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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: barb.sharanowski@ucf.edu
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(which was not certified by peer review) is the author/funder.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
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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.
(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
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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
(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
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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
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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
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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
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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
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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
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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
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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
(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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
(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
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
(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
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
(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
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
(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
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
(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
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
(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
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
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(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
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
(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
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
(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
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
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(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
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
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(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
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
(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
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
(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
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
(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
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
(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
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)
(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
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