Mitochondrial Phylogenomics of the Hymenoptera T ⁎ Pu Tanga,B,C, Jia-Chen Zhua, Bo-Yin Zhenga, Shu-Jun Weid, Michael Sharkeye, Xue-Xin Chena,B, , Alfried P

Molecular Phylogenetics and Evolution 131 (2019) 8–18

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Mitochondrial phylogenomics of the Hymenoptera T ⁎ Pu Tanga,b,c, Jia-chen Zhua, Bo-yin Zhenga, Shu-jun Weid, Michael Sharkeye, Xue-xin Chena,b, , Alfried P. Voglerc,f a Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China b State Key Laboratory of Rice Biology and Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, China c Department of Life Sciences, Natural History Museum, London SW7 5BD, UK d Institute of Plant and Environmental Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China e Department of Entomology, University of Kentucky, Lexington, KY 40546-0091, USA f Department of Life Sciences, Silwood Park Campus, Imperial College London, Ascot SL5 7PY, UK

ARTICLE INFO ABSTRACT

Keywords: The insect order Hymenoptera presents marvelous morphological and ecological diversity. Higher-level hyme- Mitogenomics nopteran relationships remain controversial, even after recent phylogenomic analyses, as their taxon sampling Hymenoptera was limited. To shed light on the origin and diversification of Hymenoptera, in particular the poorly studied Evaniomorpha basal relationships Parasitica, we undertook phylogenetic analyses of 40 newly and 43 previously sequenced mitochondrial gen- Dated tree omes representing all major clades of Hymenoptera. Various Bayesian inferences using different data partitions and phylogenetic methods recovered similar phylogenetic trees with strong statistical support for almost all nodes. Novel findings of the mitogenomic phylogeny mainly affected the three infraorders Ichneumonomorpha, Proctotrupomorpha and Evaniomorpha, the latter of which was split into three clades. Basal relationships of Parasitica recovered Stephanoidea + (Gasteruptiidae + Aulacidae) as the sister group to Ichneumonomorpha + (Trigonalyoidea + Megalyroidea). This entire clade is sister to Proctotrupomorpha, and Ceraphronoidea + Evaniidae is sister to Aculeata (stinging wasps). Our divergence time analysis indicates that major hymenopteran lineages originated in the Mesozoic. The radiation of early apocritans may have been triggered by the Triassic–Jurassic mass extinction; all extant families were present by the Cretaceous.

1. Introduction (UCEs) (Branstetter et al., 2017) and transcriptomes (Peters et al., 2017) did not fully resolve this problem due to uneven and un- Hymenoptera are one of the four hyperdiverse orders of holometa- representative sampling of taxa. In particular, these studies were lim- bolan insects, which are traditionally subdivided into two suborders, ited in the diversity of taxa traditionally assigned to the suborders Symphyta and Apocrita. The Symphyta has been considered a para- Evaniomorpha and Proctotrupomorpha (Tang and Vogler, 2017). The phyletic lineage for a long time. Members of the Symphyta are mostly Parasitica, a paraphyletic concept, is one of two suborders of the phytophagous, with the exception of the Orussidae, which is the only Apocrita, and is highly species-rich. The other suborder, Aculeata parasitoid symphytan family and in most analyses is grouped as the (stinging wasps) is well studied (Branstetter et al., 2017; Peters et al., sister to Apocrita (wasps, bees, and ants) (Sharkey, 2007; Song et al., 2017). Based on morphological and fossil evidence, Rasnitsyn (1988) 2016b). More than 90% of the Hymenoptera are Apocrita, which is suggested the Parasitica can be divided into three lineages, Ichneu- monophyletic and comprises parasitic and aculeate (stinging) wasps monomorpha, Proctotrupomorpha and Evaniomorpha, the latter two of (Huber, 2009; Sharkey, 2007). Apocritan species exhibit a wide range which are considered sister groups. This system is currently used as the of lifestyles, including parasitoidism, predation, eusociality, pollen framework for grouping major lineages outside of the well-studied, feeding and gall-making (Huber, 2009). They play vital roles in biolo- robustly monophyletic Aculeata (Castro and Dowton, 2006; Dowton, gical control, ecosystem function, and agriculture. 2001; Dowton and Austin, 1994; Dowton et al., 1997; Heraty et al., Higher-level relationships among parasitic wasps are still con- 2011; Klopfstein et al., 2013; Mao et al., 2015; Ronquist et al., 1999; troversial. Genomic data sets based on Ultra-Conserved Elements Sharkey et al., 2011; Vilhelmsen et al., 2010; Wei et al., 2014).

⁎ Corresponding author at: Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China. E-mail address: [email protected] (X.-x. Chen). https://doi.org/10.1016/j.ympev.2018.10.040 Received 14 June 2018; Received in revised form 2 September 2018; Accepted 30 October 2018 Available online 03 November 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved. P. Tang et al. Molecular Phylogenetics and Evolution 131 (2019) 8–18

Ichneumonomorpha has been considered as the sister of Aculeata in Celera Assembler v7.0 (Myers et al., 2000), IDBA_tran (Peng et al., some morphological and early molecular studies (Dowton and Austin, 2012) and Ray (Boisvert et al., 2012) for assembling each DNA library 1994; Dowton et al., 1997; Rasnitsyn and Zhang, 2010; Vilhelmsen and subsequently combined contigs from the three assemblies in Gen- et al., 2010). Other studies recovered the Aculeata nested within the eious (Biomatters Ltd) to obtain a non-redundant set. The position of paraphyletic Evaniomorpha, whereas a sister group relationship be- tRNA genes was conducted on the tRNAscan-SE search server (Lowe tween the Ichneumonomorpha and the Proctotrupomorpha was sug- and Eddy, 1997), with a Cove cutoff score of 5, and on the Mitos gested by most subsequent molecular analyses (Dowton, 2001; Heraty WebServer (Bernt et al., 2013). When not detected by either approach, et al., 2011; Klopfstein et al., 2013; Mao et al., 2015; Sharkey et al., we confirmed the tRNA positions by sequence alignment with their 2011). Another scenario places Ichneumonomorpha together with the homologs in related species. Protein-coding and rRNA genes were in- evaniomorph superfamilies Trigonalyoidea + Megalyroidea (Mao itially annotated with Mitos and edited in Geneious through compar- et al., 2015). Similarly, Proctotrupomorpha was not recovered as ison to other hymenopteran mitochondrial genomes. Mitogenomes monophyletic in early studies (Dowton et al., 1997; Ronquist et al., from the sequencing mixtures were assigned to individual species using 1999). Subsequently, more comprehensive analyses supported Procto- the cox1 region against sequences in the BOLD system, which could trupomorpha, while the internal relationships remained equivocal connect barcode sequences to the specimen identifications provided by (Heraty et al., 2011; Klopfstein et al., 2013; Mao et al., 2015; Peters taxonomists responsible for the database entries (Ratnasingham and et al., 2017; Sharkey et al., 2011). The most volatile clade is Evanio- Hebert, 2007), which unequivocally placed each mitogenome at least to morpha, whose monophyly has not been consistently supported in ei- family level. All other partial or complete mtDNAs downloaded from ther morphological or molecular analyses (Branstetter et al., 2017; GenBank were re-annotated following the approach described above for Heraty et al., 2011; Klopfstein et al., 2013; Mao et al., 2014, 2015; uniform annotations. Peters et al., 2017; Rasnitsyn and Zhang, 2010; Sharkey et al., 2011). Mitochondrial genomes of the Hymenoptera exhibit many unique 2.2. Mitochondrial genome feature analysis characteristics, such as an exceptionally high A + T content (Wei et al., 2014), frequent gene rearrangement (Dowton et al., 2009a), high sub- Gene rearrangements were inferred using CREx which establishes stitution rates (Oliveira et al., 2008) and a strong base composition bias the steps on a phylogenetic tree to transform one gene order into an- (Dowton and Austin, 1997). Gene rearrangements are usually confined other (Bernt et al., 2007). The assessment was based on the analysis of to specific lineages, which may be useful for phylogenetic reconstruc- breakpoints and changes in orientation determined in pairwise com- tion at lower taxonomic levels, e.g., at the subfamily level in Braco- parisons of protein coding, tRNA and rRNA genes and AT regions in the nidae. However, compositional heterogeneity across lineages and con- hymenopteran mitochondrial genomes, relative to the ancestral insect vergent evolution of gene rearrangements (Dowton et al., 2009b; Wei gene order. We calculated the proportion of GC-rich amino acid codons et al., 2014) confound phylogenetic inferences in higher-level phylo- (GARP) and GC content in MEGA version 7.0 (Kumar et al., 2016), and genetic studies of Hymenoptera. Several studies were compromised due the mean Ka (non-synonymous substitution rate) and Ks (synonymous to sparse taxon sampling (Mao et al., 2015; Wei et al., 2014). The substitution rate) values between each taxon and all other taxa in number of available mitochondrial genomes remains lower than for DNASP version 5.0 (Librado and Rozas, 2009). We obtained tip-to-root other large orders (Coleoptera and Lepidoptera) and biased towards distances with Dendroscope 3 (Huson and Scornavacca, 2012). certain groups (bees and ants). Mitochondrial genome sequencing of Apocrita has been difficult with conventional Sanger technology due to high A + T content and frequent gene rearrangements. 2.3. Phylogenetic analyses In this study, we used a genome skimming approach for low-cost sequencing of mitogenomes from mixed total genomic DNA for dense The data matrices used in the phylogenetic analyses were generated sampling of major hymenopteran lineages to infer basal relationships, in eight combinations, based on first and second codon positions only especially in the unstable clades, and to date the origin of the main (P12), the P12 and two rRNA genes (P12RNA), all codon positions hymenopteran lineages. The resulting phylogenetic hypothesis will (P123), and the P123 and RNA genes (P123RNA); these four schemes enable insights into the origin and diversity of lifestyles in were analyzed with and without the inclusion of outgroup taxa. Hymenoptera. Protein-coding genes and two rRNA genes were aligned using the G- INS-i and Q-INS-i algorithms implemented in MAFFT version 7.205, 2. Materials and methods respectively (Golubchik et al., 2007; Katoh and Standley, 2013). The alignment of nucleotide sequences was guided by the amino acid se- 2.1. Mitochondrial genome sequencing and NGS data processing quence alignment using the Perl script TranslatorX version 1.1 (Abascal et al., 2010). We concatenated the aligned sequences using the Perl DNA extractions were performed on 40 species which were firstly script FASconCAT-G version 1.0 (Kück and Longo, 2014), then used identified at least to family level by taxonomic specialists (Table S1), PartitionFinder version 1.1.1 (Lanfear et al., 2012) to simultaneously concentrating on clades of Hymenoptera in which sampling has been choose the best partition schemes and substitution models for each scarce or whose placement was controversial (Evaniomorpha and matrix. The maximum possible partition schemes inputted into the Proctotrupomorpha). We extracted genomic DNA from individual adult PartitionFinder software was defined by codon position for nucleotide specimens using a DNeasy tissue kit (Qiagen, Hilden, Germany) and sequences of protein-coding genes and RNA genes. The search of determined the DNA concentration for each sample using the Qubit models was set to be “Mrbayes” and “BIC”, using the greedy algorithm, dsDNA high-sensitivity kit (Invitrogen). DNAs were combined in with branch lengths estimated, to search for the best-fit partitioning roughly equimolar concentrations and split into two pools for con- model. The branch lengths were set as linked. struction of Illumina TruSeq Nano libraries, separating close relatives Bayesian inference (BI) was conducted with MrBayes (Ronquist (same family). The libraries were sequenced on an Illumina HiSeq se- et al., 2012b) using the chosen partitions and model parameters (Table quencer (2*250 bp). S2). Four simultaneous Markov chains were run for 10 million gen- Mitogenome assembly mostly followed a bioinformatics pipeline of erations, with tree sampling occurring every 1000 generations and a Crampton-Platt et al. (2015), starting with the FastqExtract script to burn-in of 25% of trees. Maximum Likelihood analyses were performed filter out mitochondrial sequences against a reference dataset including with RAxML (Stamatakis, 2014) under the GTRGAMMA model. For the 144 hymenopteran mitochondrial genomes from 106 different species analysis of each dataset, 200 runs were conducted to find the highest- (downloaded from the Genbank database on 25 April 2016). We used likelihood tree, followed by analysis of 1000 bootstrap replicates.

9 P. Tang et al. Molecular Phylogenetics and Evolution 131 (2019) 8–18

2.4. Phylogenetic hypothesis tests

Critical nodes deﬁning basal relationships in Hymenoptera were M M M M – – analyzed by four-cluster likelihood mapping in TREE-PUZZLE version P M 5.2 (Schmidt et al., 2002; Schmidt and von Haeseler, 2007). The method produces a graph of the tree-likeness for the three possible quartet topologies, shown as a triangle whose three corners represent – M M – the three alternative topologies. The data points represent the support for each topology based on quartets of terminals drawn from the tree at random (but representing one terminal from each of the focal taxa), and M – the graph can be represented as three or seven areas that record the – distribution of topologies. The matrix of protein coding genes was used With outgroup MMM MMM MMM – M M for this test. The GTR model was applied to the two nucleotide align- PPPMM ments (4 Gamma rates and invariable sites). Rate heterogeneity was set as mixed for all models.

2.5. Divergence time estimation M M M M – P M

We analyzed the concatenated DNA sequence alignment using BEAST 1.8.3 (Drummond et al., 2012). Partitioning of data and model – selection was chosen as previously identiﬁed by PartitionFinder. We M used the best topology from the MrBayes analyses as ﬁxed input topology and used BEAST to estimate branch lengths under uncorrelated M relaxed clocks and a Yule speciation process. We performed two in- – dependent MCMC runs for 50 million generations, sampled every 5000 generations. Burn-in sample sizes and convergence stationarity were Without outgroup MMM MMM ML MMM –– – – –– – – – –– – – –– – – –– – – –– – – –– – – examined using Tracer 1.6. The consensus tree was generated using PPPMMM Tree Annotator 1.8.3. Trees were calibrated using 19 fossils (Table 2). For each calibration point we chose a separate prior selecting 'uniform' with a given minimum and maximum age. M M M M P M 3. Results

Out of 42 morphologically identified species included in the two shotgun sequencing libraries, we obtained 40 full or partial mitochondrial genome assemblies. Two specimens belonging to Mymaridae and Mymarommatoidea failed to produce mitogenomes, presumably due to their small body size and insufficient yield of DNA reads. All newly sequenced mitogenomes contained the full set of 13 protein-coding fi With outgroup MMM MMM MMM MMM MM M M MM MMM M M M M MM M M genes, but the tRNA genes were missing in a few cases. Identi cation MM MPPPMMM M using barcodes and morphology were identical at least to the family level.

3.1. Mitochondrial genome organization M M M M – – – – – P M

Gene arrangements were assessed for the 40 newly and 106 previously sequenced unique mitochondrial genomes relative to the pu- M M tative ancestral insect gene order. We scored mitochondrial gene orders M for each of 15 higher taxonomic groups of Hymenoptera (Table S3). Gene arrangements occur in all of these lineages, showing at least one translocated tRNA. Prior to this study, identical gene rearrangement – – – erent dataset and analytical approach. only had been observed within the same family or genus. However, two ff distantly related species, the sawfly Xyela sp. (Xyelidae) and the ant MMM MMM MMMMMMM MMMM BI Without outgroup PCG PCG12MMM PCGRNA PCG12RNA PCG PCG12 PCGRNA PCG12RNA PCG PCG12 PCGRNA PCG12RNA PCG PCG12 PCGRNA PCG12RNA Camponotus atrox (Formicidae), exhibited the same rearrangement due PPPMMM to the convergent shuffling of two tRNAs (trnQ and trnM). Gene rearrangements in the Symphyta, Stephanoidea, Aulacidae, Megalyroidea, Platygastroidea, Proctotrupoidea, Diaproidea and Evaniidae only affected the position of tRNA genes. In addition, Gasteruptiidae, Trigonaloidea, Cynipoidea, Chalcidoidea and Ceraphronoidea exhibited rearrangements of protein coding genes, while Ichneumonoidea and Aculeata included two (Cotesia vestalis and Venturia canescens) and one species (Cephalonomia gallicola), respectively, with such gene order changes. Interestingly, rearrangements of , not recovered; M, Monophyly; P, paraphyly; PCG, 1st, 2nd and 3rd codon positions of 13 protein-coding genes; PCG12, 1st and 2nd codon positions of 13 protein-coding genes; BI, Bayesian analysis; ML,

the rRNA genes only occurred in one species, Megalyra sp. – (Megalyroidea). Consistency of mitochondrial rearrangements with the Evan-b Clade Ichn Ichn + Evan-b Procto Evan-c Evan-a Evan-c + Acul (Ichn + Evan-b) + Evan-a ((Ichn + Evan-b) + Evan-a) + Procto M Symp Acul

phylogenetic tree was tested with the CREx software. Some major types Table 1 Summary of the major clades recovered by di Note: maximum likelihoodProctotrupomorpha; Acul, analysis; Aculeata; Symp, Symphyta. Ichn, Ichneumonomorpha; Evan-a, Stephanoidea + (Gasteruptiidae + Aulacidae); Evan-b, Trigonalyoidea + Megalyroidea; Evan-c, Ceraphronoidea + Evaniidae; Procto,

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Table 2 Fossils used in this study for estimation of divergence time of major clades.

Species Age (Ma)/minimum age constraint for Calibration group Soft maximum bound (97.5% Reference group probability)

Pseudoxyelocerus bascharagensis 178 Tenthredinoidea 174.1–182.7 Nel et al. (2004) Kronostephanus zigrasi 97 Stephanoidea 93.9–100.5 Engel and Grimaldi (2013) Baissa magna 119 Aulacidae 113–125 Rasnitsyn (1991) Manlaya anglica 142 Gasteruptiidae 139.8–145 Rasnitsyn and Jarzembowski (1998) Radiophron ibericus 106 Ceraphronoidea 100.5–113 Ortega-Blanco et al. (2010) Cretevania concordia 131 Evaniidae 129.4–132.9 Rasnitsyn and Jarzembowski (1998) Cleistogaster buriatica 160 Megalyroidea 163.5–157.3 Rasnitsyn (1975) Turgonalus cooperi 127 Trigonaloidea 125–129.4 Rasnitsyn and Jarzembowski (1998) Cretobraconus maculatus 148 Braconidae 145–152.1 Rasnitsyn and Sharkey (1988) Amplicella beipiaoensis 131 Ichneumonidae 129.4–132.9 Zhang and Rasnitsyn (2003) Khutelchalcis gobiensis 127 Chalcidoidea 125–129.4 Rasnitsyn et al. (2004) Coramia minuta 142 Diaprioidea 139.8–145 Rasnitsyn and Jarzembowski (1998) Dahurocynips dahurica 118 Cynipoidea 113–125 Rasnitsyn and Kovalev (1988) Archaeopelecinus tebbei 160 Proctotrupoidea 157.3–163.5 Shih et al. (2009) Proteroscelio gravatus 127 Platygastroidea 125–129.4 Johnson et al. (2008) Priorvespa longiceps 123 Vespidae 122.5–125 Carpenter and Rasnitsyn (1990) Haidomyrmodes mammuthus 103 Formicidae 99.6–105.3 Perrichot et al. (2007) Pompilopterus wimbledoni 142 Apoidea 139.8–145 Rasnitsyn and Jarzembowski (1998) Melittosphex burmensis 96 Anthophila 93.9–100.5 Poinar and Danforth (2006) of rearrangements involving protein coding genes were limited to specifically interested in possible nucleotide compositional biases and particular clades, such as the inversion of the cox1–nad2–cox2 segment rate heterogeneity that might group the three Evaniomorpha clades in Gasteruptiidae and of cox3–atp6–atp8–cox2–cox1 in Chalcidoidea, falsely with other clades, preventing their recovery as a monophyletic and a translocation of cox1 to a position between cox3 and nad5 group. GC content (ranging from 18% to 32%) and the corresponding (nad3–cox1–nad5) in Trigonaloidea. However, most major clades were preponderance of GARP amino acids differed substantially among taxa not diagnosable by rearranged regions. (Fig. 3). If considered in the context of the phylogenetic tree, these parameters varied greatly even between close relatives, but there were also lineage-wide patterns. Specifically, the clade composed of Eva- 3.2. Phylogenetic analysis niomorpha-c and Aculeata showed a generally increased level of GC

and Ks against most of the other clades, including Evaniomorpha-a and We conducted BI and ML analyses on the eight concatenated nu- Evaniomorpha-b that resembled more strongly the values for Ichneu- cleotide sequences (PCG123, PCG123RNA, PCG12 and PCG12RNA with monomorpha to which they were allied in the tree. or without outgroup) (Table 1). All analyses robustly supported the Finally, we used Aligroove to test for data heterogeneity within and paraphyly of Symphytaand the monophyly of Ichneumonomorpha, as among the major lineages (Fig. 4). Several taxa were highlighted by this expected. All BI analyses and most of the ML analyses supported test, more so when 3rd codon positions were included and in particular Proctotrupomorpha and Aculeata as monophyletic groups, whereas when adding the rRNA genes into the analysis, but generally these Evaniomorpha were polyphyletic and separated into three major clades. outliers affected individual terminals only, indicating the lack of con- One of these evaniomorph lineages, Trigonalyoidea + Megalyroidea founding signal at the level of major lineages based on this test. (denoted Evaniomorpha-b), was recovered consistently as sister group to Ichneumonomorpha, corroborating the results of Mao et al. (2015). 3.3. Divergence time estimation The second evaniomorph lineage grouped Gasteruptiidae with Aula- cidae, as sister to Stephanoidea (= Evaniomorpha-a). This was found in A chronogram for Hymenoptera divergence dates and topologies almost all BI analyses except the PCG12RNA dataset without outgroups, based on whole mitochondrial protein coding genes is shown in Fig. 5. as well as in all the ML analyses in the PCG123 matrix. The third clade The base of the Hymenoptera tree was dated to 276 Million years ago of Evaniomorpha was Ceraphronoidea + Evaniidae (= Evaniomorpha- (Mya) (266–286 Mya 95% CI). The clade comprising Or- c), which was recovered in most BI analyses, except for the PCG12 ussidae + Apocrita diverged from the other Symphyta 239 Mya dataset without outgroups, but none of the ML analyses supported this (232–247 Mya 95% CI) and Apocrita diverged from Orussoidea 232 result (Fig. 1; Figs. S1–S15). Mya (225–239 Mya 95% CI). Apocrita diverged into two clades 224 The relationships of the three Evaniomorpha clades and their re- Mya (217–230 Mya) and then all the major lineages within Apocrita lationships with the Proctotrupomorpha and Ichneumonomorpha were diverged around 200 Mya. The ‘eusocial clade’ comprising ants, vespids assessed by four-cluster likelihood mapping (on the protein-coding and bees diverged from its sister (other aculeates) between 184 Mya genes). First, we tested relationships among at the deepest level, col- (stem age; 177–192 Mya) and 170 Mya (crown age; 178–159 Mya). The lapsing the Evaniomorpha-a and Evaniomorpha-b together with most recent common ancestor of bees was estimated to have arisen 100 Ichneumonomorpha into a single clade, which weakly supported the Mya (98–100 Mya). sister relationships with the Proctotrupomorpha over the two other possible groupings with either Evaniomorpha-c or Aculeata (Fig. 2), in accordance the findings from BI analyses (Fig. 1). Secondly, we con- 4. Discussion ducted four-cluster analysis within the Evaniomorpha-Ichneumono- morpha clade, which supported the Megalyroidea + Trigonaloidea 4.1. Phylogeny of Hymenoptera (Evaniomorpha-b) as sister to Ichneumonomorpha. This arrangement was preferred over the sister relationship with Evaniidae + Cer- Mitochondrial genomes were at times thought to be an ineffective aphronoidea (Evaniomorpha-c) or with Ichneumonomorpha (Fig. 2). tool for phylogenetics due to compositional heterogeneity or high rates The evidence against the monophyly of Evaniomorpha was further of change, but denser sampling could solve these problems as shown examined for possible artifacts from long-branch attraction. We were previously for beetles (Timmermans et al., 2015), termites

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Fig. 1. Phylogenetic tree of Hymenoptera. The tree is based on protein coding genes and Bayesian inference with an optimal partitioning scheme selected with PartitionFinder. Branch labels are the Bayesian posterior probabilities.

(Bourguignon et al., 2015) and true bugs (Li et al., 2017). Our study which is identical to the ML analysis of Heraty et al. (2011). Although now shows that mitochondrial genomes are a useful tool for con- Mao et al. (2015) proposed Cynipoidea as the sister group to the Dia- structing robust trees of Hymenoptera, despite their extremely high rate prioidea + Chalcidoidea, most of the recent studies place Cynipoidea of change compared to virtually all other insect groups (Song et al., closer to Platygastroidea (Castro et al., 2006; Dowton, 2001; Klopfstein 2016a). The new data add to recent genomic studies of this order by et al., 2013). The infraorder Evaniomorpha is polyphyletic, agreeing increasing the sampling density in key parts of the tree to address with most previous studies (Castro and Dowton, 2006; Gibson, 1999; questions in particular about the monophyly and relative positions of Heraty et al., 2011; Klopfstein et al., 2013; Mao et al., 2014, 2015; the three apocritan infraorders Ichneumonomorpha, Procto- Rasnitsyn and Zhang, 2010; Ronquist et al., 1999; Sharkey et al., 2011; trupomorpha and Evaniomorpha. Wei et al., 2014). Recently, Rasnitsyn and Zhang (2010) proposed that We concentrated our sequencing on the poorly sampled Parasitica, Evaniomorpha (sensu lato) be divided into three distinct groups, Ste- and added only a few sequences to the existing entries for Symphyta phanomorpha (Stephanoidea), Ceraphronomorpha (Ceraphronoidea, and Aculeata. The phylogenetic tree recovers the expected paraphyly of Megalyroidea and Trigonaloidea), and a reduced Evaniomorpha (sensu the Symphyta, while Orussoidea forms a sister lineage with the sub- stricto, Evanioidea). Our results also show that Evaniomorpha (sensu order Apocrita, as shown in most previous studies (Dowton et al., lato) is split into three parts, supported by the four-clusters likelihood 2009a; Mao et al., 2015; Sharkey et al., 2011; Song et al., 2016b; Wei mapping test, i.e., (1) Trigonalyoidea + Megalyroidea (= Evanio- et al., 2014). The suborder Apocrita is a well-established monophyletic morpha-b), as the sister group to Ichneumonomorpha, as already pro- group, but its internal relationships have been controversial (Ashmead, posed in Mao et al. (2015); (2) Ceraphronoidea + Evaniidae (= Eva- 1896; Dowton and Austin, 1994; Dowton et al., 2009a; Heraty et al., niomorpha-c), which combined are the sister group of Aculeata. This 2011; Klopfstein et al., 2013; Mao et al., 2015; Ronquist et al., 1999; grouping diﬀers from Mao et al. (2014, 2015) who proposed Cer- Sharkey et al., 2011; Wei et al., 2014). Three infraorders Ichneumo- aphronoidea + Evanioidea; (3) Stephanoidea + (Gaster- nomorpha, Proctotrupomorpha and Vespomorpha (Aculeata) are well- uptiidae + Aulacidae) (= Evaniomorpha-a), which is well supported, supported by our results, as recovered in most previous studies. Within unlike in some previous studies that recognized Stephanoidea as an the Proctotrupomorpha, our results strongly support Platygastroidea + early branch of the apocritan lineage (Heraty et al., 2011; Mao et al., (Cynipoidea + (Proctotrupoidea + (Diaprioidea + Chalcidoidea))), 2014, 2015; Peters et al., 2011; Sharkey et al., 2011; Vilhelmsen et al.,

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Fig 2. Results from four-clusters likelihood mapping showing the support for the hypotheses of two conﬂict nodes (A and B). This analysis is based on matrix that 13 PCG sequences. A. Possible relationships of Evaniomorpha-a, Ichneumonomorpha, Evaniomorpha-b, Proctotrupomorpha, Evaniomorpha-c and Aculeata are displayed on the edges of the triangle. B. Possible relationships of Evaniomorpha-a, Ichneumonomorpha, Evaniomorpha-b and Proctotrupomorpha displayed on the edges of the triangle. The deeper colors on the region represent the higher support values of the relationship on this triangle.

2010; Wei et al., 2014). for a wide sampling of insects. Our initial attempts using re- Tree topologies were generally well supported by all Bayesian and presentatives of multiple orders (Coleoptera, Diptera, Lepidoptera and ML analyses (Table 1), whether analyzed with or without the inclusion Neuroptera) produced confusing topologies, presumably due to diﬃ- of outgroup taxa. Outgroup selection is problematic because the rooting culties with alignment and the highly divergent nature of these taxa. In of Hymenoptera, as sister to all other holometabolan orders, would call line with a previous study (Peters et al., 2017), we chose only the

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Fig. 3. Heterogeneity in nucleotide composition and substitution rates of selected Hymenoptera. For each taxon the bar charts display: droot, the root-to-tip distance obtained from the tree topology in Fig. 1; garp, the proportion of GC-rich amino acid codons; gccont, the GC content (%); ka, the mean Ka (non-synonymous substitution rate) value; ks, the mean Ks (synonymous substitution rate) in pairwise comparisons in all selected taxa. All values except root-to-tip distances were obtained from the protein coding genes. members of Neuropteridia, which had a less drastic effect on the in- correlated with biases in nucleotide variation. Instead, there seems a group relationships. The inclusion or exclusion of these outgroups affect general tendency that the inclusion of 3rd codon positions and rRNA predominantly various nodes within the Apocrita but not the most basal genes in the analysis increases the chance of recovering these nodes, nodes. The analysis corroborated that the Symphyta at the base of indicating that greater amounts of data are responsible, which may Hymenoptera is a paraphyletic grade, with Orussidae as the sister group provide an argument for retaining all nucleotide positions. Tests of data to Apocrita. When outgroups were excluded, these basal arrangements heterogeneity with the Aligroove method only indicate anomalous remained unchanged but the positioning of the various subclades of patterns affecting the tip level (individual terminals), i.e. it does not Evaniomorpha and their relationships to other infraorders differed provide evidence of biases affecting the position of major clades. (Table 1), as the trees without outgroups often did not recover the However, we note the general trend towards higher GC content and expected relationships. Thus we confirmed previous analyses that per- lower Ks in the Aculeata, compared to the other apocritan branches, formed better when recovering hymenopteran phylogenetic relation- while the same is true also for the Evaniomorpha-c, i.e. the sister taxon ships using mitochondrial genomes with outgroups (Castro and of Aculeata in our analysis. While these similarities in nucleotide Dowton, 2007). composition and evolutionary rates cement these sister relationships, Our study used a variety of tests to examine potential biases in the there is no reason to think that this is not due to common ancestry. data that might be responsible for spurious associations of taxa. The Branstetter et al. (2017) and Peters et al. (2017) also found some mean GC content and the corresponding preponderance of GARP amino lineage of Evaniomorpha (Trigonaloidea) as sister to Aculeata, sup- acids differed substantially among taxa. Thus, problems of nucleotide porting the current findings. However, taxon sampling was limited in heterogeneity (which we did not examine explicitly) might be directly both studies and was missing the Megalyroidea whose presence had linked to phenomena of long-branch attraction, but they should be less great impact on this relationship even with more completely sampled prevalent using amino acid sequences or nucleotide data after removal data sets. Alternatively, the non-monophyly of the Evaniomorpha might of 3rd codon positions, although other biases could affect the non-sy- result from convergent biases for lower GC content and faster rates in nonymous character changes. The different analyses with inclusion and the other two clades (Evaniomorpha-a and Evaniomorpha-c) producing exclusion of 3rd positions had no consistent effects; while convergent biases with their respective sister group (mainly Ichneu- Evaniomorpha-b was detected in all cases, Evaniomorpha-a and monomorpha), but this seems equally unlikely given the findings of Evaniomorpha-c were not universally recovered, but this was not Branstetter et al. (2017) and Peters et al. (2017) who find some

14 P. Tang et al. Molecular Phylogenetics and Evolution 131 (2019) 8–18

Fig. 4. AliGROOVE analysis for various datasets. The mean similarity score between pairs of sequences is represented by a colored square, based on AliGROOVE scores from −1, indicating great diﬀerence in sequence composition from the remainder of the data set (red coloring), to +1, indicating high similarity to all other comparisons (blue coloring). PCG, protein coding genes; PCG12, 1st and 2nd codon positions; PCGRNA with rRNA genes included.

Evaniomorpha grouped near Ichneumonomorpha (albeit again in stu- Peters et al., 2017), although slightly older (by ∼20 Ma) than the two dies with much lower sampling). Even our greatly expanded taxon other calibrations (Branstetter et al., 2017; Misof et al., 2014). Apocrita sampling of these groups remains a limited set chosen from the huge diverged from Orussoidea at 232 (225–239) Mya, which implies the diversity of parasitic wasps. The relative ease of mitogenome sequen- maximum age for the origin of parasitoidism as well. Contrary to cing should lead to the rapid addition of further taxa and thus improve ﬁndings of previous studies, major clades of Parasitica diverged earlier the support for basal relationships and the delimitation of major clades than clades of Aculeata (O'Reilly and Donoghue, 2016; Ronquist et al., within the “Evaniomorpha”. 2012a). All major clades within Apocrita, including the Aculeata con- stituting one of these lineages, diverged almost at the same time around 200 Mya, and the Aculeata started to diverge shortly thereafter. This 4.2. Dating of major clades and diversiﬁcation among Hymenoptera period coincides with the Triassic-Jurassic extinction event, which places the radiation and early divergence of Apocrita in its pa- The phylogenetic chronogram dates the most recent hymenopteran laeoenvironmental context. The origin of this lineage marks the be- common ancestor to the Permian, to 276 (266–286) Mya, which is ginning of the spectacular evolutionary radiation in post-Triassic and consistent with several previous studies (O'Reilly and Donoghue, 2016;

15 P. Tang et al. Molecular Phylogenetics and Evolution 131 (2019) 8–18

Fig. 5. Chronogram showing hymenopteran phylogeny and divergence time estimates. The tree presents divergence dates produced by the Beast analysis of the PCG dataset using 19 fossil calibration points. Blue bars indicate 95% mean confidence intervals of each node. A geological timescale is shown at the bottom. early-Jurassic ecosystems, when Apocrita acquired an astonishing range Funds for the Central Universities, China [2018QNA6025]; the National of life styles. Most modern families of Aculeata were proposed to have Key R&D Program of China, China [2017YFD0201000]; National originated during the Late Cretaceous when angiosperms radiated and Natural Science Foundation of China, China [31101661] and the diversified (Wilson et al., 2013), which is confirmed here. This supports Biodiversity Initiative of the Natural History Museum, London, UK. the idea that flourishing angiosperm diversity and herbivorous insects feeding on them increased the diet diversity and habitat complexity, as Appendix A. Supplementary material potential key factors driving diversification in predatory wasps (Johnson et al., 2013; Wilson et al., 2013). Our study suggests that this Supplementary data to this article can be found online at https:// might also apply to the contemporaneously diversifying Parasitica, to- doi.org/10.1016/j.ympev.2018.10.040. gether with their rapidly diversifying hosts. The evolutionary shifts in life histories strategies of Hymenoptera are only slowly being resolved, References given their huge diversity, but the combination of genome and mitogenome data, the latter permitting dense sampling, will provide an Abascal, F., Zardoya, R., Telford, M.J., 2010. TranslatorX: multiple alignment of nu- increasingly accurate and comprehensive tree to more fully resolve the cleotide sequences guided by amino acid translations. Nucleic. Acids Res. 38, W7–W13. evolutionary history of Hymenoptera. Ashmead, W.H., 1896. The phylogeny of the Hymenoptera. Proc. Entomol. Soc. Wash. 323–336. Acknowledgments Bernt, M., Donath, A., Juhling, F., Externbrink, F., Florentz, C., Fritzsch, G., Putz, J., Middendorf, M., Stadler, P.F., 2013. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319. We would like to thank van C. Achterberg (Naturalis Biodiversity Bernt, M., Merkle, D., Ramsch, K., Fritzsch, G., Perseke, M., Bernhard, D., Schlegel, M., Center, Netherlands), J.S. Noyes (Natural History Museum, UK), Stadler, P.F., Middendorf, M., 2007. CREx: inferring genomic rearrangements based – Junhua He (Zhejiang University, Hangzhou, China), Meicai Wei on common intervals. Bioinformatics 23, 2957 2958. Boisvert, S., Raymond, F., Godzaridis, E., Laviolette, F., Corbeil, J., 2012. Ray Meta: (Central South University of Forestry and Technology, Changsha, scalable de novo metagenome assembly and profiling. Genome Biol. 13, R122. China) and Jingxian Liu (South China Agricultural University, Bourguignon, T., Lo, N., Cameron, S.L., Sobotnik, J., Hayashi, Y., Shigenobu, S., Guangzhou, China) for donating and identifying specimens. This work Watanabe, D., Roisin, Y., Miura, T., Evans, T.A., 2015. The evolutionary history of termites as inferred from 66 mitochondrial genomes. Mol. Biol. Evol. 32, 406–421. was supported by the State Key Program of National Natural Science Branstetter, M.G., Danforth, B.N., Pitts, J.P., Faircloth, B.C., Ward, P.S., Buffington, M.L., Foundation of China, China [31230068]; National Natural Science Gates, M.W., Kula, R.R., Brady, S.G., 2017. Phylogenomic insights into the evolution – Foundation of China, China [31401996]; the Fundamental Research of stinging wasps and the origins of ants and bees. Curr. Biol. 27, 1019 1025.

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