authors · pichon et al., 2015; strand and burke, 2012). given their immense diversity,...

1

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

(which was not certified by peer review) is the author/funder.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

https://doi.org/10.1101/2020.06.17.157719

2

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.


https://doi.org/10.1101/2020.06.17.157719

3

1. Introduction 1

Ichneumonoidea is one of 15 superfamilies of parasitoid Hymenoptera, wasps whose larvae feed on 2

other arthropods. As a result of an exceptional rate of species diversification, this lineage comprises 3

~48,000 valid species, 33% of all Hymenoptera (145,000 spp.; (Huber, 2009)) and 3% of all known life 4

(Chapman, 2009; Yu et al., 2016). The true diversity may be many times higher when undescribed species 5

are considered. For example, estimates of known and unknown diversity in a single polydnavirus-carrying 6

subfamily (Microgastrinae) within the group might exceed the total described species for the superfamily 7

(Rodriguez et al., 2013). Ichneumonoidea has been challenging for higher level systematics because of its 8

rapid radiation and relatively high levels of morphological convergence (Quicke, 2015; Sharkey, 2007). 9

Further, the sheer size of the lineage and the relative dearth of taxonomic experts in this group has 10

presented many challenges for large phylogenetic studies. 11

The diversity of Ichneumonoidea may be due to the varied methods the wasps use to attack and 12

kill their hosts as the evolution of new mechanisms to exploit hosts may have opened up new niches, 13

enabling adaptive radiations (Price, 1980). Wasp species may be external (ectoparasitic) or internal 14

(endoparasitic) parasitoids, although the latter often leave the host to pupate (Austin and Dowton, 2000). 15

Some ichneumonoids are idiobionts, arresting the development of their host upon oviposition; whereas 16

others are koinobionts, allowing the host to continue development, often allowing the host to molt to 17

other life stages (Askew and Shaw, 1986). Further, ichneumonoids attack their arthropod hosts in all life 18

stages, from eggs to adults, and hosts in different habitats, from exposed to concealed (Quicke, 2015). 19

Understanding core phylogenetic relationships in Ichneumonoidea is pertinent to research across 20

multiple fields. Ichneumonoid parasitoid wasps are beneficial insects, providing natural management of 21

pests in crops and forests, and are estimated to provide >$17 billion in these ecosystem services (Pimentel 22

et al., 1997). Many ichneumonoids have been released in biological control programs against major 23

invasive pests, such as the Emerald ash borer (Bauer et al., 2008; Kula et al., 2010) and the Mediterranean 24

fruit fly (Vargas et al., 2001). Several ichneumonoid wasps are important models for understanding 25

parasitoid physiology (Ballesteros et al., 2017; Webb et al., 2006), interactions with host immune 26

response (Lovallo et al., 2002; Schmidt et al., 2001; Strand, 2012), development of novel transgenic 27

control methods for insect pests (Beckage and Gelman, 2004; Webb et al., 2017), and the process and 28

evolution of viral endogenization to produce beneficial viruses (Bézier et al., 2009; Burke et al., 2018a; 29

Pichon et al., 2015; Strand and Burke, 2012). Given their immense diversity, Ichneumonoidea also 30

present an exceptional opportunity to understand the factors that promote diversification, a critically 31

important question as we face increasing species extinctions with continued habitat destruction and global 32

climate change. 33


https://doi.org/10.1101/2020.06.17.157719

4

34

1.1 Phylogenetic history of Ichneumonoidea 35

Ichneumonoidea is robustly monophyletic (Sharanowski et al., 2011; Sharkey et al., 2010) and 36

currently comprises the monophyletic families: Ichneumonidae, Braconidae and Trachypetidae (Quicke et 37

al., 2020b; Sharkey, 2007). Previously in Braconidae, Trachypetidae was recently elevated to family 38

status (Quicke et al., 2020b), however the data supporting this taxonomic status is not robust, and as we 39

do not sample this lineage here, we do not discuss it further. The classification of the other two families 40

has also been in flux, with Braconidae having anywhere from 17 to 50 subfamilies and Ichneumonidae 41

with 24 to 44 subfamilies (Belshaw and Quicke, 2002; Bennett et al., 2019; Quicke et al., 2020a). 42

Braconidae, represented by over 21,000 valid species (Yu et al., 2016), 41 currently recognized 43

subfamilies (Sharanowski et al., 2011), and approximately 109 tribes, are highly valued for their use in 44

biocontrol of pestiferous hosts (Austin and Dowton, 2000; Peixoto et al., 2018; Zhang et al., 2017). A 45

molecular phylogeny of Braconidae using 7 gene regions was completed by Sharanowski et al. (2011). 46

Hypotheses of deep level relationships were well supported, allowing for a revised classification, but 47

relationships within one of the three major lineages (cyclostomes) remain largely unresolved 48

(Sharanowski et al., 2011; Zaldivar-Riverón et al., 2006), and will require greater taxon representation to 49

allow more comprehensive reclassification. 50

Although Ichneumonidae is currently larger in terms of number of described species than 51

Braconidae, the family is much less understood phylogenetically. Ichneumonidae is comprised of over 52

25,000 valid species, 38-43 subfamilies, and approximately 57 tribes (Bennett et al., 2019; Broad et al., 53

2018; Quicke et al., 2020a; Quicke, 2015; Quicke et al., 2009; Yu et al., 2012). Until recently, the three 54

molecular studies that examined higher-level relationships within Ichneumonidae (Belshaw et al., 1998; 55

Quicke et al., 2000; Quicke et al., 2009) were all based on one gene (28S rDNA), severely limiting 56

phylogenetic resolution. Phylogenetic and revisionary studies have been attempted for some lineages (e.g. 57

Bennett, 2001; Gauld et al., 2002; Gauld, 1985; Wahl and Gauld, 1998), with only a few based on 58

molecular data (Klopfstein et al., 2010; e.g. Laurenne et al., 2006; Matsumoto, 2016; Rousse et al., 2016; 59

Santos, 2017). There are three informal lineages in Ichneumonidae that have been recognized for many 60

years, namely Ichneumoniformes, Ophioniformes and Pimpliformes (Wahl, 1986, 1991; Wahl, 1993a; 61

Wahl and Gauld, 1998). These definitions were largely kept but redefined in 2009 with Quicke et al.’s 62

(2009) combined 28S and morphological analyses based on dense taxonomic sampling (1001 wasps). 63

Quicke et al. (2000) also added three additional informal groupings for single subfamily lineages 64

(Xoridiformes, Labeniformes, Orthopelmatiformes), the latter two with uncertain placement, even when 65


https://doi.org/10.1101/2020.06.17.157719

5

the taxonomic sampling was robust (Quicke et al., 2009). This classification, based on a single gene, was 66

largely followed in Quicke’s (2015) comprehensive and seminal book on Ichneumonoidea. 67

Several recent studies have been published with more comprehensive taxon, genetic, and 68

morphological sampling. Santos (2017) examined relationships among Cryptinae and higher-level 69

relationships within Ichneumoniformes using extensive taxon sampling, morphological characters, and 70

seven genes and revised the taxonomy for some lineages. Klopfstein et al. (2019) analyzed relationships 71

among the Pimpliformes using anchored hybrid enrichment with 93 genes and 723 genes from a reduced 72

taxon transcriptome dataset. They showed the evolutionary history of Pimpliformes was confounded by 73

rapid radiations across several lineages. Finally, Bennett et al. (2019) examined relationships among the 74

whole family using extensive morphological characters and three loci, and currently represents the most 75

detailed analysis and summary of ichneumonid relationships to date. Here we follow the revised higher-76

level groupings for Ichneumonidae proposed by Bennett et al. (2019). 77

Given the immense diversity on both Braconidae and Ichneumonidae, continued research on their 78

phylogenetic relationships, evolution, and taxonomy are warranted given their importance in pest control 79

and interesting evolutionary history with endogenous viruses. Further, members of both families have 80

been underutilized in biological control due to numerous cryptic species (Derocles et al., 2016; Peixoto et 81

al., 2018) and limited taxonomic resources facilitating accurate identification of many groups, particularly 82

for species (Broad et al., 2018; Quicke, 2015; Veijalainen et al., 2012; Wahl, 1993b). 83

84

1.2 Evolution of viral symbiosis in Ichneumonoidea 85

Some ichneumonoids overcome host defenses through the use of viruses produced by genes that 86

are integrated within the wasp genome and modify host physiology and immunity (Kroemer and Webb, 87

2004; Lavine and Beckage, 1995; Strand and Burke, 2012). The acquisition of these endogenous virus 88

elements (EVEs) in Ichneumonoidea promotes reproductive success and may be a factor promoting 89

diversity (Burke et al., 2018a; Burke and Strand, 2014; Federici and Bigot, 2003; Gauthier et al., 2017; 90

Whitfield, 1990). These viruses in the family Polydnaviridae (PDV, genera Bracovirus, Ichnovirus) are 91

only known from a few clades across Ichneumonoidea. Within Ichneumonidae, viruses are known from 92

two subfamilies (Banchinae and Campopleginae) that are both members of the Ophioniformes (Quicke, 93

2015; Quicke et al., 2009). In both lineages, the viruses are ichnoviruses (PDVs) that have some 94

homologous genes, but whether this represents one or two independent acquisitions remains unclear 95

(Béliveau et al., 2015; Lapointe et al., 2007; Volkoff et al., 2012; Volkoff et al., 2010), largely due to a 96

lack of knowledge on Ichneumonid relationships and viral presence across the family. There is one known 97

case of an ichneumonid, Venturia canescens (Campopleginae), which does not produce an ichnovirus but 98


https://doi.org/10.1101/2020.06.17.157719

6

instead has an alphanudivirus-like EVE that no longer packages DNA but produces virus-like particles 99

(VLPs) (Pichon et al. 2015). Thus, for Ichneumonidae, there have been at least 2 or 3 independent 100

acquisitions of EVEs. 101

Within Braconidae, EVEs are best known within the diverse microgastroid complex, in which all 102

known viruses are bracoviruses (Polydnaviridae) that were likely derived from a common ancient (~100 103

mya) betanudivirus (Nudiviridae) (Bézier et al., 2009; Bézier et al., 2013; Murphy et al., 2008; Whitfield, 104

1997, 2002; Whitfield and O’Connor, 2012). The latest discovery of an independent and recent 105

acquisition of endogenous virus related to alphanudiviruses (FaENV - Nudiviridae) in a different lineage 106

of Braconidae (some species of Fopius (Opiinae)) suggests that viral endogenization may be more 107

common than previously thought (Burke et al., 2018a). Unlike bracoviruses and ichnoviruses, FaENV in 108

species of Fopius, like in Venturia, lost the ability to package DNA and instead produce VLPs. Thus, both 109

Ichneumonidae and Braconidae have at least two subfamilies that produce viruses (PDVs and VLPs), but 110

virion morphology, virus replication machinery, genome rearrangement and gene homology suggest these 111

endogenization events likely represent four or five independent acquisitions (Béliveau et al., 2015; Burke 112

et al., 2018a; Herniou et al., 2013; Thézé et al., 2011). 113

114

1.3 Importance of a stable phylogenetic framework for Ichneumonoidea 115

Unfortunately, understanding of viral acquisition and evolution, as well as parasitoid evolution, 116

host use, development, diversification, and host immune disruption, is severely limited by the lack of a 117

robust phylogenetic framework (Desjardins et al., 2008; Gauld, 1988; Lapointe et al., 2007; Pennacchio 118

and Strand, 2006; Whitfield and O’Connor, 2012). Outside of low genetic sampling of previous studies, 119

the difficulty of resolving clades in Ichneumonoidea is likely due in part to several rapid radiations 120

(Banks and Whitfield, 2006; Klopfstein et al., 2019; Whitfield and Kjer, 2008). Further, previous 121

phylogenetic analyses based on morphology (Belshaw et al., 2003; Dowton et al., 2002; Pitz et al., 2007; 122

Quicke et al., 2000; Sharkey and Wahl, 1992; van Achterberg and Quicke, 1992; Wahl and Gauld, 1998; 123

Wharton et al., 1992) have discovered high levels of convergence, thereby obfuscating true relationships 124

(Gauld and Mound, 1982; Quicke, 2015; Whitfield, 2002; Wild et al., 2013). Thus, deep genetic sampling 125

will likely be necessary to understand relationships in Ichneumonoidea. Several recent studies of other 126

insect groups that likely had rapid radiations have shown the power of a phylogenomics approach using 127

hundreds of genes (Branstetter et al., 2017; Peters et al., 2017; Piekarski et al., 2018), even if taxonomic 128

sampling was relatively low (Johnson et al., 2013; Misof et al., 2014; Savard et al., 2006; Simon et al., 129

2009). 130

In this study we resolve several deep phylogenetic relationships for Ichneumonoidea using 131

anchored hybrid enrichment (AHE) (Lemmon et al., 2012) and focusing more on the poorly resolved 132


https://doi.org/10.1101/2020.06.17.157719

7

ichneumonids. We sequenced whole genomes of 11 ichneumonoid wasps and utilize these genomes along 133

with previously sequenced Hymenopteran genomes to design a novel probe set targeted for 134

Ichneumonoidea, but capable of capturing genes across multiple lineages of Hymenoptera. We sequenced 135

479 genes for 80 ingroup exemplars, one of the largest molecular datasets for this immensely diverse 136

superfamily to date. These data provide a robust phylogenetic framework for Ichneumonoidea and 137

establish molecular tools for future sampling in the lineage. We reconstruct ancestral states for two traits 138

related to parasitism: idiobiosis versus koinobiosis, and ecto- versus endoparasitism. Further we examine 139

the utility of probes to capture polydnaviruses across a broad array of taxa. Our data help resolve previous 140

phylogenetic uncertainties and improve the ability to address interesting biological questions in this rapid 141

radiation. 142

143

2. METHODS 144

2.1 Genomic Sequencing and Probe Design 145

The goals for probe design for AHE (Lemmon et al., 2012) were to specifically target 146

ichneumonoids, but also to have broader applicability across all of Hymenoptera. Thus, we utilized 147

published Hymenoptera sequences and sequenced several new genomes to design the probe set. The 148

probe design was generated by optimizing across published genomes from the bees Apis mellifera 149

(Honeybee Genome Sequencing Consortium, 2006) and Bombus impatiens (Sadd et al., 2015), the 150

parasitoid wasp Nasonia vitripennis (Werren et al., 2010), seven ant species (Gadau et al., 2012), and two 151

braconid genomes from i5k (http://arthropodgenomes.org/wiki/i5K): (Microgastrinae: Microplitis 152

demolitor (Burke et al., 2018b) and Opiinae: Diachasma alleoleum (Tvedte et al., 2019)). To these we 153

added whole genome sequences from 11 additional ichneumonoid wasps, including four braconids and 154

seven ichneumonids that represent diverse lineages across the ichneumonoid phylogeny (Table 1). For 155

whole genome sequencing of these 11 individuals, DNA was extracted from whole specimens using a 156

DNeasy Blood and Tissue Kit with RNase treatment. DNA was only used if good quality scores and 157

DNA concentration were obtained from the Nanodrop, and gel electrophoresis showed minimal DNA 158

degradation. Indexed libraries with ~250 bp inserts were prepared for sequencing following Lemmon et 159

al. (2012), and sequenced at 100bp paired end to achieve ~15x genomic coverage on two HiSeq2500 160

sequencing lanes (79 Gb total output). Reads were submitted to the NCBI Sequence Read Archive 161

(FigShare 10.6084/m9.figshare.12497468). 162

163


https://doi.org/10.1101/2020.06.17.157719

8

Raw reads from newly sequenced taxa were combined with assembled genome data to design the 164

Hymenoptera probe set. In general, we followed locus selection procedures outlined in Hamilton et al. 165

(2016). Initial Hymenoptera probe design involved finding orthologs to an optimal set of 941 insect loci 166

established in Coleoptera (Haddad et al., 2018). Using the red flour beetle (Tribolium castaneum) 167

sequence as a reference, we scanned the assembled genomes of two divergent hymenopteran species 168

(Bombus impatiens and Nasonia vitripennis) for each of the Coleoptera anchored hybrid enrichment loci. 169

The best matching region in each of the two Hymenoptera genomes was identified and a 4000 bp region 170

containing each locus was extracted and aligned using MAFFT v7.023b (Katoh and Standley, 2013b) 171

with –genafpair and –maxiterate 1000 flags. We then scanned the 12 assembled and raw reads from 11 172

unassembled genomes outlined above for targeted anchor regions using Nasonia vitripennis sequences. 173

For the 12 assembled genomes, we isolated up to 4000 bp surrounding the region that best matched the 174

reference. For the unassembled genomes, reads were merged and trimmed following Rokyta et al. (2012), 175

and then mapped to Nasonia vitripennis. The consensus sequences from the resulting assemblies were 176

extended in flanking regions to produce up to a 4000 bp sequence for each species at each locus following 177

protocols outlines in Hamilton et al. (2016). The resulting loci from N. vitripennis and the 23 target 178

species were then aligned using MAFFT. These were visually inspected to confirm adequate alignment in 179

Geneious (Kearse et al., 2012), and then trimmed and masked to isolate well-aligned regions. To ensure 180

sufficient enrichment efficiency, we removed loci that contained fewer than 50% of the taxa, resulting in 181

528 anchor loci. 182

We also added a few custom selected genes into the probeset: five recognized venom proteins 183

(calreticulin, phenoloxidases, chitinase, trehalase, aspartylglucosaminidase); two genes (ACC and CAD) 184

used in a previous braconid phylogeny (Sharanowski et al., 2011); and five polydnavirus genes (see 185

below). For each of these, rather than use the genomes, probes were designed from a reference set of 186

previously published gene sequences. 187

Polydnaviruses were included to enable comparison of taxon incidence, number of origins, age of 188

virus endogenization events and evolution of polydnaviruses among the Ichneumonoidea while also 189

assessing the ability of the anchored enrichment approach to capture more rapidly evolving adaptive 190

genes. Furthermore, this capture could increase the numbers of these genes across taxa to enable an 191

improved framework for examining their evolution in future. The PDV genes included two markers 192

identified from bracoviruses (Bézier et al., 2009) from three species: HzNVorf128, a gene of viral origin 193

associated with replication, and p74 per os infectivity factor, a viral envelope gene. We also designed 194

probes for two ichnovirus replication or structural proteins that occur across three species of 195

Campopleginae, including EST data derived from Sharanowski et al. (2010): IVSP4 and U1 (Burke et al., 196


https://doi.org/10.1101/2020.06.17.157719

9

2013; Lorenzi et al., 2019; Volkoff et al., 2012; Volkoff et al., 2010). These genes are part of the 197

replication machinery maintained within the host genome and were selected because they are more likely 198

to be single copy or involve just a few copies. In addition, we included sequences representing the 199

vankyrin gene family across all PDV lineages to test the ability to capture gene family representatives 200

(Kroemer and Webb, 2005). Unlike the other loci, vankyrins are packaged within PDVs and are likely not 201

of viral origin (Huguet et al., 2012). 202

Repetitive Kmer alignment regions among all loci were masked following Hamilton et al. (2016). To 203

build the final probe set we tiled 120 bp probes at 2X density across the 24 taxon-specific sequences for 204

each locus. The final probe set included 57,066 probes from 541 loci covering 212,392 total base pairs. 205

Loci included in the probe design consisted of a diverse array of genes, with no significant enrichment of 206

any particular class of genes based on Blast2GO (Conesa et al., 2005) analysis. 207

2.2 Taxon Sampling 208

To provide a deeper phylogenetic framework for systematic revision in Ichneumonoidea, we 209

focused our taxon sampling on the lesser-understood ichneumonids, but also included sampling of 210

braconids to enable comparison with a previously published 7-gene phylogeny on Braconidae 211

(Sharanowski et al., 2011). We utilized 59 Ichneumonidae species representing 25 subfamilies and 21 212

Braconidae species representing 17 subfamilies (Table 1). For Ichneumonidae, taxa were selected to 213

maximize the number of represented lineages and provide denser sampling for lineages that were 214

suspected to be paraphyletic based on previous studies (Quicke et al., 2009) or contain PDVs (Bigot et al., 215

2008; Strand and Burke, 2015). For Braconidae, taxa were selected to include representatives of major 216

lineages to assess how our results obtained with the anchored hybrid enrichment compare to a more 217

traditional molecular study completed by Sharanowski et al. (2011). Species level identifications were not 218

possible for the majority of taxa, as many genera require revisionary works or there are not adequate keys 219

or descriptions of species to ensure accuracy. Outgroups consisted of genomic data from published model 220

genomes that were included in our probe design (Table 1). We follow the higher-level groupings of 221

Sharanowski et al. (2011) for Braconidae and Bennett et al. (2019) for Ichneumonidae, except we keep 222

the grouping Labeniformes for members of Labeninae, and do not include this lineage within 223

Ichneumoniformes s.l. All specimens were deposited in the University of Central Florida Collection of 224

Arthropods (UCFC) except specimens from Sweden, which were deposited at Station Linné, Swedish 225

Malaise Trap Project (SMTP). 226

2.3 Hybrid Enrichment Genome Capture & Data Processing 227


https://doi.org/10.1101/2020.06.17.157719

10

The remaining sequence data were obtained at Florida State University’s Center for Anchored 228

Phylogenomics (www.anchoredphylogeny.com) using hybrid enrichment sequence capture with 229

SureSelect. For this approach, genomic DNA samples were extracted using the Qiagen DNEasy kit with 230

quality assessed using gel electrophoresis and a Nanodrop. Indexed libraries were prepared following a 231

protocol modified from Meyer & Kircher (2010) but modified to accommodate the Beckmann-Coulter 232

FXP liquid handling robot. Libraries were pooled and enriched using an Agilent Custom SureSelect kit 233

containing all 57,066 probes and were sequenced using PE 100bp PE sequencing on the Illumina HiSeq 234

2000. 235

The resulting paired Illumina anchored hybrid enrichment reads were demultiplexed with no 236

mismatches tolerated after quality filtering using the Illumina Casava high chastity filter. Read pairs were 237

then merged using a Bayesian approach that identifies the sequence overlap, removes adapter sequences, 238

and corrects sequencing errors in overlapping regions (Rokyta et al. 2012). The resulting reads were then 239

assembled using a quasi de novo assembler described by Hamilton et al (2016), with the following 240

references (Table 1): Megachile rotundata, Microplitis demolitor, Aleiodes sp. (PSUC_FEM 10030003), 241

Lissonota sp. (PSUCFEM_10030012), and Odontocolon sp. (PSUC_FEM 10030002). The effects of low-242

level contamination and misindexing were avoided by filtering out assembly clusters with fewer than 100 243

reads. To establish orthology, homologous consensus sequences from each assembly cluster passing the 244

coverage filter were evaluated to compute a pairwise distance matrix that was used to identify orthosets 245

using a neighbor-joining approach (following Hamilton et al., 2016). After assembly and preliminary 246

trimming using the pipeline, 479 loci were retained. 247

2.4 Alignment and Dataset Filtering 248

Orthologous sequences were aligned using MAFFT (v7.023b, with –genafpair and –maxiterate 249

1000 flags (Katoh and Standley, 2013a). Alignments were manually inspected for errors after 250

autotrimming (mingoodsites=12, minpropsame=0.4 and 20 missing taxa allowed) following the approach 251

outlined in Hamilton et al. (2016). Alignments were then manually checked to ensure the proper reading 252

frame was retained after auto-trimming by using the gene maps of Apis mellifera, Nasonia vitripennis, 253

and Solenopsis invicta as a guide, and where necessary, non-coding regions were removed (7 genes 254

captured parts of introns and 3 genes captured parts of untranslated regions (UTRs). 255

2.5 Model testing and Phylogenetic Analyses 256

Each locus alignment was translated into a protein dataset and both protein and nucleotide 257

datasets were concatenated using SequenceMatrix 1.8 (Vaidya et al., 2011) and analyzed separately. We 258


https://doi.org/10.1101/2020.06.17.157719

11

analyzed the datasets using Maximum Likelihood with IQ-Tree v.1.6.10 (Nguyen et al., 2014) on either 259

the Stokes HPC at the University of Central Florida Advanced Research Computing Center or on the 260

CIPRES Science Gateway (Miller et al., 2009; Miller et al., 2010). Bayesian analyses were also 261

performed on the concatenated nucleotide dataset with the third codon removed using MrBayes 3.2.6 262

(Ronquist et al., 2012). The full nucleotide or protein dataset was not completed using MrBayes, as 263

analyses were taking longer than 3 months and often did not converge, even when constraints of known 264

monophyletic lineages were applied. For the Nucleotide Bayesian analysis with the third codon position 265

removed, we constrained monophyly for Braconidae, Ichneumonidae, Ants, and Bees. PartitionFinder 266

V.2.1.1 (Lanfear et al., 2012; Lanfear et al., 2016) was used to find the best-fit models of molecular 267

evolution and partitioning scheme using each locus as a priori subsets for the nucleotide dataset. For each 268

treatment, an hcluster search with branch lengths linked was performed and model selection was based on 269

the Bayesian Information Criterion (Schwarz, 1978). For the protein dataset, the best fit models of 270

molecular evolution and partitioning scheme was inferred using ModelFinder (Chernomor et al., 2016; 271

Kalyaanamoorthy et al., 2017) within IQ-Tree v.1.6.10, limited to the following models with the option 272

for invariant and gamma parameters: Blosum62, Dayhoff, JTT, LG, VT, and WAG. All alignment 273

datasets can be found on FigShare (10.6084/m9.figshare.12497468). 274

We also ran saturation, relative synonymous codon usage, and long branch exclusion tests and ran 275

the nucleotide dataset with the 3rd position removed using IQ-tree using the methods outlined above. 276

Nodal support in IQ-tree analyses were assessed with 1000 pseudo-replicates of both the ultrafast 277

bootstrap (Hoang et al., 2017) and Shimodaira-Hasegawa-like approximate Likelihood Ratio Test (SH-278

aLRT) (Guindon et al., 2010). The Bayesian analysis was run with 45 million generations sampling every 279

1000 generations with a 25% burn-in. The analysis consisted of two independent runs with four chains 280

each. Convergence of the Bayesian analysis was assessed and confirmed using diagnostics in Tracer v.1.6 281

(Rambaut et al., 2013). To test for saturation, we calculated the number of pairwise differences and 282

Tajima Nei genetic distance for each codon position using MEGA v.7.0 (Kumar et al., 2016), and plotted 283

against each other using R (R Team, 2016) and the package “ggplot2”(Wickham, 2016). To test for codon 284

bias in the nucleotide dataset we performed a principal component analysis using relative synonymous 285

codon usage (RSCU) and amino acid composition, and performed a phylogenetic ANOVA using the 286

effective number of codons (ENC) in R with the following packages: “seqinr” (Charif and Lobry, 2007), 287

“phangorn” (Schliep, 2011), “geiger” (Harmon et al., 2008), “vhica” (Wallau et al., 2016), and “dplyr” 288

(Wickham et al., 2015). In addition to this we calculated GC content for each taxon and synonymous 289

skew on a scale of 0 to 1 for each of the six-fold degenerate amino acids (Rota-Stabelli et al., 2013) using 290

the formula introduced by Perna and Kocher (1995), and mapped these metrics onto the nucleotide 291


https://doi.org/10.1101/2020.06.17.157719

12

phylogeny using the R packages “ape” (Paradis et al., 2004) and “phytools” (Revell, 2012). As there were 292

differences in some relationships between the nucleotide and amino acid analyses, we tested stability of 293

the relationships recovered in the amino acid analyses by performing a series of taxon exclusion tests 294

(Bergsten, 2005). For each analysis, one or more taxa were removed and the analysis rerun in IQ-tree 295

following the methods outlined above. Choices of taxa to exclude were based on length of branches and 296

instability in phylogenetic position between the nucleotide and amino acid datasets. 297

Successfully captured PDV genes were aligned along with the available GenBank sequences used 298

to facilitate capture (FigShare 10.6084/m9.figshare.12497468) using Geneious with the native alignment 299

algorithm and default parameters. These alignments were run through jmodeltest2 (Darriba et al., 2012) to 300

assign models of evolution and were then run in MrBayes v.3.2.6 (10 M generations, 3 runs) using 301

CIPRES Science Gateway (Miller et al., 2010). All were confirmed to have converged with Tracer v. 1.6 302

(Rambaut et al., 2013). As no taxa were captured with U1, these analyses included three genes: six taxa 303

for Bracovirus HzNVorf128 at 1035 aligned bases and run under a GTR+G model, four taxa for 304

Bracovirus per os infectivity factor p74 run with 2144 bps under a GTR+I model, and ten sequences for 305

the Ichnovirus IVSP4, 1198 bp run under a GTR+I model. 306

2.6 Ancestral State Reconstructions 307

To examine evolutionary transitions of parasitoid biology, ancestral states were reconstructed for 308

two traits: ectoparasitism (0) versus endoparasitism (1); and idiobiosis (0) versus koinobiosis (1) 309

(Supplementary Table S1). Reconstructions were completed using the IQ-tree phylogeny of the amino 310

acid analysis and repeated with Labeninae removed (due to instability in this lineage’s phylogenetic 311

position; see results). To assess robustness of results across phylogenetic uncertainty, the analysis was 312

repeated with the IQ-tree phylogeny based on nucleotide data with the third position removed (nt-3out) 313

and repeated with Labeninae removed. Binary characters were analyzed using Maximum Likelihood 314

using Mesquite version 3.2 (Maddison and Maddison, 2019). Two models were tested for likelihood 315

reconstructions: the Markov k-state one parameter (Mk1), which has an equal rate for character gains and 316

losses, and the asymmetrical two-parameter Markov k-state (Assym2), which applies a separate rate. To 317

test whether the characters evolved in drastically different ways between Braconidae and Ichneumonidae, 318

we tested whether the same model was a better fit when only one family was included for analysis. 319

3. Results 320

3.1 Sequence Data 321


https://doi.org/10.1101/2020.06.17.157719

13

Selective enrichment was good, with 27.3% of reads on average (range 14.6–49.0%) mapped to 322

loci. Most of the loci were recovered across individuals, with an average of 478 of the 541 genes captured 323

per individual. This is nearly the same number of genes that were recovered from blasts of taxa sequenced 324

from whole genome data against references for each gene (n=480), suggesting that nearly all genes 325

present with close affinity to reference sequences were captured. Many of the loci had comprehensive or 326

nearly comprehensive coverage, with 206 of the loci having high read numbers (>100) mapped across all 327

35 taxa (1 taxon failed in the sequencing run) and 297 loci having high read mapping across at least 33 of 328

the 35 taxa. Average length per locus was 990 bps (Supplementary Fig. 1). After trimming only seven 329

genes had introns partially captured and three had portions of untranslated regions captured, which were 330

manually trimmed out of the dataset. The concatenated datasets had a total of 91 taxa and 479 genes with 331

lengths of 150,435 base pairs and 50,145 amino acids for the nucleotide and amino acid datasets, 332

respectively. The nucleotide dataset with the third position removed had 100,290 base pairs. 333

3.2 Evolutionary Relationships among Ichneumonoidea 334

Saturation was indicated in the third codon position of the concatenated dataset (Fig. S2), and 335

thus the maximum likelihood phylogeny with all data included (Fig. S3) is not discussed further given the 336

likelihood for systematic error. The maximum likelihood amino acid phylogeny is depicted in Fig. 1A-C. 337

Nodal supports for the nucleotide dataset with the 3rd codon position removed (nt-3out) for the maximum 338

likelihood (Fig. S4) and Bayesian (Fig. S5) phylogenies are also indicated on Fig. 1A-C where 339

relationships were identical. All analyses recovered a maximally supported and monophyletic 340

Ichneumonoidea, Braconidae, and Ichneumonidae (Fig 1a). Maximum support indicates a value of 100 341

for Shimodaira-Hasegawa approximate Likelihood Ratio Tests (aLRT), 100 for Ultra-Fast bootstraps 342

(UFb), and 1.0 for posterior probability (PP). 343

Within Braconidae, several higher-level lineages were also maximally supported across all 344

analyses, including the cyclostomes s.l., cyclostomes s.s., non-cyclostomes, the Aphidioid, Helconoid and 345

Microgastroid complexes, and the Alysioid subcomplex (Fig. 1B), similar to results reported in 346

Sharanowski et al. (2011). Relationships among all cyclostomes were also consistent with Sharanowski et 347

al. (2011), despite the low taxonomic sampling: the Aphidioid complex was sister to the cyclostomes s.s., 348

Rhyssalinae was recovered as sister to the remaining cyclostomes s.s., and Braconinae was recovered as 349

sister to the Alysioid complex with maximal support (Fig. 1B). The placements of Rogadinae and 350

Pambolinae were labile between the amino acid (Fig. 1B) and nt-3out analyses (Figs. S4-5); however, 351

support for Rogadinae as sister to Braconinae + the Alysioid complex increased when different outgroups 352

were used (Fig. S6, Table S2) and when Aphidiinae was removed (Fig. S7). Many relationships within 353


https://doi.org/10.1101/2020.06.17.157719

14

the non-cyclostomes were identical to results in Sharanowski et al. (2011). For example, Euphorinae was 354

consistently recovered as sister to the Helconoid complex (Fig.1b): Acampsohelconinae (Brachistinae 355

(Helconinae + Macrocentroid subcomplex)). One notable exception was the placement of Ichneutinae, 356

which was variably placed as sister to Agathidinae + the Microgastroid complex (Fig. 1B) or sister to 357

Agathidinae (Figs S4-5) with variable support. Most previous studies have recovered Ichneutinae as a 358

paraphyletic group sister to the Microgastroid complex s.s. (Belshaw et al., 2000; Belshaw and Quicke, 359

2002; Dowton et al., 2002; Quicke and van Achterberg, 1990; Sharanowski et al., 2011). When 360

Agathidinae was removed, Ichneutinae was recovered as sister to the Microgastroid complex (Fig. S8) 361

with very high support (≥ 99), possibly indicating that low taxonomic sampling or long branch attraction 362

may have been an issue for the unexpected placement of Ichneutinae. 363

Within Ichneumonidae, Xoridinae was always recovered as sister to all remaining ichneumonids 364

with strong support (>95, Fig. 1C), consistent with previous analyses (Klopfstein et al., 2019; Quicke et 365

al., 1999b) where Xoridinae was not set as the root (but see Bennett et al., 2019). The next basal lineage 366

was Labeninae in the protein analysis (Fig. 1C), but this placement had very low support. In the nt-3out 367

analyses, Labeninae was recovered as sister to Orthopelmatinae with relatively strong support (Figs. S4-368

S5), and these taxa were sister to Ichneumoniformes + Pimpliformes. Other recent studies with large 369

taxon or gene sampling also had variable placement of Labeninae (Bennett et al., 2019; Klopfstein et al., 370

2019; Santos, 2017). These variable results may reflect the low taxonomic sampling, especially non-371

sampling of potentially related subfamilies, such as Brachycyrtinae, which has been recovered as the 372

sister group to Labeninae (Bennett et al., 2019). In general, Labeninae was the most labile lineage across 373

analyses and when different taxa were excluded (Table S2, Figs. S6-S17). When Xoridinae was excluded, 374

the branch support for Labeninae as the most basal taxon is increased (Fig. S9). Further when Labeninae 375

was excluded (Fig. S10), the support for all remaining Ichneumonids (except Xoridinae) as a 376

monophyletic lineage was substantially increased (≥ 99). 377

Several higher-level relationships among Ichneumonidae were maximally supported, including 378

Ichneumoniformes, Pimpliformes, and Ophioniformes (Fig. 1C). Ophioniformes was recovered as sister 379

to all remaining ichneumonids (excluding Xoridinae and Labeninae) with the following relationships: 380

Pimpliformes (Orthopelmatinae + Ichneumoniformes). The amino acid analysis recovered 381

Orthopelmatinae as sister to the Ichneumoniformes, but with low support (Fig. 1C); however, in the 382

outgroup test (Fig. S6) and several taxon exclusion tests, support for this relationship increased (Table S2, 383

Figs. S6, S13-14, S17). Although Orthopelmatinae and Eucerotinae had very long branches, when 384

Eucerotinae was excluded (Fig. S13), Orthopelmatinae was recovered as sister to Ichneumoniformes s.l. 385

with increased support. Interestingly, when both Labeninae and Orthopelmatinae were excluded (Table 386


https://doi.org/10.1101/2020.06.17.157719

15

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

(Ichneumoniformes + Pimpliformes). 388

Eucerotinae was always recovered as sister to a clade containing Adelognathinae, Cryptinae, and 389

Ichneumoninae with maximal support (Fig. 1C). Despite our low taxonomic sampling for this lineage, 390

these results support Eucerotinae within Ichneumoniformes s.l. (following Bennett et al., 2019). However, 391

our lack of members of several subfamilies within Ichneumoniformes s.l. (Brachycyrtinae, Claseinae, 392

Agriotypinae, Microleptinae, Pedunculinae, Ateleutinae, Phygadeuontinae) limits understanding of 393

phylogenetic relationships in this lineage. The representatives we included for Ichneumoninae and 394

Cryptinae were always recovered as monophyletic with maximum support, however, the placement of 395

Adelognathinae varied with respect to these subfamilies across the amino-acid (Fig. 1C) and Bayesian nt-396

3out analysis (Fig. S5). 397

Similar to Klopfstein et al. (2019), relationships within Pimpliformes varied widely across the 398

amino acid (Fig. 1C) analysis and nt-3out analyses (Figs. S4-5). In the amino acid analysis, Acaenitinae 399

was recovered as sister to the remaining Pimpliformes with strong support (Fig. 1C), whereas 400

Diplazontinae + Orthocentrinae were recovered at the base of the radiation in the nucleotide analyses with 401

strong support (Figs S4-5). Interestingly, the relationships recovered in the amino acid analysis (Fig. 1C) 402

remained stable across all outgroup and taxon exclusion tests (Table S2, Figs. S6-17) and were largely 403

consistent with relationships proposed in a comprehensive morphological study by Wahl and Gauld 404

(1998). We investigated codon use biases and observed that Diplazontinae has more similar codon usage 405

to Ichneumoniformes s.s. than the rest of Pimpliformes (Fig. S18), and this may account for its more basal 406

position in the nt-3out analyses (Figs. S4-5). Like long branch attraction (Bergsten, 2005), false 407

homologies created through the codon use patterns may pull convergent taxa falsely together. 408

Consistent and well supported relationships within Pimpliformes included: Diplazontinae + 409

Orthocentrinae (consistent with Wahl, 1990; Wahl and Gauld, 1998); Collyriinae + Cyllocerinae; and 410

monophyly of the Ephialtini and Pimplini tribes of Pimplinae, consistent with the findings of Klopfstein 411

et al. (2019) (Fig. 1C). Pimplinae was recovered as paraphyletic when nucleotide data was analyzed, but 412

monophyletic when the amino acid data was analyzed (a similar issue was found in Klopfstein et al., 413

2019). There was also a distinct difference in codon usage between the Ephialtini and Pimplini clades of 414

Pimplinae (Fig. S19). Thus, the amino acid analysis probably more accurately reflects the true monophyly 415

of Pimplinae, which has been supported by some morphological studies (Gauld et al., 2002; Wahl and 416

Gauld, 1998). The lone specimen of Poemeniinae (Neoxorides sp.) was recovered as sister to the 417

Pimplinae, suggesting its close affiliation (Bennett et al., 2019; Quicke et al., 2009; Wahl and Gauld, 418


https://doi.org/10.1101/2020.06.17.157719

16

1998). The inclusion of Collyriinae in Pimpliformes supports previous studies (Bennett et al., 2019; 419

Klopfstein et al., 2019; Quicke et al., 2009; Sheng et al., 2012). 420

Within Ophioniformes, the following subfamilies (that had more than one exemplar) were 421

recovered as monophyletic across all analyses with maximum support (Fig. 1C): Tryphoninae, Banchinae, 422

Tersilochinae, Metopiinae, Anomaloninae, Cremastinae, and Campopleginae. Tryphoninae was 423

consistently recovered and maximally supported as the sister to all remaining Ophioniformes. Banchinae 424

was the next most basal lineage (consistent with Klopfstein et al., 2019; Quicke et al., 2009), but this 425

placement was poorly supported (Fig. 1C). However, this placement was largely consistent across the 426

outgroup and taxon exclusion analyses (Table S2), except when Mesochorinae was removed (Fig. S16). 427

Further, in the nt-3out analyses (Figs S4-5), Banchinae was recovered as sister to Tersilochinae with 428

strong support, and both were sister to the remaining Ophioniformes (except Tryphoninae) (Figs. S4-5). 429

The remaining Ophioniformes were consistently recovered in a maximally supported clade, including: 430

Mesochorinae, Oxytorinae, Ctenopelmatinae, Metopiinae, Cremastinae, Hybrizontinae, Anomaloninae, 431

Ophioninae, and Campopleginae. Of these, Mesochorinae was recovered as sister to the rest, but with 432

variable support (Fig. 1C). Ctenopelmatinae was unsurprisingly paraphyletic as was found previously 433

(Bennett et al., 2019; Quicke et al., 2009). Metopiinae was consistently recovered as sister to a maximally 434

supported clade that roughly corresponds to Quicke et al.’s (2009) “higher Ophioniformes,” including 435

Cremastinae, Hybrizontinae, Anomaloninae, Ophioninae, and Campopleginae. Interestingly, 436

Hybrizontinae (a group that has varied widely in placement across all previous analyses) was recovered as 437

sister to Cremastinae with strong but variable support across all analyses (Fig. 1C). When Hybrizontinae 438

was excluded (Fig. S15), Anomaloninae was recovered as sister to Cremastinae with relatively high 439

support (>93). Ophioninae was consistently recovered as the sister group to Campopleginae with maximal 440

support across all analyses. 441

3.3 Ancestral State Reconstructions 442

Ancestral biological states were reconstructed on phylogenies that varied in the data type they 443

were estimated from (amino acid or nt-3out) and taxon representation: either all taxa were included, all 444

taxa but Labeninae (due to its uncertain placement), and with or without outgroups included. Further, 445

each family was tested individually based on the amino acid analysis, with all other taxa removed. As our 446

sampling was not comprehensive, we only discuss ancestral state reconstructions for higher-level 447

relationships, including Ichneumonoidea, Braconidae and Ichneumonidae. Within Braconidae we also 448

discuss reconstructions for the ancestors of the Cyclostomes s.l., Cyclostomes s.s., and Non-cyclostomes, 449

and within Ichneumonidae, ancestors of Ichneumoniformes s.l., Pimpliformes, Ophioniformes, and 450


https://doi.org/10.1101/2020.06.17.157719

17

Ophioniformes excluding Tryphoninae (Fig. 2A and B). The full reconstructions for each trait and input 451

phylogeny can be found on FigShare (10.6084/m9.figshare.12497468). 452

The Assym2 model (separate parameters for gains and reversals) consistently outperformed the 453

Mk1 model (equal rates of transition between character states) for character 1 (idiobiont vs koinobiont) 454

when all Ichneumonoidea were analyzed together (Table 2). Under these analyses, the ancestors of 455

Ichneumonoidea, Ichneumonidae, and Braconidae were reconstructed as idiobionts, although this was 456

only consistently significant for Ichneumonidae (Fig. 2A, Table 3). Within Braconidae, the ancestors of 457

the Cyclostomes s.l. and s.s. were also recovered as idiobionts, but this was only consistently significant 458

for the latter. Unsurprisingly, the ancestor of the non-cyclostomes was significantly koinobiont as all 459

known members are koinobiont (Fig. 2A, Table 3). Within Ichneumonidae, the Ichneumoniformes s.l. and 460

Pimpliformes were ancestrally idiobiont, whereas Ophioniformes was koinobiont, and all reconstructions 461

were significant (Fig. 2A, Table 3). When the families were analyzed individually, the above results 462

remained consistent for Ichneumonidae, but not Braconidae as the Mk1 model was preferred for 463

Braconidae (Table 2). Under this model all major braconid nodes were reconstructed as koinobiont and 464

significant for all lineages except the cyclostomes s.s. (Fig. 2A, Table 3), suggesting this character is 465

sensitive to the data used to infer the ancestral states for Braconidae. 466

For character 2 (ecto- vs. endoparasitism), the ancestral states were very sensitive to the 467

phylogeny utilized in the analysis (Fig. 2B). The Mk1 model was supported for character 2 when 468

Labeninae and outgroups were both excluded, when the nt-3out phylogeny was used, or when the 469

Braconidae or Ichneumonidae were analyzed individually (Table 2). Under the Mk1 model, all major 470

nodes were reconstructed as endoparasitoid and this consistently significant for most reconstructions 471

except the Ichneumonoidea, Ichneumonidae, Ophioniformes, and cyclostomes s.s. (Fig. 2B, Table 3). For 472

Ichneumonidae, these reconstructions suggested that the Ichneumonidae, Ichneumoniformes s.l., and 473

Pimpliformes had idiobiont endoparasitoid ancestors. When the Assym2 model was most appropriate 474

(Table 2), all major braconid nodes were reconstructed as ectoparasitoid, except the non-cyclostomes, and 475

only the cyclostomes s.s. had significant reconstructions (Table 3). There was also inconsistency across 476

the Ichneumonidae, where under the Assym2 model, only the Pimpliformes and Ophioniformes without 477

Tryphoninae were recovered as endoparasitoid and these reconstructions were not significant (Fig. 2B, 478

Table 3). These data suggest that ancestral reconstruction, particularly of ecto- vs. endoparasitism, is 479

sensitive to the taxon representation and type of data used in the analysis. 480

3.4 Capture of Polydnavirus Genes 481


https://doi.org/10.1101/2020.06.17.157719

18

Capture of polydnavirus genes had limited success. The U1 ichnovirus replication gene was not 482

captured in any of the taxa in our dataset including the campoplegines most likely to harbor a copy of it. 483

The IVSP4 gene was represented in the probe set by two paralogs from Hyposoter didymator (IVSP4-1 484

and IVSP4-2) and one gene copy from Campoletis sonorensis (Fig. 3A). Glypta fumiferanae (Banchinae) 485

was not used as the probes were designed prior to the publication of this genome (Béliveau et al., 2015). 486

We were able to capture matches to both IVSP4-1 and IVSP4-2 in another Hyposoter species with 487

considerable divergence from the other Hyposoter (I9995), suggesting these genes may be rapidly 488

evolving. We were also able to capture several copies of this gene family from Campoletis: two 489

individuals of Campoletis (I2339, I10007, Table 1) matched very closely to the original Campoletis 490

sonorensis sequence and Campoletis sp. 1 (I10007) exhibited two additional paralogs (copy 2, 3) that 491

were more closely related to the other Campoletis sp. 1 paralog than to paralogs of Hyposoter (Fig. 3A). 492

Casinaria sp. showed affinities to a copy in Campoletis (copy 3). This demonstrates that this ichnovirus 493

replication gene is part of a gene family that likely has lineage specific diversification. Hyposoter, 494

Campoletis sp. 1 and 2, and Casinaria sp. 1 are recovered in a maximally supported lineage and represent 495

just one lineage of Campopleginae (Fig. 1C). Interestingly, these genes were not captured in species of 496

Dusona and Campoplex, suggesting either these genera do not have ichnoviruses, or the ichnovirus genes 497

are highly divergent from other lineages within Campopleginae. No IVSP4 family members were 498

captured in Lissonota despite being present in the genome (Burke et al., 2020), but this may be due to not 499

having a banchine sequence in the original alignment used to design the probes. Although in Hyposoter 500

both IVSP4 copies were captured, it is interesting that not all copies were captured in each case, which 501

again could be a complication of this approach for capturing gene copies successfully or for indication of 502

potential gains and losses of ancestrally attained gene copies. 503

In the bracovirus replication genes, HzNVorf128 was represented by a Chelonus (Cheloninae) and 504

a Cotesia (Microgastrinae) species in the original probe design and was effectively captured and located 505

for all taxa in our sampling thought to include bracoviruses (Fig. 3B). The relationships inferred from 506

HzNVorf128 were congruent with the expected phylogenetic relationships (Fig. 1B), suggesting the 507

expected co-phylogeny of the replication machinery with the taxa they harbor (Fig. 3B). Also, there was 508

no indication of gene duplications in this gene, making it a good candidate to evaluate further in PDV 509

analysis. The per os infectivity factor p74, however, was captured/identified in the genomes of the two 510

closely related microgastroids to the probe sequences, with Schoenlandella (Cardiochilinae) not captured 511

and the gene not found in the Phanerotoma genomic sequence (Fig. 3C). It likely is evolving more 512

rapidly, thus eluding successful identification beyond close relatives. We included the vankyrin genes to 513

test if we could delineate members of a gene family using hybrid capture and because they are present in 514


https://doi.org/10.1101/2020.06.17.157719

19

each of the lineages that have PDVs (bracoviruses and ichnoviruses). Some individuals exhibited an 515

overabundance of vankyrins in the capture, e.g., 2% of the Australoglypta (Banchinae) sequence was 516

comprised solely of vankyrins, and other individuals had very low and likely non-specific capture. Given 517

the complexity of these results and establishing paralogy among divergent gene family members, we 518

excluded vankyrins from further analysis. 519

520

4. Discussion 521

4.1 Utility of Probe Set for Phylogenetics 522

The designed 541 probes set worked very well for capturing genes across Ichneumonoidea, with 523

an average of 478 genes captured per individual and 491 genes used in the final dataset analyzed here. 524

The probes were designed from alignments across taxa within the Holometabola and thus genes were 525

largely conserved. Further, the vast majority of genes captured only coding regions after trimming, 526

making them appropriate genes for examining macroevolutionary relationships, such as for 527

Ichneumonoidea, which has been estimated to have diverged in either the late Jurassic or early Triassic 528

(Peters et al., 2017; Zhang et al., 2015). These probes have also been successfully used to capture loci for 529

examining phylogenetic relationships and the evolution of eusociality in Vespidae (Piekarski et al., 2018), 530

indicating they have wide applicability across different hymenopteran taxa. 531

4.2 Evolutionary Relationships among Ichneumonoidea 532

Sampling across the superfamily was sparse relative to the total number of described species 533

within Ichneumonoidea (> 44,000, Yu et al., 2012), however, the inferred phylogenies were largely 534

stable. This was true across different analytical methods and datasets and when taxa with suspected long 535

branches or labile placement were excluded. Numerous maximally supported higher-level relationships 536

were inferred using the 491 gene dataset, providing a robust higher-level evolutionary picture for 537

Ichneumonoidea. 538

Although the taxon sampling for Braconidae was very low, the core higher-level relationships 539

recovered from traditional molecular phylogenetic approaches with a handful of genes (Sharanowski et 540

al., 2011) are largely recapitulated using a phylogenomics approach with denser genetic sampling. 541

Interestingly, regions of the Braconidae phylogeny with low support were similar to regions of low 542

support from Sharanowski et al. (2011), a symptom typical of rapid radiations (Giarla and Esselstyn, 543

2015; Xi et al., 2012), including in Braconidae (Banks and Whitfield, 2006; Zaldivar-Riverón et al., 544


https://doi.org/10.1101/2020.06.17.157719

20

2008). Thus, a combined dense taxonomic and genetic sampling approach will be necessary to fully 545

understand the phylogenetic history of this extremely diverse family of parasitoid wasps. 546

The relationships of Ichneumonidae were also largely stable and highly supported with a few 547

exceptions. All analyses and datasets recovered Xoridinae as the sister to all remaining Ichneumonids, an 548

important finding since some previous analyses have used Xoridinae to root the Ichneumonid phylogeny 549

(Quicke et al., 2000; Quicke et al., 2009) and its placement impacts the understanding of the evolution of 550

parasitism (Belshaw and Quicke, 2002; Gauld, 1988). Subfamilies with previously ambiguous placement 551

include Orthopelmatinae and Eucerotinae (Quicke et al., 2000; Quicke et al., 2009). In our analyses, 552

Orthopelmatinae was recovered as sister to Ichneumoniformes s.l. with low support in the amino acid 553

analysis. However, support for this relationship increased when different outgroups were used or when 554

specific taxa were individually excluded (e.g. Eucerotinae, Adelognathinae). This relationship was not 555

recovered in the nt-3out relationship because of the placement of Labeninae, but still suggested an affinity 556

with Ichneumoniformes s.l. Orthopelmatinae has been considered an aberrant group, sometimes placed in 557

its own Orthopelmatiformes group (Quicke et al., 2000; Quicke et al., 2009), of which the placement has 558

been unstable across numerous previous analyses, either as sister to Ophioniformes (Quicke et al., 2000; 559

Quicke et al., 2009), sister to Pimpliformes (Quicke et al., 2000, morphological analysis), as the second 560

most basal lineage next to Xoridinae (under parsimony, Bennett et al., 2019), or within the Ophioniformes 561

(Quicke et al., 2009). Whether or not Orthopelmatinae is sister to Ichneumoniformes s.l. will require 562

denser taxonomic sampling, specifically across more basal lineages. 563

Eucerotinae has also been variably treated due to its unique (within Ichneumonoidea) larval 564

biology and stalked eggs (see Bennett et al., 2019 for detailed review), but has more recently been 565

associated with taxa not sampled here, including Agriotypinae, Claseinae, Pedunculinae, Brachycyrtinae, 566

and/or Labeninae, along with Ichneumoniformes (Bennett et al., 2019; Gauld and Wahl, 2002; Quicke et 567

al., 2009). Here, Eucerotinae was consistently recovered as sister to Ichneumoniformes with maximum 568

support, consistent with recent studies with more extensive taxonomic sampling (Bennett et al., 2019; 569

Santos, 2017). Further our data supports the assertion of Bennett et al. (2019) that Eucerotinae should be 570

placed within the Ichneumoniformes (Bennett et al.’s Ichneumoniformes s.l.). 571

While monophyletic, the placement of Labeninae varied widely across analyses and taxon 572

exclusion schemes, and this impacted the support values of a handful of deeper nodes within 573

Ichneumonidae. When labile taxa were removed or different outgroups were used, support values tended 574

to increase across the backbone of the ichneumonid phylogeny. We recovered Labeninae as sister to the 575

rest of the Ichneumonidae, except Xoridinae, similar to some analyses in other previous studies 576


https://doi.org/10.1101/2020.06.17.157719

21

(Klopfstein et al., 2019; Quicke et al., 2009; Santos, 2017). The placement of Labeninae has varied 577

widely across these and other studies (Bennett et al., 2019; Quicke et al., 2000) and its true placement 578

represents an important future challenge for understanding ichneumonid evolution. 579

Quicke et al. (2009) separated Ophioniformes into the ‘higher Ophioniformes’ (Campopleginae, 580

Cremastinae, Anomaloninae, Ophioninae, Mesochorinae, Nesomesochorinae, and Tatogastrinae, the last 581

two not sampled here) and a ‘basal grade’ of other subfamilies (Tryphoninae, Banchinae, Tersilochinae, 582

Oxytorinae, Ctenopelmatinae, Metopiinae, Neorhacodinae, Sisyrostolinae, and Stilbopinae, the last three 583

not sampled here). Alternatively, Bennett et al. (2019) defined the ‘higher Ophioniformes’ as including 584

only Anomaloninae, Campopleginae, Cremastinae, Ophioninae, and Nesomesochorinae, but did not state 585

why. Perhaps the smaller set of taxa relates to support for this grouping across some of their analyses, or 586

maybe due to the somewhat contradictory statement of the groups’ membership in the discussion of 587

Quicke et al. (2009): “The probable monophyly of the “higher” ophioniformes (Anomaloninae, 588

Campopleginae, Cremastinae, Nesomesochorinae, Ophioninae, but not Tersilochinae nor Tatogastrinae) 589

implies a very large ichneumonid radiation associated with koinobiont endoparasitism of Lepidoptera 590

larvae (with some secondary host shifts within these groups). The hosts of Nesomesochorinae are 591

completely unknown and the Hybrizontinae may or may not be associated with this clade (p. 1355)”. 592

Further, in some of their analyses and other studies (Belshaw and Quicke, 2002; Quicke et al., 2000) 593

Metopiinae is recovered with several of these subfamilies. Here we recovered a maximally supported 594

group including Metopiinae, Hybrizontinae, Cremastinae, Anomaloninae, Ophioninae, and 595

Campopleginae. The latter six subfamilies were also maximally supported across all analyses. 596

The placement of Hybrizontinae has varied widely across different analyses, including with 597

affiliation to Ctenopelmatinae (Bennett et al., 2019), Lycorininae, Anomaloninae, and Ophioninae 598

(Quicke et al., 2009) among others (see Bennett et al., 2019; Quicke, 2015 for a detailed review). Here, 599

Hybrizontinae is consistently (albeit variably supported) as sister to Cremastinae and falls firmly within 600

the ‘higher Ophioniformes’, contrary to (Bennett et al., 2019). Ophioninae was consistently recovered as 601

the sister group to Campopleginae with maximal support across all analyses, a relationship espoused by 602

Gauld (1985) and also recovered by Klopfstein et al. (2019). This finding is contrary to Bennett et al. 603

(2019) and Wahl (1991) who recovered Cremastinae as sister to Campopleginae. Although the study of 604

Klopfstein et al. (2019) was focused on Pimpliformes, they recovered very similar relationships among 605

the Ophioniformes using an independent probe set. 606

There were some differences between the two datasets (amino acid and nt-3out), particularly for 607

Ichneumonidae. However, several of these differences could be explained by further analysis of the data. 608


https://doi.org/10.1101/2020.06.17.157719

22

For example, the relationships among Pimpliformes were completely different between the protein and 609

nucleotide datasets, as was found in a recent phylogenomics study of this lineage (Klopfstein et al., 2019). 610

Examination of codon biases showed that there were distinct differences across several lineages. 611

Diplazontinae has a codon use pattern very similar to Ichneumoniformes, and thus, this lineage may have 612

been pulled to the base of the Pimpliformes in the nt-3out analyses due to convergence. Similarly, 613

Pimplinae was only recovered as monophyletic with the amino acid analysis due to distinct codon use 614

patterns between the two tribes sampled here: Ephialtini and Pimplini (Delomeristini and Theroniini were 615

not sampled). Pimplinae has been recovered as monophyletic with morphological data (Gauld et al., 2002; 616

Wahl and Gauld, 1998), but paraphyletic with molecular data (Bennett et al., 2019; Klopfstein et al., 617

2019; Quicke et al., 2009). Interestingly, our amino acid results of Pimpliformes are most consistent with 618

the relationships proposed by Wahl and Gauld (1998), but as mentioned by Klopfstein et al. (2019), the 619

evolution of this lineage is likely the result of a rapid radiation which may require extensive genetic and 620

taxonomic sampling to resolve. 621

Although nucleotide bias has been observed before between Braconidae and Ichneumonidae in 622

select genes, mostly mitochondrial (Li et al., 2016; Quicke et al., 2020b; Sharanowski et al., 2010), here 623

we note distinct codon use patterns across hundreds of genes. Further, the outgroups used here were most 624

similar to Braconidae, suggesting that careful examination of codon biases, outgroup choice, and 625

performing differential outgroup analyses are important for phylogenetic inference of Ichneumonoidea. 626

Thus, it may not be appropriate to root braconid or ichneumonid phylogenies with its sister family. We 627

also suggest that amino acid analyses are more useful for examining relationships among 628

Ichneumonoidea, and particularly Ichneumonidae because of codon use patterns. 629

4.3 Evolution of Modes of Parasitism 630

Although the taxon sampling is low in this study, we did recover interesting patterns when 631

inferring the ancestral states for major lineages in Ichneumonoidea. In general, it appears that the ancestor 632

of Ichneumonoidea was an idiobiont ectoparasitoid, although the result was only occasionally significant 633

for idiobiosis and never for ectoparasitism. Within Braconidae, the results varied depending on the data 634

used or taxa included in the analyses. The ancestor of Braconidae was inferred as either an idiobiont 635

ectoparasitoid or a koinobiont endoparasitoid. However, only the latter inferred biology was significant, a 636

result that is consistent with Sharanowski (2009), which had denser taxonomic sampling for Braconidae. 637

Koinobiont endoparasitism is the most common biology within Braconidae (Gauld, 1988), as this biology 638

is common to several subfamilies within the cyclostomes s.l. and all of the non-cyclostomes. However, 639

the non-cyclostomes were always recovered with a koinobiont endoparasitoid ancestor, whereas the 640


https://doi.org/10.1101/2020.06.17.157719

23

cyclostomes s.s had the most support for an idiobiont ectoparasitoid ancestor, although the result varied 641

across analyses. If the ancestor of Braconidae was a koinobiont endoparasitoid, then there may have been 642

one or more transitions to idiobiosis and ectoparasitism within the cyclostomes s.s. 643

Within Ichneumonidae, the results for idiobiosis/koinobiosis were consistent across all analyses. 644

Ichneumonidae, Ichneumoniformes s.l., and Pimpliformes were all significantly reconstructed as 645

idiobionts, whereas the ancestors for Ophioniformes, with or without Tryphoninae, were significantly 646

reconstructed as koinobionts. As all Ophioniformes are koinobionts, this makes intuitive sense and 647

demonstrates that koinobiosis developed early in ichneumonid evolution. For the ecto- versus 648

endoparasitism, there was a great deal of inconsistency across the analyses. The only consistent results 649

were for ancestors of Pimpliformes and Ophioniformes (excluding Tryphoninae), which were always 650

reconstructed as endoparasitoids with varying statistical support. This suggests that Pimpliformes had an 651

idiobiont endoparasitoid ancestor. Gauld (1988) theorized that idiobiont parasitoids transitioned from 652

ectoparasitism to endoparasitism by attacking cocooned hosts, allowing for enough time to develop before 653

the host metamorphoses into an adult. However, previous analyses (Klopfstein et al., 2019) have been 654

equivocal with respect to the ancestral state of idiobiosis or koinobiosis for Pimpliformes, and the 655

diversity of biological strategies in this group may make it one of the most interesting lineages to examine 656

to understand the evolution of parasitism. The ancestor of all Ophioniformes may have had a koinobiont 657

ecto- or endoparasitoid ancestor, as results varied depending on analysis. As Tryphoninae are koinobiont 658

ectoparasitoids, this may have been a transitory step from idiobiosis to koinobiont endoparasitism, as 659

suggested by (Gauld, 1988), or it could represent a reversal in this lineage to ectoparasitism (Quicke, 660

2015). 661

The results presented here suggests that strong conclusions cannot be drawn from these data. 662

Teasing apart the full nature of how different parasitoid strategies evolved will require a very detailed 663

look at host taxa and host biology and the stage attacked (Quicke et al., 1999a; Quicke, 2015), which is 664

sorely lacking for most Ichneumonoidea. Although the sampling is likely too low to draw strong 665

conclusions about the number of transitions across Braconidae and Ichneumonidae, there certainly have 666

been transitions from koinobiosis back to idiobiosis and from ecto– to endoparasitism in both families. 667

This was advocated as well by Bennett et al. (2019) who also suggested mechanisms for transition from 668

koinobiosis back to idiobiosis, challenging the prevailing notion that this transition was rare or non-669

existent (Gauld, 1988; Quicke, 2015; Quicke et al., 2000). 670

4.4 Evolution of Endogenous Virus Elements in Wasp Genomes 671


https://doi.org/10.1101/2020.06.17.157719

24

Endogenous virus elements that produce virions (PDVs) or VLPs have been described in both 672

braconids and ichneumonids. Until recently, Braconidae was thought to have a single origin of 673

endogenous viruses in the microgastroid lineage (Beckage et al., 1994; Bézier et al., 2009; Huguet et al., 674

2012; Strand and Burke, 2015; Whitfield, 1997, 2002). A new endogenous virus descended from 675

pathogenic alphanudiviruses was discovered in some species of Fopius, opiine braconids, representing a 676

second integration event and a relatively recent one (Burke, 2019; Burke et al., 2018a). In Ichneumonidae, 677

PDVs with separate evolutionary origins from braconid PDVs have been discovered in members of 678

Banchinae and Campopleginae (Espagne et al., 2004; Lapointe et al., 2007; Norton et al., 1975; Robin et 679

al., 2019; Stoltz et al., 1981; Stoltz and Whitfield, 1992; Tanaka et al., 2007; Volkoff et al., 2010). 680

Further, at least one species of campoplegine has acquired a VLP related to nudiviruses (Pichon et al., 681

2015; Reineke et al., 2006; Rotheram, 1973). Given the robust phylogeny here, it is clear that banchines 682

and campoplegines are not closely related, and the intervening taxa have not been verified to possess 683

PDVs (but see Cummins et al., 2011). Although the ichnoviruses in Campopleginae and Banchinae come 684

from closely related virus ancestors (Béliveau et al., 2015), our data here provide strong evidence that the 685

Campopleginae and Banchinae are not sister subfamilies within the Ichneumonidae. Two possibilities 686

exist: first, that there have been two independent origins of ichnoviruses in the Ichneumonidae, or second, 687

that there was a single origin and ichnoviruses were lost in some other subfamilies between the 688

ichnovirus-producing families (Beliveau et al. 2015). In a companion study (Burke et al., 2020), a more 689

extensive search for PDVs in intervening taxa using a genome sequencing approach revealed that there 690

are no ichnovirus genes in several intervening taxa, consistent with the failure to capture ichnovirus genes 691

within these taxa using the anchored hybrid enrichment approach used in this study. 692

PDV genes are known to evolve very rapidly, presenting challenges for finding these genes in the 693

genome with targeted approaches such as PCR. Targeted enrichment approaches hold some promise for 694

sampling across a broader array of PDV genes specific to certain taxa to determine whether they may be 695

present in previously untested species and how these genes have evolved. We have found ichnovirus 696

genes in the Hyposoter and Campoletis sampled here, but they are not identified to species. However, 697

Campoletis sp. 1 was collected in Australia, where C. sonorensis is not known to occur, and thus 698

represents at least one new species with an ichnovirus in this genus. Further, we provide the first evidence 699

of PDVs in the genus Casinaria. The gene used, IVSP4, appears to undergo within lineage gene 700

expansions or contractions, which the AHE pipelines were able to detect, but will require more data from 701

more taxa to better understand their evolution. These genes were not captured in species of Dusona and 702

Campoplex, which were recovered as sister taxa, which suggests these taxa either do not have 703

ichnoviruses or they have very divergent copies. Without richer taxonomic sampling their sister 704


https://doi.org/10.1101/2020.06.17.157719

25

relationship cannot be certain. In another recent study, Dusona was recovered as sister to Casinaria 705

(Bennett et al., 2019), and a study with deeper sampling recovered conflicting placement and non-706

monophyly for several campoplegine genera (Quicke et al., 2009). 707

For the bracovirus replication gene HzNVorf128 we were able to fully capture the gene from all 708

expected bracovirus-harboring taxa and reconstruct a pattern of evolution that is consistent with species 709

relationships determined using other non-viral molecular markers. Thus, capture in Phanerotoma and 710

Schoenlandella represent confirmations in new taxa, but these were expected given that microgastroids 711

are all assumed to have bracoviruses (Whitfield, 1997, 2002). However, the other bracovirus gene (per os 712

infectivity factor p74) was not captured in full across likely bracovirus harboring taxa, most likely due to a 713

more rapid evolutionary rate. In general, these genes appear to be evolving rapidly, thus limiting the 714

ability to capture some viral genes using probes from known sequences. It is conceivable that more 715

targeted approaches that iteratively design probes focused upon single-copy genes with high sequence 716

conservation may be more effective for cross-lineage capture of divergent viral genes. 717

5. Conclusions 718

Here we developed a 541 gene probe set for anchored hybrid enrichment that was largely 719

successful for examining relationships in Ichneumonoidea and with wide utility across Hymenoptera. 720

Although we also targeted polydnavirus genes (PDVs), capture was not entirely successful across taxa 721

known to have viruses endogenized in their genomes. It is possible that a more lineage-specific targeted 722

approach to probe design could be more successful, but genome sequencing may be a more appropriate 723

method for discovering virus association and endogenization given the continually decreasing cost. 724

Higher-level relationships across Ichneumonoidea are mostly stable and well supported relationships and 725

reinforce various findings from other studies. While we focused on Ichneumonidae, the braconid 726

relationships were largely consistent with previous studies. Within Ichneumonidae, Xoridinae was the 727

most basal taxon, and the placements of Labeninae and Orthopelmatinae remain uncertain. Excluding 728

these three taxa, we recovered Ophioniformes (Ichneumoniformes (including Eucerotinae) + 729

Pimpliformes). Higher-level relationships among Ichneumonoidea have been historically difficult to 730

resolve, but anchored hybrid enrichment offers a valuable tool to recovering robust relationships in this 731

highly diverse lineage. However, our data demonstrate that systematic biases need to be thoroughly 732

investigated to prevent erroneous relationships and outgroup selection must be vetted. We found codon 733

use bias present in some lineages, suggesting amino acid data may be better suited to understanding 734

evolution in Ichneumonoidea. 735


https://doi.org/10.1101/2020.06.17.157719

26

Ancestral state reconstructions were sensitive to the taxa included or data used to infer the 736

character states. The data suggest that the ancestor of Ichneumonoidea was an idiobiont ectoparasitoid. 737

While analyses for Ichneumonidae were stable for idiobiosis/koinobiosis, they varied for ecto-versus 738

endoparasitism. Braconidae may have had a koinobiont endoparasitoid ancestor, although reconstructions 739

varied widely for the family preventing strong conclusions. The results suggest many transitions of these 740

traits within Ichneumonoidea and full understanding of the evolution of parasitoid strategies will require 741

deep taxonomic sampling and increased knowledge on the biology of this diverse lineage. At least two 742

independent acquisitions of endogenous polydnaviruses (PDVs) within Ichneumonidae are well supported 743

by the data due to the distant relationship between Banchinae and Campopleginae and lack of evidence of 744

PDVs in intervening taxa. However, probes designed to capture polydnavirus genes likely require a more 745

targeted lineage-specific approach. 746

Acknowledgements: 747

We are deeply thankful for specimens from Michael Sharkey, David Smith, and Dave Karlsson with the 748

Swedish Malaise Trap Project (SMTP). We thank Geoff Allen (University of Tasmania), Andrew Austin 749

(University of Adelaide), James Adams (La Ceiba, Honduras), and Bery Josue Almendares Romero and 750

entomological researchers at the Universidad Nacional Autónoma de Honduras for hosting during 751

collecting trips and assisting with permits. Our grateful thanks to Davide Dal Pos and Andrés Herrera-752

Flórez for some specimen identifications and to Davide Dal Pos for valuable comments on manuscript 753

drafts. A special thank-you to Andrew Bennett for many discussions about relationships across the 754

Ichneumonidae. We thank Terry Wheeler (deceased) and Chris Buddle for organizing a collecting trip to 755

Yukon through the Northern Biodiversity Program (Canada). We would like to thank John Heraty for 756

providing alignments for the probe design, which increased their utility across Hymenoptera. We are 757

grateful to Michelle Kortyna and Alyssa Bigelow at FSU’s Center for Anchored Phylogenomics for 758

assistance with sample processing. Funding support for this project was provided by the Natural Sciences 759

and Engineering Research Council (NSERC) Discovery Program to BJS and the National Science 760

Foundation (NSF) (Award Number: 1916914) to BJS and GRB. 761

References Cited: 762

Askew, R.R., Shaw, M.R., 1986. Parasitoid communities: their size, structure and development. In: 763

Waage, J., Greathead, D. (Eds.), Insect Parasitoids, 13th Symposium of Royal Entomological 764

Society of London. Academic Press, London, pp. 225-264. 765

Austin, A., Dowton, M., 2000. Hymenoptera: Evolution, Biodiversity, and Biological Control. CSIRO, 766

Canberra. 767


https://doi.org/10.1101/2020.06.17.157719

27

Ballesteros, G.I., Gadau, J., Legeai, F., Gonzalez-Gonzalez, A., Lavandero, B., Simon, J.-C., Figueroa, 768

C.C., 2017. Expression differences in Aphidius ervi (Hymenoptera: Braconidae) females reared 769

on different aphid host species. PeerJ 5, e3640. 770

Banks, J.C., Whitfield, J.B., 2006. Dissecting the ancient rapid radiation of microgastrine wasp genera 771

using additional nuclear genes. Mol. Phylogen. Evol. 41, 690–703. 772

Bauer, L.S., Liu, H., Miller, D., Gould, J., 2008. Developing a classical biological control program for 773

Agrilus planipennis (Coleoptera: Buprestidae), an invasive ash pest in North America. 774

Beckage, N.E., Gelman, D.B., 2004. Wasp parasitoid disruption of host development: Implications for 775

new biologically based strategies for insect control. Annu. Rev. Entomol. 49, 299-330. 776

Béliveau, C., Cohen, A., Stewart, D., Periquet, G., Djoumad, A., Kuhn, L., Stoltz, D., Boyle, B., Volkoff, 777

A.-N., Herniou, E.A., 2015. Genomic and proteomic analyses indicate that banchine and 778

campoplegine polydnaviruses have similar, if not identical, viral ancestors. J. Virol. 89, 8909-779

8921. 780

Belshaw, R., Dowton, M., Quicke, D.L.J., Austin, A., 2000. Estimating ancestral geographical 781

distributions: A gondwanan origin for aphid parasitoids? Proceedings of the Royal Society of 782

London B 267, 491-496. 783

Belshaw, R., Fitton, M., Herniou, E., Gimeno, C., Quicke, D.L.J., 1998. A phylogenetic reconstruction of 784

the Ichneumonoidea (Hymenoptera) based on the D2 variable region of 28S ribosomal RNA. 785

Syst. Entomol. 23, 109-123. 786

Belshaw, R., Grafen, A., Quicke, D.L.J., 2003. Inferring life history from ovipositor morphology in 787

parasitoid wasps using phylogenetic regression and discriminant analysis. Zool. J. Linn. Soc. 139, 788

213-228. 789

Belshaw, R., Quicke, D.L.J., 2002. Robustness of ancestral state estimates: evolution of life history 790

strategy in Ichneumonoid parasitoids. Syst. Biol. 51, 450-477. 791

Bennett, A.M.R., 2001. Phylogeny of Agriotypinae (Hymenoptera: Ichneumonidae), with comments on 792

the subfamily relationships of the basal Ichneumonidae. Syst. Entomol. 26, 329-356. 793

Bennett, A.M.R., Cardinal, S., Gauld, I.D., Wahl, D.B., 2019. Phylogeny of the subfamilies of 794

Ichneumonidae (Hymenoptera). J. Hymenoptera Res. 71, 1-156. 795

Bergsten, J., 2005. A review of long-branch attraction. Cladistics 21, 163-193. 796

Bézier, A., Annaheim, M., Herbiniere, J., Wetterwald, C., Gyapay, G., Bernard-Samain, S., Wincker, P., 797

Roditi, I., Heller, M., Belghazi, M., Pfister-Wilhem, R., Periquet, G., Dupuy, C., Huguet, E., 798

Volkoff, A.-N., Lanzrein, B., Drezen, J.-M., 2009. Polydnaviruses of braconid wasps derive from 799

an ancestral nudivirus. Science 323, 926-930. 800

Bézier, A., Louis, F., Jancek, S., Periquet, G., Thézé, J., Gyapay, G., Musset, K., Lesobre, J., Lenoble, P., 801

Dupuy, C., Gundersen-Rindal, D., Herniou, E.A., Drezen, J.-M., 2013. Functional endogenous 802

viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the 803

evolutionary dynamics of bracoviruses. Philosophical Transactions of the Royal Society of 804

London B: Biological Sciences 368. 805

Bigot, Y., Samain, S., Auge-Gouillou, C., Federici, B., 2008. Molecular evidence for the evolution of 806

ichnoviruses from ascoviruses by symbiogenesis. BMC Evol. Biol. 8, 253. 807

Branstetter, M.G., Danforth, B.N., Pitts, J.P., Faircloth, B.C., Ward, P.S., Buffington, M.L., Gates, M.W., 808

Kula, R.R., Brady, S.G., 2017. Phylogenomic insights into the evolution of stinging wasps and 809

the origins of ants and bees. Curr. Biol. 27, 1019-1025. 810

Broad, G., Shaw, M., Fitton, M., 2018. The Ichneumonid Wasps of Britain and Ireland (Hymenoptera: 811

Ichneumonidae). Royal Entomological Society, Telford. 812

Burke, G.R., Hines, H.M., Sharanowski, B.J., 2020. Endogenization from diverse viral ancestors is 813

common and widespread in parasitoid wasps. Genome Biology and Evolution, Submitted. 814

Burke, G.R., Simmonds, T.J., Sharanowski, B.J., Geib, S.M., 2018a. Rapid viral symbiogenesis via 815

changes in parasitoid wasp genome architecture. Mol. Biol. Evol. 35, 2463–2474. 816

Burke, G.R., Strand, M.R., 2014. Systematic analysis of a wasp parasitism arsenal. Mol. Ecol. 23, 890-817

901. 818


https://doi.org/10.1101/2020.06.17.157719

28

Burke, G.R., Thomas, S.A., Eum, J.H., Strand, M.R., 2013. Mutualistic polydnaviruses share essential 819

replication gene functions with pathogenic ancestors. PLoS Path. 9, e1003348. 820

Burke, G.R., Walden, K.K., Whitfield, J.B., Robertson, H.M., Strand, M.R., 2018b. Whole genome 821

sequence of the parasitoid wasp Microplitis demolitor that harbors an endogenous virus mutualist. 822

G3: Genes, Genomes, Genetics 8, 2875-2880. 823

Chapman, A.D., 2009. Numbers of Living Species in Australia and the World. In: Department of 824

Sustainability, E., Water, Population and Communities (Ed.). Australian Biological Resources 825

Study, Canberra, pp. 1-84. 826

Charif, D., Lobry, J.R., 2007. SeqinR 1.0-2: a contributed package to the R project for statistical 827

computing devoted to biological sequences retrieval and analysis. Structural approaches to 828

sequence evolution. Springer, pp. 207-232. 829

Chernomor, O., Von Haeseler, A., Minh, B.Q., 2016. Terrace aware data structure for phylogenomic 830

inference from supermatrices. Systematic biology 65, 997-1008. 831

Darriba, D., Taboada, G.L., Doallo, R.n., Posada, D., 2012. jModelTest 2: more models, new heuristics 832

and parallel computing. Nat. Methods 9, 772-772. 833

Derocles, S.A., Plantegenest, M., Rasplus, J.Y., Marie, A., Evans, D.M., Lunt, D.H., Le Ralec, A., 2016. 834

Are generalist Aphidiinae (Hym. Braconidae) mostly cryptic species complexes? Syst. Entomol. 835

41, 379-391. 836

Desjardins, C.A., Gundersen-Rindal, D.E., Hostetler, J.B., Tallon, L.J., Fadrosh, D.W., Fuester, R.W., 837

Pedroni, M.J., Haas, B.J., Schatz, M.C., Jones, K.M., Crabtree, J., Forberger, H., Nene, V., 2008. 838

Comparative genomics of mutualistic viruses of Glyptapanteles parasitic wasps. Genome Biology 839

9, 1-17. 840

Dowton, M., Belshaw, R., Austin, A.D., Quicke, D.L.J., 2002. Simultaneous molecular and 841

morphological analysis of Braconid relationships (Insecta: Hymenoptera: Braconidae) indicates 842

independent mt-tRNA gene inversions within a single wasp family. J. Mol. Evol. 54, 210-226. 843

Federici, B.A., Bigot, Y., 2003. Origin and evolution of polydnaviruses by symbiogenesis of insect DNA 844

viruses in endoparasitic wasps. J Insect Physiol 49. 845

Gadau, J., Helmkampf, M., Nygaard, S., Roux, J., Simola, D.F., Smith, C.R., Suen, G., Wurm, Y., Smith, 846

C.D., 2012. The genomic impact of 100 million years of social evolution in seven ant species. 847

Trends Genet. 28, 14-21. 848

Gauld, I., Wahl, D.B., Broad, G.R., 2002. The suprageneric groups of the Pimplinae (Hymenoptera: 849

Ichneumonidae): a cladistic re-evaluation and evolutionary biological study. Zool. J. Linn. Soc. 850

136, 421-485. 851

Gauld, I.D., 1985. The phylogeny, classification and evolution of parasitic wasps of the subfamily 852

Ophioninae (Ichneumonidae). Bulletin of the British Museum (Natural History), Entomology 853

Series 51, 61-185. 854

Gauld, I.D., 1988. Evolutionary patterns of host utilization by ichneumonoid parasitoids (Hymenoptera: 855

Ichneumonidae and Braconidae). Biol. J. Linn. Soc. 35, 351-377. 856

Gauld, I.D., Mound, L.A., 1982. Homoplasy and the delineation of holophyletic genera in some insect 857

groups. Syst. Entomol. 7, 73-86. 858

Gauld, I.D., Wahl, D.B., 2002. The Eucerotinae: a Gondwanan origin for a cosmopolitan group of 859

Ichneumonidae? J. Nat. Hist. 36, 2229-2248. 860

Gauthier, J., Drezen, J.-M., Herniou, E.A., 2017. The recurrent domestication of viruses: major 861

evolutionary transitions in parasitic wasps. Parasitology, 1-13. 862

Giarla, T.C., Esselstyn, J.A., 2015. The challenges of resolving a rapid, recent radiation: empirical and 863

simulated phylogenomics of Philippine shrews. Syst. Biol. 64, 727-740. 864

Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms 865

and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 866

3.0. Syst. Biol. 59, 307-321. 867


https://doi.org/10.1101/2020.06.17.157719

29

Haddad, S., Shin, S., Lemmon, A.R., Lemmon, E.M., Svacha, P., Farrell, B., ŚLIPIŃSKI, A., Windsor, 868

D., McKenna, D.D., 2018. Anchored hybrid enrichment provides new insights into the phylogeny 869

and evolution of longhorned beetles (Cerambycidae). Syst. Entomol. 43, 68-89. 870

Hamilton, C.A., Lemmon, A.R., Lemmon, E.M., Bond, J.E., 2016. Expanding anchored hybrid 871

enrichment to resolve both deep and shallow relationships within the spider tree of life. BMC 872

Evol. Biol. 16, 212. 873

Harmon, L.J., Weir, J.T., Brock, C.D., Glor, R.E., Challenger, W., 2008. GEIGER: investigating 874

evolutionary radiations. Bioinformatics 24, 129-131. 875

Herniou, E.A., Huguet, E., Thézé, J., Bézier, A., Periquet, G., Drezen, J.-M., 2013. When parasitic wasps 876

hijacked viruses: genomic and functional evolution of polydnaviruses. Philosophical Transactions 877

of the Royal Society of London B: Biological Sciences 368. 878

Hoang, D.T., Chernomor, O., Von Haeseler, A., Minh, B.Q., Vinh, L.S., 2017. UFBoot2: improving the 879

ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518-522. 880

Honeybee Genome Sequencing Consortium, 2006. Insights into social insects from the genome of the 881

honeybee Apis mellifera. Nature 443, 931-949. 882

Huber, J.T., 2009. Biodiversity of Hymenoptera. In: Foottit, R.G., Adler, P.H. (Eds.), Insect Biodiversity: 883

Science and Society. Blackwell Publishing, London, pp. 303-323. 884

Huguet, E., Serbielle, C., Moreau, S.J., 2012. Evolution and origin of polydnavirus virulence genes. In: 885

Beckage, N.E., Drezen, J.-M. (Eds.), Parasitoid Viruses: Symbionts and Pathogens. Academic 886

Press, San Diego, CA, pp. 63-78. 887

Johnson, B.R., Borowiec, M.L., Chiu, J.C., Lee, E.K., Atallah, J., Ward, P.S., 2013. Phylogenomics 888

resolves evolutionary relationships among ants, bees, and wasps. Curr. Biol. 23, 2058-2062. 889

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K., von Haeseler, A., Jermiin, L.S., 2017. ModelFinder: fast 890

model selection for accurate phylogenetic estimates. Nature methods 14, 587. 891

Katoh, K., Standley, D.M., 2013a. MAFFT multiple sequence alignment software version 7: 892

improvements in performance and usability. Mol. Biol. Evol. 30, 772-780. 893

Katoh, K., Standley, D.M., 2013b. MAFFT multiple sequence alignment software version 7: 894

Improvements in performance and usability. Mol Biol Evol 30, 772–780. 895

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., 896

Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., Drummond, A., 2012. Geneious 897

Basic: An integrated and extendable desktop software platform for the organization and analysis 898

of sequence data. Bioinformatics 28, 1647-1649. 899

Klopfstein, S., Kropf, C., Quicke, D.L.J., 2010. An Evaluation of phylogenetic informativeness profiles 900

and the molecular phylogeny of Diplazontinae (Hymenoptera, Ichneumonidae). Syst. Biol. 59, 901

226-241. 902

Klopfstein, S., Langille, B., Spasojevic, T., Broad, G.R., Cooper, S.J., Austin, A.D., Niehuis, O., 2019. 903

Hybrid capture data unravel a rapid radiation of pimpliform parasitoid wasps (Hymenoptera: 904

Ichneumonidae: Pimpliformes). Systematic Entomology, Online Early View. 905

doi:10.1111/syen.12333 44, 361-383. 906

Kroemer, J.A., Webb, B.A., 2004. Polydnavirus genes and genomes: emerging gene families and new 907

insights into polydnavirus replication. Annu. Rev. Entomol. 49, 431. 908

Kula, R.R., Knight, K.S., Rebbeck, J., Bauer, L.S., Cappaert, D.L., Gandhi, K.J., 2010. Leluthia astigma 909

(Ashmead)(Hymenoptera: Braconidae: Doryctinae) as a parasitoid of Agrilus planipennis 910

Fairmaire (Coleoptera: Buprestidae: Agrilinae), with an assessment of host associations for 911

Nearctic species of Leluthia Cameron. Proc. Entomol. Soc. Wash. 112, 246-257. 912

Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 913

for bigger datasets. Molecular biology and evolution 33, 1870-1874. 914

Lanfear, R., Calcott, B., Ho, S.Y.W., Guindon, S., 2012. PartitionFinder: combined selection of 915

partitioning schemes and substitution models for phylogenetic analyses. Mol. Biol. Evol. 29, 916

1695-1701. 917


https://doi.org/10.1101/2020.06.17.157719

30

Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B., 2016. PartitionFinder 2: new methods 918

for selecting partitioned models of evolution for molecular and morphological phylogenetic 919

analyses. Mol. Biol. Evol. 34, 772-773. 920

Lapointe, R., Tanaka, K., Barney, W.E., Whitfield, J.B., Banks, J.C., Béliveau, C., Stoltz, D., Webb, 921

B.A., Cusson, M., 2007. Genomic and morphological features of a banchine polydnavirus: 922

comparison with Bracoviruses and Ichnoviruses. J. Virol. 81, 6491-6501. 923

Laurenne, N.M., Broad, G.R., Quicke, D.L.J., 2006. Direct optimization and multiple alignment of 28S 924

D2–D3 rDNA sequences: problems with indels on the way to a molecular phylogeny of the 925

cryptine ichneumon wasps (Insecta: Hymenoptera). Cladistics 22, 442-473. 926

Lavine, M.D., Beckage, N.E., 1995. Polydnaviruses: potent mediators of host insect immune dysfunction. 927

Parasitol. Today 11, 368-378. 928

Lemmon, A.R., Emme, S.A., Lemmon, E.M., 2012. Anchored hybrid enrichment for massively high-929

throughput phylogenomics. Syst. Biol. 62, 1-18. 930

Li, Q., Wei, S.-J., Tang, P., Wu, Q., Shi, M., Sharkey, M.J., Chen, X.-X., 2016. Multiple lines of evidence 931

from mitochondrial genomes resolve phylogenetic relationships of parasitic wasps in Braconidae. 932

Genome Biology and Evolution 8, 2651-2662. 933

Lorenzi, A., Ravallec, M., Eychenne, M., Jouan, V., Robin, S., Darboux, I., Legeai, F., Gosselin-Grenet, 934

A.-S., Sicard, M., Stoltz, D., Volkoff, A.-N., 2019. RNA interference identifies domesticated viral 935

genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps. 936

PLoS Path. 15, e1008210. 937

Lovallo, N., McPheron, B., Cox-Foster, D., 2002. Effects of the polydnavirus of Cotesia congregata on 938

the immune system and development of non-habitual hosts of the parasitoid. J. Insect Physiol. 48, 939

517-526. 940

Maddison, W.P., Maddison, D.R., 2019. Mesquite: a modular system for evolutionary analysis. Version 941

3.61. http://mesquiteproject.org. 942

Matsumoto, R., 2016. Molecular phylogeny and systematics of the Polysphincta group of genera 943

(Hymenoptera, Ichneumonidae, Pimplinae). Syst. Entomol. 41, 854-864. 944

Meyer, M., Kircher, M., 2010. Illumina sequencing library preparation for highly multiplexed target 945

capture and sequencing. Cold Spring Harbor Protocols 6, pdb.prot5448. 946

Miller, M.A., Holder, M.T., Vos, R., Midford, P.E., Liebowitz, T., Chan, L., Hoover, P., Warnow, T., 947

2009. The CIPRES Portals. URL:http://www.webcitation.org/5imQlJeQa. Accessed: 2009-08-04. 948

Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of 949

large phylogenetic trees. Gateway Computing Environments Workshop (GCE), 2010. IEEE, pp. 950

1-8. 951

Misof, B., Liu, S., Meusemann, K., Peters, R.S., Donath, A., Mayer, C., Frandsen, P.B., Ware, J., Flouri, 952

T., Beutel, R.G., 2014. Phylogenomics resolves the timing and pattern of insect evolution. 953

Science 346, 763-767. 954

Murphy, N., Banks, J., Whitfield, J.B., Austin, A., 2008. Phylogeny of the parasitic microgastroid 955

subfamilies (Hymenoptera: Braconidae) based on sequence data from seven genes, with an 956

improved time estimate of the origin of the lineage. Mol. Phylogen. Evol. 47, 378-395. 957

Nguyen, L.-T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2014. IQ-TREE: a fast and effective 958

stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268-959

274. 960

Paradis, E., Claude, J., Strimmer, K., 2004. APE: Analyses of Phylogenetics and Evolution in R language. 961

Bioinformatics 20, 289-290. 962

Peixoto, L., Allen, G.R., Ridenbaugh, R.D., Quarrell, S.R., Withers, T.M., Sharanowski, B.J., 2018. 963

When taxonomy and biological control researchers unite: species delimitation of Eadya 964

parasitoids (Braconidae) and consequences for biological control of invasive paropsine pests of 965

Eucalyptus. PLoS One 13, e0201276. 966

Pennacchio, F., Strand, M.R., 2006. Evolution of developmental strategies in parasitic Hymenoptera. 967

Annu. Rev. Entomol. 51, 233-258. 968


https://doi.org/10.1101/2020.06.17.157719

31

Perna, N.T., Kocher, T.D., 1995. Patterns of nucleotide composition at fourfold degenerate sites of animal 969

mitochondrial genomes. Journal of molecular evolution 41, 353-358. 970

Peters, R.S., Krogmann, L., Mayer, C., Donath, A., Gunkel, S., Meusemann, K., Kozlov, A., 971

Podsiadlowski, L., Petersen, M., Lanfear, R., 2017. Evolutionary history of the Hymenoptera. 972

Curr. Biol. 27, 1013-1018. 973

Pichon, A., Bézier, A., Urbach, S., Aury, J.-M., Jouan, V., Ravallec, M., Guy, J., Cousserans, F., Thézé, 974

J., Gauthier, J., 2015. Recurrent DNA virus domestication leading to different parasite virulence 975

strategies. Science Advances 1, e1501150. 976

Piekarski, P.K., Carpenter, J.M., Lemmon, A.R., Moriarty Lemmon, E., Sharanowski, B.J., 2018. 977

Phylogenomic evidence overturns current conceptions of social evolution in wasps (Vespidae). 978

Mol. Biol. Evol. 35, 2097-2109. 979

Pimentel, D., Wilson, C., McCullum, C., Huang, R., Dwen, P., Flack, J., Tran, Q., Saltman, T., Cliff, B., 980

1997. Economic and environmental benefits of biodiversity. Bioscience 47, 747-757. 981

Pitz, K., Dowling, A.P.G., Sharanowski, B.J., Boring, C.A.B., Seltmann, K.C., Sharkey, M.J., 2007. 982

Phylogenetic relationships among the Braconidae (Hymenoptera: Ichneumonoidea) as proposed 983

by Shi et al.: a reassessment. Mol. Phylogen. Evol. 43, 338-343. 984

Price, P.W., 1980. Evolutionary Biology of Parasites. Princeton University Press. 985

Quicke, D., LeRalec, A., Vilhelmsen, L., 1999a. Ovipositor structure and function in the parasitic 986

Hymenoptera. Rendiconti, 197-239. 987

Quicke, D.L., Austin, A.D., Fagan�Jeffries, E.P., Hebert, P.D., Butcher, B.A., 2020a. Molecular 988

phylogeny places the enigmatic subfamily Masoninae within the Ichneumonidae, not the 989

Braconidae. Zool. Scr. 49, 64-71. 990

Quicke, D.L., Austin, A.D., Fagan�Jeffries, E.P., Hebert, P.D., Butcher, B.A., 2020b. Recognition of the 991

Trachypetidae stat. n. as a new extant family of Ichneumonoidea (Hymenoptera), based on 992

molecular and morphological evidence. Syst. Entomol. Early online view: 993

doi:10.1111/syen.12426. 994

Quicke, D.L.J., 2015. The Braconid and Ichneumonid Parasitoid Wasps: Biology, Systematics, Evolution 995

And Ecology. Wiley-Blackwell, Chichester. 996

Quicke, D.L.J., Fitton, M.G., Notton, D.G., Broad, G.R., 2000. Phylogeny of the subfamilies of 997

Ichneumonidae (Hymenoptera): A simultaneous molecular and morphological analysis. In: 998

Austin, A.D., Dowton, M. (Eds.), Hymenoptera: Evolution, Biodiversity and Biocontrol. CSIRO 999

Publishing, Collingwood, VIC, pp. 74-83. 1000

Quicke, D.L.J., Laurenne, N.M., Fitton, M.G., Broad, G.R., 2009. A thousand and one wasps: a 28S 1001

rDNA and morphological phylogeny of the Ichneumonidae (Insecta: Hymenoptera) with an 1002

investigation into alignment parameter space and elision. J. Nat. Hist. 43, 1305-1421. 1003

Quicke, D.L.J., Lopez-Vaamonde, C., Belshaw, R., 1999b. The basal Ichneumonidae (Insecta, 1004

Hymenoptera): 28S D2 rDNA considerations of the Brachycyrtinae, Labeninae, Paxylommatinae 1005

and Xoridinae. Zool. Scr. 28, 203-210. 1006

Quicke, D.L.J., van Achterberg, C., 1990. Phylogeny of the subfamilies of the family Braconidae 1007

(Hymenoptera: Ichneumonoidea). Zoologische Verhandelingen Leiden 258, 1-180. 1008

Rambaut, A., Suchard, M.A., Drummond, A.J., 2013. Tracer v1.6. 1009

http://tree.bio.ed.ac.uk/software/tracer/ [Accessed Oct 2015]. 1010

Revell, L.J., 2012. phytools: an R package for phylogenetic comparative biology (and other things). 1011

Methods in Ecology and Evolution 3, 217-223. 1012

Rodriguez, J.J., Fernández-Triana, J.L., Smith, M.A., Janzen, D.H., Hallwachs, W., Erwin, T.L., 1013

Whitfield, J.B., 2013. Extrapolations from field studies and known faunas converge on 1014

dramatically increased estimates of global microgastrine parasitoid wasp species richness 1015

(Hymenoptera: Braconidae). Insect Conservation and Diversity 6, 530-536. 1016

Rokyta, D.R., Lemmon, A.R., Margres, M.J., Aronow, K., 2012. The venom-gland transcriptome of the 1017

eastern diamondback rattlesnake (Crotalus adamanteus). BMC Genomics 13, 1-23. 1018


https://doi.org/10.1101/2020.06.17.157719

32

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., 1019

Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference 1020

and model choice across a large model space. Syst. Biol. 61, 539-542. 1021

Rota-Stabelli, O., Lartillot, N., Philippe, H., Pisani, D., 2013. Serine codon-usage bias in deep 1022

phylogenomics: pancrustacean relationships as a case study. Systematic biology 62, 121-133. 1023

Rousse, P., Quicke, D.L., Matthee, C.A., Lefeuvre, P., Noort, S., 2016. A molecular and morphological 1024

reassessment of the phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae). 1025

Zool. J. Linn. Soc. 178, 128-148. 1026

Sadd, B.M., Barribeau, S.M., Bloch, G., de Graaf, D.C., Dearden, P., Elsik, C.G., Gadau, J., 1027

Grimmelikhuijzen, C.J., Hasselmann, M., Lozier, J.D., Robertson, H.M., Smagghe, G., Stolle, E., 1028

Van Vaerenbergh, M., Waterhouse, R.M., Bornberg-Bauer, E., Klasberg, S., Bennett, A.K., 1029

Câmara, F., Guigó, R., Hoff, K., Mariotti, M., Munoz-Torres, M., Murphy, T., Santesmasses, D., 1030

Amdam, G.V., Beckers, M., Beye, M., Biewer, M., Bitondi, M.M., Blaxter, M.L., Bourke, A.F., 1031

Brown, M.J., Buechel, S.D., Cameron, R., Cappelle, K., Carolan, J.C., Christiaens, O., 1032

Ciborowski, K.L., Clarke, D.F., Colgan, T.J., Collins, D.H., Cridge, A.G., Dalmay, T., Dreier, S., 1033

du Plessis, L., Duncan, E., Erler, S., Evans, J., Falcon, T., Flores, K., Freitas, F.C., Fuchikawa, T., 1034

Gempe, T., Hartfelder, K., Hauser, F., Helbing, S., Humann, F.C., Irvine, F., Jermiin, L.S., 1035

Johnson, C.E., Johnson, R.M., Jones, A.K., Kadowaki, T., Kidner, J.H., Koch, V., Köhler, A., 1036

Kraus, F.B., Lattorff, H.M.G., Leask, M., Lockett, G.A., Mallon, E.B., Antonio, D.S.M., Marxer, 1037

M., Meeus, I., Moritz, R.F., Nair, A., Näpflin, K., Nissen, I., Niu, J., Nunes, F.M., Oakeshott, 1038

J.G., Osborne, A., Otte, M., Pinheiro, D.G., Rossié, N., Rueppell, O., Santos, C.G., Schmid-1039

Hempel, R., Schmitt, B.D., Schulte, C., Simões, Z.L., Soares, M.P., Swevers, L., Winnebeck, 1040

E.C., Wolschin, F., Yu, N., Zdobnov, E.M., Aqrawi, P.K., Blankenburg, K.P., Coyle, M., 1041

Francisco, L., Hernandez, A.G., Holder, M., Hudson, M.E., Jackson, L., Jayaseelan, J., Joshi, V., 1042

Kovar, C., Lee, S.L., Mata, R., Mathew, T., Newsham, I.F., Ngo, R., Okwuonu, G., Pham, C., Pu, 1043

L.-L., Saada, N., Santibanez, J., Simmons, D., Thornton, R., Venkat, A., Walden, K.K., Wu, Y.-1044

Q., Debyser, G., Devreese, B., Asher, C., Blommaert, J., Chipman, A.D., Chittka, L., Fouks, B., 1045

Liu, J., O’Neill, M.P., Sumner, S., Puiu, D., Qu, J., Salzberg, S.L., Scherer, S.E., Muzny, D.M., 1046

Richards, S., Robinson, G.E., Gibbs, R.A., Schmid-Hempel, P., Worley, K.C., 2015. The 1047

genomes of two key bumblebee species with primitive eusocial organization. Genome Biology 1048

16, 76. 1049

Santos, B.F., 2017. Phylogeny and reclassification of Cryptini (Hymenoptera, Ichneumonidae, 1050

Cryptinae), with implications for ichneumonid higherâ level classification. Syst. Entomol. 1051

Savard, J., Tautz, D., Richards, S., Weinstock, G.M., Gibbs, R.A., Werren, J.H., Tettelin, H., Lercher, 1052

M.J., 2006. Phylogenomic analysis reveals bees and wasps (Hymenoptera) at the base of the 1053

radiation of Holometabolous insects. Genome Res. 16, 1334-1338. 1054

Schliep, K.P., 2011. phangorn: phylogenetic analysis in R. Bioinformatics 27, 592-593. 1055

Schmidt, O., Theopold, U., Strand, M., 2001. Innate immunity and its evasion and suppression by 1056

hymenopteran endoparasitoids. Bioessays 23, 344. 1057

Schwarz, G., 1978. Estimating the dimension of a model. The Annals of Statistics 6, 461-464. 1058

Sharanowski, B.J., 2009. Hymenopteran molecular phylogenetics: From Apocrita to Braconidae 1059

(Ichneumonidae). Entomology. University of Kentucky, Lexington, p. 113. 1060

http://archive.uky.edu/bitstream/10225/11013/Sharanowski_Dissertation_10205_12009.pdf. 1061

Sharanowski, B.J., Dowling, A.P.G., Sharkey, M.J., 2011. Molecular phylogenetics of Braconidae 1062

(Hymenoptera: Ichneumonoidea) based on multiple nuclear genes, and its implications for 1063

classification. Syst. Entomol. 36, 549-572. 1064

Sharanowski, B.J., Robbertse, B., Walker, J., Voss, S.R., Yoder, R., Spatfora, J., Sharkey, M.J., 2010. 1065

Expressed sequence tags reveal Proctotrupomorpha (minus Chalcidoidea) as sister to Aculeata 1066

(Hymenoptera: Insecta). Mol. Phylogen. Evol. 57, 101-112. 1067

Sharkey, M.J., 2007. Phylogeny and classification of Hymenoptera. Zootaxa 1668, 521-548. 1068


https://doi.org/10.1101/2020.06.17.157719

33

Sharkey, M.J., Carpenter, J.M., Vilhelmsen, L., Heraty, J., Ronquist, F., Deans, A.R., Dowling, A.P.G., 1069

Hawks, D., Schulmeister, S., Wheeler, W.C., 2010. Morphology and molecules, the first 1070

comprehensive, total evidence, phylogenetic analysis of the Hymenoptera. Seventh Internal 1071

Congress of Hymenopterists, Koszeg, Hungary. 1072

Sharkey, M.J., Wahl, D.B., 1992. Cladistics of the Ichneumonoidea (Hymenoptera). J. Hymenoptera Res. 1073

1, 15-24. 1074

Sheng, M.-L., Broad, G., Sun, S.-P., 2012. A new genus and species of Collyriinae (Hymenoptera, 1075

Ichneumonidae). J. Hymenoptera Res. 25, 103-125. 1076

Simon, S., Strauss, S., von Haeseler, A., Hadrys, H., 2009. A phylogenomic approach to resolve the basal 1077

pterygote divergence. Mol. Biol. Evol. 26, 2719-2730. 1078

Strand, M.R., 2012. Polydnavirus gene products that interact with the host immune system. In: Beckage, 1079

N.E., Drezen, J.-M. (Eds.), Parasitoid Viruses: Symbionts and Pathogens. Elsevier, London, UK, 1080

pp. 149-161. 1081

Strand, M.R., Burke, G.R., 2012. Polydnaviruses as symbionts and gene delivery systems. PLoS Path. 8, 1082

e1002757. 1083

Strand, M.R., Burke, G.R., 2015. Polydnaviruses: From discovery to current insights. Virology 479, 393-1084

402. 1085

Team, R.C., 2016. R: A language and environment for statistical computing. R Core Team. R foundation 1086

for statistical computing, Vienna, Austria. 1087

Thézé, J., Bézier, A., Periquet, G., Drezen, J.-M., Herniou, E.A., 2011. Paleozoic origin of insect large 1088

dsDNA viruses. Proceedings of the National Academy of Sciences 108, 15931-15935. 1089

Tvedte, E., Walden, K.K., McElroy, K.E., Werren, J., Forbes, A.A., Hood, G.R., Logsdon, J.M., Feder, 1090

J.L., Robertson, H.M., 2019. Genome of the parasitoid wasp Diachasma alloeum, an emerging 1091

model for ecological speciation and transitions to asexual reproduction. bioRxiv, 545277. 1092

Vaidya, G., Lohman, D.J., Meier, R., 2011. SequenceMatrix: concatenation software for the fast assembly 1093

of multi�gene datasets with character set and codon information. Cladistics 27, 171-180. 1094

van Achterberg, C., Quicke, D.L.J., 1992. Phylogeny of the subfamilies of the family Braconidae: A 1095

reassessment assessed. Cladistics 8, 237-264. 1096

Vargas, R.I., Peck, S.L., McQuate, G.T., Jackson, C.G., Stark, J.D., Armstrong, J.W., 2001. Potential for 1097

areawide integrated management of Mediterranean fruit fly (Diptera: Tephritidae) with a braconid 1098

parasitoid and a novel bait spray. J. Econ. Entomol. 94, 817-825. 1099

Veijalainen, A., Wahlberg, N., Broad, G.R., Erwin, T.L., Longino, J.T., Sääksjärvi, I.E., 2012. 1100

Unprecedented ichneumonid parasitoid wasp diversity in tropical forests. Proceedings of the 1101

Royal Society B: Biological Sciences 279, 4694-4698. 1102

Volkoff, A.-N., Drezen, J.-M., Cusson, M., Webb, B.A., 2012. The organization of genes encoding 1103

ichnovirus structural proteins. In: Beckage, N.E., Drezen, J.-M. (Eds.), Parasitoid Viruses: 1104

Symbionts and Pathogens. Elsevier, London, pp. 33-45. 1105

Volkoff, A.-N., Jouan, V., Urbach, S., Samain, S., Bergoin, M., Wincker, P., Demettre, E., Cousserans, 1106

F., Provost, B., Coulibaly, F., 2010. Analysis of virion structural components reveals vestiges of 1107

the ancestral ichnovirus genome. PLoS Path. 6, e1000923. 1108

Wahl, D., 1990. A review of the mature larvae of Diplazontinae, with notes on larvae of Acaenitinae and 1109

Orthocentrinae and proposal of two new subfamilies (Insecta: Hymenoptera, Ichneumonidae). J. 1110

Nat. Hist. 24, 27-52. 1111

Wahl, D.B., 1986. Larval structures of oxytorines and their significance for the higher classification of 1112

some Ichneumonidae (Hymenoptera). Syst. Entomol. 11, 117-127. 1113

Wahl, D.B., 1991. The status of Rhimphoctona, with special reference to the higher categories within 1114

Campopleginae and the relationships of the subfamily (Hymenoptera: Ichneumonidae). 1115

Transactions of the American Entomological Society (1890-) 117, 193-213. 1116

Wahl, D.B., 1993a. Cladistics of the ichneumonid subfamily Labeninae (Hymenoptera: Ichneumonidae). 1117

Entomologica Generalis 18, 91-105. 1118


https://doi.org/10.1101/2020.06.17.157719

34

Wahl, D.B., 1993b. Family Ichneumonidae. In: Goulet, H., Huber, J.T. (Eds.), Hymenoptera of the 1119

World: an Identification Guide to Families. Research Branch, Agriculture Canada and Canada 1120

Communications Group, Ottawa, Canada, pp. 395-442. 1121

Wahl, D.B., Gauld, I.D., 1998. The cladistics and higher classification of the Pimpliformes 1122

(Hymenoptera: Ichneumonidae). Syst. Entomol. 23, 265-298. 1123

Wallau, G.L., Capy, P., Loreto, E., Le Rouzic, A., Hua-Van, A., 2016. VHICA, a new method to 1124

discriminate between vertical and horizontal transposon transfer: Application to the mariner 1125

family within Drosophila. Molecular biology and evolution 33, 1094-1109. 1126

Webb, B., Steele, K.H., Fath-Goodin, A., 2017. Compositions and methods for pest control management. 1127

U.S. Patent No. 9,770,033. Washington, DC: U.S. Patent and Trademark Office, Issued Sept 26, 1128

2017. 1129

Webb, B.A., Strand, M.R., Dickey, S.E., Beck, M.H., Hilgarth, R.S., Barney, W.E., Kadash, K., Kroemer, 1130

J.A., Lindstrom, K.G., Rattanadechakul, W., Shelby, K.S., Thoetkiattikul, H., Turnbull, M.W., 1131

Witherell, R.A., 2006. Polydnavirus genomes reflect their dual roles as mutualists and pathogens. 1132

Virology 347. 1133

Werren, J.H., Richards, S., Desjardins, C.A., Niehuis, O., Gadau, J., Colbourne, J.K., The Nasonia 1134

Genome Working, G., 2010. Functional and evolutionary insights from the genomes of three 1135

parasitoid Nasonia Species. Science 327, 343-348. 1136

Wharton, R.A., Shaw, M.R., Sharkey, M.J., Wahl, D.B., Woolley, J.B., Whitfield, J.B., Marsh, P.M., 1137

Johnson, W., 1992. Phylogeny of the subfamilies of the family Braconidae (Hymenoptera: 1138

Ichneumonoidea): A reassessment. Cladistics 8, 199-235. 1139

Whitfield, J., 1990. Parasitoids, polydnaviruses and endosymbiosis. Parasitol. Today 6, 381-384. 1140

Whitfield, J.B., 1997. Molecular and morphological data suggest a common origin for the polydnaviruses 1141

among braconid wasps. Naturwissenschaften 84, 502-507. 1142

Whitfield, J.B., 2002. Estimating the age of the polydnavirus/braconid wasp symbiosis. Proc. Natl. Acad. 1143

Sci. USA 99, 7508-7513. 1144

Whitfield, J.B., Kjer, K.M., 2008. Ancient rapid radiations of insects: challenges for phylogenetic 1145

analysis. Annu. Rev. Entomol. 53, 449-472. 1146

Whitfield, J.B., O’Connor, J.M., 2012. Molecular systematics of wasp and polydnavirus genomes and 1147

their coevolution. In: Beckage, N.E., Drezen, J.-M. (Eds.), Parasitoid Viruses: Symbionts and 1148

Pathogens. Elsevier, London, UK, pp. 89-97. 1149

Wickham, H., 2016. ggplot2: elegant graphics for data analysis. Springer. 1150

Wickham, H., Francois, R., Henry, L., Müller, K., 2015. dplyr: A grammar of data manipulation. R 1151

package version 0.4 3. 1152

Wild, A.L., Marsh, P.M., Whitfield, J.B., 2013. Fast-evolving homoplastic traits are best for species 1153

identification in a group of Neotropical wasps. PloS One 8, e74837. 1154

Xi, Z., Ruhfel, B.R., Schaefer, H., Amorim, A.M., Sugumaran, M., Wurdack, K.J., Endress, P.K., 1155

Matthews, M.L., Stevens, P.F., Mathews, S., 2012. Phylogenomics and a posteriori data 1156

partitioning resolve the Cretaceous angiosperm radiation Malpighiales. Proceedings of the 1157

National Academy of Sciences 109, 17519-17524. 1158

Yu, D., Van Achterberg, C., Horstmann, K., 2016. Taxapad 2016. Ichneumonoidea 2015 (Biological and 1159

taxonomical information), Taxapad Interactive Catalogue Database on flash-drive. 1160

www.taxapad.com. Nepean, Ontario, Canada. 1161

Yu, D.S., Horstmann, K., van Achterberg, C., 2012. Taxapad: scientific names for information 1162

management. Biological and Taxonomical Information: Ichneumonoidea 2011. CD. Taxapad, 1163

Vancouver. 1164

Zaldivar-Riverón, A., Mori, M., Quicke, D.L.J., 2006. Systematics of the cyclostome subfamilies of 1165

braconid parasitic wasps (Hymenoptera: Ichneumonoidea): A simultaneous molecular and 1166

morphological Bayesian approach. Mol. Phylogen. Evol. 38, 130-145. 1167


https://doi.org/10.1101/2020.06.17.157719

35

Zaldivar-Riverón, A., Shaw, M.R., Sáez, A.G., Mori, M., Belokobylskij, S.A., Shaw, S.R., Quicke, 1168

D.L.J., 2008. Evolution of the parasitic wasp subfamily Rogadinae (Braconidae): phylogeny and 1169

evolution of lepidopteran host ranges and mummy characteristics. BMC Evol. Biol. 8, 329. 1170

Zhang, C., Stadler, T., Klopfstein, S., Heath, T.A., Ronquist, F., 2015. Total-evidence dating under the 1171

fossilized birth–death process. Syst. Biol. 65, 228-249. 1172

Zhang, M.Y., Ridenbaugh, R.D., Sharanowski, B.J., 2017. Integrative taxonomy improves understanding 1173

of native beneficial fauna: revision of the Nearctic Peristenus pallipes complex (Hymenoptera: 1174

Braconidae) and implications for release of exotic biocontrol agents. Syst. Entomol. 42, 596-608. 1175

1176

1177

1178

1179

1180

1181

1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194


https://doi.org/10.1101/2020.06.17.157719

36

Table 1. Taxon sampling used in this study, including voucher codes (where appropriate), 1195

taxonomy, and country of origin. Genome indicates publicly available genomes used in the study. 1196

Bolded taxa are genomes sequenced in this study. 1197

Outgroups

Voucher Code FSU

Code

Family Subfamily Genus species Country

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

Code

Higher-Group Subfamily Genus species Country

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


https://doi.org/10.1101/2020.06.17.157719

37

Ichneumonidae

Voucher Code FSU

Code

Higher Group Subfamily Genus species Country

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


https://doi.org/10.1101/2020.06.17.157719

38

Table 2. Model tests for maximum likelihood ancestral state reconstructions for character 1 1201

(idiobiosis vs. koinobiosis) and character 2 (ecto- vs. endoparasitism). Two models were tested, the 1202

Markov k-state one parameter (Mk1), which has an equal rate for character gains and losses, and the 1203

asymmetrical two-parameter Markov k-state (Assym2), which applies a separate rate to gains and losses. 1204

Likelihood ratio tests were performed to assess which model better fit the data, which are chi-square (χ2) 1205

distributed and critical values was used to assess significance at p<0.01. The best supported model for 1206

each source phylogeny is highlighted in gray. Source phylogenies include the amino acid maximum 1207

likelihood phylogeny (AA w/ OGs) with all taxa (Fig 1. A-C); and with outgroups (OGs) trimmed prior to 1208

analysis (AA w/o OGs); the amino acid phylogeny with Labeninae excluded (AA w/ OGs Lab-out, Fig. 1209

S10); and with outgroups trimmed (AA w/o OGs Lab-out); the nucleotide dataset with the third position 1210

removed (nt-3out, Fig. S4). The other three source phylogenies are based on the amino acid dataset (Fig. 1211

1A-C), either with (AA Bracs only; AA Ichs only) or without Labeninae excluded (Fig. S10; AA Ichs 1212

only Lab-out), and all taxa were trimmed except the family of interest: Braconidae (Bracs) or 1213

Ichneumonidae (Ichs). 1214

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


https://doi.org/10.1101/2020.06.17.157719

39

Table 3. Summary of ancestral state reconstructions across several source phylogenies for 1220

character 1 (idiobiosis vs. koinobiosis) and character 2 (ecto- vs. endoparasitism). Color coding and 1221

the proportional likelihoods (PLH) are listed for the most likely character state. Source phylogenies are 1222

detailed in Table 2. 1223

1224

AA

AA

(no OGs)

AA

(Lab-

out)

AA

(no OGs,

Lab-out) nt-3out

AA Bracs

only

AA Ichs

only

AA Ichs

only

(Lab-

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


https://doi.org/10.1101/2020.06.17.157719

40

Figure Legends: 1231

Figure 1, A-C. Maximum-likelihood (ML) phylogeny of Ichneumonoidea based on 479 genes 1232

analyzed as amino acids in IQ-tree. A) Summary tree with Braconidae and Ichneumonidae collapsed 1233

for viewing; B) Braconidae; C) Ichneumonidae. Branch supports are summarized in a 5 cell box, with the 1234

first two cells representing the amino acid analysis, the second two representing the ML phylogeny 1235

inferred from nucleotide data with the 3rd position removed (nt-3out, see also Fig. S4), and the last box 1236

representing the Bayesian analysis of the nt-3out dataset (see also Fig. S5). For the ML analyses, two 1237

supports are given in the first and second boxes, respectively: the Shimodaira-Hasegawa approximate 1238

Likelihood Ratio Test statistic (aLRT) and the Ultra-Fast bootstrap value (UFb). The Bayesian posterior 1239

probability is reported in the last cell (PP). See branch support legend for color scheme of values for 1240

nodal supports. Taxa with an asterisk after the name have an uncertain identification at the lowest 1241

taxonomic rank indicated. The scale bar indicates the number of substitutions per site for branch lengths. 1242

Figure 2, A-B. Summary of two ancestral state reconstructions (ASR) using maximum likelihood 1243

for A) Idiobiosis versus koinobiosis and B) Ecto- versus endoparasitism. The collapsed tree visualized 1244

here is based on the maximum likelihood phylogeny of amino acid data with Labeninae removed (Fig. 1245

S10). Pie charts show the proportional likelihood (PLH) for each character state and an asterisk near the 1246

chart indicates the reconstruction as significant for the state with the highest PLH (see also Table 3). Pie 1247

charts above the nodes have all taxa included, whereas pie charts below the nodes were ASRs run with all 1248

taxa removed except the family of interest for both Braconidae and Ichneumonidae. 1249

Figure 3, A-C. Bayesian phylogenies of three polydnavirus (PDV) genes: A) IVSP4, B) 1250

HzNVorf128, and C) per os infectivity factor p74. Sequences were either captured with 1251

anchored hybrid enrichment probes (probe), obtained from newly sequenced genomes (genome-1252

Illumina), or were used as original reference sequences for probe capture and are available on 1253

GenBank (GenBank ID provided). Taxon voucher codes are listed next to genus name for newly 1254

sequenced taxa (FSU code, Table 1). 1255

1256

1257

1258

1259

1260

1261


https://doi.org/10.1101/2020.06.17.157719

0.02

98/86

67/53

Tryphoninae

Other Ophioniformes

Pimpliformes

Ichneumoniformes s.l.

OrthopelmatinaeXoridinae

NonCyclostomes

Cyclostomes s.s.

Aphidioid

Ophioniformes

Cyclostomes s.l.

0.02

98/86

67/53

Other Ophioniformes

Pimpliformes

Xoridinae

NonCyclostomes

Aphidioid

Ophioniformes

Cyclostomes s.l.

Tryphoninae

Ichneumoniformes s.l.

Orthopelmatinae

Cyclostomes s.s.

Idiobiont

Koinobiont

Ectoparasitism

Endoparasitism

A

B

*

*

*

*

***

*

**

*

*

* *

*

*

*

*

*

*

*

*

*

*

*

Braconidae

Ichneumonidae

Braconidae

Ichneumonidae


https://doi.org/10.1101/2020.06.17.157719

Aphidiinae_PauesiaAphidiinae_Praon

Opiinae_Diachasma_alloeumAlysiinae_Chorebus

Helconinae_Eumacrocentrus_americanus

Braconinae_BraconRogadinae_Aleiodes

Rhyssalinae_RhyssalusPambolinae_Pambolus

Ichneutinae_Ichneutes

Cheloninae_Phanerotoma

Agathidinae_BassusMicrogastrinae_Microplitis_demolitor

Orgilinae_Orgilus_ablusus

Cardiochilinae_Schoenlandella

Brachistinae_Diospilus

Euphorinae_Meteorus

Brachistinae_ApoblacusBrachistinae_Blacus

Acampsohelconinae_Urosigalphus

Cheloninae_Chelonus

Apidae_Apis_mellifera

Harpegnathos_saltator

Megachilidae_Megachile_rotundaPteromalidae_Nasonia_vitripennis

Apidae_Bombus_impatiens

Solenopsis_invictaPogonomyrmex_barbatus

Acromyrmex_echinatior

Camponotus_floridanus

Atta_cephalotes

Linepithema_humile

Proteinnt-3out

Branch support legend

Branch support scale 99 - 100

95 - < 99

80 - < 9550 - < 800 - < 50

branch not recovered

Braconidae

Ichneumonidae

Aphidioid

Cyclostome s.s.

Alysioid

Microgastroid

Helconoid

A. Summary Tree With Outgroups

0.02

LRT UFb LRT UFb PP

ML B

Formicidae(Ants)

Non-cyclostomes

B. BraconidaeCyclostome s.l.

Bees

Ichneumonoidea


https://doi.org/10.1101/2020.06.17.157719

0.02

Xoridinae_OdontocolonLabeninae_Labium

Labeninae_Certonotus1Labeninae_Certonotus2

Orthopelmatinae_OrthopelmaEucerotinae_Euceros

Adelognathinae_AdelognathusCryptinae_Mesostenus

Ichneumoninae_IchneumoniniIchneumoninae_Gavrana1

Cryptinae_Echthrus

Ichneumoninae_AkymichenumonIchneumoninae_GavranaAcaenitinae_Spilopteron

Collyriinae_Collyria

Pimplinae_Dolichomitus

Pimplinae_Ecthromorpha

Pimplinae_Scambus

Pimplinae_Pimpla_pPimplinae_Pimpla_m

Pimplinae_Eriostethus

Tryphoninae_ExentenusTryphoninae_NeteliaTryphoninae_Phytodietus

Banchinae_Lissonota

Banchinae_Lissonota1*

Banchinae_Meniscomorpha1Banchinae_Meniscomorpha2

Banchinae_GlyptaBanchinae_Australglypta

Campopleginae_Hyposoter*Campopleginae_Casinaria1

Ophioninae_Ophion

Campopleginae_Campoletis1Campopleginae_Campoletis2

Campopleginae_Campoplex2Campopleginae_Dusona2

Campopleginae_Dusona1

Campopleginae_Campoplex1*

Campopleginae_Dusona3*

Cyllocerinae_CylloceriaDiplazontinae_Homotropus

Orthocentrinae_OrthocentrusPoemeniinae_Neoxorides

Tersilochinae_Phradis*Tersilochinae_Stethantyx

Oxytorinae_OxytorusCtenopelmatinae_MegaceriaCtenopelmatinae_AnoncusCtenopelmatinae_Mesoleius*

Mesochorinae_Mesochorus

Metopiinae_SpudaeusMetopiinae_Metopius

Hybrizontinae_HybrizonCremastinae_Cremastus

Cremastinae_PristomerusAnomaloninae_Habronyx

Anomaloninae_Agrypon

Metopiinae_Exochus

Pimpliformes

Ophioniformes

HigherOphioniformes

Ichneumoniformes

C. Ichneumonidae

Labeniformes

Orthopelmatiformes

Xoridiformes

Proteinnt-3out

Branch support legend

Branch support scale 99 - 100

95 - < 99

80 - < 9550 - < 800 - < 50

branch not recovered

LRT UFb LRT UFb PP

ML B

Pimplini

Ephialtini


https://doi.org/10.1101/2020.06.17.157719

Casinaria sp.1 I10005 (probe)

Campoletis sp.1 I10007 Copy 2 (probe)

Campoletis sp.1 Copy 1 I10007 (probe)

Hyposoter sp.* I9995 Copy 2 (probe)

Campoletis sp.1 I10007 Copy 3 (probe)

Hyposoter sp.* I9995 Copy 1 (probe)

Campoletis sp.2 I2339 (probe)

Campoletis sonorensis (HO082184)

Hyposoter didymator IVSPER2 (ADI40475.1)

Hyposoter didymator IVSPER3 (ADI40477.1)

1 1

0.99

0.99

1

0.81

1

1

A. IVSP4

Chelonus sp. I2351 (probe)

Chelonus inanitus (CAR40202.1)

Cotesia congregata (CAR82249.1)

Phanerotoma sp. I15714 (genome - Illumina)

Schoenlandella sp. I2349 (probe)

Microplitus demolitor (EZA45377.1)1

1

1

B. HzNVorf128

Chelonus inanitus (CAR40192.1)

Cotesia congregata (CAR31575.1)

Chelonus sp. I2351 (probe)

1

1

C. per os infectivity factor p74

0.06 0.08

0.05

Microplitus demolitor (EZA44986.1)


https://doi.org/10.1101/2020.06.17.157719

authors · pichon et al., 2015; strand and burke, 2012). given their immense diversity,...

Documents