 implies a very large ichneumonid radiation associated with koinobiont endoparasitism of Lepidoptera 591 larvae (with some secondary host shifts within these groups). The hosts of Nesomesochorinae are 592 completely unknown and the Hybrizontinae may or may not be associated with this clade (p. 1355)”. 593 Further, in some of their analyses and other studies (Belshaw and Quicke, 2002; Quicke et al., 2000) 594 Metopiinae is recovered with several of these subfamilies. Here we recovered a maximally supported 595 group including Metopiinae, Hybrizontinae, Cremastinae, Anomaloninae, Ophioninae, and 596 Campopleginae. The latter six subfamilies were also maximally supported across all analyses.

597 The placement of Hybrizontinae has varied widely across different analyses, including with 598 affiliation to Ctenopelmatinae (Bennett et al., 2019), Lycorininae, Anomaloninae, and Ophioninae 599 (Quicke et al., 2009) among others (see Bennett et al., 2019; Quicke, 2015 for a detailed review). Here, 600 Hybrizontinae is consistently (albeit variably supported) as sister to Cremastinae and falls firmly within 601 the ‘higher Ophioniformes’, contrary to (Bennett et al., 2019). Ophioninae was consistently recovered as 602 the sister group to Campopleginae with maximal support across all analyses, a relationship espoused by 603 Gauld (1985) and also recovered by Klopfstein et al. (2019). This finding is contrary to Bennett et al. 604 (2019) and Wahl (1991) who recovered Cremastinae as sister to Campopleginae. Although the study of 605 Klopfstein et al. (2019) was focused on Pimpliformes, they recovered very similar relationships among 606 the Ophioniformes using an independent probe set.

607 There were some differences between the two datasets (amino acid and nt-3out), particularly for 608 Ichneumonidae. However, several of these differences could be explained by further analysis of the data.

609 For example, the relationships among Pimpliformes were completely different between the protein and 610 nucleotide datasets, as was found in a recent phylogenomics study of this lineage (Klopfstein et al., 2019). 611 Examination of codon biases showed that there were distinct differences across several lineages. 612 Diplazontinae has a codon use pattern very similar to Ichneumoniformes, and thus, this lineage may have 613 been pulled to the base of the Pimpliformes in the nt-3out analyses due to convergence. Similarly, 614 Pimplinae was only recovered as monophyletic with the amino acid analysis due to distinct codon use 615 patterns between the two tribes sampled here: Ephialtini and Pimplini (Delomeristini and Theroniini were 616 not sampled). Pimplinae has been recovered as monophyletic with morphological data (Gauld et al., 2002; 617 Wahl and Gauld, 1998), but paraphyletic with molecular data (Bennett et al., 2019; Klopfstein et al., 618 2019; Quicke et al., 2009). Interestingly, our amino acid results of Pimpliformes are most consistent with 619 the relationships proposed by Wahl and Gauld (1998), but as mentioned by Klopfstein et al. (2019), the 620 evolution of this lineage is likely the result of a rapid radiation which may require extensive genetic and 621 taxonomic sampling to resolve.

622 Although nucleotide bias has been observed before between Braconidae and Ichneumonidae in 623 select genes, mostly mitochondrial (Li et al., 2016; Quicke et al., 2020b; Sharanowski et al., 2010), here 624 we note distinct codon use patterns across hundreds of genes. Further, the outgroups used here were most 625 similar to Braconidae, suggesting that careful examination of codon biases, outgroup choice, and 626 performing differential outgroup analyses are important for phylogenetic inference of Ichneumonoidea. 627 Thus, it may not be appropriate to root braconid or ichneumonid phylogenies with its sister family. We 628 also suggest that amino acid analyses are more useful for examining relationships among 629 Ichneumonoidea, and particularly Ichneumonidae because of codon use patterns.

630 4.3 Evolution of Modes of Parasitism

631 Although the taxon sampling is low in this study, we did recover interesting patterns when 632 inferring the ancestral states for major lineages in Ichneumonoidea. In general, it appears that the ancestor 633 of Ichneumonoidea was an idiobiont ectoparasitoid, although the result was only occasionally significant 634 for idiobiosis and never for ectoparasitism. Within Braconidae, the results varied depending on the data 635 used or taxa included in the analyses. The ancestor of Braconidae was inferred as either an idiobiont 636 ectoparasitoid or a koinobiont endoparasitoid. However, only the latter inferred biology was significant, a 637 result that is consistent with Sharanowski (2009), which had denser taxonomic sampling for Braconidae. 638 Koinobiont endoparasitism is the most common biology within Braconidae (Gauld, 1988), as this biology 639 is common to several subfamilies within the cyclostomes s.l. and all of the non-cyclostomes. However, 640 the non-cyclostomes were always recovered with a koinobiont endoparasitoid ancestor, whereas the

641 cyclostomes s.s had the most support for an idiobiont ectoparasitoid ancestor, although the result varied 642 across analyses. If the ancestor of Braconidae was a koinobiont endoparasitoid, then there may have been 643 one or more transitions to idiobiosis and ectoparasitism within the cyclostomes s.s.

644 Within Ichneumonidae, the results for idiobiosis/koinobiosis were consistent across all analyses. 645 Ichneumonidae, Ichneumoniformes s.l., and Pimpliformes were all significantly reconstructed as 646 idiobionts, whereas the ancestors for Ophioniformes, with or without Tryphoninae, were significantly 647 reconstructed as koinobionts. As all Ophioniformes are koinobionts, this makes intuitive sense and 648 demonstrates that koinobiosis developed early in ichneumonid evolution. For the ecto- versus 649 endoparasitism, there was a great deal of inconsistency across the analyses. The only consistent results 650 were for ancestors of Pimpliformes and Ophioniformes (excluding Tryphoninae), which were always 651 reconstructed as endoparasitoids with varying statistical support. This suggests that Pimpliformes had an 652 idiobiont endoparasitoid ancestor. Gauld (1988) theorized that idiobiont parasitoids transitioned from 653 ectoparasitism to endoparasitism by attacking cocooned hosts, allowing for enough time to develop before 654 the host metamorphoses into an adult. However, previous analyses (Klopfstein et al., 2019) have been 655 equivocal with respect to the ancestral state of idiobiosis or koinobiosis for Pimpliformes, and the 656 diversity of biological strategies in this group may make it one of the most interesting lineages to examine 657 to understand the evolution of parasitism. The ancestor of all Ophioniformes may have had a koinobiont 658 ecto- or endoparasitoid ancestor, as results varied depending on analysis. As Tryphoninae are koinobiont 659 ectoparasitoids, this may have been a transitory step from idiobiosis to koinobiont endoparasitism, as 660 suggested by (Gauld, 1988), or it could represent a reversal in this lineage to ectoparasitism (Quicke, 661 2015).

662 The results presented here suggests that strong conclusions cannot be drawn from these data. 663 Teasing apart the full nature of how different parasitoid strategies evolved will require a very detailed 664 look at host taxa and host biology and the stage attacked (Quicke et al., 1999a; Quicke, 2015), which is 665 sorely lacking for most Ichneumonoidea. Although the sampling is likely too low to draw strong 666 conclusions about the number of transitions across Braconidae and Ichneumonidae, there certainly have 667 been transitions from koinobiosis back to idiobiosis and from ecto– to endoparasitism in both families. 668 This was advocated as well by Bennett et al. (2019) who also suggested mechanisms for transition from 669 koinobiosis back to idiobiosis, challenging the prevailing notion that this transition was rare or non- 670 existent (Gauld, 1988; Quicke, 2015; Quicke et al., 2000).

671 4.4 Evolution of Endogenous Virus Elements in Wasp Genomes

672 Endogenous virus elements that produce virions (PDVs) or VLPs have been described in both 673 braconids and ichneumonids. Until recently, Braconidae was thought to have a single origin of 674 endogenous viruses in the microgastroid lineage (Beckage et al., 1994; Bézier et al., 2009; Huguet et al., 675 2012; Strand and Burke, 2015; Whitfield, 1997, 2002). A new endogenous virus descended from 676 pathogenic alphanudiviruses was discovered in some species of Fopius, opiine braconids, representing a 677 second integration event and a relatively recent one (Burke, 2019; Burke et al., 2018a). In Ichneumonidae, 678 PDVs with separate evolutionary origins from braconid PDVs have been discovered in members of 679 Banchinae and Campopleginae (Espagne et al., 2004; Lapointe et al., 2007; Norton et al., 1975; Robin et 680 al., 2019; Stoltz et al., 1981; Stoltz and Whitfield, 1992; Tanaka et al., 2007; Volkoff et al., 2010). 681 Further, at least one species of campoplegine has acquired a VLP related to nudiviruses (Pichon et al., 682 2015; Reineke et al., 2006; Rotheram, 1973). Given the robust phylogeny here, it is clear that banchines 683 and campoplegines are not closely related, and the intervening taxa have not been verified to possess 684 PDVs (but see Cummins et al., 2011). Although the ichnoviruses in Campopleginae and Banchinae come 685 from closely related virus ancestors (Béliveau et al., 2015), our data here provide strong evidence that the 686 Campopleginae and Banchinae are not sister subfamilies within the Ichneumonidae. Two possibilities 687 exist: first, that there have been two independent origins of ichnoviruses in the Ichneumonidae, or second, 688 that there was a single origin and ichnoviruses were lost in some other subfamilies between the 689 ichnovirus-producing families (Beliveau et al. 2015). In a companion study (Burke et al., 2020), a more 690 extensive search for PDVs in intervening taxa using a genome sequencing approach revealed that there 691 are no ichnovirus genes in several intervening taxa, consistent with the failure to capture ichnovirus genes 692 within these taxa using the anchored hybrid enrichment approach used in this study.

693 PDV genes are known to evolve very rapidly, presenting challenges for finding these genes in the 694 genome with targeted approaches such as PCR. Targeted enrichment approaches hold some promise for 695 sampling across a broader array of PDV genes specific to certain taxa to determine whether they may be 696 present in previously untested species and how these genes have evolved. We have found ichnovirus 697 genes in the Hyposoter and Campoletis sampled here, but they are not identified to species. However, 698 Campoletis sp. 1 was collected in Australia, where C. sonorensis is not known to occur, and thus 699 represents at least one new species with an ichnovirus in this genus. Further, we provide the first evidence 700 of PDVs in the genus Casinaria. The gene used, IVSP4, appears to undergo within lineage gene 701 expansions or contractions, which the AHE pipelines were able to detect, but will require more data from 702 more taxa to better understand their evolution. These genes were not captured in species of Dusona and 703 Campoplex, which were recovered as sister taxa, which suggests these taxa either do not have 704 ichnoviruses or they have very divergent copies. Without richer taxonomic sampling their sister

705 relationship cannot be certain. In another recent study, Dusona was recovered as sister to Casinaria 706 (Bennett et al., 2019), and a study with deeper sampling recovered conflicting placement and non- 707 monophyly for several campoplegine genera (Quicke et al., 2009).

708 For the bracovirus replication gene HzNVorf128 we were able to fully capture the gene from all 709 expected bracovirus-harboring taxa and reconstruct a pattern of evolution that is consistent with species 710 relationships determined using other non-viral molecular markers. Thus, capture in Phanerotoma and 711 Schoenlandella represent confirmations in new taxa, but these were expected given that microgastroids 712 are all assumed to have bracoviruses (Whitfield, 1997, 2002). However, the other bracovirus gene (per os 713 infectivity factor p74) was not captured in full across likely bracovirus harboring taxa, most likely due to a 714 more rapid evolutionary rate. In general, these genes appear to be evolving rapidly, thus limiting the 715 ability to capture some viral genes using probes from known sequences. It is conceivable that more 716 targeted approaches that iteratively design probes focused upon single-copy genes with high sequence 717 conservation may be more effective for cross-lineage capture of divergent viral genes.

718 5. Conclusions

719 Here we developed a 541 gene probe set for anchored hybrid enrichment that was largely 720 successful for examining relationships in Ichneumonoidea and with wide utility across Hymenoptera. 721 Although we also targeted polydnavirus genes (PDVs), capture was not entirely successful across taxa 722 known to have viruses endogenized in their genomes. It is possible that a more lineage-specific targeted 723 approach to probe design could be more successful, but genome sequencing may be a more appropriate 724 method for discovering virus association and endogenization given the continually decreasing cost. 725 Higher-level relationships across Ichneumonoidea are mostly stable and well supported relationships and 726 reinforce various findings from other studies. While we focused on Ichneumonidae, the braconid 727 relationships were largely consistent with previous studies. Within Ichneumonidae, Xoridinae was the 728 most basal taxon, and the placements of Labeninae and Orthopelmatinae remain uncertain. Excluding 729 these three taxa, we recovered Ophioniformes (Ichneumoniformes (including Eucerotinae) + 730 Pimpliformes). Higher-level relationships among Ichneumonoidea have been historically difficult to 731 resolve, but anchored hybrid enrichment offers a valuable tool to recovering robust relationships in this 732 highly diverse lineage. However, our data demonstrate that systematic biases need to be thoroughly 733 investigated to prevent erroneous relationships and outgroup selection must be vetted. We found codon 734 use bias present in some lineages, suggesting amino acid data may be better suited to understanding 735 evolution in Ichneumonoidea.

736 Ancestral state reconstructions were sensitive to the taxa included or data used to infer the 737 character states. The data suggest that the ancestor of Ichneumonoidea was an idiobiont ectoparasitoid. 738 While analyses for Ichneumonidae were stable for idiobiosis/koinobiosis, they varied for ecto-versus 739 endoparasitism. Braconidae may have had a koinobiont endoparasitoid ancestor, although reconstructions 740 varied widely for the family preventing strong conclusions. The results suggest many transitions of these 741 traits within Ichneumonoidea and full understanding of the evolution of parasitoid strategies will require 742 deep taxonomic sampling and increased knowledge on the biology of this diverse lineage. At least two 743 independent acquisitions of endogenous polydnaviruses (PDVs) within Ichneumonidae are well supported 744 by the data due to the distant relationship between Banchinae and Campopleginae and lack of evidence of 745 PDVs in intervening taxa. However, probes designed to capture polydnavirus genes likely require a more 746 targeted lineage-specific approach.

747 Acknowledgements:

748 We are deeply thankful for specimens from Michael Sharkey, David Smith, and Dave Karlsson with the 749 Swedish Malaise Trap Project (SMTP). We thank Geoff Allen (University of Tasmania), Andrew Austin 750 (University of Adelaide), James Adams (La Ceiba, Honduras), and Bery Josue Almendares Romero and 751 entomological researchers at the Universidad Nacional Autónoma de Honduras for hosting during 752 collecting trips and assisting with permits. Our grateful thanks to Davide Dal Pos and Andrés Herrera- 753 Flórez for some specimen identifications and to Davide Dal Pos for valuable comments on manuscript 754 drafts. A special thank-you to Andrew Bennett for many discussions about relationships across the 755 Ichneumonidae. We thank Terry Wheeler (deceased) and Chris Buddle for organizing a collecting trip to 756 Yukon through the Northern Biodiversity Program (Canada). We would like to thank John Heraty for 757 providing alignments for the probe design, which increased their utility across Hymenoptera. We are 758 grateful to Michelle Kortyna and Alyssa Bigelow at FSU’s Center for Anchored Phylogenomics for 759 assistance with sample processing. Funding support for this project was provided by the Natural Sciences 760 and Engineering Research Council (NSERC) Discovery Program to BJS and the National Science 761 Foundation (NSF) (Award Number: 1916914) to BJS and GRB.

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1168 Zaldivar-Riverón, A., Shaw, M.R., Sáez, A.G., Mori, M., Belokobylskij, S.A., Shaw, S.R., Quicke, 1169 D.L.J., 2008. Evolution of the parasitic wasp subfamily Rogadinae (Braconidae): phylogeny and 1170 evolution of lepidopteran host ranges and mummy characteristics. BMC Evol. Biol. 8, 329. 1171 Zhang, C., Stadler, T., Klopfstein, S., Heath, T.A., Ronquist, F., 2015. Total-evidence dating under the 1172 fossilized birth–death process. Syst. Biol. 65, 228-249. 1173 Zhang, M.Y., Ridenbaugh, R.D., Sharanowski, B.J., 2017. Integrative taxonomy improves understanding 1174 of native beneficial fauna: revision of the Nearctic Peristenus pallipes complex (Hymenoptera: 1175 Braconidae) and implications for release of exotic biocontrol agents. Syst. Entomol. 42, 596-608.

1176

1177

1178

1179

1180

1181

1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

1195 Table 1. Taxon sampling used in this study, including voucher codes (where appropriate), 1196 taxonomy, and country of origin. Genome indicates publicly available genomes used in the study. 1197 Bolded taxa are genomes sequenced in this study.

Outgroups Voucher Code FSU Family Subfamily Genus species Country Code N/A I15712 Pteromalidae Pteromalinae Nasonia vitripennis N/A N/A I15697 Apidae Apinae Apis mellifera N/A N/A I15699 Apidae Apinae Bombus impatiens N/A N/A I15707 Megachilidae Megachilinae Megachile rotunda N/A N/A I15693 Formicidae Myrmicinae Acromyrmex echinatior N/A N/A I15698 Formicidae Myrmicinae Atta cephalotes N/A N/A I15700 Formicidae Formicinae Camponotus floridanus N/A N/A I15705 Formicidae Ponerinae Harpegnathos saltator N/A N/A I15706 Formicidae Dolichoderinae Linepithema humile N/A N/A I15715 Formicidae Myrmicinae Pogonomyrmex barbatus N/A N/A I15716 Formicidae Myrmicinae Solenopsis invicta N/A Braconidae Voucher Code FSU Higher-Group Subfamily Genus species Country Code PSUCFEM10030041 I2347 Aphidioid Aphidiinae Pauesia sp. Canada: Manitoba PSUCFEM10030046 I2352 Aphidioid Aphidiinae Praon sp. Canada: Manitoba PSUCFEM10030015 I2325 Cyclostomes s.s. Rhyssalinae Rhyssalus sp. USA: California PSUCFEM10030003 I15695 Cyclostomes s.s. Rogadinae Aleiodes sp. Canada: Manitoba PSUCFEM10030006 I2358 Cyclostomes s.s. Pambolinae Pambolus sp. USA: Arizona PSUCFEM10030050 I2355 Cyclostomes s.s. Braconinae Bracon sp. Canada: Manitoba PSUCFEM10030007 I2359 Cyclostomes s.s. Alysiinae Chorebus sp. Canada: Manitoba Genome I15701 Cyclostomes s.s. Opiinae Diachasma alloeum Genome PSUCFEM10030043 I2349 Microgastroid Cardiochilinae Schoenlandella sp. Thailand: Loei Genome I15711 Microgastroid Microgastrinae Micropilitus demoliter Genome PSUCFEM10030045 I2351 Microgastroid Cheloninae Chelonus sp. Canada: Manitoba PSUCFEM10030009 I15714 Microgastroid Cheloninae Phanerotoma sp. Costa Rica: Guanacaste PSUCFEM10030035 I2343 Microgastroid Ichneutinae Ichneutes sp. Canada: Yukon PSUFEM10030037 I2344 Sigalphoid Agathidinae Bassus sp. USA: Arizona PSUCFEM10030008 I15710 Euphoroid Euphorinae Meterous sp. Costa Rica: Guanacaste PSUCFEM10030032 I2342 Helconoid Orgilinae Orgilus ablusus* Canada: Manitoba PSUCFEM10030004 I15704 Helconoid Helconinae Eumacrocentrus americanus USA: West Virginia PSUCFEM10030049 I2354 Helconoid Acampsohelconinae Urosigalphus sp. USA: Kansas PSUCFEM10030039 I2346 Helconoid Brachistinae Apoblacus sp. Chile: X Region PSUCFEM10030038 I2345 Helconoid Brachistinae Blacus sp. French Guiana: Patawa PSUCFEM10030048 I2353 Helconoid Brachistinae Diospilus sp. USA: West Virginia Ichneumonidae PSUCFEM10030002 I15713 Xoridiformes Xoridinae Odontocolon sp. Canada: Manitoba PSUCFEM10030023 I9984 Orthopelmatiformes Orthopelmatinae Orthopelma sp. Canada: Manitoba PSUCFEM000070305 I9986 Unknown Eucerotinae Euceros sp. Sweden: Uppland PSUCFEM000070330 I10002 Labeniformes Labeninae Labium sp. Australia: Tasmania PSUCFEM000070316 I9990 Labeniformes Labeninae Certonotus nitidulus Australia: Tasmania PSUCFEM000070320 I9994 Labeniformes Labeninae Certonotus nitidulus Australia: Tasmania PSUCFEM10030001 I15694 Ichneumoniformes Adelognathinae Adelognathus sp. Canada: Manitoba PSUCFEM10030018 I2328 Ichneumoniformes Cryptinae Mesostenus thoracicus Canada: Manitoba PSUCFEM10030019 I2329 Ichneumoniformes Cryptinae Echthrus abdominalis Canada: Manitoba PSUCFEM000070324 I9997 Ichneumoniformes Ichneumoninae Gavrana sp. Australia: Queensland PSUCFEM000070340 I10010 Ichneumoniformes Ichneumoninae Ichneumonini (genus ?) Australia: Tasmania PSUCFEM000070315 I9989 Ichneumoniformes Ichneumoninae Akymichneumon sp. Australia: Tasmania PSUCFEM000070327 I9999 Ichneumoniformes Ichneumoninae Gavrana maculipes s.g. Australia: Tasmania

Ichneumonidae Voucher Code FSU Higher Group Subfamily Genus species Country Code PSUCFEM000070326 I9998 Pimpliformes Pimplinae Echthromorpha intricatoria Australia: Tasmania PSUCFEM10030020 I2330 Pimpliformes Pimplinae Pimpla pedalis Canada: Manitoba PSUCFEM000070323 I9996 Pimpliformes Pimplinae Pimpla molesta Honduras: FM PSUCFEM10030005 I15702 Pimpliformes Pimplinae Dolichomitus sp. Canada: Manitoba PSUCFEM10030052 I2356 Pimpliformes Pimplinae Scambus sp. Canada: Manitoba PSUCFEM000070333 I10004 Pimpliformes Pimplinae Eriostethus pulcherrimus Australia: Tasmania PSUCFEM10030023 I2333 Pimpliformes Orthocentrinae Orthocentrus sp. Canada: Manitoba PSUCFEM10030021 I2331 Pimpliformes Poemeniinae Neoxorides borealis Canada: Manitoba PSUCFEM10030016 I2326 Pimpliformes Acaenitinae Spilopteron occiputale Canada: Manitoba PSUCFEM000070310 I9988 Pimpliformes Cylloceriinae Cylloceria sp. Sweden: Halland PSUCFEM000070308 I9987 Pimpliformes Collyriinae Collyria sp. Sweden: Oland PSUCFEM10030022 I2332 Pimpliformes Diplazontinae Homotropus sp. Canada: Manitoba PSUCFEM10030014 I2324 Ophioniformes Tryphoninae Exenterus sp. Canada: Manitoba PSUCFEM10030025 I2335 Ophioniformes Tryphoninae Netelia (Netelia) sp. Canada: Manitoba PSUCFEM000070300 I9983 Ophioniformes Tryphoninae Phytodietus sp. Sweden: Uppland PSUCFEM10030031 I2341 Ophioniformes Tersilochinae Phradis? USA: Florida PSUCFEM000070318 I9992 Ophioniformes Tersilochinae Stethantyx sp. Honduras: FM PSUCFEM10030028 I2338 Ophioniformes Banchinae Glypta sp. Canada: Manitoba PSUCFEM000070337 I10008 Ophioniformes Banchinae Australoglypta sp. Australia: Tasmania PSUCFEM000070329 I10001 Ophioniformes Banchinae Meniscomorpha sp.1 Honduras: FM PSUCFEM10030012 I15708 Ophioniformes Banchinae Lissonota sp. Canada: Manitoba PSUCFEM000070332 I10003 Ophioniformes Banchinae Meniscomorpha sp.2 Honduras: FM PSUCFEM000070348 I10013 Ophioniformes Banchinae Lissonota sp.* Australia: Tasmania PSUCFEM000070303 I9985 Ophioniformes Oxytorinae Oxytorus sp. Sweden: Östergötland PSUCFEM000070328 I10000 Ophioniformes Ctenopelmatinae Megaceria sp. Australia: Tasmania PSUCFEM10030000 I15696 Ophioniformes Ctenopelmatinae Anoncus sp. Canada: Manitoba PSUCFEM10030030 I2340 Ophioniformes Ctenopelmatinae Mesoleius sp.* Canada: Manitoba PSUCFEM10030011 I15709 Ophioniformes Mesochorinae Mesochorus sp. Canada: Manitoba PSUCFEM10030024 I2334 Ophioniformes Metopiinae Exochus sp. Canada: Manitoba PSUCFEM10030053 I2357 Ophioniformes Metopiinae Spudaeus sp. Canada: Manitoba PSUCFEM000070319 I9993 Ophioniformes Metopiinae Metopius sp. Australia: Tasmania PSUCFEM10030017 I2327 Ophioniformes Ophioninae Ophion sp. Canada: Manitoba PSUCFEM000070291 I9982 Ophioniformes Hybrizontinae Hybrizon buccatus Sweden: Gotland PSUCFEM10030026 I2336 Ophioniformes Cremastinae Cremastus sp. Canada: Manitoba PSUCFEM000070281 I9981 Ophioniformes Cremastinae Pristomerus sp.* Australia: Tasmania PSUCFEM10030027 I2337 Ophioniformes Anomaloninae Agrypon sp. Canada: Manitoba PSUCFEM000070338 I10009 Ophioniformes Anomaloninae Habronyx sp.* Australia: Tasmania PSUCFEM10030013 I15703 Ophioniformes Campopleginae Dusona sp.2 Canada: Manitoba PSUCFEM10030029 I2339 Ophioniformes Campopleginae Campoletis sp.2 Canada: Manitoba PSUCFEM000070317 I9991 Ophioniformes Campopleginae Campoplex sp.2 Australia: Tasmania PSUCFEM000070322 I9995 Ophioniformes Campopleginae Hyposoter sp.* Australia: Tasmania PSUCFEM000070334 I10005 Ophioniformes Campopleginae Casinaria sp.1 Australia: Tasmania PSUCFEM000070335 I10006 Ophioniformes Campopleginae Campoplex sp.1* Australia: Tasmania PSUCFEM000070341 I10011 Ophioniformes Campopleginae Dusona sp.3* Australia: Tasmania PSUCFEM000070336 I10007 Ophioniformes Campopleginae Campoletis sp.1 Australia: Tasmania PSUCFEM000070347 I10012 Ophioniformes Campopleginae Dusona sp.1 Australia: Tasmania 1198

1199

1200

1201 Table 2. Model tests for maximum likelihood ancestral state reconstructions for character 1 1202 (idiobiosis vs. koinobiosis) and character 2 (ecto- vs. endoparasitism). Two models were tested, the 1203 Markov k-state one parameter (Mk1), which has an equal rate for character gains and losses, and the 1204 asymmetrical two-parameter Markov k-state (Assym2), which applies a separate rate to gains and losses. 1205 Likelihood ratio tests were performed to assess which model better fit the data, which are chi-square (χ2) 1206 distributed and critical values was used to assess significance at p<0.01. The best supported model for 1207 each source phylogeny is highlighted in gray. Source phylogenies include the amino acid maximum 1208 likelihood phylogeny (AA w/ OGs) with all taxa (Fig 1. A-C); and with outgroups (OGs) trimmed prior to 1209 analysis (AA w/o OGs); the amino acid phylogeny with Labeninae excluded (AA w/ OGs Lab-out, Fig. 1210 S10); and with outgroups trimmed (AA w/o OGs Lab-out); the nucleotide dataset with the third position 1211 removed (nt-3out, Fig. S4). The other three source phylogenies are based on the amino acid dataset (Fig. 1212 1A-C), either with (AA Bracs only; AA Ichs only) or without Labeninae excluded (Fig. S10; AA Ichs 1213 only Lab-out), and all taxa were trimmed except the family of interest: Braconidae (Bracs) or 1214 Ichneumonidae (Ichs).

Log Likelihood

Source Phylogeny Character Mk1 Assym2 χ2 P-value AA w/ OGs 1 -34.216 -27.230 13.973 0.00019 2 -33.670 -29.275 8.790 0.003 AA w/o OGs 1 -33.267 -25.052 16.430 0.00005 2 -33.180 -29.142 8.076 0.0045 AA w/ OGs Lab-out 1 -31.663 -25.924 11.478 0.0007 2 -30.970 -27.608 6.724 0.0095 AA w/o OGs Lab-out 1 -30.556 -23.539 14.033 0.00018 2 -30.585 -27.501 6.169 0.013 nt-3out 1 -35.345 -27.692 15.307 0.000092 2 -30.625 -33.386 -5.522 0.019 AA Bracs only 1 -10.262 -8.207 4.110 0.043 2 -10.262 -8.207 4.110 0.043 AA Ichs only 1 -22.721 -17.510 10.423 0.0012 2 -22.571 -21.315 2.511 0.119 AA Ichs only Lab-out 1 -20.590 -15.865 9.450 0.0021 2 -20.594 -19.594 1.999 0.16 1215

1216

1217

1218

1219

1220 Table 3. Summary of ancestral state reconstructions across several source phylogenies for 1221 character 1 (idiobiosis vs. koinobiosis) and character 2 (ecto- vs. endoparasitism). Color coding and 1222 the proportional likelihoods (PLH) are listed for the most likely character state. Source phylogenies are 1223 detailed in Table 2.

1224

AA Ichs AA AA only AA (Lab- (no OGs, AA Bracs AA Ichs (Lab- AA (no OGs) out) Lab-out) nt-3out only only out) Node PLH PLH PLH PLH PLH PLH PLH PLH Ichneumonoidea 0.941 0.877 0.899 0.848 0.928 N/A N/A N/A Braconidae 0.871 0.832 0.828 0.807 0.826 0.979 N/A N/A Cyclostomes s.l. 0.892 0.864 0.857 0.845 0.849 0.968 N/A N/A Cyclostomes s.s. 0.986 0.986 0.976 0.98 0.974 0.762 N/A N/A Non-cyclostomes 0.815 0.784 0.777 0.748 0.836 0.999 N/A N/A Ichneumonidae 0.999 0.999 0.989 0.999 0.996 N/A 0.96 0.994 Ichneumoniformes s.l. 0.997 0.998 0.991 0.994 0.998 N/A 0.983 0.996 Pimpliformes 0.98 0.986 0.974 0.983 0.997 N/A 0.971 0.981 Ophioniformes 0.956 0.945 0.939 0.925 0.96 N/A 0.912 0.943 Ophioniformes (no Try) 0.986 0.98 0.978 0.97 0.99 N/A 0.96 0.979 Ichneumonoidea 0.788 0.794 0.724 0.987 0.861 N/A N/A N/A Braconidae 0.714 0.715 0.66 0.995 0.96 0.979 N/A N/A Cyclostomes s.l. 0.763 0.762 0.717 0.979 0.94 0.968 N/A N/A Cyclostomes s.s. 0.945 0.943 0.918 0.768 0.732 0.762 N/A N/A Non-cyclostomes 0.805 0.81 0.772 0.999 0.999 0.999 N/A N/A Ichneumonidae 0.994 0.994 0.959 0.97 0.84 N/A 0.807 0.637 Ichneumoniformes s.l. 0.965 0.961 0.932 0.998 0.958 N/A 0.989 0.942 Pimpliformes 0.745 0.762 0.664 0.999 0.999 N/A 0.999 0.998 Ophioniformes 0.97 0.968 0.945 0.958 0.884 N/A 0.906 0.706 Ophioniformes (no Try) 0.647 0.655 0.598 0.998 0.995 N/A 0.994 0.979 1225

1226

1227

1228

1229

1230

1231 Figure Legends:

1232 Figure 1, A-C. Maximum-likelihood (ML) phylogeny of Ichneumonoidea based on 479 genes 1233 analyzed as amino acids in IQ-tree. A) Summary tree with Braconidae and Ichneumonidae collapsed 1234 for viewing; B) Braconidae; C) Ichneumonidae. Branch supports are summarized in a 5 cell box, with the 1235 first two cells representing the amino acid analysis, the second two representing the ML phylogeny 1236 inferred from nucleotide data with the 3rd position removed (nt-3out, see also Fig. S4), and the last box 1237 representing the Bayesian analysis of the nt-3out dataset (see also Fig. S5). For the ML analyses, two 1238 supports are given in the first and second boxes, respectively: the Shimodaira-Hasegawa approximate 1239 Likelihood Ratio Test statistic (aLRT) and the Ultra-Fast bootstrap value (UFb). The Bayesian posterior 1240 probability is reported in the last cell (PP). See branch support legend for color scheme of values for 1241 nodal supports. Taxa with an asterisk after the name have an uncertain identification at the lowest 1242 taxonomic rank indicated. The scale bar indicates the number of substitutions per site for branch lengths.

1243 Figure 2, A-B. Summary of two ancestral state reconstructions (ASR) using maximum likelihood 1244 for A) Idiobiosis versus koinobiosis and B) Ecto- versus endoparasitism. The collapsed tree visualized 1245 here is based on the maximum likelihood phylogeny of amino acid data with Labeninae removed (Fig. 1246 S10). Pie charts show the proportional likelihood (PLH) for each character state and an asterisk near the 1247 chart indicates the reconstruction as significant for the state with the highest PLH (see also Table 3). Pie 1248 charts above the nodes have all taxa included, whereas pie charts below the nodes were ASRs run with all 1249 taxa removed except the family of interest for both Braconidae and Ichneumonidae.

1250 Figure 3, A-C. Bayesian phylogenies of three polydnavirus (PDV) genes: A) IVSP4, B) 1251 HzNVorf128, and C) per os infectivity factor p74. Sequences were either captured with 1252 anchored hybrid enrichment probes (probe), obtained from newly sequenced genomes (genome- 1253 Illumina), or were used as original reference sequences for probe capture and are available on 1254 GenBank (GenBank ID provided). Taxon voucher codes are listed next to genus name for newly 1255 sequenced taxa (FSU code, Table 1).

1256

1257

1258

1259

1260

1261

A Aphidioid * Cyclostomes s.l. * Cyclostomes s.s. Braconidae * Non * Cyclostomes * Xoridinae Orthopelmatinae 67/53 * Ichneumoniformes s.l. * * Idiobiont * 98/86 * Koinobiont Pimpliformes *

Tryphoninae Ichneumonidae * * 0.02 * Ophioniformes Other * Ophioniformes

B Aphidioid * Cyclostomes s.l. * Cyclostomes s.s.

* Braconidae Non * Cyclostomes Xoridinae Orthopelmatinae 67/53 * Ichneumoniformes s.l. * * Ectoparasitism 98/86 Endoparasitism Pimpliformes *

Tryphoninae Ichneumonidae *

0.02 Ophioniformes Other * Ophioniformes bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.157719; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A. Summary Tree With Outgroups Pteromalidae_Nasonia_vitripennis Megachilidae_Megachile_rotunda Apidae_Apis_mellifera Bees Apidae_Bombus_impatiens Harpegnathos_saltator Linepithema_humile Camponotus_floridanus Formicidae Pogonomyrmex_barbatus Solenopsis_invicta (Ants) Acromyrmex_echinatior Atta_cephalotes Ichneumonoidea Branch support legend Braconidae nt-3out Protein ML B

LRT UFb LRT UFb PP

Branch support scale 0.02 99 - 100 95 - < 99 80 - < 95 Ichneumonidae 50 - < 80 0 - < 50 branch not recovered B. Braconidae Cyclostome s.l. Aphidiinae_Pauesia Aphidioid Aphidiinae_Praon Rhyssalinae_Rhyssalus Pambolinae_Pambolus Cyclostome s.s. Rogadinae_Aleiodes Braconinae_Bracon Opiinae_Diachasma_alloeum Alysiinae_Chorebus Alysioid Ichneutinae_Ichneutes Agathidinae_Bassus Microgastrinae_Microplitis_demolitor Cardiochilinae_Schoenlandella Cheloninae_Phanerotoma Cheloninae_Chelonus Euphorinae_Meteorus Microgastroid Acampsohelconinae_Urosigalphus Helconinae_Eumacrocentrus_americanus Orgilinae_Orgilus_ablusus Helconoid Brachistinae_Apoblacus Brachistinae_Blacus Non-cyclostomes Brachistinae_Diospilus bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.157719; this version posted June 18, 2020. The copyright holder for this preprint C. Ichneumonidae(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Xoridinae_Odontocolon Xoridiformes Labeninae_Labium Labeninae_Certonotus1 Labeniformes Labeninae_Certonotus2 Orthopelmatinae_Orthopelma Orthopelmatiformes Eucerotinae_Euceros Adelognathinae_Adelognathus Cryptinae_Mesostenus Cryptinae_Echthrus Ichneumoninae_Ichneumonini Ichneumoniformes Ichneumoninae_Gavrana1 Ichneumoninae_Akymichenumon Ichneumoninae_Gavrana Acaenitinae_Spilopteron Collyriinae_Collyria Cyllocerinae_Cylloceria Diplazontinae_Homotropus Orthocentrinae_Orthocentrus Pimpliformes Poemeniinae_Neoxorides Pimplinae_Dolichomitus Pimplinae_Eriostethus Ephialtini Pimplinae_Scambus Pimplinae_Ecthromorpha Pimplinae_Pimpla_p Pimplini Pimplinae_Pimpla_m Tryphoninae_Exentenus Tryphoninae_Netelia Tryphoninae_Phytodietus Banchinae_Lissonota Banchinae_Meniscomorpha1 Banchinae_Meniscomorpha2 Banchinae_Glypta Banchinae_Australglypta Banchinae_Lissonota1* Tersilochinae_Phradis* Tersilochinae_Stethantyx Mesochorinae_Mesochorus Ophioniformes Oxytorinae_Oxytorus Ctenopelmatinae_Megaceria Ctenopelmatinae_Anoncus Ctenopelmatinae_Mesoleius* Metopiinae_Exochus Metopiinae_Spudaeus Metopiinae_Metopius Hybrizontinae_Hybrizon Branch support legend Cremastinae_Cremastus nt-3out Cremastinae_Pristomerus Protein ML B Anomaloninae_Habronyx LRT UFb LRT UFb PP Anomaloninae_Agrypon Ophioninae_Ophion Branch support scale Campopleginae_Hyposoter* 99 - 100 Higher Campopleginae_Casinaria1 95 - < 99 Ophioniformes Campopleginae_Campoletis1 80 - < 95 Campopleginae_Campoletis2 50 - < 80 Campopleginae_Campoplex1* 0 - < 50 Campopleginae_Campoplex2 branch not recovered Campopleginae_Dusona2 Campopleginae_Dusona3* 0.02 Campopleginae_Dusona1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.157719; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A. IVSP4 Hyposoter sp.* I9995 Copy 1 (probe) 1 Hyposoter didymator IVSPER2 (ADI40475.1)

Hyposoter sp.* I9995 Copy 2 (probe) 1 Hyposoter didymator IVSPER3 (ADI40477.1)

Campoletis sp.1 I10007 Copy 3 (probe) 1 1 Casinaria sp.1 I10005 (probe) Campoletis sp.1 I10007 Copy 2 (probe) 0.99 Campoletis sp.2 I2339 (probe) 0.99 0.81 Campoletis sp.1 Copy 1 I10007 (probe) 1 Campoletis sonorensis (HO082184) 0.05

B. HzNVorf128 C. per os infectivity factor p74 Phanerotoma sp. I15714 (genome - Illumina) Schoenlandella sp. I2349 (probe) Chelonus sp. I2351 (probe)

1 Cotesia congregata (CAR82249.1) 1 Chelonus inanitus (CAR40192.1) 1 Microplitus demolitor (EZA45377.1) Microplitus demolitor (EZA44986.1) Chelonus sp. I2351 (probe) 1 Cotesia congregata (CAR31575.1) 1 Chelonus inanitus (CAR40202.1) 0.06 0.08