bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 Compositional heterogeneity and outgroup choice

2 influence the internal phylogeny of the

*a b c d c 3 Marek L. Borowiec , Christian Rabeling , Seán G. Brady , Brian L. Fisher , Ted R. Schultz , and a 4 Philip S. Ward

a 5 Department of Entomology and Nematology, One Shields Avenue, University of California at 6 Davis, Davis, California, 95616, USA b 7 School of Life Sciences, Social Research Group, Arizona State University, Tempe, 8 Arizona, 85287, USA c 9 Department of Entomology, National Museum of Natural History, Smithsonian Institution, 10 Washington, D.C., USA d 11 Department of Entomology, California Academy of Sciences, San Francisco, California, 94118, 12 USA

13 Abstract

14 Knowledge of the internal phylogeny and evolutionary history of ants (Formicidae), the world’s 15 most -rich of eusocial organisms, has dramatically improved since the advent of 16 molecular . A number of relationships at the subfamily level, however, remain uncer- 17 tain. Key unresolved issues include placement of the root of the tree of life and the relationships 18 among the so-called poneroid subfamilies. Here we assemble a new data set to attempt a resolution 19 of these two problems and carry out divergence dating, focusing on the age of the root node of 20 crown Formicidae. For the phylogenetic analyses we included data from 110 ant species, including 21 the key species . We focused taxon sampling on non-formicoid lineages of ants 22 to gain insight about deep nodes in the ant phylogeny. For divergence dating we retained a subset 23 of 62 extant taxa and 42 fossils in order to approximate diversified sampling in the context of the 24 fossilized birth-death process. We sequenced 11 nuclear gene fragments for a total of ~7.5 kb and 25 investigated the DNA sequence data for the presence of among-taxon compositional heterogeneity, 26 a property known to mislead phylogenetic inference, and for its potential to affect the rooting of 27 the ant phylogeny. We found sequences of the and several outgroup taxa to be rich in 28 adenine and thymine (51% average AT content) compared to the remaining ants (45% average). To 29 investigate whether this heterogeneity could bias phylogenetic inference we performed outgroup 30 removal experiments, analysis of compositionally homogeneous sites, and a simulation study. We

*Corresponding author. Current address: School of Life Sciences, Social Insect Research Group, Arizona State University, Tempe, Arizona, 85287, USA. E-mail address: [email protected] (M. L. Borowiec).

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31 found that compositional heterogeneity indeed appears to affect the placement of the root of the 32 ant tree but has limited impact on more recent nodes. We put forward a novel hypothesis regarding 33 the rooting of the ant phylogeny, in which Martialis and the Leptanillinae together constitute a 34 clade that is sister to all other ants. After correcting for compositional heterogeneity this emerges 35 as the best-supported hypothesis of relationships at deep nodes in the ant tree. The results of our 36 divergence dating under the fossilized birth-death process and diversified sampling suggest that the 37 crown Formicidae originated during the Albian or Aptian ages of the Lower (103–124 38 Ma). In addition, we found support for monophyletic poneroids comprising the subfamilies Agroe- 39 comyrmecinae, , Apomyrminae, Paraponerinae, , and , and 40 well-supported relationships among these subfamilies except for the placement of Proceratiinae and 41 (Amblyoponinae + Apomyrminae). Our phylogeny also highlights the non-monophyly of several 42 ant genera, including and in the Leptanillinae, in the Procerati- 43 inae, and Cryptopone, Euponera, and Mesoponera within the Ponerinae. 44 Key words: Systematic bias, phylogenetics, systematics, diversified sampling, fossilized birth- 45 death process

46 1. Introduction

47 Ants are among the world’s dominant social , with more species and greater ecological im- 48 pact than any other group of eusocial (Hölldobler and Wilson, 1990). Knowledge of ant 49 phylogeny is vital to understanding the processes driving the evolution of these ubiquitous and 50 diverse organisms. 51 About 90% of extant ant diversity, or almost 11,000 species and 9 of 16 subfamilies, belongs to 52 a group dubbed the ”formicoid clade” (Brady et al., 2006). The relationships among the subfam- 53 ilies of the formicoid clade are well-resolved. This contrasts with the uncertain branching order 54 of lineages outside of formicoids. Two enduring issues in higher ant phylogeny, highlighted by 55 recent studies (Moreau et al., 2006; Brady et al., 2006; Rabeling et al., 2008) and a review by Ward 56 (2014), include the identity and composition of the lineage that is sister to all other extant ants, and 57 whether the so-called poneroid subfamilies (, Amblyoponinae, Apomyrminae, 58 Paraponerinae, Ponerinae, Proceratiinae) form a clade or a grade. 59 In molecular phylogenetic studies published to date, two ant subfamilies, the Martialinae and 60 Leptanillinae (recently redefined to include the Opamyrma; Ward and Fisher (2016)), have 61 been competing for the designation as the sister group to all other ants (Figure 1)(Rabeling et al., 62 2008; Kück et al., 2011; Moreau and Bell, 2013; Ward and Fisher, 2016). Rabeling et al. (2008) 63 discovered and described Martialis heureka, and inferred that this sole species of the morphologi- 64 cally divergent Martialinae is the sister species to all other ants. Subsequent phylogenies, however, 65 proposed that either the subfamily Leptanillinae is the lineage first branching from the rest of the 66 ants (Kück et al., 2011), or were ambiguous about the placement of the ant root (Moreau and Bell, 67 2013). 68 The monophyly of the poneroid subfamilies was recovered by Moreau et al. (2006) but it was 69 later contested by Brady et al. (2006) and Rabeling et al. (2008). Brady et al. (2006) pointed out that 70 long-branch attraction may be responsible for poneroid monophyly and found that non-monophyly 71 of poneroids could not be rejected based on their data set of seven nuclear gene fragments. Most 72 subsequent phylogenetic studies failed to satisfactorily resolve this issue (reviewed in Ward (2014))

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73 although poneroid monophyly was recovered with strong support by Ward and Fisher (2016). A 74 recently published phylogenomic study also supported poneroid monophyly (Branstetter et al., 75 2017b). The phylogenomic data were apparently insufficient, however, to resolve relationships 76 among poneroid lineages because of low taxon sampling (Branstetter et al., 2017b). 77 In addition to the phylogenetic uncertainty present in the above mentioned studies, there is also 78 potential for systematic bias to preclude correct inference of ant relationships, especially near the 79 root of the ant tree (Ward, 2014). This is because most ants are relatively guanine and cytosine- 80 (GC) rich compared to many aculeate outgroups and species of the Leptanillinae, which are un- 81 usually adenine and thymine- (AT) rich. Such compositional heterogeneity is known to mislead 82 phylogenetic inference (Jermiin et al., 2004) and most of the commonly used models of sequence 83 evolution do not take it into account. This and other potential violations may lead to poor model 84 fit, which was demonstrated in phylogenetics in general (Brown, 2014) and in an ant phylogeny 85 specifically (Rabeling et al., 2008). It is thus possible that the position of the Leptanillinae 86 appearing in some studies is an artifact resulting from model misspecification. 87 To address these issues of uncertainty and potential bias near the base of the ant tree of life, we 88 assembled a new comprehensive data set that included all ant subfamilies, with sampling focused on 89 non-formicoid lineages. The amount of sequence data we gathered is significantly greater than in 90 previous studies that included the pivotal species Martialis heureka. We investigated the potential 91 of these data to be biased by base-frequency heterogeneity and implemented strategies aimed at 92 minimizing such bias. 93 Another outstanding question concerns the age of the most-recent common ancestor of ants, 94 which has been variously estimated to have lived as recently as ca. 115 Ma (Brady et al., 2006) or as 95 early as 168 Ma (Moreau et al., 2006), with the oldest undisputed crown formicid fossil Kyromyrma 96 neffi dated at 92 Ma (Grimaldi and Agosti, 2000; Barden, 2017). Efforts to infer the age of origin 97 of crown ants have been conducted thus far using either penalized likelihood (Sanderson, 2002) or 98 node dating in a Bayesian framework (Drummond and Rambaut, 2007). Here we take advantage 99 of the fossilized birth-death process framework (Heath et al., 2014), which was recently extended 100 to accommodate diversified taxon sampling (Zhang et al., 2016). 101 The recently developed fossilized birth-death process (FBD) approach to divergence dating pro- 102 vides several advantages over the node-dating approach, as it explicitly treats both extant and fossil 103 taxa as parts of the same underlying diversification process. This is different from the node-dating 104 approach, where fossils only provide clues about the probability-density distributions of ages for 105 certain splits (Heath et al., 2014). FBD is thus able to avoid some of the challenges documented 106 for node-dating, such as formulation of the statistical problem accidentally precluding reasonable 107 age estimate for a node of interest (Brown and Smith, 2017). Early implementations of the FBD, 108 however, shared the assumption of other Bayesian divergence-time estimation methods that taxon 109 sampling reflects complete or random sampling of the diversification process that created the phy- 110 logeny. Because phylogenies of higher taxa aim for maximizing phylogenetic diversity (Höhna 111 et al., 2011), this assumption is often violated and is known to cause biased age estimates (Beaulieu 112 et al., 2015). The most recent implementation of the FBD accounts for this by explicitly allowing 113 modeling under a diversified sampling scheme (Zhang et al., 2016).

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Figure 1: Comparison of alternative hypotheses for the root of the ant phylogeny. A, Martialis sister to all other ants (Rabeling et al., 2008); B, Leptanillinae sister to all other ants, including Martialis (Kück et al., 2011); C, Martialis plus Leptanillinae sister to all other ants (this study).

114 2. Methods

115 2.1. Taxon sampling and data collection

116 We collected data from 11 nuclear loci for 110 ant species, including Martialis heureka, and 13 117 outgroup taxa. Our data set has extensive sampling within Leptanillinae (21 species), all major 118 lineages of the poneroids (66 species), and at least one representative of all formicoid subfamilies 119 (22 species). The sequence data comes from 28S ribosomal DNA (28S) and ten nuclear protein- 120 coding genes: abdominal-A (abdA), elongation factor 1-alpha F2 copy (EF1aF2), long wavelength 121 rhodopsin (LW Rh), arginine kinase (argK), topoisomerase I (Top1), ultrabithorax (Ubx), DNA pol- 122 delta (POLD1), NaK ATPase (NaK), antennapedia (Antp), and wingless (Wg). Sequences were 123 assembled with Sequencher v5.2.2 (Gene Codes Corporation, Ann Arbor, MI, U.S.A.), aligned 124 with Clustal X v2.1 (Thompson et al., 1997), and manually edited and concatenated with MacClade 125 v4.08 (Maddison and Maddison, 2005). We excluded introns, autapomorphic indels (9 bp abdA, 3 126 bp EF1aF2, 9 bp Ubx, 3 bp POLD1, 3 bp NaK, 75 bp Antp, 3 bp Wg), and hypervariable regions of 127 28S. The resulting data matrix is 7,451 bp long and has no missing data except for gaps introduced 128 by non-autapomorphic indels, which constitute 3% of the data. Our protocols for extraction and 129 amplification were described in detail in Ward et al. (2010) and Ward and Fisher (2016). Of the 130 1,353 sequences, 676 were newly generated for this study (GenBank accessions XXXXXXXX- 131 XXXXXXXX [submission in progress]; Supplementary Table 4 ).

132 2.2. Compositional heterogeneity

133 We assessed compositional heterogeneity using methods implemented in the p4 phylogenetics li- 134 brary (Foster, 2004). To investigate whether our data significantly departed from the assumption of 2 135 homogeneity across taxa we applied two tests: 1) a χ test for compositional heterogeneity, and 2) 136 a more sensitive, simulation-based test corrected for phylogeny. The phylogeny-corrected tests in- 137 volved inferring a neighbor-joining tree for the data, constructed with BioNJ (Gascuel, 1997), onto 138 which we simulated 1,000 replicates using GTR+4Γ model. The distribution of sequence compo- 139 sitions in the empirical data were then compared against simulated data. Following the finding that 2 140 the combined data set failed both the χ and phylogenetically-corrected tests, we further divided 141 the data set into partitions as follows: 1) each locus as its own partition, and 2) first, second, and 142 third codon position within each locus assigned to separate partitions. We then ran the two compo-

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143 sitional heterogeneity tests on each partition separately and recorded the results. We also computed 144 base frequencies for each taxon and partition using AMAS (Borowiec, 2016).

145 2.3. Data matrices

146 In addition to the full data set, we composed two different matrices by excluding the most AT-rich 147 and the most GC-rich outgroups, respectively. To this end, we first ranked non-ant taxa by AT 148 content and then removed six taxa that had either the highest or lowest AT content, which also 149 corresponded to above or below mean AT content for the entire data set. We retained moderately 150 AT-rich Pristocera MG01 as the external outgroup. 151 We also constructed a data set from which we removed all heterogeneous partitions (i.e., 28S, 152 all third codon positions, and first codon positions of POLD1 and Ubx). 153 Selected statistics of the four analyzed data matrices are in the first four rows of Table 1.

Table 1: Data properties.

Data matrix Length Percent gaps Variable sites Parsimony informative sites Full data set 7451 3.0 3902 3369 AT-rich outgroups removed 7451 3.0 3806 3237 GC-rich outgroups removed 7451 3.0 3825 3291 Homogeneous partitions 3995 2.7 1181 831 28S 845 6.8 425 317 abdA 621 3.7 311 258 argK 673 0.3 334 296 Antp 865 14.0 497 397 EF1aF2 517 0.0 206 194 LW Rh 458 0.0 287 262 NaK 955 0.0 409 376 POLD1 583 0.0 374 325 Top1 880 0.0 477 426 Ubx 630 0.7 313 280 Wg 424 3.4 269 238 All 1st codon pos. 2200 2.5 829 620 All 2nd codon pos. 2199 2.5 536 349 All 3rd codon pos. 2207 2.5 2112 2083

154 2.4. Partitioning

155 In the light of recent criticism of the k-means partitioning strategy (Baca et al., 2017), for each of the 156 four data sets we used two different strategies for partitioning: a ’greedy’ algorithm on predefined 157 user partitions and the k-means partitioning algorithm (Frandsen et al., 2015) as implemented in 158 PartitionFinder 2 pre-release v13 (Lanfear et al., 2017). The greedy strategy relies on user-defined 159 sets of characters as input and our predefined sets constituted sites from the three codon positions 160 in each of the loci used, except for 28S which was defined as a single set. We used a maximum- 161 likelihood tree generated with the fast RAxML algorithm (”-f E” option) (Stamatakis, 2014) as

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162 the starting tree for each PartitionFinder run, which then used PhyML for subsequent steps of the 163 algorithm (Guindon et al., 2010). Because of our use of MrBayes in downstream analyses, we 164 restricted models to be evaluated by PartitionFinder to those available in that program.

165 2.5. Phylogenetics

166 We constructed phylogenies for all empirical data sets using Bayesian inference with MrBayes 167 v3.2.6 (Ronquist et al., 2012) and maximum-likelihood with IQ-TREE v1.4.2 beta (Nguyen et al., 168 2014). For the Bayesian analyses, we ran two separate runs, four chains each, for 20 to 80 million 169 generations for each of the eight analyses. We used a 20% burnin fraction and determined mix- 170 ing and convergence by examining output of MrBayes ”sump” command, including average stan- 171 dard deviation of split frequencies (below < 0.01), effective sample size (ESS) for each parameter 172 (minimum 200), and potential scale reduction factor (PSRF) near 1.0. In the maximum-likelihood 173 analyses we specified the same partitioning schemes and substitution models as for the Bayesian 174 inference. We changed the default IQ-TREE settings by using the slow nearest-neighbor inter- 175 change search (”-allnni” option) and setting the number of unsuccessful iterations to stop at 1,000 176 instead of the default 100 (”-numstop” option). We assessed support by running 2,000 ultrafast 177 boostrap replicates (Minh et al., 2013). The authors of this fast bootstrap approximation point out 178 that this algorithm tends to overestimate probability of a correct relationship under 70% support 179 but is more unbiased than RAxML’s rapid bootstrapping above that threshold, resulting in 95% 180 support being approximately equal to 95% probability of a relationship being true under the true 181 model. Therefore, support at or above 95% should be interpreted as significant (Minh et al., 2013). 182 We also attempted analyses under tree-heterogeneous models implemented in p4 (Foster, 2004) 183 and nhPhyloBayes (Blanquart and Lartillot, 2008) on the full data matrix, but these turned out to 184 be prohibitively computationally expensive, requiring by our estimate a minimum of five months 185 to reach convergence.

186 2.6. Simulation

187 To further assess the sensitivity of the ant phylogeny to bias we simulated a data set with com- 188 positional heterogeneity comparable to that present in our data set. In particular, we were inter- 189 ested in investigating whether the position of Martialis could be incorrectly inferred as sister to 190 the poneroids plus formicoids clade even if the data were simulated on a topology where Martialis 191 is sister to Leptanillinae. To create the simulated data set we first split our empirical alignment, 192 excluding ribosomal 28S, into alignments of first, second, and third codon positions. We then used 193 a fixed topology which had Martialis as sister to Leptanillinae, in this case the Bayesian posterior 194 consensus from the full data set analysis under k-means partitioning, to estimate branch lengths 195 for each of the three alignments. To estimate the branch lengths we used IQ-TREE (Nguyen et al., 196 2015) under the general time-reversible model with rate heterogeneity described by a proportion 197 of invariant sites and a gamma distribution discretized into four bins (GTR+pinv+4Γ). For each 198 of the codon position alignments we also calculated the proportion of invariant sites and base fre- 199 quencies for all taxa using AMAS (Borowiec, 2016). Furthermore, we calculated average base 200 frequencies for alignments composed of first and second positions. For the third codon positions 201 alignment we calculated two average base frequencies: one for the 25 most AT-rich taxa, repre- 202 sented almost exclusively by Leptanillinae species and some of the outgroups, and the other for

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203 all remaining taxa, thus approximating mean base frequencies for AT-rich and GC-rich taxa, re- 204 spectively. We then used the topologies with branch lengths, proportion of invariant sites, and 205 empirical base frequencies to simulate three separate alignments, each 2,200 sites long, similar to 206 the empirical data, under GTR+pinv+4Γ using p4 (Foster, 2004). For the alignments imitating first 207 and second codon positions we simulated the data under a tree-homogeneous model, but for the 208 third codon position alignment we used two composition vectors corresponding to the two empiri- 209 cal means of AT-rich (A: 0.24, C: 0.24, G: 0.23, T: 0.29) and GC-rich sequences (A: 0.16, C: 0.33, 210 G: 0.31, T: 0.20) at that position. The AT-rich frequencies were applied to the Leptanillinae and 211 outgroup taxa considered AT-rich in our outgroup taxa removal experiments outlined above. We 212 replicated the simulation 100 times for each alignment under different starting seed numbers. We 213 then performed maximum-likelihood analyses under GTR+pinv+4Γ (using IQ-TREE settings as 214 described above) for the concatenated simulated data as well as each of the three simulated align- 215 ments separately. Following the inference we constructed majority-rule consensus trees using all 216 100 maximum-likelihood trees for all codon position simulations and the concatenated alignments 217 in order to visualize the topology recovered from simulated alignments.

218 2.7. Divergence time estimation

219 We performed divergence dating under the fossilized birth-death process (Heath et al., 2014) and 220 diversified sampling, as implemented in MrBayes v3.2.6 (Ronquist et al., 2012; Zhang et al., 2016). 221 To obtain a taxon sample that most closely approximates diversified sampling sensu Höhna et al. 222 (2011), i.e., one extant descendant sampled for each branch that was present at a given time in 223 the past, we pruned our original alignment down to 62 species. To calibrate the analysis we used 224 42 fossil calibrations (See Supplementary Table 3) and a diffuse root node exponential prior with a 225 mean of 250 Ma and offset at 150 Ma (just older than the oldest fossil calibration used). Because we 226 did not include morphological data in our analysis to place the fossils (”total evidence” dating sensu 227 Ronquist et al. (2012)), we assigned them to appropriate groups via monophyly constraints (Heath 228 et al., 2014). For this analysis we also constrained the topology of outgroup taxa to correspond 229 to the aculeate phylogeny recovered by a recent phylogenomic study (Branstetter et al., 2017a). 230 We chose the topology from this study over that of Peters et al. (2017) because the latter did not 231 include Rhopalosomatidae and Sierolomorphidae and thus arguably had insufficient taxon sampling 232 to correctly place Vespidae. 233 We ran the analysis with four runs, each with six incrementally heated chains, for 100 million 234 generations and discarded 10% of the samples as burn-in. The analysis was unpartitioned, with 235 the GTR+6Γ substitution model and a relaxed independent clock model with rate drawn from a 236 gamma distribution. We checked for convergence by examining the average standard deviation of 237 split frequencies towards the end of the run (<0.006), potential scale reduction factor values for 238 each parameter (maximum 1.028, average 1.001), and effective sample sizes (>500 for combined 239 runs). We examined MCMC trace files with Tracer v1.5 to confirm that the two runs converged on 240 all parameters and to compare posterior distributions to the analysis without data (i.e., under the 241 prior).

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242 2.8. Data availability

243 All data matrices, configurations for and output from PartitionFinder, Bayesian, and maximum- 244 likelihood analyses, as well as custom scripts used are available the Zenodo data repository, DOI: 245 10.5281/zenodo.838799.

Pristocera MG01 Mischocyttarus flavitarsis Metapolybia cingulata Sapyga pumila Dasymutilla aureola Aporus niger Chyphotes mellipes outgroups Brachycistis sp Chalybion californicum Apis mellifera Ampulex compressa Scolia verticalis Apterogyna ZA01 1.00 Martialis heureka Opamyrma hungvuong Martialinae Leptanilla TH03 0.99 Yavnella argamani MM01 1.00 Leptanilla TH08 Leptanilla TH06 Leptanilla TH04 Leptanilla TH05 Leptanilla TH02 Leptanilla TH01 Leptanilla TH09 Leptanilla ZA01 Leptanillinae 0.78 Leptanilla GR02 Leptanilla GR01 Protanilla TH02 Protanilla TH01 Protanilla JP01 Protanilla VN03 Protanilla VN01 Protanilla TH03 boltoni Leptanilloides femoralis Labidus praedator Formicidae Cerapachys jacobsoni Tetraponera rufonigra

1.00 Tetraponera punctulata formicoids macrops pyriformis 1.00 Liometopum occidentale Dolichoderus pustulatus Aneuretus simoni Myrmelachista flavocotea Lasius californicus Camponotus maritimus Pogonomyrmex vermiculatus Myrmica tahoensis Manica bradleyi Stenamma expolitum Myrmicaria carinata Cyphomyrmex longiscapus cf Crematogaster kelleri chalybaea Acanthoponera minor stygia Apomyrma CF02 Xymmer muticus Apomyrminae Myopopone castanea 1.00 boltoni Mystrium voeltzkowi Stigmatomma pallipes Stigmatomma denticulatum caputleae Amblyoponinae Fulakora saundersi Fulakora mystriops Prionopelta amabilis Onychomyrmex hedleyi 0.59 Amblyopone australis Proceratium stictum Proceratium avium Proceratium silaceum Proceratium SC02 tani Probolomyrmex brujitae cf Proceratiinae testacea Discothyrea SC03 1.00 Discothyrea SC02 Discothyrea MG07 clavata Paraponerinae 1.00 tatusia coronacantha Agroecomyrmecinae poneroids Platythyrea punctata Platythyrea mocquerysi Platythyrea lamellosa Diacamma pallidum 1.00 swezeyi Pseudoponera stigma Parvaponera darwinii Emeryopone MY01 Ectomomyrmex leeuwenhoeki Cryptopone gilva Simopelta pergandei cf Neoponera unidentata Dinoponera longipes Thaumatomyrmex atrox Belonopelta HN01 Hypoponera sakalava Hypoponera opacior Harpegnathos saltator Psalidomyrmex procerus Plectroctena ugandensis Ponerinae Loboponera politula angolensis tenuis Brachyponera obscurans Mesoponera melanaria Leptogenys diminuta 0.1 Asphinctopone silvestrii silvestrii Odontoponera transversa Streblognathus peetersi Mesoponera ambigua Euponera sikorae Euponera brunoi 0.407 AT content 0.533 Fisheropone ambigua Cryptopone hartwigi Bothroponera wasmannii Phrynoponera pulchella coquereli Anochetus madagascarensis Figure 2: Preferred phylogenetic hypothesis for the ingroup (Formicidae), with AT content indi- cated for each terminal taxon. Tree topology with branch lengths from the Bayesian analysis of the full data matrix under k-means partitioning strategy. See Supplementary Figure 2 for support val- ues on all nodes. Warmer branch colors signify higher AT content. Scale in expected substitutions per site.

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246 3. Results

247 We first summarize results regarding compositional heterogeneity, followed by a discussion of the 248 placement of the formicid root as it was impacted by the different methods. Second, we present phy- 249 logenetic findings which were less sensitive to different analytical treatments, including poneroid 250 monophyly, the relationships within poneroids, and non-monophyly of some of the non-formicoid 251 genera.

252 3.1. Compositional heterogeneity

253 Within the ingroup, ants in the Leptanillinae stands out as particularly AT-rich at 50.9% on average 254 compared to a mean of 46.2% for all taxa (or 44.9% for non-leptanilline ants). At 46.6%, Martialis 255 is close to the mean (Figure 2, Supplementary Table 1). 256 We found considerable compositional heterogeneity among taxa in our data set, mostly confined 257 to third codon positions. Overall difference in AT content among taxa was 12.6% across the entire 258 data set. This difference was equal to 37.3% at third codon positions, compared to only 3.5% at 259 first codon positions and 1.1% at the second codon positions. Third codon positions also accounted 260 for 56.7% variable sites and 61.8% parsimony informative sites of the data set. 261 Consistent with this pattern, the phylogeny-corrected test identified all third codon partitions 262 as those for which the homogeneity assumption could be rejected with a p < 0.05 (Supplementary 263 Table 2). In addition to the third codon positions, 28S, and first codon positions of POLD1 and 264 Ubx were found to violate the homogeneity assumption using this test. Similar to the phylogeny- 2 265 corrected test, the χ test identified all of the third codon positions as heterogeneous but it did not 266 reject the homogeneity assumption for 28S and first codon positions of POLD1 and Ubx (Supple- 267 mentary Table 2). 268 Compositional heterogeneity was also high among the outgroups. Those that fell above the 269 mean AT content for the entire alignment included (in order of decreasing AT content) Mischocyt- 270 tarus flavitarsis, Metapolybia cingulata, Brachycistis sp., Apis mellifera, Dasymutilla aureola, Sco- 271 lia verticalis, and Pristocera MG01. The outgroup species that were more GC-rich than average 272 were (in order of decreasing AT content) Chyphotes mellipes, Ampulex compressa, Apterogyna 273 ZA01, Aporus niger, Chalybion californicum, and Sapyga pumila (Supplementary Table 1).

274 3.2. Analyses of the full data matrix

275 With all taxa retained and no attempt at reducing the compositional heterogeneity of the data, the 276 partitioning strategy has a strong effect on the results. Under the k-means strategy Martialis heureka 277 is sister to the Leptanillinae with strong support of pp = 0.99 in the Bayesian analysis (Figure 278 2; Supplementary Figure 2) and low support of 87% bootstrap in the maximum-likelihood tree 279 (Supplementary Figure 4). Under the greedy analyses, both Bayesian and maximum-likelihood 280 trees recover a topology where the Leptanillinae are sister to the remaining Formicidae including 281 Martialis, although support for this topology is below significance (pp = 0.81 and bootstrap 93%; 282 Supplementary Figures 1 and 3).

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283 3.3. Effects of outgroup removal

284 Relative to the full data matrix with all 13 outgroup species, removal of the AT-rich outgroups 285 (Apis mellifera, Brachycistis sp., Dasymutilla aureola, Scolia verticalis, Metapolybia cingulata, 286 and Mischocyttarus flavitarsis) shifts support towards a tree where Martialis and the Leptanillinae 287 together form a clade that is sister to all other ants. In consensus Bayesian trees, the support for 288 this clade is at pp = 0.91 under greedy (Supplementary Figure 5) and pp = 1.0 under the k-means 289 partitioning strategy (Supplementary Figure 6). Under maximum-likelihood, the effect is less ob- 290 vious, as the ML tree under greedy partitioning has Martialis sister to formicoids and poneroids 291 but now with only 50% bootstrap support (Supplementary Figure 7). In the k-means maximum- 292 likelihood tree, Martialis and Leptanillinae form a clade supported in 99% bootstrap replicates 293 (Supplementary Figure 8). 294 Removal of GC-rich outgroups (Ampulex compressa, Aporus niger, Apterogyna ZA01, Chaly- 295 bion californicum, and Chyphotes mellipes) reinforces support for the topology where Leptanillinae 296 are sister to Martialis plus formicoids plus poneroids with pp = 1.0 under greedy partitioning (Sup- 297 plementary Figure 9) and pp = 0.98 under k-means in Bayesian analyses (Supplementary Figure 298 10). The same pattern is present in maximum-likelihood trees, which both show Leptanillinae sis- 299 ter to Martialis plus formicoids and poneroids. Under greedy analyses bootstrap support for this 300 clade is 99% (Supplementary Figure 11) and under k-means it is 96% (Supplementary Figure 12).

301 3.4. Compositionally homogeneous matrix

302 The analyses of homogeneous data partitions result in a tree where Martialis is sister to the Lep- 303 tanillinae with strong support regardless of partitioning scheme and inference method (Table 2). 304 Bayesian trees under both greedy and k-means partitioning strategies show pp = 1.00 (Supple- 305 mentary Figures 13 and 14). Under maximum-likelihood this node is supported in 100% bootstrap 306 replicates under both greedy k-means partitioning strategies (Supplementary Figures 15 and 16).

307 3.5. Simulation

308 In the maximum-likelihood analyses of simulated alignments imitating first codon positions the 309 Martialis plus Leptanillinae clade, i.e., the topology under which the data were simulated, is recov- 310 ered consistently (Supplementary Figure 17). For the alignments imitating second codon positions 311 Martialis emerges as sister to the Leptanillinae in 96 out of 100 trees Supplementary Figure 18), 312 and in the tree derived from the matrix imitating third codon positions Martialis is sister to the 313 Leptanillinae in only 64 trees out of 100 (Supplementary Figure 19). The remaining 36 trees show 314 Martialis either as sister to the poneroid plus formicoid clade, with Leptanillinae being sister to 315 all other ants, or, alternatively as the sister group to all ants. The combined data set supports the 316 Martialis plus Leptanillinae clade in 99 out of 100 trees (Supplementary Figure 20).

317 3.6. Relationships among poneroid subfamillies

318 The so-called poneroid ant subfamillies that include Agroecomyrmecinae, Amblyoponinae, Apomyr- 319 minae, Paraponerinae, Ponerinae, and Proceratiinae form a well supported clade. This result ap- 320 pears more robust to different analytics than the placement of the root of the tree. Support for this 321 clade is often maximum in Bayesian analyses and generally above 90% bootstrap proportion in the

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322 maximum-likelihood analyses, except for the data set with GC-rich outgroups removed, where the 323 support is only 75% (Table 2). 324 Within the poneroid clade, another set of relationships that is well-supported across the anal- 325 yses is the sister relationship of Agroecomyrmecinae and Paraponerinae, which is significantly 326 supported in all analyses except in the maximum-likelihood trees inferred from the homogeneous 327 data matrix. This clade is in turn sister to the Ponerinae in all analyses, although support varies. 328 This relationship receives maximum support in all Bayesian analyses except for the data matrix 329 with GC-rich outgroups removed. In maximum-likelihood trees support varies between 74% and 330 97% bootstrap replicates (Table 2). 331 The most problematic is placement of Proceratiinae and (Amblyoponinae + Apomyrminae), 332 which in some analyses form a clade, and in others form a grade where Amblyoponinae plus 333 Apomyrminae are the sister clade to all other poneroids and Proceratiinae is sister to the remaining 334 subfamilies. The support for both of these alternatives is never significant, however (Table 2).

335 3.7. Non-monophyly of currently recognized genera

336 Several shallow nodes, well-supported regardless of the data set and analysis method, highlight 337 non-monophyly of genera outside of the formicoid clade (Figure 2; Supplementary Figures 1–16). 338 In the Leptanillinae, the morphologically derived genus Anomalomyrma is nested within Protanilla, 339 and two genera known only from males, Phaulomyrma and Yavnella, are nested within Leptanilla. 340 Within the small subfamily Proceratiinae, the four species of Proceratium included in our data 341 matrices were paraphyletic with respect to Probolomyrmex. 342 Under all analyses we recover three non-monophyletic genera within Ponerinae: Cryptopone 343 gilva and Cryptopone hartwigi included here are only very distantly related, the genus Euponera 344 is paraphyletic with respect to the Cryptopone hartwigi plus Fisheropone clade, and Mesoponera 345 is polyphyletic, here represented by M. melanaria, which is sister to Leptogenys, and M. ambigua, 346 here sister to Streblognathus peetersi.

347 3.8. Divergence time analyses

348 Our divergence time analysis recovers a relatively young age for the most common ancestor of 349 crown-group ants, estimated to have lived during the Albian or Aptian ages of the Lower Cretaceous 350 (Figure 3, Table 3; median age 112 Ma, 95% highest posterior density interval 103–123 Ma). The 351 crown formicoids are estimated to have arisen ~101 Ma, closely followed by the split of Martialis 352 from the Leptanillinae around 99 Ma, and the origin of poneroids at 92 Ma. The median ages 353 inferred for the subfamilies where our taxon sampling spanned the root node are as follows: 45 for 354 Agroecomyrmecinae, 75 Ma for Ambyloponinae plus Apomyrminae, 55 Ma for Dolichoderinae, 355 60 Ma for Formicinae, 66 Ma for Leptanillinae, 45 Ma for Myrmeciinae, 61 Ma for Myrmicinae, 356 73 Ma for Ponerinae, and 65 Ma for Proceratiinae.

357 4. Discussion

358 4.1. Compositional heterogeneity and the rooting of the ant tree

359 Earlier studies recognized the difficulty in rooting the ant tree of life (Brady et al., 2006; Rabeling 360 et al., 2008) and our analyses confirm the supposition (Ward, 2014) that the effects of composi-

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Table 2: Support for selected relationships in Bayesian consensus and maximum-likelihood (ML) trees. Support for Bayesian analyses is expressed in posterior probabilities rounded to two decimal places and for ML in percentage of bootstrap replicates. Ag: Agroe- comyrmecinae, Am: Amblyoponinae plus Apomyrminae, for: formicoids, Le: Leptanillinae, Ma: Martialinae, Pa: Paraponerinae, Po: Ponerinae, pon: poneroids, Pr: Proceratiinae. NA signifies a case where the relationship was not recovered in the consensus or maximum-likelihood tree. ; a this versionpostedAugust8,2017. Matrix Method Partitioning Ma + Le Ma + (for pon Ag + Pa Po + (Ag Pr + Am Pr + Am + CC-BY-NC 4.0Internationallicense + pon) monophyl. + Pa) (Ag/Pa/Po) (Ag/Pa/Po) Full ML greedy NA 93 100 100 97 NA 77 NA Full ML k-means 87 NA 92 98 88 71 NA NA Full Bayes greedy NA 0.81 1.00 1.00 1.00 NA 0.54 NA 12 Full Bayes k-means 0.99 NA 1.00 1.00 1.00 0.59 NA NA AT-rich outgr. removed ML greedy NA 50 99 100 92 NA 70 NA AT-rich outgr. removed ML k-means 99 NA 97 99 83 64 NA NA AT-rich outgr. removed Bayes greedy 0.91 NA 1.00 1.00 1.00 0.5 NA NA AT-rich outgr. removed Bayes k-means 1.00 NA 1.00 1.00 1.00 0.64 NA NA GC-rich outgr. removed ML greedy NA 99 99 100 95 NA 86 NA . The copyrightholderforthispreprint(whichwasnot GC-rich outgr. removed ML k-means NA 96 75 96 73 NA 45 NA GC-rich outgr. removed Bayes greedy NA 1.00 1.00 1.00 1.00 NA 0.87 NA GC-rich outgr. removed Bayes k-means NA 0.98 0.88 0.99 0.88 NA 0.54 NA Homogeneous ML greedy 100 NA 98 81 99 NA NA 79 Homogeneous ML k-means 100 NA 98 78 100 NA NA 72 Homogeneous Bayes greedy 1.00 NA 1.00 0.99 1.00 NA NA 0.67 Homogeneous Bayes k-means 1.00 NA 1.00 0.93 1.00 0.50 0.50 NA bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Metapolybia cingulata 24.34 Mischocyttarus flavitarsis 162.89 Brachycistis sp 128.32 Chyphotes mellipes 148.62 Aporus niger 128.59 Dasymutilla aureola 111.02 Sapyga pumila Apterogyna ZA01 171.09 137.88 Scolia verticalis Ampulex compressa 135.99 Apis mellifera 110.87 Chalybion californicum 165.64 Martialis heureka 98.96 Opamyrma hungvuong Anomalomyrma boltoni 65.97 28.42 Protanilla JP01 48.43 Leptanilla TH01 28.27 157.5 Yavnella argamani Leptanilloides femoralis 55.13 Cerapachys jacobsoni 41.68 Labidus praedator Aneuretus simoni 82.85 Dolichoderus pustulatus 55.45 100.71 Liometopum occidentale 91.99 112.21 44.82 Nothomyrmecia macrops 81.73 Tetraponera punctulata 42.01 97.63 Tetraponera rufonigra Myrmelachista flavocotea 60.28 Camponotus maritimus 51.76 Lasius californicus 94.18 Acanthoponera minor 54.68 Rhytidoponera chalybaea 81.22 Manica bradleyi 39.25 Myrmica tahoensis 61.22 Pogonomyrmex vermiculatus 105.76 55.56 Stenamma expolitum 47.61 Crematogaster kelleri 42.49 Cyphomyrmex longiscapus cf 34.64 Myrmicaria carinata Discothyrea testacea 65.19 Proceratium stictum 49.01 Probolomyrmex tani 36.62 83.06 Proceratium silaceum Apomyrma stygia Amblyopone australis 74.64 34.37 Prionopelta amabilis 66.81 Stigmatomma mystriops 91.52 48.32 Mystrium mysticum 40.79 Stigmatomma denticulatum Paraponera clavata 70.74 Ankylomyrma coronacantha 44.99 Tatuidris tatusia 83.91 Platythyrea mocquerysi Simopelta pergandei cf 72.68 50.59 Diacamma pallidum 42.73 Ponera MG01 57.66 Harpegnathos saltator 43.37 Hypoponera opacior 51.36 Myopias tenuis 40.6 Anochetus madagascarensis 28.58 Odontomachus coquereli

180 160 140 120 100 8 0 6 0 4 0 2 0 0 Figure 3: Chronogram from the divergence dating analysis under the fossilized birth-death process with diversified sampling, in MrBayes. Scale is in Ma. Bars depict the 95% highest posterior probability density of each estimate.

361 tional heterogeneity play a role. The outgroup removal experiments, exclusion of compositionally 362 heterogeneous sites, and simulations all suggest that with greater compositional heterogeneity in 363 the data the abnormally AT-rich Leptanillinae species are drawn more strongly to the base of the ant 364 tree. As a result of this, the more GC-rich Martialis can erroneously cluster with the clade of formi- 365 coids and poneroids, as in Kück et al. (2011). When compositional heterogeneity is accounted for, 366 as in the homogeneous data matrix, the Leptanillinae and Martialis emerge as sister taxa forming 367 a strongly supported clade that is sister to all other ants.

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Table 3: Comparison of divergence time estimates. Numbers for this study refer to median node age (95% highest posterior density) in Ma. Numbers from other studies are ranges of means/medians from across all analyses presented.

Crown taxon This study Brady et al. 2006 Moreau et al. 2006 Moreau and Bell 2013 Schmidt 2013 Formicidae 112 (103–123) 111–137 141–169 139–158 116–141 Leptanillinae (excl. Opamyrma) 48 (34–64) 68–89 102–123 72–104 NA Amblyoponinae + Apomyrminae 75 (62–87) 92–118 113–143 NA 85–100 Proceratiinae 65 (51–79) 78–98 111–132 NA 74–88 Ponerinae 73 (61–84) 79–103 111–132 56–60 85–102 Dolichoderinae 55 (52–62) 71–76 86–97 53–66 NA Myrmeciinae 45 (37–57) 46–52 NA NA NA Formicinae 60 (51–71) 77–82 92–104 75–90 65–70 Myrmicinae 61 (51–72) 82–89 100–114 78–90 73–76 Dorylinae >= 55 (39–72) 76–87 99–117 78–95 78–88

368 The selective outgroup removal experiment shows a trend in which support for the AT-rich Lep- 369 tanillinae as sister to all other ants including Martialis is the strongest when only AT-rich outgroups 370 are retained, moderate or weak when all outgroups (those that are AT- and GC-rich) are retained, 371 and the weakest when all the AT-rich outgroups are removed. This suggests that the AT content of 372 outgroup taxa indeed causes attraction of the above-average AT-rich Leptanillinae to the base of the 373 ant tree. This finding has interesting implications for outgroup choice in phylogenetics in general, 374 as it suggests that choice of outgroups should be made not only on the basis of their close rela- 375 tionship to the ingroup taxa, but also taking into account sequence divergence and other properties 376 relative to the ingroup (Takezaki and Nishihara, 2016) 377 Finally, our simulations show that compositional heterogeneity similar to that in our empiri- 378 cal data matrix has the potential to cause bias resulting in the incorrect placement of Leptanillinae 379 as sister to Martialis plus poneroids plus formicoids. The matrix designed to imitate third codon 380 positions was simulated on a tree where Martialis was sister to Leptanillinae, but the maximum- 381 likelihood trees inferred from this alignment often recover Martialis sister to all other ants or sister 382 to the poneroids plus formicoids, both topologies recovered in previous studies (Rabeling et al., 383 2008; Kück et al., 2011). The tree topology used for the simulation is correctly recovered from the 384 alignments imitating first and second codon positions in most cases. These alignments were sim- 385 ulated with base frequencies drawn from our empirical alignment and under a tree-homogeneous 386 model. On a combined simulated data set the negative effect of sites emulating third codon posi- 387 tions is overwhelmed by the homogeneous data and the inferred tree is consistent with the topology 388 on which the data were simulated in 99 out of 100 maximum-likelihood trees obtained from the 389 simulated concatenated data. These effects appear not to be as strong as seen in our empirical 390 data, but the simulation is a simplistic scenario that is likely to involve fewer confounding factors. 391 In particular, 1) the same substitution model that generated the data could be used for inference 392 (minus compositional heterogeneity), 2) the simulations attempted to capture only one dimension 393 of the process heterogeneity, and 3) in our empirical data matrix compositionally heterogeneous 394 sites were actually over-represented compared to the simulated matrix because of the heteroge- 395 neous 28S partition, which was not taken into account when simulating the matrix imitating the 396 non-homogenous third codon positions. The results from simulations imitating compositionally 397 heterogeneous third codon position data demonstrate that compositional heterogeneity, at least in 398 principle, has the potential to impact the position of Martialis.

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399 If our interpretation of these results is correct, the species-poor clade of blind and subterranean 400 Martialinae and Leptanillinae is the sister group to the remaining 99.5% species of the Formicidae. 401 As pointed out before (Brady et al., 2006; Rabeling et al., 2008; Pie and Feitosa, 2015), this does not 402 necessarily mean that the most-recent common ancestor of the ants was blind and lived underground 403 (Lucky et al., 2013). Rather, this fact may reflect lower relative extinction rates experienced by 404 these ants, perhaps due to the long-term stability of their subterranean environments, or different 405 relative probabilities of evolutionary transitions between subterranean and epigaeic habits.

406 4.2. Relationships of poneroid subfamillies

407 In addition to further insight into the placement of the root of the ant phylogeny, we find evidence 408 for poneroid monophyly. The question of poneroid monophyly vs. paraphyly was the second 409 outstanding issue in higher ant phylogeny highlighted in a recent review (Ward, 2014). All our em- 410 pirical analyses suggest poneroid monophyly, and in most instances this clade receives significant 411 support, with the notable exception of analyses of the data matrix from which GC-rich outgroups 412 were removed, i.e., where the phylogeny was potentially more susceptible to bias. Poneroid mono- 413 phyly was first recovered in Moreau et al. (2006) but this result was questioned as doubtful by Brady 414 et al. (2006), who emphasized contradictory results from their Bayesian and maximum-likelihood 415 analyses of which the former supported monophyletic poneroids but the latter did not. Brady et al. 416 (2006) also conducted ingroup-only analyses which supported topologies where no possible root- 417 ing could result in poneroid monophyly. Although we did not perform ingroup-only analyses here, 418 taking into account more comprehensive taxon sampling, the higher amount of sequence data, and 419 insensitivity of poneroid monophyly to the different data treatments, we interpret the support for 420 the poneroid clade as strong. Poneroid monophyly has also been recovered in other phylogenetic 421 studies of ants, including Moreau et al. (2006), some analyses of Brady et al. (2006), Ward and 422 Fisher (2016), and a phylogenomic study (Branstetter et al., 2017b). 423 Similarly strongly-supported results involve the relationships among the poneroid clade sub- 424 families Agroecomyrmecinae, Paraponerinae, and Ponerinae. Although very disparate morpho- 425 logically, Agroecomyrmecinae emerge as sister to Paraponerinae with significant support in all 426 analyses, and this clade is in turn sister to the Ponerinae. 427 Our data are inconclusive on the relative position of Proceratiinae and (Amblyoponinae plus 428 Apomyrminae), which sometimes emerge as a clade and at other times as a grade relative to the 429 clade composed of Agroecomyrmecinae, Paraponerinae, and Ponerinae. If other results presented 430 here are confirmed, the placement of (Ambyloponinae plus Apomyrminae) and Proceratiinae within 431 the poneroid group would remain the last unsolved subfamily-level relationship in ants.

432 4.3. Non-monophyly of currently recognized genera

433 Our analyses recover several of the currently recognized ant genera as para- or polyphyletic. Al- 434 though we do not favor inclusion of non-monophyletic groups in a classification, here we are only 435 highlighting existing problems without proposing any formal taxonomic changes. We feel that 436 proposing satisfactory resolutions requires additional research, as explained for each case below. 437 Among the Leptanillinae, we find the morphologically derived genus Anomalomyrma nested 438 within samples identified by us as Protanilla. A more comprehensive evaluation of Protanilla-like 439 leptanillines, including both males and workers, should be carried out for a better understanding

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440 of diversity within the group. We find two other leptanilline genera, Phaulomyrma and Yavnella, 441 nested within Leptanilla. Both these genera were described based on males not associated with 442 workers (Wheeler and Wheeler, 1930; Kugler, 1987). Because the characters defining and differ- 443 entiating leptanilline lineages based on males are not well understood (Ogata et al., 1995) and all of 444 our Leptanilla specimens were males, we feel it would be premature to propose taxonomic changes. 445 A critical reappraisal of leptanilline using both morphology and molecular phylogenetics 446 is clearly needed. 447 Our analyses find the proceratiine genus Probolomyrmex nested within the larger genus Procer- 448 atium. Several species currently in Proceratium were classified in the erstwhile genus Sysphingta. 449 The differentiation between the two taxa was mostly based on the structure of the clypeus and 450 shape of the petiole. Based on these characters, two of the Proceratium species included in our 451 phylogeny, P. avium and P. stictum, would fit the old concept of Sysphingta, while the two other, 452 Proceratium silaceum and Proceratium SC02, match Proceratium sensu stricto (P. silaceum is the 453 type species of the genus). Previous authors (Brown, 1958; Urbani and Andrade, 2003), however, 454 showed that considerable variation exists with regard to the characters originally used to distinguish 455 Proceratium from Sysphingta. Proceratium taxonomy would thus benefit from a focused study and 456 a re-evaluation of morphology under a modern phylogenetic framework. 457 Despite recent comprehensive taxonomic and phylogenetic work focusing on the Ponerinae 458 (Schmidt, 2013; Schmidt and Shattuck, 2014), our analyses reveal three non-monophyletic genera 459 within the subfamily. 460 Cryptopone is a case of a polyphyletic genus. The two species included here, C. gilva and C. 461 hartwigi, are only very distantly related. The former is a part of the Ponera genus-group and the 462 latter sister to Fisheropone and a part of the Odontomachus genus-group, as defined by Schmidt 463 (2013). As noted by Schmidt and Shattuck (2014), the resolution of Cryptopone taxonomy would 464 require a more thorough revision and sampling of all species attributed to this genus. Notably, our 465 phylogeny did not include any species placed in the erstwhile genus Wadeura, which may well turn 466 out to be yet another lineage unrelated to the type species C. testacea. 467 Euponera, which was recognized to form two morphologically distinct species groups by Schmidt 468 and Shattuck (2014), is represented by E. brunoi and E. sikorae in our data set. In our phylogeny, 469 E. brunoi is more closely related to ”Cryptopone” hartwigi and Fisheropone than E. sikorae. To- 470 gether, the paraphyletic Euponera, the species ”Cryptopone” hartwigi, and Fisheropone ambigua 471 form a well-supported group with well-resolved internal relationships. Euponera is divisible into 472 two groups based on morphology but there are several species that cannot be placed with certainty 473 even within the genus as presently defined (Schmidt and Shattuck, 2014). Assignment of Euponera 474 species into two different genera should thus be postponed until more evidence is available. 475 Mesoponera, also found to be polyphyletic in our analyses, presents a particularly taxonomi- 476 cally challenging genus that would require a more comprehensive reexamination of morphology 477 and inclusion of more species in a phylogeny for satisfactory resolution. See Schmidt and Shattuck 478 (2014) for a more thorough discussion.

479 4.4. The age of extant ants

480 Our divergence-time analysis indicates that the most recent common ancestor of living ants origi- 481 nated during the Lower Cretaceous (103–124 Ma; median 113 Ma), an age estimate considerably 482 younger than those obtained by some other recent studies. Moreau et al. (2006) concluded that ants

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483 most likely arose 140–169 Ma while Moreau and Bell (2013) arrived at an estimate of 139–158 484 Ma. In contrast, Brady et al. (2006) proposed a younger age for the crown ants, 116–133 Ma. 485 Our study parallels the pattern for age estimates of the order , which has often 486 been inferred to be much older than the oldest hymenopteran fossils (Ronquist et al., 2012; Peters 487 et al., 2017), but under fossilized birth-death process with diversified sampling its estimated age 488 fell to within 20 Ma from the oldest known fossils (Zhang et al., 2016). In our study, the median 489 age for the crown Formicidae, at 113 Ma, is also about 20 Ma older than the oldest undisputed 490 crown-group fossils (Grimaldi and Agosti, 2000; Barden, 2017). 491 The ages we recovered for ant subfamilies where either few old crown-group fossils are known 492 or only a few taxa were sampled are almost certainly underestimated (e.g. Dolichoderinae at 55 Ma, 493 cf. Ward et al. (2010); Formicinae at 60 Ma, cf. Blaimer et al. (2015)). Future studies including 494 more genus-level sampling of extant and extinct taxa are likely to modify these estimates in the 495 direction of older dates.

496 5. Concluding remarks

497 Although more sequence data have often been shown to help resolve difficult phylogenetic ques- 498 tions, our study of ant phylogeny shows that systematic bias not accounted for by the commonly 499 used tree-homogeneous models may adversely affect phylogenetic inference. Simply increasing 500 the amount of data can in fact be detrimental if added sequences have properties that violate model 501 assumptions (Huelsenbeck and Hillis, 1993), such as the substantial among-taxon compositional 502 heterogeneity present in third codon positions and ribosomal 28S in our data set. The ideal solution 503 to this problem would be use of substitution models that take into account process heterogeneity 504 across the tree. Such models have been proposed (Foster, 2004; Blanquart and Lartillot, 2008; 505 Jayaswal et al., 2014), but unfortunately their current implementations do not scale well for larger 506 data sets, even for the modest amount of data present in our alignment. Alternatively, one can as- 507 sess model adequacy through simulation-based tests of compositional heterogeneity (Foster, 2004), 508 as in the current study. 509 The phylogenetic hypothesis presented here for deep nodes of the ant tree of life will soon be 510 tested with genomic-scale data. Recent advances in sequencing and analysis has already produced 511 data matrices with hundreds or even thousands of loci (Faircloth et al., 2014; Blaimer et al., 2015; 512 Branstetter et al., 2017b). As the amount of available sequence data increases, it is important that 513 the potential for model violation is carefully evaluated, as large data sets will likely be less prone 514 to uncertainty but instead may give strongly-supported results that are wrong instead (Philippe and 515 Roure, 2011). Tests of compositional heterogeneity, posterior predictive approaches to assessing 516 model fit (Bollback, 2002; Brown and ElDabaje, 2008; Doyle et al., 2015), or sensitivity of results 517 to removal of sites likely to introduce bias (Goremykin et al., 2015) should become a part of the 518 standard phylogenomics toolkit. 519 Our understanding of the timeline of ant evolution will also likely benefit from more biologi- 520 cally realistic models resulting from recent developments in divergence-dating, such as placement 521 of fossils using explicit information about morphology (Ronquist et al., 2012; Zhang et al., 2016), 522 and from inclusion of more sequence data as well as more comprehensive taxon sampling.

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523 6. Acknowledgements

524 For providing specimens used in this study we are grateful to Bob Anderson, Himender Bharti, 525 Lech Borowiec, Bui Tuan Viet, Jim Carpenter, Katsuyuki Eguchi, Juergen Gadau, Dave General, 526 Benoit Guénard, Nihara Gunawardene, Peter Hawkes, Armin Ionesco-Hirsch, Bob Johnson, Yeo 527 Kolo, John LaPolla, John Lattke, Jack Longino, Randy Morgan, Michael Ohl, Christian Peeters, 528 James Pitts, Julian Resasco, Keve Ribardo, Riitta Savolainen, Chris Schmidt, Justin Schmidt, Mike 529 Sharkey, Andy Suarez, Simon van Noort, and Masashi Yoshimura. This research was supported 530 by U.S. National Science Foundation grants EF-0431330, DEB-1402432, DEB-0842204, DEB- 531 1354996, DEB-1456964, and DEB-1654829.

532 References

533 Andrade, M. L. D. (1994). Fossil Odontomachiti ants from the Dominican Republic (Amber Col- 534 lection Stuttgart: Hymenoptera, Formicidae. VII: Odontomachiti). Stuttg. Beitr. Naturkd. Ser. B 535 (Geol. Paläontol.), 199:1–28.

536 Antropov, A. V. (2000). Digger wasps (Hymenoptera, Sphecidae) in Burmese amber. Bull. Nat. 537 Hist. Mus. Geol. Ser., 56(1):59–77.

538 Antropov, A. V. (2011). A new tribe of fossil digger wasps (Hymenoptera: Crabronidae) from 539 the Upper Cretaceous New Jersey amber and its place in the subfamily Pemphredoninae. Russ. 540 Entomol. J., 20(3):229–240.

541 Archibald, S. B., Cover, S. P., and Moreau, C. S. (2006). Bulldog ants of the Okana- 542 gan Highlands and history of the subfamily (Hymenoptera: Formicidae: Myrmeciinae). Ann. 543 Entomol. Soc. Am., 99(3):487–523.

544 Aria, C., Perrichot, V., and Nel, A. (2011). Fossil Ponerinae (Hymenoptera: Formicidae) in Early 545 Eocene amber of France. Zootaxa, 2870:53–62.

546 Baca, S. M., Toussaint, E. F., Miller, K. B., and Short, A. E. (2017). Molecular phylogeny of the 547 aquatic beetle family Noteridae (Coleoptera: Adephaga) with an emphasis on data partitioning 548 strategies. Mol. Phylogenet. Evol., 107:282–292.

549 Barden, P. (2017). Fossil ants (Hymenoptera: Formicidae): ancient diversity and the rise of modern 550 lineages. Myrmecol. News, 24:1–30.

551 Barden, P. and Grimaldi, D. A. (2016). Adaptive radiation in socially advanced stem-group ants 552 from the Cretaceous. Curr. Biol., 26(4):515–521.

553 Baroni Urbani, C. (2000). Rediscovery of the Baltic amber ant genus (Hymenoptera, 554 Formicidae) and its taxonomic consequences. Eclogae Geol. Helv., 93:471–480.

555 Beaulieu, J. M., O’Meara, B. C., Crane, P., and Donoghue, M. J. (2015). Heterogeneous rates of 556 molecular evolution and diversification could explain the age estimate for angiosperms. 557 Syst. Biol., 64(5):869–878.

558 Bennett, D. J. and Engel, M. S. (2005). A primitive sapygid wasp in Burmese amber (Hymenoptera: 559 Sapygidae). Acta Zool. Cracoviensia, 48(3-1):1–9.

18 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

560 Blaimer, B. B., Brady, S. G., Schultz, T. R., Lloyd, M. W., Fisher, B. L., and Ward, P. S. (2015). 561 Phylogenomic methods outperform traditional multi-locus approaches in resolving deep evolu- 562 tionary history: a case study of formicine ants. BMC Evol. Biol., 15(1):e271.

563 Blanquart, S. and Lartillot, N. (2008). A site- and time-heterogeneous model of amino acid re- 564 placement. Mol. Biol. Evol., 25(5):842–858.

565 Bollback, J. P. (2002). Bayesian model adequacy and choice in phylogenetics. Mol. Biol. Evol., 566 19(7):1171–1180.

567 Borowiec, M. L. (2016). AMAS: a fast tool for alignment manipulation and computing of summary 568 statistics. PeerJ, 4:e1660.

569 Boudinot, B. E., Probst, R. S., Brandão, C. R. F., Feitosa, R. M., and Ward, P. S. (2016). Out of 570 the Neotropics: newly discovered relictual species sheds light on the biogeographical history of 571 spider ants (, Dolichoderinae, Formicidae). Syst. Entomol., 41(3):658–671.

572 Brady, S. G., Fisher, B. L., Schultz, T. R., and Ward, P. S. (2014). The rise of army ants and their 573 relatives: diversification of specialized predatory doryline ants. BMC Evol. Biol., 14(1):93.

574 Brady, S. G., Schultz, T. R., Fisher, B. L., and Ward, P. S. (2006). Evaluating alternative hypotheses 575 for the early evolution and diversification of ants. Proc. Natl. Acad. Sci. U.S.A., 103:18172– 576 18177.

577 Branstetter, M. G., Danforth, B. N., Pitts, J. P., Faircloth, B. C., Ward, P. S., Buffington, M. L., 578 Gates, M. W., Kula, R. R., and Brady, S. G. (2017a). Phylogenomic insights into the evolution 579 of stinging wasps and the origins of ants and bees. Curr. Biol., 27(7):1019–1025.

580 Branstetter, M. G., Longino, J. T., Ward, P. S., and Faircloth, B. C. (2017b). Enriching the ant tree 581 of life: enhanced UCE bait set for genome-scale phylogenetics of ants and other Hymenoptera. 582 Methods Ecol. Evol., 9:768–776.

583 Brothers, D. J. (1974). The first recent species of Protomutilla (Hymenoptera: Mutillidae; Myr- 584 mosinae). Psyche (Cambridge), 81(2):268–271.

585 Brown, W. L., J. (1958). Contributions toward a reclassification of the Formicidae. II. Tribe Ec- 586 tatommini (Hymenoptera). Bull. Mus. Comp. Zool., 118:173–362.

587 Brown, J. M. (2014). Predictive approaches to assessing the fit of evolutionary models. Syst. Biol., 588 63(3):289–292.

589 Brown, J. M. and ElDabaje, R. (2008). PuMA: Bayesian analysis of partitioned (and unpartitioned) 590 model adequacy. Bioinformatics, 25(4):537–538.

591 Brown, J. W. and Smith, S. A. (2017). The past sure is tense: on interpreting phylogenetic diver- 592 gence time estimates. bioRxiv.

593 Chomicki, G., Ward, P. S., and Renner, S. S. (2015). Macroevolutionary assembly of ant/plant 594 symbioses: ants and their ant-housing plants in the Neotropics. Proc. R. Soc. 595 Lond. Ser. B. Biol. Sci., 282(1819):20152200.

596 Darling, D. C. and Sharkey, M. J. (1990). Taxonomic names, in insects from the Santana Forma- 597 tion, Lower Cretaceous, of . Chapter 7: Order Hymenoptera. Bull. Amer. Mus. Nat. Hist.,

19 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

598 195:123–153.

599 Dlussky, G. M. (1988). Ants of Sakhalin amber (Paleocene?). Paleontolog. J., 22(1):50–61.

600 Dlussky, G. M. (1997). Genera of ants (Hymenoptera: Formicidae) from Baltic amber. Paleon- 601 tolog. J., 31:616–627.

602 Dlussky, G. M. (2009). The ant subfamilies Ponerinae, Cerapachyinae, and Pseudomyrmecinae 603 (Hymenoptera, Formicidae) in the Late Eocene Ambers of Europe. Paleontolog. J., 43(9):1043– 604 1086.

605 Dlussky, G. M. and Perkovsky, E. E. (2002). Ants (Hymenoptera, Formicidae) from the Rovno 606 Amber. [In Russian.]. Vestn. Zool., 36(5):3–20.

607 Dlussky, G. M. and Radchenko, A. G. (2011). Pristomyrmex rasnitsyni sp. n., the first known fossil 608 species of the ant genus Pristomyrmex mayr (Hymenoptera: Formicidae) from the Late Eocene 609 Danish amber. Russ. Entomol. J., 20(3):251–254.

610 Dlussky, G. M. and Rasnitsyn, A. P. (2003). Ants (Hymenoptera: Formicidae) of Formation Green 611 River and some other Middle Eocene deposits of North America. Russ. Entomol. J., 11(4):411– 612 436.

613 Dlussky, G. M. and Rasnitsyn, A. P. (2009). Ants (Insecta: Vespida: Formicidae) in the Upper 614 Eocene amber of central and eastern Europe. Paleontolog. J., 43(9):1024–1042.

615 Dlussky, G. M., Rasnitsyn, A. P., and Perfilieva, K. S. (2015). The ants (Hymenoptera: Formicidae) 616 of Bol’shaya Svetlovodnaya (Late Eocene of Sikhote-Alin, Russian Far East). Cauc. Entomol. 617 Bull., 11(1):131–152.

618 Dlussky, G. M., Wappler, T., and Wedmann, S. (2008). New middle Eocene formicid species from 619 Germany and the evolution of weaver ants. Acta Palaeontol. Polonica, 53(4):615–626.

620 Doyle, V. P., Young, R. E., Naylor, G. J. P., and Brown, J. M. (2015). Can we identify genes with 621 increased phylogenetic reliability? Syst. Biol., 64(5):824–837.

622 Drummond, A. J. and Rambaut, A. (2007). BEAST: bayesian evolutionary analysis by sampling 623 trees. BMC Evol. Biol., 7(1):214.

624 Engel, M. S. (2008). The wasp family Rhopalosomatidae in mid-Cretaceous amber from Myanmar 625 (Hymenoptera: Vespoidea). J. Kansas Entomol. Soc., 81(3):168–174.

626 Engel, M. S., Ortega-Blanco, J., and Bennett, D. J. (2009). A remarkable tiphiiform wasp in 627 mid-Cretaceous amber from Myanmar (Hymenoptera: Tiphiidae). Trans. Kans. Acad. Sci., 628 112(1/2):1–6.

629 Faircloth, B. C., Branstetter, M. G., White, N. D., and Brady, S. G. (2014). Target enrichment of 630 ultraconserved elements from provides a genomic perspective on relationships among 631 Hymenoptera. Mol. Ecol. Resour., 15(3):489–501.

632 Foster, P. (2004). Modeling compositional heterogeneity. Syst. Biol., 53(3):485–495.

633 Frandsen, P. B., Calcott, B., Mayer, C., and Lanfear, R. (2015). Automatic selection of partitioning 634 schemes for phylogenetic analyses using iterative k-means clustering of site rates. BMC Evol. 635 Biol., 15(1):13.

20 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

636 Gascuel, O. (1997). BIONJ: an improved version of the NJ algorithm based on a simple model of 637 sequence data. Mol. Biol. Evol., 14(7):685–695.

638 Goremykin, V. V., Nikiforova, S. V., Cavalieri, D., Pindo, M., and Lockhart, P. (2015). The root of 639 flowering plants and total evidence. Syst. Biol., 64(5):879–891.

640 Grimaldi, D. and Agosti, D. (2000). A formicine in New Jersey Cretaceous amber (Hymenoptera: 641 Formicidae) and early evolution of the ants. Proc. Natl. Acad. Sci. U.S.A., 97:13678–13683.

642 Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W., and Gascuel, O. (2010). 643 New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the per- 644 formance of PhyML 3.0. Syst. Biol., 59(3):307–321.

645 Heath, T. A., Huelsenbeck, J. P., and Stadler, T. (2014). The fossilized birth-death process for 646 coherent calibration of divergence-time estimates. Proc. Natl. Acad. Sci. U.S.A., 111(29):E2957– 647 E2966.

648 Höhna, S., Stadler, T., Ronquist, F., and Britton, T. (2011). Inferring speciation and extinction rates 649 under different sampling schemes. Mol. Biol. Evol., 28(9):2577–2589.

650 Hölldobler, B. and Wilson, E. O. (1990). The ants. Harvard University Press.

651 Huelsenbeck, J. P. and Hillis, D. M. (1993). Success of phylogenetic methods in the four-taxon 652 case. Syst. Biol., 42(3):247–264.

653 Jayaswal, V., Wong, T. K. F., Robinson, J., Poladian, L., and Jermiin, L. S. (2014). Mixture models 654 of nucleotide sequence evolution that account for heterogeneity in the substitution process across 655 sites and across lineages. Syst. Biol., 63(5):726–742.

656 Jermiin, L., Ho, S. Y., Ababneh, F., Robinson, J., and Larkum, A. W. (2004). The biasing effect 657 of compositional heterogeneity on phylogenetic estimates may be underestimated. Syst. Biol., 658 53(4):638–643.

659 Kugler, J. (1987). The Leptanillinae (Hymenoptera: Formicidae) of Israel and a description of a 660 new species from India. Isr. J. Entomol., 20:45–57.

661 Kück, P., Hita Garcia, F., Misof, B., and Meusemann, K. (2011). Improved phylogenetic anal- 662 yses corroborate a plausible position of Martialis heureka in the ant tree of life. PLoS ONE 663 (doi:10.1371/journal.pone.0021031), 6:e21031.

664 Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T., and Calcott, B. (2017). PartitionFinder 665 2: new methods for selecting partitioned models of evolution for molecular and morphological 666 phylogenetic analyses. Mol. Biol. Evol., 34(3):772–773.

667 Lapolla, J. S. and Greenwalt, D. E. (2015). Fossil ants (Hymenoptera: Formicidae) of the Middle 668 Eocene Kishenehn Formation. Sociobiology, 62(2):163–174.

669 Lelej, A. S. (1986). Males of the genus Protomutilla (Hymenoptera, Mutillidae) from Baltic amber. 670 Paleontol. Zh., 4:104–106.

671 Lucky, A., Trautwein, M. D., Guénard, B. S., Weiser, M. D., and Dunn, R. R. (2013). Tracing the 672 rise of ants - out of the ground. PLoS ONE, 8(12):e84012.

673 MacKay, W. P. (1991). Anochetus brevidentatus, new species, a second fossil Odontomachiti ant

21 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

674 (Hymenoptera: Formicidae). J. N. Y. Entomol. Soc., 99(1):138–140.

675 Maddison, D. R. and Maddison, W. P. (2005). MacClade 4., v. 4.08 for OSX.

676 Mayr, G. (1868). Die Ameisen des baltischen Bernsteins. Beitr. Naturkd. Preuss., 1:1–102.

677 McKellar, R. C., Glasier, J. R. N., and Engel, M. S. (2013). New ants (Hymenoptera: Formicidae: 678 Dolichoderinae) from Canadian Late Cretaceous amber. Bull. Geosci., 88(3):583–594.

679 Minh, B. Q., Nguyen, M. A. T., and von Haeseler, A. (2013). Ultrafast approximation for phylo- 680 genetic bootstrap. Mol. Biol. Evol., 30(5):1188–1195.

681 Moreau, C. S. and Bell, C. D. (2013). Testing the museum versus cradle tropical biological diversity 682 hypothesis: phylogeny, diversification, and ancestral biogeographic range evolution of the ants. 683 Evolution, 67(8):2240–2257.

684 Moreau, C. S., Bell, C. D., Vila, R., Archibald, S. B., and Pierce, N. E. (2006). Phylogeny of the 685 ants: diversification in the age of angiosperms. Science, 312:101–104.

686 Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., and Minh, B. Q. (2014). IQ-TREE: a fast and 687 effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol., 688 32(1):268–274.

689 Nguyen, N. D., Mirarab, S., Kumar, K., and Warnow, T. (2015). Ultra-large alignments using 690 phylogeny-aware profiles. Genome Biol., 16(1):124.

691 Ogata, K., Terayama, M., and Masuko, K. (1995). The ant genus Leptanilla: discovery of the 692 worker-associated male of L. japonica, and a description of a new species from Taiwan (Hy- 693 menoptera: Formicidae: Leptanillinae). Syst. Entomol., 20:27–34.

694 Ohl, M. (2004). The first fossil representative of the wasp genus Dolichurus, with a review of fossil 695 Ampulicidae (Hymenoptera: Apoidea). J. Kansas Entomol. Soc., 77(4):332–342.

696 Perrichot, V. (2015). A new species of Baikuris (Hymenoptera: Formicidae: ) in 697 mid-Cretaceous amber from France. Cretaceous Res., 52(B):585–590.

698 Perrichot, V., Lacau, S., Néraudeau, D., and Nel, A. (2008). Fossil evidence for the early ant 699 evolution. Naturwissenschaften, 95(2):85–90.

700 Peters, R. S., Krogmann, L., Mayer, C., Donath, A., Gunkel, S., Meusemann, K., Kozlov, A., 701 Podsiadlowski, L., Petersen, M., Lanfear, R., Diez, P. A., Heraty, J., Kjer, K. M., Klopfstein, 702 S., Meier, R., Polidori, C., Schmitt, T., Liu, S., Zhou, X., Wappler, T., Rust, J., Misof, B., and 703 Niehuis, O. (2017). Evolutionary history of the hymenoptera. Curr. Biol., 27(7):1013–1018.

704 Philippe, H. and Roure, B. (2011). Difficult phylogenetic questions: more data, maybe; better 705 methods, certainly. BMC Biol., 9(1):91.

706 Pie, M. R. and Feitosa, R. M. (2015). Relictual ant lineages (Hymenoptera: Formicidae) and their 707 evolutionary implications. Myrmecol. News, 22:55–58.

708 Pilgrim, E. M., Dohlen, C. D. V., and Pitts, J. P. (2008). Molecular phylogenetics of Vespoidea 709 indicate paraphyly of the superfamily and novel relationships of its component families and 710 subfamilies. Zool. Scripta, 37(5):539–560.

22 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

711 Poinar, G. O. (2009). Melittosphex (Hymenoptera: Melittosphecidae), a primitive bee and not a 712 wasp. Palaeontology, 52(2):483–484.

713 Poinar, G. O. and Danforth, B. N. (2006). A fossil bee from Early Cretaceous Burmese amber. 714 Science, 314(5799):614.

715 Rabeling, C., Brown, J. M., and Verhaagh, M. (2008). Newly discovered sister lineage sheds light 716 on early ant evolution. Proc. Natl. Acad. Sci. U.S.A., 105:14913–14917.

717 Radchenko, A. G., Elmes, G. W., and Dlussky, G. (2007). The ants of the genus Myrmica 718 (Hymenoptera, Formicidae) from Baltic and Saxonian amber (Late Eocene). J. Paleontol., 719 81(6):1494–1501.

720 Rasnitsyn, A. P. and Jarzembowski, E. A. (1998). Systematic part, in wasps (Insecta: Vespida 721 = Hymenoptera) from the Purbeck and Wealden Supergroups (Lower Cretaceous) of southern 722 England and their biostratigraphical and palaeoenvironmental significance. Cretaceous Res., 723 19(3/4):329–391.

724 Rasnitsyn, A. P. and Martínez-Delclòs, X. (2000). Wasps (Insecta: Vespida = Hymenoptera) from 725 the Early Cretaceous of Spain. Acta Geol. Hisp., 35(1-2):65–96.

726 Rodriguez, J., Waichert, C., Dohlen, C. D. V., Jr, G. P., and Pitts, J. P. (2016). Eocene and not 727 Cretaceous origin of spider wasps: fossil evidence from amber. Acta Palaeontol. Polonica, 728 61(1):89–96.

729 Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, 730 L., Suchard, M. A., and Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient bayesian phylogenetic 731 inference and model choice across a large model space. Syst. Biol., 61(3):539–542.

732 Rust, J. and Andersen, N. M. (1999). Giant ants from the Paleogene of Denmark with a discussion 733 of the fossil history and early evolution of ants (Hymenoptera: Formicidae). Zool. J. Linn. Soc., 734 125(3):331–348.

735 Sanderson, M. J. (2002). Estimating absolute rates of molecular evolution and divergence times: a 736 penalized likelihood approach. Mol. Biol. Evol., 19(1):101–109.

737 Schmidt, C. (2013). Molecular phylogenetics of ponerine ants (Hymenoptera: Formicidae: Poner- 738 inae). Zootaxa, 3647:201–250.

739 Schmidt, C. A. and Shattuck, S. O. (2014). The higher classification of the ant subfamily Ponerinae 740 (Hymenoptera: Formicidae), with a review of ponerine ecology and behavior. Zootaxa, 3817:1– 741 242.

742 Stamatakis, A. (2014). RAxML version 8: a tool for phylogenet. anal. and post-analysis of large 743 phylogenies. Bioinformatics, 30(9):1312–1313.

744 Takezaki, N. and Nishihara, H. (2016). Resolving the phylogenetic position of coelacanth: the 745 closest relative is not always the most appropriate outgroup. Genome Biol. Evol., 8(4):1208– 746 1221.

747 Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997). The 748 CLUSTAL_x windows interface: flexible strategies for multiple sequence alignment aided by 749 quality analysis tools. Nucleic Acids Res., 25(24):4876–4882.

23 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

750 Urbani, C. B. (1980). Anochetus corayi n. sp., the first fossil Odontomachiti ant. (Amber Collection 751 Stuttgart: Hymenoptera, Formicidae. II: Odontomachiti). Stuttg. Beitr. Naturkd. Ser. B (Geol. 752 Paläontol.), (55):1–6.

753 Urbani, C. B. and Andrade, M. L. D. (2003). The ant genus Proceratium in the extant and fossil 754 rec. (Hymenoptera: Formicidae). Mus. Reg. Sci. Nat. Monogr. (Turin), 36:1–492.

755 Wappler, T., Dlussky, G. M., Engel, M. S., Prokop, J., and Knor, S. (2014). A new trap-jaw 756 ant species of the genus Odontomachus (Hymenoptera: Formicidae: Ponerinae) from the Early 757 Miocene (Burdigalian) of the Czech Republic. Paläntolog. Z., 88(4):495–502.

758 Ward, P. S. (2014). The phylogeny and evolution of ants. Annu. Rev. Ecol. Evol. Syst., 45:23–43.

759 Ward, P. S., Brady, S. G., Fisher, B. L., and Schultz, T. R. (2010). Phylogeny and biogeography of 760 dolichoderine ants: effects of data partitioning and relict taxa on historical inference. Syst. Biol., 761 59:342–362.

762 Ward, P. S., Brady, S. G., Fisher, B. L., and Schultz, T. R. (2015). The evolution of myrmicine ants: 763 phylogeny and biogeography of a hyperdiverse ant clade (Hymenoptera: Formicidae). Syst. 764 Entomol., 40(1):61–81.

765 Ward, P. S. and Fisher, B. L. (2016). Tales of dracula ants: the evolutionary history of the ant 766 subfamily Amblyoponinae (Hymenoptera: Formicidae). Syst. Entomol., 41(3):683–693.

767 Wheeler, G. C. and Wheeler, E. W. (1930). Two new ants from Java. Psyche (Cambridge), 37:193– 768 201.

769 Wheeler, W. M. (1915). The ants of the Baltic amber. Schr. Phys.-Ökon. Ges. Königsb., (55):1–142.

770 Wilson, E. O. (2004). The biogeography of the West Indian ants (Hymenoptera: Formicidae). In 771 Liebherr, J. K., editor, Zoogeography of Caribbean insects, chapter 10, pages 266–290. Cornell 772 University Press, Ithaca, New York.

773 Zhang, C., Stadler, T., Klopfstein, S., Heath, T. A., and Ronquist, F. (2016). Total-evidence dating 774 under the fossilized birth–death process. Syst. Biol., 65(2):228–249.

24 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Brachycistis_sp 1 Chyphotes_mellipes 1 Aporus_niger 1 1 Dasymutilla_aureola 0.5958 Sapyga_pumila Apterogyna_ZA01 1 Scolia_verticalis 1 Ampulex_compressa 1 Apis_mellifera 1 Chalybion_californicum Opamyrma_hungvuong Protanilla_JP01 0.9416 1 Protanilla_TH01 1 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9984 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 0.9868 Leptanilla_TH01 1 Leptanilla_TH09 1 Leptanilla_ZA01 1 Leptanilla_GR01 0.9994 1 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani 1 Leptanilla_TH08 Phaulomyrma_MM01 1 Leptanilla_TH02 1 1 Leptanilla_TH05 1 Leptanilla_TH04 0.9634 Leptanilla_TH06 Martialis_heureka Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.8206 Labidus_praedator Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 1 Tetraponera_rufonigra 0.8121 Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus Acanthoponera_minor 1 1 Rhytidoponera_chalybaea 0.997 Pogonomyrmex_vermiculatus 0.7133 Manica_bradleyi 1 1 Myrmica_tahoensis Stenamma_expolitum 1 Crematogaster_kelleri 0.9864 Cyphomyrmex_longiscapus_cf 1 0.9061 Myrmicaria_carinata Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 1 1 Amblyopone_australis 0.9424 Onychomyrmex_hedleyi Fulakora_mystriops 0.9122 1 Fulakora_saundersi Myopopone_castanea 1 Mystrium_voeltzkowi Stigmatomma_boltoni 1 Stigmatomma_denticulatum Stigmatomma_pallipes Adetomyrma_caputleae 0.5152 1 Xymmer_muticus Discothyrea_testacea 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 1 Proceratium_silaceum 0.5385 Paraponera_clavata 1 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_mocquerysi 1 Platythyrea_lamellosa 0.9319 Platythyrea_punctata 1 Simopelta_pergandei_cf Belonopelta_HN01 1 0.666 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki 1 Emeryopone_MY01 0.5317 Ponera_swezeyi 0.9801 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9897 Hypoponera_opacior 1 0.9562 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 Plectroctena_ugandensis 1 Brachyponera_obscurans 0.7228 Myopias_tenuis Bothroponera_wasmannii Phrynoponera_pulchella 1 Anochetus_madagascarensis 1 Odontomachus_coquereli Mesoponera_ambigua 0.9336 0.9892 Streblognathus_peetersi Odontoponera_transversa 0.8174 Promyopias_silvestrii Asphinctopone_silvestrii 0.6729 Leptogenys_diminuta 1 Mesoponera_melanaria Euponera_sikorae 1 Euponera_brunoi 1 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.08 Supplementary Figure 1: Bayesian consensus tree inferred under greedy partitioning strategy for full data set.

25 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Brachycistis_sp 1 Chyphotes_mellipes 1 Aporus_niger 1 1 Dasymutilla_aureola 0.9825 Sapyga_pumila Ampulex_compressa 1 Apis_mellifera 1 Chalybion_californicum 1 Apterogyna_ZA01 1 Scolia_verticalis Martialis_heureka Opamyrma_hungvuong 0.9901 Protanilla_JP01 1 Protanilla_TH01 0.9996 1 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9867 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 Leptanilla_TH01 1 Leptanilla_TH09 0.7817 1 Leptanilla_ZA01 1 Leptanilla_GR01 0.9863 1 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani Leptanilla_TH08 1 0.6402 Phaulomyrma_MM01 1 Leptanilla_TH02 1 Leptanilla_TH05 1 Leptanilla_TH04 0.8636 Leptanilla_TH06 Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.9991 1 Labidus_praedator Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 1 Tetraponera_rufonigra Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus Acanthoponera_minor 1 1 Rhytidoponera_chalybaea 0.9997 Pogonomyrmex_vermiculatus 0.643 Manica_bradleyi 1 1 Myrmica_tahoensis Stenamma_expolitum 1 Crematogaster_kelleri 0.6688 Cyphomyrmex_longiscapus_cf 0.5256 Myrmicaria_carinata Discothyrea_testacea 1 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 0.5856 1 Proceratium_silaceum Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 1 1 Amblyopone_australis 0.9988 Onychomyrmex_hedleyi 0.769 Fulakora_mystriops 1 Fulakora_saundersi 1 Adetomyrma_caputleae Stigmatomma_denticulatum 1 0.9999 1 Stigmatomma_pallipes 0.9735 Mystrium_voeltzkowi 0.7218 Stigmatomma_boltoni Myopopone_castanea 0.6959 Xymmer_muticus Paraponera_clavata 1 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_lamellosa 1 Platythyrea_mocquerysi 0.6824 0.9999 Platythyrea_punctata Simopelta_pergandei_cf Belonopelta_HN01 1 0.9965 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum 1 Emeryopone_MY01 Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki Ponera_swezeyi 0.991 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9995 Hypoponera_opacior 1 0.9999 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 1 Plectroctena_ugandensis Myopias_tenuis Brachyponera_obscurans 1 Asphinctopone_silvestrii 0.9234 Leptogenys_diminuta 0.9990.9989 5 Mesoponera_melanaria Phrynoponera_pulchella 0.9039 0.9863 Anochetus_madagascarensis 1 Odontomachus_coquereli 0.8269 Bothroponera_wasmannii Mesoponera_ambigua 1 Streblognathus_peetersi 0.8961 Odontoponera_transversa 0.8748 Promyopias_silvestrii Euponera_sikorae 1 Euponera_brunoi 1 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.06 Supplementary Figure 2: Bayesian consensus tree inferred under k-means partitioning strategy for full data set.

26 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Brachycistis_sp 8 9 Chyphotes_mellipes 9 7 Aporus_niger 8 8 Dasymutilla_aureola 7 4 Sapyga_pumila Apterogyna_ZA01 9 8 Scolia_verticalis 9 3 Ampulex_compressa 9 8 Apis_mellifera 100 Chalybion_californicum Opamyrma_hungvuong Protanilla_JP01 8 3 100 Protanilla_TH01 100 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 9 Protanilla_VN03 100Protanilla_TH03 100 100 Protanilla_VN01 7 8 Leptanilla_TH01 100 Leptanilla_TH09 100 Leptanilla_ZA01 100 Leptanilla_GR01 9 2 100 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani Leptanilla_TH08 10808 Phaulomyrma_MM01 9 8 Leptanilla_TH02 100 100 Leptanilla_TH05 100 Leptanilla_TH04 9 6 Leptanilla_TH06 Martialis_heureka Cerapachys_jacobsoni 100 Labidus_praedator 9 3 Leptanilloides_femoralis Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 100 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 Tetraponera_punctulata 100 100 Tetraponera_rufonigra 9 3 Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus 100 Acanthoponera_minor 100 Rhytidoponera_chalybaea Manica_bradleyi 9 8 100 Myrmica_tahoensis 100 Pogonomyrmex_vermiculatus 8 6 Stenamma_expolitum 100 Crematogaster_kelleri 9 3 Cyphomyrmex_longiscapus_cf 100 8 8 Myrmicaria_carinata Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 9 8 100 Amblyopone_australis 9 8 Onychomyrmex_hedleyi 9 7 Fulakora_mystriops 100 Fulakora_saundersi Stigmatomma_denticulatum 100 9 5 Stigmatomma_pallipes Myopopone_castanea 10403 Stigmatomma_boltoni 4 4 Mystrium_voeltzkowi 4 4 Adetomyrma_caputleae 7 3 100 Xymmer_muticus Discothyrea_testacea 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 100 Proceratium_silaceum 7 7 Paraponera_clavata 100 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_mocquerysi 100 Platythyrea_lamellosa 9 9 Platythyrea_punctata 9 7 Simopelta_pergandei_cf Belonopelta_HN01 100 9 2 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 Neoponera_unidentata 100 Diacamma_pallidum Emeryopone_MY01 1007 6 Cryptopone_gilva 9 5 100 Ectomomyrmex_leeuwenhoeki Ponera_swezeyi 8 5 Parvaponera_darwinii 100 Pseudoponera_stigma 100 Harpegnathos_saltator 9 5 Hypoponera_opacior 100 9 3 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 100 Plectroctena_ugandensis Asphinctopone_silvestrii 9 9 5 5 Anochetus_madagascarensis 100 Odontomachus_coquereli 3 4 Brachyponera_obscurans 8 8 Myopias_tenuis 4 1 Leptogenys_diminuta 9 3 Mesoponera_melanaria 100 Bothroponera_wasmannii 4 5 Promyopias_silvestrii 2 9 Odontoponera_transversa 7 8 Phrynoponera_pulchella 4 0 Mesoponera_ambigua 9 0 Streblognathus_peetersi 6 5 Euponera_sikorae 100 Euponera_brunoi 9 8 Cryptopone_hartwigi 100 Fisheropone_ambigua

0.08 Supplementary Figure 3: Maximum-likelihood tree inferred under greedy partitioning strategy for full data set.

27 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Brachycistis_sp 8 7 Chyphotes_mellipes 9 1 Aporus_niger 8 4 Dasymutilla_aureola 8 0 Sapyga_pumila Ampulex_compressa 9 0 Apis_mellifera 100 Chalybion_californicum 9 5 Apterogyna_ZA01 9 5 Scolia_verticalis Martialis_heureka Opamyrma_hungvuong 8 7 Protanilla_JP01 100 Protanilla_TH01 7 6 100 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 9 Protanilla_VN03 100 Protanilla_TH03 100 100 Protanilla_VN01 Leptanilla_TH01 100 Leptanilla_TH09 6 5 100 Leptanilla_ZA01 100 Leptanilla_GR01 9 3 100 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani Leptanilla_TH08 10903 Phaulomyrma_MM01 9 7 Leptanilla_TH02 100 Leptanilla_TH05 100 Leptanilla_TH04 9 4 Leptanilla_TH06 Leptanilloides_femoralis 100 Cerapachys_jacobsoni 9 6 100 Labidus_praedator Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 100 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 Tetraponera_punctulata 100 100 Tetraponera_rufonigra Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus 100 Acanthoponera_minor 100 Rhytidoponera_chalybaea Manica_bradleyi 9 9 100 Myrmica_tahoensis 100 Pogonomyrmex_vermiculatus 5 8 Stenamma_expolitum 100 Crematogaster_kelleri 6 8 Cyphomyrmex_longiscapus_cf 6 2 Myrmicaria_carinata Discothyrea_testacea 100 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 7 1 100 Proceratium_silaceum Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 9 1 100 Amblyopone_australis 9 2 Onychomyrmex_hedleyi 9 2 Fulakora_mystriops 100 Fulakora_saundersi 100 Adetomyrma_caputleae Stigmatomma_denticulatum 1009 6 9 2 Stigmatomma_pallipes 8 3 Mystrium_voeltzkowi 5 5 Stigmatomma_boltoni 5 1 Myopopone_castanea 8 3 Xymmer_muticus Paraponera_clavata 9 8 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_lamellosa 100 Platythyrea_mocquerysi 9 4 8 8 Platythyrea_punctata Simopelta_pergandei_cf Belonopelta_HN01 100 9 8 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 100 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 100 100 Ectomomyrmex_leeuwenhoeki 100 Emeryopone_MY01 9 1 Ponera_swezeyi 9 3 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 8 Hypoponera_opacior 100 9 8 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 9 9 100 Plectroctena_ugandensis Myopias_tenuis Brachyponera_obscurans 100 Asphinctopone_silvestrii 5 0 Leptogenys_diminuta 9 7 9 6 Mesoponera_melanaria 5 0 Phrynoponera_pulchella 7 1 Anochetus_madagascarensis 100 Odontomachus_coquereli 3 8 Odontoponera_transversa 7 1 Promyopias_silvestrii Mesoponera_ambigua 3 59 1 Streblognathus_peetersi 2 5 Bothroponera_wasmannii 2 9 Euponera_sikorae 100 Euponera_brunoi 100 Cryptopone_hartwigi 100 Fisheropone_ambigua

0.06 Supplementary Figure 4: Maximum-likelihood tree inferred under k-means partitioning strategy for full data set.

28 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Sapyga_pumila 0.719 Aporus_niger 0.9472 Chyphotes_mellipes Ampulex_compressa 1 Chalybion_californicum Apterogyna_ZA01 1 Martialis_heureka Opamyrma_hungvuong 0.9143 Protanilla_JP01 1 Protanilla_TH01 1 0.9997 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9993 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 Leptanilla_TH01 1 Leptanilla_TH09 1 1 Leptanilla_ZA01 1 Leptanilla_GR01 0.9991 1 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani 1 Leptanilla_TH08 Phaulomyrma_MM01 1 Leptanilla_TH02 1 Leptanilla_TH05 1 Leptanilla_TH04 0.9979 Leptanilla_TH06 Cerapachys_jacobsoni 1 Labidus_praedator 0.5425 1 Leptanilloides_femoralis Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 1 Tetraponera_rufonigra Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus Acanthoponera_minor 1 1 Rhytidoponera_chalybaea 0.9768 Pogonomyrmex_vermiculatus 0.5679 Manica_bradleyi 1 1 Myrmica_tahoensis Stenamma_expolitum 1 Crematogaster_kelleri 0.9694 Cyphomyrmex_longiscapus_cf 0.7484 Myrmicaria_carinata Discothyrea_testacea 1 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 0.5001 1 Proceratium_silaceum Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 1 1 Amblyopone_australis 0.9986 Onychomyrmex_hedleyi Fulakora_mystriops 0.8375 1 Fulakora_saundersi 1 Myopopone_castanea Mystrium_voeltzkowi 1 1 Stigmatomma_boltoni Adetomyrma_caputleae 0.9339 Xymmer_muticus Stigmatomma_denticulatum 0.5891 Stigmatomma_pallipes Paraponera_clavata 1 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_mocquerysi 1 Platythyrea_lamellosa 0.7049 Platythyrea_punctata 1 Simopelta_pergandei_cf Belonopelta_HN01 1 0.7468 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum 1 Emeryopone_MY01 Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki Ponera_swezeyi 0.9768 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9964 Hypoponera_opacior 1 0.9821 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 1 Plectroctena_ugandensis Brachyponera_obscurans 0.9437 Myopias_tenuis Phrynoponera_pulchella 0.7265 Mesoponera_ambigua 1 0.8012 Streblognathus_peetersi Odontoponera_transversa 0.9155 0.984 Promyopias_silvestrii 0.7332 Asphinctopone_silvestrii 0.705 Anochetus_madagascarensis 1 0.7897 Odontomachus_coquereli Bothroponera_wasmannii 0.7538 Leptogenys_diminuta 1 0.6444 Mesoponera_melanaria Euponera_sikorae 1 Euponera_brunoi 0.9999 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.07 Supplementary Figure 5: Bayesian consensus tree inferred under greedy partitioning strategy for datset with AT-rich outgroups removed.

29 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Sapyga_pumila 0.7461 Aporus_niger 0.6422 Chyphotes_mellipes Ampulex_compressa 0.9999 Chalybion_californicum Apterogyna_ZA01 1 Martialis_heureka Opamyrma_hungvuong 0.9999 Protanilla_JP01 1 Protanilla_TH01 1 0.9473 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9939 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 Leptanilla_TH01 1 Leptanilla_TH09 0.9495 1 Leptanilla_ZA01 1 Leptanilla_GR01 0.9833 1 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani Leptanilla_TH08 1 0.6119 Phaulomyrma_MM01 1 Leptanilla_TH02 1 Leptanilla_TH05 1 Leptanilla_TH04 0.8911 Leptanilla_TH06 Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.9973 1 Labidus_praedator Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 1 Tetraponera_rufonigra Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus 1 Acanthoponera_minor 1 Rhytidoponera_chalybaea Manica_bradleyi 0.9973 1 Myrmica_tahoensis 1 Pogonomyrmex_vermiculatus 0.6186 Stenamma_expolitum 1 Crematogaster_kelleri 0.745 Cyphomyrmex_longiscapus_cf 0.5708 Myrmicaria_carinata Discothyrea_testacea 1 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 0.6395 1 Proceratium_silaceum Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 1 1 Amblyopone_australis 0.9979 Onychomyrmex_hedleyi 0.8513 Fulakora_mystriops 1 Fulakora_saundersi 1 Adetomyrma_caputleae Mystrium_voeltzkowi 1 1 Stigmatomma_boltoni Myopopone_castanea 0.836 Xymmer_muticus 0.806 Stigmatomma_denticulatum 0.9948 Stigmatomma_pallipes Paraponera_clavata 1 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_lamellosa 1 Platythyrea_mocquerysi 0.629 0.9961 Platythyrea_punctata Simopelta_pergandei_cf Belonopelta_HN01 1 0.9951 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum 1 Emeryopone_MY01 Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki 0.5241 Ponera_swezeyi 0.9931 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9993 Hypoponera_opacior 1 0.9998 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 1 Plectroctena_ugandensis Myopias_tenuis Bothroponera_wasmannii 1 Euponera_sikorae 0.99981 Euponera_brunoi 1 Cryptopone_hartwigi 1 Fisheropone_ambigua 0.8448 Mesoponera_ambigua 1 Streblognathus_peetersi Odontoponera_transversa 0.7590.9892 3 Promyopias_silvestrii Phrynoponera_pulchella 0.7675 0.9899 Anochetus_madagascarensis 1 0.8348 Odontomachus_coquereli Asphinctopone_silvestrii 0.8253 Brachyponera_obscurans Leptogenys_diminuta 0.9982 Mesoponera_melanaria

0.07 Supplementary Figure 6: Bayesian consensus tree inferred under k-means partitioning strategy for datset with AT-rich outgroups removed.

30 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Chyphotes_mellipes Aporus_niger 5 8 Sapyga_pumila Ampulex_compressa 8 5 Chalybion_californicum 3 9 Apterogyna_ZA01 Opamyrma_hungvuong Protanilla_JP01 100 Protanilla_TH01 100 7 7 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 9 Protanilla_VN03 100Protanilla_TH03 100 100 Protanilla_VN01 9 0 Leptanilla_TH01 100 Leptanilla_TH09 100 Leptanilla_ZA01 100 Leptanilla_GR01 100 9 0 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani 100 Leptanilla_TH08 9 7 Phaulomyrma_MM01 Leptanilla_TH02 9 2100 100 Leptanilla_TH05 100 Leptanilla_TH04 9 5 Leptanilla_TH06 Martialis_heureka Cerapachys_jacobsoni 100 Labidus_praedator 9 7 Leptanilloides_femoralis Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 9 9 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 9 9 Tetraponera_punctulata 100 100 Tetraponera_rufonigra 5 0 Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus 100 Acanthoponera_minor 100 Rhytidoponera_chalybaea Manica_bradleyi 9 6 100 Myrmica_tahoensis 100 Pogonomyrmex_vermiculatus 6 9 Stenamma_expolitum 100 Crematogaster_kelleri 7 0 Cyphomyrmex_longiscapus_cf 100 5 5 Myrmicaria_carinata Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 9 7 100 Amblyopone_australis 100 Onychomyrmex_hedleyi 9 7 Fulakora_mystriops 100 Fulakora_saundersi Myopopone_castanea 100 8 2 Mystrium_voeltzkowi 100 Stigmatomma_boltoni Adetomyrma_caputleae 7 78 1 Xymmer_muticus 6 7 Stigmatomma_denticulatum 9 4 9 9 Stigmatomma_pallipes Discothyrea_testacea 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 100 Proceratium_silaceum 7 0 Paraponera_clavata 100 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_lamellosa 100 Platythyrea_mocquerysi 9 4 Platythyrea_punctata 9 2 Simopelta_pergandei_cf Belonopelta_HN01 100 9 2 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 100 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 100 9 7 Ectomomyrmex_leeuwenhoeki 100 Emeryopone_MY01 9 0 Ponera_swezeyi 9 0 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 6 Hypoponera_opacior 100 9 2 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 100 9 8 Plectroctena_ugandensis Brachyponera_obscurans 8 9 Myopias_tenuis Asphinctopone_silvestrii 5 0 Leptogenys_diminuta 100 9 3 Mesoponera_melanaria Anochetus_madagascarensis 100 6 2 Odontomachus_coquereli 5 0 Odontoponera_transversa 7 5 Phrynoponera_pulchella 4 6 Bothroponera_wasmannii 5 7 Promyopias_silvestrii Mesoponera_ambigua 2 98 7 Streblognathus_peetersi 6 8 Euponera_sikorae 100 Euponera_brunoi 9 9 Cryptopone_hartwigi 100 Fisheropone_ambigua

0.09 Supplementary Figure 7: Maximum-likelihood tree inferred under greedy partitioning strategy for datset with AT-rich outgroups removed.

31 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Sapyga_pumila 8 0 Aporus_niger 6 0 Chyphotes_mellipes Ampulex_compressa 7 6 Chalybion_californicum Apterogyna_ZA01 Martialis_heureka Opamyrma_hungvuong 9 9 Protanilla_JP01 100 Protanilla_TH01 100 5 2 100 Protanilla_TH02 100 Anomalomyrma_boltoni 100 Protanilla_VN03 100Protanilla_TH03 100 100 Protanilla_VN01 Leptanilla_TH01 100 Leptanilla_TH09 6 6 100 Leptanilla_ZA01 100 Leptanilla_GR01 9 2 100 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani Leptanilla_TH08 10901 Phaulomyrma_MM01 9 7 Leptanilla_TH02 100 Leptanilla_TH05 100 Leptanilla_TH04 9 4 Leptanilla_TH06 Leptanilloides_femoralis 100 Cerapachys_jacobsoni 9 6 100 Labidus_praedator Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 100 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 Tetraponera_punctulata 100 100 Tetraponera_rufonigra Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus 100 Acanthoponera_minor 100 Rhytidoponera_chalybaea Manica_bradleyi 9 9 100 Myrmica_tahoensis 100 Pogonomyrmex_vermiculatus 5 6 Stenamma_expolitum 100 Crematogaster_kelleri 5 5 Cyphomyrmex_longiscapus_cf 5 0 Myrmicaria_carinata Discothyrea_testacea 9 8 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 6 4 100 Proceratium_silaceum Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 9 6 100 Amblyopone_australis 9 3 Onychomyrmex_hedleyi 9 1 Fulakora_mystriops 100 Fulakora_saundersi 100 Mystrium_voeltzkowi Adetomyrma_caputleae 10405 9 7 Stigmatomma_boltoni 4 2 Myopopone_castanea 6 5 Xymmer_muticus 6 5 Stigmatomma_denticulatum 9 1 Stigmatomma_pallipes Paraponera_clavata 9 9 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_lamellosa 100 Platythyrea_mocquerysi 9 4 8 3 Platythyrea_punctata Simopelta_pergandei_cf Belonopelta_HN01 100 9 7 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 100 Neoponera_unidentata Diacamma_pallidum 100 Emeryopone_MY01 Cryptopone_gilva 1009 9 Ectomomyrmex_leeuwenhoeki 7 7 Ponera_swezeyi 9 3 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 9 Hypoponera_opacior 100 9 8 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 9 9 100 Plectroctena_ugandensis Myopias_tenuis Bothroponera_wasmannii 100 Euponera_sikorae 9 7 100 Euponera_brunoi 9 9 Cryptopone_hartwigi 100 Fisheropone_ambigua 7 7 Mesoponera_ambigua 8 8 Streblognathus_peetersi Odontoponera_transversa 6 47 5 Promyopias_silvestrii 5 4 Phrynoponera_pulchella 6 7 Anochetus_madagascarensis 100 5 6 Odontomachus_coquereli Asphinctopone_silvestrii 6 5 Brachyponera_obscurans 6 8 Leptogenys_diminuta 9 6 Mesoponera_melanaria

0.06 Supplementary Figure 8: Maximum-likelihood tree inferred under k-means partitioning strategy for datset with AT-rich outgroups removed.

32 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Scolia_verticalis 1 Brachycistis_sp 1 Dasymutilla_aureola 0.9846 Apis_mellifera Opamyrma_hungvuong Protanilla_JP01 1 Protanilla_TH01 1 0.9786 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9981 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 0.9785 Leptanilla_TH01 1 Leptanilla_TH09 1 Leptanilla_ZA01 1 Leptanilla_GR01 0.9961 1 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani 1 Leptanilla_TH08 Phaulomyrma_MM01 1 Leptanilla_TH02 1 1 Leptanilla_TH05 1 Leptanilla_TH04 0.9988 Leptanilla_TH06 Martialis_heureka Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.8158 Labidus_praedator Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 1 Tetraponera_rufonigra 0.9999 Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus Acanthoponera_minor 1 1 Rhytidoponera_chalybaea 0.996 Pogonomyrmex_vermiculatus 0.7314 Manica_bradleyi 1 1 Myrmica_tahoensis Stenamma_expolitum 1 Crematogaster_kelleri 0.9897 Cyphomyrmex_longiscapus_cf 1 0.8933 Myrmicaria_carinata Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 1 1 Amblyopone_australis 0.833 Onychomyrmex_hedleyi 0.9288 Fulakora_mystriops 1 Fulakora_saundersi 1 Adetomyrma_caputleae Stigmatomma_denticulatum 1 0.5286 Stigmatomma_pallipes 0.8341 Mystrium_voeltzkowi 0.5006 Stigmatomma_boltoni Myopopone_castanea 0.5657 1 Xymmer_muticus Discothyrea_testacea 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 1 Proceratium_silaceum 0.8714 Paraponera_clavata 1 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_mocquerysi 1 Platythyrea_lamellosa 0.9531 Platythyrea_punctata 1 Simopelta_pergandei_cf Belonopelta_HN01 1 0.7794 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki 1 Emeryopone_MY01 0.6299 Ponera_swezeyi 0.9693 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9434 Hypoponera_opacior 1 0.9181 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 Plectroctena_ugandensis 1 Brachyponera_obscurans 0.8434 Myopias_tenuis Asphinctopone_silvestrii Phrynoponera_pulchella 1 Anochetus_madagascarensis 1 Odontomachus_coquereli Mesoponera_ambigua 0.8496 0.9991 Streblognathus_peetersi Odontoponera_transversa 0.7133 Promyopias_silvestrii Bothroponera_wasmannii 0.5677 Leptogenys_diminuta 1 Mesoponera_melanaria Euponera_sikorae 1 Euponera_brunoi 1 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.08 Supplementary Figure 9: Bayesian consensus tree inferred under greedy partitioning strategy for datset with GC-rich outgroups removed.

33 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Scolia_verticalis 1 Brachycistis_sp 0.9998 Dasymutilla_aureola 1 Apis_mellifera Opamyrma_hungvuong Protanilla_JP01 1 Protanilla_TH01 1 0.5104 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9987 Protanilla_VN03 1 Protanilla_TH03 1 1 Protanilla_VN01 0.6293 Leptanilla_TH01 1 Leptanilla_TH09 1 Leptanilla_ZA01 1 Leptanilla_GR01 1 0.9619 Leptanilla_GR02 Leptanilla_TH03 1 Yavnella_argamani 1 Leptanilla_TH08 1 Phaulomyrma_MM01 Leptanilla_TH02 0.5631 5 1 Leptanilla_TH05 1 Leptanilla_TH04 0.8848 Leptanilla_TH06 Martialis_heureka Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.9829 Labidus_praedator Aneuretus_simoni 1 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 1 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 Tetraponera_rufonigra 0.9833 1 Myrmelachista_flavocotea 1 Camponotus_maritimus 1 Lasius_californicus 1 Acanthoponera_minor 1 Rhytidoponera_chalybaea Manica_bradleyi 0.9974 1 Myrmica_tahoensis 1 Pogonomyrmex_vermiculatus 0.5222 Stenamma_expolitum 1 Crematogaster_kelleri 0.8397 Cyphomyrmex_longiscapus_cf 1 0.7353 Myrmicaria_carinata Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 0.882 1 Amblyopone_australis 0.9976 Onychomyrmex_hedleyi 0.8402 Fulakora_mystriops 1 Fulakora_saundersi 1 Adetomyrma_caputleae 1 Mystrium_voeltzkowi Stigmatomma_boltoni 0.741 Myopopone_castanea 0.8418 Xymmer_muticus 0.7684 Stigmatomma_denticulatum 0.9973 0.882 Stigmatomma_pallipes Discothyrea_testacea 1 Discothyrea_MG07 1 Discothyrea_SC02 1 1 Discothyrea_SC03 Proceratium_avium 1 Proceratium_stictum 1 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 1 0.5389 Proceratium_silaceum Paraponera_clavata 0.9922 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_lamellosa 1 Platythyrea_mocquerysi 0.7688 Platythyrea_punctata 0.8816 Simopelta_pergandei_cf Belonopelta_HN01 1 0.9994 Thaumatomyrmex_atrox 1 Dinoponera_longipes 1 1 1 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 1 1 Ectomomyrmex_leeuwenhoeki 1 Emeryopone_MY01 0.6218 Ponera_swezeyi 0.992 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9991 Hypoponera_opacior 1 0.9999 Hypoponera_sakalava Centromyrmex_angolensis 1 Psalidomyrmex_procerus 1 Loboponera_politula 1 1 Plectroctena_ugandensis Myopias_tenuis Bothroponera_wasmannii 1 Euponera_sikorae 1 1 Euponera_brunoi 1 Cryptopone_hartwigi 1 Fisheropone_ambigua 0.9293 Mesoponera_ambigua 0.9997 Streblognathus_peetersi Odontoponera_transversa 0.8320.9368 8 Promyopias_silvestrii 0.6763 Phrynoponera_pulchella 0.9913 Anochetus_madagascarensis 1 0.9021 Odontomachus_coquereli Asphinctopone_silvestrii 0.8996 Brachyponera_obscurans 0.6714 Leptogenys_diminuta 0.9923 Mesoponera_melanaria

0.07 Supplementary Figure 10: Bayesian consensus tree inferred under k-means partitioning strategy for datset with GC-rich outgroups removed.

34 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Scolia_verticalis Brachycistis_sp 9 1 Dasymutilla_aureola 8 3 Apis_mellifera Opamyrma_hungvuong Protanilla_JP01 100 Protanilla_TH01 100 5 6 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 9 Protanilla_VN03 100Protanilla_TH03 100 100 Protanilla_VN01 6 1 Leptanilla_TH01 100 Leptanilla_TH09 100 Leptanilla_ZA01 100 Leptanilla_GR01 100 9 1 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani 100 Leptanilla_TH08 9 7 Phaulomyrma_MM01 Leptanilla_TH02 8 9 100 100 Leptanilla_TH05 100 Leptanilla_TH04 9 5 Leptanilla_TH06 Martialis_heureka Cerapachys_jacobsoni 100 Labidus_praedator 8 8 Leptanilloides_femoralis Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 100 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 9 9 Tetraponera_punctulata 100 100 Tetraponera_rufonigra 9 9 Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus Acanthoponera_minor 100 100 Rhytidoponera_chalybaea 9 6 Pogonomyrmex_vermiculatus 8 5 Manica_bradleyi 100 100 Myrmica_tahoensis Stenamma_expolitum 100 Crematogaster_kelleri 9 8 Cyphomyrmex_longiscapus_cf 100 9 1 Myrmicaria_carinata Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 9 9 100 Amblyopone_australis 9 6 Onychomyrmex_hedleyi 9 6 Fulakora_mystriops 100 Fulakora_saundersi 100 Adetomyrma_caputleae Stigmatomma_denticulatum 1009 0 Stigmatomma_pallipes 7 8 Stigmatomma_boltoni 7 4 Mystrium_voeltzkowi 5 5 Myopopone_castanea 6 6 9 9 Xymmer_muticus Discothyrea_testacea 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 100 Proceratium_silaceum 8 6 Paraponera_clavata 100 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_mocquerysi 100 Platythyrea_lamellosa 9 7 Platythyrea_punctata 9 5 Simopelta_pergandei_cf Belonopelta_HN01 100 9 4 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 100 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 100 9 3 Ectomomyrmex_leeuwenhoeki 100 Emeryopone_MY01 7 5 Ponera_swezeyi 8 8 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 6 Hypoponera_opacior 100 9 4 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 9 9 9 9 Plectroctena_ugandensis Brachyponera_obscurans 8 5 Myopias_tenuis Bothroponera_wasmannii 4 9 Leptogenys_diminuta 100 9 2 Mesoponera_melanaria Anochetus_madagascarensis 100 7 7 Odontomachus_coquereli 4 1 Asphinctopone_silvestrii 3 7 Promyopias_silvestrii 2 8 Odontoponera_transversa 7 8 2 0 Phrynoponera_pulchella Mesoponera_ambigua 8 7 Streblognathus_peetersi 5 2 Euponera_sikorae 100 Euponera_brunoi 9 9 Cryptopone_hartwigi 100 Fisheropone_ambigua

0.09 Supplementary Figure 11: Maximum-likelihood tree inferred under greedy partitioning strategy for datset with GC-rich outgroups removed.

35 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Apis_mellifera Scolia_verticalis 4 1 Brachycistis_sp 8 5 Dasymutilla_aureola Opamyrma_hungvuong Protanilla_JP01 8 5 100 Protanilla_TH01 100 100 Protanilla_TH02 100 Anomalomyrma_boltoni 100 Protanilla_VN03 100Protanilla_TH03 100 100 Protanilla_VN01 4 4 Leptanilla_TH01 100 Leptanilla_TH09 100 Leptanilla_ZA01 100 Leptanilla_GR01 100 9 0 Leptanilla_GR02 Leptanilla_TH03 100 Yavnella_argamani 100 Leptanilla_TH08 9 8 Phaulomyrma_MM01 Leptanilla_TH02 9 5 9 9 100 Leptanilla_TH05 100 Leptanilla_TH04 9 4 Leptanilla_TH06 Martialis_heureka Leptanilloides_femoralis 100 Cerapachys_jacobsoni 9 3 Labidus_praedator Aneuretus_simoni 100 Dolichoderus_pustulatus 100 100 Liometopum_occidentale 100 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 Tetraponera_punctulata 100 Tetraponera_rufonigra 9 6 100 Myrmelachista_flavocotea 100 Camponotus_maritimus 100 Lasius_californicus 100 Acanthoponera_minor 100 Rhytidoponera_chalybaea Manica_bradleyi 9 9 100 Myrmica_tahoensis 100 Pogonomyrmex_vermiculatus 8 3 Stenamma_expolitum 100 Crematogaster_kelleri 9 0 Cyphomyrmex_longiscapus_cf 100 8 6 Myrmicaria_carinata Apomyrma_CF02 100 Apomyrma_stygia Prionopelta_amabilis 7 4 100 Amblyopone_australis 9 3 Onychomyrmex_hedleyi 9 5 Fulakora_mystriops 100 Fulakora_saundersi 100 Adetomyrma_caputleae Mystrium_voeltzkovi 10301 Stigmatomma_boltoni 5 7 Myopopone_castanea 5 9 Xymmer_muticus 6 1 Stigmatomma_denticulatum 8 9 7 5 Stigmatomma_pallipes Discothyrea_testacea 100 Discothyrea_MG07 100 Discothyrea_SC02 100 100 Discothyrea_SC03 Proceratium_avium 100 Proceratium_stictum 100 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 100 Proceratium_SC02 100 4 5 Proceratium_silaceum Paraponera_clavata 9 6 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_lamellosa 100 Platythyrea_mocquerysi 9 5 Platythyrea_punctata 7 3 Simopelta_pergandei_cf Belonopelta_HN01 100 9 9 Thaumatomyrmex_atrox 100 Dinoponera_longipes 100 100 100 Neoponera_unidentata Diacamma_pallidum Cryptopone_gilva 100 9 9 Ectomomyrmex_leeuwenhoeki 100 Emeryopone_MY01 9 1 Ponera_swezeyi 9 4 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 8 Hypoponera_opacior 100 9 7 Hypoponera_sakalava Centromyrmex_angolensis 100 Psalidomyrmex_procerus 100 Loboponera_politula 9 8 100 Plectroctena_ugandensis Myopias_tenuis Bothroponera_wasmannii 100 Euponera_sikorae 9 7 100 Euponera_brunoi 100 Cryptopone_hartwigi 100 Fisheropone_ambigua 8 3 Mesoponera_ambigua 8 5 Streblognathus_peetersi Odontoponera_transversa 6 76 8 Promyopias_silvestrii 4 9 Phrynoponera_pulchella 6 9 Anochetus_madagascarensis 100 5 9 Odontomachus_coquereli Asphinctopone_silvestrii 6 7 Brachyponera_obscurans 8 0 Leptogenys_diminuta 9 4 Mesoponera_melanaria

0.07 Supplementary Figure 12: Maximum-likelihood tree inferred under k-means partitioning strategy for datset with GC-rich outgroups removed.

36 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Dasymutilla_aureola 1 Brachycistis_sp 0.5799 Sapyga_pumila 0.8277 Aporus_niger 1 1 Chyphotes_mellipes Ampulex_compressa 0.9909 Apis_mellifera 0.9999 Chalybion_californicum 0.8226 Apterogyna_ZA01 1 Scolia_verticalis Martialis_heureka Opamyrma_hungvuong 0.9982 Protanilla_JP01 1 Protanilla_TH01 1 1 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9933 Protanilla_VN03 1 Protanilla_TH03 0.9499 0.8925 Protanilla_VN01 Leptanilla_TH01 0.9972 Leptanilla_TH09 0.9473 1 Leptanilla_ZA01 1 Leptanilla_GR01 1 0.6842 Leptanilla_GR02 Leptanilla_TH03 1 Leptanilla_TH08 1 Yavnella_argamani 0.9993 Phaulomyrma_MM01 0.6008 Leptanilla_TH05 1 Leptanilla_TH02 0.7124 Leptanilla_TH04 0.9999 Leptanilla_TH06 Acanthoponera_minor 1 1 Rhytidoponera_chalybaea Cerapachys_jacobsoni 1 Labidus_praedator Leptanilloides_femoralis 0.7319 Aneuretus_simoni 0.9979 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 0.8683 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 Tetraponera_rufonigra Myrmelachista_flavocotea 1 Camponotus_maritimus 0.9768 Lasius_californicus 0.6498 Manica_bradleyi 1 Myrmica_tahoensis Myrmicaria_carinata 1 0.9595 Pogonomyrmex_vermiculatus 0.9811 Crematogaster_kelleri 0.9998 Cyphomyrmex_longiscapus_cf 0.5917 1 Stenamma_expolitum Discothyrea_testacea 1 Discothyrea_MG07 1 Discothyrea_SC02 1 0.9583 Discothyrea_SC03 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 0.9999 Proceratium_SC02 1 Proceratium_silaceum 0.7975 Proceratium_avium 1 Proceratium_stictum Apomyrma_CF02 1 Apomyrma_stygia 0.9999 Prionopelta_amabilis 0.8999 0.9993 Amblyopone_australis 1 Onychomyrmex_hedleyi Fulakora_mystriops 0.9222 1 Fulakora_saundersi 1 Myopopone_castanea Mystrium_voeltzkowi 0.8332 Stigmatomma_boltoni Adetomyrma_caputleae 0.7193 0.6714 Xymmer_muticus Stigmatomma_denticulatum 0.6658 Stigmatomma_pallipes Paraponera_clavata 0.9938 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_mocquerysi 1 Platythyrea_lamellosa 0.9712 Platythyrea_punctata 0.9996 Diacamma_pallidum 0.6884 Simopelta_pergandei_cf 0.9921 Thaumatomyrmex_atrox 0.9993 Belonopelta_HN01 0.952 Dinoponera_longipes 0.9944 0.998 1 Neoponera_unidentata Ectomomyrmex_leeuwenhoeki 0.9995 Cryptopone_gilva Emeryopone_MY01 0.9537 Ponera_swezeyi Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9986 Hypoponera_opacior 1 1 Hypoponera_sakalava Centromyrmex_angolensis 1 Loboponera_politula 0.9984 Plectroctena_ugandensis 0.7301 0.879 Psalidomyrmex_procerus Bothroponera_wasmannii 0.9132 Myopias_tenuis Asphinctopone_silvestrii 1 Brachyponera_obscurans Leptogenys_diminuta Odontoponera_transversa 0.8528 Anochetus_madagascarensis 0.9996 Odontomachus_coquereli Phrynoponera_pulchella 0.6863 Promyopias_silvestrii Mesoponera_ambigua 0.769 Mesoponera_melanaria 0.684 Streblognathus_peetersi Euponera_sikorae 1 Euponera_brunoi 0.6845 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.02 Supplementary Figure 13: Bayesian consensus tree inferred under greedy partitioning strategy for compositionally homogeneous datset.

37 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 1 Mischocyttarus_flavitarsis Dasymutilla_aureola 1 Brachycistis_sp 0.6349 Sapyga_pumila 0.9088 Aporus_niger 1 0.9999 Chyphotes_mellipes Ampulex_compressa Apis_mellifera 0.851 0.9995 Chalybion_californicum Apterogyna_ZA01 1 Scolia_verticalis Martialis_heureka 1 Opamyrma_hungvuong 0.9987 Protanilla_JP01 1 Protanilla_TH01 1 1 Protanilla_TH02 1 Anomalomyrma_boltoni 0.9917 Protanilla_VN03 1 Protanilla_TH03 0.9795 0.9555 Protanilla_VN01 Leptanilla_TH01 0.9978 Leptanilla_TH09 0.9951 0.9997 Leptanilla_GR01 1 Leptanilla_GR02 1 0.651 Leptanilla_ZA01 Leptanilla_TH03 1 Leptanilla_TH08 1 Yavnella_argamani 0.9803 Phaulomyrma_MM01 0.5091 Leptanilla_TH05 1 Leptanilla_TH02 0.757 Leptanilla_TH04 0.999 Leptanilla_TH06 Acanthoponera_minor 1 1 Rhytidoponera_chalybaea Leptanilloides_femoralis 1 Cerapachys_jacobsoni 0.5835 Labidus_praedator 0.522 Aneuretus_simoni 0.9968 Dolichoderus_pustulatus 1 1 Liometopum_occidentale 0.7649 Myrmecia_pyriformis 1 Nothomyrmecia_macrops 1 Tetraponera_punctulata 1 Tetraponera_rufonigra Myrmelachista_flavocotea 1 Camponotus_maritimus 0.9193 Lasius_californicus 0.6373 Manica_bradleyi 1 Myrmica_tahoensis Myrmicaria_carinata 1 0.9547 Pogonomyrmex_vermiculatus 0.984 Crematogaster_kelleri 0.9999 Cyphomyrmex_longiscapus_cf 0.6491 Stenamma_expolitum 1 Discothyrea_testacea 1 Discothyrea_MG07 1 Discothyrea_SC02 1 0.9597 Discothyrea_SC03 Probolomyrmex_brujitae_cf 1 Probolomyrmex_tani 1 Proceratium_SC02 1 Proceratium_silaceum 0.5456 Proceratium_avium 1 Proceratium_stictum Apomyrma_CF02 1 Apomyrma_stygia Prionopelta_amabilis 0.949 1 Amblyopone_australis 0.9981 Onychomyrmex_hedleyi Fulakora_mystriops 1 0.9243 1 Fulakora_saundersi 1 Myopopone_castanea Mystrium_voeltzkowi 0.8294 Stigmatomma_boltoni Adetomyrma_caputleae 0.7434 Xymmer_muticus Stigmatomma_denticulatum 0.6768 Stigmatomma_pallipes Paraponera_clavata 0.9255 Ankylomyrma_coronacantha 1 Tatuidris_tatusia Platythyrea_mocquerysi 1 Platythyrea_lamellosa 0.9363 Platythyrea_punctata 0.9998 Diacamma_pallidum 0.6587 Simopelta_pergandei_cf 0.9945 Thaumatomyrmex_atrox 0.9998 Belonopelta_HN01 0.91 Dinoponera_longipes 0.9239 0.9997 1 Neoponera_unidentata Emeryopone_MY01 0.9984 Cryptopone_gilva 0.7012 Ectomomyrmex_leeuwenhoeki Ponera_swezeyi 0.7972 Parvaponera_darwinii 1 1 Pseudoponera_stigma Harpegnathos_saltator 0.9977 Hypoponera_opacior 1 0.9987 Hypoponera_sakalava Centromyrmex_angolensis 1 Loboponera_politula 0.9995 Plectroctena_ugandensis 0.8212 0.7873 Psalidomyrmex_procerus Bothroponera_wasmannii 0.9663 Myopias_tenuis 0.9998 Leptogenys_diminuta Asphinctopone_silvestrii 0.9858 Brachyponera_obscurans Anochetus_madagascarensis 0.732 1 Odontomachus_coquereli Odontoponera_transversa Phrynoponera_pulchella 0.8349 0.6258 Promyopias_silvestrii Mesoponera_melanaria 0.7142 Mesoponera_ambigua 0.7502 0.6872 Streblognathus_peetersi Euponera_sikorae 1 Euponera_brunoi 0.8345 Cryptopone_hartwigi 1 Fisheropone_ambigua

0.02 Supplementary Figure 14: Bayesian consensus tree inferred under k-means partitioning strategy for compositionally homogeneous datset.

38 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Brachycistis_sp 6 0 Sapyga_pumila 5 7 Dasymutilla_aureola 5 7 Aporus_niger 9 6 Chyphotes_mellipes Ampulex_compressa 9 5 Apis_mellifera 9 5 9 9 Chalybion_californicum Apterogyna_ZA01 9 7 Scolia_verticalis Martialis_heureka Opamyrma_hungvuong 100 Protanilla_JP01 100 Protanilla_TH01 9 4 100 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 7 Protanilla_VN03 100 Protanilla_TH03 100 100 Protanilla_VN01 Leptanilla_TH01 9 6 Leptanilla_TH09 9 0 100 Leptanilla_ZA01 100 Leptanilla_GR01 100 8 1 Leptanilla_GR02 Leptanilla_TH03 100 Leptanilla_TH08 100 Yavnella_argamani 9 5 Phaulomyrma_MM01 9 5 Leptanilla_TH05 100 Leptanilla_TH02 9 8 Leptanilla_TH04 9 6 Leptanilla_TH06 Leptanilloides_femoralis 100 100 Cerapachys_jacobsoni 9 6 Labidus_praedator 9 5 Aneuretus_simoni 9 9 Dolichoderus_pustulatus 100 Liometopum_occidentale 9 6 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 100 Tetraponera_punctulata 100 Tetraponera_rufonigra Acanthoponera_minor 9 9 Rhytidoponera_chalybaea Myrmelachista_flavocotea 8 8 100 Camponotus_maritimus 9 1 Lasius_californicus 9 4 Manica_bradleyi 100 Myrmica_tahoensis Myrmicaria_carinata 100 9 7 Pogonomyrmex_vermiculatus 9 9 Crematogaster_kelleri 100 Cyphomyrmex_longiscapus_cf 9 8 9 9 Stenamma_expolitum Discothyrea_testacea 9 8 Discothyrea_MG07 100 Discothyrea_SC02 100 8 5 Discothyrea_SC03 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 9 7 Proceratium_SC02 100 Proceratium_silaceum 8 8 Proceratium_avium 100 Proceratium_stictum Apomyrma_CF02 100 Apomyrma_stygia 9 8 Prionopelta_amabilis 8 1 9 9 Amblyopone_australis 9 9 Onychomyrmex_hedleyi 9 1 Fulakora_mystriops 100 Fulakora_saundersi Stigmatomma_denticulatum 9 9 7 7 Stigmatomma_pallipes Myopopone_castanea 9 3 2 8 Xymmer_muticus 6 0 7 9 Stigmatomma_boltoni 2 7 Adetomyrma_caputleae 4 2 Mystrium_voeltzkowi Paraponera_clavata 8 1 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_mocquerysi 100 Platythyrea_lamellosa 9 8 Platythyrea_punctata 9 9 Diacamma_pallidum 9 4 Simopelta_pergandei_cf 9 6 Thaumatomyrmex_atrox 100 Belonopelta_HN01 100 Dinoponera_longipes 9 7 100 8 0 Neoponera_unidentata Ectomomyrmex_leeuwenhoeki 9 7 Cryptopone_gilva Emeryopone_MY01 9 7 6 1 Ponera_swezeyi 5 4 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 8 Hypoponera_opacior 100 9 8 Hypoponera_sakalava Centromyrmex_angolensis 100 Loboponera_politula 100 Plectroctena_ugandensis 9 0 Psalidomyrmex_procerus 9 9 Bothroponera_wasmannii 9 1 Myopias_tenuis Anochetus_madagascarensis 9 5 Odontomachus_coquereli 9 8 6 3 Leptogenys_diminuta 6 2 Asphinctopone_silvestrii 7 8 Brachyponera_obscurans 8 5 Odontoponera_transversa 3 4 Streblognathus_peetersi 3 8 Phrynoponera_pulchella 7 2 7 3 Promyopias_silvestrii Mesoponera_ambigua 4 6 Mesoponera_melanaria 4 3 Cryptopone_hartwigi 100 Fisheropone_ambigua 100 Euponera_brunoi 7 9 Euponera_sikorae

0.02 Supplementary Figure 15: Maximum-likelihood tree inferred under greedy partitioning strategy for compositionally homogeneous datset.

39 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata 100 Mischocyttarus_flavitarsis Dasymutilla_aureola Brachycistis_sp 5 7 Sapyga_pumila 9 9 7 0 Aporus_niger 9 4 Chyphotes_mellipes Apis_mellifera 9 7 6 9 Chalybion_californicum Ampulex_compressa Apterogyna_ZA01 9 7 Scolia_verticalis 9 8 Martialis_heureka Opamyrma_hungvuong 100 Protanilla_JP01 100 Protanilla_TH01 6 2 100 100 Protanilla_TH02 100 Anomalomyrma_boltoni 9 7 Protanilla_VN03 100 Protanilla_TH03 100 100 Protanilla_VN01 Leptanilla_TH01 100 Leptanilla_TH09 9 6 100 Leptanilla_GR01 100 Leptanilla_GR02 100 8 9 Leptanilla_ZA01 Leptanilla_TH03 100 Leptanilla_TH08 100 Yavnella_argamani 9 6 Phaulomyrma_MM01 9 2 Leptanilla_TH05 9 9 Leptanilla_TH02 9 9 Leptanilla_TH04 9 7 Leptanilla_TH06 Leptanilloides_femoralis 100 100 Cerapachys_jacobsoni 9 9 Labidus_praedator 8 2 Aneuretus_simoni 9 8 Dolichoderus_pustulatus 100 Liometopum_occidentale 9 1 Myrmecia_pyriformis 100 Nothomyrmecia_macrops 100 100 Tetraponera_punctulata 100 Tetraponera_rufonigra Acanthoponera_minor 100 Rhytidoponera_chalybaea Myrmelachista_flavocotea 8 0 100 Camponotus_maritimus 9 2 Lasius_californicus 9 1 Manica_bradleyi 100 Myrmica_tahoensis Myrmicaria_carinata 100 9 5 Pogonomyrmex_vermiculatus 100 Crematogaster_kelleri 100 Cyphomyrmex_longiscapus_cf 100 9 9 Stenamma_expolitum Discothyrea_testacea 9 9 Discothyrea_MG07 100 Discothyrea_SC02 100 9 3 Discothyrea_SC03 Probolomyrmex_brujitae_cf 100 Probolomyrmex_tani 9 8 Proceratium_SC02 100 Proceratium_silaceum 8 1 Proceratium_avium 100 Proceratium_stictum Apomyrma_CF02 100 Apomyrma_stygia 9 8 Prionopelta_amabilis 8 2 9 9 Amblyopone_australis 9 5 Onychomyrmex_hedleyi Fulakora_mystriops 9 3 9 9 Fulakora_saundersi Stigmatomma_boltoni 9 7 8 2 Adetomyrma_caputleae 9 1 Xymmer_muticus 9 6 Myopopone_castanea 6 0 Mystrium_voeltzkowi 7 2 6 7 Stigmatomma_denticulatum 8 0 Stigmatomma_pallipes Paraponera_clavata 7 8 Ankylomyrma_coronacantha 100 Tatuidris_tatusia Platythyrea_mocquerysi 100 Platythyrea_lamellosa 9 6 Platythyrea_punctata 100 Diacamma_pallidum 8 3 Simopelta_pergandei_cf 9 3 Thaumatomyrmex_atrox 100 Belonopelta_HN01 9 7 100 Dinoponera_longipes 7 6 100 Neoponera_unidentata Emeryopone_MY01 9 4 Ectomomyrmex_leeuwenhoeki 7 5 Cryptopone_gilva 6 6 Ponera_swezeyi 8 2 Parvaponera_darwinii 100 100 Pseudoponera_stigma Harpegnathos_saltator 9 8 Hypoponera_opacior 100 9 7 Hypoponera_sakalava Centromyrmex_angolensis 100 Loboponera_politula 9 9 Plectroctena_ugandensis 9 8 9 0 Psalidomyrmex_procerus Bothroponera_wasmannii 9 6 Myopias_tenuis Leptogenys_diminuta 9 9 Asphinctopone_silvestrii 1 7 9 4 Brachyponera_obscurans 3 2 Anochetus_madagascarensis 9 7 Odontomachus_coquereli 6 2 Odontoponera_transversa 4 4 Phrynoponera_pulchella 8 8 Promyopias_silvestrii 5 7 Mesoponera_melanaria 8 6 Mesoponera_ambigua 8 2 6 0 Streblognathus_peetersi Euponera_sikorae 100 Euponera_brunoi 9 2 Cryptopone_hartwigi 100 Fisheropone_ambigua

0.02 Supplementary Figure 16: Maximum-likelihood tree inferred under k-means partitioning strategy for compositionally homogeneous datset.

40 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata [100] Mischocyttarus_flavitarsis Aporus_niger Brachycistis_sp [97] [82] Chyphotes_mellipes Sapyga_pumila [87] Dasymutilla_aureola Ampulex_compressa [96] Apis_mellifera [100] Chalybion_californicum [100] Apterogyna_ZA01 [100] Scolia_verticalis Martialis_heureka Opamyrma_hungvuong [100] Protanilla_JP01 [100] Protanilla_TH01 [99] [100] [100] Protanilla_TH02 [100] Anomalomyrma_boltoni [100] Protanilla_VN03 [100] Protanilla_TH03 [94] [99] Protanilla_VN01 Leptanilla_TH01 [97] Leptanilla_TH09 [100] [100] Leptanilla_ZA01 [100] Leptanilla_GR01 [98] [100] Leptanilla_GR02 Leptanilla_TH03 [100] Yavnella_argamani Leptanilla_TH08 [100] [54] Phaulomyrma_MM01 [69] Leptanilla_TH02 [100] Leptanilla_TH05 Leptanilla_TH04 [95] Leptanilla_TH06 Leptanilloides_femoralis [100] Cerapachys_jacobsoni [96] [100] Labidus_praedator Aneuretus_simoni [100] Dolichoderus_pustulatus [100] [100] Liometopum_occidentale [100] Tetraponera_punctulata [100] Tetraponera_rufonigra [97] Myrmecia_pyriformis [100] [51] Nothomyrmecia_macrops Myrmelachista_flavocotea [100] Camponotus_maritimus [95] Lasius_californicus Acanthoponera_minor [100] [100] Rhytidoponera_chalybaea Pogonomyrmex_vermiculatus [65] [88] Manica_bradleyi [100] [100] Myrmica_tahoensis Crematogaster_kelleri [98] Stenamma_expolitum Cyphomyrmex_longiscapus_cf [53] Myrmicaria_carinata Discothyrea_testacea [100] [100] Discothyrea_MG07 [100] Discothyrea_SC02 [100] Discothyrea_SC03 [100] Proceratium_avium [100] Proceratium_stictum Probolomyrmex_brujitae_cf [100] [100] Probolomyrmex_tani Proceratium_SC02 [74] [100] Proceratium_silaceum Apomyrma_CF02 [100] Apomyrma_stygia Prionopelta_amabilis [97] [100] Amblyopone_australis [99] Onychomyrmex_hedleyi [100] Stigmatomma_mystriops [100] Stigmatomma_saundersi [100] Adetomyrma_caputleae Stigmatomma_denticulatum [97] [95] [100] Stigmatomma_pallipes [77] Mystrium_mysticum [53] Stigmatomma_boltoni Myopopone_castanea [84] Xymmer_muticus Paraponera_clavata [81] Ankylomyrma_coronacantha [100] Tatuidris_tatusia Platythyrea_lamellosa [100] Platythyrea_mocquerysi [99] [100] Platythyrea_punctata Simopelta_pergandei_cf Belonopelta_HN01 [99] [80] Thaumatomyrmex_atrox [94] Dinoponera_longipes [100] [99] [100] Neoponera_unidentata Diacamma_pallidum [97] Emeryopone_MY01 Cryptopone_gilva [53] [100] Ectomomyrmex_leeuwenhoeki Ponera_swezeyi [97] Parvaponera_darwinii [100] [100] Pseudoponera_stigma Harpegnathos_saltator [98] Hypoponera_opacior [100] [100] Hypoponera_sakalava Centromyrmex_angolensis [100] Psalidomyrmex_procerus [100] Loboponera_politula [99] [95] Plectroctena_ugandensis Myopias_tenuis [100] Brachyponera_obscurans Asphinctopone_silvestrii [94] Leptogenys_diminuta [85] Mesoponera_melanaria [81] Odontoponera_transversa Promyopias_silvestrii Bothroponera_wasmannii Mesoponera_ambigua [89] [99] Streblognathus_peetersi Phrynoponera_pulchella [71] Anochetus_madagascarensis [99] Odontomachus_coquereli Euponera_sikorae [100] Euponera_brunoi [99] Cryptopone_hartwigi [100] Fisheropone_ambigua

Supplementary Figure 17: Majority-rule consensus of maximum-likelihood trees inferred from 100 simulated alignments imitating first codon positions.

41 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata [100] Mischocyttarus_flavitarsis Brachycistis_sp [75] Chyphotes_mellipes [84] Aporus_niger [92] Sapyga_pumila Dasymutilla_aureola Ampulex_compressa Apis_mellifera [84] [88] Chalybion_californicum Apterogyna_ZA01 [78] Scolia_verticalis Martialis_heureka [96] Opamyrma_hungvuong [96] Protanilla_JP01 [100] Protanilla_TH01 [100] [75] Protanilla_TH02 [100] Anomalomyrma_boltoni [81] Protanilla_VN03 [100] Protanilla_TH03 [70] [84] Protanilla_VN01 Leptanilla_TH01 [80] Leptanilla_TH09 [76] [98] Leptanilla_GR01 [100] Leptanilla_GR02 [100] Leptanilla_ZA01 Leptanilla_TH03 [100] Yavnella_argamani [100] Leptanilla_TH08 [93] Phaulomyrma_MM01 Leptanilla_TH02 [100] Leptanilla_TH05 Leptanilla_TH04 [95] Leptanilla_TH06 Aneuretus_simoni [100] Dolichoderus_pustulatus [100] Liometopum_occidentale Acanthoponera_minor [88] Rhytidoponera_chalybaea Cerapachys_jacobsoni [100] Labidus_praedator [100] Leptanilloides_femoralis Myrmelachista_flavocotea [100] Camponotus_maritimus [97] Lasius_californicus Tetraponera_punctulata [100] Tetraponera_rufonigra [99] Myrmecia_pyriformis [100] Nothomyrmecia_macrops Pogonomyrmex_vermiculatus Manica_bradleyi [100] [100] Myrmica_tahoensis Crematogaster_kelleri [95] Stenamma_expolitum Cyphomyrmex_longiscapus_cf [77] [73] Myrmicaria_carinata Discothyrea_testacea [100] Discothyrea_MG07 [95] Discothyrea_SC02 [100] Discothyrea_SC03 Proceratium_avium [100] Proceratium_stictum [99] Proceratium_SC02 [100] Proceratium_silaceum [93] Probolomyrmex_brujitae_cf [100] Probolomyrmex_tani Apomyrma_CF02 [100] Apomyrma_stygia [83] Prionopelta_amabilis [84] [92] Amblyopone_australis [99] Onychomyrmex_hedleyi Stigmatomma_mystriops [88] [99] Stigmatomma_saundersi [98] Adetomyrma_caputleae Stigmatomma_boltoni [65] Mystrium_mysticum Stigmatomma_denticulatum [97] Stigmatomma_pallipes Myopopone_castanea [83] Xymmer_muticus Paraponera_clavata [100] Ankylomyrma_coronacantha [93] Tatuidris_tatusia Platythyrea_lamellosa [100] Platythyrea_mocquerysi [97] Platythyrea_punctata Myopias_tenuis Diacamma_pallidum Simopelta_pergandei_cf [99] [52] Belonopelta_HN01 [100] Thaumatomyrmex_atrox Dinoponera_longipes [100] Neoponera_unidentata Emeryopone_MY01 Ponera_swezeyi [94] [94] Parvaponera_darwinii [97] Pseudoponera_stigma Cryptopone_gilva [67] Ectomomyrmex_leeuwenhoeki Harpegnathos_saltator [77] Hypoponera_opacior [100] [68] Hypoponera_sakalava Centromyrmex_angolensis [100] Psalidomyrmex_procerus [91] Loboponera_politula [83] Plectroctena_ugandensis Leptogenys_diminuta Mesoponera_melanaria Brachyponera_obscurans [92] Asphinctopone_silvestrii Phrynoponera_pulchella [59] Anochetus_madagascarensis [99] Odontomachus_coquereli Bothroponera_wasmannii Promyopias_silvestrii [79] Odontoponera_transversa [74] Mesoponera_ambigua [92] Streblognathus_peetersi Euponera_sikorae [100] Euponera_brunoi [93] Cryptopone_hartwigi [100] Fisheropone_ambigua

Supplementary Figure 18: Majority-rule consensus of maximum-likelihood trees inferred from 100 simulated alignments imitating second codon positions.

42 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata [100] Mischocyttarus_flavitarsis Ampulex_compressa [99] Apis_mellifera [100] Chalybion_californicum Brachycistis_sp [100] Chyphotes_mellipes [100] Aporus_niger [100] Dasymutilla_aureola [98] Sapyga_pumila Apterogyna_ZA01 [100] [100] Scolia_verticalis Martialis_heureka Opamyrma_hungvuong [64] Protanilla_JP01 [100] Protanilla_TH01 [100] [100] Protanilla_TH02 [100] Anomalomyrma_boltoni [93] Protanilla_VN03 [100] Protanilla_TH03 [100] [100] Protanilla_VN01 Leptanilla_TH01 [99] Leptanilla_TH09 [94] [100] Leptanilla_ZA01 [100] Leptanilla_GR01 [100] [100] Leptanilla_GR02 Leptanilla_TH03 [100] Yavnella_argamani Leptanilla_TH08 [100] [76] Phaulomyrma_MM01 [100] Leptanilla_TH06 [100] Leptanilla_TH04 Leptanilla_TH02 [100] Leptanilla_TH05 Leptanilloides_femoralis [100] Cerapachys_jacobsoni [92] [100] Labidus_praedator Aneuretus_simoni [100] Dolichoderus_pustulatus [100] [100] Liometopum_occidentale [99] Tetraponera_punctulata [100] Tetraponera_rufonigra [97] Myrmecia_pyriformis [100] [100] Nothomyrmecia_macrops Myrmelachista_flavocotea [100] Camponotus_maritimus [100] Lasius_californicus Acanthoponera_minor [100] [100] Rhytidoponera_chalybaea [100] Pogonomyrmex_vermiculatus [86] Manica_bradleyi [100] [100] Myrmica_tahoensis Stenamma_expolitum [100] Crematogaster_kelleri [98] Cyphomyrmex_longiscapus_cf [100] Myrmicaria_carinata Discothyrea_testacea [100] [100] Discothyrea_MG07 [100] Discothyrea_SC02 [100] [100] Discothyrea_SC03 Proceratium_avium [100] Proceratium_stictum [100] Proceratium_SC02 [100] Proceratium_silaceum [100] Probolomyrmex_brujitae_cf [99] [100] Probolomyrmex_tani Apomyrma_CF02 [100] Apomyrma_stygia Prionopelta_amabilis [99] [100] Amblyopone_australis [96] Onychomyrmex_hedleyi [95] Stigmatomma_mystriops [100] Stigmatomma_saundersi [100] Adetomyrma_caputleae Stigmatomma_denticulatum [100] [89] [100] Stigmatomma_pallipes [100] Mystrium_mysticum [100] Stigmatomma_boltoni Myopopone_castanea [81] Xymmer_muticus Paraponera_clavata [99] Ankylomyrma_coronacantha [100] Tatuidris_tatusia Platythyrea_lamellosa [100] Platythyrea_mocquerysi [93] Platythyrea_punctata [100] Simopelta_pergandei_cf Belonopelta_HN01 [100] [98] Thaumatomyrmex_atrox [100] Dinoponera_longipes [100] [100] [100] Neoponera_unidentata Diacamma_pallidum [100] Emeryopone_MY01 Cryptopone_gilva [100] [100] Ectomomyrmex_leeuwenhoeki Ponera_swezeyi [100] Parvaponera_darwinii [100] [100] Pseudoponera_stigma Harpegnathos_saltator [99] Hypoponera_opacior [100] [92] Hypoponera_sakalava Centromyrmex_angolensis [100] Psalidomyrmex_procerus [100] Loboponera_politula [100] [100] Plectroctena_ugandensis Myopias_tenuis Brachyponera_obscurans [100] Asphinctopone_silvestrii [96] Leptogenys_diminuta [94] [100] Mesoponera_melanaria Phrynoponera_pulchella [100] [83] Anochetus_madagascarensis [100] Odontomachus_coquereli [68] Bothroponera_wasmannii Mesoponera_ambigua [96] Streblognathus_peetersi [84] Odontoponera_transversa [98] Promyopias_silvestrii Euponera_sikorae [100] Euponera_brunoi [100] Cryptopone_hartwigi [100] Fisheropone_ambigua

Supplementary Figure 19: Majority-rule consensus of maximum-likelihood trees inferred from 100 simulated alignments imitating third codon positions.

43 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pristocera_MG01 Metapolybia_cingulata [100] Mischocyttarus_flavitarsis Brachycistis_sp [100] Chyphotes_mellipes [100] Aporus_niger [100] Dasymutilla_aureola [100] Sapyga_pumila Ampulex_compressa [100] Apis_mellifera [100] Chalybion_californicum [100] Apterogyna_ZA01 [100] Scolia_verticalis Martialis_heureka Opamyrma_hungvuong [99] Protanilla_JP01 [100] Protanilla_TH01 [100] [100] [100] Protanilla_TH02 [100] Anomalomyrma_boltoni [100] Protanilla_VN03 [100] Protanilla_TH03 [100] [100] Protanilla_VN01 Leptanilla_TH01 [100] Leptanilla_TH09 [100] [100] Leptanilla_ZA01 [100] Leptanilla_GR01 [100] [100] Leptanilla_GR02 Leptanilla_TH03 [100] Yavnella_argamani Leptanilla_TH08 [100] [80] Phaulomyrma_MM01 [100] Leptanilla_TH04 [100] Leptanilla_TH06 [100] Leptanilla_TH02 [100] Leptanilla_TH05 Leptanilloides_femoralis [100] Cerapachys_jacobsoni [98] [100] Labidus_praedator Aneuretus_simoni [100] Dolichoderus_pustulatus [100] [100] Liometopum_occidentale [100] Tetraponera_punctulata [100] Tetraponera_rufonigra [100] Myrmecia_pyriformis [100] [100] Nothomyrmecia_macrops Myrmelachista_flavocotea [100] Camponotus_maritimus [100] Lasius_californicus Acanthoponera_minor [100] [100] Rhytidoponera_chalybaea [100] Pogonomyrmex_vermiculatus [95] Manica_bradleyi [100] [100] Myrmica_tahoensis Stenamma_expolitum [100] Crematogaster_kelleri [98] Cyphomyrmex_longiscapus_cf [99] Myrmicaria_carinata Discothyrea_testacea [100] [100] Discothyrea_MG07 [100] Discothyrea_SC02 [100] [100] Discothyrea_SC03 Proceratium_avium [100] Proceratium_stictum [100] Proceratium_SC02 [100] Proceratium_silaceum [100] Probolomyrmex_brujitae_cf [99] [100] Probolomyrmex_tani Apomyrma_CF02 [100] Apomyrma_stygia Prionopelta_amabilis [100] [100] Amblyopone_australis [100] Onychomyrmex_hedleyi [100] Stigmatomma_mystriops [100] Stigmatomma_saundersi [100] Adetomyrma_caputleae Stigmatomma_denticulatum [100] [100] [100] Stigmatomma_pallipes [99] Mystrium_mysticum [100] Stigmatomma_boltoni Myopopone_castanea [98] Xymmer_muticus Paraponera_clavata [100] Ankylomyrma_coronacantha [100] Tatuidris_tatusia Platythyrea_lamellosa [100] Platythyrea_mocquerysi [100] Platythyrea_punctata [100] Simopelta_pergandei_cf Belonopelta_HN01 [100] [100] Thaumatomyrmex_atrox [100] Dinoponera_longipes [100] [100] [100] Neoponera_unidentata Diacamma_pallidum [100] Emeryopone_MY01 Cryptopone_gilva [100] [100] Ectomomyrmex_leeuwenhoeki Ponera_swezeyi [100] Parvaponera_darwinii [100] [100] Pseudoponera_stigma Harpegnathos_saltator [100] Hypoponera_opacior [100] [100] Hypoponera_sakalava Centromyrmex_angolensis [100] Psalidomyrmex_procerus [100] Loboponera_politula [100] [100] Plectroctena_ugandensis Myopias_tenuis Brachyponera_obscurans [100] Asphinctopone_silvestrii [96] Leptogenys_diminuta [100] [100] Mesoponera_melanaria Phrynoponera_pulchella [100] [99] Anochetus_madagascarensis [100] Odontomachus_coquereli [98] Bothroponera_wasmannii Mesoponera_ambigua [100] Streblognathus_peetersi [95] Odontoponera_transversa [99] Promyopias_silvestrii Euponera_sikorae [100] Euponera_brunoi [100] Cryptopone_hartwigi [100] Fisheropone_ambigua

Supplementary Figure 20: Majority-rule consensus of maximum-likelihood trees inferred from 100 simulated alignments imitating first, second, and third codon positions combined.

44 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Table 1: Taxon properties in the full data set. AT and GC contents are expressed as proportions and nucleotide and gap columns contain character counts. Alignment has 7,451 sites total.

Taxon name AT content GC content A C G T Gap Acanthoponera minor 0.446 0.554 1792 2043 1967 1431 217 Adetomyrma caputleae 0.455 0.545 1822 2005 1933 1460 231 Amblyopone australis 0.452 0.548 1819 2008 1954 1448 222 Ampulex compressa 0.440 0.560 1790 2074 1958 1380 249 Aneuretus simoni 0.424 0.576 1711 2112 2054 1360 214 Ankylomyrma coronacantha 0.463 0.537 1839 1939 1943 1511 219 Anochetus madagascarensis 0.449 0.551 1833 2041 1949 1421 207 Anomalomyrma boltoni 0.514 0.486 2023 1745 1765 1696 222 Apis mellifera 0.491 0.509 1956 1813 1865 1590 225 Apomyrma CF02 0.483 0.517 1917 1883 1857 1575 219 Apomyrma stygia 0.478 0.522 1904 1906 1865 1553 223 Aporus niger 0.436 0.564 1756 2060 2012 1397 226 Apterogyna ZA01 0.438 0.562 1739 2025 2020 1413 254 Asphinctopone silvestrii 0.447 0.553 1801 2028 1976 1433 213 Belonopelta HN01 0.416 0.584 1686 2158 2072 1324 205 Bothroponera wasmannii 0.453 0.547 1833 2011 1944 1447 216 Brachycistis sp 0.502 0.498 2006 1781 1806 1614 204 Brachyponera obscurans 0.438 0.562 1781 2089 1983 1390 207 Camponotus maritimus 0.459 0.541 1823 1975 1925 1492 236 Centromyrmex angolensis 0.443 0.557 1773 2014 1998 1424 213 Cerapachys jacobsoni 0.452 0.548 1807 2002 1958 1464 220 Chalybion californicum 0.428 0.572 1753 2121 2015 1342 220 Chyphotes mellipes 0.452 0.548 1833 2021 1937 1428 232 Crematogaster kelleri 0.464 0.536 1843 1954 1923 1508 223 Cryptopone gilva 0.436 0.564 1766 2073 2013 1387 212 Cryptopone hartwigi 0.443 0.557 1791 2049 1968 1410 216 Cyphomyrmex longiscapus cf 0.469 0.531 1866 1934 1907 1530 214 Dasymutilla aureola 0.488 0.512 1965 1839 1854 1560 233 Diacamma pallidum 0.446 0.554 1801 2031 1982 1429 208 Dinoponera longipes 0.429 0.571 1723 2075 2051 1383 213 Discothyrea MG07 0.459 0.541 1856 1998 1914 1465 218 Discothyrea SC02 0.446 0.554 1802 2020 1988 1421 220 Discothyrea SC03 0.454 0.546 1835 2001 1947 1448 219 Discothyrea testacea 0.424 0.576 1737 2126 2048 1332 208 Dolichoderus pustulatus 0.456 0.544 1807 1982 1954 1488 217 Ectomomyrmex leeuwenhoeki 0.436 0.564 1764 2082 1999 1387 219 Emeryopone MY01 0.428 0.572 1748 2116 2029 1359 199 Euponera brunoi 0.441 0.559 1784 2065 1979 1410 207 Euponera sikorae 0.444 0.556 1797 2057 1966 1415 210 Fisheropone ambigua 0.444 0.556 1803 2054 1968 1413 213 Fulakora mystriops 0.491 0.509 1921 1824 1843 1620 243 Fulakora saundersi 0.454 0.546 1824 2002 1951 1467 207 Harpegnathos saltator 0.451 0.549 1815 2002 1973 1451 210 Hypoponera opacior 0.453 0.547 1799 1979 1978 1476 219 Hypoponera sakalava 0.450 0.550 1808 2006 1967 1445 219 Labidus praedator 0.463 0.537 1823 1951 1932 1525 219 Lasius californicus 0.452 0.548 1791 2012 1947 1478 223 Leptanilla GR01 0.528 0.472 2064 1698 1709 1741 239 Leptanilla GR02 0.529 0.471 2074 1692 1707 1740 238 Leptanilla TH01 0.515 0.485 2011 1732 1761 1704 243 Leptanilla TH02 0.506 0.494 1989 1775 1788 1659 240 Leptanilla TH03 0.512 0.488 2014 1742 1782 1684 229 Leptanilla TH04 0.505 0.495 1990 1783 1786 1652 240 Leptanilla TH05 0.508 0.492 1985 1757 1791 1679 239 Leptanilla TH06 0.504 0.496 1987 1786 1791 1650 237 Leptanilla TH08 0.506 0.494 1990 1779 1789 1663 230 Leptanilla TH09 0.532 0.468 2064 1661 1716 1776 234 Leptanilla ZA01 0.533 0.467 2079 1678 1689 1769 236 Leptanilloides femoralis 0.424 0.576 1730 2146 2021 1343 211 Leptogenys diminuta 0.454 0.546 1811 1997 1953 1480 210 Liometopum occidentale 0.442 0.558 1763 2043 1988 1432 225 Loboponera politula 0.431 0.569 1741 2078 2037 1379 216

45 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Table 1: Continued. Taxon properties in the full data set. AT and GC contents are expressed as proportions and nucleotide and gap columns contain character counts. Alignment has 7,451 sites total.

Taxon name AT content GC content A C G T Gap Manica bradleyi 0.440 0.560 1766 2060 1990 1415 219 Martialis heureka 0.466 0.534 1849 1960 1901 1520 221 Mesoponera ambigua 0.444 0.556 1792 2047 1973 1420 219 Mesoponera melanaria 0.455 0.545 1846 2013 1924 1440 222 Metapolybia cingulata 0.516 0.484 2022 1781 1745 1738 165 Mischocyttarus flavitarsis 0.530 0.470 2095 1718 1701 1757 180 Myopias tenuis 0.421 0.579 1703 2138 2055 1340 209 Myopopone castanea 0.471 0.529 1877 1935 1883 1525 231 Myrmecia pyriformis 0.464 0.536 1851 1971 1902 1500 227 Myrmelachista flavocotea 0.451 0.549 1784 2001 1961 1474 231 Myrmica tahoensis 0.438 0.562 1762 2062 1997 1406 223 Myrmicaria carinata 0.457 0.543 1821 1986 1942 1483 219 Mystrium voeltzkowi 0.476 0.524 1902 1915 1865 1535 234 Neoponera unidentata 0.443 0.557 1787 2042 1988 1420 199 Nothomyrmecia macrops 0.451 0.549 1811 2035 1935 1446 224 Odontomachus coquereli 0.445 0.555 1787 2029 1989 1436 204 Odontoponera transversa 0.456 0.544 1829 1989 1946 1464 216 Onychomyrmex hedleyi 0.463 0.537 1850 1961 1923 1495 222 Opamyrma hungvuong 0.473 0.527 1887 1935 1874 1535 220 Paraponera clavata 0.472 0.528 1873 1922 1896 1538 222 Parvaponera darwinii 0.416 0.584 1706 2173 2056 1308 208 Phaulomyrma MM01 0.507 0.493 2000 1775 1781 1661 234 Phrynoponera pulchella 0.440 0.560 1783 2062 1992 1406 207 Platythyrea lamellosa 0.460 0.540 1816 1975 1934 1516 210 Platythyrea mocquerysi 0.443 0.557 1792 2053 1977 1418 211 Platythyrea punctata 0.466 0.534 1837 1968 1896 1530 220 Plectroctena ugandensis 0.445 0.555 1791 2025 1986 1426 216 Pogonomyrmex vermiculatus 0.465 0.535 1857 1963 1903 1507 219 Ponera swezeyi 0.440 0.560 1766 2049 2007 1416 213 Prionopelta amabilis 0.481 0.519 1903 1894 1857 1569 228 Pristocera MG01 0.468 0.532 1863 1923 1907 1512 228 Probolomyrmex brujitae cf 0.413 0.587 1673 2151 2094 1309 224 Probolomyrmex tani 0.444 0.556 1777 2034 1979 1427 234 Proceratium avium 0.443 0.557 1787 2041 1979 1412 215 Proceratium SC02 0.461 0.539 1836 1957 1941 1501 216 Proceratium silaceum 0.458 0.542 1834 1968 1950 1483 216 Proceratium stictum 0.447 0.553 1794 2024 1971 1430 215 Promyopias silvestrii 0.457 0.543 1839 1985 1923 1456 219 Protanilla JP01 0.499 0.501 1964 1817 1798 1643 229 Protanilla TH01 0.517 0.483 2038 1768 1718 1699 228 Protanilla TH02 0.518 0.482 2037 1759 1724 1703 228 Protanilla TH03 0.490 0.510 1946 1861 1836 1604 204 Protanilla VN01 0.491 0.509 1941 1849 1836 1615 210 Protanilla VN03 0.491 0.509 1943 1853 1832 1607 214 Psalidomyrmex procerus 0.436 0.564 1776 2078 2005 1383 209 Pseudoponera stigma 0.419 0.581 1704 2138 2066 1324 219 Rhytidoponera chalybaea 0.452 0.548 1814 2005 1961 1458 213 Sapyga pumila 0.407 0.593 1689 2193 2105 1257 205 Scolia verticalis 0.484 0.516 1939 1894 1833 1554 231 Simopelta pergandei cf 0.441 0.559 1777 2058 1998 1422 196 Stenamma expolitum 0.451 0.549 1787 1981 1979 1466 237 Stigmatomma boltoni 0.474 0.526 1884 1926 1869 1535 237 Stigmatomma denticulatum 0.454 0.546 1826 2007 1938 1455 225 Stigmatomma pallipes 0.456 0.544 1833 1997 1930 1460 231 Streblognathus peetersi 0.438 0.562 1768 2074 1997 1399 213 Tatuidris tatusia 0.478 0.522 1886 1888 1879 1559 239 Tetraponera punctulata 0.434 0.566 1729 2064 2017 1401 240 Tetraponera rufonigra 0.448 0.552 1788 2021 1961 1440 241 Thaumatomyrmex atrox 0.438 0.562 1752 2054 2010 1415 214 Xymmer muticus 0.472 0.528 1871 1925 1891 1538 225 Yavnella argamani 0.511 0.489 1993 1754 1775 1688 241

46 bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Table 2: Results of compositional homogeneity tests for all data partitions. P-values below 0.05 considered significant violation of homogeneity assumption.

Partition Corrected test p-value Chi-squared p-value Full data set 0.000 0.000 28S 0.000 1.000 abdA 0.000 0.990 abdA 1st pos 0.905 1.000 abdA 2nd pos 0.262 1.000 abdA 3rd pos 0.000 0.000 argK 0.000 0.000 argK 1st pos 0.698 1.000 argK 2nd pos 0.987 1.000 argK 3rd pos 0.000 0.000 Antp 0.000 0.000 Antp 1st pos 0.847 1.000 Antp 2nd pos 0.520 1.000 Antp 3rd pos 0.000 0.000 EF1aF2 0.000 0.000 EF1aF2 1st pos 1.000 1.000 EF1aF2 2nd pos 0.555 1.000 EF1aF2 3rd pos 0.000 0.000 LW Rh 0.000 0.000 LW Rh 1st pos 1.000 1.000 LW Rh 2nd pos 0.926 1.000 LW Rh 3rd pos 0.000 0.000 NaK 0.000 0.025 NaK 1st pos 0.558 1.000 NaK 2nd pos 0.460 1.000 NaK 3rd pos 0.000 0.000 POLD1 0.000 0.000 POLD1 1st pos 0.004 1.000 POLD1 2nd pos 0.766 1.000 POLD1 3rd pos 0.000 0.000 Top 1 0.000 0.000 Top 1 1st pos 0.106 1.000 Top 1 2nd pos 0.964 1.000 Top 1 3rd pos 0.000 0.000 Ubx 0.000 0.009 Ubx 1st pos 0.038 1.000 Ubx 2nd pos 0.202 1.000 Ubx 3rd pos 0.000 0.000 Wg 0.000 0.000 Wg 1st pos 0.749 1.000 Wg 2nd pos 0.923 1.000 Wg 3rd pos 0.000 0.000 All 1st pos 0.028 1.000 All 2nd pos 1.000 1.000 All 3rd pos 0.000 0.000

47 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

Supplementary Table 3: Calibration fossils used in divergence dating analysis.

Least inclusive clade Least inclusive clade Fossil genus Fossil species (total group) in this Deposit Age Reference(s) Notes (total group) study Pristomyrmex rasnitsyni Pristomyrmex Crematogaster Danish amber 36 Pristomyrmex in late Eocene Danish amber (Dlussky and Radchenko, 2011). Node calibration in Ward et al. (2015): 42 (offset), 52 (median), 73 (95% quantile)

Aphaenogaster sensu doi: Aphaenogaster antiqua Stenamma Rovno amber 36 Aphaenogaster antiqua from Rovno amber (Dlussky and Perkovsky, 2002). Node calibration in Ward et al. (2015): 42 (offset), 52 (median), 73 (95% quantile) stricto Four Myrmica species (longispinosa, rudis, intermedia, eocenica) from Baltic amber

Myrmica longispinosa Myrmica Myrmica Baltic amber 36 (Dlussky and Rasnitsyn, 2009), not definitely assignable to modern species groups, Node calibration in Ward et al. (2015): 42 (offset), 60 (median), 85 (95% quantile) https://doi.org/10.1101/173393 although similar to the plesiotypic ritae-group (Radchenko et al., 2007). europaea Gnamptogenys Rhytidoponera Baltic amber 36 Two species of Gnamptogenys in Baltic amber (Dlussky, 2009). Myrmelachista plus New Jersey Kyromyrma neffi Formicinae Camponotus plus 92 Kyromyrma in New Jersey amber (Grimaldi and Agosti, 2000). Node calibration in Ward et al. (2015): 92 (offset), 95 (median), 120 (95% quantile) amber Lasius Kishenehn Lasius glom Lasius Lasius 46 Lasius glom in Kishenehn Formation (Lapolla and Greenwalt, 2015) Formation Grube Messel, Oecophylla longiceps from Grube Messel, Darmstadt, Germany; middle Eocene, Oecophylla longiceps Oecophylla Camponotus 47 Germany (ca. 47 Ma) (Dlussky et al., 2008). Node calibration in Chomicki et al. (2015) of crown Pseudomyrmecinae with truncated Tetraponera sp Oise Tetraponera Tetraponera Oise amber 52 Three species of Tetraponera in Oise amber (Aria et al. (2011); Aria pers. comm.). normal prior, mean 55, sd 5; truncated at 55 Ma. Tetraponera simplex in Baltic amber (Dlussky and Rasnitsyn, 2009), with some Considered crown Tetraponera, given mandible structure (reduced teeth) and loss of Tetraponera simplex Tetraponera crown Tetraponera punctulata Baltic amber 36 features of T. nigra group. ocelli. Myrmecia plus Mo-Clay, Ypresiomyrma rebekkae from Mo-clay, Stolleklint, Isle of Fur, Denmark; Eocene, Ypresiomyrma rebekkae Myrmeciinae 53.5 Nothomyrmecia Denmark Ypresian (Rust and Andersen (1999); Archibald et al. (2006); Dlussky et al. (2015)). Prionomyrmex janzeni Nothomyrmecia Nothomyrmecia Baltic amber 36 Two species of Prionomyrmex in Baltic amber (Mayr, 1868; Baroni Urbani, 2000). Green River Dolichoderus kohlsi Dolichoderus Dolichoderus 51.5 Dolichoderus kohlsi from Green River Formation (Dlussky and Rasnitsyn, 2003). Second most abundant species of ant in Green River Formation. Formation Liometopum oligocenicum in Baltic amber (Dlussky, 1997), but rare (Dlussky and Node calibration in Ward et al. (2010) (ame calibration for Liometopum and Tapinoma): ; Liometopum oligocenicum Liometopum Liometopum Baltic amber 36 a

Rasnitsyn, 2009). 42 (offset), 54 (median), 70 (95% quantile); in Boudinot et al. (2016): 42, 59, 77. this versionpostedAugust8,2017. CC-BY-NC 4.0Internationallicense Dolichoderus plus Canadian Grassy Node calibration in Boudinot et al. (2016): 78 (offset), 95 (median), 110 (95% Chronomyrmex medicinehatensis Dolichoderinae 78 Chronomyrmex in Grassy Lake amber (McKellar et al. (2013); Boudinot et al. (2016)). Liometopum Lake amber quantile). Aneuretinae plus Aneuretinae plus Because of possible paraphyly of Aneuretinae we assume a conservative assignment to Aneuretellus deformis Sakhalin amber 57.5 Aneuretellus deformis from Sakhalin amber (Dlussky, 1988). Dolichoderinae Dolichoderinae the total group of (Aneuretinae plus Dolichoderinae), i.e. crown dolichoderomorphs. Labidus plus Procerapachys favosus Dorylinae Cerapachys plus Baltic amber 36 Three species of Procerapachys in Baltic amber (Dlussky, 2009). Node calibration in (Brady et al., 2014): 42 (offset), 80 (median), 100 (95% quantile) Leptanilloides Platythyrea dlusskyi Platythyrea Platythyrea Oise amber 52 Platythyrea dlusskyi in Oise amber (Aria et al., 2011). Different calibration (Baltic amber) in Ward et al. (2015). Hypoponera atavia Hypoponera Hypoponera Baltic amber 36 Hypoponera atavia in Baltic amber (Dlussky, 1997; Dlussky and Rasnitsyn, 2009). Bílina, Czech Odontomachus paleomyagra from Bílina, Czech Republic; Early Miocene Odontomachus paleomyagra Odontomachus Odontomachus 18 48 Republic (Burdigalian, 16-20 Ma) (Wappler et al., 2014). Dominican Anochetus lucidus Anochetus Anochetus 17 Anochetus in Dominican amber (Urbani, 1980; Andrade, 1994; MacKay, 1991). amber Ponera mayri Ponera Ponera swezeyi Baltic amber 36 Dlussky and Rasnitsyn (2009) recognizes three Ponera species from Baltic amber. Stigmatomma Stigmatomma Stigmatomma groehni in Baltic amber, said to be member of S. denticulatum group Stigmatomma groehni Baltic amber 36 Node calibration in Ward and Fisher (2016): 42 (offset), 55 (median), 80 (95% quantile) denticulatum group denticulatum (Dlussky, 2009). Stigmatomma Stigmatomma Stigmatomma electrina Baltic amber 36 Stigmatomma electrina (male) in Baltic amber (Dlussky, 2009). (XMMAS clade) mystriops Dominican Prionopelta sp Dominican Prionopelta Prionopelta 17 Undescribed Prionopelta species from Dominican amber (Wilson, 2004). Node calibration in Ward and Fisher (2016): 15 (offset), 40 (median), 60 (95% quantile) amber Discothyrea plus Bradoponera electrina Proceratiinae Proceratium plus Baltic amber 36 Three species of Bradoponera in Baltic amber (Urbani and Andrade, 2003)

Probolomyrmex . The copyrightholderforthispreprint(whichwasnot Proceratium plus Proceratium eocenicum Proceratium Baltic amber 36 Proceratium eocenicum in Baltic amber (Dlussky, 2009). Probolomyrmex Agroecomyrmex duisburgi (Agroecomyrmecinae) in Baltic amber (Wheeler, 1915; Agroecomyrmex duisburgi Tatuidris Tatuidris Baltic amber 36 Node calibration in Ward et al. (2015): 42 (offset), 60 (median), 90 (95% quantile) Dlussky and Rasnitsyn, 2009). Camelomecia janovitzi Formicidae Formicidae Myanmar amber 97 (Barden and Grimaldi, 2016). Charentese Gerontoformica cretacica and G. occidentalis in Charentese amber (Perrichot et al., Gerontoformica cretacica Formicidae Formicidae 99.5 Sphecomyrmodes now considered a junior synonym (Barden and Grimaldi, 2016). amber 2008; Perrichot, 2015; Barden and Grimaldi, 2016). Lulworth Pompilopterus difficilis Apoidea Apoidea Formation, 143 Pompilopterus difficilis (Rasnitsyn and Jarzembowski, 1998). Age range: 145.5 to 140.2 Ma. England Crato formation, Said to be Anthoboscinae. Note that anthoboscines are Thynnidae, not Tiphiidae (sensu Architiphia rasnitsyni Anthoboscinae Chyphotes 117.6 Darling and Sharkey (1990). Date from fossilworks (midpoint of 122.46 to 112.6) Ceará, Brazil Pilgrim et al. (2008)). Tiphioidea plus Chyphotes plus Thanatotiphia nyx Myanmar amber 97 Engel et al. (2009). Date from fossilworks (midpoint of 99.7 to 94.3). Not definitively assigned to thynnid or tiphiid (s.s.) groups. Thynnoidea Brachycistis Metapolybia plus Eorhopalosoma gorgyra Rhopalosomatidae Myanmar amber 97 Engel (2008). Date from fossilworks (midpoint of 99.7 to 94.3). Engel (2008) argues that this is a crown rhopalosomatid. Mischocyttarus Mesorhopalo- Metapolybia plus Crato formation, cearae Rhopalosomatidae 117.6 Darling and Sharkey (1990). Date from fossilworks (midpoint of 122.46 to 112.6). Mesorhopalosoma likely a stem rhopalosomatid. soma Mischocyttarus Ceará, Brazil Rodriguez et al. (2016). Note that these authors remove Bryopompilus (Myanmar Paleogenia wahisi Pepsinae Aporus Baltic amber 36 amber) from Pompilidae. Cretosapyga resinicola Sapygidae Sapyga Myanmar amber 97 Bennett and Engel (2005)). Date from fossilworks (midpoint of 99.7 to 94.3). archaeotricho- Protomutilla Myrmosidae Dasymutilla Baltic amber 36 Lelej (1986), Brothers (1974). soma Rasnitsyn and Martínez-Delclòs (2000). Date from fossilworks (midpopint of 130.0 to At least seven other species of Cretoscolia have been described, all from the Cretoscolia conquensis Scoliidae Scolia Barremian, Spain 127.7 125.45). Cretaceous. Crato formation, Cariridris bipetiolata Ampulicidae Ampulex 117.6 Ohl (2004). Date from fossilworks (midpoint of 122.46 to 112.6) Ceará, Brazil Cretampulex gracilis Ampulicidae Ampulex Myanmar amber 97 Antropov (2000). Date from fossilworks (midpoint of 99.7 to 94.3). Cretospilomena familiaris Pemphredoninae Apis Myanmar amber 97 Antropov (2000, 2011). Date from fossilworks (midpoint of 99.7 to 94.3). There are many other Cretaceous pemphredonines. Poinar and Danforth (2006), Poinar (2009). Date from fossilworks (midpoint of 99.7 to Melittosphex burmensis Anthophila Apis Myanmar amber 97 94.3). bioRxiv preprint doi: https://doi.org/10.1101/173393; this version posted August 8, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Table 4: Taxa included in the study with GenBank accession numbers and specimen voucher information. Detailed collection data for each species is available by searching for the specimen code on AntWeb (www.antweb.org).

Taxon Voucher 28S Wg AA LR F2 ArgK Top1 Ubx DP NK AP Acanthoponera minor CASENT0039772 EF012953 EF013661 EF013081 EF013533 EF013371 KJ861140 KJ861751 KJ860480 XXXXXXXX XXXXXXXX XXXXXXXX Adetomyrma caputleae CASENT0491481 EF012956 EF013664 EF013084 EF013536 EF013375 KU671604 KU671667 KU671730 KU671870 KU671947 KU671793 Amblyopone australis CASENT0106229 KJ860056 KJ861944 KJ861331 KJ861515 KJ859868 KJ861141 KJ861752 KJ860481 KU671871 KU671948 KU671794 Aneuretus simoni CASENT0007014 EF012961 EF013669 EF013089 EF013541 EF013382 FJ939840 KJ523645 KJ523562 XXXXXXXX XXXXXXXX XXXXXXXX Ankylomyrma coronacantha CASENT0406734 KJ860057 KJ861945 KJ861332 KJ861516 KJ859869 KJ861142 KJ861753 KJ860482 KU671924 KU672001 KU671847 Anochetus madagascarensis CASENT0498593 EF012962 EF013670 EF013090 EF013542 EF013383 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Anomalomyrma boltoni CASENT0217032 KU671445 KU671598 KU672069 KU671547 KU671496 KU671656 KU671719 KU671782 KU671925 KU672002 KU671848 Apomyrma CF02 CASENT0086073 KU671402 KU671555 KU672026 KU671504 KU671453 KU671607 KU671670 KU671733 KU671874 KU671951 KU671797 Apomyrma stygia CASENT0007017 EF012967 EF013675 EF013095 EF013547 EF013390 KU671608 KU671671 KU671734 KU671875 KU671952 KU671798 Asphinctopone silvestrii CASENT0417123 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Belonopelta HN01 CASENT0235904 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Bothroponera wasmannii CASENT0076787 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Brachyponera obscurans CASENT0059658 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Camponotus maritimus CASENT0106083 AY867464 AY867433 AY867480 AY867495 EF013400 KT443562 KT443642 KT443722 XXXXXXXX XXXXXXXX XXXXXXXX Centromyrmex angolensis CASENT0417147 EF012979 EF013687 EF013107 EF013559 EF013403 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Cerapachys jacobsoni CASENT0106233 KJ523194 KJ523510 KJ523841 KJ523731 KJ523786 KJ523310 KJ523655 KJ523572 KU671927 KU672004 KU671850 Crematogaster kelleri CASENT0498885 KJ859905 KJ861793 KJ861182 KJ861367 KJ859719 KJ860961 KJ861563 KJ860292 XXXXXXXX XXXXXXXX XXXXXXXX Cryptopone gilva CASENT0106309 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Cryptopone hartwigi CASENT0217039 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Cyphomyrmex longiscapus cf CASENT0052777 KJ859911 KJ861799 KJ861187 KJ861372 KJ859724 KJ860967 KJ861569 KJ860298 XXXXXXXX XXXXXXXX XXXXXXXX Diacamma pallidum CASENT0106139 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Dinoponera longipes CASENT0004663 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Discothyrea MG07 CASENT0042927 EF012987 EF013695 EF013115 EF013567 EF013415 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Discothyrea SC02 CASENT0159387 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Discothyrea SC03 CASENT0161605 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Discothyrea testacea CASENT0270725 KU671446 KU671599 KU672070 KU671548 KU671497 KU671658 KU671721 KU671784 KU671929 KU672006 KU671852 Dolichoderus pustulatus CASENT0106164 FJ939792 FJ940028 FJ939824 FJ939995 FJ939963 FJ939859 KJ523685 KJ523602 KU671930 KU672007 KU671853 Ectomomyrmex leeuwenhoeki CASENT0106306 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Emeryopone MY01 CASENT0226984 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Euponera brunoi CASENT0297649 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Euponera sikorae CASENT0487847 EF013032 EF013740 EF013160 EF013612 EF013475 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Fisheropone ambigua CASENT0289205 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Harpegnathos saltator CASENT0179535 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Hypoponera opacior CASENT0106093 AY703555 AY703622 AY703689 AY703756 EF013428 KU671659 KU671722 KU671785 KU671931 KU672008 KU671854 Hypoponera sakalava CASENT0494141 EF012997 EF013705 EF013125 EF013577 EF013429 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Labidus praedator CASENT0106227 KJ523222 KJ523538 KJ523869 KJ523759 KJ523814 KJ523345 KJ523691 KJ523608 XXXXXXXX XXXXXXXX XXXXXXXX Lasius californicus CASENT0106045 EF012998 EF013706 EF013126 EF013578 EF013430 KJ523346 KJ523692 KJ523609 KU671932 KU672009 KU671855 Leptanilla GR01 CASENT0106236 EF012999 EF013707 JN967846 JN967889 JN967829 JN967880 JN967820 JN967809 XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla GR02 CASENT0106067 JN967864 JN967856 JN967848 JN967891 JN967831 JN967883 JN967823 JN967812 XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH01 CASENT0119792 KU671447 KU671600 KU672071 KU671549 KU671498 KU671660 KU671723 KU671786 KU671933 KU672010 KU671856 Leptanilla TH02 CASENT0119531 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH03 CASENT0129721 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH04 CASENT0129695 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH05 CASENT0134656 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH06 CASENT0129609 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH08 CASENT0227555 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla TH09 CASENT0227556 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilla ZA01 CASENT0106085 AY867452 AY867421 AY867468 AY867483 EF013432 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Leptanilloides femoralis CASENT0106180 KJ523223 KJ523539 KJ523870 KJ523760 KJ523815 KJ523347 KJ523693 KJ523610 XXXXXXXX XXXXXXXX XXXXXXXX Leptogenys diminuta CASENT0106010 EF013000 EF013708 EF013128 EF013580 EF013435 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Liometopum occidentale CASENT0106078 AY867465 AY867434 AY867481 AY867496 EF013441 FJ939875 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Loboponera politula CASENT0003095 EF013005 EF013713 EF013133 EF013585 EF013442 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Manica bradleyi CASENT0106022 EF013006 EF013714 EF013134 EF013586 EF013443 FJ939877 KJ523698 KJ523615 KU671934 KU672011 KU671857 Martialis heureka CASENT0106181 KU671448 KU671601 KU672072 KU671550 KU671499 KU671661 KU671724 KU671787 KU671935 KU672012 KU671858 Mesoponera ambigua CASENT0441714 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Mesoponera melanaria CASENT0145371 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Myopias tenuis CASENT0106254 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Myopopone castanea CASENT0106147 KU671404 KU671557 KU672028 KU671506 KU671455 KU671611 KU671674 KU671737 KU671878 KU671955 KU671801 Myrmecia pyriformis CASENT0106088 AY703567 AY703634 AY703701 AY703768 EF013454 FJ939878 KJ523699 KJ523616 KU671936 KU672013 KU671859 Myrmelachista flavocotea CASENT0106049 EF013017 EF013725 EF013145 EF013597 EF013457 KJ861144 KJ861755 KJ860484 XXXXXXXX XXXXXXXX XXXXXXXX Myrmica tahoensis CASENT0106091 AY703562 AY703629 AY703696 AY703763 EF013459 GU085791 KJ861640 KJ860369 XXXXXXXX XXXXXXXX XXXXXXXX Myrmicaria carinata CASENT0106282 KJ859969 KJ861857 KJ861244 KJ861429 KJ859781 KJ861034 KJ861641 KJ860370 XXXXXXXX XXXXXXXX XXXXXXXX Mystrium voeltzkowi CASENT0076622 EF013022 EF013730 EF013150 EF013602 EF013463 KU671613 KU671676 KU671739 KU671880 KU671957 KU671803 Neoponera unidentata CASENT0106307 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Nothomyrmecia macrops CASENT0106089 AY703568 AY703635 AY703702 AY703769 EF013466 FJ939880 KJ523703 KJ523620 XXXXXXXX XXXXXXXX XXXXXXXX Odontomachus coquereli CASENT0499525 EF013026 EF013734 EF013154 EF013606 EF013469 KU671662 KU671725 KU671788 KU671937 KU672014 KU671860 Odontoponera transversa CASENT0010127 EF013027 EF013735 EF013155 EF013607 EF013470 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Onychomyrmex hedleyi CASENT0106018 EF013029 EF013737 EF013157 EF013609 EF013472 KU671615 KU671678 KU671741 KU671882 KU671959 KU671805 Opamyrma hungvuong CASENT0178347 KU671407 KU671560 KU672031 KU671509 KU671458 KU671616 KU671679 KU671742 KU671883 KU671960 KU671806 Paraponera clavata CASENT0106092 AY703556 AY703623 AY703690 AY703757 EF013477 KJ523355 KJ523704 KJ523621 KU671938 KU672015 KU671861 Parvaponera darwinii CASENT0484950 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Phaulomyrma MM01 CASENT0179537 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Phrynoponera pulchella CASENT0217034 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Platythyrea lamellosa CASENT0106138 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Platythyrea mocquerysi CASENT0106094 AY867453 AY867422 AY867469 AY867484 EF013484 KJ861145 KJ861756 KJ860485 KU671939 KU672016 KU671862 Platythyrea punctata CASENT0006819 EF013040 EF013748 EF013168 EF013620 EF013485 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Plectroctena ugandensis CASENT0003063 EF013041 EF013749 EF013169 EF013621 EF013486 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Pogonomyrmex vermiculatus CASENT0106213 KJ859996 KJ861884 KJ861271 KJ861456 KJ859808 KJ861069 KJ861676 KJ860405 XXXXXXXX XXXXXXXX XXXXXXXX Ponera swezeyi CASENT0047257 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Prionopelta amabilis CASENT0052775 KU671409 KU671562 KU672033 KU671511 KU671460 KU671618 KU671681 KU671744 KU671885 KU671962 KU671808 Probolomyrmex brujitae cf CASENT0106137 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Probolomyrmex tani CASENT0041507 EF013050 EF013758 EF013178 EF013630 EF013495 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Proceratium avium CASENT0059014 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Proceratium SC02 CASENT0159684 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Proceratium silaceum CASENT0235164 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Proceratium stictum CASENT0106095 AY703557 AY703624 AY703691 AY703758 EF013497 KJ861146 KJ861757 KJ860486 KU671940 KU672017 KU671863 Promyopias silvestrii CASENT0178751 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Protanilla JP01 CASENT0007002 EF013053 EF013761 EF013181 EF013633 EF013499 KU671663 KU671726 KU671789 KU671941 KU672018 KU671864 Protanilla TH01 CASENT0119776 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Protanilla TH02 CASENT0128922 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Protanilla TH03 CASENT0119791 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Protanilla VN01 CASENT0179564 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Protanilla VN03 CASENT0179565 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Psalidomyrmex procerus CASENT0003082 EF013054 EF013762 EF013182 EF013634 EF013500 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Pseudoponera stigma CASENT0106311 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Rhytidoponera chalybaea CASENT0106000 EF013058 EF013766 EF013186 EF013638 EF013505 FJ939885 KJ523705 KJ523622 KU671942 KU672019 KU671865 Simopelta pergandei cf CASENT0052744 EF013061 EF013769 EF013189 EF013641 EF013508 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Stenamma expolitum CASENT0052774 KJ860019 KJ861907 KJ861294 KJ861478 KJ859831 GU085793 KJ861704 KJ860433 XXXXXXXX XXXXXXXX XXXXXXXX Stigmatomma boltoni CASENT0179570 KU671413 KU671566 KU672037 KU671515 KU671464 KU671623 KU671686 KU671749 KU671890 KU671967 KU671813 Stigmatomma denticulatum CASENT0906827 KU671419 KU671572 KU672043 KU671521 KU671470 KU671629 KU671692 KU671755 KU671896 KU671973 KU671819 Stigmatomma mystriops INB0003690672 KU671430 KU671583 KU672054 KU671532 KU671481 KU671640 KU671703 KU671766 KU671907 KU671984 KU671830 Stigmatomma pallipes CASENT0106070 AY703554 AY703621 AY703688 AY703755 EF013381 KJ523366 KJ523716 KJ523633 KU671908 KU671985 KU671831 Stigmatomma saundersi CASENT0006813 KU671432 KU671585 KU672056 KU671534 KU671483 KU671642 KU671705 KU671768 KU671910 KU671987 KU671833 Streblognathus peetersi CASENT0260406 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Tatuidris tatusia CASENT0423526 EF013067 EF013775 EF013195 EF013647 EF013516 KJ861147 KJ861758 KJ860487 KU671944 KU672021 KU671867 Tetraponera punctulata CASENT0106098 AY703581 AY703648 AY703715 AY703782 EF013523 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Tetraponera rufonigra CASENT0106099 AY703582 AY703649 AY703716 AY703783 EF013524 FJ939892 KJ523718 KJ523635 XXXXXXXX XXXXXXXX XXXXXXXX Thaumatomyrmex atrox CASENT0010121 EF013074 EF013782 EF013202 EF013654 EF013525 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Xymmer muticus CASENT0006827 EF012960 EF013668 EF013088 EF013540 EF013380 KU671653 KU671716 KU671779 KU671921 KU671998 KU671844 Yavnella argamani CASENT0235253 KU671449 KU671602 KU672073 KU671551 KU671500 KU671665 KU671728 KU671791 KU671945 KU672022 KU671868 Ampulex compressa CASENT0217334 KU671444 KU671597 KU672068 KU671546 KU671495 KU671655 KU671718 KU671781 KU671923 KU672000 KU671846 Apis mellifera CASENT0106100 AY703551 AY703618 AY703685 AY703752 EF013389 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Aporus niger CASENT0106104 EF012968 EF013676 EF013096 EF013548 EF013391 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Apterogyna ZA01 CASENT0106304 KJ860058 KJ861946 KJ861333 KJ861517 KJ859870 KJ861143 KJ861754 KJ860483 KU671926 KU672003 KU671849 Brachycistis sp CASENT0106122 EF012959 EF013667 EF013087 EF013539 EF013379 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Chalybion californicum CASENT0106103 EF012981 EF013689 EF013109 EF013561 EF013407 KU671657 KU671720 KU671783 KU671928 KU672005 KU671851 Chyphotes mellipes CASENT0106101 AY703552 AY703619 AY703686 AY703753 EF013409 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Dasymutilla aureola CASENT0106123 EF012986 EF013694 EF013114 EF013566 EF013414 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Metapolybia cingulata CASENT0106106 EF013011 EF013719 EF013139 EF013591 EF013448 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Mischocyttarus flavitarsis CASENT0106102 AY703553 AY703620 AY703687 AY703754 EF013451 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Pristocera MG01 CASENT0006958 EF013049 EF013757 EF013177 EF013629 EF013494 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Sapyga pumila CASENT0106105 EF013059 EF013767 EF013187 EF013639 EF013506 XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX Scolia verticalis CASENT0106107 EF013060 EF013768 EF013188 EF013640 EF013507 KU671664 KU671727 KU671790 KU671943 KU672020 KU671866

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