Convergent Evolution of the Army Ant Syndrome and Congruence in Big-Data Phylogenetics

bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.

Convergent evolution of the army ant syndrome and congruence in big-data phylogenetics

Marek L. Borowiec

Department of Entomology and Nematology, One Shields Avenue, University of California at Davis, Davis, California, 95616, USA Current address: School of Life Sciences, Social Insect Research Group, Arizona State University, Tempe, Arizona, 85287, USA E-mail: [email protected]

Abstract The evolution of the suite of morphological and behavioral adaptations underlying the ecological success of army ants has been the subject of considerable debate. This ”army ant syndrome” has been argued to have arisen once or multiple times within the ant subfamily Do- rylinae. To address this question I generated data from 2,166 loci and a comprehensive taxon sampling for a phylogenetic investigation. Most analyses show strong support for convergent evolution of the army ant syndrome in the Old and New World but certain relationships are sensitive to analytics. I examine the signal present in this data set and find that conflict is di- minished when only loci less likely to violate common phylogenetic model assumptions are considered. I also provide a temporal and spatial context for doryline evolution with time- calibrated, biogeographic, and diversification rate shift analyses. This study underscores the need for cautious analysis of phylogenomic data and calls for more efficient algorithms em- ploying better-fitting models of molecular evolution.

Key words: Dorylinae, phylogenomics, k-means, partitioning Significance Recent interpretation of army ant evolution holds that army ant behavior and morphology originated only once within the subfamily Dorylinae. An inspection of phylogenetic signal in a large new data set shows that support for this hypothesis may be driven by bias present in the data. Convergent evolution of the army ant syndrome is consistently supported when sequences violating assumptions of a commonly used model of sequence evolution are excluded from the analysis. This hypothesis also fits with a simple scenario of doryline biogeography. These results highlight the importance of careful evaluation of signal and conflict within phylogenomic data sets, even when taxon sampling is comprehensive.

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Introduction

Army ants (Figure 1) are a charismatic group of organisms that inspire research in such disparate fields as behavioral ecology (Schöning et al., 2005), biodiversity conservation (Peters et al., 2008), and computational biology (Garnier et al., 2013). These ants are distributed throughout warm tem- perate and tropical regions of the world and belong to a more inclusive clade known as the subfamily Dorylinae (Brady et al., 2014). They are characterized by a suite of morphological and behavioral adaptations, together dubbed the army ant syndrome (Brady, 2003). This syndrome includes obligate collective foraging, frequent colony relocation, and highly specialized wingless queens. In contrast to many other ant species, army ants never forage individually. Nests of army ants are temporary and in some species colonies undergo cycles of stationary and nomadic phases (Schneirla, 1945). Unable to fly, the queens must disperse on foot and are adapted to producing enormous quantities of brood (Raignier et al., 1955). Other peculiarities of army ants include colony repro- duction by fission and highly derived male morphology. No army ant species are known to lack any of the components of the syndrome (Gotwald, 1995). Because of its antiquity and persistence, the army ant syndrome has been cited as an example of remarkable long-term evolutionary stasis (Brady, 2003). Several distantly related lineages of ants evolved one or more of the components of the army ant syndrome but did not reach the degree of social complexity or ecological dominance of the ”true army ants” in the Dorylinae (Kronauer, 2009). A major question of army ant biology is whether the army ant syndrome originated only once or if it arose independently in New World and Old World army ants (Kronauer, 2009). The an- swer requires a robust phylogenetic framework, but Dorylinae are an example of an ancient rapid radiation and elucidating its phylogeny has proven difficult (Brady et al., 2014). Before the ad- vent of quantitative phylogenetic methods, the army ants were thought to have arisen more than once within the subfamily (Brown, 1975; Gotwald, 1979; Bolton, 1990). This view was based on the observation that army ants are poor at dispersal and on the assumption that they evolved after the breakup of Gondwana around 100 Ma ago. More recent studies (Baroni Urbani et al., 1992; Brady, 2003), however, reported monophyly of army ant lineages, even though statistical support for this grouping was often low (Brady et al., 2014). Furthermore, the most recent divergence time estimates suggest that army ants indeed originated after the breakup of Gondwana, implying either independent origins or long-distance dispersal (Kronauer, 2009). Here I reassess the phylogeny of the Dorylinae including New World and Old World army ants. I use a large phylogenomic data set and taxon sampling increased twofold compared to a previous phylogeny (Brady et al., 2014), including 155 taxa representing more than 22% of described doryline species diversity and all 27 currently recognized extant genera (Borowiec, 2016b). The sequence data comes from a total of 2,166 loci centered around Ultraconserved Elements or UCEs (Faircloth et al., 2012; Branstetter et al., 2017) distributed throughout the ant genomes, comprising 892,761 nucleotide sites with only 15% of missing data and gaps. Analyses of the complete data set and loci with high average bootstrap support or ”high phylogenetic signal” (Salichos and Rokas, 2013) produce results that are sensitive to partitioning scheme and inference method. Analysis of data less prone to systematic bias, such as slow-evolving (Rodríguez-Ezpeleta et al., 2007; Betancur-R. et al., 2013; Goremykin et al., 2015) or compositionally homogeneous loci (Jermiin et al., 2004) and amino acid sequences (Hasegawa and Hashimoto, 1993), consistently support the hypothesis of independent origins of the army ant syndrome in the Old and New World.

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A B

C D

Figure 1: Striking morphological similarity of Old World (left) and New World (right) army ants. A: Aenictus male, B: Neivamyrmex male, C: Dorylus soldier, D: Labidus soldier. Photographs courtesy of Alex Wild/alexanderwild.com.

Results

Relationships Among Doryline Lineages Inferred From Combined Data Matrix The concatenated dataset produces different topologies depending on the partitioning scheme and method used to infer the phylogeny (Figure 2A–D, P). The true army ants are not monophyletic in the maximum likelihood tree under partitioning by locus. The tree shows a clade that includes almost all New World dorylines, including New World army ants (hereafter called ”New World Clade”; Figure 2A; Supplementary Figure 1). The latter include the genera Cheliomyrmex, Eciton, Labidus, Neivamyrmex, and Nomamyrmex. Apart from New World army ants, the New World Clade unites all exclusively New World lineages of the Dorylinae and includes Acanthostichus, Cylindromyrmex, Leptanilloides, Neocerapachys, and Sphinctomyrmex. Old World driver ants Dorylus and the poorly known genus Aenictogiton are sister to Aenictus. Other well-supported clades comprising multiple genera include a clade of South-East Asian and Malagasy lineages Cerapachys, Chrysapace, and Yunodorylus, a well-resolved clade of several Old World genera (Lioponera, Lividopone, Parasyscia, Zasphinctus) that was also recovered in (Brady et al., 2014). Another South-East Asian clade consists of the mostly subterranean genera Eusphinctus, Ooceraea, and Syscia. The backbone of the tree shows multiple short internodes and generally lower resolution, consistent with a previous study (Brady et al., 2014). The maximum likelihood tree inferred from the same data set but using k-means partitioning (Frandsen et al., 2015) shows a strongly- supported Old World Aenictus and New World army ants clade (Figure 2B; Supplementary Figure 2). Army ants are not monophyletic in this tree either, because Dorylus and Aenictogiton are not sister to the Aenictus plus New World army ants clade. Unpartitioned analysis results in a similar topology (Figure 2C; Supplementary Figure 3). Coalescent species tree inferred from all loci is similar to the concatenation results with respect

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to poor resolution at deep nodes (Figure 2D; Supplementary Figure 4). The species tree resembles the unpartitioned and k-means partitioned concatenated trees because of moderate support for the Aenictus plus New World army ants clade, and contrast most with both concatenated trees by sup- porting army ant monophyly, the hypothesis favored by previous studies (Brady, 2003; Brady and Ward, 2005; Brady et al., 2014). Overall support for the deep nodes is lower in species trees and this approach does not recover some of the clades present across all concatenated analyses, namely the New World or South-East Asian/Malagasy groups mentioned above.

Evidence for Bias in Maximum Likelihood Tree Based on All and ”High Signal” Loci The conflict among ML topologies derived from different partitioning schemes and between concatenated and species tree warranted further investigation. I constructed five data sets in addition to the combined data matrix: matrix of ”high signal” loci (Salichos and Rokas, 2013) equivalent in length to 1/5 of the sites in combined data set matrix, matrix composed of slow-evolving loci equivalent in length to 1/5 of the sites in combined data set matrix, matrix composed of only compositionally homogeneous loci, and matrix that excluded the long-branched Aenictus species but is otherwise identical to combined data matrix (See Supplementary Table 2 for more information on these matrices). Additionally, I developed a workflow to extract putatively protein-coding regions from the UCE data set (see Extended Methods) and analyzed those regions separately, coded as amino acids. Analyses of loci whose trees have highest mean bootstrap support (high phylogenetic signal sensu Salichos and Rokas (2013)) shows even more discordance among analyses than that of the combined data set (Figure 2E–H; Supplementary Figures 5–8). Army ant lineages show different topologies in each of the three used partitioning schemes. Using only the slowest evolving one-fifth of the data (”slow-evolving” hereafter), there is universal support for a clade that unites all exclusively New World lineages, including the New World army ants under all partitioning schemes (Figure 2I–K; Supplementary Figures 9–11). Species tree analyses of slow-evolving loci also support New World Clade and the Aenictus plus (Aenictogi- ton+Dorylus) clade (Figure 2L; Supplementary Figure 12). Examining locus properties in different rate bins reveals that more rapidly evolving sequences and ”high signal” loci exhibit qualities previously associated with systematic bias, namely higher compositional heterogeneity (Lockhart et al., 1994; Jermiin et al., 2004) and saturation (Philippe and Forterre, 1999). The most slowly evolving loci have lower overall among-taxon sequence heterogeneity than more rapidly evolving and high bootstrap loci (Supplementary Figure 28A; RCFV two-sample t-test slow-evolving vs. all other loci: t = 28.564, slow-evolving mean = 0.0358, all other loci mean = 0.0575, p ≪ 0.01; slow-evolving vs. high bootstrap: t = 27.538 high bootstrap mean = 0.0647, p ≪ 0.01). Saturation is also greater in more rapidly evolving and high bootstrap loci ((Supplementary Figure 28B; slope of regression two-sample t-test slow-evolving vs. all other loci: t = 21.682, slow-evolving mean = 0.474, all other loci mean = 0.361, p ≪ 0.01; slow- evolving vs. high bootstrap: t = 10.129, high bootstrap mean = 0.393, p ≪ 0.01). Of the 379 loci that pass the homogeneity test (Foster, 2004), 244 are found among slow-evolving loci and zero are present among the high bootstrap loci. Analysis of loci that pass the phylogeny-corrected compositional homogeneity test (Foster, 2004) shows universal support for the New World Clade, regardless of partitioning scheme (Fig- ure 2M; Supplementary Figure 13–15). Among-taxon compositional heterogeneity is a relatively

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A B C D ML combined data ML combined data ML combined data ASTRAL all supergenes partitioned by locus k-means partitions unpartitioned E E E * E+N * * A A A

D D * D D A N N N E F G H ML high bootstrap ML high bootstrap ML high bootstrap ASTRAL high bootstrap partitioned by locus k-means partitions unpartitioned supergenes

E E A E * * A A E A * *

D D D D

* N * N N N I J K ML slow-evolving ML slow-evolving ML slow-evolving L ASTRAL slow-evolving partitioned by locus k-means partitions unpartitioned supergenes

E+N E+N E+N E+N

* D D D D * A A A A M N ML homogeneous loci ML amino acids O ML combined data P Bayesian combined data all partitioning schemes partitioned by locus k-means partitions k-means partitions no Aenictus

E+N * E+N E+N * E+N

D D D * * D A A A

Figure 2: Summary of phylogenetic position of army ant lineages recovered in different analyses. In each figure, letter A signifies Aenictus, D: Aenictogiton+Dorylus, E: New World army ants (Cheliomytmex, Eciton, Labidus, Neivamyrmex, Nomamyrmex), N: all other New World dorylines (Acanthostichus, Cylindromyrmex, Leptanilloides, Neocerapachys, Sphinctomyrmex) except Syscia. Note increased incongruence in analysis of combined data set (A–D) and ”high signal” data set (E–H) compared to slow-evolving (I–L) and compositionally homogeneous (M) loci. Asterisk indicates support ≤ 99%. Dashed lines signify polyphyly. See Supplementary Figures 1–18 for complete trees.

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well-understood source of bias in phylogenetic analysis (Jermiin et al., 2004) and is not accounted for by the general time-reversible model of sequence evolution used in RAxML, the program used here for maximum likelihood inference. Amino acid alignments are also known to be more robust against compositional bias (Hasegawa and Hashimoto, 1993) and saturation, although not free from it when distantly related lineages are considered (Foster and Hickey, 1999). In this study the amino acid matrix of protein-coding sequences is highly conserved compared to the nucleotide matrix of combined data with 14% proportion of parsimony informative sites in the former compared to 48% in the latter. The amino acid matrix analyzed under maximum likelihood recovers monophyletic New World Clade and Old World army ants (Figure 2N; Supplementary Figure 16). More evidence for the artefactual nature of the grouping of Old World and New World army ants comes from an analysis where Aenictus is removed from the combined data matrix partitioned under the k-means scheme. If Aenictus was not affecting the position of New World army ants on the tree, one would expect to see no change of the position of the latter if the former is removed. This is not the case here; the phylogeny recovered from the alignment without Aenictus has New World army ants nested within the New World Clade (Figure 2O; Supplementary Figure 17). Bayesian analysis of k-means partitioned combined data matrix strongly supports the New World Clade and monophyly of Aenictogiton+Dorylus, unlike the ML analysis of the same dataset under the same partitioning scheme and sequence evolution model (Figure 2P; Supplementary Fig- ure 18). In summary, although concatenated ML and species tree analysis of combined data matrix and high-bootstrap loci support grouping of Old World army ants Aenictus with New World army ants under some conditions, analyses using more reliable data are congruent in their support for independent origins of the army ant syndrome in New World and Old World.

The Timeline of Doryline Evolution and Diversification Fossilized birth-death (FBD) process divergence dating (Heath et al., 2014) employed here shows that crown doryline ants started diversifying in the Cretaceous, around 74 Ma (53–101 Ma 95% highest probability density interval or HPD) ago according to Bayesian inference (97 Ma under penalized likelihood or PL; Supplementary Figure 19) (Figure 3). The FBD results near the root are characterized by high uncertainty but the mean age suggests a younger crown age than 87 Ma recovered in a previous study (Brady et al., 2014). The difference is likely at least in part due to a different calibration approach used in the present study (FBD versus node dating) and a revised, younger age estimate of Baltic amber (Aleksandrova and Zaporozhets, 2008). Similar to concatenation, divergence dating shows a tree highly compressed during early evolution, with 16 splits occurring within the first 20 Ma. According to penalized likelihood this period of early diversification lasted longer, accounting for about 35 Ma. The most recent common ancestor of the New World Clade is resolved at 60 Ma (43–83 Ma 95% HPD interval; 60 Ma under PL). The split of Old World army ant lineages of Aenictus and Dorylus+Aenictogiton is apparently very ancient at 58 Ma (41–79 Ma 95% HPD; 54 Ma under PL) and occurred during the initial diversification period. The old age of this node and long branch subtending extant Aenictus help explain why this relationship is difficult to recover. The five currently recognized genera of New World army ants share an ancestor at about 28 Ma (20–37 Ma 95% HPD; 27 Ma under PL) ago. These dates are younger than those inferred for several of the non-army ant doryline genera such as Eburopone or

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Neivamyrmex texanus M274 Neivamyrmex californicus M272

Neivamyrmex kiowapache M275 NW ARMY AN T Neivamyrmex opacithorax M276 Neotropical Neivamyrmex sumichrasti M151 Neivamyrmex asper M233 Neivamyrmex compressinodis M23 Nearctic 13.4 Neivamyrmex cf nyensis M296 Neivamyrmex melanocephalus M15 Palearctic Neivamyrmex impudens M237 Neivamyrmex gibbatus M139 Neivamyrmex distans M242 Afrotropical Neivamyrmex alfaroi M240 Neivamyrmex adnepos M236 Malagasy Neivamyrmex EC M175 Neivamyrmex swainsonii M169 Eciton rapax M145 Indomalayan Eciton hamatum M293 Eciton burchellii M273 Australasian 27.5 Eciton lucanoides M239 Eciton vagans M184 Eciton quadriglume M254 Eciton mexicanum M238 Nomamyrmex hartigii M220 Nomamyrmex esenbecki M129

Labidus spininodis M172 S Labidus praedator M152 Labidus coecus M173 Cheliomyrmex cf morosus M217 Cheliomyrmex cf andicola M174 Cheliomyrmex PE M146 Leptanilloides gracilis M187 Leptanilloides erinys M246 Leptanilloides femoralis M188 Leptanilloides nubecula M167 Leptanilloides mckennae D0228 Sphinctomyrmex stali M253 Sphinctomyrmex marcoyi M202 Acanthostichus serratulus M166 Acanthostichus GF M208 60.4 Acanthostichus BR M252 Acanthostichus AZ M278 Acanthostichus AZ M277 Cylindromyrmex whymperi M271 Cylindromyrmex darlingtoni M21 Cylindromyrmex brasiliensis M1 Cylindromyrmex meinerti D778 Neocerapachys cf splendens M25 Neocerapachys BR M295 Neocerapachys neotropicus M134 Neocerapachys CR M209 Aenictus hoelldobleri M200 Aenictus fuchuanensis M201 Aenictus turneri M135 Aenictus gracilis M199 Aenictus cornutus M230 OW ARMY AN T Aenictus currax M245 Aenictus rotundicollis M232 Aenictus laeviceps M269 Aenictus hodgsoni M198 Aenictus fergusoni M223 Aenictus ZA M228 Aenictus UG M143 Aenictus camposi M222 Aenictus levior M231 23.3 Aenictus bobaiensis M197 Aenictus latifemoratus M266 Aenictus yamanei M265 Aenictus silvestrii M221 Dorylus rubellus M287 Dorylus UG M153 Dorylus molestus M290 Dorylus mayri M291 Dorylus braunsi M289 57.1 Dorylus affinis M177 Dorylus kohli M180 Dorylus spininodis M292 Dorylus fulvus M179

Dorylus cf fulvus M149 S 15.6 Dorylus fimbriatus M288 Dorylus orientalis M244 Dorylus conradti M178 Dorylus laevigatus M157 Aenictogiton ZM02 M181 Aenictogiton UG M189 Yunodorylus eguchii M247 Yunodorylus TH M191 Yunodorylus paradoxus M190 Yunodorylus TH M160 Chrysapace cf crawleyi M262 Chrysapace TH M156 Chrysapace cf sauteri M261 Chrysapace MG M155 Cerapachys antennatus M263 Cerapachys TH M203 Cerapachys MY M264 Cerapachys sulcinodis M243 Eburopone MG M214 Eburopone MG M213 Eburopone MG M195 Eburopone MG M130 Eburopone UG M259 Eburopone CM02 D0788 Ooceraea PG M248 Ooceraea FJ06 M168 Ooceraea MY M270 Ooceraea biroi Genome Ooceraea australis M138 Ooceraea fragosa D0842 Syscia MY M176 Syscia MY M147 Syscia typhla D0841 Syscia augustae M196 Syscia GT M127 Eusphinctus TH M158 Simopone trita M185 Simopone rex M133 Simopone marleyi M171 Simopone conradti M144 Simopone dryas M224 Simopone grandidieri M186 Simopone cf oculata D0792 Tanipone hirsuta M279 Tanipone aglandula M280 Tanipone zona M182 Tanipone scelesta M159 Vicinopone conciliatrix M128 Parasyscia wittmeri M282 Parasyscia UG M218 Parasyscia UG M219 Parasyscia dohertyi M142 Parasyscia PG M204 Parasyscia VN M207 73.8 Parasyscia MG M212 Parasyscia UG M281 Zasphinctus imbecilis M170 Zasphinctus PG M285 Zasphinctus trux M136 Zasphinctus KE M227 Zasphinctus TH M192 Zasphinctus MZ M229 Lividopone MG M216 Lividopone MG M194 Lividopone MG M294 Lividopone MG M132 Lioponera ruficornis M165 Lioponera princeps M137 Lioponera marginata M249 Lioponera PG M250 Lioponera cf suscitata M267 Lioponera MY M268 Lioponera nr mayri M215 Lioponera nr kraepelinii M131 Lioponera vespula M210 Lioponera longitarsus M193

80 60 40 20 0 Ma Figure 3: Timeline of doryline evolution and biogeographic history. The highest relative probabilities of ancestral ranges are shown as inferred using BioGeoBEARS under DEC+J, averaged over 100 trees from the BEAST posterior. The tree is the BEAST consensus. Selected mean divergence time estimates are given at nodes. Stars signify rate shifts. All dates in Ma. See Supplementary Figures 20, 21, and 26 for average node dates, posterior probabilities from the BEAST analysis, and pie charts of relative probabilities of all possible ancestral ranges, respectively.

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Leptanilloides. The conspicuous above-ground foraging seen in some Dorylus driver ants and in New World Eciton is remarkably young, as it appears that these groups diversified within the last 5–6 Ma, a result robust across different dating analyses (see Supplementary Figures 19-21). The driver ant genus Dorylus is shown to be young relative to earlier analyses (Brady, 2003; Kronauer et al., 2007) at ca. 16 Ma (11–22 95% HPD; 15 Ma under PL). The Bayesian divergence time estimates, especially those early in the tree and outside the New World Clade, are associated with considerable uncertainty. This is likely due to both topological uncertainty and the fact that only seven fossil calibrations were available for the dorylines, all except one located within the New World Clade (Supplementary Table 4; Supplementary Figure 20). Diversification shift analyses in BAMM (Rabosky, 2014) identified a three shift-scenario for dorylines, with shifts occurring separately on the branches subtending Aenictus, Dorylus, and Neivamyrmex (Figure 3). Rate shift configurations in which only two shifts were identified, however, were also common in the posterior sampling (Supplementary Figure 25), either on branches leading to Aenictus and Neivamyrmex only, or on the branches subtending the clade of Aenictus, Aenictogiton, and Dorylus and Neivamyrmex (see Supplementary Figures 22-25).

Biogeographic History Strong geographic affinities are apparent within the doryline phylogeny. Large clades are mostly confined to only one or two adjacent realms, although movement within both the Old and New World appears to have been common (Figure 3). There is strong evidence for Old World origins of the dorylines and the analyses summarized across a sample of trees from the posterior suggest an Afrotropical ancestral range (Figure 3; Supplementary Figure 26), although the analysis under BEAST consensus only results in high uncertainty and combined Afrotropical-Malagasy-Oriental as the most likely ancestral range (Supplementary Figure 27). Two lineages are confined to the New World. One gave rise to the radiation of almost all extant New World forms, including New World army ants and their kin. The dates estimated for the origin of this New World Clade coin- cide with warm climatic conditions and multiple land bridges connecting the Old and New World in northern latitudes (Brikiatis, 2014). The other New World group is much younger and appeared to arrive some time after 28 Ma ago. The presumably SE Asian or Palearctic ancestor of the New World species of Syscia either crossed the Beringian land bridge or dispersed across the Pacific further south, since by that time North Atlantic connections were closed (Sanmartín et al., 2001). The Old World Aenictus and New World genus Neivamyrmex, although superficially similar in ap- pearance and biology, illustrate different scenarios of biogeographic history for generic lineages within Dorylinae. Crown Aenictus is older at 23 Ma (34 Ma under PL) and originated in the Indo- malayan region. It then subsequently moved into the Afrotropics, dispersed back into Indomalaya, and moved into Australasia with possible movement back into Indomalaya. Some species also range into the Palearctic. In contrast, Neivamyrmex remained largely confined to the Neotropics where it originated around 13 Ma (20 Ma under PL) ago, with at least one clade moving into and diversifying in the southern Nearctic and with some species returning to the Neotropics or strad- dling the boundary of the two adjacent regions. Madagascar is a center of doryline diversity with seven overall and two endemic genera but no true army ants (Figure 3).

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Concluding Remarks

Doryline Biology and Evolution Need Further Study The new phylogeny presented here reveals that the army ant syndrome can be viewed as both an example of long-term evolutionary stasis and a remarkable case of convergent evolution. Brady (2003) argued that the army ant syndrome originated only once around 100 Ma ago and has since persisted without loss in any descendant lineages. While the present study suggests that this set of behavioral and morphological traits evolved at least twice in the Dorylinae, it also shows that the syndrome has been conserved within Old and New World army ants. The alternative scenario of single origin requires multiple losses on lineages leading to both Old and New World army ants (Figure 3), an unlikely proposition given that no species are known to have lost any of the syndrome components in the large and diverse genera such as Aenictus, Dorylus, or Neivamyrmex. Despite the improved resolution of the army ant tree, much work remains to be done with the regard to doryline evolution. A particularly vexing matter is poor knowledge of the Afrotropical genus Aenictogiton. Although based on male morphology and its phylogenetic affinity to Dory- lus it has been assumed that it is a subterranean army ant, there is no direct evidence of army ant behavior or queen morphology (Borowiec, 2016b). If Aenictogiton is not an army ant, our views on the evolution of the army ant syndrome have to be adjusted. In general, the current knowledge of doryline ecology and behavior is mostly limited to the minority of species that are conspicuous above-ground foragers, although the clonal Ooceraea biroi is a notable exception (Tsuji and Yamauchi, 1995; Ravary and Jaisson, 2002; Oxley et al., 2014). Better understanding of doryline behavior and morphology will undoubtedly yield further insights into the evolution of the army ant syndrome. For example, independent evolution of army ants is perhaps less surprising given that certain components of the syndrome (e.g., derived queen and male morphologies) appear multiple times within the subfamily. Unfortunately, too little data exists for a rigorous study of these trends in a comparative framework (Borowiec, 2016b). Early anatomical research implied army ant polyphyly by emphasizing differences in sting and Dufour gland morphologies (Hermann, 1969; Billen, 1985; Billen and Gotwald, 1988) between the Old and New World army ants. The new phylogenetic and taxonomic (Borowiec, 2016b) framework should reinvigorate comparative work on doryline morphology and biology.

Densely Sampled Phylogenomic Data Sets Are Not Robust to Artefacts and Bias Genome-scale data offers powerful tools for reconstructing phylogenies. This study, however, demonstrates that caution is necessary when evaluating hypotheses generated by these new data sets, even when taxon sampling is comprehensive. Empirical studies that emphasize the importance of bias often recommend improving taxon sampling but usually also deal with cases where sampling could be increased relative to first attempts, such as in broad-scale phylogenies of eukary- otes or Metazoa (Delsuc et al., 2005). The doryline phylogeny represents a case where the scope for improvement by additional sampling of currently known lineages is limited. This is because adding more species is likely to fail at significantly shortening long branches in the tree (Borowiec, 2016b). Researchers should be thus wary of systematic bias whenever combination of very short and very long branches is encountered, regardless of taxon sampling. Two general strategies are available for exploring sensitivity to model mis-specifications: using only data less prone to bias or applying better-fitting heterogeneous models (Rodríguez-Ezpeleta et al., 2007). The latter ap-

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proach is ideal but computationally expensive, becoming prohibitive with data sets such as the one used here, including over 150 taxa and thousands of loci. In this study, reduction of phylogenetic noise resulting from compositional heterogeneity and saturation increased congruence among topologies obtained using different analytics. In the case of the complete data set, mutually exclusive and strongly supported results were be obtained depending on the statistical framework (e.g., maximum likelihood vs. Bayesian) or sequence evolution model (e.g., k-means partitioning vs. partitioning by locus) chosen for analysis. Using only ”high signal” loci (Salichos and Rokas, 2013) with highest average bootstrap exacerbated incongruence among analyses. These loci also exhibited undesirable properties such as higher potential for saturation and violation of the among-taxon compositional heterogeneity. This indicates that using additional measures of data quality is needed in phylogenomics, such as direct and indirect measures of model mis-specification (Brown, 2014). Other recent research suggests that analysis results can be strongly affected by a tiny proportion of highly biased loci or sites (Shen et al., 2017). In conclusion, phylogenomic studies should always perform sensitivity analyses to test the robustness of the result to different analytics.

Acknowledgments

I would like to thank Phil Ward for guidance, support, and valuable conversations related to this project. Many thanks to Michael Branstetter who allowed me to work with an unpublished probe set and provided wet lab protocol training. Alex Wild, Corrie Moreau, and Daniel Kronauer gave important feedback during early stages of conceiving this study. Alex also provided photographs of live army ants used in Figure 1. Brian Johnson and Christian Rabeling kindly gave access to computing resources. Karl Kjer and Kimiora Ward provided comments that helped to improve this manuscript. Thank you to everyone who contributed specimens for sequencing or helped with field work: Fidèle Bemaeva, Brendon Boudinot, Júlio Chaul, Katsuyuki Eguchi, Flávia Esteves, Rodrigo Feitosa, Brian Fisher, Paco Hita-Garcia, Milan Janda, Jack Longino, Andrea Lucky, Sean McKenzie, Matt Prebus, Andry Rakotomalala, Caspar Schöning, Michael Staab, Andy Suarez, and Phil Ward. This research was funded by NSF Doctoral Dissertation Improvement Grant 1402432, a Young Explorers Grant from National Geographic, Microsoft Azure Research Award, Henry A. Jastro awards from the Department of Entomology and Nematology at UC Davis, and facilitated by Ernst Mayr Travel Grants in Animal Systematics and SYNTHESYS awards FR-TAF-594 and GB-TAF-303.

Methods

Taxon Sampling and Data Generation For a detailed description of methods and additional references see Supplementary Materials. I chose doryline species for the analyses based on a recent generic revision of the subfamily (Borowiec, 2016b) and other recent taxonomic and phylogenetic work. I maximized the breadth of sampling by including at least one representative from each biogeographic region in which a genus occurs and aiming to sample across mor- phologically disparate groups within genera. I extracted the genomic DNA, prepared libraries and enriched them with molecular probes designed as described in (Faircloth et al., 2014), targeting 2,524 loci (Branstetter et al., 2017). Dual-indexed (Faircloth and Glenn, 2012), enriched sequences were sequenced on two lanes

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of Illumina HiSeq 2500 platform. I cleaned demultiplexed reads using Illumiprocessor (Faircloth, 2011) and performed assembly with Trinity (Grabherr et al., 2011). I then mapped resulting contigs to probe sequences and mined published genomes for outgroup and one ingroup sequence using the Phyluce pipeline (Faircloth, 2015), aligned orthologous sequences with Upp (Nguyen et al., 2015), and trimmed using Gblocks (Talav- era and Castresana, 2007) with stringency settings relaxed from the default. I discarded any sequences with fewer than 114 taxa (70%) from further analyses. This resulted in 2,166 orthologous loci, 412 bases long on average. Extraction of protein-coding sequences was done by blasting (Camacho et al., 2009) UCE loci against published proteins of three reference ant species (Acromyrmex echinatior, Harpegnathos saltator, and Oocer- aea biroi), followed by collecting of protein queries and their nucleotide equivalents from best BLASTX hits for each locus using a custom bioinformatics pipeline. Separate alignment and trimming of resulting data set to include no fewer than 70% of all taxa produced an amino acid matrix of 1,103 loci 89.5 k amino acids long.

Phylogenetics I first estimated a gene tree for each locus using RAxML (Stamatakis, 2014) under GTR+4Γ model with 200 rapid bootstrap replicates. I then used individual locus characteristics to assess whether loci with different properties produce different phylogenies. Most basic statistics, such as alignment length, proportion of variable sites, and missing data were calculated using AMAS (Borowiec, 2016a). Using a custom R script I computed average branch lengths and average bootstrap support, used here as a proxy for rate of evolution and phylogenetic signal, respectively. With a custom Python script I calculated RCFV (Zhong et al., 2011), a measure of compositional heterogeneity, and performed a simulation-based compositional heterogeneity test with the Python p4 phylogenetics toolkit (Foster, 2004). Following this, I constructed a concatenated matrix of 271 loci with highest average bootstrap support, equal in length to 1/5 sites present in the combined data set. I also prepared a matrix of 379 compositionally homogeneous loci only and an alignment of identical to the combined data matrix but with Aenictus removed. I also constructed an amino acid matrix from extracted protein-coding sequences. For each of the concatenated nucleotide UCE data sets I used three partitioning schemes: 1) partitioning by UCE locus, 2) partitioning using the k-means algorithm (Frandsen et al., 2015) implemented in PartitionFinder2 (Lanfear et al., 2017), and 3) an unpartitioned model. The protein-coding sequences were analyzed as partitioned by locus. I estimated the phylogeny with RAxML under the partitioned GTR+4Γ model and 500 rapid bootstrap replicates (see Extended Methods) for all nucleotide matrices except for combined data partitioned by locus, which was analyzed with 100 bootstrap replicates. The amino acid matrix was analyzed under JTT+4Γ model and 100 bootstrap replicates. In addition to maximum likelihood analysis on all matrices, I also ran Bayesian analysis using ExaBayes (Aberer et al., 2014) on combined dataset. Consistent with the recent criticism of the k-means algorithm (Baca et al., 2017), this partitioning scheme appears to result in incorrect topology under ML analysis of the combined data and ”high signal” loci matrices but not in analyses of slow-evolving or compositionally homogeneous loci or the Bayesian analysis of combined data. The concatenated analyses were done on CIPRES (Miller et al., 2010) and on the University of Rochester Center for Integrated Research Computing BlueHive computer cluster. I also used the statistical binning pipeline (Bayzid et al., 2015) and ASTRAL (Mirarab and Warnow, 2015) to estimate summary species trees on the combined data matrix, ”high signal”, and slow-evolving loci.

Divergence Time Estimation I employed two different strategies to estimate the time-calibrated tree. The first approach used penalized likelihood and the fixed tree topology and branch length from the slow-evolving data. I used penalized

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likelihood as implemented in the chronos function of the R package ape (Paradis, 2013). I used an outgroup- rooted tree from the concatenated analysis of slow-evolving loci and included calibrations for nodes in the outgroup. Hard bounds were placed on node calibrations using fossil ages for the upper bound and previous estimates for node ages as lower bounds (For more details see Supplementary Table 3). The strict molecular clock could not be rejected for this tree, and I performed the analysis for 100 replicates in order to assess robustness to random starting values of the algorithm. The second approach utilized Bayesian inference with only ingroup taxa included, with several clades constrained as monophyletic and the root position and backbone relationships of the tree sampled from the posterior. Because Bayesian divergence time estimation is computationally very expensive, that approach was limited to 109 loci evenly distributed across the spectrum of rate of evolution. I used BEAST2 (Bouckaert et al., 2014) under the unpartitioned GTR+4Γ model, uncorrelated molecular clock with branch lengths drawn from a lognormal distribution, and fossilized birth- death process (Heath et al., 2014) conditioning on the root to obtain divergence time estimates and a posterior sample of trees for biogeographic analyses. Calibrations included seven doryline fossils (see Supplementary Table 4 for more details): A Neivamyrmex sp. from Chiapas Amber, five species from Dominican Amber (Acanthosticus hispaniolicus, Cylindromyrmex inopinatus, C. electrinus, C. antillanus, and Neivamyrmex ectopus), and Chrysapace sp. from Baltic Amber. Another Baltic Amber fossil, Procerapachys spp. was not used because of uncertainty in its placement among crown or stem dorylines. I ran four independent MCMC chains for over 250 million generations until convergence was reached. Combined effective sample size (ESS) for all parameters was above 200.

Diversification Analyses To assess diversification rate shifts on the doryline phylogeny I used BAMM v2.5 (Rabosky, 2014). For input I used the consensus tree from BEAST analyses and sampling probabilities based on extant species diversity estimates (Borowiec, 2016b) for each genus in order to correct for uneven sampling of extant taxa across the phylogeny. I ran the MCMC for 20 million generations, sampling every 2,000 generations.

Biogeographic History Estimation I used the R package BioGeoBEARS (Matzke, 2013, 2014) for model selection and estimation of biogeographic history of the dorylines. As input I used the consensus tree from BEAST runs and a sample of 100 trees from the posterior of the same analysis. I compared six models commonly used for biogeographic inference on the consensus tree, with the DEC+J model emerging as the best-fitting. I then estimated biogeographic history on the 100 trees under this model to account for uncertainty in the deeper relationships and the timeline of early divergences. These results were then summarized on the consensus tree.

Extended Methods

Data availability Trimmed reads generated for this study are available at the NCBI Sequence Read Archive (to be submitted upon acceptance for publication). Sequence files, alignments, configuration files, and output of analyses, including phylogenetic trees, are available on Zenodo: https://doi.org/10.5281/zenodo.569071. Cus- tom scripts used in this study are available on GitHub: https://doi.org/10.5281/zenodo.571246. Pipeline for extracting protein-coding sequences is available in a separate GitHub repository at https: //doi.org/10.5281/zenodo.571247.

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Taxon sampling Taxon sampling included 154 newly sequenced ingroup species from all 27 currently recognized genera of Dorylinae ants and was guided by previous taxonomic and phylogenetic studies (De Andrade, 1998a; Borgmeier, 1955; Borowiec and Longino, 2011; Brady, 2003; Brady and Ward, 2005; Brady et al., 2014; Jaitrong and Yamane, 2011; MacKay, 1996; Bolton and Fisher, 2012) and expertise acquired preparing the global genus-level taxonomic revision of the group (Borowiec, 2016b). In addition to sampling all doryline genera, species from all biogeographic regions (as defined in (Cox, 2001) but treating the Malagasy region separate from Afrotropical) were included for each major lineage. Nine outgroup and one ingroup species (Ooceraea biroi) were also included based on publicly available ant genomes: Atta cephalotes (Suen et al., 2011), Camponotus foridanus (Bonasio et al., 2010), Cardiocondyla obscurior (Schrader et al., 2014), Harpegnathos saltator (Bonasio et al., 2010), Linepithema humile (Smith et al., 2011a), Ooceraea biroi (Ox- ley et al., 2014), Pogonomyrmex barbatus (Smith et al., 2011b), Solenopsis invicta (Wurm et al., 2011), and Vollenhovia emeryi (Smith et al., 2015).

Molecular data collection and sequencing I extracted DNA from all newly sequenced specimens (Supplementary Table 1) using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Most specimens were extracted non-destructively, with extraction voucher retained. For several extractions the DNA collection was done destructively and a voucher specimen from the same colony was kept. I quantified DNA for each extraction using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and sheared <5–50 ng of DNA to a target size of approximately 400–600 bp. The shearing was done by sonication on a Bioruptor machine (Diagenode Inc., Philadelphia, PA, USA). The library preparation protocol that follows was slightly modified from Blaimer et al. (2015). I used a KAPA Hyper Prep Library Kit (Kapa Biosystems, Inc., Wilmington, MA, USA) with magnetic bead cleanup (Fisher et al., 2011) and a SPRI substitute (Rohland and Reich, 2012) as described in (Faircloth et al., 2014). I used TruSeq adapters (Faircloth and Glenn, 2012) for ligation followed by PCR amplification of the library using a mix of HiFi HotStart polymerase reaction mix (Kapa Biosystems), Illumina TruSeq primers, and nuclease-free water. The following thermal cycler program was used for the PCR: 98 ºC for 45 s; 13 or 14 cycles of 98 ºC for 15 s, 65 ºC for 30 s, 72 ºC for 60 s, and final extension at 72 ºC for 5 m. After rehydrating in 23 µl pH 8 Elution Buffer (EB hereafter) and purifying reactions using 1.1–1.2× speedbeads, I pooled nine to eleven libraries at equimolar ratios for final concentrations of 132–212 n/µl. I enriched each pool with 9,446 custom-designed probes (MYcroarray, Inc.) targeting 2,524 UCE loci in Hymenoptera (Branstetter et al., 2017). I followed library enrichment procedures for the MYcroarray MYBaits kit (Blumenstiel et al., 2010), except I used a 0.1× of the standard MYBaits concentration, and added 0.7 µl of 500 µmol custom blocking oligos designed against the custom sequence tags. I ran the hybridization reaction for 24 h at 65 ºC, subsequently bound all pools to streptavidin beads (MyOne C1; Life Technologies), and washed bound libraries according to a standard target enrichment protocol (Blumenstiel et al., 2010). I used the with-bead approach for PCR recovery of enriched libraries as described in (Faircloth, 2015). I combined 15 µL of streptavidin bead-bound, enriched library with 25 µL HiFi HotStart Taq (Kapa Biosystems), 5 µL of Illumina TruSeq primer mix (5 µmol each) and 5 µL of nuclease-free water. Post- enrichment PCR had the following profile: 98 ºC for 45 s; 18 cycles of 98 ºC for 15 s, 60 ºC for 30 s, 72 ºC for 60 s; and a final extension of 72 ºC for 5 m. I purified resulting reactions using 1.1–1.2× speedbeads, and rehydrated the enriched pools in 22 µL EB. Following enrichment I quantified 2 µL of each pool using a Qubit fluorometer (broad range kit). I verified if the enrichment was successful by amplifying four UCE loci targeted by the probe set. I set up a relative qPCR by amplifying two replicates of 1 ng of enriched DNA from each pool for the four loci and

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comparing those results to two replicates of 1 ng unenriched DNA from each pool. I performed qPCR using a SYBR FAST qPCR kit (Kapa Biosystems) on CFX Connect Real-Time PCR Detection System (Bio-Rad). Following data collection, I calculated fold-enrichment values, assuming an efficiency of 1.78 and using the formula 1.78 × abs(enrichedCp − unenrichedCp). I then performed qPCR quantification by creating dilutions of each pool (1:200,000, 1:800,000, 1:1.000,000, 1:10.000,000) and assuming an average library fragment length of 600 bp. Based on the size-adjusted concentrations estimated by qPCR, I pooled libraries at equimolar concentrations. The pooled libraries were then subjected to further quality control on Bioanalyzer and sequenced using one full and one partial lane of a HiSeq 125 Cycle Paired-End Sequencing v4 run. QC and sequencing were performed at the University of Utah High Throughput Genomics Core Facility. Quality-trimmed sequence reads generated as part of this study are available from the NCBI Sequence Read Archive (to be submitted upon acceptance for publication).

Processing of UCE data Read cleaning, assembly, matching of contigs to probes, construction of the unaligned data set, and alignment trimming were done using the Phyluce pipeline scripts (Faircloth, 2015). I trimmed the FASTQ data using illumiprocessor, a wrapper around Trimmomatic (Bolger et al., 2014), with default settings (LEADING:5, TRAILING:15, SLIDINGWINDOW:4:15, MINLEN:40). Assemblies were done using Trin- ity v20140717 (Grabherr et al., 2011) with the phyluce_assembly_assemblo_trinity wrapper. The orthology assessment was then done by matching the assembled contigs to enrichment probe sequences with phyluce_assembly_match_contigs_to_probes (min_coverage=50, min_identity=80). This step generated a sqlite database which was then used to build FASTA files for the 2,524 orthologous loci with phyluce_assembly_get_match_counts, phyluce_assembly_get_fastas_from_match_counts, and phyluce_assembly_explode_get_fastas_file.

Extraction of protein-coding sequences For the purpose of extracting protein-coding data from the sequenced UCE loci I developed a custom bioinformatics workflow that consists of three major components: using NCBI BLASTX (Camacho et al., 2009) to match unaligned UCE sequences to reference proteins and 2) choosing the best hit for each sequence followed by 3) extraction of protein queries and their nucleotide equivalents from those hits. Using makeblastdb program of the BLAST package I prepared a database from three publicly available collections of protein sequences of Acromyrmex echinatior (Nygaard et al., 2011), Harpegnathos saltator (Bonasio et al., 2010), and Ooceraea biroi (Oxley et al., 2014). Using BLASTX against this database resulted in one BLAST output file per UCE locus, containing multiple matches (hits) for each UCE sequence (taxon). Each hit, in turn, may be composed of one or more ranges that correspond to protein fragments (exons). I used BLAST scores for those ranges to identify best hits for each taxon and UCE. This was done with custom Python code using Biopython’s (Cock et al., 2009) module for parsing BLAST XML output and the following logic: For each sequence (taxon) and hit within, both total and maximum scores are tallied. If a hit’s total score is equal to the maximum score of its ranges and it corresponds to the maximum score for the taxon, such hit is considered best and is kept. This means that this hit was composed of a single range and its score was not exceeded by any other hits, composed of one or multiple ranges. If a hit’s total score is higher than the maximum score of any one of its ranges, and that hit’s total score is the best hit score for a species, this hit is kept unless it contains overlapping ranges. Finally, if the total hit score is equal to its maximum score but not to the best hit score for a species, its total score is checked to see if it corresponds to the highest individual range score for a species. If this is true, the hit is kept. Such hit would be composed of single range and considered best even if hits with higher total scores but overlapping ranges are present. If composed of

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multiple ranges, a best hit is concatenated into a query in the order based on its coordinates and its presence on either forward or reverse strand. These protein queries are then matched to corresponding input nucleotide sequence or its reverse complement. If introns that do not change reading frame are present, translations of the query sequence may span across them. Because of this I performed additional trimming if long (4 sites or more) gaps in the subject protein sequence were found. All sites corresponding to those long gaps were trimmed from the protein query and its nucleotide equivalent. If at this point there is still a stop codon in the protein query, such record was discarded. For each record the resulting protein queries and their corresponding nucleotides were considered ready for downstream analyses.

Alignment and trimming Assembly and contig matching resulted in sequences of varying lengths across taxa, as evidenced by sum- maries produced with phyluce_assembly_get_fasta_lengths. Because of this I used UPP (Nguyen et al., 2015), a phylogeny-aware alignment tool designed to align fragmentary sequences to a backbone of longer sequences. Based on the performance (recovered final post-trimming alignment length) of different settings, for the backbone I chose the cutoff of 30% of the longest sequences present in each locus. Because the UPP version used did not have an option to specify a fixed number of longest sequences in the backbone, a locus-specific command was printed for each locus based on its fragment size distribution with UPP’s -M option set to longest sequence and threshold (option -T) calculated to encompass 30% of taxa. UPP was also set to filter sequences from the backbone if their branches were 5 times longer than the median for all backbone sequences (-l 5 argument): run_upp.py -s [input_alignment] -M [longest_sequence_length] -T [locus_threshold] -d [alignment_output_directory] -o [output] -p [temporary_output_directory] -l 5 These custom commands were printed with print_upp_command.py, a custom script utilizing code from phyluce_assembly_get_fasta_lengths. Although alignment trimming has been recently criticized (Tan et al., 2015), the untrimmed alignments contained on average more than 75% of gaps and missing data. Because of the substantial computational burden of gap-rich data analysis, I trimmed the alignments using Gblocks (Talavera and Castresana, 2007) under settings relaxed from the default (b1=0.5 b2=0.5 b3=12 b4=7). I calculated alignment statistics and manipulated the files using AMAS v0.98 (Borowiec, 2016a). All loci with fewer than 114 taxa (less than 70%) were discarded, resulting in 2,166 out of 2,524 loci for downstream analyses. These loci had on average 151.7 (92.5%) taxa, were 412.2 nucleotides long, and had 7.7% missing data (gaps). Due to computing time constraints, loci with protein-coding sequences extracted were aligned using MAFFT v7.300b with --leavegappyout setting turned on. These alignments were trimmed using Gblocks with settings as above and further trimmed for outlier taxa using a custom R script and AMAS, removing any ingroup sequences whose uncorrected p-distance was more than 3σ from the mean for a locus.

Partitioning PartitionFinder 2 (Lanfear et al., 2017) was used to partition concatenated alignments using the k-means clustering of sites based on evolutionary rates (Frandsen et al., 2015). A starting tree for model fit and site clustering algorithm was generated with RAxML Pthreads v8.2.3 (Stamatakis, 2014) using the fast tree search algorithm (-f E flag). Because of recent criticism of the k-means algirithm (Baca et al., 2017), I performed additional ML analyses for combined data set under unpartitioned and partitioned by locus schemes. Protein-coding sequences were analyzed as partitioned by locus. K-means tends to result in relatively low

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number of partitions and other partitioning by locus scheme was not computationally feasible for Bayesian inference.

Phylogenetic analyses using maximum likelihood For each locus I estimated a gene tree with RAxML Pthreads v8.2.3 under a general time-reversible model of sequence evolution with rate modeled using a gamma distribution discretized into four bins (GTR+4Γ model). 200 rapid bootstraps were followed by a thorough search of the maximum likelihood tree: raxml -T [no_cores] -m GTRGAMMA -f a -# 200 -p 12345 -x 12345 -s [input_alignment] -n [output_file_name] The same mode of inference was applied to supergenes created by the statistical binning pipeline (see Species Tree analyses below) but each supergene was partitioned by constituent loci using a partitions file (-q) and the -M flag was added for a fully partitioned analysis with branch lengths optimized separately for each partition (Bayzid et al., 2015): raxml -T [no_cores] -m GTRGAMMA -f a -# 200 -p 12345 -x 12345 -M -s [input_alignment] -q [partitions_file] -n [output_file_name] I ran the analyses of concatenated matrices with RAxML Hybrid v8.2.4 and v8.2.9 on CIPRES Portal (Miller et al., 2010) using the same model and bootstrap settings but with a pre-defined partitioning scheme (see above), no -M option due to the high number of partitions, and either 100 (amino acid analysis and combined data partitioned by locus) or 500 bootstrap replicates (all other analyses). The amino acid analysis was ran under the JTT+4Γ model.

Phylogenetic analyses using Bayesian inference I used ExaBayes v1.4.1 (Aberer et al., 2014) to perform analysis on k-means partitioned matrix of combined data set under GTR+4Γ. The analysis was ran with two runs, four MCMC chains each for 5 million generations. I disabled the default of parsimony starting tree such that analysis was initiated with a random topology. Convergence and mixing of the MCMC were determined by monitoring average standard devia- tion of split frequencies, which are considered acceptable below 5% (final value 1.75% for combined data set) and effective sample sizes (ESS) for all parameters, considered acceptable above 200 (min ESS: 900). I used 25% burn-in to construct consensus trees.

Species tree analyses In addition to phylogenetic inference on concatenated loci I performed species tree analyses that attempt to reconcile gene tree incongruencies arising due to incomplete lineage sorting (Edwards, 2009). I used Accurate Species TRee Algorithm, ASTRAL v4.10.12 (Mirarab and Warnow, 2015; Sayyari and Mirarab, 2016). I used a weighted statistical binning pipeline (Bayzid et al., 2015) to create supergene alignments and trees. Locus trees used as input for the pipeline were considered under 75 bootstrap support threshold. Summary methods for species tree inference such as those used here have been shown to be negatively impacted by error in estimated input gene trees (Roch and Warnow, 2015). The binning approach was devised to alleviate this (Mirarab et al., 2014). The data sets analyzed with species tree approaches included binned supergenes of all loci (514 supergenes), supergenes identified in the high bootstrap loci (147 supergenes), and supergenes from the slow-evolving loci (100 supergenes).

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Measures of compositional heterogeneity Most models commonly used for phylogenetic inference, including the partitioned GTR+4Γ model used here, assume that alignments are compositionally homogeneous among taxa (Moran et al., 2015). To quan- tify among-taxon compositional heterogeneity of the data, I used two approaches: 1) statistical tests of compositional heterogeneity and 2) a continuous measure of relative composition frequency variability (RCFV) (Zhong et al., 2011). The former included a phylogeny-corrected statistical test that compares compositions in data sets simulated under a model (the null distribution) to the compositions in the observed alignment (Foster, 2004). For the purpose of this study the test was done on 200 simulated alignments for each observed alignment, assuming a GTR+4Γ model and a neighbor joining tree calculated using BioNJ (Gascuel, 1997). The often used but less appropriate χ2 test for compositional heterogeneity was also performed for comparison. The two tests were carried out using the p4 program for phylogenetic inference (Foster, 2004). Relative composition of frequency variability (RCFV) is the other measure used here to compare compositional heterogeneity among data (Zhong et al., 2011). RCFV is the sum of absolute values of differences observed among frequencies of all four nucleotides, divided by the number of taxa. The differences are calculated by subtracting the overall frequency of the character in a matrix from the frequency of that character in an individual sequence (taxon). The sum of these differences is then divided by the number of taxa and this number in turn is summed for each sequence/taxon in the alignment:

∑m ∑n |A j − A | RCFV = i i n i=1 j=1

where m is the number of distinct character states (four for nucleotide sequences), Aij is the frequency of nucleotide i in taxon j and Ai is the frequency of character (nucleotide) i across n taxa. RCFV thus gives a relative measure of compositional heterogeneity for a data set, and as the sum of differences between frequencies is calculated for each sequence, it also allows for comparison among taxa within an alignment.

Tree-based locus statistics Following maximum likelihood estimation of gene trees, I computed average branch length for each of the 2,166 loci. The average branch length is a proxy for the rate of evolution of each locus. Saturation is another property that is potentially correlated with poor model fit. This I calculated by plotting p-distances of an alignment against distances on the tree from model-based maximum likelihood inference (Philippe and Forterre, 1999). These distances would show a perfect fit to simple linear regression in the absence of saturation. When there is a need of correction for multiple substitutions, however, the curve will depart from linearity. I sorted each locus by slope of regression for a relative measure of saturation. I computed all the tree-based measures with a custom R (v3.0.2 (2013-09-25)) script leveraging packages ape v3.1-1 (Paradis et al., 2004) and seqinr v3.1-2 (Charif and Lobry, 2007), modified from code originally developed for Borowiec et al. (2015).

Divergence time estimation To build a time-calibrated chronogram of the Dorylinae I used the R (v3.2.3 (Team, 2014) package ape v3.4 and its function chronos (Paradis et al., 2004; Paradis, 2013). Chronos uses the penalized likelihood method (Sanderson, 2002) and allows selection of the molecular clock model best fitting the data using an information criterion introduced in (Paradis, 2013). I used the maximum likelihood tree with branch lengths, rooted with Harpegnathos saltator estimated from slow-evolving loci partitioned under k-means as input. The method requires that nodes are calibrated with hard bounds of minimum and maximum ages. The

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calibration scheme is given in Supplementary Table 3. The information criterion implemented in chronos identified the strict molecular clock as the best fitting. Because unknown dates are initialized with a random algorithm it is possible to assess the robustness of the node ages to these initial ages by running multiple independent analyses. I ran 100 chronos replicates and summarized output trees using the sumtrees.py script distributed as a part of Denropy package v4.0.3, (Sukumaran and Holder, 2010). The summarized tree has mean branch lengths mean node ages, as well as uncertainty captured as node age ranges obtained across the 100 replicates. I visualized the tree with mean node ages and age ranges using FigTree v1.4.2. Because the information criterion implemented was shown to be poor at distinguishing the strict clock from a model with a small fixed number of rates (Paradis, 2013), I repeated the procedure for a discrete clock model with 10 categories. The results are presented in Supplementary Figure 19. Because calibrations requiring hard minimum and maximum ages may be considered prone to bias and because penalized likelihood does not utilize sequence data, I also performed Bayesian divergence dating using the recently developed birth-death process (Heath et al., 2014). This method assumes no prior belief on calibrated node ages, instead relying on a single recovery age of a fossil that is assumed to be a descendant of the calibrated node. The method simulates tree topology via a birth-death process and treats fossils as a part of the diversification process with variable attachment points on the tree and fixed recovery ages. In addition to assuming no prior beliefs on calibrated node ages, this method is not limited to using only the oldest fossils known for a given node. For these analyses I used BEAST v2.3.2 (Bouckaert et al., 2014) with Sampled Ancestors package (Gavryushkina et al., 2014). Because Bayesian divergence time estimation is computationally expensive with 150+ taxa, these analyses were limited to 109 loci (5% of all loci) sampled at even intervals throughout the rate spectrum. I set up the BEAST analyses under unpartitioned GTR+4Γ model of sequence evolution and uncorrelated clock sampling rates from a lognormal distribution. The analysis was set up with four independent runs for >300 million generations. I determined convergence and adequate sample size (all parameters sampled at ESS > 200) using Tracer v1.5. The calibration scheme included seven fossils with fixed sampling times obtained by drawing a random number from a uniform distribution (runif function in R base) bounded by minimum and maximum ages of the deposit in which the fossil is found (deposit ages follow the Fossilworks website (Alroy, 2016); Supplementary Table 4). I used conditioning on the root age with a prior.

Diversification analyses BAMM v2.5 (Rabosky, 2014) analyses used the consensus BEAST chronogram and a table of sampling probabilities based on extant species diversity estimates for each genus (Borowiec, 2016b). The sampling proportions were set as follows: Acanthostichus: 0.17, Aenictogiton: 0.2, Aenictus: 0.09, Cerapachys: 0.5, Cheliomyrmex: 0.6, Chrysapace: 0.8, Cylindromyrmex: 0.4, Dorylus: 0.2, Eburopone: 0.12, Eciton: 0.58, Eusphinctus: 0.5, Labidus: 0.6, Leptanilloides: 0.17, Lioponera: 0.08, Lividopone: 0.13, Neivamyrmex: 0.11, Neocerapachys: 0.4, Nomamyrmex: 1, Ooceraea: 0.2, Parasyscia: 0.08, Simopone: 0.18, Sphincto- myrmex: 0.4, Syscia: 0.13, Tanipone: 0.4, Vicinopone: 1, Yunodorylus: 0.4, Zasphinctus: 0.15. I used the ”setBAMMpriors” function in the R package BAMMtools (Rabosky et al., 2014) to create priors used for the analysis. I ran the MCMC for 20 million generations, sampling every 2,000 generations. I checked the convergence and plotted the analysis results using BAMMtools and CODA (Plummer et al., 2006).

Biogeographic analyses I used the maximum likelihood functions available in BioGeoBEARS R package (Matzke, 2013, 2014) to compare fit and select from among models commonly used for estimation of biogeographic histories. I discretized species distributions into six biogeographic regions following (Cox, 2001) and treating Malagasy,

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as the seventh separate region. The regions included Neotropical, Nearctic, Palearctic, Afrotropical, Mala- gasy, Indomalayan, and Australasian. The boundary between Indomalayan and Australasian regions is the Wallace Line. I set up a time-stratified analysis that assumed different region adjacency and dispersal probabilities between 110-50 Ma and 50 Ma-present (M3_stratified-type model of Matzke (2014)) and compared likelihoods and AICc scores under six models: DEC, DEC+J, DIVALIKE, DIVALIKE+J, BAYAREALIKE, and BAYAREALIKE+J.

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Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Atta_cephalotes_Genome 100 Acromyrmex_echinatior_Genome Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_hirsuta_M279 100 Tanipone_aglandula_M280 Lioponera_longitarsus_M193 Lioponera_vespula_M210 100100 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 100 Lioponera_MY_M268 100 Lioponera_cf_suscitata_M267 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 87 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 100 Lividopone_MG_M194 100Lividopone_MG_M216 66 Lividopone_MG_M294 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 Zasphinctus_imbecilis_M170 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_UG_M219 100 100 Parasyscia_UG_M218 100 100 Parasyscia_wittmeri_M282 83 Parasyscia_VN_M207 100 Parasyscia_PG_M204 100 Parasyscia_dohertyi_M142 Vicinopone_conciliatrix_M128 Simopone_grandidieri_M186 83 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100 Simopone_conradti_M144 93 Simopone_rex_M133 100 Simopone_trita_M185 Syscia_augustae_M196 100 Syscia_GT_M127 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Eusphinctus_TH_M158 83 93 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100 100 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_PG_M248 100 Ooceraea_FJ06_M168 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M213 100 Eburopone_MG_M214 Yunodorylus_TH_M160 100 98 Yunodorylus_paradoxus_M190 100 Yunodorylus_TH_M191 100 Yunodorylus_eguchii_M247 100 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 84 Cerapachys_TH_M203 Chrysapace_TH_M156 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_MG_M155 100 Chrysapace_cf_sauteri_M261 Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 97 100 Dorylus_laevigatus_M157 Dorylus_orientalis_M244 99 100100 Dorylus_conradti_M178 100 Dorylus_fimbriatus_M288 Dorylus_cf_fulvus_M149 100 100 Dorylus_fulvus_M179 100 Dorylus_spininodis_M292 Dorylus_braunsi_M289 100100 Dorylus_affinis_M177 97 100Dorylus_kohli_M180 100Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_rubellus_M287 93 Dorylus_UG_M153 Aenictus_silvestrii_M221 100 Aenictus_yamanei_M265 99 Aenictus_latifemoratus_M266 49 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 99 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_turneri_M135 100Aenictus_fuchuanensis_M201 100 100 Aenictus_hoelldobleri_M200 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 Aenictus_gracilis_M199 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 97 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 98 Acanthostichus_serratulus_M166 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 Leptanilloides_erinys_M246 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 98 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 96 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 97 Labidus_praedator_M152 Nomamyrmex_hartigii_M220 100 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 61Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 100Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 100 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 0.04 100 Neivamyrmex_melanocephalus_M154 Neivamyrmex_cf_nyensis_M296 combined data matrix 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 100100 maximum likelihood tree Neivamyrmex_asper_M233 100Neivamyrmex_kiowapache_M275 RAxML 100Neivamyrmex_opacithorax_M276 96Neivamyrmex_texanus_M274 partitioned by locus GTR+4Gamma model 100 Neivamyrmex_californicus_M272 500 bootstrap replicates

Supplementary Figure 1: Maximum likelihood tree obtained from the combined data matrix partitioned by locus. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

28 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Atta_cephalotes_Genome 100 Acromyrmex_echinatior_Genome Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 100 Dorylus_laevigatus_M157 Dorylus_orientalis_M244 100100 Dorylus_conradti_M178 100 Dorylus_fimbriatus_M288 Dorylus_fulvus_M179 100 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100Dorylus_kohli_M180 100Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_rubellus_M287 100 Dorylus_UG_M153 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 10090 Aenictus_yamanei_M265 96 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_ZA_M228 100 100 Aenictus_UG_M143 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_turneri_M135 100Aenictus_fuchuanensis_M201 100 100 100 Aenictus_hoelldobleri_M200 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 100 Aenictus_gracilis_M199 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 Labidus_praedator_M152 100 Nomamyrmex_hartigii_M220 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 Eciton_vagans_M184 100100 Eciton_quadriglume_M254 100 100 Eciton_lucanoides_M239 100Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 99 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 99 Neivamyrmex_melanocephalus_M154 Neivamyrmex_cf_nyensis_M296 82 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 100100 Neivamyrmex_asper_M233 100Neivamyrmex_kiowapache_M275 100Neivamyrmex_opacithorax_M276 100Neivamyrmex_texanus_M274 100 Neivamyrmex_californicus_M272 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_TH_M191 100 Yunodorylus_eguchii_M247 Chrysapace_TH_M156 100 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_MG_M155 100 89 Chrysapace_cf_sauteri_M261 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 Cerapachys_TH_M203 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 Leptanilloides_nubecula_M167 96 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 100 Leptanilloides_erinys_M246 100 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 96 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 100 Acanthostichus_serratulus_M166 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M213 100 Eburopone_MG_M214 Syscia_GT_M127 100 Syscia_augustae_M196 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 83 100 Syscia_MY_M147 Eusphinctus_TH_M158 96 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100 100 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 100 Vicinopone_conciliatrix_M128 Simopone_grandidieri_M186 64 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 Simopone_rex_M133 100 100 Simopone_trita_M185 100 Simopone_conradti_M144 100 Simopone_marleyi_M171 88 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_hirsuta_M279 100 Tanipone_aglandula_M280 Lioponera_longitarsus_M193 Lioponera_vespula_M210 100100 Lioponera_nr_kraepelinii_M131 100 64 Lioponera_nr_mayri_M215 100 Lioponera_MY_M268 100 Lioponera_cf_suscitata_M267 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 41 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M194 100Lividopone_MG_M216 100 Lividopone_MG_M294 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 Zasphinctus_imbecilis_M170 0.07 Parasyscia_UG_M281 100 Parasyscia_MG_M212 combined data matrix Parasyscia_UG_M219 100 100 Parasyscia_UG_M218 100 maximum likelihood tree 100 Parasyscia_wittmeri_M282 Parasyscia_VN_M207 RAxML 100 Parasyscia_PG_M204 100 k-means partitions GTR+4Gamma model Parasyscia_dohertyi_M142 500 bootstrap replicates

Supplementary Figure 2: Maximum likelihood tree obtained from the combined data matrix under k-means partitions. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

29 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

HARPEGNATHOS_SALTATOR_GENOME LINEPITHEMA_HUMILE_GENOME 100 CAMPONOTUS_FLORIDANUS_GENOME 100 POGONOMYRMEX_BARBATUS_GENOME 100 VOLLENHOVIA_EMERYI_GENOME 100 CARDIOCONDYLA_OBSCURIOR_GENOME 100 SOLENOPSIS_INVICTA_GENOME 100 ACROMYRMEX_ECHINATIOR_GENOME 100 ATTA_CEPHALOTES_GENOME AENICTOGITON_UG_M189 100AENICTOGITON_ZM02_M181 100 DORYLUS_LAEVIGATUS_M157 DORYLUS_ORIENTALIS_M244 100100 DORYLUS_CONRADTI_M178 100 DORYLUS_FIMBRIATUS_M288 DORYLUS_CF_FULVUS_M149 100 100DORYLUS_FULVUS_M179 100 DORYLUS_SPININODIS_M292 100DORYLUS_AFFINIS_M177 100DORYLUS_BRAUNSI_M289 100DORYLUS_KOHLI_M180 100DORYLUS_MAYRI_M291 100DORYLUS_MOLESTUS_M290 100DORYLUS_UG_M153 98DORYLUS_RUBELLUS_M287 AENICTUS_SILVESTRII_M221 100 AENICTUS_YAMANEI_M265 100 AENICTUS_LATIFEMORATUS_M266 56 AENICTUS_LEVIOR_M231 100 100 AENICTUS_BOBAIENSIS_M197 AENICTUS_CAMPOSI_M222 AENICTUS_UG_M143 100 100 AENICTUS_ZA_M228 AENICTUS_FERGUSONI_M223 100 100 AENICTUS_HODGSONI_M198 100AENICTUS_ROTUNDICOLLIS_M232 100AENICTUS_LAEVICEPS_M269 100 AENICTUS_CURRAX_M245 100AENICTUS_CORNUTUS_M230 100 100 100 AENICTUS_GRACILIS_M199 AENICTUS_TURNERI_M135 100AENICTUS_HOELLDOBLERI_M200 99 100AENICTUS_FUCHUANENSIS_M201 CHELIOMYRMEX_PE_M146 100CHELIOMYRMEX_CF_ANDICOLA_M174 99CHELIOMYRMEX_CF_MOROSUS_M217 LABIDUS_COECUS_M173 100 100 LABIDUS_SPININODIS_M172 100LABIDUS_PRAEDATOR_M152 100 NOMAMYRMEX_ESENBECKI_M129 100 NOMAMYRMEX_HARTIGII_M220 100 ECITON_MEXICANUM_M238 48ECITON_VAGANS_M184 100 100ECITON_QUADRIGLUME_M254 ECITON_LUCANOIDES_M239 100 100ECITON_BURCHELLII_M273 100ECITON_RAPAX_M145 100ECITON_HAMATUM_M293 NEIVAMYRMEX_DISTANS_M242 100 NEIVAMYRMEX_ALFAROI_M240 98 NEIVAMYRMEX_SWAINSONII_M169 100 NEIVAMYRMEX_EC_M175 100NEIVAMYRMEX_ADNEPOS_M236 100 NEIVAMYRMEX_GIBBATUS_M139 100 NEIVAMYRMEX_IMPUDENS_M237 100NEIVAMYRMEX_MELANOCEPHALUS_M154 99 98 NEIVAMYRMEX_CF_NYENSIS_M296 100 NEIVAMYRMEX_COMPRESSINODIS_M235 100 NEIVAMYRMEX_ASPER_M233 100NEIVAMYRMEX_SUMICHRASTI_M151 100NEIVAMYRMEX_KIOWAPACHE_M275 100NEIVAMYRMEX_OPACITHORAX_M276 96NEIVAMYRMEX_CALIFORNICUS_M272 100NEIVAMYRMEX_TEXANUS_M274 YUNODORYLUS_PARADOXUS_M190 100 YUNODORYLUS_TH_M160 100 YUNODORYLUS_EGUCHII_M247 100YUNODORYLUS_TH_M191 100 CERAPACHYS_SULCINODIS_M243 100 CERAPACHYS_MY_M264 100 CERAPACHYS_ANTENNATUS_M263 100 86 CERAPACHYS_TH_M203 CHRYSAPACE_TH_M156 100 CHRYSAPACE_CF_CRAWLEYI_M262 100 CHRYSAPACE_MG_M155 100 CHRYSAPACE_CF_SAUTERI_M261 SPHINCTOMYRMEX_STALI_M253 100 SPHINCTOMYRMEX_MARCOYI_M202 92 LEPTANILLOIDES_NUBECULA_M167 100 LEPTANILLOIDES_MCKENNAE_D0228 100 LEPTANILLOIDES_FEMORALIS_M188 99 100 LEPTANILLOIDES_ERINYS_M246 100 LEPTANILLOIDES_GRACILIS_M187 100 NEOCERAPACHYS_CR_M209 100 NEOCERAPACHYS_NEOTROPICUS_M134 100 NEOCERAPACHYS_BR_M295 100NEOCERAPACHYS_CF_SPLENDENS_M251 91 CYLINDROMYRMEX_MEINERTI_D778 100 CYLINDROMYRMEX_BRASILIENSIS_M140 100 CYLINDROMYRMEX_WHYMPERI_M271 100 100 CYLINDROMYRMEX_DARLINGTONI_M211 ACANTHOSTICHUS_AZ_M277 100 ACANTHOSTICHUS_AZ_M278 100 ACANTHOSTICHUS_BR_M252 99 100ACANTHOSTICHUS_GF_M208 100ACANTHOSTICHUS_SERRATULUS_M166 EBUROPONE_CM02_D0788 100 EBUROPONE_UG_M259 100 EBUROPONE_MG_M195 100EBUROPONE_MG_M130 100EBUROPONE_MG_M214 100EBUROPONE_MG_M213 SYSCIA_GT_M127 100SYSCIA_AUGUSTAE_M196 100 SYSCIA_TYPHLA_D0841 96 100 SYSCIA_MY_M147 100 100SYSCIA_MY_M176 EUSPHINCTUS_TH_M158 98 OOCERAEA_FRAGOSA_D0842 100 OOCERAEA_AUSTRALIS_M138 100 OOCERAEA_BIROI_GENOME 100 OOCERAEA_MY_M270 100 OOCERAEA_FJ06_M168 100 94 OOCERAEA_PG_M248 VICINOPONE_CONCILIATRIX_M128 TANIPONE_SCELESTA_M159 100 TANIPONE_ZONA_M182 100 TANIPONE_HIRSUTA_M279 100 63 TANIPONE_AGLANDULA_M280 SIMOPONE_CF_OCULATA_D0792 100 88 SIMOPONE_GRANDIDIERI_M186 100 SIMOPONE_DRYAS_M224 100 SIMOPONE_MARLEYI_M171 100 SIMOPONE_CONRADTI_M144 91 SIMOPONE_REX_M133 100SIMOPONE_TRITA_M185 51 LIOPONERA_LONGITARSUS_M193 LIOPONERA_VESPULA_M210 100100 LIOPONERA_NR_KRAEPELINII_M131 100LIOPONERA_NR_MAYRI_M215 100 LIOPONERA_CF_SUSCITATA_M267 100 LIOPONERA_MY_M268 100 LIOPONERA_PG_M250 100 LIOPONERA_MARGINATA_M249 93 100 LIOPONERA_PRINCEPS_M137 100 LIOPONERA_RUFICORNIS_M165 LIVIDOPONE_MG_M132 100 LIVIDOPONE_MG_M194 100LIVIDOPONE_MG_M294 62LIVIDOPONE_MG_M216 ZASPHINCTUS_MZ_M229 100 100 ZASPHINCTUS_KE_M227 100 ZASPHINCTUS_TH_M192 100 ZASPHINCTUS_TRUX_M136 100 100 ZASPHINCTUS_PG_M285 100 ZASPHINCTUS_IMBECILIS_M170 0.04 PARASYSCIA_UG_M281 combined data matrix 100 PARASYSCIA_MG_M212 PARASYSCIA_VN_M207 maximum likelihood tree 100 100 PARASYSCIA_PG_M204 100 100 PARASYSCIA_DOHERTYI_M142 RAxML PARASYSCIA_UG_M219 100 PARASYSCIA_UG_M218 unpartitioned GTR+4Gamma model 100 500 bootstrap replicates PARASYSCIA_WITTMERI_M282

Supplementary Figure 3: Maximum likelihood tree obtained from unpartitioned combined data matrix. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

30 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

HARPEGNATHOS_SALTATOR_GENOME LINEPITHEMA_HUMILE_GENOME 1 CAMPON OTUS_F L O R I DANUS_G ENO M E 1 POGONOMYRMEX_BARBATUS_GENOME VOLLENHOVIA_EMERYI_GENOME 1 1 CARDIOCONDYLA_OBSCURIOR_GENOME 1 SOLENOPSIS_INVICTA_GENOME 1 A CRO MYRME X_ECHI N ATIOR_G ENO M E 1 ATTA_CEPHALOTES_GENOME VICINOPONE_CONCILIATRIX_M128 SYSCIA_GT_M127 1 SYSCIA_AUGUSTAE_M196 1 S Y SCIA_ T Y P HLA_D0841 1 SYSC IA_MY _M176 1 1 SYSC IA_MY _M147 1 EUSPHINCTUS_TH_M158 1 OOCERAEA_FRAGOSA_D0842 OOCERAEA_AUSTRALIS_M138 1 1 OOCERAEA_BIROI_GENOME 1 OOCERAEA_MY_M270 1 OOC E RAEA_ FJ06_M168 1 OOCERAEA_PG_M248 1 SIMOPONE_CF_OCULATA_D0792 1 SIMOPONE_GRANDIDIERI_M186 1 SIMOPONE_DRYAS_M224 SIM OPONE_CO NRA D TI_M144 1 0 . 7 9 SIMOPONE_MARLEYI_M171 1 SIMOPONE_TRITA_M185 1 SIMOPONE_REX_M133 TAN I P O N E _ S C E L ESTA_M159 1 1 TANIPONE_ZONA_M182 1 T ANI P O NE_HI RSUT A_M279 1 TANIPONE_AGLANDULA_M280 LIOPONERA_LONGITARSUS_M193 L IOP O NERA_VES PULA_M210 11 L IOPONERA_NR_MAY R I _M215 0 . 5 7 1 LIOPONERA_NR_KRAEPELINII_M131 1 LIOPONERA_MY_M268 1 LIOPONERA_CF_SUSCITATA_M267 1 LIOPONERA_MARGINATA_M249 1 LIOPONERA_PG_M250 0 . 9 4 L IOP O NERA_P R I NCEPS_M137 1 1 LIOPONERA_RUFICORNIS_M165 1 LIVIDOPONE_MG_M132 1 LIVIDOPONE_MG_M194 1 LIVIDOPONE_MG_M216 1 LIVIDOPONE_MG_M294 ZAS P H I NCT US_MZ _M229 1 ZASPHINCTUS_TH_M192 1 1 ZASPHINCTUS_KE_M227 1 ZASPHINCTUS_TRUX_M136 1 ZASPHINCTUS_PG_M285 1 1 ZASPHINCTUS_IMBECILIS_M170 PARASYSC IA_UG _M281 1 PARASYSCIA_MG_M212 PARASYSCIA_VN_M207 1 1 PARASYSCIA_PG_M204 1 1 PARASYSCIA_DOHERTYI_M142 PAR A S YSC IA_UG _M219 1 PARASYSCIA_WITTMERI_M282 1 PARASYSC IA_UG _M218 EBUROPONE_CM02_D0788 1 EBUROPONE_UG_M259 EBUROPONE_MG_M213 1 1 EBUROPONE_MG_M214 1 EBUROPONE_MG_M195 1 EBUROPONE_MG_M130 CERAPACHYS_SULCINODIS_M243 1 CERAPACHYS_MY_M264 1 C E R APACHYS_ T H_M203 1 01. 9 7 CERAPACHYS_ANTENNATUS_M263 YUNODORYLUS_TH_M160 1 Y UNO D O R Y LUS _ PAR A D OXU S _M190 1 YUNODORYLUS_TH_M191 1 Y UNO D O R Y LUS _ EGUCHII_M247 LEPT ANI LLOIDES_MCK ENNA E_D0228 1 LEPTANILLOIDES_NUBECULA_M167 1 LEPTANILLOIDES_FEMORALIS_M188 1 1 LEPTANILLOIDES_ERINYS_M246 1 LEPTANILLOIDES_GRACILIS_M187 SPH I NCTOM Y RMEX _ STAL I _M253 1 SPHINCTOMYRMEX_MARCOYI_M202 NEOCERAPACHYS_CR_M209 1 1 NEOCERAPACHYS_NEOTROPICUS_M134 1 NEO CERAPACHYS_CF _SPLENDENS_M251 1 NEOCERAPACHYS_BR_M295 11 CYLINDROMYRMEX_MEINERTI_D778 1 CYLINDROMYRMEX_BRASILIENSIS_M140 1 CYLI NDRO MYRMEX_DARLI N GTON I _M211 1 1 CYLINDROMYRMEX_WHYMPERI_M271 ACANTHOSTICHUS_AZ_M278 1 ACANTHOSTICHUS_AZ_M277 1 ACA N T H OSTICHUS _ B R_M252 1 ACANTHOSTICHUS_GF_M208 1 ACANTHOSTICHUS_SERRATULUS_M166 0 . 7 1 CHRYSAPACE_MG_M155 1 CHRYSAPACE_CF _SAUT ERI _M261 1 CHRYSAP A CE_T H_M156 1 CHRYSAPACE_CF_CRAWLEYI_M262 AENICTOGITON_UG_M189 1 AENICTOGITON_ZM02_M181 1 DORYLUS_LAEVIGATUS_M157 DORYLUS_CONRADTI_M178 1 1 DORYLUS_ORIENTALIS_M244 1 DORYLUS_FIMBRIATUS_M288 DORYLUS_CF_FULVUS_M149 1 1 DORYLUS_FULVUS_M179 1 1 DORYLUS_SPININODIS_M292 D O RYLUS_B RAUNSI_M289 1 1 DORYLUS_AFFINIS_M177 1 DORYLUS_KOHLI_M180 1 DORYLUS_MAYRI_M291 1 DORYLUS_MOLESTUS_M290 1 DORYLUS_UG_M153 0 . 6 2 DORYLUS_RUBELLUS_M287 AENICTUS_SILVESTRII_M221 AENICTUS_LATIFEMORATUS_M266 1 10 . 9 9 AEN I C T U S _ YAM A N EI_M265 1 AENICTUS_LEVIOR_M231 1 1 AENI C T US_BO BAI ENSI S_M197 AENICTUS_CAMPOSI_M222 AENICTUS_UG_M143 1 1 AENICTUS_ZA_M228 AENICTUS_FERGUSONI_M223 1 1 AENICTUS_HODGSONI_M198 1 AENICTUS_LAEVICEPS_M269 1 AENICTUS_ROTUNDICOLLIS_M232 1 AENICTUS_CURRAX_M245 1 AENICTUS_CORNUTUS_M230 1 1 AENICTUS_GRACILIS_M199 AENI C T US_T URNERI _M135 1 AENICTUS_FUCHUANENSIS_M201 1 0 . 8 4 AENICTUS_HOELLDOBLERI_M200 CHELIOMYRMEX_PE_M146 1 CHELIOMYRMEX_CF_MOROSUS_M217 1 CHELIOMYRMEX_CF_ANDICOLA_M174 LABIDUS_COECUS _M173 1 1 LABIDUS_PRAEDATOR_M152 1 LABIDUS_SPININODIS_M172 1 NOMAMYRMEX_ESENBECKI_M129 1 N O MAMYRME X_HA R TIGII_M220 1 ECITON_MEXICANUM_M238 0 . 4 ECITON_Q U A DRIGLUME _M254 1 1 E C ITON_VAGAN S _M184 ECITON_LUCANOIDES_M239 1 1 ECITON_BURCHELLII_M273 1 E C ITON_HA MAT UM_M293 1 ECITON_RAPAX_M145 NEIVAMYRMEX_DISTANS_M242 1 NEI V A MYRMEX _ALFAR OI_M240 0 . 9 9 NEIVAMYRMEX_SWAINSONII_M169 1 NEIVAMYRMEX_ADNEPOS_M236 1 NEIVAMYRMEX_EC_M175 1 NEIVAMYRMEX_GIBBATUS_M139 1 NEIVAMYRMEX_IMPUDENS_M237 1 N EIVAMY RME X_MELA N O C E PHA LUS _M154 1 NEI VAMYRMEX_CF _NY ENSI S_M296 1 N EIVAM Y RME X_CO M P R ESSIN O D IS_M235 NEIVAMYRMEX_SUMICHRASTI_M151 1 1 3 . 0 NEIVAMYRMEX_ASPER_M233 all loci 1 NEIVAMYRMEX_KIOWAPACHE_M275 1 NEIVAMYRMEX_OPACITHORAX_M276 weighted statistical binning 1 NEIVAMYRMEX_TEXANUS_M274 1 ASTRAL species tree NEIVAMYRMEX_CALIFORNICUS_M272

Supplementary Figure 4: Summary species tree obtained from all 2,166 loci using ASTRAL. Scale is in coalescent units. Nodal support in local posterior probabilities.

31 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Lioponera_longitarsus_M193 Lioponera_vespula_M210 10095 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 98 Lioponera_cf_suscitata_M267 100 Lioponera_MY_M268 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 48 Lioponera_ruficornis_M165 100 100 Lioponera_princeps_M137 Lividopone_MG_M132 100 Lividopone_MG_M294 100 Lividopone_MG_M194 67 Lividopone_MG_M216 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 94 Zasphinctus_PG_M285 Parasyscia_MG_M212 100 Parasyscia_UG_M281 Parasyscia_VN_M207 82 100 Parasyscia_dohertyi_M142 100 100 Parasyscia_PG_M204 Parasyscia_UG_M219 100 100 Parasyscia_UG_M218 99 Parasyscia_wittmeri_M282 Vicinopone_conciliatrix_M128 62 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_hirsuta_M279 100 53 Tanipone_aglandula_M280 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 Simopone_trita_M185 100 100 Simopone_rex_M133 100 Simopone_conradti_M144 66 Simopone_marleyi_M171 Eusphinctus_TH_M158 Syscia_GT_M127 100 Syscia_augustae_M196 100 100 39 Syscia_typhla_D0841 94 Syscia_MY_M147 100 77 Syscia_MY_M176 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100 75 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M214 58 100 100 Eburopone_MG_M213 100 Eburopone_MG_M195 100 Eburopone_MG_M130 Sphinctomyrmex_marcoyi_M202 100 Sphinctomyrmex_stali_M253 Leptanilloides_mckennae_D0228 76 100 Leptanilloides_nubecula_M167 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 Leptanilloides_erinys_M246 64 Neocerapachys_CR_M209 100 57 Neocerapachys_neotropicus_M134 100 Neocerapachys_BR_M295 100 Neocerapachys_cf_splendens_M251 91 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 Acanthostichus_serratulus_M166 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 39 100 Cerapachys_antennatus_M263 100 Cerapachys_TH_M203 Chrysapace_TH_M156 88 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_MG_M155 100 Chrysapace_cf_sauteri_M261 37 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 100 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 100 Dorylus_laevigatus_M157 Dorylus_conradti_M178 100100 Dorylus_orientalis_M244 45 95 Dorylus_fimbriatus_M288 Dorylus_cf_fulvus_M149 100 100 Dorylus_fulvus_M179 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100Dorylus_kohli_M180 90Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_UG_M153 91 Dorylus_rubellus_M287 Aenictus_silvestrii_M221 Aenictus_bobaiensis_M197 50 100 100 Aenictus_levior_M231 99 Aenictus_latifemoratus_M266 56 100 Aenictus_yamanei_M265 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_currax_M245 100Aenictus_gracilis_M199 100 100 Aenictus_cornutus_M230 Aenictus_turneri_M135 100Aenictus_hoelldobleri_M200 98 41 Aenictus_fuchuanensis_M201 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_morosus_M217 100 Cheliomyrmex_cf_andicola_M174 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 Labidus_praedator_M152 Nomamyrmex_esenbecki_M129 100 100 Nomamyrmex_hartigii_M220 100 Eciton_mexicanum_M238 87Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 100Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 65 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 83 Neivamyrmex_melanocephalus_M154 Neivamyrmex_cf_nyensis_M296 0.05 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_asper_M233 high bootstrap loci 100100 Neivamyrmex_sumichrasti_M151 maximum likelihood tree 100Neivamyrmex_opacithorax_M276 RAxML 100Neivamyrmex_kiowapache_M275 57Neivamyrmex_californicus_M272 100 partitioned by locus GTR+4Gamma model Neivamyrmex_texanus_M274 500 bootstrap replicates

Supplementary Figure 5: Maximum likelihood tree obtained from high-bootstrap data matrix partitioned by locus. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

32 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_praedator_M152 100 Labidus_spininodis_M172 100 Nomamyrmex_hartigii_M220 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 Eciton_vagans_M184 100100 Eciton_quadriglume_M254 100 100 Eciton_lucanoides_M239 100Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 58 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 83 Neivamyrmex_melanocephalus_M154 Neivamyrmex_cf_nyensis_M296 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 100100 Neivamyrmex_asper_M233 100Neivamyrmex_opacithorax_M276 100Neivamyrmex_kiowapache_M275 31Neivamyrmex_californicus_M272 100 100 Neivamyrmex_texanus_M274 Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 100 Dorylus_laevigatus_M157 Dorylus_conradti_M178 100100 Dorylus_orientalis_M244 100 Dorylus_fimbriatus_M288 Dorylus_cf_fulvus_M149 100 100 Dorylus_fulvus_M179 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100Dorylus_kohli_M180 90Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_UG_M153 92 Dorylus_rubellus_M287 45 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 10070 Aenictus_yamanei_M265 98 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_ZA_M228 100 100 Aenictus_UG_M143 Aenictus_fergusoni_M223 100100 Aenictus_hodgsoni_M198 100Aenictus_rotundicollis_M232 100 Aenictus_laeviceps_M269 100 Aenictus_currax_M245 100Aenictus_gracilis_M199 100 100 Aenictus_cornutus_M230 71 Aenictus_turneri_M135 100Aenictus_hoelldobleri_M200 100 Aenictus_fuchuanensis_M201 Chrysapace_MG_M155 100 Chrysapace_cf_sauteri_M261 100 Chrysapace_TH_M156 100 Chrysapace_cf_crawleyi_M262 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 100 Cerapachys_TH_M203 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 100 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 Sphinctomyrmex_marcoyi_M202 94 100 Sphinctomyrmex_stali_M253 Leptanilloides_nubecula_M167 91 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 100 Leptanilloides_gracilis_M187 78 Neocerapachys_CR_M209 85 100 Neocerapachys_neotropicus_M134 100 Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 91 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_darlingtoni_M211 100 100 Cylindromyrmex_whymperi_M271 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_serratulus_M166 89 100 Acanthostichus_GF_M208 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M213 100 100 Eburopone_MG_M214 100 Eburopone_MG_M130 100 Eburopone_MG_M195 Eusphinctus_TH_M158 Syscia_GT_M127 100 100 Syscia_augustae_M196 100 Syscia_typhla_D0841 100 Syscia_MY_M176 41 100 30 Syscia_MY_M147 Ooceraea_fragosa_D0842 100 Ooceraea_australis_M138 100 Ooceraea_biroi_Genome 99 Ooceraea_MY_M270 100 Ooceraea_PG_M248 100 Ooceraea_FJ06_M168 Vicinopone_conciliatrix_M128 98 66 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_hirsuta_M279 100 49 Tanipone_aglandula_M280 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 Simopone_trita_M185 100 100 Simopone_rex_M133 100 Simopone_conradti_M144 100 Simopone_marleyi_M171 99 Lioponera_longitarsus_M193 Lioponera_vespula_M210 10058 Lioponera_nr_mayri_M215 100 Lioponera_nr_kraepelinii_M131 96 Lioponera_cf_suscitata_M267 100 Lioponera_MY_M268 100 Lioponera_marginata_M249 100 Lioponera_PG_M250 100 Lioponera_ruficornis_M165 100 100 Lioponera_princeps_M137 Lividopone_MG_M132 100 Lividopone_MG_M194 100Lividopone_MG_M294 100 Lividopone_MG_M216 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 Zasphinctus_imbecilis_M170 0.08 Parasyscia_MG_M212 100 Parasyscia_UG_M281 high bootstrap loci Parasyscia_VN_M207 100 100 Parasyscia_PG_M204 maximum likelihood tree 100 100 Parasyscia_dohertyi_M142 RAxML Parasyscia_UG_M219 100 Parasyscia_wittmeri_M282 100 k-means partitions GTR+4Gamma model Parasyscia_UG_M218 500 bootstrap replicates

Supplementary Figure 6: Maximum likelihood tree obtained from high-bootstrap data matrix under k-means partitions. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

33 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Atta_cephalotes_Genome 100 Acromyrmex_echinatior_Genome Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 10054 Aenictus_yamanei_M265 98 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_ZA_M228 100 100 Aenictus_UG_M143 Aenictus_fergusoni_M223 100100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 100 Aenictus_gracilis_M199 Aenictus_turneri_M135 100Aenictus_fuchuanensis_M201 98 Aenictus_hoelldobleri_M200 Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 100 Dorylus_laevigatus_M157 Dorylus_orientalis_M244 100100 Dorylus_conradti_M178 97 Dorylus_fimbriatus_M288 Dorylus_fulvus_M179 100 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100Dorylus_kohli_M180 85Dorylus_mayri_M291 100 100Dorylus_molestus_M290 100Dorylus_rubellus_M287 92 65 Dorylus_UG_M153 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_praedator_M152 100 Labidus_spininodis_M172 Nomamyrmex_esenbecki_M129 100 100 Nomamyrmex_hartigii_M220 100 Eciton_mexicanum_M238 86Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 100Eciton_burchellii_M273 100Eciton_rapax_M145 100 Eciton_hamatum_M293 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 64 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_EC_M175 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 80 Neivamyrmex_melanocephalus_M154 Neivamyrmex_cf_nyensis_M296 59 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_asper_M233 100100 Neivamyrmex_sumichrasti_M151 100Neivamyrmex_opacithorax_M276 100Neivamyrmex_kiowapache_M275 62Neivamyrmex_californicus_M272 100 Neivamyrmex_texanus_M274 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 71 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 56 Cerapachys_TH_M203 Chrysapace_cf_sauteri_M261 100 Chrysapace_MG_M155 100 Chrysapace_TH_M156 100 Chrysapace_cf_crawleyi_M262 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 Leptanilloides_nubecula_M167 90 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 80 100 Leptanilloides_gracilis_M187 64 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100 Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 94 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_serratulus_M166 74 100 Acanthostichus_GF_M208 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M130 100 100 Eburopone_MG_M195 100 Eburopone_MG_M213 100 Eburopone_MG_M214 Eusphinctus_TH_M158 Syscia_augustae_M196 100 Syscia_GT_M127 100 100 Syscia_typhla_D0841 94 Syscia_MY_M176 29 100 63 Syscia_MY_M147 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100 72 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Vicinopone_conciliatrix_M128 77 67 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_aglandula_M280 100 44 Tanipone_hirsuta_M279 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 Simopone_trita_M185 100 100 Simopone_rex_M133 100 Simopone_conradti_M144 59 Simopone_marleyi_M171 80 Lioponera_longitarsus_M193 Lioponera_vespula_M210 10096 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 99 Lioponera_cf_suscitata_M267 100 Lioponera_MY_M268 100 Lioponera_marginata_M249 100 Lioponera_PG_M250 56 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M294 100Lividopone_MG_M216 59 Lividopone_MG_M194 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 93 Zasphinctus_PG_M285 Parasyscia_MG_M212 0.04 100 Parasyscia_UG_M281 high bootstrap loci Parasyscia_VN_M207 81 100 Parasyscia_PG_M204 100 maximum likelihood tree 100 Parasyscia_dohertyi_M142 Parasyscia_UG_M219 RAxML 100 Parasyscia_wittmeri_M282 100 unpartitioned GTR+4Gamma model Parasyscia_UG_M218 500 bootstrap replicates

Supplementary Figure 7: Maximum likelihood tree obtained from unpartitioned high-bootstrap data matrix. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

34 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnat hos_salt a t or_G enome Linepit hema_humile_G enome 1 Camponot u s _ f loridanus _ G enome 1 Pogonomyrmex_barbat u s _ G enome V ollenhov ia_emery i_G enome 1 0 . 9 9 Cardioc ondy la_obs curior_G enome 1 Solenops is_inv ict a_G enome 1 A cromy rmex _ec hinat ior_G enome 1 Atta_cephalot e s _ G enome Chry sapace_cf _saut eri_M261 1 Chrysapace_MG _M155 1 Chrysapac e_cf _ c rawleyi_M262 1 Chrysapac e_T H_M156 A enictogit on_UG _M189 1 Aenict ogit on_Z M02_M181 1 Dorylus_laevigat us_M157 Dorylus_c onradt i_M178 1 1 Dorylus_orient alis_M244 0 . 7 6 Dory lus _ f imbriat u s _M288 Dorylus_f ulvus_M179 1 1 Dory lus _ cf_ f ulv u s _M149 0 . 9 2 1 Dorylus _spininodis _M292 Dorylus_braunsi_M289 1 1 Dory lus_affinis _M177 1 Dory lus_k ohli_M180 0 . 9 6 Dory lus _mayri_M291 1 Dorylus_molest us_M290 1 Dorylus_UG _M153 0 . 4 5 Dorylus_rubellus_M287 A enict u s _ s ilvestrii_M221 0 . 9 5 1 A enict us_lat i f emorat u s _M266 0 . 9 7Aenict u s _ y amanei_M265 0 . 5 Aenict u s _lev ior_M231 1 1 A enictu s _bobaiens i s _M197 Aenict u s _ c amposi_M222 A enict u s _UG _M143 1 1 A enictus_ZA_M228 A enictu s _ f ergusoni_M223 1 1 A enictu s _hodgs oni_M198 1 Aenict us_laev iceps_M269 1 A enict u s _rot undic ollis_M232 1 A enictu s _ t urneri_M135 1 A enict us_f uchuanensis _M201 1 1 A enictus_hoelldobleri_M200 A enictu s _currax _M245 1 A enict us_grac ilis _M199 1 0 . 5 4 A enictu s _ c ornut u s _M230 Cheliomyrmex _PE_M146 1 Cheliomy rmex_cf_moros u s _M217 1 Cheliomy rmex_cf_andic ola_M174 1 Labidus_c oecus _M173 1 Labidus_spininodis_M172 1 Labidus _praedat or_M152 1 Nomamy rmex _hart igii_M220 1 1 Nomamy rmex _es enbeck i_M129 E c i t on_quadriglume_M254 1 1 Eci t on_v agans _M184 1 Ecit on_mexicanum_M238 0 . 4 7 E cit on_lucanoides_M239 1 1 Ecit on_burchellii_M273 1 E c i t on_hamat um_M293 1 Eci t on_rapax _M145 Neivamy rmex _alf aroi_M240 1 Neivamy rmex _dist ans _M242 0 . 9 2 Neivamy rmex _swains onii_M169 1 Neiv amy rmex _adnepos _M236 1 Neivamyrmex_EC_M175 1 Neiv amy rmex _gibbat u s _M139 1 Neiv amyrmex_melanocephalus_M154 1 Neivamyrmex_impudens_M237 0 . 9 1 Neiv amyrmex_cf _ny ens i s _M296 1 Neivamy rmex _ c ompressinodis _M235 Neiv amyrmex_s umichrast i_M151 1 0 . 9 9 Neivamyrmex_asper_M233 1 Neiv amy rmex _ k iowapac he_M275 1 Neiv amy rmex _opac i t horax _M276 0 . 5 6 Neivamyrmex _ t exanus_M274 1 Neivamyrmex_c alif ornicus_M272 Cerapac h ys_sulc inodis_M243 1 Cerapac h y s_MY_M264 1 Cerapac h ys_ T H_M203 1 Cerapac h ys_ant ennat u s _M263 Neoc erapachy s_CR_M209 1 Neoc erapac h ys_neot ropic u s _M134 1 Neoc erapac hys _cf _splendens_M251 1 Neoc erapac h y s_BR_M295 00. .9967 Cylindromyrmex_meinert i_D778 1 Cylindromyrmex_brasiliens i s _M140 1 Cylindromy rmex_whymperi_M271 1 1 Cylindromyrmex _darlingt oni_M211 A cant host i c hus_AZ_M278 1 A cant host i c hus_AZ_M277 1 Acant host ichus_BR_M252 1 Acant host i c hus _ GF_M208 0 . 8 6 1 A cant hostichus_s errat ulus_M166 E buropone_CM02_D0788 1 Eburopone_UG _M259 Eburopone_MG _M214 1 1 Eburopone_MG _M213 1 Eburopone_MG _M195 1 Eburopone_MG _M130 0 . 8 9 S phinct omyrmex _st ali_M253 1 S phinctomy rmex _marc o y i_M202 Y unodory lus _ T H_M191 1 Yunodorylus _eguc hii_M247 1 Yunodorylus _paradoxus _M190 1 0 . 8 7 Y unodory lus _ T H_M160 Lept anilloides _mckennae_D0228 1 Lept anilloides _nubec ula_M167 1 Lept anilloides _ f emoralis_M188 0 . 6 3 1 Lept anilloides_erinys _M246 1 Lept anilloides _gracilis_M187 S y s cia_august ae_M196 1 Sys c ia_GT_M127 1 Syscia_t yphla_D0841 1 Syscia_MY _M147 0 . 8 8 1 1 Syscia_MY _M176 E u s phinctu s _ T H_M158 0 . 8 3 O oceraea_f ragos a_D0842 1 O oceraea_australis _M138 1 O o c eraea_biroi_G enome 0 . 6 1 O oceraea_MY _M270 1 1 O oceraea_FJ06_M168 1 O o c eraea_PG_M248 V i c inopone_c onciliat rix _M128 T anipone_s celest a_M159 1 T anipone_z ona_M182 1 T anipone_hirsut a_M279 1 0 . 8 5 T anipone_aglandula_M280 Simopone_cf _oculat a_D0792 1 S imopone_grandidieri_M186 1 Simopone_dryas_M224 0 . 9 9 Simopone_conradt i_M144 1 0 . 6 Simopone_marleyi_M171 1 S imopone_t rit a_M185 1 Simopone_rex _M133 Lioponera_longit ars u s _M193 Lioponera_ves pula_M210 0 . 4 6 1 0 . 9 6 Lioponera_nr_mayri_M215 1 Lioponera_nr_k raepelinii_M131 1 Lioponera_MY _M268 1 Lioponera_cf _ s u s cit a t a_M267 1 Lioponera_PG_M250 1 Lioponera_marginat a_M249 0 . 7 7 Lioponera_princeps _M137 1 1 Lioponera_ruf i c ornis _M165 Liv idopone_MG _M132 1 Liv idopone_MG _M194 1 Liv idopone_MG _M216 0 . 5 2 Liv idopone_MG _M294 Z asphinct us_MZ _M229 1 Z a s phinctu s _KE_M227 1 1 Z asphinct us_T H_M192 1 Z asphinct us_t rux_M136 1 Z asphinct us_imbecilis_M170 1 0 . 9 7 Z a s phinctus_PG _M285 P arasyscia_UG _M281 1 Parasy s c ia_MG _M212 Parasyscia_VN_M207 0 . 7 1 1 Paras yscia_dohertyi_M142 3 . 0 1 1 P arasyscia_PG_M204 high bootstrap loci P arasyscia_UG _M219 1 Parasysc ia_wittmeri_M282 0 . 9 9 weighted statistical binning P arasyscia_UG _M218 ASTRAL species tree

Supplementary Figure 8: Summary species tree obtained from 271 highest bootstrap loci using ASTRAL. Scale is in coalescent units. Nodal support in local posterior probabilities.

35 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Vicinopone_conciliatrix_M128 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100Tanipone_aglandula_M280 100 86 Tanipone_hirsuta_M279 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100Simopone_conradti_M144 83 Simopone_trita_M185 100 Simopone_rex_M133 35 Lioponera_vespula_M210 91 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 100 Lioponera_longitarsus_M193 Lioponera_cf_suscitata_M267 38 100 Lioponera_MY_M268 100 Lioponera_PG_M250 100 100 Lioponera_marginata_M249 98 Lioponera_ruficornis_M165 100 100 Lioponera_princeps_M137 Lividopone_MG_M132 100 Lividopone_MG_M294 100Lividopone_MG_M216 63 Lividopone_MG_M194 Zasphinctus_MZ_M229 100 Zasphinctus_TH_M192 100 100 Zasphinctus_KE_M227 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 Zasphinctus_imbecilis_M170 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_VN_M207 100100 Parasyscia_dohertyi_M142 100 100 Parasyscia_PG_M204 Parasyscia_UG_M219 100 Parasyscia_wittmeri_M282 40 100 Parasyscia_UG_M218 Syscia_augustae_M196 100 Syscia_GT_M127 100 Syscia_typhla_D0841 100 Syscia_MY_M147 100 100 Syscia_MY_M176 Eusphinctus_TH_M158 92 Ooceraea_fragosa_D0842 Ooceraea_australis_M138 100100 Ooceraea_biroi_Genome 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M214 100 100 Eburopone_MG_M213 100 Eburopone_MG_M195 100 Eburopone_MG_M130 Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 100 Dorylus_laevigatus_M157 Dorylus_orientalis_M244 68 100100 Dorylus_conradti_M178 98 Dorylus_fimbriatus_M288 62 Dorylus_fulvus_M179 98 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100 100Dorylus_kohli_M180 93Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_UG_M153 57 Dorylus_rubellus_M287 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 10078 Aenictus_yamanei_M265 59 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100 78 100 Aenictus_hodgsoni_M198 100Aenictus_rotundicollis_M232 100 Aenictus_laeviceps_M269 100 Aenictus_turneri_M135 100 Aenictus_hoelldobleri_M200 100 100 Aenictus_fuchuanensis_M201 Aenictus_currax_M245 100Aenictus_gracilis_M199 100 Aenictus_cornutus_M230 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 Cerapachys_TH_M203 Yunodorylus_TH_M160 100 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 85 Chrysapace_cf_sauteri_M261 100 Chrysapace_MG_M155 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_TH_M156 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 63 100Neocerapachys_BR_M295 100 Neocerapachys_cf_splendens_M251 72 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 100 Acanthostichus_serratulus_M166 Sphinctomyrmex_marcoyi_M202 100 Sphinctomyrmex_stali_M253 Leptanilloides_mckennae_D0228 100 Leptanilloides_nubecula_M167 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 100 Leptanilloides_erinys_M246 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_morosus_M217 69 Cheliomyrmex_cf_andicola_M174 100 Labidus_coecus_M173 62 100 Labidus_praedator_M152 100 Labidus_spininodis_M172 100 Nomamyrmex_esenbecki_M129 100 Nomamyrmex_hartigii_M220 100 Eciton_mexicanum_M238 Eciton_vagans_M184 100100 Eciton_quadriglume_M254 94 100 Eciton_lucanoides_M239 98 Eciton_burchellii_M273 100Eciton_rapax_M145 100 Eciton_hamatum_M293 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 82 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_EC_M175 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_melanocephalus_M154 100 94 Neivamyrmex_impudens_M237 0.02 Neivamyrmex_cf_nyensis_M296 100 Neivamyrmex_compressinodis_M235 slow-evolving loci Neivamyrmex_asper_M233 10099 Neivamyrmex_sumichrasti_M151 maximum likelihood tree 100Neivamyrmex_opacithorax_M276 RAxML 100Neivamyrmex_kiowapache_M275 52Neivamyrmex_californicus_M272 100 partitioned by locus GTR+4Gamma model Neivamyrmex_texanus_M274 500 bootstrap replicates

Supplementary Figure 9: Maximum likelihood tree obtained from slow-evolving data matrix partitioned by locus. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

36 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M214 100 Eburopone_MG_M213 Vicinopone_conciliatrix_M128 Syscia_augustae_M196 100 Syscia_GT_M127 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Eusphinctus_TH_M158 94 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100100 Ooceraea_australis_M138 100 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Tanipone_scelesta_M159 100 Tanipone_zona_M182 31 100Tanipone_hirsuta_M279 100 91 Tanipone_aglandula_M280 Simopone_cf_oculata_D0792 100 Simopone_grandidieri_M186 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100Simopone_conradti_M144 38 Simopone_rex_M133 100 Simopone_trita_M185 45 Lioponera_longitarsus_M193 Lioponera_vespula_M210 35 100100 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 98 Lioponera_MY_M268 100 Lioponera_cf_suscitata_M267 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 100 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M194 100Lividopone_MG_M216 100 Lividopone_MG_M294 Zasphinctus_MZ_M229 100 Zasphinctus_TH_M192 100 100 Zasphinctus_KE_M227 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 100 Zasphinctus_PG_M285 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_UG_M219 100100 Parasyscia_UG_M218 100 100 Parasyscia_wittmeri_M282 Parasyscia_VN_M207 100 Parasyscia_PG_M204 100 24 Parasyscia_dohertyi_M142 Yunodorylus_TH_M191 100 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 Chrysapace_cf_sauteri_M261 100 100 Chrysapace_MG_M155 100 Chrysapace_cf_crawleyi_M262 100 31 Chrysapace_TH_M156 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_TH_M203 100 Cerapachys_antennatus_M263 Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 68 100 Dorylus_laevigatus_M157 Dorylus_orientalis_M244 100100 Dorylus_conradti_M178 92 Dorylus_fimbriatus_M288 Dorylus_fulvus_M179 92 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_braunsi_M289 100100 Dorylus_affinis_M177 100 100Dorylus_kohli_M180 95Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_UG_M153 69 Dorylus_rubellus_M287 Aenictus_latifemoratus_M266 81 Aenictus_yamanei_M265 100 Aenictus_silvestrii_M221 24 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 69 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_turneri_M135 100 Aenictus_hoelldobleri_M200 100 100 Aenictus_fuchuanensis_M201 Aenictus_currax_M245 100Aenictus_gracilis_M199 100 Aenictus_cornutus_M230 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 88 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 100 Acanthostichus_serratulus_M166 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 100 Leptanilloides_gracilis_M187 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 100 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 65 Labidus_praedator_M152 100 Nomamyrmex_esenbecki_M129 100 Nomamyrmex_hartigii_M220 100 Eciton_mexicanum_M238 Eciton_vagans_M184 100100 Eciton_quadriglume_M254 97 100 Eciton_lucanoides_M239 97 Eciton_burchellii_M273 100Eciton_rapax_M145 100 Eciton_hamatum_M293 Neivamyrmex_alfaroi_M240 100 Neivamyrmex_distans_M242 78 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 97 Neivamyrmex_melanocephalus_M154 0.04 Neivamyrmex_cf_nyensis_M296 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 slow-evolving loci 10099 Neivamyrmex_asper_M233 maximum likelihood tree 100Neivamyrmex_opacithorax_M276 RAxML 100Neivamyrmex_kiowapache_M275 88Neivamyrmex_californicus_M272 100 k-means partitions GTR+4Gamma model Neivamyrmex_texanus_M274 500 bootstrap replicates

Supplementary Figure 10: Maximum likelihood tree obtained from slow-evolving data matrix under k-means partitions. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

37 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

HARPEGNATHOS_SALTATOR_GENOME LINEPITHEMA_HUMILE_GENOME 100 CAMPONOTUS_FLORIDANUS_GENOME 100 POGONOMYRMEX_BARBATUS_GENOME 100 VOLLENHOVIA_EMERYI_GENOME 100 CARDIOCONDYLA_OBSCURIOR_GENOME 100 SOLENOPSIS_INVICTA_GENOME 100 ACROMYRMEX_ECHINATIOR_GENOME 100 ATTA_CEPHALOTES_GENOME VICINOPONE_CONCILIATRIX_M128 TANIPONE_SCELESTA_M159 100 TANIPONE_ZONA_M182 100TANIPONE_HIRSUTA_M279 100 87 TANIPONE_AGLANDULA_M280 SIMOPONE_GRANDIDIERI_M186 100 SIMOPONE_CF_OCULATA_D0792 100 SIMOPONE_DRYAS_M224 100 SIMOPONE_MARLEYI_M171 100 SIMOPONE_CONRADTI_M144 80 SIMOPONE_TRITA_M185 100SIMOPONE_REX_M133 32 LIOPONERA_LONGITARSUS_M193 LIOPONERA_VESPULA_M210 10095 LIOPONERA_NR_MAYRI_M215 100LIOPONERA_NR_KRAEPELINII_M131 85 LIOPONERA_MY_M268 100 LIOPONERA_CF_SUSCITATA_M267 100 LIOPONERA_PG_M250 100 100 LIOPONERA_MARGINATA_M249 100 100 LIOPONERA_RUFICORNIS_M165 100 LIOPONERA_PRINCEPS_M137 LIVIDOPONE_MG_M132 100 LIVIDOPONE_MG_M294 100 LIVIDOPONE_MG_M194 56LIVIDOPONE_MG_M216 ZASPHINCTUS_MZ_M229 100 100 ZASPHINCTUS_KE_M227 100 ZASPHINCTUS_TH_M192 100 ZASPHINCTUS_TRUX_M136 100 100 ZASPHINCTUS_IMBECILIS_M170 100 ZASPHINCTUS_PG_M285 PARASYSCIA_UG_M281 100 PARASYSCIA_MG_M212 PARASYSCIA_VN_M207 100100 PARASYSCIA_DOHERTYI_M142 100 100 PARASYSCIA_PG_M204 PARASYSCIA_UG_M219 33 100 PARASYSCIA_UG_M218 96 PARASYSCIA_WITTMERI_M282 SYSCIA_AUGUSTAE_M196 100SYSCIA_GT_M127 100 SYSCIA_TYPHLA_D0841 100 SYSCIA_MY_M147 100 100 SYSCIA_MY_M176 EUSPHINCTUS_TH_M158 90 OOCERAEA_FRAGOSA_D0842 100 OOCERAEA_AUSTRALIS_M138 100 OOCERAEA_BIROI_GENOME 100 OOCERAEA_MY_M270 100 OOCERAEA_FJ06_M168 100 OOCERAEA_PG_M248 EBUROPONE_CM02_D0788 100 EBUROPONE_UG_M259 100 EBUROPONE_MG_M214 100EBUROPONE_MG_M213 100 EBUROPONE_MG_M195 100EBUROPONE_MG_M130 AENICTOGITON_ZM02_M181 100AENICTOGITON_UG_M189 100 DORYLUS_LAEVIGATUS_M157 38 DORYLUS_CONRADTI_M178 100100 DORYLUS_ORIENTALIS_M244 96 DORYLUS_FIMBRIATUS_M288 55 DORYLUS_CF_FULVUS_M149 96 100DORYLUS_FULVUS_M179 100 DORYLUS_SPININODIS_M292 100DORYLUS_AFFINIS_M177 100DORYLUS_BRAUNSI_M289 100 100DORYLUS_KOHLI_M180 95DORYLUS_MAYRI_M291 100DORYLUS_MOLESTUS_M290 100DORYLUS_RUBELLUS_M287 57DORYLUS_UG_M153 AENICTUS_YAMANEI_M265 73 AENICTUS_LATIFEMORATUS_M266 100 AENICTUS_SILVESTRII_M221 31 AENICTUS_LEVIOR_M231 100 100 AENICTUS_BOBAIENSIS_M197 AENICTUS_CAMPOSI_M222 AENICTUS_ZA_M228 100 100 AENICTUS_UG_M143 AENICTUS_FERGUSONI_M223 100 AENICTUS_HODGSONI_M198 50 100 100AENICTUS_ROTUNDICOLLIS_M232 100AENICTUS_LAEVICEPS_M269 100 AENICTUS_TURNERI_M135 100 AENICTUS_HOELLDOBLERI_M200 100 100 AENICTUS_FUCHUANENSIS_M201 AENICTUS_CURRAX_M245 100AENICTUS_CORNUTUS_M230 100AENICTUS_GRACILIS_M199 CERAPACHYS_SULCINODIS_M243 100 CERAPACHYS_MY_M264 100 CERAPACHYS_ANTENNATUS_M263 100 CERAPACHYS_TH_M203 100 CHRYSAPACE_CF_SAUTERI_M261 100 CHRYSAPACE_MG_M155 100 CHRYSAPACE_CF_CRAWLEYI_M262 100 CHRYSAPACE_TH_M156 91 YUNODORYLUS_TH_M160 100 YUNODORYLUS_PARADOXUS_M190 100 YUNODORYLUS_EGUCHII_M247 100YUNODORYLUS_TH_M191 NEOCERAPACHYS_CR_M209 100 NEOCERAPACHYS_NEOTROPICUS_M134 31 100NEOCERAPACHYS_BR_M295 100NEOCERAPACHYS_CF_SPLENDENS_M251 85 CYLINDROMYRMEX_MEINERTI_D778 100 CYLINDROMYRMEX_BRASILIENSIS_M140 100 CYLINDROMYRMEX_WHYMPERI_M271 100 100 CYLINDROMYRMEX_DARLINGTONI_M211 ACANTHOSTICHUS_AZ_M277 100 ACANTHOSTICHUS_AZ_M278 100 ACANTHOSTICHUS_BR_M252 100ACANTHOSTICHUS_GF_M208 100 100ACANTHOSTICHUS_SERRATULUS_M166 SPHINCTOMYRMEX_MARCOYI_M202 100 SPHINCTOMYRMEX_STALI_M253 LEPTANILLOIDES_MCKENNAE_D0228 100 LEPTANILLOIDES_NUBECULA_M167 100 LEPTANILLOIDES_FEMORALIS_M188 100 LEPTANILLOIDES_GRACILIS_M187 100 100 LEPTANILLOIDES_ERINYS_M246 CHELIOMYRMEX_PE_M146 100CHELIOMYRMEX_CF_ANDICOLA_M174 78CHELIOMYRMEX_CF_MOROSUS_M217 100 LABIDUS_COECUS_M173 65 100 LABIDUS_SPININODIS_M172 100LABIDUS_PRAEDATOR_M152 100 NOMAMYRMEX_ESENBECKI_M129 100 NOMAMYRMEX_HARTIGII_M220 100 ECITON_MEXICANUM_M238 100ECITON_VAGANS_M184 100ECITON_QUADRIGLUME_M254 96 ECITON_LUCANOIDES_M239 100 98 ECITON_BURCHELLII_M273 100ECITON_HAMATUM_M293 100ECITON_RAPAX_M145 NEIVAMYRMEX_ALFAROI_M240 100 NEIVAMYRMEX_DISTANS_M242 76 NEIVAMYRMEX_SWAINSONII_M169 100 NEIVAMYRMEX_EC_M175 100NEIVAMYRMEX_ADNEPOS_M236 100 NEIVAMYRMEX_GIBBATUS_M139 100 NEIVAMYRMEX_IMPUDENS_M237 100NEIVAMYRMEX_MELANOCEPHALUS_M154 96 NEIVAMYRMEX_CF_NYENSIS_M296 0.03 100 NEIVAMYRMEX_COMPRESSINODIS_M235 slow-evolving loci 100 NEIVAMYRMEX_ASPER_M233 99NEIVAMYRMEX_SUMICHRASTI_M151 maximum likelihood tree 100NEIVAMYRMEX_OPACITHORAX_M276 RAxML 100NEIVAMYRMEX_KIOWAPACHE_M275 53NEIVAMYRMEX_CALIFORNICUS_M272 100 unpartitioned GTR+4Gamma model NEIVAMYRMEX_TEXANUS_M274 500 bootstrap replicates

Supplementary Figure 11: Maximum likelihood tree obtained from unpartitioned slow-evolving data matrix. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

38 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

HARPEGNATHOS_SALTATOR_GENOME LINEPITHEMA_HUMILE_GENOME 1 CAMPO N OTUS_F L O R I DANUS_G ENO M E 1 POGONOMYRMEX_BARBATUS_GENOME CARDIOCONDYLA_OBSCURIOR_GENOME 1 1 VOLLENHOVIA_EMERYI_GENOME 1 SOLENOPSIS_INVICTA_GENOME 1 A CRO MYRME X_ECHI NATIOR_GEN O M E 1 ATTA_CEPHALOTES_GENOME EBUROPONE_CM02_D0788 1 EBUROPONE_UG_M259 EBUROPONE_MG_M213 1 1 EBUROPONE_MG_M214 1 EBUROPONE_MG_M195 1 EBUROPONE_MG_M130 0 . 5 2 VICINOPONE_CONCILIATRIX_M128 TAN IPON E _ S C E L ESTA_M159 1 TANIPONE_ZONA_M182 1 T ANI P O N E _HI RSUTA_M279 1 0 . 2 8 TANIPONE_AGLANDULA_M280 SIMOPONE_GRANDIDIERI_M186 1 SIMOPONE_CF_OCULATA_D0792 1 SIMOPONE_DRYAS_M224 0 . 4 8 1 SIMOPONE_MARLEYI_M171 1 SIM OPONE_CO NRA D TI_M144 0 . 9 9 SIMOPONE_REX_M133 1 SIMOPONE_TRITA_M185 LIOPONERA_LONGITARSUS_M193 L IOP O NERA _VESP ULA _M210 0 . 4 4 11 L IOPONERA_NR_MAY R I _M215 1 LIOPONERA_NR_KRAEPELINII_M131 0 . 8 7 LIOPONERA_MY_M268 1 LIOPONERA_CF_SUSCITATA_M267 1 LIOPONERA_PG_M250 1 LIOPONERA_MARGINATA_M249 0 . 9 5 L IOP O NERA_P R I NCEPS_M137 1 1 LIOPONERA_RUFICORNIS_M165 LIVIDOPONE_MG_M132 1 LIVIDOPONE_MG_M294 1 LIVIDOPONE_MG_M216 0 . 9 6 LIVIDOPONE_MG_M194 ZAS P H I NCT US_MZ _M229 1 ZASPHINCTUS_TH_M192 1 1 ZASPHINCTUS_KE_M227 1 1 ZASPHINCTUS_TRUX_M136 1 ZASPHINCTUS_PG_M285 1 1 ZASPHINCTUS_IMBECILIS_M170 PARASYSC IA_UG _M281 1 PARASYSCIA_MG_M212 PARASYSC IA_UG _M218 0 . 9 3 1 PARASYSCIA_WITTMERI_M282 0 . 8 4 1 PARASYSC IA_UG _M219 PARASYSCIA_VN_M207 1 PARASYSCIA_PG_M204 1 PARASYSCIA_DOHERTYI_M142 SYSCIA_GT_M127 1 SYSCIA_AUGUSTAE_M196 1 S Y SCIA_ T Y P HLA_D0841 1 SYSC IA_MY _M147 1 1 SYSC IA_MY _M176 EUSPHINCTUS_TH_M158 1 OOCERAEA_FRAGOSA_D0842 OOCERAEA_AUSTRALIS_M138 1 1 OOCERAEA_BIROI_GENOME 1 OOCERAEA_MY_M270 1 OOCERAEA_PG_M248 1 OOC E RAEA_ FJ06_M168 CERAPACHYS_SULCINODIS_M243 1 CERAPACHYS_MY_M264 1 C E R APACHYS_ T H_M203 1 CERAPACHYS_ANTENNATUS_M263 CHRYSA PAC E _ T H_M156 1 1 CHRYSAPACE_CF_CRAWLEYI_M262 1 CHRY SAPACE _CF _ S AUT ERI _M261 0 . 6 5 1 CHRYSAPACE_MG_M155 0 . 8 6 YUNODORYLUS_TH_M191 1 Y UNO D O R Y LUS _EG UCHII_M247 1 Y UNO D O R Y LUS _ PAR A D OXU S _M190 1 YUNODORYLUS_TH_M160 AENICTOGITON_UG_M189 1 AENICTOGITON_ZM02_M181 1 DORYLUS_FIMBRIATUS_M288 1 DORYLUS_LAEVIGATUS_M157 DORYLUS_ORIENTALIS_M244 1 0 . 8 7 DORYLUS_CONRADTI_M178 DORYLUS_FULVUS_M179 1 1 DORYLUS_CF_FULVUS_M149 1 DORYLUS_SPININODIS_M292 1 DORYLUS_AFFINIS_M177 1 1 D O RYLUS_BRA UNSI _M289 0 . 9 7 1 DORYLUS_KOHLI_M180 0 . 9 9 DORYLUS_MAYRI_M291 1 DORYLUS_UG_M153 1 DORYLUS_RUBELLUS_M287 0 . 6 9 DORYLUS_MOLESTUS_M290 AENICTUS_SILVESTRII_M221 1 AENICTUS_LATIFEMORATUS_M266 1 AEN I C T U S _ YAM A N EI_M265 0 . 3 7 AENICTUS_LEVIOR_M231 1 1 AENI C T US_BO BAI ENSI S_M197 AENICTUS_CAMPOSI_M222 AENICTUS_ZA_M228 1 1 AENICTUS_UG_M143 AENICTUS_FERGUSONI_M223 1 1 AENICTUS_HODGSONI_M198 0 . 9 7 1 AENICTUS_ROTUNDICOLLIS_M232 1 AENICTUS_LAEVICEPS_M269 1 AENICTUS_CURRAX_M245 1 AENICTUS_GRACILIS_M199 1 1 AENICTUS_CORNUTUS_M230 AENI C T US_T URNERI _M135 1 AENICTUS_FUCHUANENSIS_M201 1 AENICTUS_HOELLDOBLERI_M200 NEOCERAPACHYS_CR_M209 1 NEOCERAPACHYS_NEOTROPICUS_M134 1 NEO CERAPACHYS_CF _SPLENDENS _M251 1 NEOCERAPACHYS_BR_M295 CYLINDROMYRMEX_MEINERTI_D778 1 CYLINDROMYRMEX_BRASILIENSIS_M140 1 CYLINDROMYRMEX_WHYMPERI_M271 1 1 1 CYLI NDRO MYRMEX_DARLI N GTON I _M211 ACANTHOSTICHUS_AZ_M278 1 ACANTHOSTICHUS_AZ_M277 1 ACA N T H OSTICHUS _ B R_M252 1 ACANTHOSTICHUS_GF_M208 1 ACANTHOSTICHUS_SERRATULUS_M166 0 . 4 9 SPHINCTOMYRMEX_MARCOYI_M202 1 S PHI NCTOMYRME X_ST ALI _M253 LEPT ANI LLOIDES_MCK ENNA E_D0228 1 LEPTANILLOIDES_NUBECULA_M167 1 LEPTANILLOIDES_FEMORALIS_M188 1 LEPTANILLOIDES_GRACILIS_M187 1 1 LEPTANILLOIDES_ERINYS_M246 CHELIOMYRMEX_PE_M146 1 CHELIOMYRMEX_CF_ANDICOLA_M174 1 CHELIOMYRMEX_CF_MOROSUS_M217 1 LABIDUS_COECUS _M173 0 . 9 9 1 LABIDUS_PRAEDATOR_M152 1 LABIDUS_SPININODIS_M172 1 N O MAMYRME X_HA R TIGII_M220 1 NOMAMYRMEX_ESENBECKI_M129 1 ECITON_MEXICANUM_M238 ECITON_Q U A DRIGLUME_M254 1 1 E C ITON_VAGAN S _M184 1 0 . 4 9ECITON_LUCANOIDES_M239 1 ECITON_BURCHELLII_M273 1 E C ITON_HA MAT UM_M293 1 ECITON_RAPAX_M145 NEIVAMYRMEX_DISTANS_M242 1 NEI V A MYRMEX _ALFAR OI_M240 NEIVAMYRMEX_SWAINSONII_M169 11 NEIVAMYRMEX_EC_M175 1 NEIVAMYRMEX_ADNEPOS_M236 0 . 6 4 NEIVAMYRMEX_GIBBATUS_M139 1 N EIVAMY RME X_MELA N O C E PHA LUS _M154 1 NEIVAMYRMEX_IMPUDENS_M237 1 NEI VAMYRMEX_CF _NYE N SIS_M296 1 N EIVAM Y RME X_CO M P R ESSIN O D IS_M235 NEIVAMYRMEX_ASPER_M233 1 1 4 . 0 NEIVAMYRMEX_SUMICHRASTI_M151 slow-evolving loci 1 NEIVAMYRMEX_OPACITHORAX_M276 1 NEIVAMYRMEX_KIOWAPACHE_M275 weighted statistical binning 1 NEIVAMYRMEX_CALIFORNICUS_M272 1 ASTRAL species tree NEIVAMYRMEX_TEXANUS_M274

Supplementary Figure 12: Summary species tree obtained from 580 slow-evolving loci using AS- TRAL. Scale is in coalescent units. Nodal support in local posterior probabilities.

39 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 99 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 100 Atta_cephalotes_Genome 100 Acromyrmex_echinatior_Genome Aenictogiton_ZM02_M181 100 Aenictogiton_UG_M189 100 Dorylus_fimbriatus_M288 Dorylus_laevigatus_M157 10035 Dorylus_orientalis_M244 100 Dorylus_conradti_M178 35 Dorylus_cf_fulvus_M149 100 Dorylus_fulvus_M179 100 Dorylus_spininodis_M292 Dorylus_braunsi_M289 100100 Dorylus_affinis_M177 72 100Dorylus_kohli_M180 93Dorylus_mayri_M291 100Dorylus_rubellus_M287 100Dorylus_molestus_M290 100 Dorylus_UG_M153 Aenictus_silvestrii_M221 100 Aenictus_latifemoratus_M266 76 Aenictus_yamanei_M265 48 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_turneri_M135 100 Aenictus_fuchuanensis_M201 100 100 Aenictus_hoelldobleri_M200 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 Aenictus_gracilis_M199 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_hirsuta_M279 100 89 Tanipone_aglandula_M280 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100 100 Simopone_conradti_M144 100 Simopone_rex_M133 43 100 Simopone_trita_M185 Vicinopone_conciliatrix_M128 Lioponera_longitarsus_M193 Lioponera_vespula_M210 10099 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 67 99 Lioponera_MY_M268 100 Lioponera_cf_suscitata_M267 100 Lioponera_marginata_M249 100 Lioponera_PG_M250 100 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M294 100Lividopone_MG_M194 37 Lividopone_MG_M216 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 Zasphinctus_imbecilis_M170 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_UG_M219 100100 Parasyscia_UG_M218 98 100 Parasyscia_wittmeri_M282 Parasyscia_VN_M207 56 100 Parasyscia_PG_M204 100 Parasyscia_dohertyi_M142 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 Cerapachys_TH_M203 Chrysapace_TH_M156 100 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_MG_M155 100 Chrysapace_cf_sauteri_M261 65 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_TH_M191 100 70 Yunodorylus_eguchii_M247 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M213 100 Eburopone_MG_M214 73 Syscia_augustae_M196 100 Syscia_GT_M127 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Eusphinctus_TH_M158 78 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100100 69 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_PG_M248 100 Ooceraea_FJ06_M168 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 83 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 100 Acanthostichus_serratulus_M166 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 Leptanilloides_erinys_M246 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 100 Cheliomyrmex_cf_morosus_M217 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_PE_M146 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 85 Labidus_praedator_M152 Nomamyrmex_hartigii_M220 100 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 93 Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 97 Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 76 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_impudens_M237 100 Neivamyrmex_melanocephalus_M154 81 Neivamyrmex_cf_nyensis_M296 0.03 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 compositionally homogeneous loci 100100 Neivamyrmex_asper_M233 100Neivamyrmex_texanus_M274 maximum likelihood tree 100 Neivamyrmex_californicus_M272 100 RAxML Neivamyrmex_opacithorax_M276 61 partitioned by locus GTR+4Gamma model Neivamyrmex_kiowapache_M275 500 bootstrap replicates

Supplementary Figure 13: Maximum likelihood tree obtained from compositionally homogeneous data matrix partitioned by locus. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

40 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 100 100 Cardiocondyla_obscurior_Genome 100 Solenopsis_invicta_Genome 97 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 100 Dorylus_fimbriatus_M288 Dorylus_laevigatus_M157 10066 Dorylus_orientalis_M244 100 Dorylus_conradti_M178 66 Dorylus_fulvus_M179 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 83 100Dorylus_kohli_M180 95Dorylus_mayri_M291 100Dorylus_rubellus_M287 100Dorylus_molestus_M290 100 Dorylus_UG_M153 Aenictus_silvestrii_M221 100 Aenictus_latifemoratus_M266 66 Aenictus_yamanei_M265 47 Aenictus_levior_M231 100 100 Aenictus_bobaiensis_M197 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 100 Aenictus_gracilis_M199 Aenictus_turneri_M135 100 Aenictus_fuchuanensis_M201 100 Aenictus_hoelldobleri_M200 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_aglandula_M280 100 60 Tanipone_hirsuta_M279 Simopone_cf_oculata_D0792 100 Simopone_grandidieri_M186 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100 100 Simopone_conradti_M144 100 Simopone_rex_M133 26 100 Simopone_trita_M185 Vicinopone_conciliatrix_M128 Lioponera_longitarsus_M193 Lioponera_vespula_M210 10098 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 41 96 Lioponera_cf_suscitata_M267 100 Lioponera_MY_M268 100 Lioponera_marginata_M249 100 Lioponera_PG_M250 100 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M216 100Lividopone_MG_M194 92 Lividopone_MG_M294 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 100 Zasphinctus_PG_M285 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_UG_M219 100100 Parasyscia_UG_M218 100 100 Parasyscia_wittmeri_M282 Parasyscia_VN_M207 40 100 Parasyscia_PG_M204 100 Parasyscia_dohertyi_M142 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_TH_M160 100 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 51 Cerapachys_TH_M203 Chrysapace_MG_M155 100 Chrysapace_cf_sauteri_M261 100 Chrysapace_cf_crawleyi_M262 100 51 Chrysapace_TH_M156 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M213 100 Eburopone_MG_M214 57 Syscia_augustae_M196 100 Syscia_GT_M127 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Eusphinctus_TH_M158 95 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100100 32 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 65 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_GF_M208 100 100 Acanthostichus_serratulus_M166 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 100 Leptanilloides_gracilis_M187 Sphinctomyrmex_marcoyi_M202 100 Sphinctomyrmex_stali_M253 100 Cheliomyrmex_cf_morosus_M217 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_PE_M146 100 Labidus_coecus_M173 100 Labidus_spininodis_M172 100 91 Labidus_praedator_M152 Nomamyrmex_hartigii_M220 100 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 77 Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 98 Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 78 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_EC_M175 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_melanocephalus_M154 100 Neivamyrmex_impudens_M237 86 Neivamyrmex_cf_nyensis_M296 0.04 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_asper_M233 compositionally homogeneous loci 100100 Neivamyrmex_sumichrasti_M151 maximum likelihood tree 100Neivamyrmex_texanus_M274 100 Neivamyrmex_californicus_M272 RAxML 100 Neivamyrmex_opacithorax_M276 44 k-means partitions GTR+4Gamma model Neivamyrmex_kiowapache_M275 500 bootstrap replicates

Supplementary Figure 14: Maximum likelihood tree obtained from compositionally homogeneous data matrix under k-means partitions. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

41 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 99 Camponotus_floridanus_Genome 99 Pogonomyrmex_barbatus_Genome Cardiocondyla_obscurior_Genome 100 100 Vollenhovia_emeryi_Genome 100 Solenopsis_invicta_Genome 100 Atta_cephalotes_Genome 100 Acromyrmex_echinatior_Genome Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 100 Dorylus_fimbriatus_M288 Dorylus_laevigatus_M157 10039 Dorylus_orientalis_M244 100 Dorylus_conradti_M178 39 Dorylus_cf_fulvus_M149 100 Dorylus_fulvus_M179 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 69 100Dorylus_kohli_M180 95Dorylus_mayri_M291 100Dorylus_rubellus_M287 100Dorylus_molestus_M290 100 Dorylus_UG_M153 Aenictus_silvestrii_M221 100 Aenictus_latifemoratus_M266 69 Aenictus_yamanei_M265 39 Aenictus_bobaiensis_M197 100 100 Aenictus_levior_M231 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 100 Aenictus_turneri_M135 100 Aenictus_fuchuanensis_M201 100 100 Aenictus_hoelldobleri_M200 Aenictus_currax_M245 100Aenictus_cornutus_M230 100 Aenictus_gracilis_M199 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_aglandula_M280 100 85 Tanipone_hirsuta_M279 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100 100 Simopone_conradti_M144 100 Simopone_rex_M133 30 100 Simopone_trita_M185 Vicinopone_conciliatrix_M128 Lioponera_longitarsus_M193 Lioponera_vespula_M210 100100 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 60 100 Lioponera_cf_suscitata_M267 100 Lioponera_MY_M268 100 Lioponera_marginata_M249 100 Lioponera_PG_M250 100 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M294 100Lividopone_MG_M216 33 Lividopone_MG_M194 Zasphinctus_MZ_M229 100 Zasphinctus_TH_M192 100 100 Zasphinctus_KE_M227 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 99 Zasphinctus_PG_M285 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_VN_M207 100100 Parasyscia_PG_M204 100 100 Parasyscia_dohertyi_M142 Parasyscia_UG_M219 63 100 Parasyscia_wittmeri_M282 98 Parasyscia_UG_M218 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_TH_M203 100 Cerapachys_antennatus_M263 Yunodorylus_TH_M160 100 100 Yunodorylus_paradoxus_M190 100 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 60 Chrysapace_TH_M156 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_MG_M155 100 71 Chrysapace_cf_sauteri_M261 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M214 100 100 Eburopone_MG_M213 100 Eburopone_MG_M195 100 Eburopone_MG_M130 69 Syscia_GT_M127 100 Syscia_augustae_M196 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Eusphinctus_TH_M158 82 Ooceraea_fragosa_D0842 Ooceraea_biroi_Genome 100100 66 Ooceraea_australis_M138 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100Neocerapachys_BR_M295 100 Neocerapachys_cf_splendens_M251 78 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100 Acanthostichus_serratulus_M166 100 100 Acanthostichus_GF_M208 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_gracilis_M187 100 Leptanilloides_erinys_M246 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 100 Cheliomyrmex_cf_morosus_M217 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_PE_M146 100 Labidus_coecus_M173 100Labidus_praedator_M152 100 89 Labidus_spininodis_M172 Nomamyrmex_esenbecki_M129 100 100 Nomamyrmex_hartigii_M220 100 Eciton_mexicanum_M238 93 Eciton_vagans_M184 100 100Eciton_quadriglume_M254 Eciton_lucanoides_M239 100 98 Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 Neivamyrmex_alfaroi_M240 76 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_EC_M175 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_melanocephalus_M154 100 Neivamyrmex_impudens_M237 78 Neivamyrmex_cf_nyensis_M296 0.03 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 compositionally homogeneous loci 100100 Neivamyrmex_asper_M233 maximum likelihood tree 100Neivamyrmex_californicus_M272 100 Neivamyrmex_texanus_M274 RAxML 100 Neivamyrmex_opacithorax_M276 53 unpartitioned GTR+4Gamma model Neivamyrmex_kiowapache_M275 500 bootstrap replicates

Supplementary Figure 15: Maximum likelihood tree obtained from unpartitioned compositionally homogeneous data matrix. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

42 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Camponotus_floridanus_Genome 99 Linepithema_humile_Genome 99 Pogonomyrmex_barbatus_Genome Atta_cephalotes_Genome 100 100 Acromyrmex_echinatior_Genome 100 Vollenhovia_emeryi_Genome 33 Solenopsis_invicta_Genome 60 Cardiocondyla_obscurior_Genome Tanipone_scelesta_M159 100 Tanipone_zona_M182 100Tanipone_hirsuta_M279 100 Tanipone_aglandula_M280 Vicinopone_conciliatrix_M128 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M213 67 100 Eburopone_MG_M214 100 100 Eburopone_MG_M195 100 Eburopone_MG_M130 Lioponera_longitarsus_M193 Lioponera_vespula_M210 100100 Lioponera_nr_kraepelinii_M131 100 Lioponera_nr_mayri_M215 100 Lioponera_MY_M268 100 96 Lioponera_cf_suscitata_M267 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 100 Lioponera_princeps_M137 100 100 Lioponera_ruficornis_M165 Lividopone_MG_M132 100 Lividopone_MG_M194 100Lividopone_MG_M294 27 Lividopone_MG_M216 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 45 100 Zasphinctus_trux_M136 100 Zasphinctus_imbecilis_M170 100 100 Zasphinctus_PG_M285 Parasyscia_UG_M281 100 Parasyscia_MG_M212 Parasyscia_VN_M207 100 99 Parasyscia_PG_M204 100 100 Parasyscia_dohertyi_M142 Parasyscia_UG_M218 100 Parasyscia_wittmeri_M282 100 Parasyscia_UG_M219 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100 Neocerapachys_cf_splendens_M251 100 Neocerapachys_BR_M295 69 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_whymperi_M271 100 100 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 19 100Acanthostichus_GF_M208 100 63 Acanthostichus_serratulus_M166 Leptanilloides_nubecula_M167 100 Leptanilloides_mckennae_D0228 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 100 Leptanilloides_gracilis_M187 Sphinctomyrmex_marcoyi_M202 100 Sphinctomyrmex_stali_M253 58 Cheliomyrmex_cf_andicola_M174 100Cheliomyrmex_PE_M146 94 Cheliomyrmex_cf_morosus_M217 100 Labidus_praedator_M152 100 Labidus_coecus_M173 76 63 Labidus_spininodis_M172 Nomamyrmex_hartigii_M220 100100 Nomamyrmex_esenbecki_M129 Eciton_burchellii_M273 100 72Eciton_rapax_M145 100 Eciton_hamatum_M293 100Eciton_vagans_M184 100 51 Eciton_quadriglume_M254 58 Eciton_mexicanum_M238 69 Eciton_lucanoides_M239 Neivamyrmex_swainsonii_M169 99 Neivamyrmex_EC_M175 100 Neivamyrmex_adnepos_M236 Neivamyrmex_alfaroi_M240 88 100 Neivamyrmex_distans_M242 52 Neivamyrmex_gibbatus_M139 34 98 Neivamyrmex_melanocephalus_M154 100 31 Neivamyrmex_impudens_M237 Neivamyrmex_cf_nyensis_M296 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_asper_M233 100 100 Neivamyrmex_sumichrasti_M151 100Neivamyrmex_kiowapache_M275 100Neivamyrmex_opacithorax_M276 67Neivamyrmex_texanus_M274 44 Neivamyrmex_californicus_M272 Simopone_grandidieri_M186 100 Simopone_cf_oculata_D0792 100 Simopone_dryas_M224 100 Simopone_marleyi_M171 100 Simopone_conradti_M144 100 Simopone_rex_M133 100 40 Simopone_trita_M185 Eusphinctus_TH_M158 Syscia_augustae_M196 100 Syscia_GT_M127 100 100 Syscia_typhla_D0841 100 Syscia_MY_M176 100 100 Syscia_MY_M147 Ooceraea_fragosa_D0842 Ooceraea_australis_M138 100 100 Ooceraea_biroi_Genome 100 Ooceraea_MY_M270 100 Ooceraea_FJ06_M168 100 Ooceraea_PG_M248 21 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 99 Cerapachys_TH_M203 Chrysapace_TH_M156 94 100 Chrysapace_cf_crawleyi_M262 100 Chrysapace_cf_sauteri_M261 100 Chrysapace_MG_M155 92 Yunodorylus_eguchii_M247 100 Yunodorylus_TH_M191 100 Yunodorylus_TH_M160 100 Yunodorylus_paradoxus_M190 Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 36 100 Dorylus_laevigatus_M157 Dorylus_fimbriatus_M288 10078 Dorylus_orientalis_M244 100 Dorylus_conradti_M178 81 Dorylus_cf_fulvus_M149 100 Dorylus_fulvus_M179 100Dorylus_spininodis_M292 Dorylus_braunsi_M289 100100 Dorylus_affinis_M177 73 100Dorylus_kohli_M180 100Dorylus_mayri_M291 100Dorylus_rubellus_M287 100Dorylus_UG_M153 91 Dorylus_molestus_M290 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 10047 Aenictus_yamanei_M265 31 Aenictus_levior_M231 100 100 Aenictus_bobaiensis_M197 Aenictus_camposi_M222 Aenictus_UG_M143 100 100 Aenictus_ZA_M228 Aenictus_fergusoni_M223 100 100 Aenictus_hodgsoni_M198 100Aenictus_laeviceps_M269 100 Aenictus_rotundicollis_M232 amino acid alignment 100 Aenictus_currax_M245 100Aenictus_gracilis_M199 maximum likelihood tree 97 100Aenictus_cornutus_M230 RAxML Aenictus_hoelldobleri_M200 100 Aenictus_turneri_M135 76 partitioned by locus GTR+4Gamma model Aenictus_fuchuanensis_M201 100 bootstrap replicates

0.02 Supplementary Figure 16: Maximum likelihood tree obtained from amino acid data matrix partitioned by locus. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

43 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 100 Camponotus_floridanus_Genome 100 Pogonomyrmex_barbatus_Genome Cardiocondyla_obscurior_Genome 100 100 Vollenhovia_emeryi_Genome 100 Solenopsis_invicta_Genome 100 Acromyrmex_echinatior_Genome 100 Atta_cephalotes_Genome Aenictogiton_UG_M189 100 Aenictogiton_ZM02_M181 100 Dorylus_laevigatus_M157 Dorylus_conradti_M178 100100 Dorylus_orientalis_M244 100 Dorylus_fimbriatus_M288 Dorylus_fulvus_M179 100 100 Dorylus_cf_fulvus_M149 100 Dorylus_spininodis_M292 Dorylus_affinis_M177 100100 Dorylus_braunsi_M289 100Dorylus_kohli_M180 100Dorylus_mayri_M291 100Dorylus_molestus_M290 100Dorylus_rubellus_M287 98 Dorylus_UG_M153 Yunodorylus_paradoxus_M190 100 Yunodorylus_TH_M160 100 Yunodorylus_TH_M191 100 Yunodorylus_eguchii_M247 Chrysapace_cf_sauteri_M261 100 100 Chrysapace_MG_M155 100 Chrysapace_cf_crawleyi_M262 100 100 98 Chrysapace_TH_M156 Cerapachys_sulcinodis_M243 100 Cerapachys_MY_M264 100 Cerapachys_antennatus_M263 100 Cerapachys_TH_M203 Neocerapachys_CR_M209 100 Neocerapachys_neotropicus_M134 100 Neocerapachys_BR_M295 100 Neocerapachys_cf_splendens_M251 97 Cylindromyrmex_meinerti_D778 100 Cylindromyrmex_brasiliensis_M140 100 Cylindromyrmex_darlingtoni_M211 100 100 Cylindromyrmex_whymperi_M271 Acanthostichus_AZ_M277 100 Acanthostichus_AZ_M278 100 Acanthostichus_BR_M252 100Acanthostichus_serratulus_M166 100 97 Acanthostichus_GF_M208 Leptanilloides_mckennae_D0228 100 96 Leptanilloides_nubecula_M167 100 Leptanilloides_femoralis_M188 100 Leptanilloides_erinys_M246 100 Leptanilloides_gracilis_M187 Sphinctomyrmex_stali_M253 100 Sphinctomyrmex_marcoyi_M202 97 Cheliomyrmex_PE_M146 100Cheliomyrmex_cf_andicola_M174 100 Cheliomyrmex_cf_morosus_M217 100 Labidus_coecus_M173 100 Labidus_praedator_M152 100 97 Labidus_spininodis_M172 100 Nomamyrmex_hartigii_M220 100 Nomamyrmex_esenbecki_M129 100 Eciton_mexicanum_M238 Eciton_vagans_M184 100100 Eciton_quadriglume_M254 100 100 Eciton_lucanoides_M239 100Eciton_burchellii_M273 100Eciton_hamatum_M293 100 Eciton_rapax_M145 Neivamyrmex_distans_M242 100 56 Neivamyrmex_alfaroi_M240 99 Neivamyrmex_swainsonii_M169 100 Neivamyrmex_EC_M175 100 Neivamyrmex_adnepos_M236 100 Neivamyrmex_gibbatus_M139 100 Neivamyrmex_melanocephalus_M154 100 Neivamyrmex_impudens_M237 100 Neivamyrmex_cf_nyensis_M296 100 Neivamyrmex_compressinodis_M235 Neivamyrmex_sumichrasti_M151 100100 Neivamyrmex_asper_M233 100Neivamyrmex_kiowapache_M275 100Neivamyrmex_opacithorax_M276 100Neivamyrmex_californicus_M272 100 Neivamyrmex_texanus_M274 Eburopone_CM02_D0788 100 Eburopone_UG_M259 Eburopone_MG_M195 100 100 Eburopone_MG_M130 100 Eburopone_MG_M213 100 Eburopone_MG_M214 Syscia_GT_M127 100 Syscia_augustae_M196 100 Syscia_typhla_D0841 100 Syscia_MY_M147 57 100 100 Syscia_MY_M176 Eusphinctus_TH_M158 96 Ooceraea_fragosa_D0842 Ooceraea_australis_M138 100 100 Ooceraea_biroi_Genome 100 Ooceraea_MY_M270 100 Ooceraea_PG_M248 100 Ooceraea_FJ06_M168 98 Vicinopone_conciliatrix_M128 Simopone_cf_oculata_D0792 63 100 Simopone_grandidieri_M186 100 Simopone_dryas_M224 Simopone_rex_M133 100 100 Simopone_trita_M185 100 Simopone_conradti_M144 100 Simopone_marleyi_M171 82 Tanipone_scelesta_M159 100 Tanipone_zona_M182 100 Tanipone_aglandula_M280 100 Tanipone_hirsuta_M279 Lioponera_longitarsus_M193 Lioponera_vespula_M210 100100 Lioponera_nr_mayri_M215 100 62 Lioponera_nr_kraepelinii_M131 100 Lioponera_MY_M268 100 Lioponera_cf_suscitata_M267 100 Lioponera_PG_M250 100 Lioponera_marginata_M249 32 Lioponera_ruficornis_M165 100 100 Lioponera_princeps_M137 Lividopone_MG_M132 100 Lividopone_MG_M194 100Lividopone_MG_M294 100 Lividopone_MG_M216 Zasphinctus_MZ_M229 100 Zasphinctus_KE_M227 100 100 Zasphinctus_TH_M192 100 Zasphinctus_trux_M136 100 Zasphinctus_PG_M285 100 100 0.07 Zasphinctus_imbecilis_M170 Parasyscia_UG_M281 combined data matrix 100 Parasyscia_MG_M212 no Aenictus Parasyscia_UG_M219 100 100 Parasyscia_wittmeri_M282 100 maximum likelihood tree 100 Parasyscia_UG_M218 Parasyscia_VN_M207 RAxML 100 Parasyscia_PG_M204 100 k-means partitions GTR+4Gamma model Parasyscia_dohertyi_M142 500 bootstrap replicates

Supplementary Figure 17: Maximum likelihood tree obtained under from complete data matrix with Aenictus removed under k-means partitioning. Scale is in number of substitutions per site. Nodal support in percent bootstrap.

44 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Harpegnathos_saltator_Genome Linepithema_humile_Genome 1 Camponotus_floridanus_Genome 1 Pogonomyrmex_barbatus_Genome Vollenhovia_emeryi_Genome 1 1 Cardiocondyla_obscurior_Genome 1 Solenopsis_invicta_Genome 1 Atta_cephalotes_Genome 1 Acromyrmex_echinatior_Genome Vicinopone_conciliatrix_M128 Tanipone_scelesta_M159 1 Tanipone_zona_M182 1 Tanipone_hirsuta_M279 1 0.52 Tanipone_aglandula_M280 Simopone_cf_oculata_D0792 1 0.98 Simopone_grandidieri_M186 1 Simopone_dryas_M224 1 Simopone_marleyi_M171 1 Simopone_conradti_M144 1 Simopone_rex_M133 1 Simopone_trita_M185 0.52 Lioponera_longitarsus_M193 Lioponera_vespula_M210 1 1 Lioponera_nr_mayri_M215 1 Lioponera_nr_kraepelinii_M131 1 Lioponera_cf_suscitata_M267 1 Lioponera_MY_M268 1 Lioponera_PG_M250 1 Lioponera_marginata_M249 1 Lioponera_ruficornis_M165 1 1 Lioponera_princeps_M137 Lividopone_MG_M132 1 Lividopone_MG_M194 1 Lividopone_MG_M294 1 Lividopone_MG_M216 Zasphinctus_MZ_M229 1 Zasphinctus_TH_M192 1 1 Zasphinctus_KE_M227 1 Zasphinctus_trux_M136 1 Zasphinctus_PG_M285 1 1 Zasphinctus_imbecilis_M170 1 Parasyscia_UG_M281 1 Parasyscia_MG_M212 Parasyscia_VN_M207 1 1 Parasyscia_dohertyi_M142 1 1 Parasyscia_PG_M204 Parasyscia_UG_M219 1 Parasyscia_wittmeri_M282 1 Parasyscia_UG_M218 Syscia_augustae_M196 1 Syscia_GT_M127 1 Syscia_typhla_D0841 1 Syscia_MY_M147 1 1 Syscia_MY_M176 Eusphinctus_TH_M158 1 Ooceraea_fragosa_D0842 Ooceraea_australis_M138 1 1 Ooceraea_biroi_Genome 1 Ooceraea_MY_M270 1 Ooceraea_FJ06_M168 1 Ooceraea_PG_M248 Eburopone_CM02_D0788 1 Eburopone_UG_M259 Eburopone_MG_M195 1 1 Eburopone_MG_M130 1 Eburopone_MG_M213 1 Eburopone_MG_M214 Yunodorylus_TH_M191 1 0.98 Yunodorylus_eguchii_M247 1 Yunodorylus_paradoxus_M190 1 Yunodorylus_TH_M160 Chrysapace_TH_M156 1 1 Chrysapace_cf_crawleyi_M262 1 Chrysapace_cf_sauteri_M261 1 1 Chrysapace_MG_M155 Cerapachys_sulcinodis_M243 1 Cerapachys_MY_M264 1 Cerapachys_antennatus_M263 1 Cerapachys_TH_M203 Aenictogiton_ZM02_M181 1 Aenictogiton_UG_M189 1 1 Dorylus_laevigatus_M157 Dorylus_conradti_M178 1 1 1 Dorylus_orientalis_M244 0.98 Dorylus_fimbriatus_M288 Dorylus_fulvus_M179 1 1 Dorylus_cf_fulvus_M149 1 Dorylus_spininodis_M292 Dorylus_braunsi_M289 1 1 Dorylus_affinis_M177 1 1 Dorylus_kohli_M180 1 Dorylus_mayri_M291 1Dorylus_molestus_M290 1Dorylus_rubellus_M287 1 Dorylus_UG_M153 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 11 Aenictus_yamanei_M265 1 Aenictus_levior_M231 1 1 Aenictus_bobaiensis_M197 Aenictus_camposi_M222 Aenictus_ZA_M228 1 1 Aenictus_UG_M143 1 Aenictus_fergusoni_M223 1 1 Aenictus_hodgsoni_M198 1 Aenictus_laeviceps_M269 1 Aenictus_rotundicollis_M232 1 Aenictus_turneri_M135 1 Aenictus_fuchuanensis_M201 1 1 Aenictus_hoelldobleri_M200 Aenictus_currax_M245 1 Aenictus_cornutus_M230 1 Aenictus_gracilis_M199 Neocerapachys_CR_M209 1 Neocerapachys_neotropicus_M134 1 Neocerapachys_BR_M295 1 Neocerapachys_cf_splendens_M251 1 Cylindromyrmex_meinerti_D778 1 Cylindromyrmex_brasiliensis_M140 1 Cylindromyrmex_whymperi_M271 1 1 Cylindromyrmex_darlingtoni_M211 Acanthostichus_AZ_M277 1 Acanthostichus_AZ_M278 1 Acanthostichus_BR_M252 1 Acanthostichus_serratulus_M166 1 1 Acanthostichus_GF_M208 Leptanilloides_nubecula_M167 1 Leptanilloides_mckennae_D0228 1 Leptanilloides_femoralis_M188 1 Leptanilloides_erinys_M246 1 Leptanilloides_gracilis_M187 Sphinctomyrmex_stali_M253 1 Sphinctomyrmex_marcoyi_M202 1 Cheliomyrmex_PE_M146 1 Cheliomyrmex_cf_morosus_M217 1 Cheliomyrmex_cf_andicola_M174 1 Labidus_coecus_M173 1 Labidus_spininodis_M172 1 1 Labidus_praedator_M152 Nomamyrmex_esenbecki_M129 1 1 Nomamyrmex_hartigii_M220 1 Eciton_mexicanum_M238 1 Eciton_vagans_M184 1 1 Eciton_quadriglume_M254 Eciton_lucanoides_M239 1 1 Eciton_burchellii_M273 1 Eciton_rapax_M145 1 Eciton_hamatum_M293 Neivamyrmex_distans_M242 1 Neivamyrmex_alfaroi_M240 1 Neivamyrmex_swainsonii_M169 1 Neivamyrmex_EC_M175 1 Neivamyrmex_adnepos_M236 1 Neivamyrmex_gibbatus_M139 1 Neivamyrmex_impudens_M237 1 1 Neivamyrmex_melanocephalus_M154 0.04 Neivamyrmex_cf_nyensis_M296 1 Neivamyrmex_compressinodis_M235 combined data matrix Neivamyrmex_asper_M233 1 1 Bayesian posterior consensus tree Neivamyrmex_sumichrasti_M151 1 Neivamyrmex_kiowapache_M275 ExaBayes 1 Neivamyrmex_opacithorax_M276 1 Neivamyrmex_texanus_M274 k-means partitions GTR+4Gamma model 1 Neivamyrmex_californicus_M272

Supplementary Figure 18: Bayesian posterior consensus tree obtained from complete data matrix under k-means partitioning. Scale is in number of substitutions per site. Nodal support in posterior probability.

45 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Neivamyrmex_californicus_M272 2.1 2.71 Neivamyrmex_texanus_M274 2.89 Neivamyrmex_kiowapache_M275 3.88 Neivamyrmex_opacithorax_M276 Neivamyrmex_sumichrasti_M151 2.85 6.43 Neivamyrmex_asper_M233 8.7 Neivamyrmex_compressinodis_M235 Neivamyrmex_cf_nyensis_M296 14.44 Neivamyrmex_impudens_M237 4.81 10.52 Neivamyrmex_melanocephalus_M154 Neivamyrmex_gibbatus_M139 19.51 Neivamyrmex_adnepos_M236 2.42 9.28 Neivamyrmex_EC_M175 14.37 Neivamyrmex_swainsonii_M169 Neivamyrmex_alfaroi_M240 8.91 Neivamyrmex_distans_M242 Eciton_rapax_M145 0.98 3.12 Eci t on_hamat um_M293 3.65 Eciton_burchellii_M273 27.36 4.46 Eciton_lucanoides_M239 E c i t on_vagans_M184 3.36 5.55 Eciton_quadriglume_M254 12.25 Eciton_mexicanum_M238 Nomamyrmex_esenbecki_M129 6.64 18.06 Nomamyrmex_hartigii_M220 44.36 Labidus_spininodis_M172 6.11 10.7 Labidus_praedator_M152 23.8 Labidus_coecus_M173 Cheliomyrmex_cf_andicola_M174 5.87 9.78 Cheliomy rmex _ cf_moros u s _M217 51.98 Cheliomyrmex_PE_M146 Sphinctomyrmex_stali_M253 19.98 Sphinct omyrmex_marc o y i_M202 Leptanilloides_erinys_M246 12.36 18.03 Leptanilloides_gracilis_M187 32.92 Leptanilloides_femoralis_M188 Leptanilloides_nubecula_M167 15.1 Leptanilloides_mckennae_D0228 A c ant hostichus _ GF_M208 60.11 6.07 11.67 Acanthostichus_serratulus_M166 25.63 Acanthostichus_BR_M252 Acanthostichus_AZ_M278 12.35 Acanthostichus_AZ_M277 39.72 Cylindromyrmex_whymperi_M271 8.45 20.62 Cylindromyrmex_darlingtoni_M211 28.25 Cylindromyrmex_brasiliensis_M140 51.52 Cylindromyrmex_meinerti_D778 Neocerapachys_cf_splendens_M251 7.39 15.7 Neocerapachys_BR_M295 22.75 Neocerapachys_neotropicus_M134 Neoc erapac h ys_CR_M209 Aenict us_grac ilis _M199 1.99 3.21 Aenictus_cornutus_M230 Aenictus_currax_M245 8.09 Aenictus_hoelldobleri_M200 3.2 4.18 Aenictus_fuchuanensis_M201 9.67 Aenictus_turneri_M135 Aenictus_laeviceps_M269 1.79 2.84 Aenictus_rotundicollis_M232 13.08 5.36 Aenictus_hodgsoni_M198 70.42 Aenictus_fergusoni_M223 19.31 Aenictus_UG_M143 6.43 Aenictus_ZA_M228 Aenictus_camposi_M222 33.52 Aenictus_bobaiensis_M197 6.58 13.32 Aenictus_levior_M231 23.45 Aenictus_silvestrii_M221 Aenictus_latifemoratus_M266 13.2 Aenictus_yamanei_M265 Dorylus _UG _M153 1.14 1.4Dorylus_rubellus_M281 7 1.9Dorylus_molestus_M295 0 2.65Dorylus_mayri_M291 54.18 2.87 Dorylus _ k ohli_M180 Dorylus_braunsi_M289 1.84 4.11 Dorylus_affinis_M177 6.49 Dory lus_spininodis_M292 Dorylus_fulvus_M179 8.46 1.05 Dorylus_cf _ f ulvus_M149 11.27 Dorylus _ f imbriat u s _M288 Dory lus_orient alis _M244 14.68 6.66 Dorylus_conradti_M178 62.67 37.53 Dorylus_laevigatus_M157 Aenictogiton_UG_M189 13.71 Aenictogiton_ZM02_M181 Cerapachys_T H_M203 8.34 13.05 Cerapachys_antennatus_M263 24.95 Cerapachys_MY_M264 Cerapachys_sulcinodis_M243 43.37 Chrysapace_cf_crawleyi_M262 14.26 Chrysapace_T H_M156 26.22 Chrysapace_cf_sauteri_M261 14.45 54.73 Chrysapace_MG_M155 Y unodory lus _ T H_M160 18.04 Yunodorylus_paradoxus_M190 35.76 Y unodory lus _ T H_M191 16.15 Yunodory lus_eguchii_M247 80.43 Parasyscia_PG_M204 3.67 5.86 Paras ysc ia_dohert yi_M142 Parasyscia_VN_M207 6.93 Parasyscia_UG_M218 6.01 8.26.35 6 Parasyscia_wittmeri_M282 Parasyscia_UG_M219 9.33 Parasyscia_MG_M212 Parasyscia_UG_M281 Zasphinctus_imbecilis_M170 18.57 3.15 3.93 Zasphinctus_PG_M285 7.44 Zasphinctus_trux_M136 Zasphinctus_TH_M192 9.62 2.54 23.09 Z asphinct us_KE_M227 Zasphinctus_MZ_M229 Lividopone_MG_M216 3.18 4.13 Lividopone_MG_M294 11.8 Lividopone_MG_M194 Lividopone_MG_M132 Lioponera_princeps_M137 33.4 5.45 6.27 Lioponera_ruficornis_M165 6.81 Lioponera_marginata_M249 8.52 Lioponera_PG_M250 Lioponera_MY_M268 6.34 11.79 Lioponera_cf_suscitata_M267 Lioponera_nr_kraepelinii_M131 2.66 91.03 14.61 8.43 Lioponera_nr_mayri_M215 Lioponera_vespula_M210 46.34 Lioponera_longit ars us_M193 S imopone_rex _M133 1.85 5.58 Simopone_trita_M185 6.53 Simopone_conradti_M144 9.92 Simopone_marleyi_M171 18.5 Simopone_dryas_M224 Simopone_cf_oculata_D0792 11.07 Simopone_grandidieri_M186 36.06 T anipone_hirsut a_M279 4.76 60.98 8.79 Tanipone_aglandula_M280 14.21 Tanipone_zona_M182 T anipone_sc elest a_M159 Ooceraea_FJ06_M168 5.77 Ooceraea_PG_M248 97.48 6.56 8.27 Ooceraea_MY_M270 Ooceraea_biroi_Genome 6.66 11.2 Ooceraea_australis_M138 22.72 O o c eraea_f ragos a_D0842 Eusphinctus_TH_M158 Syscia_MY_M176 41.35 5.6 14.03 Syscia_MY_M147 25.34 Syscia_typhla_D0841 Sys cia_august ae_M196 12.22 Syscia_GT_M127 Vicinopone_conciliatrix_M128 Eburopone_MG_M214 118.62 7.5 Eburopone_MG_M213 14.98 Eburopone_MG_M195 8.23 27.58 Eburopone_MG_M130 50.21 Eburopone_UG_M259 E buropone_CM02_D0788 Acromyrmex_echinatior_Genome 10.91 28.47 Atta_cephalotes_Genome 47.67 Solenopsis_invicta_Genome Vollenhovia_emeryi_Genome 28.07 70.34 Cardiocondyla_obscurior_Genome 103.75 Pogonomy rmex _barbat us_G enome 108.04 Camponot u s _ f loridanus_G enome Linepit hema_humile_G enome

120.0 100.0 80.0 60.0 40.0 20.0 0.0 Supplementary Figure 19: Summary tree with mean ages from 100 analyses under penalized likelihood in Chronos.

46 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Neivamyrmex_impudens_M237 4.52 10.31 Neivamyrmex_melanocephalus_M154 12.52 Neivamyrmex_gibbatus_M139 Neivamyrmex_alfaroi_M240 10.57 13.04 Neivamyrmex_distans_M242 Neivamyrmex_EC_M175 1.33 11.6 Neivamyrmex_adnepos_M236 Neivamyrmex_swainsonii_M169 Neivamyrmex_californicus_M272 2.13 13.37 3.01 Neivamyrmex_texanus_M274 3.44 Neivamyrmex_kiowapache_M275 4.3 Neivamyrmex_opacithorax_M276 Neivamyrmex_asper_M233 3.86 9.34 Neivamyrmex_sumichrasti_M151 11.29 Neivamyrmex_compressinodis_M235 Neivamyrmex_cf_nyensis_M296 Eci t on_hamat um_M293 0.79 3.37 Eciton_rapax_M145 27.5 4.34 Eciton_burchellii_M273 4.71 Eciton_lucanoides_M239 Eciton_quadriglume_M254 4.15 4.91 E c i t on_vagans_M184 13.23 Eciton_mexicanum_M238 Nomamyrmex_esenbecki_M129 7.92 18.46 Nomamyrmex_hartigii_M220 Labidus_praedator_M152 56.66 5.21 6.91 Labidus_spininodis_M172 23.31 Labidus_coec u s _M173 Cheliomyrmex_cf_andicola_M174 3.53 4.36 Cheliomy rmex _cf _moros u s _M217 Cheliomyrmex_PE_M146 59.16 Leptanilloides_erinys_M246 20.07 24.35 Leptanilloides_gracilis_M187 34.69 Leptanilloides_femoralis_M188 Leptanilloides_mckennae_D0228 8.26 Leptanilloides_nubecula_M167 Sphinct omyrmex _marc oyi_M202 23.24 Sphinctomyrmex_stali_M253 A c ant hostichus_GF_M208 60.35 3.17 4.94 Acanthostichus_serratulus_M166 19.46 Acanthostichus_BR_M252 Acanthostichus_AZ_M277 8.14 Acanthostichus_AZ_M278 41.84 Cylindromyrmex_darlingtoni_M211 7.33 22.57 Cylindromyrmex_whymperi_M271 27.43 Cylindromyrmex_brasiliensis_M140 58.83 Cylindromyrmex_meinerti_D778 Neocerapachys_BR_M295 3.33 7.32 Neocerapachys_cf_splendens_M251 9.85 Neocerapachys_neotropicus_M134 Neoc erapachys_CR_M209 Aenictus_cornutus_M230 2.09 3.96 Aenict us_gracilis_M199 Aenictus_currax_M245 10.55 Aenictus_fuchuanensis_M201 3.86 4.73 Aenictus_hoelldobleri_M200 Aenictus_turneri_M135 13.25 Aenictus_laeviceps_M269 2.26 3.55 Aenictus_rotundicollis_M232 15.61 7.81 Aenictus_hodgsoni_M198 Aenictus_fergusoni_M223 61.53 19.9 Aenictus_UG_M143 7 Aenictus_ZA_M228 Aenictus_camposi_M222 23.25 Aenictus_bobaiensis_M197 7.06 13.07 Aenictus_levior_M231 13.88 Aenictus_latifemoratus_M266 14.9 Aenictus_yamanei_M265 Aenictus_silvestrii_M221 Dorylus _UG _M153 1.14 1.4Dorylus_rubellus_M286 7 3.29 Dorylus_molestus_M290 Dorylus_mayri_M291 57.71 4.94 Dorylus_affinis _M177 2.49 6.654.52 Dorylus_braunsi_M289 Dorylus _kohli_M 180 10.6 Dory lus_spininodis_M292 Dorylus_cf _ f ulv u s _M149 14.1 0.88 Dorylus_fulvus_M179 15.6 Dorylus _ f imbriat u s _M288 Dorylus_conradti_M178 8.79 63.23 Dory lus_orient alis _M244 44.38 14.18 60.29 Dorylus_laevigatus_M157 Aenictogiton_UG_M189 4.68 Aenictogiton_ZM02_M181 Chrysapace_MG_M155 15.52 Chrysapace_cf_sauteri_M261 25.97 Chrysapace_T H_M156 16.85 Chrysapace_cf_crawleyi_M262 56.83 Y unodory lus _ T H_M160 14.88 Yunodorylus_paradoxus_M190 29.08 Y unodory lus _ T H_M191 3.52 58.56 Yunodory lus_eguchii_M247 Cerapachys_T H_M203 8.97 11.03 Cerapachys_antennatus_M263 15.12 Cerapachys_MY_M264 65.27 Cerapachys_sulcinodis_M243 Eburopone_MG_M130 3.76 Eburopone_MG_M195 6.32 Eburopone_MG_M213 1.64 28.7 Eburopone_MG_M214 33.58 Eburopone_UG_M259 E buropone_CM02_D0788 Ooceraea_FJ06_M168 12.25 15.01 Ooceraea_PG_M248 20.19 Ooceraea_MY_M270 Ooceraea_australis_M138 17.85 27.44 Ooceraea_biroi_Genome O o c eraea_f ragos a_D0842 67.14 48.76 Syscia_MY_M147 2.94 16.18 Syscia_MY_M176 Syscia_typhla_D0841 50.96 19.56 Syscia_GT_M127 5.31 Sys cia_august ae_M196 Eusphinctus_TH_M158 Simopone_conradti_M144 9.38 Simopone_marleyi_M171 10.44 S imopone_rex _M133 69.16 3.46 12.54 Simopone_trita_M185 21.7 Simopone_dryas_M224 Simopone_cf_oculata_D0792 13.63 Simopone_grandidieri_M186 63.2 Tanipone_aglandula_M280 3.41 7.73 T anipone_hirs u t a_M279 19.28 Tanipone_zona_M182 T anipone_scelest a_M159 Vicinopone_conciliatrix_M128 Parasyscia_PG_M204 6.88 13.33 Paras ysc ia_dohertyi_M142 Parasyscia_VN_M207 16.86 Parasyscia_UG_M218 12.84 Parasyscia_wittmeri_M282 73.8 22.53 14.19 Parasyscia_UG_M219 25.37 Parasyscia_MG_M212 Parasyscia_UG_M281 Zasphinctus_PG_M285 52.49 8.74 11.01 Zasphinctus_imbecilis_M170 22.42 Zasphinctus_trux_M136 Zasphinctus_TH_M192 6.34 27.67 Z asphinct u s _KE_M227 58.53 Zasphinctus_MZ_M229 Lividopone_MG_M194 5.03 6.34 Lividopone_MG_M216 26.65 Lividopone_MG_M294 Lividopone_MG_M132 Lioponera_princeps_M137 69.58 10.89 13.12 Lioponera_ruficornis_M165 13.87 Lioponera_marginata_M249 16.6 Lioponera_PG_M250 Lioponera_MY_M268 12.46 20.87 Lioponera_cf_suscitata_M267 Lioponera_nr_kraepelinii_M131 6.12 24.48 19.78 Lioponera_nr_mayri_M215 Lioponera_vespula_M210 Lioponera_longit arsus_M193

80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Supplementary Figure 20: Mean ages and their 95% confidence intervals on the consensus BEAST tree inferred under fossilized birth-death process. All ages in Ma.

47 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Neivamyrmex_impudens_M237 1 1 Neivamyrmex_melanocephalus_M154 0.9862 Neivamyrmex_gibbatus_M139 Neivamyrmex_alfaroi_M240 1 0.6657 Neivamyrmex_distans_M242 Neivamyrmex_EC_M175 1 1 Neivamyrmex_adnepos_M236 Neivamyrmex_swainsonii_M169 Neivamyrmex_californicus_M272 1 1 0.9998 Neivamyrmex_texanus_M274 1 Neivamyrmex_kiowapache_M275 1 Neivamyrmex_opacithorax_M276 Neivamyrmex_asper_M233 1 1 Neivamyrmex_sumichrasti_M151 1 Neivamyrmex_compressinodis_M235 Neivamyrmex_cf_nyensis_M296 Eci t on_hamat um_M293 1 1 Eciton_rapax_M145 1 1 Eciton_burchellii_M273 0.8854 Eciton_lucanoides_M239 Eciton_quadriglume_M254 1 1 E c i t on_vagans_M184 1 Eciton_mexicanum_M238 Nomamyrmex_esenbecki_M129 1 1 Nomamyrmex_hartigii_M220 Labidus_praedator_M152 0.9999 1 1 Labidus_spininodis_M172 1 Labidus_coecus_M173 Cheliomyrmex_cf_andicola_M174 0.8933 1 Cheliomy rmex _ cf_moros u s _M217 Cheliomyrmex_PE_M146 1 Leptanilloides_erinys_M246 1 1 Leptanilloides_gracilis_M187 1 Leptanilloides_femoralis_M188 Leptanilloides_mckennae_D0228 1 Leptanilloides_nubecula_M167 Sphinct omyrmex_marc o y i_M202 1 Sphinctomyrmex_stali_M253 A c ant hostichus _ GF_M208 1 1 1 Acanthostichus_serratulus_M166 Acanthostichus_BR_M252 1 Acanthostichus_AZ_M277 1 Acanthostichus_AZ_M278 1 Cylindromyrmex_darlingtoni_M211 1 1 Cylindromyrmex_whymperi_M271 1 Cylindromyrmex_brasiliensis_M140 1 Cylindromyrmex_meinerti_D778 Neocerapachys_BR_M295 1 1 Neocerapachys_cf_splendens_M251 1 Neocerapachys_neotropicus_M134 Neoc erapac h ys_CR_M209 Aenictus_cornutus_M230 1 1 Aenict us_grac ilis _M199 Aenictus_currax_M245 1 Aenictus_fuchuanensis_M201 1 1 Aenictus_hoelldobleri_M200 Aenictus_turneri_M135 1 Aenictus_laeviceps_M269 1 1 Aenictus_rotundicollis_M232 1 1 Aenictus_hodgsoni_M198 Aenictus_fergusoni_M223 1 1 Aenictus_UG_M143 1 Aenictus_ZA_M228 Aenictus_camposi_M222 1 Aenictus_bobaiensis_M197 1 0.569 Aenictus_levior_M231 0.9999 Aenictus_latifemoratus_M266 1 Aenictus_yamanei_M265 Aenictus_silvestrii_M221 Dorylus _UG _M153 0.9974 1 Dorylus_rubellus_M287 1 Dorylus_molestus_M290 Dorylus_mayri_M291 1 1 Dorylus_affinis_M177 1 1 0.9998 Dorylus_braunsi_M289 Dorylus _ k ohli_M180 1 Dory lus_spininodis_M292 Dorylus_cf _ f ulvus_M149 0.9373 1 Dorylus_fulvus_M179 1 Dorylus _ f imbriat u s _M288 Dorylus_conradti_M178 1 1 Dory lus_orient alis _M244 1 0.8747 1 Dorylus_laevigatus_M157 Aenictogiton_UG_M189 1 Aenictogiton_ZM02_M181 Chrysapace_MG_M155 1 Chrysapace_cf_sauteri_M261 1 Chrysapace_T H_M156 1 Chrysapace_cf_crawleyi_M262 0.5869 Y unodory lus _ T H_M160 1 Yunodorylus_paradoxus_M190 1 Y unodory lus _ T H_M191 1 1 Yunodory lus_eguchii_M247 Cerapachys_T H_M203 1 1 Cerapachys_antennatus_M263 1 Cerapachys_MY_M264 0.9997 Cerapachys_sulcinodis_M243 Eburopone_MG_M130 1 Eburopone_MG_M195 1 Eburopone_MG_M213 1 1 Eburopone_MG_M214 1 Eburopone_UG_M259 E buropone_CM02_D0788 Ooceraea_FJ06_M168 1 1 Ooceraea_PG_M248 1 Ooceraea_MY_M270 Ooceraea_australis_M138 1 1 Ooceraea_biroi_Genome O o c eraea_f ragos a_D0842 0.9523 0.999 Syscia_MY_M147 1 1 Syscia_MY_M176 Syscia_typhla_D0841 1 1 Syscia_GT_M127 1 Sys cia_august ae_M196 Eusphinctus_TH_M158 Simopone_conradti_M144 0.9983 Simopone_marleyi_M171 1 S imopone_rex _M133 0.7991 1 1 Simopone_trita_M185 1 Simopone_dryas_M224 Simopone_cf_oculata_D0792 1 Simopone_grandidieri_M186 0.97 Tanipone_aglandula_M280 1 1 T anipone_hirsut a_M279 1 Tanipone_zona_M182 T anipone_sc elest a_M159 Vicinopone_conciliatrix_M128 Parasyscia_PG_M204 1 1 Paras ysc ia_dohert yi_M142 Parasyscia_VN_M207 1 Parasyscia_UG_M218 1 Parasyscia_wittmeri_M282 1 1 1 Parasyscia_UG_M219 1 Parasyscia_MG_M212 Parasyscia_UG_M281 Zasphinctus_PG_M285 1 1 1 Zasphinctus_imbecilis_M170 Zasphinctus_trux_M136 1 Zasphinctus_TH_M192 1 1 Z asphinct us_KE_M227 1 Zasphinctus_MZ_M229 Lividopone_MG_M194 1 1 Lividopone_MG_M216 1 Lividopone_MG_M294 Lividopone_MG_M132 Lioponera_princeps_M137 1 1 0.9991 Lioponera_ruficornis_M165 1 Lioponera_marginata_M249 1 Lioponera_PG_M250 Lioponera_MY_M268 1 1 Lioponera_cf_suscitata_M267 Lioponera_nr_kraepelinii_M131 1 1 0.9997 Lioponera_nr_mayri_M215 Lioponera_vespula_M210 Lioponera_longit ars us_M193

70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0

Supplementary Figure 21: Posterior probabilities on the consensus BEAST tree inferred under fossilized birth-death process. Red dots reflect monophyly constraints used in the dating analysis. All ages in Ma.

48 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

Neivamyrmex_texanus_M274 Neivamyrmex_californicus_M272 Neivamyrmex_kiowapache_M275 Neivamyrmex_opacithorax_M276 Neivamyrmex_sumichrasti_M151 Neivamyrmex_asper_M233 Neivamyrmex_compressinodis_M235 Neivamyrmex_cf_nyensis_M296 Neivamyrmex_melanocephalus_M154 Neivamyrmex_impudens_M237 Neivamyrmex_gibbatus_M139 Neivamyrmex_distans_M242 Neivamyrmex_alfaroi_M240 Neivamyrmex_adnepos_M236 Neivamyrmex_EC_M175 Neivamyrmex_swainsonii_M169 Eciton_rapax_M145 Eciton_hamatum_M293 Eciton_burchellii_M273 Eciton_lucanoides_M239 Eciton_vagans_M184 Eciton_quadriglume_M254 Eciton_mexicanum_M238 Nomamyrmex_hartigii_M220 Nomamyrmex_esenbecki_M129 Labidus_spininodis_M172 Labidus_praedator_M152 Labidus_coecus_M173 Cheliomyrmex_cf_morosus_M217 Cheliomyrmex_cf_andicola_M174 Cheliomyrmex_PE_M146 Leptanilloides_gracilis_M187 Leptanilloides_erinys_M246 Leptanilloides_femoralis_M188 Leptanilloides_nubecula_M167 Leptanilloides_mckennae_D0228 Sphinctomyrmex_stali_M253 Sphinctomyrmex_marcoyi_M202 Acanthostichus_serratulus_M166 Acanthostichus_GF_M208 Acanthostichus_BR_M252 Acanthostichus_AZ_M278 Acanthostichus_AZ_M277 Cylindromyrmex_whymperi_M271 Cylindromyrmex_darlingtoni_M211 Cylindromyrmex_brasiliensis_M140 Cylindromyrmex_meinerti_D778 Neocerapachys_cf_splendens_M251 Neocerapachys_BR_M295 Neocerapachys_neotropicus_M134 Neocerapachys_CR_M209 Aenictus_hoelldobleri_M200 Aenictus_fuchuanensis_M201 Aenictus_turneri_M135 Aenictus_gracilis_M199 Aenictus_cornutus_M230 Aenictus_currax_M245 Aenictus_rotundicollis_M232 Aenictus_laeviceps_M269 Aenictus_hodgsoni_M198 Aenictus_fergusoni_M223 Aenictus_ZA_M228 Aenictus_UG_M143 Aenictus_camposi_M222 Aenictus_levior_M231 Aenictus_bobaiensis_M197 Aenictus_latifemoratus_M266 Aenictus_yamanei_M265 Aenictus_silvestrii_M221 Dorylus_rubellus_M287 Dorylus_UG_M153 Dorylus_molestus_M290 Dorylus_mayri_M291 Dorylus_braunsi_M289 Dorylus_affinis_M177 Dorylus_kohli_M180 Dorylus_spininodis_M292 Dorylus_fulvus_M179 Dorylus_cf_fulvus_M149 Dorylus_fimbriatus_M288 Dorylus_orientalis_M244 Dorylus_conradti_M178 Dorylus_laevigatus_M157 Aenictogiton_ZM02_M181 Aenictogiton_UG_M189 Yunodorylus_eguchii_M247 Yunodorylus_TH_M191 Yunodorylus_paradoxus_M190 Yunodorylus_TH_M160 Chrysapace_cf_crawleyi_M262 Chrysapace_TH_M156 Chrysapace_cf_sauteri_M261 Chrysapace_MG_M155 Cerapachys_antennatus_M263 Cerapachys_TH_M203 Cerapachys_MY_M264 Cerapachys_sulcinodis_M243 Eburopone_MG_M214 Eburopone_MG_M213 Eburopone_MG_M195 Eburopone_MG_M130 Eburopone_UG_M259 Eburopone_CM02_D0788 Ooceraea_PG_M248 Ooceraea_FJ06_M168 Ooceraea_MY_M270 Ooceraea_biroi_Genome Ooceraea_australis_M138 Ooceraea_fragosa_D0842 Syscia_MY_M176 Syscia_MY_M147 Syscia_typhla_D0841 Syscia_augustae_M196 Syscia_GT_M127 Eusphinctus_TH_M158 Simopone_trita_M185 Simopone_rex_M133 Simopone_marleyi_M171 Simopone_conradti_M144 Simopone_dryas_M224 Simopone_grandidieri_M186 Simopone_cf_oculata_D0792 Tanipone_hirsuta_M279 Tanipone_aglandula_M280 Tanipone_zona_M182 Tanipone_scelesta_M159 Vicinopone_conciliatrix_M128 Parasyscia_wittmeri_M282 Parasyscia_UG_M218 Parasyscia_UG_M219 Parasyscia_dohertyi_M142 Parasyscia_PG_M204 Parasyscia_VN_M207 Parasyscia_MG_M212 Parasyscia_UG_M281 Zasphinctus_imbecilis_M170 Zasphinctus_PG_M285 Zasphinctus_trux_M136 Zasphinctus_KE_M227 Zasphinctus_TH_M192 Zasphinctus_MZ_M229 Lividopone_MG_M216 Lividopone_MG_M194 Lividopone_MG_M294 Lividopone_MG_M132 Lioponera_ruficornis_M165 Lioponera_princeps_M137 Lioponera_marginata_M249 Lioponera_PG_M250 Lioponera_cf_suscitata_M267 Lioponera_MY_M268 Lioponera_nr_mayri_M215 Lioponera_nr_kraepelinii_M131 Lioponera_vespula_M210 Lioponera_longitarsus_M193

Supplementary Figure 22: BAMM rate shift tree showing the overall best fit configuration. Red- filled circles signify placement of the shifts.

49 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

0.41 A 0.3 D

0.19

0.085

Supplementary Figure 23: BAMM rate shift tree showing net diversification rates. A: Aenictus, D: Dorylus, L: Lioponera, N: Neivamyrmex.

50 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

f = 0.21 f = 0.19 f = 0.16 N N N

A A A D D D

L L L

f = 0.047 f = 0.043 f = 0.035 N N N

A A A D D D

L L L

f = 0.033 f = 0.025 f = 0.02 N N N

A A A D D D

L L L

Supplementary Figure 24: BAMM plot showing nine most common shift configurations in the credible set. The ”f” number corresponds to the proportion of the posterior samples in which this configuration is present. A: Aenictus, D: Dorylus, L: Lioponera, N: Neivamyrmex.

51 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

L D A N

N 1

A 0.5 D

Supplementary Figure 25: BAMM cohort plot. Blocks signify comparisons of shift regimes among species and clades, except across the diagonal which represents the comparison of a species to itself. A: Aenictus, D: Dorylus, L: Lioponera, N: Neivamyrmex.

52 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

N Neivamyrmex texanus M274 N Neivamyrmex californicus M272 N Neivamyrmex kiowapache M275 TN Neivamyrmex opacithorax M276 T Neivamyrmex sumichrasti M151 T Neivamyrmex asper M233 T Neivamyrmex compressinodis M235 N Neivamyrmex cf nyensis M296 TN Neivamyrmex melanocephalus M154 T Neivamyrmex impudens M237 T Neivamyrmex gibbatus M139 T Neivamyrmex distans M242 T Neivamyrmex alfaroi M240 T Neivamyrmex adnepos M236 T Neivamyrmex EC M175 TN Neivamyrmex swainsonii M169 T T Eciton rapax M145 T Eciton hamatum M293 N T Eciton burchellii M273 T Eciton lucanoides M239 P T Eciton vagans M184 T Eciton quadriglume M254 E T Eciton mexicanum M238 T Nomamyrmex hartigii M220 M TN Nomamyrmex esenbecki M129 O T Labidus spininodis M172 T Labidus praedator M152 A TN Labidus coecus M173 T Cheliomyrmex cf morosus M217 TN T Cheliomyrmex cf andicola M174 T Cheliomyrmex PE M146 TP T Leptanilloides gracilis M187 T Leptanilloides erinys M246 TE T Leptanilloides femoralis M188 TM T Leptanilloides nubecula M167 T Leptanilloides mckennae D0228 TO T Sphinctomyrmex stali M253 T Sphinctomyrmex marcoyi M202 TA T Acanthostichus serratulus M166 T Acanthostichus GF M208 NP T Acanthostichus BR M252 N Acanthostichus AZ M278 NE N Acanthostichus AZ M277 NM T Cylindromyrmex whymperi M271 T Cylindromyrmex darlingtoni M211 NO T Cylindromyrmex brasiliensis M140 T Cylindromyrmex meinerti D778 NA T Neocerapachys cf splendens M251 T Neocerapachys BR M295 PE T Neocerapachys neotropicus M134 T Neocerapachys CR M209 PM O Aenictus hoelldobleri M200 O Aenictus fuchuanensis M201 PO A Aenictus turneri M135 PA O Aenictus gracilis M199 O Aenictus cornutus M230 EM A Aenictus currax M245 O Aenictus rotundicollis M232 EO O Aenictus laeviceps M269 O Aenictus hodgsoni M198 EA POAenictus fergusoni M223 E Aenictus ZA M228 MO E Aenictus UG M143 O Aenictus camposi M222 MA O Aenictus levior M231 OA O Aenictus bobaiensis M197 O Aenictus latifemoratus M266 TNP O Aenictus yamanei M265 O Aenictus silvestrii M221 TNE E Dorylus rubellus M287 E Dorylus UG M153 TNM E Dorylus molestus M290 E Dorylus mayri M291 TNO E Dorylus braunsi M289 TNA E Dorylus affinis M177 E Dorylus kohli M180 TPE E Dorylus spininodis M292 PE Dorylus fulvus M179 TPM E Dorylus cf fulvus M149 E Dorylus fimbriatus M288 TPO O Dorylus orientalis M244 E Dorylus conradti M178 TPA O Dorylus laevigatus M157 E Aenictogiton ZM02 M181 TEM E Aenictogiton UG M189 TEO O Yunodorylus eguchii M247 O Yunodorylus TH M191 TEA O Yunodorylus paradoxus M190 O Yunodorylus TH M160 TMO O Chrysapace cf crawleyi M262 O Chrysapace TH M156 TMA O Chrysapace cf sauteri M261 TOA M Chrysapace MG M155 O Cerapachys antennatus M263 NPE O Cerapachys TH M203 O Cerapachys MY M264 NPM O Cerapachys sulcinodis M243 M Eburopone MG M214 NPO M Eburopone MG M213 M Eburopone MG M195 NPA M Eburopone MG M130 E Eburopone UG M259 NEM E Eburopone CM02 D0788 NEO A Ooceraea PG M248 A Ooceraea FJ06 M168 NEA O Ooceraea MY M270 O Ooceraea biroi Genome NMO A Ooceraea australis M138 O Ooceraea fragosa D0842 NMA O Syscia MY M176 NOA O Syscia MY M147 O Syscia typhla D0841 PEM N Syscia augustae M196 T Syscia GT M127 PEO O Eusphinctus TH M158 M Simopone trita M185 PEA M Simopone rex M133 E Simopone marleyi M171 PMO E Simopone conradti M144 E Simopone dryas M224 PMA M Simopone grandidieri M186 POA O Simopone cf oculata D0792 M Tanipone hirsuta M279 EMO M Tanipone aglandula M280 M Tanipone zona M182 EMA M Tanipone scelesta M159 E Vicinopone conciliatrix M128 EOA E Parasyscia wittmeri M282 E Parasyscia UG M218 MOA E Parasyscia UG M219 O Parasyscia dohertyi M142 A Parasyscia PG M204 O Parasyscia VN M207 M Parasyscia MG M212 E Parasyscia UG M281 A Zasphinctus imbecilis M170 A Zasphinctus PG M285 A Zasphinctus trux M136 E Zasphinctus KE M227 O Zasphinctus TH M192 E Zasphinctus MZ M229 M Lividopone MG M216 M Lividopone MG M194 M Lividopone MG M294 M Lividopone MG M132 A Lioponera ruficornis M165 A Lioponera princeps M137 O Lioponera marginata M249 A Lioponera PG M250 O Lioponera cf suscitata M267 O Lioponera MY M268 M Lioponera nr mayri M215 M Lioponera nr kraepelinii M131 E Lioponera vespula M210 POLioponera longitarsus M193

80 60 40 20 0 Ma Supplementary Figure 26: Relative likelihoods of ranges estimations from BioGeoBEARS under DEC+J, averaged over 100 posterior BEAST trees. Pie charts at the nodes correspond to ancestral state estimations and pie charts on the corners correspond to ranges immediately following speciation. The region names are abbreviated as follows: Neotropical (T), Nearctic (N), Palearctic (P), Afrotropical (E), Malagasy (M), Indomalayan (O), and Australasian (A). All ages in Ma.

53 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

N Neivamyrmex texanus M274 N Neivamyrmex californicus M272 N Neivamyrmex kiowapache M275 TNNeivamyrmex opacithorax M276 T Neivamyrmex sumichrasti M151 T Neivamyrmex asper M233 T Neivamyrmex compressinodis M235 N Neivamyrmex cf nyensis M296 TN Neivamyrmex melanocephalus M154 T Neivamyrmex impudens M237 T Neivamyrmex gibbatus M139 T Neivamyrmex distans M242 T Neivamyrmex alfaroi M240 T Neivamyrmex adnepos M236 T Neivamyrmex EC M175 TN Neivamyrmex swainsonii M169 T Eciton rapax M145 T Eciton hamatum M293 T Eciton burchellii M273 T Eciton lucanoides M239 T Eciton vagans M184 T Eciton quadriglume M254 T Eciton mexicanum M238 T Nomamyrmex hartigii M220 TN Nomamyrmex esenbecki M129 T Labidus spininodis M172 T Labidus praedator M152 TN Labidus coecus M173 T Cheliomyrmex cf morosus M217 T Cheliomyrmex cf andicola M174 T Cheliomyrmex PE M146 T Leptanilloides gracilis M187 T Leptanilloides erinys M246 T Leptanilloides femoralis M188 T Leptanilloides nubecula M167 T Leptanilloides mckennae D0228 T Sphinctomyrmex stali M253 T Sphinctomyrmex marcoyi M202 T Acanthostichus serratulus M166 T Acanthostichus GF M208 T Acanthostichus BR M252 N Acanthostichus AZ M278 N Acanthostichus AZ M277 T Cylindromyrmex whymperi M271 T Cylindromyrmex darlingtoni M21 T T Cylindromyrmex brasiliensis M140 N T Cylindromyrmex meinerti D778 P T Neocerapachys cf splendens M251 T Neocerapachys BR M295 E T Neocerapachys neotropicus M134 M T Neocerapachys CR M209 O O Aenictus hoelldobleri M200 O Aenictus fuchuanensis M201 A A Aenictus turneri M135 TN O Aenictus gracilis M199 TP O Aenictus cornutus M230 A Aenictus currax M245 TE O Aenictus rotundicollis M232 TM O Aenictus laeviceps M269 TO O Aenictus hodgsoni M198 POAenictus fergusoni M223 TA E Aenictus ZA M228 NP E Aenictus UG M143 NE O Aenictus camposi M222 O Aenictus levior M231 NM O Aenictus bobaiensis M197 NO O Aenictus latifemoratus M266 NA O Aenictus yamanei M265 O Aenictus silvestrii M221 PE E Dorylus rubellus M287 PM E Dorylus UG M153 PO E Dorylus molestus M290 E Dorylus mayri M291 PA E Dorylus braunsi M289 EM E Dorylus affinis M177 EO E Dorylus kohli M180 E Dorylus spininodis M292 EA PE Dorylus fulvus M179 MO E Dorylus cf fulvus M149 MA E Dorylus fimbriatus M288 O Dorylus orientalis M244 OA E Dorylus conradti M178 TNP O Dorylus laevigatus M157 TNE E Aenictogiton ZM02 M181 E Aenictogiton UG M189 TNM O Yunodorylus eguchii M247 TNO O Yunodorylus TH M191 TNA O Yunodorylus paradoxus M190 O Yunodorylus TH M160 TPE O Chrysapace cf crawleyi M262 TPM O Chrysapace TH M156 TPO O Chrysapace cf sauteri M261 M Chrysapace MG M155 TPA O Cerapachys antennatus M263 TEM O Cerapachys TH M203 TEO O Cerapachys MY M264 O Cerapachys sulcinodis M243 TEA M Eburopone MG M214 TMO M Eburopone MG M213 TMA M Eburopone MG M195 M Eburopone MG M130 TOA E Eburopone UG M259 NPE E Eburopone CM02 D0788 NPM A Ooceraea PG M248 A Ooceraea FJ06 M168 NPO O Ooceraea MY M270 NPA O Ooceraea biroi Genome NEM A Ooceraea australis M138 O Ooceraea fragosa D0842 NEO O Syscia MY M176 NEA O Syscia MY M147 NMO O Syscia typhla D0841 N Syscia augustae M196 NMA T Syscia GT M127 NOA O Eusphinctus TH M158 PEM M Simopone trita M185 M Simopone rex M133 PEO E Simopone marleyi M171 PEA E Simopone conradti M144 PMO E Simopone dryas M224 M Simopone grandidieri M186 PMA O Simopone cf oculata D0792 POA M Tanipone hirsuta M279 EMO M Tanipone aglandula M280 M Tanipone zona M182 EMA M Tanipone scelesta M159 EOA E Vicinopone conciliatrix M128 MOA E Parasyscia wittmeri M282 E Parasyscia UG M218 E Parasyscia UG M219 O Parasyscia dohertyi M142 A Parasyscia PG M204 O Parasyscia VN M207 M Parasyscia MG M212 E Parasyscia UG M281 A Zasphinctus imbecilis M170 A Zasphinctus PG M285 A Zasphinctus trux M136 E Zasphinctus KE M227 O Zasphinctus TH M192 E Zasphinctus MZ M229 M Lividopone MG M216 M Lividopone MG M194 M Lividopone MG M294 M Lividopone MG M132 A Lioponera ruficornis M165 A Lioponera princeps M137 O Lioponera marginata M249 A Lioponera PG M250 O Lioponera cf suscitata M267 O Lioponera MY M268 M Lioponera nr mayri M215 M Lioponera nr kraepelinii M131 E Lioponera vespula M210 POLioponera longitarsus M193

80 60 40 20 0 Ma Supplementary Figure 27: Relative likelihoods of ranges from BioGeoBEARS under DEC+J estimated on the BEAST consensus tree. Pie charts at the nodes correspond to ancestral state estimations and pie charts on the corners correspond to ranges immediately following speciation. The region names are abbreviated as follows: Neotropical (T), Nearctic (N), Palearctic (P), Afrotropical (E), Malagasy (M), Indomalayan (O), and Australasian (A). All ages in Ma.

54 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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.

0.09

CFV 0.06 R

0.03

B slow evolving homogeneous high bootstrap

0.8

0.6 ession

0.4 Slope of r eg

0.2

0.0

slow evolving homogeneous high bootstrap Loci

Supplementary Figure 28: Box plots comparison of properties of slow-evolving, compositionally homogeneous, and ”high signal” or high average bootstrap loci. A: Relative composition frequency variability (RCFV), B: Slope of regression of p-distances against distances on ML tree from a locus. Higher RCFV signifies more compositional heterogeneity and higher slope of regression signifies less potential for saturation.

55 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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: Voucher specimens used in this study. CASENT numbers correspond to records on AntWeb.org.

Taxon Name Specimen Code Country Year Collected Acanthostichus AZ M277 CASENT0324689 United States 2010 Acanthostichus AZ M278 CASENT0324880 United States 2009 Acanthostichus BR M252 CASENT0732106 Brazil 2014 Acanthostichus GF M208 CASENT0732074 French Guiana 2006 Acanthostichus serratulus M166 CASENT0732042 Ecuador 2003 Aenictogiton UG M189 CASENT0317577 Uganda 2012 Aenictogiton ZM02 M181 CASENT0732056 Zambia 2005 Aenictus bobaiensis M197 CASENT0732068 China 2014 Aenictus camposi M222 CASENT0732087 Malaysia 2014 Aenictus cornutus M230 CASENT0732089 Malaysia 2014 Aenictus currax M245 CASENT0732104 Papua New Guinea 2010 Aenictus fergusoni M223 CASENT0732088 India 2001 Aenictus fuchuanensis M201 CASENT0732072 China 2013 Aenictus gracilis M199 CASENT0732070 Malaysia 2008 Aenictus hodgsoni M198 CASENT0732069 China 2012 Aenictus hoelldobleri M200 CASENT0732071 China 2013 Aenictus laeviceps M269 CASENT0702955 Malaysia 2014 Aenictus latifemoratus M266 CASENT0392370 Malaysia 2014 Aenictus levior M231 CASENT0732090 Malaysia 2014 Aenictus rotundicollis M232 CASENT0732091 Malaysia 2014 Aenictus silvestrii M221 CASENT0732086 Malaysia 2014 Aenictus turneri M135 CASENT0732016 Australia 2004 Aenictus UG M143 CASENT0732021 Uganda 2012 Aenictus yamanei M265 CASENT0385898 Malaysia 2014 Aenictus ZA M228 CASENT0764132 South Africa 2011 Cerapachys antennatus M263 CASENT0384548 Malaysia 2014 Cerapachys MY M264 CASENT0384921 Malaysia 2014 Cerapachys sulcinodis M243 CASENT0732102 China 2015 Cerapachys TH M203 CASENT0268115 Thailand 2006 Cheliomyrmex cf andicola M174 CASENT0732049 Ecuador 2009 Cheliomyrmex cf morosus M217 CASENT0732082 Honduras 2010 Cheliomyrmex sp M146 CASENT0732024 Peru 2013 Chrysapace cf crawleyi M262 CASENT0391566 Malaysia 2014 Chrysapace cf sauteri M261 CASENT0385026 Malaysia 2014 Chrysapace MG M155 CASENT0304584 Madagascar 2013 Chrysapace TH M156 CASENT0278856 Thailand 2006 Cylindromyrmex brasiliensis M140 CASENT0731132 Venezuela 2008 Cylindromyrmex brasiliensis M72 CASENT0731132 Venezuela 2008 Cylindromyrmex darlingtoni M211 CASENT0756069 Dominican Republic 2015 Cylindromyrmex whymperi M271 CASENT0732116 Costa Rica 2003 Dorylus affinis M177 CASENT0732052 Kenya 2004 Dorylus braunsi M289 CASENT0732037 Kenya 2004 Dorylus cf fulvus M149 CASENT0732026 Uganda 2012 Dorylus conradti M178 CASENT0732053 Kenya 2004 Dorylus fimbriatus M288 CASENT0732036 Kenya 2004 Dorylus fulvus M179 CASENT0732054 Kenya 2008 Dorylus kohli M180 CASENT0732055 Kenya 2007 Dorylus laevigatus M157 CASENT0202245 Malaysia 2010 Dorylus mayri M291 CASENT0732123 Nigeria 2005 Dorylus molestus M290 CASENT0732122 Uganda 2012 Dorylus orientalis M244 CASENT0732103 China 2015

56 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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. Voucher specimens used in this study. CASENT numbers correspond to records on AntWeb.org.

Taxon Name Specimen Code Country Year Collected Dorylus rubellus M287 CASENT0732035 Nigeria 2005 Dorylus spininodis M292 CASENT0732124 Uganda 2012 Dorylus UG M153 CASENT0732030 Uganda 2012 Eburopone CM02 D0788 CASENT0178891 Cameroon 2000 Eburopone MG M130 CASENT0732012 Madagascar 2013 Eburopone MG M195 CASENT0732066 Madagascar 2013 Eburopone MG M213 CASENT0732077 Madagascar 2013 Eburopone MG M214 CASENT0732078 Madagascar 2013 Eburopone UG M259 CASENT0352799 Uganda 2012 Eciton burchellii M273 CASENT0732118 United States 2007 Eciton hamatum M293 CASENT0732125 Mexico 2014 Eciton lucanoides M239 CASENT0732098 Panama 2015 Eciton mexicanum M238 CASENT0732097 Costa Rica 2013 Eciton quadriglume M254 CASENT0732108 Brazil 2011 Eciton rapax M145 CASENT0732023 Peru 2013 Eciton vagans M184 CASENT0732059 Costa Rica 2004 Eusphinctus TH M158 CASENT0285681 Thailand 2006 Labidus coecus M173 CASENT0732048 Ecuador 2009 Labidus praedator M152 CASENT0732029 Mexico 2014 Labidus spininodis M172 CASENT0732047 Ecuador 2009 Leptanilloides erinys M246 CASENT0234600 Ecuador 2009 Leptanilloides femoralis M188 CASENT0732063 Venezuela 2008 Leptanilloides gracilis M187 CASENT0732062 Mexico 2008 Leptanilloides mckennae D0228 CASENT0106086 Costa Rica 2003 Leptanilloides nubecula M167 CASENT0732043 Ecuador 2004 Lioponera cf suscitata M267 CASENT0386895 Malaysia 2014 Lioponera longitarsus M193 CASENT0732064 Bangladesh 2014 Lioponera marginata M249 CASENT0219054 Papua New Guinea 2009 Lioponera MY M268 CASENT0389090 Malaysia 2014 Lioponera nr kraepelinii M131 CASENT0732013 Madagascar 2013 Lioponera nr mayri M215 CASENT0732079 Madagascar 2013 Lioponera PG M250 CASENT0215868 Papua New Guinea 2009 Lioponera princeps M137 CASENT0732018 Australia 2006 Lioponera ruficornis M165 CASENT0732041 Australia 2005 Lioponera vespula M210 CASENT0234847 Uganda 2012 Lividopone MG M132 CASENT0732014 Madagascar 2013 Lividopone MG M194 CASENT0732065 Madagascar 2013 Lividopone MG M216 CASENT0732080 Madagascar 2013 Lividopone MG M294 CASENT0732126 Madagascar 2013 Neivamyrmex adnepos M236 CASENT0732095 Costa Rica 2006 Neivamyrmex alfaroi M240 CASENT0732099 Costa Rica 2013 Neivamyrmex asper M233 CASENT0732092 Costa Rica 2014 Neivamyrmex californicus M272 CASENT0732117 United States 2004 Neivamyrmex cf nyensis M296 CASENT0249496 United States 2003 Neivamyrmex compressinodis M235 CASENT0732094 Nicaragua 2011 Neivamyrmex distans M242 CASENT0732101 Costa Rica 1998 Neivamyrmex EC M175 CASENT0732050 Ecuador 2009 Neivamyrmex gibbatus M139 CASENT0732019 Venezuela 2008 Neivamyrmex impudens M237 CASENT0732096 Guatemala 2009 Neivamyrmex kiowapache M275 CASENT0732120 United States 2015 Neivamyrmex melanocephalus M154 CASENT0732031 Mexico 2014

57 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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. Voucher specimens used in this study. CASENT numbers correspond to records on AntWeb.org.

Taxon Name Specimen Code Country Year Collected Neivamyrmex opacithorax M276 CASENT0732121 United States 2015 Neivamyrmex sumichrasti M151 CASENT0732028 Mexico 2014 Neivamyrmex swainsonii M169 CASENT0732045 United States 2004 Neivamyrmex texanus M274 CASENT0732119 United States 2015 Neocerapachys BR M295 CASENT0732127 Brazil 2014 Neocerapachys cf splendens M251 CASENT0732105 Brazil 2013 Neocerapachys neotropicus M134 CASENT0732015 Costa Rica 2014 Neocerapachys sp M209 CASENT0732075 Costa Rica 2004 Nomamyrmex esenbecki M129 CASENT0732011 Mexico 2013 Nomamyrmex hartigii M220 CASENT0732085 Costa Rica 2006 Ooceraea australis M138 CASENT0106146 Australia 2006 Ooceraea FJ06 M168 CASENT0732044 Fiji 2006 Ooceraea fragosa D0842 CASENT0106215 Sri Lanka 2005 Ooceraea MY M270 CASENT0722573 Malaysia 2014 Ooceraea PG M248 CASENT0187824 Papua New Guinea 2009 Parasyscia dohertyi M142 CASENT0732020 Malaysia 2010 Parasyscia MG M212 CASENT0732076 Madagascar 2013 Parasyscia PG M204 CASENT021652 Papua New Guinea 2009 Parasyscia UG M218 CASENT0732083 Uganda 2012 Parasyscia UG M219 CASENT0732084 Uganda 2012 Parasyscia UG M281 CASENT0352569 Uganda 2012 Parasyscia VN M207 CASENT0731211 Vietnam 2007 Parasyscia wittmeri M282 CASENT0263905 Saudi Arabia 2011 Simopone cf oculata D0792 CASENT0139634 Thailand 2006 Simopone conradti M144 CASENT0732022 Uganda 2012 Simopone dryas M224 CASENT0764130 Kenya 2006 Simopone grandidieri M186 CASENT0732061 Madagascar 2009 Simopone marleyi M171 CASENT0249324 South Africa 1986 Simopone rex M133 CASENT0731175 Madagascar 2013 Simopone trita M185 CASENT0732060 Madagascar 2003 Sphinctomyrmex marcoyi M202 CASENT073146 Argentina 2013 Sphinctomyrmex stali M253 CASENT0732107 Brazil 2013 Syscia augustae M196 CASENT0732067 United States 2015 Syscia GT M127 CASENT0732010 Guatemala 2009 Syscia MY M147 CASENT0732025 Malaysia 2014 Syscia MY M176 CASENT0732051 Malaysia 2009 Syscia typhla D0841 CASENT0106214 Sri Lanka 2006 Tanipone aglandula M280 CASENT0052407 Madagascar 2003 Tanipone hirsuta M279 CASENT0147732 Madagascar 2008 Tanipone scelesta M159 CASENT0229662 Madagascar 2010 Tanipone zona M182 CASENT0732057 Madagascar 2003 Vicinopone conciliatrix M128 CASENT0731168 Uganda 2012 Yunodorylus eguchii M247 CASENT0731166 Vietnam 2004 Yunodorylus paradoxus M190 CASENT0731165 Malaysia 2006 Yunodorylus TH M160 CASENT0139796 Thailand 2006 Yunodorylus TH M191 CASENT0134717 Thailand 2006 Zasphinctus imbecilis M170 CASENT0732046 Australia 2005 Zasphinctus MZ M229 MCZENT00512765 Mozambique 2012 Zasphinctus PG M285 CASENT0732033 Papua New Guinea 2000 Zasphinctus KE M227 CASENT0764121 Kenya 2012 Zasphinctus TH M192 CASENT0131746 Thailand 2006 Zasphinctus trux M136 CASENT0732017 Australia 2006

58 bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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: Statistics of data matrices used in this study.

Proportion of Number Alignment Percent Alignment Name Parsimony AT Content of Taxa Length Missing Informative Sites Combined Data 164 892,761 15.476 0.478 0.530 ”High-Signal” Loci 164 177,947 17.534 0.565 0.578 Slow-Evolving Loci 164 178,662 10.245 0.348 0.486 Homogeneous Loci 164 97,849 10.177 0.369 0.488 No Aenictus 146 892,761 15.006 0.469 0.528 BEAST Matrix 155 44,079 16.489 0.466 0.531 Amino Acids 164 89,483 16.104 0.138 -

59 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint doi: Supplementary Table 3: Calibration scheme used for penalized likelihood analyses in chronos. MRCA column refers to the most recent common ancestor of two tip names in the maximum likelihood tree obtained from slow-evolving loci matrix. https://doi.org/10.1101/134064

Clade MRCA Minimum Age (Ma) Maximum Age (Ma) Notes This calibration assumes that poneroids and formicoids shared the last common ancestor at least as early as Burmomyrma, a 94.3-99.7 Ma fossil from Burmese Amber. Harpeg- This taxon was has originally assigned to Aneuretinae (Dlussky, 1996) but recently its nathos_saltator_Genome, Root 94 135 placement has been questioned (LaPolla et al., 2013). Here I take a conservative view Acanthos- that it is at least a representative of stem formicoids. Maximum age is based on the tichus_serratulus_M166 upper bound of 95% confidence interval for this node in the time-calibrated phylogeny of the Formicidae by Moreau and Bell (2013). Kyromyrma neffi (Grimaldi and Agosti, 2000) is a 89.3-94.3 Ma old fossil from New

Jersey Amber that shows a clearly preserved acidopore, a unique formicine feature. a ; CC-BY-NC 4.0Internationallicense crown Campono- Kyromyrma is universally treated as a stem Formicinae. Although the upper 95% this versionpostedMay4,2017. Formicinae plus tus_floridanus_Genome, 89.3 120 confidence interval for this node was only 95 Ma in Moreau and Bell (2013), the Myrmicinae Atta_cephalotes_Genome maximum age assumed here is more conservatively placed at 120 Ma to accommodate the fact that Ward et al. (2015) inferred ages of one of the constituent clades, the Myrmicinae, up to 110 Ma. Crown Mymicinae are here calibrated with the Baltic amber Myrmica (Radchenko 60 et al., 2007) from the Priabonian age of Eocene (33.9-37.2 Ma). Although Pogono- Pogonomyrmex was a lineage branching after Myrmica in the phylogeny by Ward et al. crown myrmex_barbatus_Genome, 33.9 110 (2015), the latest UCE-based results (Branstetter et al. (2016), M.G. Branstetter pers. Myrmicinae Atta_cephalotes_Genome comm.) suggest that Pogonomyrmex and Myrmica form a clade within the Myrmicinae. The maximum age is based on the upper bound of 95% confidence interval in time-calibrated phylogeny of (Ward et al., 2015). A recently discovered Baltic Amber fossil recognizable as Chrysapace (author’s The copyrightholderforthispreprint(whichwasnot

personal observation) is used to calibrate the node of Cerapachys, Yunodorylus, and . Cerapachys, Chrysapace_TH_M156, Chrysapace. Although the maximum likelihood tree places Cerapachys as sister to Chrysapace, and 33.9 85 Yunodorylus_TH_M191 Chrysapace, this relationship has low support, so a conservative approach calibrates the Yunodorylus next node down. Maximum age corresponds to the upper bound of 95% confidence interval in the time-calibrated tree of Brady et al. (2014). Cylin- Cylindromyrmex inopinatus from Dominican Amber is considered a sister species of crown dromyrmex_meinerti_D778, extant C. longiceps (De Andrade, 2001) and thus used here to calibrate crown 13.7 43 Cylindromyrmex Cylin- Cylindromyrmex. Maximum age is the upper bound of 95% confidence interval for dromyrmex_darlingtoni_M211 Cylindromyrmex age in Brady et al. (2014). Neivamyrmex ectopus (Wilson, 1985) is a Dominican Amber species that is somewhat difficult to place in the extant Neivamyrmex diversity. A potentially older fossil of crown New Eciton_hamatum_M293, Neivamyrmex comes from Mexican Amber, 16-23 Ma (Coty et al., 2014). The ancestor 16 46 World army ants Neivamyrmex_californicus_M272 of New World army ants is here dated with a minimum age of 16 Ma. Maximum age is based on upper bound of 95% confidence interval age for this clade in (Brady et al., 2014). bioRxiv preprint doi: https://doi.org/10.1101/134064; this version posted May 4, 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: Calibration scheme used for fossilized birth-death process analyses in BEAST. Total group refers to fossil that could be placed in either stem or crown group.

Sampling Deposit Species Group Calibrated Notes Time (Ma) Age (Ma) Chrysapace sp. total Chrysapace 36.6 33.9-37.2 See notes in Supplementary Table 2. Neivamyrmex total 19.2 13.7-20.4 See notes in Supplementary Table 2 ectopus Neivamyrmex Cylindromyrmex total This species was described together with C. electrinus by De Andrade (1998a). Both 19.5 13.7-20.4 antillanus Cylindromyrmex are used to calibrate total group Cylindromyrmex. This fossil was described by De Andrade (1998b) as similar to A. skwarrae, A. quirozi Acanthostichus total and A. arizonensis. Although it is possible that one of the male Acanthostichus 14.5 13.7-20.4 hispaniolicus Acanthostichus specimens sequenced here from Arizona corresponds to A. arizonensis, this fossil is used here to calibrate the total group Acanthostichus. Cylindromyrmex total 18.1 13.7-20.4 See C. antillanus notes above. electrinus Cylindromyrmex crown Cylindromyrmex Cylindromyrmex 14.2 13.7-20.4 See notes in Supplementary Table 2. inopinatus excl. C. meinerti total Neivamyrmex sp. 19.3 16-23 Coty et al. (2014) reported a Neivamyrmex species from Chiapas Amber. Neivamyrmex