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1 Title: 2 Phylogeny, evolution, and classification of the ant genus Lasius, the tribe Lasiini, and the 3 subfamily Formicinae (Hymenoptera: Formicidae) 4 5 Authors and affiliations: 6 B. E. Boudinot1,2, M. L. Borowiec1,3,4, M. M. Prebus1,5* 7 1Department of Entomology & Nematology, University of California, Davis CA 8 2Friedrich-Schiller-Universität Jena, Institut für Spezielle Zoologie, Jena, Germany 9 3Department of Plant Pathology, Entomology and Nematology, University of Idaho, Moscow ID 10 4Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow ID 11 5School of Life Sciences, Arizona State University, Tempe AZ 12 *Corresponding author. 13 14 Author ZooBank LSIDs: 15 Borowiec: http://zoobank.org/urn:lsid:zoobank.org:author:411B711F-605B-4C4B-ABDB- 16 D4D96075CE48 17 Boudinot: http://zoobank.org/urn:lsid:zoobank.org:author:919F03B0-60BA-4379-964D- 18 A56EB582E16D 19 Prebus: http://zoobank.org/urn:lsid:zoobank.org:author:1A6494C7-795E-455C-B66F- 20 7F6C32F76584 21 22 ZooBank Article LSID: http://zoobank.org/urn:lsid:zoobank.org:pub:016059BA-33C3-43B2- 23 ADAD-6807DC5CB6D8 24 25 Running head: Phylogeny and evolution of Lasius and the Lasiini 26 27 Keywords: Integrated taxonomy, morphology, biogeography, convergent evolution, character 28 polarity, total-evidence. 29 30 Count of figures: 10 main text, 20 supplementary. 31 Count of tables: 7 main text, 3 supplementary. 32 Count of references: 182. 33 Count of appendices: 2. 34 35 Taxonomic actions proposed herein are disclaimed until the final published version is 36 released. 37 38 Summary of taxonomic actions: 39 1. Subgenera of Lasius synonymized: Lasius = Acanthomyops syn. rev. = Austrolasius syn. 40 n. = Cautolasius syn. n. = Chthonolasius syn. n. = Dendrolasius syn. n. 41 2. Lasius myrmidon transferred to XXX gen. n. (Lasiini, XXX genus group). 42 3. †Lasius pumilus transferred to †XXX gen. n. (Lasiini, XXX genus group). 43 4. †Pseudolasius boreus transferred to † XXX gen. n. (incertae sedis in Formicinae) (tribal 44 transfer). 45 5. †Glaphyromyrmex transferred to the Formicini from the Lasiini (tribal transfer). bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

46 6. Lasius escamole Reza, 1925 synonymized with Liometopum apiculatum Mayr, 1870, 47 syn. n. (subfamilial transfer). 48 7. Paratrechina kohli (Forel, 1916) transferred to Anoplolepis (Plagiolepidini) (genus and 49 tribal transfer). 50 51 Abstract 52 53 Within the Formicidae, the higher classification of nearly all subfamilies has been recently 54 revised due to the findings of molecular phylogenetics. Here, we integrate morphology and 55 molecular data to holistically address the evolution and classification of the ant genus Lasius, its 56 tribe Lasiini, and their subfamily Formicinae. We accomplish this through a critical re- 57 examination of morphology of extant and fossil taxa, molecular phylogenetic analyses, total- 58 evidence dating under fossilized birth-death process, phylogeography, and ancestral state 59 estimation. We use these results to provide revised taxonomic definitions for the Lasiini and 60 select genera, and we provide a key to the genera of the Lasiini with emphasis on the Lasius 61 genus group. We find that the crown Lasiini originated around the end of the Cretaceous on the 62 Eurasian continent and is divisible into four morphologically distinct clades: Cladomyrma, the 63 Lasius genus group, the Prenolepis genus group, and a previously undetected lineage we name 64 XXX gen. n. The crown of the Lasius genus group is considerably younger than that of the 65 Prenolepis genus group, indicating that extinction has played a major role in the evolution of the 66 former clade. Lasius itself is divided into two well-supported monophyletic groups which are 67 approximately equally speciose. We present evidence that temporary social parasitism and 68 fungiculture arose in Lasius two times independently. Additionally, we recover the paraphyly of 69 three Lasius subgenera and propose replacing all subgenera with an informal species group 70 classification: Lasius = Acanthomyops syn. rev., = Austrolasius syn. n., = Cautolasius syn. n., = 71 Chthonolasius syn. n., = Dendrolasius syn. n. Total-evidence analysis reveals that the Baltic- 72 region amber fossil species †Lasius pumilus and †Pseudolasius boreus are misplaced to genus; 73 we therefore designate †XXX gen. n. for the former and †XXX gen. n. for the latter. Further, we 74 transfer †XXX and †Glaphyromyrmex out of the tribe, considering the former to be incertae sedis 75 in the subfamily, and the latter a member of the Formicini (tribal transfer). Two final 76 taxonomic actions are deemed necessary: synonymy of Lasius escamole Reza, 1925 with 77 Liometopum apiculatum Mayr, 1870 syn. n. (subfamilial transfer), and transfer of Paratrechina 78 kohli to Anoplolepis (tribal transfer, forming A. kohli (Forel, 1916) n. comb.). 79 80 81 Introduction 82 83 Ant taxonomy is a rapidly transforming field, incorporating studies across data types and 84 systematic methodologies. Built from the synthesis of all species-level and higher taxonomic 85 works on the Formicidae, the morphology-based systematic hypotheses of Brown (e.g., 1954; see 86 also Keller 2011) and Bolton (e.g., 1990a,b,c, 1994, 1995, 2003) were first tested using 87 parsimony methods (e.g., Ward 1990, 1994; Agosti 1991; Baroni Urbani et al. 1992; Shattuck 88 1992, 1995; Lattke 1994; Grimaldi et al. 1997; Agosti et al. 1999, Keller 2000) followed by 89 likelihood-based and Bayesian phylogenetics (e.g., Felsenstein 1983, 1988, 2001, 2003). 90 Whereas a minority of ant phylogenetic studies combined morphological and multi-locus data 91 (Ward & Brady 2003; Astruc et al. 2004; Janda et al. 2004; Ward & Downie 2005; Maruyama et

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92 al. 2008) and/or included fossils as terminals (Barden et al. 2017a, Prebus 2017), most 93 phylogenetic studies have only utilized data from Sanger-sequenced loci (e.g., Moreau et al. 94 2006; Brady et al. 2006; Ward et al. 2010, 2015; Ward & Fisher 2016; Borowiec et al. 2019). 95 More recently, high-throughput sequencing platforms enabled genomic approaches to ant 96 systematics, such as targeted enrichment of ultraconserved elements (UCEs) (e.g., Faircloth et al. 97 2015; Blaimer et al. 2015, 2016, 2018; Jesovnik et al. 2017; Branstetter et al. 2017a,b,c; Prebus 98 2017, 2021; Sosa-Calvo et al. 2018; Borowiec 2019; Branstetter & Longino 2019), RADseq 99 (Fischer et al. 2015, Darwell et al. 2019, Liu et al. 2020), and transcriptomics (Jesovnik et al. 100 2016, Romiguier et al. 2018). Here, we employ an array of phylogenetic methods, including 101 traditional comparative morphology, total-evidence divergence dating (Ronquist et al. 2012a, 102 2016), phylogeographic analysis (Bouckaert et al. 2014; Matzke 2014), ancestral state estimation 103 (Paradis et al. 2004, Paradis & Schliep 2018), and species tree estimation (Mirarab & Warnow 104 2015) to holistically address the phylogeny, evolution, and classification of the genus Lasius F., 105 1904, its tribe Lasiini Ashmead, 1905, and the subfamily Formicinae Lepeletier de Saint- 106 Fargeau, 1934 (see Table 1 for a summary of our questions and methods). 107 Lasius is a northern temperate ant genus that is important in terms of its relative ecological 108 impact and as a model for studying social insect biology. For example, Lasius was a frequent 109 choice in both early and recent work on pheromone communication (Bergström & Löfqvist 110 1970; Holman et al. 2013), dominance hierarchy, competition, and succession in ant 111 communities (Pontin 1961; Traniello & Levings 1986; Markó & Czechowski 2004; Parr & Gibb 112 2010), and the evolution of social parasitism (Hasegawa 1998; Janda et al. 2004), among other 113 topics (Hölldobler & Wilson 1990; Quque & Bles 2020). Lasius ants are particularly known for 114 well-documented symbioses with honeydew producing insects and for temporary social 115 parasitism. The latter phenomenon is a mode of colony foundation for many species of Lasius, 116 and involves parasite queens invading nests of other Lasius, killing the host queen, and using 117 subordinated workers to raise the first batch of their own workers. This is followed by attrition of 118 the host and eventually a colony consisting solely of parasite conspecifics. Social parasitism is 119 known in species currently classified in subgenera Austrolasius, Acanthomyops, Chthonolasius, 120 and Dendrolasius (Hölldobler & Wilson 1990; Buschinger 2009; Raczkowski & Luque 2011). 121 Certain species of the subgenus Chthonolasius are known to use ascomycete fungi to bind 122 masticated wood and soil to reinforce nest walls. This association is considered an example of 123 fungiculture because the ants provide the fungi with honeydew and prevent overgrowing by 124 competing fungal species (Schlick-Steiner et al. 2008). 125 Historically, Lasius was split by Fabricius (1804) from Linnaeus’s (1758) all-encompassing 126 genus Formica based on features of the mouthparts; Latreille (1809) soon after erected the 127 family Formicidae for the ant genera known at the time. The first tribal classification of the 128 family is attributed to the hymenopterist Amédée Michel Louis Lepeletier de Saint-Fargeau 129 (1835), who included les Formicites, a precursor to the modern Formicinae. Lepeletier de Saint- 130 Fargeau’s classification of ants incorporated information on wing venation (Lepeletier de Saint- 131 Fargeau 1836). Later investigators relied on natural history, external, as well as internal 132 morphology, especially of the proventriculus, which is a valve located between the crop and 133 midgut of ants and other insects (Eisner 1957). A relatively stable supra-generic classification of 134 the subfamily emerged around the turn of the prior century (Forel 1878, 1886, 1912; Emery 135 1895, 1925), which was used into the beginning of the 1990s. Agosti (1991) provided a critical 136 reassessment of this system and used new exoskeletal characters to diagnose four informal genus 137 groups in the Formicinae. Expanding on Agosti’s skepticism and system, Bolton (2003) divided

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138 the Formicinae into a total of 11 morphologically defined tribes, which were placed in the 139 “lasiine” and “formicine” tribe groups, with three and six tribes, respectively, plus two unplaced 140 tribes. 141 Ant classification has increasingly become more reliant on phylogenetic trees inferred from 142 molecular data, with subsequent morphological revisionary work. The Formicinae is no 143 exception: Blaimer et al. (2015) provided a comprehensive molecular phylogenetic study of the 144 subfamily which was used for a morphological reassessment of its tribal classification, 145 emphasizing the hyperdiverse Camponotini (Ward et al. 2016; Ward & Boudinot 2021). Like 146 Bolton (2003), Ward et al. (2016) also recognized 11 tribes, for which the generic composition of 147 most remained largely unchanged. The notable exceptions included tribes Lasiini, Melophorini, 148 and Plagiolepidini, whose scope was substantially revised but for which no updated 149 morphological circumscription was provided. Within the Lasiini, the modern taxonomy of Lasius 150 was initiated by Wilson’s revision of the genus (Wilson 1955), for which six subgenera have 151 been recognized (Ward 2005). Monophyly of the subgenera has not been questioned except for 152 the nominotypical subgenus (Janda et al. 2004; Maruyama et al. 2008; see Fig. 1A, B, C), but the 153 monophyly of Lasius was cast into doubt by Blaimer et al. (2015), whose analysis of Sanger data 154 suggested paraphyly of the genus with respect to Myrmecocystus. A more recent study using 155 UCEs and a limited taxonomic sampling of the genus recovered Lasius as monophyletic (van 156 Elst et al. 2021). 157 In this contribution we focus on the genus Lasius and the tribe Lasiini, integrating molecular 158 and morphological methods to address several questions about their classification and evolution 159 (Table 1). In addition to the monophyly of Lasius and its subgenera (Table 1, Questions 1 & 2), 160 we are also interested in the fossil history of the genus and the Lasiini (e.g., Mayr 1868; Wheeler 161 1915; Wilson 1955; LaPolla et al. 2013; Barden 2017a), from both taxonomic (Table 1, Question 162 3) and evolutionary perspectives (Table 1, Questions 4 & 5). We propose a revised 163 morphological definition of Lasius, the Lasiini, and a number of newly recognized nodes on the 164 formicine phylogeny, resulting from explicit integration of morphology of both extant and fossil 165 lineages into our phylogenetic analyses. We also provide a systematic, illustrated key to the 166 extant genera of the tribe (Table 1, Question 6). Our overarching hope is to encourage the 167 reconciliation of morphological and molecular systematics . 168 169 Materials and methods 170 Study design 171 Our study addresses six questions outlined in Table 1. A summary of methods is provided below 172 with additional details available as supplementary text. In brief, our objective is to holistically 173 evaluate the phylogeny and evolution of extant and extinct Lasius and Lasiini in the context of 174 the Formicinae. We employed an array of tools to three datasets comprising a broad sample of 175 the Formicinae and selected non-formicine outgroups: (1) a set of previously published 959 176 individual UCE locus alignments for 89 extant terminals (Blaimer et al. 2015, Question 1); (2) a 177 set of 11 Sanger loci for 135 extant terminals (Questions 1, 3–5); (3) a set of 11 Sanger loci for 178 55 extant terminals in the Lasiini (Question 2); and (4) a set of 110 binary morphological 179 characters for 154 terminals (136 extant and 18 extinct) (Question 3). Additionally, we tested the 180 polarity of six morphological characters traditionally used in the classification of the Formicinae 181 (Question 5). In order to revise the generic classification of the Lasiini and to provide a new key, 182 we comparatively evaluated several qualitative traits, and took six linear measurements for 109 183 specimens of 56 species (Question 6). The three new genera we recognize are registered with

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184 ZooBank (LSIDs in respective taxonomic accounts below). All Bayesian inference (BI) and 185 maximum likelihood (ML) analyses were conducted on the CIPRES Science Gateway V. 3.3 186 (Miller et al. 2010, accessed at http://www.phylo.org/portal2). 187 188 Sanger sequence data 189 New sequence data were generated from a selection of 20 Lasius and two Myrmecocystus 190 species. Our sampling of Lasius was designed to encompass the morphological, geographical, 191 and life history variation within the genus (Table 2), spanning all currently recognized subgenera 192 (Wilson 1955; Bolton 2003; Ward 2005), and was partially guided by previous molecular 193 phylogenies (Janda et al. 2004, Maruyama et al. 2008). Notably, we included a species closely 194 related to or conspecific with the aberrant and previously unsampled species, Lasius atopus, 195 from California, U.S.A. as well as another unsampled species, Lasius myrmidon, described from 196 Greece (Mei 1998). Although we included only three species of Myrmecocystus, they were 197 chosen to span the root node of the genus (O’Meara 2008, van Elst et al. 2021). We also included 198 data from all genera recognized in the Prenolepis genus group (LaPolla et al. 2010, LaPolla et al. 199 2012), the majority of formicine genera, and a sample of formicoid outgroups. Voucher 200 specimens from DNA extractions made for this study are deposited in the University of 201 California Bohart Museum of Entomology collection (UCDC) and collection data associated 202 with these specimens can be found in Table 2 and on AntWeb at http://antweb.org/. 203 We used the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA) to conduct 204 non-destructive DNA extractions by piercing the cuticle of each specimen, then soaking the 205 specimens overnight in a solution of proteinase K and the lysis buffer provided with the kit. For 206 the remainder of the extraction, we followed the manufacturer’s protocol except for eluting the 207 extract with sterilized water rather than the supplied buffer. With these 22 extractions, we 208 amplified and sequenced fragments of eight nuclear protein coding genes: abdominal-A (abdA), 209 arginine kinase (ArgK), rudimentary (CAD), elongation factor 1α copy F2 (EF1aF2), long-wave 210 rhodopsin (LW Rh), DNA topoisomerase 1 (Top1), ultrabithorax (Ubx), and wingless (wg). 211 Amplifications of desired gene fragments were performed with primers listed in Table S1 using 212 standard PCR methods described in Ward & Downie (2005). Sequencing reactions were carried 213 out on an ABI 3730 Capillary Electrophoresis Genetic Analyzer with ABI Big-Dye Terminator 214 v3.1 Cycle Sequencing chemistry (Applied Biosystems Inc., Foster City, CA). 215 Sequence base calling was performed in Sequencher v5.0 (Gene Codes Corporation, Ann 216 Arbor, MI). Because it has been suggested that arbitrary choice of alleles can bias phylogenetic 217 results in some data sets (Weisrock et al. 2012), we retained ambiguous base calls (R or Y) for 218 any potentially heterozygous sites. To expand our taxon sampling, we downloaded sequences 219 from GenBank. For 18 Prenolepis genus-group taxa from LaPolla et al. (2010, 2012) we 220 obtained five nuclear loci (ArgK, CAD, EF1aF1, EF1aF2, and wg); we obtained nine nuclear 221 loci (abdA, ArgK, CAD, EF1aF1, EF1aF2, LW Rh, Top1, Ubx, and wg) and two ribosomal loci 222 (18S and 28S) for an additional 116 formicine and 19 non-formicine taxa from datasets published 223 by Brady et al. (2006), Branstetter (2012), Brady et al. (2014), Blaimer et al. (2015), Chomicki 224 et al. (2015), and Ward et al. (2015). The final data set comprised a total of eleven nuclear loci 225 for 135 taxa spanning the Formicinae and including non-formicine outgroups, with 21 Lasius 226 species, 3 Myrmecocystus species, and 30 Prenolepis genus group species. GenBank identifiers 227 for all sequences used in this study are listed in Table S2. 228

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229 Morphological data 230 Worker specimens of extant and extinct species were examined from the American Museum of 231 Natural History (AMMH) and collections housed at the University of California, Davis: the 232 Bohart Museum of Entomology (UCDC), and the personal collections of Philip S. Ward (PSWC) 233 and the authors (BEBC, MLBC, MMPC). Additionally, morphological characters were evaluated 234 from type specimens and other material imaged on AntWeb (http://antweb.org). We constructed 235 two separate datasets for our study: (1) continuous morphometric data to differentiate “L. 236 myrmidon” and “†L. pumilus” relative to other Lasius (measurements are defined in Appendix 237 S1, and data is reported in Lasiini_eye_metrics_XXX_vs_XXX.csv, available on Dryad, 238 https://doi.org/10.25338/B8B645), and (2) discretized binary morphological data to test our 239 assumptions of the taxonomic placement of fossils informative for the Lasiini (characters are 240 defined in Appendix S2, and states scored for each taxon are in 241 Lasiini_w_outgroups_w_fossils_154t_morphology.fasta, available on Dryad, 242 https://doi.org/10.25338/B8B645). For the discretized dataset, worker-based observations were 243 made of 110 morphological characters coded in binary fashion for phylogenetic analysis 244 (character list reported in Appendix S2). These data were scored for 150 terminals in total, of 245 which 18 were fossils and 132 were extant Formicinae. Some of the characters were derived 246 from prior studies, while others were interpreted de novo from comparative morphological study 247 across the Formicidae. Each character is treated as a state which is either observed to be true (1) 248 or false (0) for a given specimen. Therefore, some complex (multistate) characters were 249 separated into multiple characters, with observed absences and inapplicable states scored as 250 false, similar to the “Composite Coding” method described by Strong & Lipscomb (1999). An 251 effort was made to score characters in a homology-neutral way, i.e., that each true statement is 252 based on the morphological definition, without assuming a priori that the state must be 253 homologous when observed across the phylogeny. See the “Species group classification of 254 Lasius” section below for a record of examined Lasius species. 255 256 Phylogenetic analyses 257 Alignment and partitioning of Sanger sequence data 258 We generated two molecular datasets from our initial set of sequences: 259 ‘Lasiini_w_outgroups_135t’ contains the full taxon set, with 135 taxa spanning the Formicinae, 260 including formicoid clade outgroups (Brady et al. 2006); ‘Lasiini_55t’ is a subset of 261 ‘Lasius_w_outgroups_135t’, containing only species from the genera of the Lasiini. For both 262 datasets, individual loci were aligned with the L-INS-I algorithm in MAFFT v7.419 (Katoh et al. 263 2002; Katoh & Standley 2013), and exonic regions were inspected in Aliview (Larsson 2014) to 264 ensure that the alignments were in frame. The individual alignments were trimmed with 265 GBLOCKS v0.91b (Castresana 2000), adjusting the default settings to trim less stringently: b1 266 and b2 were set to half of the total taxon set in each locus (plus one), b3 was set to 12, b4 to 7, 267 and b5 was set to half. After trimming, the ends of each sequence alignment were further 268 trimmed such that the alignment began with the first codon position and ended with the third 269 codon position. If the locus contained introns, the alignment was then split into exonic and 270 intronic regions using AMAS (Borowiec 2016b). We concatenated the resulting data into the 271 ‘Lasius_w_outgroups_135t’ and ‘Lasiini_55t’ matrices, as well as individual gene alignments for 272 each matrix. 273 As the assumption that evolution of all sequence data is homogeneous is often violated in 274 empirical data (Buckley et al. 2001), we partitioned our matrices into subsets of similarly

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275 evolving sites for our concatenated and single gene analyses. To find the optimal partitioning 276 scheme and best substitution models we used PartitionFinder v2 (Lanfear et al. 2016) on our 277 concatenated molecular datasets as well as the individual gene alignments. As input, we used 278 blocks of data which represent codon positions within exonic regions of protein coding loci; 279 introns and ribosomal loci were treated as individual data blocks. We included JC, K80, HKY, 280 SYM, F81, and GTR, all with or without +Γ or +I, but we excluded models invoking both 281 gamma and proportion of invariable sites (i.e., +Γ+I) as it has been shown that interaction of 282 these parameters may cause anomalies in Bayesian inference (Sullivan & Swofford 2001, Yang 283 2006). To find the optimal scheme, we chose the Bayesian information criterion (BIC) and the 284 ‘greedy’ algorithm. 285 286 Phylogenetic analysis of molecular-only data 287 We used MrBayes 3.2.6 (Ronquist et al. 2012b) for BI on concatenated and single gene 288 alignments (Table 1, Questions 1–3) using the model and partition schemes estimated from the 289 PartitionFinder v2 analyses. State frequencies, substitution rates, shape of gamma distribution of 290 substitution rates at sites, and proportion of invariable sites were unlinked and allowed to vary 291 among partitions, while branch lengths remained linked. We ran each analysis for 25 million 292 generations with two runs, four chains each, and sampled parameters every 2500 generations, 293 leaving burnin at the default 25%. We used the following statistics to diagnose MCMC: PSRF 294 values approaching 1.0, proposal acceptance rates between 20–70%, and standard deviation of 295 split frequencies with values of approximately 0.01 or less. Additionally, we used the program 296 Tracer v1.6.0 (Rambaut et al. 2014) to evaluate trace plots and minimum effective sample size 297 values (should be > 200) for each parameter in the different runs. 298 For phylogenetic inference under ML (Table 1, Question 2), we used IQTREE v2 (Minh et 299 al. 2020). We used the model and partition schemes estimated from PartitionFinder v2, allowing 300 each partition to have its own rate of evolution (-p), and 1000 ultrafast bootstrap replicates. Trees 301 for both Bayesian and ML analyses were manipulated with FigTree v1.4.2 (Rambaut & 302 Drummond 2014). Additionally, we performed a gene and site concordance factor analyses for 303 each concatenated dataset using the consensus tree and single gene trees from the MrBayes 304 analyses above as input. 305 Based on the results from the above ML and BI analyses, we performed stepping-stone 306 analyses of several contrasting topological hypotheses (Table 1, Questions 1 & 2). These follow 307 Bergsten et al. (2013), who recommended comparisons of analyses under equally constrained 308 topologies (Xie et al. 2011) to estimate marginal log likelihoods in MrBayes. The hypotheses we 309 tested include: (1) the placement of Lasius myrmidon as either with Lasius and Myrmecocystus 310 or with the Prenolepis genus group; (2) monophyly of Lasius with respect to Myrmecocystus; (3) 311 monophyly of Lasius s. str.; (4) monophyly of Cautolasius; and (5) monophyly of Chthonolasius 312 (Fig. S1). For these analyses, we ran MrBayes for 25.5 million generations with two runs, four 313 chains, fifty steps, sampling every 100 generations, and with the alpha shape parameter set to 314 0.4. We took the mean marginal log likelihood of the two stepping-stone runs for each analysis, 315 subtracted the likelihood of the alternative hypothesis from the null, then doubled this number. 316 Results of these Bayes factor tests are interpreted after Kass & Raftery (1995), with these 317 specific 2lne(BF01) ranges: 0–2, “not worth explaining”; 2–6, “positive support”; 6–10, “strong 318 support”; and > 10, “very strong support”. 319 Additionally, because Lasius was recovered as paraphyletic with respect to Myrmecocystus in 320 the Sanger data analyses of Blaimer et al. (2015), we employed summary coalescent species tree

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321 analysis in ASTRAL-II (Mirarab & Warnow 2015) with individual UCE locus trees from 322 Blaimer et al. (2015) supplied by the author as input. We did not use the statistical binning 323 pipeline (Mirarab et al. 2014; Bayzid et al. 2015) due to recent criticism (Adams & Castoe 324 2019), but instead used individual gene trees as input to ASTRAL-II version 4.10.11, which we 325 ran under default settings. 326 327 Phylogenetic analysis of combined data and divergence dating 328 We analyzed a morphology-only data matrix and two combined-data matrices with MrBayes 329 3.2.6 to evaluate placement of fossil taxa and to estimate informative priors on relaxed clock 330 models (Table 1, Question 3). For morphology-only analysis, we built the 331 ‘Lasiini_w_outgroups_w_fossils_morphology_154t’ dataset, with 110 morphological characters 332 for 154 taxa. For our first combined-data matrix, we created a 136-taxon dataset containing our 333 partitioned 135-taxon 11-gene matrix and morphological data from extant taxa, adding 334 Paratrechina kohli, which was represented only by morphological data 335 (‘Lasiini_w_outgroups_136t’). For our second combined-data matrix, we added morphological 336 data from 18 fossil taxa to the previous matrix (‘Lasiini_w_outgroups_w_fossils_154t’). All 337 morphological data were included in a single partition to which we applied the Mk model (Lewis 338 2001), accounting for among character rate variation using +Γ. As rate asymmetry has been 339 shown to have important consequences for morphological analysis (Wright et al. 2016; 340 Klopfstein & Spasojevic 2019), particularly in the otherwise robust total-evidence dating 341 framework (Klopfstein et al. 2019), the assumption of equal forward and reverse transformations 342 was relaxed by setting the ‘symdirihyperpr’ to a uniform distribution (‘fixed(1.0)’) in our input 343 files, thus allowing MrBayes to estimate asymmetric stationary frequencies (π) across states. To 344 account for ascertainment bias (Lewis 2001), we set coding for the morphological partition to 345 “variable”. 346 With the three datasets above, we performed alternate analyses combining different sets of 347 parameters (Table 3), examining the effect on the placement of fossil taxa when analyzing 348 morphological data in isolation in comparison to the results of combined-data analyses in the 349 absence of a molecular clock. To estimate informative priors for the two commonly used relaxed 350 clock models in MrBayes (IGR and TK02), we followed the methods used in Ronquist et al 351 (2012a) with our Lasiini_w_outgroups_136t dataset. The two clock models with informative 352 priors were then compared against each other using stepping-stone analysis with the 353 Lasiini_w_outgroups_w_fossils_154t dataset. Because the results of the stepping-stone analyses 354 were ambiguous, we analyzed the Lasiini_w_outgroups_w_fossils_154t dataset with both clock 355 models for comparison. For the analyses of the Lasiini_w_outgroups_w_fossils_154t dataset, we 356 used the fossilized birth-death branch length (FBD) model (Heath et al. 2014) and a uniform 357 clock for comparison. All analyses included a root node topology constraint (i.e., monophyly of 358 core formicoids enforced), but were otherwise unconstrained. Because our taxon sample does not 359 meet the expectations of the “diversified sampling” approach (Zhang et al. 2016), we 360 implemented a random sample stratification for FBD analyses. For fossil terminals, uniform 361 distributions were used for stratigraphic ages (New Jersey: 94.3–89.3 mya; Canadian: 84.9–70.6 362 mya; Baltic: 37.2–33.9 mya; Dominican: 20.4–13.7 mya). The root node prior age was set to the 363 estimated age of the oldest known fossil in the Formicidae, an offset exponential distribution 364 with a minimum of 90 and mean of 99 mya. We ran each analysis for 100 million generations 365 with two runs of four chains each, with sampling every 10,000 generations, and the default

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366 burnin of 25%. MCMC diagnoses were conducted as for the molecular-only analyses described 367 above. 368 369 Historical biogeography 370 After divergence dating, the post-burnin consensus chronogram resulting from the MrBayes 371 fossilized birth-death analysis was trimmed to 55 extant taxa of the Lasiini with the ‘drop.tip’ 372 function in the R package ‘phytools’ (Revell 2012) and used as input for the likelihood-based 373 biogeographic program BioGeoBEARS (Matzke 2013). We used a coarse biogeographical 374 classification following previous studies (e.g., Ward et al. 2015): Neotropical, Nearctic, 375 Palearctic, Afrotropical, Indomalayan, and Australasian regions, with Wallace’s line dividing the 376 last two areas. The maximum ancestral species range size was set to two areas, and the matrices 377 of allowed areas and dispersal constraints were set for three periods: before 60 mya, 60–30 mya, 378 and after 30 mya, reflecting tectonic drift referenced from Scotese (2010). We conducted our 379 phylogeographic analyses using the DEC, DIVALIKE, and BAYAREALIKE models, all with or 380 without +J, but due to criticism of +J (Ree & Sanmartín 2018) we excluded results that included 381 this parameter from consideration. The script and input files used in this analysis can be found on 382 Dryad (https://doi.org/10.25338/B8B645). 383 384 Ancestral state estimation 385 We employed the ace function of the R package ape (Paradis & Schliep 2018) to estimate 386 ancestral states of six traditional morphological characters and two life history traits (Table 1, 387 Questions 5 & 6). These six characters, described in the next paragraph, were scored for the 388 MrBayes analysis above after trimming fossils from the 154-taxon tree with the ‘drop.tip’ 389 function in the R package ‘phytools’, resulting in a 135-taxon phylogeny representing the 390 Formicinae and outgroups, and a 21-taxon phylogeny representing Lasius. We contrasted two 391 evolutionary models for five characters and two traits with binary states: equal (ER) and unequal 392 rates (ARD) of forward and reverse transition. For the abdominal segment III character, which 393 had ternary states, we tested symmetrical rates (SYM) in addition to the ER and ARD models. 394 To determine if the more-complex SYM and ARD models had significantly better fit, we 395 conducted pairwise ANOVA for each character. 396 We qualitatively reinterpreted the phenotypic diagnostic traits used by Bolton (2003) for 397 classification at the tribe and ‘tribe group’ level, as well as the form of the proventriculus, a 398 classic character in formicine classification (e.g., Emery 1925). The six morphological characters 399 that we reconstructed the ancestral states for are: (1) eye placement, with eyes set at or anterior to 400 head midlength as measured from the anterolateral clypeal corners [state 1] or posterior to 401 midlength [state 0]; (2) metacoxal separation, with coxae either close-set (“formicoform”) [state 402 0] or wideset (“lasiiform”) [state 1]; (3) abdominal sternum III with transverse sulcus at base of 403 helcium [state 1] or without [state 0]; (4) propodeal spiracle circular to elliptical [state 0] or slit- 404 shaped [state 1]; (5) abdominal segment III tergosternal suture extending laterally then broadly 405 curving posteriorly or simply linear (“formicoform”/broadly shouldered) [state 0], suture curving 406 forward then narrowly arching posteriorly (“lasiiform”/narrowly-shouldered, low) [state 1], or 407 suture extended dorsally, with free tergite and sternite commencing well away from helcium 408 (“plagiolepidiform”/narrowly-shouldered, high) [state 2]; and (6) proventriculus asepalous [state 409 0] or sepalous [state 1]. Character 6, proventriculus form, was scored as generalizations based on 410 Forel (1878), Emery (1888, 1895, 1925), Eisner (1957), and Prins (1983), and therefore may be 411 inaccurate; future anatomical studies should evaluate the plausibility of our generalized scoring.

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412 Note that metacoxal separation, as scored, is also a proxy for a long petiolar foramen and U- 413 shaped petiolar sternum cross-section used by Bolton (2003) to define his “lasiine tribe group”. 414 The two life history traits for which we reconstructed ancestral states are: (1) temporary social 415 parasitism, absence [state 0] or presence [state 1]; (2) fungiculture, absence [state 0] or presence 416 [state 1]. 417 418 Results and discussion 419 Alignment and partitioning of the Sanger sequence data 420 Overall, the 11-gene 135-taxon molecular matrix is 8.3 kbp long and contains 21% missing data, 421 2902 variable sites (35%) of which 2449 are parsimony informative (30%). The 11-gene 55- 422 taxon molecular matrix is 8.8 kbp long and contains 39% missing data, 1600 variable sites (18%) 423 of which 1010 are parsimony informative (12%) (statistics calculated with AMAS, Borowiec 424 2016b). The PartitionFinder analysis resulted in a best-scoring scheme with 17 partitions for our 425 135-taxon matrix, and a 12-partition scheme for our 55-taxon matrix (Table S3). 426 427 Phylogenetic relationships 428 Results in this section pertain to Questions 1–3 of Table 1. 429 Question 1: Is Lasius monophyletic? 430 Answer: The extant fauna of Lasius is monophyletic with the exclusion of XXX myrmidon gen 431 nov., comb. nov. 432 Across all analyses there is overwhelming support for the placement of Lasius myrmidon as 433 sister to the Prenolepis genus-group (Table 4). For this reason, we transfer this species to a new 434 genus, forming XXX myrmidon gen. nov., comb. nov., which we diagnose in the Taxonomy 435 section below. With the exclusion of M. myrmidon, we find somewhat low topological support 436 for Lasius monophyly using standard measures (0.96 posterior probability [PP], 87 maximum 437 likelihood bootstrap [BS] (Fig.2); 36.6 gene concordance factor [gCF], 42.3 site concordance 438 factor [sCF] (Fig. S2). However, genome-scale coalescence analysis recovers Lasius monophyly 439 with respect to Myrmecocystus (Fig. 3), and a Bayes Factor constraint test using the Sanger loci 440 results in “very strong support” for monophyly (Table 4). For these reasons, we accept the 441 hypothesis that Lasius and Myrmecocystus are reciprocally monophyletic. 442 443 Question 2: Are the subgenera of Lasius monophyletic? 444 Answer: Three of the six subgenera of Lasius—Lasius sensu stricto, Cautolasius, and 445 Chthonolasius—are not monophyletic (Table 4, Figs. 1D, 2, 4). 446 We found that Lasius, with the exclusion of XXX myrmidon, is divided into two major clades. 447 One clade comprises members of the subgenera Lasius s. str. and Dendrolasius (here called the 448 “niger clade”), while the other includes all other sampled species (here called the “flavus clade”). 449 Critically, we found that the flavus clade is diagnosable by an enlarged metapleural gland bulla, 450 constituting a new morphological autapomorphy (see Key to extant genera of Lasiini below). 451 Within the niger clade, the socially parasitic species classified in Dendrolasius are nested within 452 taxa currently considered to belong to Lasius s. str., while another Lasius s. str. species, L. 453 pallitarsis, is a member of the flavus clade. The aberrant Lasius species near atopus, currently 454 classified in Chthonolasius, is in the flavus clade, sister to L. pallitarsis with moderate statistical 455 support. Finally, the four Cautolasius sampled here are not monophyletic but instead form two 456 well-supported clades, which we recognize as the flavus and nearcticus species groups. Because 457 of this phylogenetic arrangement and a continuing trend to abandon subgeneric classification in

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458 ants (e.g., Borowiec 2016a), we propose synonymizing Lasius subgenera and recognition of 459 informal species groups instead (see Taxonomy below). 460 461 Question 3: What are the relationships of fossil Formicinae? 462 Answer: Some previously hypothesized relationships are stable, some are sensitive to model 463 choice, and the taxa †Lasius pumilus, †Pseudolasius boreus, and †Glaphyromyrmex require 464 reclassification. 465 Overall, we find that fossil placement is sensitive the inclusion or exclusion of a clock model. 466 Under “clockless” conditions, fossils were highly nested within the phylogeny (Fig. S3 & S6). 467 However, the topology resulting from clockless morphology-only analysis (Fig. S3) had both 468 very limited support within the Formicinae and was in considerable conflict with previous and 469 present analyses using molecular data. The outgroup fossils were consistent at the subfamily 470 level, while among our formicine fossils, five of the nine Baltic-region fossil taxa were recovered 471 close to their original attributions (Figs. 4, S3, S6–S9): (1) †Lasius schiefferdeckeri may be sister 472 to or nested within the Lasius genus group; (2) †Prenolepis henschei is within the crown of the 473 Prenolepis genus group; (3) †Formica gustawi is within the crown of the Formicini; (4) 474 †Oecophylla brischkei is correctly placed to genus; (5) †Gesomyrmex hoernesi is correctly 475 placed to genus; and (6) †Camponotus mengei is a member of the crown Camponotini. 476 In contrast, we find that one generic and two tribal transfers are necessary. †Pseudolasius 477 boreus and †Glaphyromyrmex oligocenicus, both attributed to Lasiini, are recovered outside of 478 the tribe with very high support across all combined-evidence analyses (0.98–0.99 PP). We 479 transfer the former species to a new genus forming †XXX boreus gen. nov. comb. nov., which 480 we consider incertae sedis within the subfamily (tribal transfer), and we transfer 481 †Glaphyromyrmex back to the Formicini (tribal transfer; see Taxonomy below for discussion). 482 With respect to Lasius, †Lasius pumilus is recovered outside of the genus, being consistently 483 recovered as sister to XXX (Figs. 4, S7–S9); for this reason, we transfer the species to a third and 484 final new genus, forming †XXX pumilus gen. nov. comb. nov., which we place in the XXX genus 485 group (see Taxonomy below for diagnosis). 486 The placements of two key taxa, †L. schiefferdeckeri and †Kyromyrma neffi, were sensitive 487 to the choice of clock model. Under IGR (Fig. S7), †L. schiefferdeckeri was sister to the Lasius 488 genus group (0.90 PP) and †Kyromyrma was nested in the crown Formicinae to the exclusion of 489 the Myrmelachistini (0.94 PP). Under TK02 (Figs. S8, S9), †L schiefferdeckeri formed a clade 490 with Lasius (0.63–0.71 PP) and †Kyromyrma was nested in the crown Lasiini (0.91–0.94 PP). 491 Notably, the MCMC performed better under the IGR model relative to the TK02 model. Under 492 TK02, the clock rate parameter sampled poorly, which was reflected in low ESS values for tree 493 height and tree length. This, in addition to the unusually old estimations for the root node (Figs. 494 S8 & S9), led us to prefer the IGR model over TK02. Regardless, we conservatively retain †L. 495 schiefferdeckeri in Lasius and treat †Kyromyrma as incertae sedis within the Formicinae, in 496 agreement with Ward et al. (2016) and Grimaldi & Agosti (2000). 497 498 Evolutionary History 499 Results in this section pertain to Questions 4 and 5 of Table 1. 500 Question 4: Where and when did the internal clades of the Lasiini originate? 501 Answer: The Lasiini likely originated and initially diversified in the Eastern Hemisphere, with 502 modern genera present by the mid-Miocene.

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503 Our primary divergence dating findings, based on the FBD analysis with and IGR branch 504 length variation model, are as follows (Figs. 4, 5, Table 5): (1) The deepest split in the Lasiini 505 (Cladomyrma + core Lasiini) is Late Cretaceous to Paleocene; (2) the split between XXX and the 506 Prenolepis genus group crown was probably post-K/Pg and may have been during the Early 507 Eocene Climatic Optimum (part of the Ypresian Epoch, 56.0–47.8 Ma); and (3) the crown Lasius 508 genus group is young compared to that of the Prenolepis genus group, being probably Oligocene 509 to Early Miocene in origin, while the crown Prenolepis genus group arose in the Late Eocene. 510 Our date estimate for Myrmecocystus (14.6 mya) concurs with a recent study by van Elst et al. 511 (2021) (14.1 mya in that study), but the crown Lasius genus group (24.9 mya), and the crown 512 Lasius (21.9 mya) ages are slightly older (18.4 mya and 16.2 mya respectively in van Elst et al. 513 2021) but fall within the 95% HPDs estimated in that study. Our 95% HPD for Nylanderia, 514 however, does not overlap with that of Williams et al. (2020), with our range being much 515 younger (~15–30 vs. ~45–35 mya). With respect to our biogeographic reconstructions, we find 516 that DIVALIKE is the most likely model for the deep history of the Lasiini (Table 5) although 517 the second-most likely model (DEC) results in largely congruent estimated distributions (see 518 results on Dryad https://doi.org/10.25338/B8B645). Mapping the DIVALIKE inference onto our 519 chronogram and considering the climatological history of Earth (Fig. 5), we propose the 520 following narrative. 521 The Lasiini originated on the Eurasian landmass around the End Cretaceous, with the core 522 Lasiini originating in the Western or “Palearctic” portion of the ancient continent. Subsequently, 523 the Lasius genus group experienced some degree of extinction which pruned the clade down to a 524 single Nearctic ancestor, possibly due to the global cooling of the Oligocene to Pleistocene. 525 Within the crown Lasius genus group, Myrmecocystus arose as an arid-region-adapted lineage 526 from an extinct Lasius-like lineage, as evinced by the ~35 mya Baltic Lasius fossils. The 527 ancestor of Lasius dispersed back to the Palearctic, and a complicated series of range expansions 528 and dispersal events occurred throughout the Miocene and Pliocene. An early dispersal event to 529 the Nearctic from the Palearctic led to the origin of the distinctive flavus clade, wherein a 530 complex series of dispersal events between the Palearctic and the Nearctic probably occurred 531 across the Bering Land Bridge. Within the niger clade, there was at least one dispersal to the 532 Nearctic in the niger group. 533 Whereas the Lasius genus group was heavily pruned, the Prenolepis genus group began 534 radiating during the second half of the Eocene, with their depauperate sister group surviving to 535 the present day as XXX gen. nov. The ancestor of the Prenolepis genus group either adapted to, 536 or retained an ancestral physiological optimization for tropical climes, having perhaps expanded 537 into the Indomalayan region following the Eocene Optimum. Subsequently, the Prenolepis genus 538 group radiated across the global tropics through a complicated history of transcontinental 539 dispersal. Notably, the crown lineages of the Lasius genus group have failed to invade tropical 540 zones, whereas at least two invasions of the temperate zone have occurred in the Prenolepis 541 genus group. In our analyses, recovered those for the imparis clade of Prenolepis, and the 542 vividula clade of Nylanderia. A third invasion is implied by Matos-Maraví et al. 2018 for the 543 Nylanderia fulva clade. Future studies of the biogeography of Lasius may benefit from treating 544 biogeographic region as an observed state (character) in the non-genomic (phenotypic) matrix to 545 extract information from the coarse compression fossils in the Nearctic and Palearctic regions. 546

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547 Question 5: What was the evolutionary sequence of formicine morphology and life history 548 traits? 549 Answer: Our analyses recover wide-set hind coxae and sepalous proventriculi as 550 synapomorphies of the Formicinae, and show independent, convergent evolution of social 551 parasitism and fungiculture in Lasius. 552 We determined that the polarity of six characters is particularly important for understanding 553 the morphological evolution of the Lasiini and the subfamily Formicinae. In all cases, the equal 554 rates (ER) transition matrix was found to be the most likely model (Table 6). Empirical Bayesian 555 posterior probabilities are presented for all character states at selected nodes in Table 7, and 556 mapped posterior probabilities are presented in Figs. S10–15. Based on our analyses, we predict 557 that the most recent common ancestor of the Formicinae probably had, in addition to the 558 acidopore, the following states: (1) Eyes set at or anterior to head midlength [plesiomorphy]; (2) 559 wide-set hind coxae [apomorphy]; (3) absence of a transverse sulcus posterad the helcial sternite 560 [plesiomorphy]; (4) circular propodeal spiracles [plesiomorphy]; (5) broadly shouldered 561 tergosternal margins laterad the helcium [plesiomorphy]; and (6) a sepalous proventriculus 562 [apomorphy]. Roughly corresponding to expectation (Bolton 2003), slit-shaped spiracles are 563 estimated to be defining apomorphies of the Formicini, Camponotini, and the melophorines 564 Melophorus and Notostigma (Fig. S12); other results require further consideration. 565 Of the reconstructed states, the sepalous form of the proventriculus is newly detected as a 566 synapomorphy of the subfamily—if the asepalous state in Myrmelachista is interpreted as an 567 independent reversal, as implied by our results (Fig. S14). This finding contradicts the traditional 568 assumption that the sepalous form arose from the asepalous form within the Formicinae (Emery 569 1925, Eisner 1957), and hypothesis that the sepalous form is homoplastic within the subfamily 570 (Agosti 1991, Bolton 2003). Because our coding was generalized from the literature, our results 571 must be treated as tentative. We strongly recommend that future studies expand on the sampling 572 of Eisner (1957), particularly to include Leptanillinae, Martialinae, and more poneroids (sensu 573 Moreau et al. 2006, Brady et al. 2006), and to define new traits of the proventriculus for 574 evolutionary analysis because of its “social stomach” function (Eisner & Brown 1958, Wilson 575 1971). Moreover, a microanatomical study, perhaps using computed tomography, would be a 576 substantial contribution to this question. 577 Within the Formicinae, posteriorly set eyes are estimated to have been derived nine 578 independent times (Fig. S10), which is a simplistic statement, given that eye position is a 579 continuous and evolutionarily labile trait. This strongly contradicts our intuition that posteriorly 580 set eyes were ancestral, as they are observed in the Leptanillinae (where known), 581 Amblyoponinae, and stem Formicidae (Perrichot et al. 2020; Boudinot et al. 2020). If posteriorly 582 set eyes are truly retained in the various Formicinae, the Ectatomminae s. l., and the 583 Pseudomyrmecinae, then we must appreciate the low likelihood of this scenario and search for 584 corroborating evidence. Ideally, eye position should be modeled as a continuous trait (Parins- 585 Fukuchi 2018). 586 Our estimates indicate that wideset (“lasiine”) coxae are an apomorphy of the Formicinae, 587 with a subsequent reversal in the formicoform radiation (the clade that includes Camponotini, 588 Formicini, and Melophorini, along with the monotypic tribes Gesomyrmecini, Gigantiopini, 589 Oecophyllini, and Santschiellini (Fig. S11), with losses in Lasiophanes and the ancestor of 590 Teratomyrmex and Prolasius of the Plagiolepidini, as well as Myrmoteras. It may be possible to 591 evaluate the plausibility of this reconstruction through comparative µ-CT analysis of propodeal /

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592 petiolar skeletomuscular anatomy. At present, comparative data on “waist” skeletomusculature is 593 limited (e.g., Hashimoto 1996, Perrault 2004). 594 We detect three independent origins of the “plagiolepidiform” third abdominal segment (i.e., 595 raised, with free sclerites occurring high): Once in the ancestor of the Myrmelachistini, once 596 within the Lasiini (Prenolepis genus group), and once in the Plagiolepidini (Fig. S15). Our 597 results support Bolton’s (2003) hypothesis that the lasiine condition (i.e., narrowly shouldered 598 but not raised) was a precursor to the plagiolepidiform condition, as elevated shoulders arose 599 well-within the Lasiini. However, whether the narrow and low shoulders of Anoplolepis 600 (Plagiolepidini) are ancestral or a reversal (as observed for Acropyga) is uncertain. Narrowed 601 shoulders arose three times within the Melophorini (Stigmacros, Lasiophanes, and the ancestor 602 of Teratomyrmex + Prolasius). Curiously, “high and narrow shoulders” are also observed in 603 Dolichoderinae (e.g., Bolton 1994), thus comparative anatomical studies focusing on this 604 condition are warranted. 605 Finally, most of the socially parasitic species within Lasius included here (“nr. 606 subumbratus”, sabularum, carniolicus, californicus, latipes) form a strongly-supported 607 monophyletic group within the flavus clade (Fig. 2). The other two socially parasitic species, 608 fuliginosus and spathepus, are in the niger clade and are sister to one another. A subset of social 609 parasites, here represented by “nr. subumbratus”, sabularum, fuliginosus, and spathepus are also 610 known to culture fungi to bind walls of their nests (Schlick-Steiner et al. 2008). These species 611 are widely separated on the phylogeny, and it appears likely that both social parasitism and 612 fungiculture evolved at least twice within the genus (Fig. 5, S16, S17). 613 614 Taxonomy 615 Here we report the taxonomic implications of our study (Question 6 of Table 1). Note that 616 probable autapomorphies in the context of the Lasiini are italicized in the taxon definitions. 617 618 Tribe Lasiini Ashmead, 1905 619 (Figs. 6, 7, 8A–K,9A–J, 10A–D) 620 Type genus: Lasius. 621 Included genus groups: Cladomyrma, Lasius group, XXX group, Prenolepis group. 622 Definition (worker): 623 1. With characters of Formicinae (see Bolton 2003 and note 1). 624 2. Mandible triangular, with 4–11 teeth; third tooth from apex reduced. 625 3. Dorsal mandibular groove, when present, running along lateral mandibular margin as 626 seen in dorsal view (note 2). 627 4. Palp formula usually 6,4, less often 3,4. 628 5. Frontal protuberance mediad antennal toruli with frontal carinae effaced, becoming 629 broadly rounded posteriorly until continuous with face (note 3). 630 6. Frontal protuberance raised above toruli or not. 631 7. Antennal toruli near or abutting posterior clypeal margin. 632 8. Antenna 12-, 11-, or 8-merous. 633 9. Compound eyes not enormous, may be reduced to absent; long axis of eyes subparallel. 634 10. Ocelli present or absent (note 4). 635 11. Metanotum differentiated or not. 636 12. Metapleural gland present, dorsal rim of metapleural gland curved inward. 637 13. Propodeal spiracle at or near posterolateral margin of propodeum.

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638 14. Propodeal spiracle circular to elliptical, not slit-shaped. 639 15. Metacoxae wideset: Distance between mesocoxal bases less than between that between 640 bases of metacoxae with mesosoma in ventral view and coxae oriented at right-angles to 641 long axis of body. 642 16. Metatibiae without double row of ventral (inner) setae. 643 17. Petiolar foramen in profile view low, not to barely exceeding dorsal margin of 644 metapleural gland, with or without dorsal margin or lip, but lip, when present, 645 inconspicuous (note 5). 646 18. Petiolar foramen in ventral view long, anterior margin exceeding metacoxal cavities 647 anteriorly. 648 19. Petiolar node conspicuously shorter than propodeum, not reaching dorsal surface of 649 propodeum (note 6). 650 20. Petiolar apodeme (situated anteriorly on tergum) contiguous or nearly contiguous with 651 petiolar node (note 7). 652 21. Petiolar sternum U-shaped in cross-section (note 8). 653 22. Abdominal segment III transverse sulcus absent. 654 23. Base of abdominal segment III with or without complete tergosternal fusion lateral to 655 helcium, free sclerites commencing distantly up segment or near helcium; tergum III 656 overhanging petiole or not. 657 24. Proventriculus sepalous (note 9). 658 Notes on definition: 659 Note 1. The generic composition of the Lasiini has recently been reassessed following Blaimer et 660 al. (2015) and Ward et al. (2016). The tribal definition proposed here is thus revised relative 661 to Bolton (2003). It includes new characters and recognizes that several former 662 plagiolepidines belong in the Lasiini. 663 Note 2. Previous studies have not focused attention on the dorsal mandibular groove. Here it was 664 observed that the groove, as seen with the mandible in dorsal view, runs along the outer 665 margin of the mandible toward the mandibular apex in the Myrmelachistini, Lasiini, and 666 most Melophorini. This contrasts with the state observed in the “formicoform radiation” (i.e., 667 the clade that includes Camponotini, Formicini, and Melophorini, along with the monotypic 668 tribes Gesomyrmecini, Gigantiopini, Oecophyllini, and Santschiellini; see the section 669 †Kyromyrma, an ancestral formicine, under the section “Incertae sedis in the Formicinae” 670 below). When the groove is present in the “formicoform radiation”, it is very close to the 671 basal mandibular margin and is shortened, except in various plagiolepidines. The medial and 672 shortened state may have arisen multiple times of the Formicini, Plagiolepidini, and 673 Camponotini clade. 674 Note 3. The “frontal protuberance” is the region between the antennal toruli which is raised 675 relative to the regions of the face laterad the toruli. To some degree, development of this 676 condition accounts for the “laterally directed” torular condition of Formicidae (Boudinot et 677 al. 2020). In other formicine tribes, including the Melophorini (with Prolasius as an 678 exception), the frontal protuberance is carinate above the toruli (i.e., “frontal carinae are 679 present”), with these carinae continuing as a sharp ridge until their posterior terminus. Within 680 the Lasiini, the frontal carinae are effaced; they are long in all groups except Cladomyrma, 681 while in the Plagiolepidini the carinae are short in all genera except Anoplolepis. 682 Note 4. Ocellus presence / absence is a relatively weak character as expression of ocelli may be 683 variable for genera in which ocelli are observed, such as Lasius. However, this statement is

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684 included as ocellus expression is a traditional character which is easy to evaluate and may 685 have value for future works defining tribes wherein ocelli may be consistently present or 686 absent. Within the Lasiini, ocelli are always absent in Cladomyrma, Euprenolepis, 687 Nylanderia (although infrequently expressed as single, median ocellus), and Pseudolasius, 688 variably present among Lasius, Paraparatrechina, and Prenolepis species, and consistently 689 present in Myrmecocystus, Paratrechina (except P. kohli; see comments under Prenolepis 690 genus group), Zatania. 691 Note 5. The conformation of the dorsal region of the petiolar foramen is newly described here. 692 There is complex variation of the form across the subfamily, but it appears at least that the 693 form observed in the Lasiini is consistent; this form is also observed in the Myrmelachistini. 694 In various lineages within the core Formicinae, i.e., the clade that spans the node between 695 Melophorini and Camponotini, a raised and conspicuously carinate dorsal margin is 696 observed. 697 Note 6. Short petiolar nodes are also observed in the Myrmelachistini and Plagiolepidini 698 (excluding Anoplolepis). The node height is variable in the Melophorini, being short in 699 Prolasius and Myrmecorhynchus. 700 Note 7. The apodeme is clearly separated from the node in most Melophorini, except Prolasius. 701 Note 8. A U-shaped petiolar sternum was used by Bolton (2003) to diagnose the lasiine tribe 702 group. This trait also occurs in the Myrmelachistini, Myrmoteratini, Plagiolepidini, and four 703 Melophorini (Lasiophanes, Prolasius, Stigmacros, Teratomyrmex); see “Ancestral state 704 estimation” results above and Fig. S15. 705 Note 9. The form of the proventriculus forms a natural division between the Plagiolepidini and 706 the “plagiolepidiform” Prenolepis genus group, as observed by Emery (1925). Due to 707 uncertainty about the polarity of the sepalous condition—and whether this form arose 708 multiple times (Eisner 1957; Agosti 1990, 1991)—Bolton (2003) lumped the two 709 plagiolepidiform clades, plus the Myrmelachistini, into the Plagiolepidini. 710 Exclusion of †Glaphyromyrmex from the Lasiini: The Baltic amber formicine, 711 †Glaphyromyrmex Wheeler, was placed in the Formicini until recently (Wheeler 1915, 712 Donisthorpe 1943, Dlussky 1967, Dlussky & Fedoseeva 1988, Bolton 1994), when Dlussky 713 (2008) transferred the genus to the Lasiini. Placement of †Glaphyromyrmex in the Lasiini is 714 counterintuitive given presence of a ventral double-row of setae on its metatibiae, a state which 715 does not occur in any lasiine. Intuitive placement of †Glaphyromyrmex based on morphology is 716 challenging, however. †Glaphyromyrmex differs from members of all formicine tribes in which 717 double seta rows occur (Formicini, Camponotini, some Melophorini). Specifically, 718 †Glaphyromyrmex differs from the Formicini in eye position (eyes set at about head midlength), 719 from the Camponotini in having antennal toruli which abut the posterior clypeal margin (vs. 720 widely separated), and from the Melophorini, which have well-defined dorsal and ventral flaps 721 surrounding the metapleural gland, a conformation that is apparently absent in 722 †Glaphyromyrmex. Recognizing these differences, however, we transfer †Glaphyromyrmex back 723 to the Formicini (tribal transfer) based on our combined analyses (Fig. 4) and recommend 724 revised study of the fine-scale external anatomy of the fossil taxon. 725 On the identity of †Protrechina: Wilson (1985) described a genus putatively close to 726 Paratrechina sensu lato (~ Prenolepis genus group) from mid-Eocene Claiborne amber 727 (Arkansas, 40.4–37.2 Ma; Saunders et al. 1974). This genus, †Protrechina, supposedly differs 728 from Paratrechina s. l., Lepisiota, and Brachymyrmex—among other, unstated formicines—by 729 absence of standing macrosetae on the mesosomal dorsum, a state similar to that observed for

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730 †XXX and XXX as noted below. The genus has been variably treated as a lasiine (Bolton 1994, 731 1995; LaPolla & Dlussky 2010), a “prenolepidine” (Hölldobler & Wilson 1990), or as incertae 732 sedis in the subfamily where it remains at present (Wilson 1985; Dlussky & Fedoseeva 1988; 733 Bolton 2003; Ward et al. 2016). The type specimen of †Protrechina carpenteri, at the Museum 734 of Comparative Zoology (Harvard), should be reexamined to evaluate its tribal, and perhaps 735 generic, placement. This would be particularly valuable given the approximately mid-Eocene 736 origin of the Prenolepis genus group here inferred (Fig. 4; Table 5); such a study should be 737 facilitated by use of micro-CT (e.g., Barden et al. 2017b; Hita-Garcia et al. 2017), given the poor 738 condition of the specimen reported by LaPolla & Dlussky (2010). 739 740 Key to extant genera of Lasiini and primary clades of Lasius (workers) 741 1. Antenna with 8 antennomeres. Mesonotum, metanotum, and propodeum more-or-less 742 continuous, and metanotum undifferentiated (Fig. 6A). Propodeal spiracle distinctly situated 743 in dorsal third of propodeum. Frontal carinae inconspicuous, being extremely short (< 0.5 × 744 anteroposterior antennal torulus diameter), or absent … Cladomyrma (see Agosti 1991, 745 Agosti et al. 1999) 746 - Antenna with 11 or 12 antennomeres. Mesonotum, metanotum, and/or propodeum 747 discontinuous and metanotum differentiated (Figs. 6A–G, I–L), rarely mesosomal dorsum 748 continuous and metanotum undifferentiated (few Paraparatrechina) (Fig. 6H). Propodeal 749 spiracle situated somewhat below or above midheight of propodeum, but not in dorsal third. 750 Frontal carinae conspicuous (≥ 1.0 × anteroposterior antennal torulus diameter) … 2 751 752 2. Petiole posteriorly elongated. Node reduced, apex not or barely projecting dorsally above 753 metapleural gland when observed in profile view (Fig. 6F–L). Abdominal tergum IV with 754 anterolateral corners directed anterodorsally from helcium and forming lateral margins of 755 concavity which receives entire posterior region of petiole when gaster raised (Fig. 7A); 756 petiole largely obscured by abdominal tergum IV in dorsal view. Facial region between 757 antennal toruli flat, effaced frontal carinae not raised above toruli (Fig. 7C) … Prenolepis 758 genus group (see LaPolla et al. 2012, LaPolla & Fisher 2014) 759 - Petiole without posterior elongation. Node squamiform, not reduced, apex projecting dorsally 760 above metapleural gland when observed in profile view (Fig. 6B–E). Abdominal tergum IV 761 with anterolateral corners weakly or not raised anterodorsally from helcium, corners not 762 forming lateral margins of concavity, and not concealing entire posterior region of petiole 763 when gaster raised (Fig. 7B); petiole visible in dorsal view, not obscured by abdominal 764 tergum IV. Facial region between antennal toruli bulging to concave, frontal carinae raised 765 above toruli (Fig. 7D) … 3 766 767 3. Eyes situated in anterior half of head as measured in full-face view (Fig. 10A). Metapleural 768 gland orifice reduced, with opening directed posteriorly. Propodeal spiracle situated in lower 769 half of propodeum in profile view. Antennomere III broader than long. Hypostoma lacking 770 carina along lateral margins … XXX gen. n. (compare also †XXX gen. nov., below) 771 - Eyes situated in posterior half of head as measured in full-face view (Fig. 9). Metapleural gland 772 orifice small to very large, opening laterally as well as posteriorly. Propodeal spiracle situated 773 at or above midheight of propodeum in profile view. Antennomere III usually longer than 774 broad. Hypostoma with carina along lateral margins … 4 (Lasius genus group) 775

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776 4. Maxillary palpomeres III and IV strongly flattened and greatly elongated, length of each 777 exceeds length of apical antennomere. Ventral surface of head with psammophore, i.e., 778 margined by long setae (Fig. 6D). Mesothorax and metathorax elongated, mesonotum meeting 779 metanotum at very low angle (Fig. 6D). Compound eyes set at or near extreme posterior 780 margin of head in full-face view; eyes usually separated by < 1 eye diameter … 781 Myrmecocystus (see Snelling 1976, 1982) 782 - Maxillary palpomeres III and IV circular to elliptical in cross-section and not elongated, length 783 of each is less than length of apical antennomere. Ventral surface of head without 784 psammophore (Fig. 6B, C). Mesothorax not elongated and metathorax very rarely elongated, 785 mesonotum usually meeting metanotum at steep angle (Fig. 6B, C). Compound eyes set 786 further away from posterior head margin in full-face view; eyes usually separated by > 1 eye 787 diameter … 5 (Lasius) 788 789 5. Metapleural gland enlarged internally, atrium (internal chamber of gland) bulging (forming 790 bulla) and conspicuously visible from external view in profile (Fig. 7E)*. Usually, distance 791 between atrium and propodeal spiracle < 2 spiracular diameters as viewed with spiracle and 792 gland in same plane of focus. Compound eyes usually somewhat- to very-reduced; if 793 compound eye large, then offset basal tooth present on mandible and body yellowish brown 794 … Lasius flavus clade 795 - Metapleural gland small, atrium flat to concave, inconspicuous from external view in profile 796 (Fig. 7F). Usually, distance between atrium and propodeal spiracle > 2 spiracular diameters as 797 viewed with spiracle and gland in same plane of focus. Compound eyes relatively large and 798 offset basal tooth absent; if offset basal tooth present, then body jet black … Lasius niger 799 clade 800 801 *Note: The enlarged state of the metapleural gland bulla is a defining synapomorphy of the 802 Lasius flavus clade. A slightly enlarged bulla is observable in the fuliginosus species group, but 803 these jet-colored ants are otherwise highly distinct. 804 805 Genus group of Lasius 806 (Figs. 6B, C, D, 7B–F, 8A–K, 9A–J) 807 Included genera: Lasius, Myrmecocystus. 808 Definition (worker): 809 1. With characters of Lasiini. 810 2. Mandible with 4–11 teeth. 811 3. Palp formula 6,4 or 3,4. 812 4. Basal and masticatory mandibular margins meeting at a weakly oblique angle. 813 5. Frontal carinae conspicuous, > 0.5 × anteroposterior antennal torulus diameter. 814 6. Frontal region of head, between antennal toruli bulging, with frontal carinae raised above 815 toruli (note 1). 816 7. Antenna 12-merous. 817 8. Third antennomere usually longer than broad (note 2). 818 9. Compound eye set in posterior half of head (note 3). 819 10. Ocelli usually present (note 4). 820 11. Dorsum of head with at least some standing setae in addition to those on posterolateral 821 head corners.

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822 12. Mesonotum, metanotum, and/or propodeum discontinuous, metanotum differentiated. 823 13. Anterolateral margin of mesopleural area, near posteroventral pronotal margin, with 824 longitudinally oriented bosses subtending transverse groove or not (note 5). 825 14. Metapleural gland small to very large, orifice opening laterally to posterolaterally, not 826 directed posteriorly. 827 15. Propodeal spiracle situated in lower 2/3 of propodeum. 828 16. Petiole with raised squamiform node, without posterior elongation. 829 17. Abdominal tergum III vertical, without deep groove for receiving petiole; not concealing 830 petiole when gaster raised. 831 18. Tergosternal suture of abdominal segment III not raised dorsally before unfusing near 832 spiracle, rather anterior margin of abdominal sternum III directed anteriorly away from 833 helcium before narrowly curving posteriorly. 834 19. Pubescence of abdominal terga VI and VII absent, or present and linear to weakly curved. 835 Notes on definition: 836 Note 1. The polarity of this character is uncertain. Both the Lasius and XXX genus groups have 837 raised frontal regions, but neither Cladomyrma nor the Prenolepis genus group do. 838 Note 2. Antennomere III is longer than broad in most Lasius, except for the flavus species group 839 (former Cautolasius). 840 Note 3. Although we suspect that posteriorly set eyes are a plesiomorphy for the crown 841 Formicidae, our ancestral state estimation results indicate that the posteriorly set eyes of the 842 Lasius genus group are derived, and that they are homoplastic with respect to Prenolepis + 843 Zatania and Paratrechina (Fig. S10). 844 Note 4. Ocelli are not expressed in various species of the flavus clade. Generally, ocellus 845 presence is a highly variable state, even within species, thus is a weak defining feature for 846 worker ants. 847 Note 5. The development of the boss subtending the transverse mesosternal groove appears to be 848 an apomorphy of the Lasius genus group or Lasius itself, with loss in Myrmecocystus and 849 reduction in the L. fuliginosus group. The groove and shoulder (boss) are present in a reduced 850 form in XXX and are variable in the Prenolepis genus group. Specifically, they are reduced 851 such that no rim is present on the anterior mesosternal margin (e.g., Nylanderia), or the 852 groove is elongated (e.g., Zatania, Prenolepis, Paratrechina); some Paraparatrechina have 853 either the groove (narrow) or the shoulder. 854 855 Genus Lasius Fabricius, 1804 856 = Donisthorpea Morice & Durrant, 1915 857 = Acanthomyops Mayr, 1862 syn. rev. 858 = Austrolasius Faber, 1967 syn. n. 859 = Cautolasius Wilson, 1955 syn. n. 860 = Chthonolasius Ruzsky, 1912 syn. n. 861 = Dendrolasius Ruzsky, 1912 syn. n. 862 (Figs. 6B, C, 7E, F, 8A–K, 9A–J) 863 Type species: Formica nigra Linnaeus, 1758 (= Lasius niger) 864 Subgeneric classification remarks: The modern body of taxonomic work on Lasius was 865 initiated by Wilson’s revision of the genus (Wilson 1955), which was classified into four 866 subgenera at the time: Cautolasius, Chthonolasius, Dendrolasius, and Lasius s. str. In this work, 867 Wilson provided a phylogeny of Lasius (Fig. 1A), but this treatment was an intuitive account of

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868 what he considered trends in the evolution of the morphology and biogeography of the genus. 869 Subsequently, the subgenus Austrolasius was erected for a few socially parasitic species (Faber 870 1967) and the former genus Acanthomyops was included in Lasius as the sixth subgenus (Ward 871 2005). 872 The first inference based on molecular data was presented by Hasegawa (1998), who used 873 COI to investigate relationships of four Lasius species (phylogeny not figured here). Since then, 874 two major attempts at resolving the phylogeny were presented by Janda et al. (2004) and 875 Maruyama et al. (2008) (Figs. 1B, C). Both studies used a combination of morphological 876 characters and molecular data, including mitochondrial (COI, COII, tRNA-Leu) and ribosomal 877 (16S) markers. A more recent effort focused on the phylogeny of European species related to 878 Lasius niger (Lasius s. str.) and included nuclear genes LW Rh and wg in addition to 16S and 879 COI (Talavera et al. 2015). 880 To date, the monophyly of the subgenera has not been questioned except for the 881 nominotypical subgenus (Janda et al. 2004, Maruyama et al. 2008). Recently, Seifert (2020, p. 882 21) stated that there is clear justification for elevating the subgenera to generic status. Such an 883 action would considerably complicate the classification of the Lasius genus group because of the 884 robustly supported paraphyly of the subgenera that we have uncovered here (Figs. 1D, 2, S1, 885 Table 4). Specifically, in order to retain monophyly at the genus rank, four new genera would 886 need to be erected for the species groups of brunneus, nearcticus, atopus, and pallitarsis. An 887 additional issue would be the placement of the species which have not been sequenced, 888 particularly those of the niger species group and, for example, the recently described L. 889 brevipalpus which is incertae sedis in the niger clade. The strongest morphological 890 reorganization at the generic or subgeneric levels would be to recognize the reciprocally 891 monophyletic niger and flavus clades as Lasius and Acanthomyops, respectively, but we refrain 892 from doing so here (for our rationale, see “Species group classification of Lasius” below). 893 Comments on extant species: We determine that one species, Lasius escamole Reza, 1925, 894 should be excluded from Lasius and considered a junior synonym of the dolichoderine 895 Liometopum apiculatum Mayr, 1870, syn. n. Reza (1925) described Lasius escamole in the 896 context of a cultural study on the eponymous traditional Mexican dish known to be made from 897 the larvae of L. apiculatum (Hoey-Chamberlain et al. 2013). Although Reza’s description and 898 illustrations are extremely vague, it is possible to see details that point to a dolichoderine 899 identity. In the figures of the original description, the mandibles have long masticatory margin 900 and small, sharp, even denticles, the ventral metasoma is shown as a slit-like anal opening rather 901 than a formicine-like acidopore, and various figures display the fine, dense, appressed pilosity 902 characteristic of Liometopum, but no erect setae as expected for Lasius. 903 Many species have been added to Lasius since Wilson’s revision, mostly in Europe and 904 Mediterranean, while North American taxa have largely remained untreated except for a 905 thorough revision of Acanthomyops (Wing 1968). Careful research has revealed multiple 906 Palearctic Lasius species that show only subtle morphological differentiation from close relatives 907 (Seifert 1983, 1990, 1991, 1992, 2020; Schlick-Steiner et al. 2003). There is no reason to believe 908 that North America does not harbor a diverse fauna of such “cryptic species”. For example, the 909 question of the putative Holarctically distributed Lasius species was resolved by Schär et al. 910 (2018) who elevated to species the American representatives of L. alienus, L. flavus, and L. 911 umbratus, recognizing the following revived taxa, in order: L. americanus, L. brevicornis, and L. 912 aphidicola. Renewed focus on the Nearctic fauna is necessary, as is expanded sequencing at the 913 global scale.

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914 Comments on extinct species: Without having scored the Baltic Lasius fossils other than †L. 915 schiefferdeckeri, i.e., †L. punctulatus and †L. nemorivagus, we are unable to quantitatively 916 address their placement. Historically, †L. punctulatus and †L. schiefferdeckeri were considered 917 to be members of Lasius s. str. (Wilson 1955, Dlussky 2011), while the queen-based †L. 918 nemorivagus was placed in Chthonolasius (Wilson 1955) later to be implicitly considered 919 incertae sedis in the genus (Dlussky 2011). 920 Our combined-evidence dating analyses recover †Lasius schiefferdeckeri as sister to or 921 within the Lasius genus group (Figs. 4, S7–S9). As the specific relationship of the fossil to the 922 extant species of the Lasius genus group is uncertain, we conservatively consider the fossil 923 incertae sedis in Lasius. There remains the possibility that †L. schiefferdeckeri is ancestral to the 924 extant niger clade and is indicative of low rates of phenotypic transformation, as suggested by 925 Mayr (1868), Wheeler (1915), and Wilson (1955). The placement of †L. schiefferdeckeri may be 926 refined in future study by scoring characters which are explicitly derived from comparison of the 927 brunneus and niger groups within the niger clade. 928 The two differentiating traits proposed for the brunneus and niger subclades are, on average, 929 < 8 mandibular teeth in the brunneus subclade (Seifert 1992; some niger subclade species with < 930 8), and presence of a subapical cleft in the mandibles of brunneus subclade males (Wilson 1955). 931 While †L. schiefferdeckeri demonstrates both a tooth count of < 8, and presence of a subapical 932 cleft, the latter character is probably plesiomorphic of the Lasius genus group, and the polarity of 933 the former is uncertain. Notably, Wilson (1955) observed that the male mandibles of †L. 934 schiefferdeckeri are observed to vary from the “brunneus form” to the derived “niger form”. 935 With these three traits in mind, it does seem reasonable that †L. schiefferdeckeri is stem to or 936 directly ancestral to the niger clade. 937 Note on biology: Despite the interest in this genus, however, basic natural history remains 938 unknown for many species, including the morphologically aberrant Lasius atopus and the species 939 we sequence here, “L. nr. atopus”. Only a handful of recently published studies have addressed 940 the behavior of some of the more rarely encountered species (e.g., Raczkowski & Luque 2011), 941 indicating that more effort is needed to elucidate the biology of Lasius. 942 943 Species group classification of Lasius 944 To avoid following the undesirable trend where taxonomy is divorced from molecular 945 phylogenetics (Steiner et al. 2009, Ward 2011), we propose an informal species-group 946 classification of Lasius which reflects the phylogenetic structure within the genus. As the ICZN 947 rules do not apply to informal species groups, this arrangement is also more adaptable to future 948 refinements and new phylogenetic findings, thus contributing to taxonomic stability. Two main 949 clades are recognized, those of L. niger (70 spp. + 1 ssp.) and L. flavus (54 spp.), with three and 950 seven species groups, respectively. Additionally, all 21 valid extinct species are formally 951 considered incertae sedis in the genus, while 14 extant species are considered incertae sedis in 952 the genus and unidentifiable. 953 Should the resurrection of subgenera be deemed advisable and desirable, we recommend 954 dividing the genus into Lasius s. str. and Acanthomyops along the boundaries of the niger- and 955 flavus-clade split. Resurrection of Acanthomyops may be justified, as it is readily recognizable 956 by the autapomorphic condition of its metapleural gland (see couplet 5 of key to lasiine genera 957 above, Fig. 7E & F, also Appendix S1), and the relatively old split between the flavus and niger 958 clades (Figs. 4 & 5). We have elected not to do so here because: (1) our molecular sampling is 959 still fractional at the species level (9/70 niger clade, 12/54 flavus clade); (2) transfer of Lasius

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960 subgenera to Acanthomyops may be disruptive to the practicing systematic community; (3) 961 Sanger data are being rapidly superseded by UCE data for ant systematics; and (4) 962 morphological investigation is necessary to diagnose the newly recovered subclades. In either 963 case, the exceptional morphometric data being generated for Lasius (e.g., Seifert 1982, 1992, 964 2020; Seifert & Galkowski 2016) will be valuable for model-based phylogenetic analysis 965 combining phenotypic and genotypic data (e.g., Barden et al. 2017a, Prebus 2017), particularly 966 in the light of the greater information content of continuous trait data relative to discretized 967 characters (Parins-Fukuchi 2018). Ultimately, our goal is to encourage the consilience of 968 morphological and molecular data, and we view both as critical for a complete understanding of 969 evolution and systematics. 970 The following inter-group transfers are made: (1) Members of non-monophyletic former 971 Lasius s. str. are dispersed among Lasius brunneus, L. niger, and L. pallitarsis groups; (2) Lasius 972 atopus is removed from the L. umbratus group (former Chthonolasius); and (3) L. nearcticus, 973 and L. sonobei are removed from the L. flavus group (former Cautolasius). For diagnosis of the 974 niger and flavus clades, see the key to extant genera of Lasiini above. The task of 975 morphologically distinguishing two pairs of clades, those of brunneus and niger and of flavus 976 and nearcticus, is difficult. Future phylogenetic studies are expected to refine the boundaries of 977 the of these species-groups. Note that, in the list below, [e] indicates that specimen(s) of the 978 taxon have been examined while empty brackets, [], indicate that specimens were unavailable 979 and placed based on study of the literature. Species that were included in our molecular sampling 980 are bolded. Genus group type species are marked with asterisks (*). Overall, with the exclusion 981 of L. myrmidon and L. escamole, 118 of the 125 valid, extant species (124) and subspecies (1) 982 were examined either directly or indirectly (via AntWeb). For authorities and taxonomic 983 synopses, refer to Bolton (1995) or AntCat (Bolton 2021). Finally, it should be noted that some 984 groups contain additional, undescribed species. 985 986 I. Clade of niger 987 988 1. Species group of brunneus (L. s. str. part 1/3) 989 Constituent species (11): austriacus [e], brunneus [e], excavatus [e], himalayanus [e], israelicus 990 [e], lasioides [e], neglectus [e], precursor [e], silvaticus [e], tapinomoides [e], turcicus [e]. 991 Distribution: Palearctic. 992 Note: Within the brunneus species group as circumscribed here, two complexes are recognized 993 by Seifert (2020): that of brunneus comprises brunneus, excavatus, lasioides, himalayanus, 994 and silvaticus (5 spp. total); that of turcicus comprises austriacus, israelicus, neglectus, 995 precursor, tapinomoides, and turcicus (6 spp. total). 996 997 2. Species group of fuliginosus (= Dendrolasius) 998 Constituent species (8): buccatus [], capitatus [e], fuji [], fuliginosus* [e], morisitai [], 999 nipponensis [e], orientalis [e], spathepus [e]. 1000 Distribution: Palearctic. 1001 1002 3. Species group of niger (F. s. str. part 2/3) 1003 Constituent species (50), subspecies (1): alienus [e], americanus [e], balearicus [], bombycina 1004 [e], casevitzi [e], chinensis [e], cinereus [e], coloratus [e], creticus [e], cyperus [e], crypticus 1005 [e], emarginatus [e], flavescens [e], flavoniger [e], grandis [e], hayashi [e], hikosanus [],

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1006 hirsutus [], illyricus [e], japonicus [e], kabaki [e], karpinisi [e], koreanus [e], kritikos [e], 1007 lawarai [e], longipalpus [e], magnus [e], maltaeus [e], mauretanicus [e], neoniger [e], 1008 niger* [e], niger pinetorum [], nigrescens [e], obscuratus [e], paralienus [e], persicus [e], 1009 piliferus [e], platythorax [e], productus [e], psammophilus [e], sakagamii [e], schaeferi [e], 1010 schulzi [e], sichuense [e], sitiens [e], tebessae [e], tunisius [e], uzbeki [e], vostochni [e], 1011 wittmeri [e], xerophilus [e]. 1012 Distribution: Holarctic. 1013 Note 1: Our molecular sample for L. americanus represents an undescribed species. 1014 Note 2: Within the niger species group as circumscribed here, Seifert (2020) recognizes two 1015 morphometrically diagnosable complexes: that of obscuratus comprises criticus, 1016 obscuratus, piliferus, and psammophilus; that of paralienus comprises bombycina, casevitzi, 1017 kritikos, and paralienus. 1018 1019 4. Species incertae sedis in the clade of niger 1020 Species (1): brevipalpus [e]. 1021 Note: The species brevipalpus was described by Seifert (2020) who remarked that its relationship 1022 to other “sensu stricto” Lasius is uncertain due to a unique character combination, at least 1023 within the Asian fauna. We confirm placement of this species in the niger clade. 1024 1025 II. Clade of flavus 1026 1027 1. Species group of atopus (= Chthonolasius part 1/2) 1028 Constituent species (1): atopus [e]. 1029 Distribution: Restricted to western North America (California). 1030 Notes: This species was classified in the former subgenus Chthonolasius; our sequenced 1031 specimen represents a new species near atopus. 1032 1033 2. Species group of carniolicus (= Austrolasius) 1034 Constituent species (2): carniolicus* [e], reginae [e]. 1035 Distribution: Palearctic. 1036 1037 3. Species group of claviger (= Acanthomyops) 1038 Constituent species (16): arizonicus [e], bureni [e], californicus [e], claviger* [e], colei [e], 1039 coloradensis [e], creightoni [e], interjectus [e], latipes [e], mexicanus [e], murphyi [e], 1040 occidentalis [], plumopilosus [e], pogonogynus [e], pubescens [e], subglaber [e]. 1041 Distribution: Nearctic. 1042 1043 4. Species group of flavus (= Cautolasius part 1/2) 1044 Constituent species (7): alienoflavus [e], brevicornis [e], elevatus [e], fallax [e], flavus* [e], 1045 myops [e], talpa [e]. 1046 Distribution: Holarctic. 1047 Notes: This group corresponds to the former subgenus Cautolasius, in part. The inclusion of 1048 alienoflavus, elevatus, fallax, and talpa is provisional; molecular data ought to be generated for 1049 these species, and a critical reanalysis of phenotypic traits conducted. 1050 1051 5. Species group of nearcticus (= Cautolasius part 2/2)

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1052 Constituent species (2): nearcticus [e], sonobei [e]. 1053 Distribution: Holarctic. 1054 1055 6. Species group of pallitarsis (= Lasius s. str. part 3/3) 1056 Constituent species (1): pallitarsis [e]. 1057 Distribution: Nearctic. 1058 Note: This species was classified in the former subgenus Lasius s. str. and has previously been 1059 recovered elsewhere in the phylogeny by Janda et al. (2004) and Maruyama et al. (2008). 1060 1061 7. Species group of umbratus (= Chthonolasius part 2/2) 1062 Constituent species (25): aphidicola [e], balcanicus [e], bicornis [e], citrinus [e], crinitus [e], 1063 distinguendus [e], draco [], humilis [e], jensi [e], longiceps [], meridionalis [e], mikir [e], 1064 minutus [e], mixtus [e], nevadensis [e], nitidigaster [e], przewalskii [], rabaudi [e], 1065 sabularum [e], speculiventris [e], subumbratus [e], tibialis [] umbratus* [e], vestitus [e], 1066 viehmeyeri [e]. 1067 Distribution: Holarctic. 1068 Note: This group corresponds to the old genus Chthonolasius, with the exclusion of L. atopus. 1069 One of our molecular terminals of this group is an undescribed species near subumbratus. 1070 1071 III. Incertae sedis in genus 1072 1073 1. Impression fossils 1074 Species (18), subspecies (1): West Palearctic: (28.4–23.0 mya, Aix-en-Provence): †epicentrus 1075 [e]; (12.7–11.6 mya, Radoboj): †anthracinus [e], †globularis [],†longaevus [e], †longipennis 1076 [e], †occultatus [e], †occultatus parschlugianus [], †ophthalmicus [e]; (12.7–11.6 mya, 1077 Berezovsky): †tertiarius []; (11.6–5.3 mya, Schnossnitz): †oblongus []; (8.7–7.2 mya, 1078 Joursac): †crispus []; (8.7–2.6 mya, Lake Chambon): †chambonensis [e]; East Palearctic: 1079 (20.4–16.0 mya, Shanwang): †inflatus [], †mordicus [],†truncatus [], †validus []; (16.0–11.6 1080 mya, Vishnevaya): †vetulus []; Nearctic: (48.6–40.4 mya, Kishenehn): †glom [e]; (37.2–33.9 1081 mya, Florissant): †peritulus [e]. 1082 Note: All impression fossils attributed to Lasius are here considered to be incertae sedis within 1083 the genus. The detail preserved for all examined specimens is insufficient to challenge 1084 generic placement but does match the gross gestalt of Lasius. Critical study of these fossils, 1085 especially †L. glom and †L. peritulus, should be undertaken. 1086 1087 2. Amber fossils 1088 Species (3): †nemorivagus [], †punctulatus [e], †schiefferdeckeri [e]. 1089 Deposits: Palearctic: Baltic, Bitterfield, Danish-Scandinavian, and Rovno (Ukrainian). 1090 Note: These species, from coniferous ambers (Wolfe et al. 2016), are considered stem Lasius as 1091 per the “Comments on extinct species” section above. 1092 1093 IV. Extant species 1094 Species (14): alienoniger Forel, 1874, emeryi Ruzsky, 1905, exulans Fabricius, 1804, longicirrus 1095 Change & He, 2002, minimus (Kuznetsov-Ugamsky, 1928), monticola (Buckley, 1866), 1096 mixtoumbratus Forel, 1874, monticola Buckley, 1866, nigerrimus (Christ, 1791), nitidus

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1097 (Kuznetsov-Ugamsky, 1927), pannonica Röszler, 1942, rubiginosa Latreille, 1802, 1098 ruficornis Fabricius, 1804, transylvanicus Röszler, 1932. 1099 Note: The species exulans, monticola, rubiginosa, and ruficornis were rendered incertae sedis by 1100 Bolton (1995); we are doubtful that these names will be resolved. Recently, Seifert (2020) 1101 considered alienoniger, emeryi, longicirrus, minimus, nigerrimus, nitidus, pannonica, and 1102 transylvanicus to be nomina nuda. The species nigerrimus is unidentifiable to subfamily 1103 (Seifert 2020). 1104 1105 Genus group of XXX 1106 (Figs. 6E, 10A–D) 1107 Genera included: †XXX gen. n., XXX gen. n. 1108 Definition (worker): 1109 1. With characters of Lasiini. 1110 2. Mandible with 6–8 teeth. 1111 3. Palp formula 6,4. 1112 4. Basal and masticatory mandibular margins meeting at a weakly oblique angle. 1113 5. Clypeus modified for reception of labrum (specifically, clypeus with anterolateral 1114 notches; note 1). 1115 6. Frontal carinae conspicuous, > 0.5 × anteroposterior antennal torulus diameter. 1116 7. Frontal region of head, between antennal toruli strongly bulging, frontal carinae raised 1117 above toruli (note 2). 1118 8. Antenna 12-merous. 1119 9. Third antennomere broader than long (note 3). 1120 10. Compound eyes set at about head midlength. 1121 11. Ocelli absent. 1122 12. Dorsum of head completely without standing setae or with only a few small setae around 1123 each posterolateral head corner (note 4). 1124 13. Mesonotum, metanotum, and/or propodeum discontinuous, metanotum undifferentiated 1125 medially. 1126 14. Mesopleural anterodorsal margin, near posterolateral region of pronotum, 1127 inconspicuously bulging, this weak shoulder a very narrow groove which traverses the 1128 mesosternum. 1129 15. Metapleural gland very small, orifice directed almost completely posteriorly (note 5). 1130 16. Propodeal spiracle situated in lower 2/3 of propodeum. 1131 17. Legs entirely or almost entirely devoid of standing setae. 1132 18. Petiole with raised squamiform node, without posterior elongation. 1133 19. Abdominal tergum III vertical, without deep groove for receiving petiole; not concealing 1134 petiole when gaster raised (note 6). 1135 20. Tergosternal suture of abdominal segment III not raised dorsally before unfusing near 1136 spiracle, rather anterior margin of abdominal sternum III directed anteriorly away from 1137 helcium before narrowly curving posteriorly (note 6). 1138 21. Pubescence of abdominal terga VI and VII linear. 1139 Notes on definition: 1140 Note 1. Modification of the anterior clypeal margin for reception of the labrum is here interpreted 1141 as a synapomorphy of XXX plus the core Prenolepis genus group. The modification is most 1142 easily observed in anterior or anterodorsal view. In most Formicinae, the perceived anterior

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1143 clypeal margin with head in full-face view is usually a carina which runs across the clypeus 1144 from the lateral clypeal margins. This carina may be raised or otherwise modified; regardless, 1145 the carina itself or the anterior region of the clypeus is produced anteriorly, concealing the 1146 clypeolabral articulation. In the Prenolepis and XXX genus groups, the clypeolabral 1147 articulation is usually exposed or nearly exposed laterally by notches in the anterior clypeal 1148 carina, although it may be exposed medially where the anterior clypeal carina is absent. 1149 Note 2. See note 1 for the Lasius genus group. 1150 Note 3. Surprisingly, this distinguishes members of the XXX genus group from most formicine 1151 genera, with the exception of some Myrmelachista, various plagiolepidines, and Lasius 1152 (Cautolasius). Cladomyrma species have a cone-shaped third antennomere which is usually 1153 longer than the diameter of its base (and often apex), except in C. dianeae for which length 1154 and basal diameter are subequal. Some Pseudolasius and Paraparatrechina approach having 1155 subequal diameter and length. 1156 Note 4. Mei (1998), in his diagnosis of XXX myrmidon, indicated that these standing setae were 1157 absent except for one specimen. 1158 Note 5. This state may be a synapomorphy for the clade consisting of XXX plus the Prenolepis 1159 genus group. It was not possible to evaluate this with confidence for †XXX, but from the 1160 dorsal view of specimen SMFBE1226 on AntWeb, the gland does appear to be small. 1161 Note 6. These characters could not be evaluated with confidence for †XXX with the available 1162 material or descriptions. 1163 Comments: Although we could not evaluate all of these characters for †XXX, the genus is 1164 consistently recovered as sister to XXX in combined analysis (Figs. 4, S7–S9). The XXX genus 1165 group is most easily differentiated from either the Lasius or Prenolepis genus groups by the 1166 combination of mid-set eyes, the posteriorly directed and reduced metapleural gland orifice, 1167 raised frontal carinae, and almost complete lack of standing setae on the head. Additionally, both 1168 genera have broad third antennomeres and unusually small bodies relative to Lasius, being < 2 1169 mm in total length (Mei 1998, Dlussky 2011). 1170 1171 Genus XXX GEN. NOV. 1172 (Figs. 6E, 10A, B) 1173 Type and included species: Lasius myrmidon Mei, 1998: 177, figs. 1–11 (w.). Original 1174 designation. 1175 ZooBank LSID: http://zoobank.org/urn:lsid:zoobank.org:act:AC5A1489-C805-4035-A1C0- 1176 3DDBC6E1206D 1177 Definition (worker): 1178 1. With characters of XXX genus group. 1179 2. Dorsal mandibular groove absent (note 1). 1180 3. Ventromedial base of mandible without trough or impression (note 2). 1181 4. Maxillary palps short, exceeding hypostomal margin but not reaching postgenal bridge 1182 midlength. 1183 5. Maxillary palpomere 3 longest, 4 shorter but as long as 5 and 6 together (note 3). 1184 6. Clypeus, in lateral view, convex and weakly bulging anteriorly (note 3). 1185 7. Anterior tentorial pit situated lateral to midlength of the epistomal suture. 1186 8. Lateral hypostomal carina absent (note 4). 1187 9. Compound eyes absent, reduced, or vestigial, with at most 9 ommatidia (note 5). 1188 10. Propodeal spiracle situated distinctly in lower half of propodeum (note 6).

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1189 11. Legs almost entirely devoid of standing setae (note 3). 1190 12. Petiolar node weakly inclined anteriorly, node well-developed, squamiform (note 7). 1191 Notes on definition: 1192 Note 1. The dorsal mandibular groove is discernable in all examined Lasius and Prenolepis 1193 genus group taxa. 1194 Note 2. Presence of a trough on the ventromedial base of the mandible is a newly detected 1195 synapomorphy of the core Prenolepis genus group. The impression is enhanced when the 1196 ventromedial mandibular margin is carinate and/or produced medially, and best seen when 1197 the mandibles are open and with the head in lateral anteroventral view. The trough may be 1198 reduced or absent in some species. 1199 Note 3. Previously included in the original diagnosis of the species XXX myrmidon by Mei 1200 (1998). 1201 Note 4. The lateral hypostoma is usually delimited by a carina which is discontinuous with the 1202 medial hypostomal lamina. Among formicines, the lateral hypostomal carina is absent only in 1203 Acropyga (some species) and Brachymyrmex, both genera outside of Lasiini. 1204 Note 5. The specimens which were available for examination had 5–8 ommatidia; the maximum 1205 ommatidium count is from Mei (1998, p. 178). 1206 Note 6. A lowered propodeal spiracle appears sporadically replicated in only a few Prenolepis 1207 genus group members. 1208 Note 7. The petiolar node is strongly inclined anteriorly in the core Prenolepis genus group. 1209 Etymology: A combination of the Greek “meta-” (with, across, after) and “lásios” (hairy), in 1210 reference to the former placement of the type species. Homophonously forming “metal-Asius”, 1211 invoking the death of the Trojan leader Asius during the assault on the Archaean wall of Troy. 1212 Masculine. 1213 Comments: XXX is uniquely identified among the Lasiini by absence of the dorsal mandibular 1214 groove and lateral hypostomal carina, short and broad third antennomere, mid-set compound 1215 eyes (when present) which are reduced to at most 9 ommatidia, and near complete absence of 1216 standing setae on the head. High magnification may be required to evaluate the lateral 1217 hypostoma. 1218 1219 Genus †XXX GEN. NOV. 1220 (Figs. 10C, D) 1221 Type and included species: †L. pumilus Mayr, 1868. Original designation. 1222 ZooBank LSID: http://zoobank.org/urn:lsid:zoobank.org:act:3E019A26-D68E-41DE-86C9- 1223 A26EA22D20CE 1224 Definition (worker): 1225 1. With characters of XXX genus group (note 1). 1226 2. Maxillary palps long, reaching occipital foramen (note 2). 1227 3. Compound eyes well-developed, with > 20 ommatidia. 1228 4. Mesosomal dorsum devoid of setae. 1229 5. Legs entirely devoid of standing setae. 1230 6. Petiolar node weakly inclined anteriorly, node squamiform. 1231 Notes on definition: 1232 Note 1. Several characters could not be evaluated for †XXX, including the ventromedial 1233 mandibular groove, palpomere proportions, clypeal profile, and lateral hypostomal carina. 1234 Note 2. From Dlussky (2011).

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1235 Etymology: Combination of the Greek words “ēlektro-” (amber) and “lásios” (hairy), indicating 1236 the geological provenance and prior taxonomic placement of these lasiine ants. Masculine. 1237 Comments: We place †XXX with XXX together based on the results of our phylogenetic 1238 analyses, and we interpret presence of the broad third antennomere and highly reduced cranial 1239 setation as synapomorphies of this clade. Although we have not examined the neotype of †XXX 1240 pumilus, designated by Dlussky (2011) and deposited in Muzeum Ziemi Polskiej Akademii Nauk 1241 in Warsaw, the specimen we have studied is unlikely to be misidentified as it exhibits unique 1242 diagnostic traits of the species among the Lasiini, let alone of the Baltic amber fauna, including 1243 absence of setae on the head dorsum, well-developed eyes, short and broad third antennomere, 1244 and very small body size (< 2 mm). Dlussky (2011) describes the eyes of both †Lasius 1245 schiefferdeckeri and †E. pumilus as “shifted somewhat posteriorly so that the length of [the] gena 1246 [is] more than [that of the] maximum diameter of [the] eyes”. This very general statement is true 1247 of both species, however the eyes of †L. schiefferdeckeri are distinctly set in the posterior head 1248 half whereas those of †E. pumilus are situated at head midlength, distinguishing it from all 1249 Lasius genus group members. †XXX pumilus differs from M. myrmidon by the following: (1) 1250 compound eyes large; (2) maxillary palps long, reaching occipital foramen; and (3) standing 1251 setae completely absent from head dorsum and mesosoma. 1252 1253 Genus group of Prenolepis 1254 (Figs. 6F–L) 1255 Genera included: Euprenolepis, Nylanderia, Paraparatrechina, Paratrechina, Prenolepis, 1256 Zatania. 1257 Definition (worker): 1258 1. With characters of Lasiini. 1259 2. Mandible with 4–7 teeth. 1260 3. Palp formula variable: 6,4; 3,4; 5,3; 4,3; 3,3; 2,3; 2,2 (note 1). 1261 4. Basal and masticatory mandibular margins usually meeting at a strongly oblique angle. 1262 5. Clypeus modified for reception of labrum (specifically, clypeus with anterolateral 1263 notches; note 1 of XXX genus group). 1264 6. Frontal carinae conspicuous, > 0.5 × anteroposterior antennal torulus diameter. 1265 7. Frontal region of head between antennal toruli flat, frontal carinae not raised above toruli. 1266 8. Antenna 11- or 12-merous. 1267 9. Third antennomere usually longer than broad (see note 3 of XXX genus group). 1268 10. Compound eyes usually at about head midlength, sometimes set posteriorly (see note 3 of 1269 Lasius genus group). 1270 11. Ocelli present or absent. 1271 12. Dorsum of head with standing setae. 1272 13. Mesonotum, metanotum, and/or propodeum discontinuous, metanotum usually 1273 differentiated (note 2). 1274 14. Mesopleuron anterodorsal margin, near posterolateral region of pronotum, usually 1275 without raised boss nor with narrow or broad groove traversing the mesosternum (note 4 1276 of Lasius genus group). 1277 15. Metapleural gland very small, orifice directed completely to nearly completely 1278 posteriorly (note 5 of XXX genus group). 1279 16. Propodeal spiracle situated in lower 2/3 of propodeum. 1280 17. Legs with or without standing setae.

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1281 18. Petiole with very low node and posteriorly elongated. 1282 19. Abdominal tergum III inclined anteriorly, with deep groove for reception of petiole, 1283 concealing most of petiole in dorsal view when gaster raised. 1284 20. Abdominal tergum III with complete tergosternal fusion adjacent to helcium, and fusion 1285 raised anterodorsally, with sclerites becoming free high up on segment. 1286 21. Pubescence of abdominal terga VI and VII usually dense, forming a uniform layer of 1287 short and strongly curved setae (note 3). 1288 Notes on definition: 1289 Note 1. The palp formula of most genera is 6,4; Euprenolepis has a palpomere count of 3,4 and 1290 in Pseudolasius it is variable but not 3,4 (Bolton 2003). 1291 Note 2. The metanotum of many but not all Paraparatrechina is undifferentiated. 1292 Note 3. These setae may or may not completely cover tergum VI. Absence of this characteristic 1293 pubescence occurs in and may be a synapomorphy of Euprenolepis and Paratrechina. 1294 Comments: Paratrechina has recently expanded from a single species to five (LaPolla et al. 1295 2010, 2013; LaPolla & Fisher 2014). One of these recently added species, P. kohli (Forel, 1916) 1296 was originally described in Prenolepis, transferred to Nylanderia (as a subgenus of 1297 Paratrechina), back to Prenolepis, and finally to Paratrechina (sensu stricto) (LaPolla & Fisher 1298 2014). This species stood out from the remainder in its surface sculpturing, eye positioning, 1299 color, and mandibular dentition. It was observed during this study that P. kohli has a petiolar 1300 node which exceeds propodeal spiracle height, lacks the raised tergosternal fusion of abdominal 1301 segment III and posterior elongation of the petiole, and closely resembles species of Anoplolepis, 1302 specifically A. carinata (Emery, 1899) and A. tenella (Santschi, 1911). Additionally, P. kohli has 1303 11-merous antennae, an undifferentiated metanotum, has posteriorly set eyes, and lacks ocelli, as 1304 is characteristic of Anoplolepis (Fisher & Bolton 2016). We find in combined analyses that P. 1305 kohli always falls outside of the Lasiini (Figs. 4, S7–S9). For these morphological and 1306 phylogenetic reasons, we transfer the species Paratrechina kohli to Anoplolepis in the 1307 Plagiolepidini, forming A. kohli (Forel, 1916) comb. n. (tribal transfer). Expanded sampling of 1308 male characters may improve placement of the fossil taxon †Prenolepis henschei with respect to 1309 the remainder of the genus group. 1310 1311 Incertae sedis in the Formicinae 1312 1313 Genus †Kyromyrma: Comparative morphological study of †Kyromyrma (~92 mya, New Jersey 1314 amber; Grimaldi & Agosti 2000) at the gross (Figs. 8L, 9K) and fine scales reveals considerable 1315 morphological affinity to Lasius (holotype examined at AMNH). In the original description of 1316 †Kyromyrma, the authors did not address the problem of within-subfamily placement, merely 1317 noting that “the fossil bears an overall resemblance to Prolasius, mostly by virtue of the 1318 generalized morphology” (Grimaldi & Agosti 2000, p. 13681). Our combined evidence analyses 1319 resulted ambiguous support for the placement of †Kyromyrma. Results placed †Kyromyrma as 1320 sister to the Lasius genus group (Fig. S5), sister to the core Lasiini (Figs. S8 & S9), sister to all 1321 Lasiini (Fig. S7), or sister to Formicinae exclusive of Myrmelachistini (Fig. 4). Statistical 1322 support for these placements was uniformly low. 1323 1324 1325 Genus †XXX GEN. NOV. 1326 (Figs. 10E, F)

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1327 Type and included species: †Pseudolasius boreus Wheeler, 1915. Original designation. 1328 ZooBank LSID: http://zoobank.org/urn:lsid:zoobank.org:act:BFF515E7-9966-443F-8A34- 1329 555A4B41906B 1330 Definition (worker): 1331 1. With characters of the Formicinae (note 1). 1332 2. Cranium size variable, width somewhat broader than to about twice that of mesosoma 1333 (note 2). 1334 3. Mandible triangular, with 7–8 teeth. 1335 4. Dorsal mandibular groove extending along lateral mandibular margin in dorsal view. 1336 5. Palps reduced, not reaching midlength of postgenal bridge. 1337 6. Frontal carinae raised above antennal toruli. 1338 7. Antennal toruli abutting posterior clypeal margin. 1339 8. Antennomere count unknown (note 3). 1340 9. Antennomere III longer than broad. 1341 10. Compound eyes situated posterior to head midlength. 1342 11. Compound eyes not enormous and long axes subparallel. 1343 12. Ocelli minutely present or absent. 1344 13. Promesonotum domed, raised well above propodeum. 1345 14. Metanotal groove well-developed. 1346 15. Petiolar foramen in profile view clearly raised and margined with conspicuous thickened 1347 carina or lip. 1348 16. Petiolar node squamiform, tall; dorsoventral height equal to or possibly exceeding 1349 dorsum of propodeum. 1350 17. Abdominal segment III without raised tergosternal fusion. 1351 Notes on definition: 1352 Note 1. Several characters were not possible to evaluate. These include the following: Reduction 1353 of third tooth from mandibular apex; palp formula; the metapleural gland; ventral length of 1354 the petiolar foramen; relative separation of the meso- and metacoxae; presence or absence of 1355 setal double-row on ventral tibial surfaces; petiolar apodeme conformation; cross-sectional 1356 shape of the petiolar sternum; and presence or absence of the post-helcial sulcus. 1357 Note 2. The massive head of †P. boreus led Wheeler (1915) to erroneously assume that the 1358 specimens were majors of Pseudolasius; Wheeler did note the posteriorly set eyes of †P. 1359 boreus, one of the distinctions between it and Pseudolasius, but vacillated based on the 1360 insufficient knowledge of the latter genus at that time. 1361 Note 3. The examined specimens are missing their terminal antennomeres, and no previous 1362 descriptions of †P. boreus include an antennomere count. 1363 Etymology: A simplification of “pseudo-Pseudolasius” inspired by Phil Collins. Masculine. 1364 Comments: †XXX boreus comb. n. was previously placed in the Lasiini (Emery 1925, Dlussky 1365 & Fedoseeva 1988, Bolton 1994), and has been considered to be a member of the genus 1366 Pseudolasius (Wheeler 1915, LaPolla & Dlussky 2010). Based on an examination of a syntype 1367 (GZG-BST04646) and a non-type (NHMW1984-31-211) worker, as well as the original 1368 description from Wheeler (1915), we reject both placements. Neither specimen can be mistaken 1369 due to the unique combination of characters they display, including setation, high petiolar nodes, 1370 massive crania, and mesosomal form. †XXX boreus does not display the modifications of the 1371 third abdominal segment characteristic of the Prenolepis genus group, also rejecting its original 1372 placement in Pseudolasius (see diagnostic note 2 above). †XXX is excluded from the Lasiini

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1373 overall by two additional characters: (1) The petiolar node, which is completely vertical and very 1374 tall dorsoventrally, is as tall as or possibly taller than the propodeum; and (2) the raised and 1375 conspicuously lipped petiolar foramen. No lasiine, extant or extinct, has such a tall petiolar node, 1376 nor do any species display the petiolar foramen conformation observed in †XXX. We cannot 1377 confidently place †XXX in any extant tribe, therefore, we consider †XXX incertae sedis in the 1378 Formicinae. As with other fossil taxa which are of uncertain placement in the Formicinae, micro- 1379 CT scans could be used to evaluate the form of the helcium and third abdominal sternum. 1380 1381 1382 Conclusion 1383 Revisionary works have traditionally scrutinized morphological systems for the systematic 1384 classification of a given group of interest. Here, we integrate traditional morphology with 1385 molecular phylogeny to understand the ant tribe Lasiini in the context of the subfamily 1386 Formicinae. Of particular interest to us are the suite of phenotypic characteristics which are used 1387 to define taxa. After reevaluating the fossil record of the Formicinae, we were able to estimate 1388 the geological age of phylogenetic branching events in the subfamily; with this chronogram, we 1389 formally estimated the probability that key diagnostic characters are ancestral or derived for any 1390 given clade. To this end, we have tested polarity hypotheses for traits used by Bolton in his 1391 crucial morphological reclassification of the Formicidae (Bolton 2003), as well as the form of the 1392 proventriculus—a structure used by taxonomists for well over a century to classify the 1393 Formicinae and other subfamilies (e.g., Forel 1874, 1878; Emery 1888, 1925). Additionally, we 1394 find that two life history traits, temporary social parasitism and fungiculture, have both evolved 1395 twice in the genus. 1396 One key question arising from our work is how to treat the two genera of the Lasius genus 1397 group relative to the fossils of Lasius, given the problem of anagenesis and the apparent high 1398 degree of morphological conservativism of the nominotypical genus. This conservativism is 1399 evidenced by the striking similarity of the L. niger clade with the Baltic amber fossils and the 1400 Cretaceous genus †Kyromyrma. Should the Baltic amber taxa be split, even though there are not 1401 enough morphological features to differentiate them from Lasius as currently delimited? Or 1402 should the phenotypically distinct genus Myrmecocystus, like Acanthomyops before it, be 1403 synonymized with Lasius? Perhaps it will be necessary to split the Baltic taxa and to revive 1404 Acanthomyops for the Lasius flavus clade. For now, we acknowledge the difficulty of justifying 1405 our choice to retain the Baltic amber fossils in the genus Lasius given the principle that all higher 1406 taxa are to be monophyletic. We also emphasize that the unexpectedly young age of the crown 1407 Lasius genus group is not likely to be an artifact of dating method or taxon sampling. We 1408 hypothesize that the overall short branch lengths of the Lasius genus group are a result of 1409 increased species turnover associated with colonization of and radiation into the recently 1410 emerged North temperate ecosystems (Economo et al. 2018). The young crown group age of 1411 Lasius and Myrmecocystus in turn likely contributes to lack of morphological divergence and 1412 makes these ants known examples of taxonomically difficult genera (e.g., Snelling 1976, Seifert 1413 & Galkowski 2016). 1414 With respect to our evolutionary questions, we reconceive the ancestral formicine as having 1415 originated around the end of the Early Cretaceous or beginning of the Late Cretaceous with 1416 anteriorly set eyes, narrowly set coxae, circular propodeal spiracles, lacking a sulcus posterad the 1417 helcial sternite, and having low anterolateral corners of abdominal tergum III. Crucially, we find 1418 support for the “sepalous” condition of the proventriculus as an autapomorphy of the Formicinae,

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1419 suggesting that greater control of the crop valve may have been a key innovation in the origin 1420 and subsequent radiation of the subfamily during the Mesozoic. In this evolutionary 1421 morphological context, the Lasiini derived wide-set coxae and raised gastral corners 1422 independently of the Plagiolepidini and some Melophorini, the former in the Late Cretaceous and 1423 the latter probably during the Tertiary period. We find that the new genus, XXX, is a probable 1424 relict of an ancient clade which split from its common ancestor with the tropicopolitan radiation 1425 of the Prenolepis genus group during the Late Cretaceous. 1426 To date, our study provides the best-supported hypothesis of phylogenetic relationships 1427 among the major lineages of Lasius and, in addition to revising the classification of the Lasiini, 1428 contributes to a greater understanding of the tempo and mode of formicine evolution. From the 1429 phylogenetic perspective, we conclude that inclusion of a clock model in analyses of morphology 1430 results in substantial topological improvement, and that divergence dating analyses should, if 1431 possible, include both morphological and molecular data in the tip-dating framework. Despite the 1432 advances we propose, much work remains to be done for the Lasiini. Extended morphological 1433 data are required to resolve the placements of fossils more completely. Such analyses may 1434 benefit from the inclusion of males (e.g., Barden et al. 2017a) and characters specific to the 1435 Prenolepis genus group. Our work has revealed that two fossil taxa were misplaced at genus rank 1436 (†Lasius pumilus, now in †XXX gen. nov., and †Pseudolasius boreus, now †XXX gen. nov.), as 1437 were two fossil taxa misplaced to tribe rank (†Glaphyromyrmex and †XXX gen. nov.). We have 1438 great hope for the future of holistic phylogenetic studies as ever more genomic and phenomic 1439 data are made available. 1440 1441 1442 Supporting information 1443 1444 All data sets, as well as input and output of all the analyses mentioned in the Methods section are 1445 deposited on Dryad (https://doi.org/10.25338/B8B645). 1446 1447 Table S1. List of primers used to generate new sequence data. 1448 Table S2. GenBank accession numbers for all previously and newly published sequences. 1449 Table S3. PartitionFinder “best scheme” for the 9-gene matrix. 1450 Appendix S1. Morphometric differentiation of Lasius, XXX, and †XXX. 1451 Appendix S2. Morphological state definitions used for the 114-character matrices. 1452 Fig. S1. Constraint schemes used for the stepping-stone topology tests in MrBayes. 1453 Fig. S2. The results of the 55-taxon concordance factor analysis. 1454 Fig. S3. The results of the 154-taxon morphology-only MrBayes analysis. 1455 Fig. S4. The results of the 135-taxon molecular-only MrBayes analysis. 1456 Fig. S5. The results of the 135-taxon molecular-only IQ-TREE analysis. 1457 Fig. S6. The results of the 154-taxon combined morphology & molecular MrBayes analysis 1458 without a clock model. 1459 Fig. S7. The results of the 154-taxon combined morphology & molecular MrBayes analysis with 1460 a uniform IGR clock. 1461 Fig. S8. The results of the 154-taxon combined morphology & molecular MrBayes analysis with 1462 a uniform TK02 clock. 1463 Fig. S9. The results of the 154-taxon combined morphology & molecular MrBayes analysis with 1464 a FBD IGR clock.

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1465 Fig. S10. Ancestral state estimation results for relative compound eye position. 1466 Fig. S11. Ancestral state estimation results for coxal separation. 1467 Fig. S12. Ancestral state estimation results for ventral sulcus posterad helcium. 1468 Fig. S13. Ancestral state estimation results for propodeal spiracle shape. 1469 Fig. S14. Ancestral state estimation results for proventriculus form. 1470 Fig. S15. Ancestral state estimation results for abdominal segment III conformation. 1471 Fig. S16. Ancestral state estimation results for temporary social parasitism. 1472 Fig. S17. Ancestral state estimation results for fungiculture. 1473 Fig. S18. Dot plot of relative eye position data among Lasius, XXX, and †XXX. 1474 Fig. S19. Plots of eye position indices among Lasius, XXX, and †XXX. 1475 Fig. S20. Plot of relative eye size contrasting Lasius clades with XXX and †XXX. 1476 1477 1478 Acknowledgments 1479 1480 We extend our primary gratitude to Phil Ward. Phil provided us with funding, specimens, 1481 sequences, constructive discussion, and attention to the manuscript; as well, he recommended the 1482 name XXX, which we appreciate. We also appreciate the critique of an anonymous reviewer of 1483 our first manuscript, as this led to our discovery of the placement of myrmidon and 1484 morphological reevaluation of the Lasiini. Thanks to Dave Grimaldi for granting access to the 1485 amber collection at the AMNH. For his exquisite automontage images of Baltic amber material 1486 and for sharing his insights into the fossil record of the Formicidae, we thank Vincent Perrichot. 1487 Brian Fisher and Michele Esposito: Thank you for AntWeb and for maintaining Bolton’s catalog 1488 and Ward et al.’s AntBib on AntCat. We thank Barry Bolton and Brian Fisher for commenting 1489 on the manuscript prior to submission. We thank Bonnie Blaimer for discussion of the 1490 Formicinae and for sending us the UCE single-locus gene trees for coalescence analysis. We 1491 thank Adrian Richter for discussing head characters. Jill Oberski caught some mistakes in the 1492 manuscript and translated Mayr’s writing for us; thank you. Finally, we thank Lech Borowiec 1493 and Sebastian Salata for contributing specimens of XXX myrmidon and Lasius carniolicus, 1494 respectively.

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1966 Tables 1967 1968 Table 1. Overview of study design, explaining which datasets and analyses were used to answer 1969 our primary questions. 1970 1971 Table 2. Voucher specimen collection data for sequences generated de novo. Specimen codes 1972 link to more detailed collection data on AntWeb (http://www.antweb.org); all specimens were 1973 deposited in the Bohart Museum of Entomology (UCDC) except for L. sonobei, which was a 1974 destructive extraction (collection code: PSW14663, duplicates available). 1975 1976 Table 3. Datasets and analyses used for divergence dating. 1977 1978 Table 4. Bayes Factor results, testing alternative placements of XXX and the monophyly of the 1979 subgenera using the Lasiini_55t matrix. The mean value for the best-supported hypothesis for 1980 each comparison is bolded. 1981 1982 Table 5. Results of divergence dating and historical biogeography reconstruction analyses. 1983 Divergence dating compares 95% HPDs for selected nodes given alternative calibration 1984 treatments. All ages reported are in units of mya and are for crown nodes. Biogeography 1985 reconstruction provides ranges at key nodes along with their likelihood (I = Indomalayan, Na = 1986 Nearctic, P = Palearctic; combinations indicate both regions were occupied). 1987 1988 Table 6. Results of ANOVA tests on alternative ancestral state estimation models. 1989 1990 Table 7. Empirical Bayesian posterior probabilities from ancestral state estimation for selected 1991 nodes; numbers may not add up to 1.00 due to rounding error. For ease of reference, the highest 1992 probability state is bolded and lowest italicized for each node. 1993 1994 1995 Figure legends 1996 1997 Fig. 1. Comparison of phylogenetic hypotheses for Lasius; red indicates paraphyly of Lasius 1998 sensu stricto, orange of Lasius (Cautolasius), and violet of Lasius (Chthonolasius). (A) Intuition- 1999 based topology from Wilson (1955). (B) and (C) combined morphological and mitochondrial 2000 topologies of Janda et al. (2004) and Maruyama et al. (2008). (D) Consensus topology from the 2001 current study; see Table 4 for Bayes Factor tests of our constraint analyses. 2002 2003 Fig. 2. Molecular phylogeny of the Lasiini inferred from Bayesian analysis of the Lasiini_55t 2004 data matrix. Node support values are reported as Bayesian posterior probabilities (BI) vs. 2005 maximum likelihood bootstrap (ML). Filled circles at nodes indicate full support from both 2006 analyses. An asterisk (*) indicates that the topology of the ML analysis diverged from the BI 2007 analysis at the given node. 2008 2009 Fig. 3. Summary coalescence species-tree phylogeny from ASTRAL-II analysis of 959 UCEs 2010 from Blaimer et al. (2015). Node support values are given as local posterior probabilities (LPP). 2011 Filled circles at nodes indicate full support.

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2012 2013 Fig. 4. Results from phylogenetic analysis of the Lasiini_w_outgroups_w_fossils_154t combined 2014 morphological and molecular dataset of the in MrBayes using the fossilized birth-death branch 2015 length prior and the IGR clock variation prior. Fossil taxa are indicated by a dagger preceding 2016 the name. Node support values are in Bayesian posterior probability. Filled circles indicate full 2017 support. Horizontal blue bars at nodes are 95% highest posterior density (HPD) intervals. 2018 2019 Fig. 5. Results of the BioGeoBEARS and life history trait ancestral state estimation analyses on 2020 a chronogram pruned from the analysis of the Lasiini_w_outgroups_w_fossils_154t combined 2021 morphological and molecular dataset. Horizontal blue bars at nodes are 95% highest posterior 2022 density (HPD) intervals from the divergence dating. Colored circles on nodes are inferred 2023 inherited biogeographic ranges, with the top half of each corresponding to the upper branch and 2024 bottom half with the lower branch; colored circles at branch tips represent biogeographical 2025 coding for terminal; dashed lines at 60 and 30 mya indicate the boundaries of the three 2026 BioGeoBEARS dispersal rate matrix time periods. Colored boxes at branch tips represent 2027 presence or absence of two traits, in Lasius: temporary social parasitism (black = absence, red = 2028 presence) and fungiculture (black = absence, green = presence). Stars on internal branches 2029 indicate the evolution of temporary social parasitism (red) and fungiculture (green) The figure 2030 background indicates generalized major climatological regimes during Earth’s history (Royer et 2031 al. 2004; Zachos et al. 2008; Hansen et al. 2013), while the inset globes visually represent 2032 paleoclimate and landmass configuration modified from Scotese (2010). 2033 2034 Fig. 6. Gross phenotypic synopsis of the Lasiini; all images in profile view; scale bars = 1.0 mm. 2035 A, Cladomyrma hewitti (CASENT0173906); B, Lasius lasioides (CASENT0906077); C, Lasius 2036 citrinus (CASENT0103542); D, Myrmecocystus melliger (CASENT0103518); E, XXX myrmidon 2037 (CASENT0903666); F, Euprenolepis procera (CASENT0906260); G, Nylanderia amblyops 2038 (CASENT0007735); H, Paraparatrechina albipes (CASENT0178759); I, Paratrechina 2039 ankarana (CASENT0454372); J, Prenolepis imparis (CASENT0179615); K, Pseudolasius 2040 amaurops (CASENT0106005); L Zatania gibberosa (CASENT0281461). (Image credits, 2041 AntWeb: A, C, G, H = April Nobile; B = Shannon Hartman; D = Jen Fogarty; E, Will Ericson; F, 2042 Estella Ortega; I, Michele Esposito; J, Erin Prado; K, Michael Branstetter; L, Ziv Lieberman). 2043 2044 Fig. 7. Some key features of the major clades of the Lasiini. A, third abdominal tergite modified 2045 with dorsoventrally-oriented groove for reception of entire petiole (Pseudolasius breviceps); 2046 black line indicates contour of tergum, and black ellipse the spiracle; B, third abdominal tergite 2047 receiving only posterior base of petiole (Myrmecocystus mimicus); white line indicates contour 2048 of tergum, and white ellipse the spiracle; C, frontal protuberance and effaced carinae low relative 2049 to face (Pseudolasius breviceps); D, frontal protuberance and effaced carinae raised dorsally 2050 from face (Myrmecocystus mimicus); E, metapleural gland atrium grossly enlarged (Lasius 2051 subumbratus); F, metapleural gland atrium small (Lasius turcicus). 2052 2053 Fig. 8. Gross phenotypic synopsis of the extant species groups of Lasius, plus two 2054 phenotypically similar fossil species; all images in full-face view; scale bars = 0.5 mm. A, Lasius 2055 brunneus (CASENT0280440); B, Lasius fuliginosus (CASENT0179898); C, Lasius nr. niger 2056 (CASENT0106128); D, Lasius nr. atopus (CASENT0234858); E, Lasius carniolicus 2057 (CASENT0280471); F, Lasius claviger (CASENT0103542); G, Lasius brevicornis

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2058 (CASENT0280456); H, Lasius nearcticus (CASENT0104774); I, Lasius pallitarsis 2059 (CASENT0005405); J, Lasius aphidicola (CASENT0280468); K, †Lasius schiefferdeckeri 2060 (MNHNB25217); L, †Kyromyrma neffi (AMNH-NJ1029). (Image credits: D = the authors; 2061 AntWeb: A = Will Ericson; B = Erin Prado; C = Michael Branstetter; E, G, J = Shannon 2062 Hartman; F, H = April Nobile; I = unknown; K = Vincent Perrichot; L = Dave Grimaldi & 2063 Vincent Perrichot.) 2064 2065 Fig. 9. Gross phenotypic synopsis of the species groups of Lasius, plus two phenotypically 2066 similar fossil species; all images in profile view; scale bars = 1.0 mm. A, Lasius brunneus 2067 (CASENT0280440); B, Lasius fuliginosus (CASENT0179898); C, Lasius nr. niger 2068 (CASENT0106128); D, Lasius nr. atopus (CASENT0234858); E, Lasius carniolicus 2069 (CASENT0280471); F, Lasius claviger (CASENT0103542); G, Lasius brevicornis 2070 (CASENT0280456); H, Lasius nearcticus (CASENT0104774); I, Lasius pallitarsis 2071 (CASENT0005405); J, Lasius aphidicola (CASENT0280468); K, †Lasius schiefferdeckeri 2072 (HJF013); L, †Kyromyrma neffi (AMNH-NJ1029). (Image credits: D = the authors; AntWeb: A 2073 = Will Ericson; B = Erin Prado; C = Michael Branstetter; E, G, J = Shannon Hartman; F, H = 2074 April Nobile; I = unknown; K = Vincent Perrichot; L = Dave Grimaldi & Vincent Perrichot.) 2075 2076 Fig. 10. Habitus images for the three new genera recognized in this study; scale bars = 0.25 mm. 2077 A, B, XXX myrmidon gen. and comb. n. (CASENT0906115); C, D, †XXX pumilus gen. and 2078 comb. n. (SMFBE1226); E, F, †XXX gen. and comb. n. (GZG-BST04646). Views: A = full- 2079 face; B, E = profile; C = laterally oblique facial; D, F = dorsal. Observe: (1) The compound eyes 2080 of XXX and †XXX are mid-set, and (2) †XXX gen. n. has a distinctly-carinate propodeal foramen, 2081 vertical petiolar node, and non-grooved (lateromedially convex) third abdominal tergite. 2082 2083 2084 Supplementary material 2085 2086 Table S1. Primers used for amplification and sequencing abdominal-A (abdA), Arginine Kinase 2087 (ArgK), rudimentary (CAD), elongation factor 1 α F2 copy (EF1αF2), long-wavelength 2088 rhodopsin (LW Rh), Topoisomerase I (Top1), Ultrabithorax (Ub), wingless (wg). Blank spaces 2089 every three nucleotides delimit codons, except for intronic sequences, which are arbitrarily 2090 presented with spaces every three nucleotides. Most frequently used primers denoted by asterisk. 2091 2092 Table S2. GenBank data for all sequences used in this study. 2093 2094 Table S3. PartitionFinder best schemes for the Lasiini_w_outgroups_135t and Lasiini_55t 2095 datasets. 2096 2097 2098 Appendix S1. 2099 2100 Morphometric differentiation of Lasius, XXX, and †XXX. 2101 Because eye position and size are important for the classification of the Lasiini, we measured six 2102 variables for representatives of all Lasius species groups plus XXX myrmidon and †XXX pumilus. 2103 Measurements were taken using a digital dual-axis stage micrometer (Mitituyo) beneath a Leica

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2104 MZ7.5 (50 × maximum magnification); two specimens of M. myrmidon and the specimen of †E. 2105 pumilus were measured from digital micrographs using Adobe Illustrator. All measurements, 2106 except for those of †E. pumilus, were recorded to three significant figures. An effort was made to 2107 measure two individuals of each species. In total, measurements were taken for 104 specimens 2108 representing 53 species of Lasius, four specimens of XXX, and one specimen of †XXX. From the 2109 six measurements recorded for each specimen, we calculated two metrics each of eye length, eye 2110 midlength location, plus the averaged eye length. With the measurements and transformations, 2111 seven indices were calculated in order to control for size variation among the measured taxa. We 2112 used ggplot in R to visualize the results of these analyses. 2113 2114 Measurements 2115 HL1 Head length 1. Length of head in full-face view from posteromedian margin of head 2116 to an imaginary line drawn between the anterolateral clypeal corners. 2117 HL2 Head length 2. Length of head in full-face view from posteromedian margin of head 2118 to anteromedian point of clypeus. 2119 ED1 Eye distance 1. Distance from an imaginary line drawn between the anterolateral 2120 clypeal corners and the anterior margins of the compound eyes. 2121 ED2 Eye distance 2. Distance from an imaginary line drawn between the anterolateral 2122 clypeal corners and the posterior margins of the compound eyes. 2123 ED3 Eye distance 3. Distance from the anteromedian point of the clypeus to the anterior 2124 margins of the compound eyes. 2125 ED4 Eye distance 4. Distance from the anteromedian point of the clypeus to the posterior 2126 margins of the compound eyes. 2127 2128 Transformations 2129 EL1 Eye length 1. ED2-ED1. 2130 EL2 Eye length 2. ED4-ED3. 2131 ELx Eye length average. (EL1+EL2)/2. 2132 EML1 Eye midlength 1. ELx+ED1. 2133 EML2 Eye midlength 2. ELx+ED2. 2134 2135 Indices 2136 ESI Eye size index. ELx/HL2*100. 2137 ClEaI Lateroclypeus to eye anterior index. ED1/HL1×100. 2138 ClEpI Lateroclypeus to eye posterior index. ED2/HL1×100. 2139 ClEmI Lateroclypeus to eye midlength index. EML1/HL×100. 2140 CaEaI Anteroclypeus to eye anterior index. ED3/HL2 ×100. 2141 CaEpI Anteroclypeus to eye posterior index. ED4/HL2 ×100. 2142 CaEmI Anteroclypeus to eye midlength index. EML2/HL2 ×100. 2143 2144 Discussion 2145 We observe that the compound eyes of XXX and †XXX are clearly set in a more-anterior position 2146 relative to Lasius (Fig. S18). Our calculated indices of eye position (Fig. S19) demonstrate that 2147 the midlength of the compound eyes of XXX and †XXX are set anterior to head midlength as 2148 measured from the anterolateral clypeal margins (Fig. S19F) and at about midlength as measured 2149 from the anterior clypeal margin (Fig. S19G). Because of variation in eye length, we observe

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2150 overlap in indexed measurements to the posterior eye margin between XXX and Lasius (Figs. 2151 S19A, C) and near overlap of indexed measurements to the anterior eye margin between †XXX 2152 and Lasius (Figs. S19B, D). We also observe that although the two clades of Lasius approach 2153 XXX with respect to relative eye size, they do not overlap, in contrast to the relatively large-eyed 2154 †XXX. Finally, we observe that the compound eyes of the L. niger clade are proportionally larger 2155 than those of the L. flavus clade (Fig. S18, S20), with some exceptions (e.g., L. pallitarsis, very 2156 small individuals). Having reviewed the patterns, we conclude that the morphometric data 2157 provide quantitative support for our qualitative definitions of the three genera recognized in our 2158 work, as well as the two primary clades of Lasius. 2159 2160 2161 Appendix S2. 2162 2163 Character list used in combined phylogenetic analysis 2164 All character states were scored as Boolean values, with states defined as TRUE (1) or FALSE 2165 (0). Characters were composed and scored independently from that of Maruyama et al. (2008), a 2166 study which itself expanded on Janda et al. (2004). Note that character descriptions of the head 2167 assume prognathy. 2168 2169 Body size proxy. 2170 1. Head width ≥ 1 mm. — Character derived from Peeters & Ito (2015) wherein head 2171 width was used as a proxy for body size in argumentation that miniaturization was a 2172 key innovation in the early evolutionary history of the Formicidae. 2173 Compound eyes and ocelli. 2174 2. Medial eye margins strongly converging, rather than parallel. — The state of anteriorly 2175 converging compound eyes has evolved a number of times in the Formicidae, including 2176 among the formicoids the genera Turneria, Santschiella, Opisthopsis, Gesomyrmex, 2177 and Myrmoteras. Used previously by Bolton (2003) to define the Gesomyrmecini, 2178 including Gesomyrmex and Santschiella. 2179 3. Eye situated at or posterior to head midlength (if FALSE, then situated anterior to head 2180 midlength). — Note that the scoring for this state is different from that used for 2181 ancestral state estimation (Fig. S10). 2182 4. Eye hypertrophied (if FALSE, then small, reduced, or absent). — Eyes scored as 2183 enlarged for the pseudomyrmecine terminals (although not the case for the whole 2184 subfamily), Gesomyrmex, Gesomyrmex, and Myrmoteras; this state also applies for 2185 Opisthopsis, which is not in the matrix. 2186 5. All three ocelli present. — Ocellar presence is a variable state, even among taxa where 2187 they usually occur. For this analysis, if all three ocelli were observable in a worker 2188 specimen, the state was scored as TRUE. 2189 Mandibles. 2190 6. Mandibular teeth in form of massive triangles or spines. — Scored as TRUE for 2191 Myrmoteras; teeth enlarged and nearly spine-like in Myrmecia. 2192 7. At least some mandibular teeth in form of fine serration. — Applies primarily to 2193 Dolichoderinae, which were not heavily sampled. 2194 8. Smaller teeth uniformly intercalated among larger teeth along margin. — Applies 2195 primarily to Dolichoderinae, which were not heavily sampled.

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2196 9. Mandible, third tooth from apex size as large as other teeth (i.e., not reduced). — 2197 Bolton (2003) inferred that a small third tooth from the apex was probably an ancestral 2198 state for the Formicinae, with an enlarged tooth being a synapomorphy for the 2199 Camponotini. 2200 10. Basal mandibular tooth offset from rest of teeth on masticatory margin. — The tooth at 2201 the juncture of the basal and masticatory margins is either oriented similarly to other 2202 teeth on the masticatory margin or is set more proximally or is at a distinct angle; the 2203 latter two states constitute “offset”, as used by Wilson (1955) for classification in 2204 Lasius. 2205 11. Basal mandibular margin with at least one tooth. — Excludes basal tooth of 2206 masticatory margin. A tooth or teeth on the basal mandibular margin occur with some 2207 frequency in the myrmechoderine clade (myrmeciomorphs + dolichoderomorphs). Such 2208 teeth occur sporadically in the Formicinae, and only in the formicoform radiation. 2209 12. Mandible elongate, with lateral and masticatory margins parallel or nearly parallel. — 2210 This state describes the sampled Myrmeciinae. 2211 13. Masticatory mandibular margins meeting in opposition, i.e., not overlapping when 2212 mandibles fully closed. — Mandibles with non-overlapping masticatory margins are 2213 observed in †Prionomyrmex, Nothomyrmecia, and various trap-jaw ants (Bolton 1999; 2214 character 10 of Ward & Brady 2003). 2215 14. Mandible with basal and masticatory margins rounding evenly into one another. — This 2216 state occurs sporadically among the sampled core formicoids and has been used 2217 previously by Shattuck (1995) as his character 14 for the Dolichoderinae. 2218 15. Mandibular basal and masticatory margins meeting at a strongly oblique angle. — 2219 Observed among sampled taxa primarily among the Prenolepis genus group and some 2220 Plagiolepidini, where the juncture between the basal and masticatory margins is 2221 clearly defined as an angle. 2222 16. Groove present on dorsal mandibular surface. — A dorsal mandibular groove is 2223 widespread in the Aculeata (Buren 1970; Hermann et al. 1971) and is known from 2224 various Formicidae (Ettershank 1966; Gotwald 1969; Hermann et al. 1971). Here, the 2225 groove is observed principally in Formicinae. 2226 Other mouthparts. 2227 17. Prementum concealed by labrum anteriorly, maxillary stipes laterally. — 2228 Autapomorphy of the Dorylinae (as the “dorylomorph subfamilies” in Bolton 2003). 2229 18. Maxillary palps elongate, exceeding occipital foramen. — Scoring differs here from 2230 that of character 11 in Maruyama et al. (2008). 2231 Perioral sclerites. 2232 19. Hypostoma with lateral flanges which are conspicuous in profile view. — A defining 2233 feature of Dolichoderus (Shattuck, 1992, 1995). 2234 20. Clypeus, in full face view, with longitudinal carinae at the lateral margins. — 2235 Described by Ward & Brady (2003) as the lateral clypeal carina in their character 6, 2236 after Baroni Urbani (2000, character 3). Such carinae are characteristic of 2237 †Prionomyrmex and Nothomyrmecia. 2238 21. Pleurostomal condyle conspicuously large and rectangular. — A newly defined state 2239 which is characteristic of the dolichoderomorphs (Aneuretinae + Dolichoderinae). 2240 Anterior clypeal margin.

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2241 22. Medioclypeus (= “middle lobe” of clypeus, or “discal clypeus” sensu Serna & Mackay 2242 2010, Serna et al. 2011) produced anteriorly relative to lateroclypeus (= “lateral lobes” 2243 of clypeus, or “premalar space”, sensu Serna & Mackay 2010, Serna et al. 2011). — 2244 This state is observed variably across the Formicidae. 2245 23. Anterior clypeal margin with distinct, lateromedially narrow lobate anteromedian 2246 process, which is with or without additional processes such as teeth. — Anterior clypeal 2247 processes have evolved a number of times in the crown Formicidae, including the 2248 Pseudomyrmecinae, various Myrmicinae, and various Formicinae. 2249 24. Anterior portion of clypeus broadly produced anteriorly, whether convex or truncate. — 2250 25. Anterior clypeal margin crenulate (i.e., with > 2 teeth). — Crenellation is observed in 2251 various Formicinae, Tetraponera, and Ponerinae. 2252 26. Anterior clypeal margin with median notch or emargination. — Notches or 2253 emargination of the anterior clypeal margin occurs with some frequency throughout the 2254 family. 2255 27. Anterolateral notches for reception of labrum present on anterior clypeal margin 2256 (visible in full-face view). — Observed in the Prenolepis genus group. 2257 Frontoclypeal complex and toruli. 2258 28. Antennal toruli directed dorsally (if FALSE, then toruli directed more-or-less laterally). 2259 — When the antennal insertion is treated as a plane defined by the medial and lateral 2260 torular arches, the insertion is directed dorsally, rather than laterally in the Dorylinae, 2261 which is also more-or-less the case for the Leptanillinae. 2262 29. Clypeus conspicuously extending posteriorly between antennal toruli by at least 1/4 2263 anteroposterior torulus length. — States 24, 25, and 26 could be treated as three 2264 discrete categories in a single multistate character which would be a measurement of 2265 the distance of the antennal toruli relative to the posterior clypeal margin. State 24, 2266 here, is observed generally in the Dorylinae, Dolichoderinae, and Myrmicinae. The 2267 toruli of most Formicinae either indent the clypeus (state 25) or are considerably 2268 distant from the posterior clypeal margin (state 26). Within the Formicinae, some 2269 Plagiolepidini have the clypeus extending between the toruli. 2270 30. Antennal toruli nearly contacting, contacting, or indenting posterior clypeal margin. — 2271 Characterization of this state drawn from Bolton (1994, 2003). 2272 31. Antennal toruli considerably distant from posterior clypeal margin (if FALSE, then 2273 closely approximated, abutting, or indenting). — As for character 25, characterization 2274 of this state is drawn from Bolton (1994, 2003) for definition of the Camponotini. This 2275 state is also observed in Myrmoteras, Oecophylla, and Notostigma. 2276 32. Antennal sockets in full-face view: fully exposed. — States 33, 34, and 35 could be 2277 treated as three discrete categories in a single multistate character. When viewed from 2278 dorsal aspect, the antennal sockets may be concealed to some degree by either the 2279 medial torular arch (state 34) or the frontal carinae (state 35). Among sampled taxa, 2280 fully exposed sockets are observed in Vicinopone, †Procerapachys, and a Polyrhachis 2281 species. 2282 33. Antennal sockets in full-face view: partially to completely concealed by laterally 2283 produced medial torular arches (or fused carinae + medial torular arches). — Most taxa 2284 observed in this study had antennal sockets which were partially concealed by the 2285 medial torular arches.

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2286 34. Antennal sockets in full-face view: partially to completely concealed anteriorly 2287 produced frontal carinae, which also conceal the medial torular arch (this state 2288 represents presence of true "frontal lobes"). — Most Dorylinae scored for this study 2289 had expanded frontal carinae which conceal, at least in part, the antennal sockets. 2290 Such expansion is also observed in various Myrmicomorpha (ectaheteromorphs + 2291 Myrmicinae) and a few Formicinae. 2292 Facial surfaces. 2293 35. Frontal carina present. — Frontal carinae are present in the majority of formicoids 2294 and, among taxa included in this study, are absent for a few Formicinae. 2295 36. Frontal carina conspicuous, being > 0.5 × anteroposterior antennal torulus diameter. — 2296 Relatively long frontal carinae occur in most sampled taxa. In addition to Cladomyrma, 2297 a number of core Formicinae have reduced carinae. 2298 37. Frontal carina carinate, continuing as a sharp ridge until posterior terminus (if FALSE, 2299 then carina ecarinate). — Rounded frontal carinae occur among the Lasiini and various 2300 other Formicinae here sampled. Reduction of the length of the frontal carinae (state 37, 2301 above) is not always associated with loss of carination. 2302 38. Frontal carinae very closely approximated on face. — Among taxa sampled in the 2303 present study, very close-set frontal carinae are only observed in Pseudomyrmex 2304 osurus and †Pseudomyrmex vicinus. 2305 39. Parafrontal carina (genal carina laterad antennal torulus) present. — Among the 2306 formicoids, parafrontal carinae are observed in Dorylinae and the myrmicine 2307 Terataner. 2308 States of relative antennomere length. 2309 40. Scape length (SL) < 0.5 × head width (HW). — In addition to geniculation, one of the 2310 traditional diagnostic features of the Formicidae is scapes which are long relative to 2311 the flagellum (e.g., Wilson et al. 1967a,b; Bolton 2003). Here, we evaluate the overall 2312 length of the scape by comparison to the cranium, and relative length to the flagellum 2313 separately. The former continuous is divided into three mutually exclusive, additive 2314 states. 2315 41. Scape length (SL) > 0.5 × head width (HW), < 1.0 x HW or head length (HL). — See 2316 character 40. 2317 42. Scape length (SL) > head width (HW) or head length (HL). — See character 40. 2318 43. Scape length > 0. 5 × funiculus (= pedicel + flagellum) length. — See character 40. 2319 44. Antennomere 3 longer than antennomere 4. — Variable among the Formicidae, with 2320 especially long third antennomeres observed in stem Formicidae (e.g., 2321 †Sphecomyrminae). 2322 45. Antennomere 3 broader than long. — Observed in XXX and †XXX. 2323 Sensilla basiconicum on flagellum. 2324 46. Flagellum: Sensilla basiconicum socket raised above antennomere (if FALSE, then 2325 flush). — Recorded as an autapomorphy of the myrmeciomorphs (Myrmeciinae, 2326 Pseudomyrmecinae) by Bolton (2003). 2327 Cranial setation. 2328 47. Coarse, paired macrosetae present on face. — This setational pattern is observed in 2329 various Prenolepis genus group taxa, as well as Anoplolepis gracilipes. 2330 48. Standing setae present on dorsum of head, excluding posterolateral corners and clypeus. 2331 — This is equivalent to character 26 of Maruyama et al. (2008). Our scoring differs for

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2332 the L. fuliginosus group: We observe dorsal setae near the posterior margin of the 2333 head in L. fuliginosus and L. spathepus, whereas Maruyama et al. recorded these setae 2334 as absent for these two species. 2335 49. Ventral head surface margined laterally and posteriorly by a continuous line of long, 2336 curved setae (psammophore present). — Ventral cranial psammophores have evolved a 2337 number of times in the Formicidae, notably in Pogonomyrmex and Veromessor 2338 (Myrmicinae), Melophorus (Formicinae), and Myrmecocystus. 2339 Mesosomal shape. 2340 50. Pronotum with dorsolaterally situated longitudinal margination. — Pronotal 2341 margination has evolved several times across the Formicidae. 2342 51. Promesonotal articulation immobile (sutured, whether suture externally visible or not). 2343 — Among sampled taxa, promesonotal fusion is observed in Dorylinae, Ectatomminae, 2344 and Myrmicinae. See also Bolton (2003) and Bolton in Fisher & Bolton (2016). 2345 52. Mesosomal profile diagonal, with anterior portion (pronotum and mesonotum) raised, 2346 often convex or strongly humped (if FALSE then mesosoma more-or-less linear, with a 2347 boxy shape and profile). — The TRUE state is modified from Bolton’s (2003) 2348 conception of the “domed” promesonotum, used to define his Pheidolini, particularly 2349 in contrast with the “boxy” form used to define his Myrmicini. 2350 53. Mesonotum in dorsal view conspicuously anteroposteriorly elongate (if FALSE then 2351 reduced to narrow, transverse bar, or absent). — Most sampled taxa have elongate 2352 mesonota. 2353 54. Mesonotum petiolate, being constricted and separating pronotum distinctly from 2354 mesopleural area. — This “petiolation”, or constriction, results in the “hourglass” 2355 shape of various Prenolepis genus group taxa, as well as a few other Formicinae (e.g., 2356 Oecophylla). 2357 55. Mesoscutellum conspicuous, bar like (if FALSE then reduced). — The workers of some 2358 species have secondarily derived a well-developed mesoscutellum. 2359 56. Metanotum present as distinct, well-defined, transverse sclerite (if FALSE then may be 2360 represented by groove or irregular ridge). — Although most crown Formicidae lack a 2361 well-developed metanotum, as noted by Barden & Grimaldi (2016), the metanotum is 2362 secondarily well-developed in some genera. 2363 57. Metanotal groove distinct and well-developed. 2364 Metapleural glands. 2365 58. Metapleural gland present. — As with scape-to-flagellum length, presence of the 2366 metapleural gland is a canonical defining state of the Formicidae (Wilson et al. 2367 1967a,b; Brothers, 1975; Bolton 2003). The gland has been variably lost in males and 2368 has been lost multiple times in the Camponotini. 2369 59. Metapleural gland with conspicuously enlarged atrium and bulla. — Newly observed 2370 apomorphy of the Lasius flavus clade. 2371 Propodeum. 2372 60. Propodeum armed with spines or other distinct protuberances. — Propodeal armature 2373 has arisen independently among various Formicidae. 2374 61. Propodeal spiracle: slit-shaped (if FALSE then rounded or elliptical). — Used by 2375 Bolton (2003) to define his formicine tribe group. See also Agosti (1990) and Agosti & 2376 Bolton (1990).

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2377 62. Propodeal spiracle situated at or above 2/3 dorsoventral height of propodeum in lateral 2378 view. — Propodeal spiracle location is informative for defining higher groups of the 2379 Formicidae, including the family itself (Bolton 2003, Boudinot 2015). Among the 2380 sampled taxa, a “high” spiracle is observed in the Myrmechoderines, the 2381 Ectatomminae sensu lato, some Myrmicinae, and various Formicinae. 2382 63. Propodeal spiracle situated at or near posterolateral margin of propodeum (if FALSE, 2383 then spiracle situated more anteriorly. — Although not maximally consistent, a 2384 posteriorly-situated spiracle is observed in Camponotini, and Lasiini among other taxa. 2385 64. Propodeum produced posterodorsally as shelf overhanging posterior face. — Observed 2386 in Dolichoderus, and some Camponotus. 2387 65. Propodeal foramen with thick, dorsal carina. — This state roughly distinguishes the 2388 “lasiiform grade” (Myrmelachistini, Lasiini) from the “formicoform radiation” 2389 (Melophorini through Camponotini). 2390 Legs. 2391 66. Metacoxa with posterodorsal, proximal spine. — Scored as an autapomorphy of 2392 †Gnamptogenys levinates, although such spines also occur in other Gnamptogenys. 2393 Surprisingly, similar posterodorsal process are also observed in Myrmosidae (e.g., 2394 Brothers 1975). 2395 67. Metacoxae wideset, with petiolar foramen extending to mesocoxal foramina. — One of 2396 the defining states of Bolton’s (2003) lasiine tribe group, along with a U-shaped 2397 ventral petiolar cross-section. 2398 68. Metatibia with double row of ventral (inner) setae. — Among sampled taxa, these setae 2399 are observed categorically in the Formicini and sporadically in the Camponotini and 2400 Melophorini. 2401 69. Anterior mesotibial spur well-developed (if FALSE spur formula 1 or 0). — Reduction 2402 of the anterior meso- and metatibial spurs has occurred numerous times in the 2403 Formicidae (see Appendix 2 of Bolton 2003). 2404 70. Anterior metatibial spur well-developed (if FALSE spur formula 1 or 0). — See 2405 comment for prior character. 2406 Flexibility of mesosoma. 2407 71. Metasoma capable of flexion over mesosoma. — This state is a defining feature of 2408 Oecophylla and the myrmicine Crematogaster. 2409 Abdominal segment II (“petiole”). 2410 72. Petiole (abdominal segment II) with dorsolateral longitudinal carinae anterior to the 2411 node, and which may continue onto node. — Observed in poneroids, the Dorylinae, 2412 some Ectatomminae sensu lato and Myrmicinae. 2413 73. Petiole (abdominal segment II) anterior foramen completely sessile. — Pedunculation 2414 of the petiole is described by this and the following two characters. Sessile petioles are 2415 observed in most Formicinae, excluding Myrmoteras and Oecophylla. 2416 74. Petiole (abdominal segment II) anterior foramen subsessile (anterior foramen narrowed, 2417 giving petiole an anteriorly extended look in lateral or dorsolateral view). 2418 75. Petiole (abdominal segment II) anterior foramen completely pedunculate (anterior 2419 region of segment long, in comparison with posterior region, anterior region narrow). 2420 76. Petiole (abdominal segment II) posterior foramen pedunculate.

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2421 77. Petiole strongly inclined anteriorly or with posterior portion elongate. — This is a 2422 feature of those groups which have the “gaster” overhanging the petiole, including the 2423 Tapinomini (Dolichoderinae), Plagiolepidini, and Prenolepis genus group. 2424 78. Petiolar node relatively high, reaching or exceeding propodeal spiracle in lateral view. 2425 — While a reduced petiolar node is characteristic of groups with the “prenolepidine 2426 syndrome” (i.e., those mentioned for the prior state), the petiolar node varies in length 2427 throughout the Formicidae. For this reason, TRUE for this state means “tall” in 2428 distinction to “short” or “very short”. 2429 79. Dorsal apex of petiolar node transversely carinate or pointedly angular, whether 2430 anteriorly, posteriorly, or at midlength. — Transverse carination appears sporadically 2431 across the Formicidae but is by no means consistent. 2432 80. Petiolar node anteroposteriorly narrow, thus scale-like. — The “squamiform” node of 2433 various Formicidae. 2434 81. Petiolar node with dorsal armature (spine or spines present). — Dorsal armature has 2435 arisen infrequently. 2436 82. Petiolar laterotergite present (if false, then reduced to inconspicuousness or absent 2437 altogether). — This is probably an ancestral feature of the Formicidae which has been 2438 lost in parallel several times. Such loss is observed during in taxa with tergosternal 2439 fusion. 2440 83. Petiole (abdominal segment II) tergosternal fusion present. — This represents the first 2441 step toward complete tergosternal fusion. 2442 84. Petiole (abdominal segment II) tergosternal fusion complete (if FALSE, then may be 2443 partial or absent). 2444 Abdominal segment III. 2445 85. Abdominal segment III, transverse sulcus present posterad helcial sternite. — Bolton 2446 (2003) recorded the sulcus as present for the Camponotini, “Notostigmatini”, 2447 Formicini, and some “Melophorini”. 2448 86. Differentiated presternite of abdominal segment III (helcial sternite), in profile, 2449 concealed by tergite. — This and the following two states were described by Bolton 2450 (1990a) in his crucial study of the abdominal characters. This state is probably 2451 ancestral for the Formicidae. 2452 87. Differentiated presternite of abdominal segment III (helcial sternite), in profile, 2453 exposed, bulging ventrally and overlapped by tergite. — An autapomorphy of the 2454 Dorylinae discovered by Bolton (1990a,b). 2455 88. Differentiated presternite of abdominal segment III (helcial sternite), in profile, 2456 exposed, continuous with tergite. — An autapomorphy of the Myrmicinae discovered 2457 by Bolton (1990a,b). 2458 89. Anterior articulatory surfaces of abdominal segment III infraaxial, i.e., below segment 2459 midheight. — The characterization of this and the following two states was derived 2460 from Keller (2011). The state of having an infraaxial helcium is widespread among the 2461 crown Formicidae. 2462 90. Anterior articulatory surfaces of abdominal segment III axial, i.e., at segment 2463 midheight. — In the present taxon set, this state is observed in the Dorylinae and all of 2464 the myrmeciomorphs except Myrmecia. 2465 91. Anterior articulatory surfaces of abdominal segment III supraaxial, i.e., above segment 2466 midheight. — Although supraaxiality occurs in various poneroids, such as most

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2467 Amblyoponinae, and some Myrmicinae, Aneuretus is the only formicoid in present 2468 matrix for which the supraaxial state is true. 2469 92. Abdominal sternum III with raised ridge or bosses (= prora) anteriorly. 2470 93. Abdominal segment III tergum and sternum fused. — In addition to tergosternal fusion 2471 of abdominal segment IV (character 101), tergosternal fusion of segment III is a feature 2472 of metasomal “tubulation”, sensu Taylor (1978). See also Bolton (1994, 2003) and 2473 Fisher & Bolton (2016). 2474 94. Abdominal segment III dorsoventrally petiolated (reduced in size, forming 2475 "postpetiole"). — “Petiolation” is a syndrome of modifications which are frequently 2476 referred to, but rarely defined. Here, we score Dorylinae, Myrmecia, 2477 Pseudomyrmecinae, and Myrmicinae as having petiolation of the third abdominal 2478 segment. Characters 95 and 96 also pertain to petiolation of AIII. 2479 95. Abdominal segment III moderately lateromedially narrowed relative to segment IV in 2480 dorsal view (i.e., AIII width > 0.5 × but < 0.90 × AIV width). — Lateromedial 2481 narrowing of the third abdominal segment relative to the fourth is another feature 2482 observed in taxa which display the “petiolization” syndrome. A narrow third segment 2483 is observed in the Myrmeciomorpha (Pseudomyrmecinae, Myrmeciinae), and is scored 2484 as such. Note that this applies to Nothomyrmecia, despite the fact that the third 2485 abdominal segment of this genus is conical and lacks a muscular node. 2486 96. Abdominal segment III strongly lateromedially narrowed relative to segment IV in 2487 dorsal view (i.e., AIII width < 0.5 × AIV width). — This is the extreme state of 2488 lateromedial narrowing of abdominal segment III associated with the “petiolization” 2489 syndrome. This was scored as TRUE for the Myrmicinae. 2490 97. Abdominal segment III tergosternal margins forming narrow shoulder laterad helcium. 2491 — “Shouldering” of abdominal segment III was used by Bolton (2003) to diagnose 2492 various groups of the Myrmicinae (e.g., comment iii of the solenopsidine tribe group), 2493 but is also observed in Formicinae, such as the Prenolepis genus group and the 2494 Plagiolepidini. 2495 98. Abdominal segment III tergosternal margins raised high above helcium. — This state, 2496 plus the preceding, were used by Bolton (2003) to define his Plagiolepidini. 2497 99. Abdominal segment III elongate, comprising most of gaster (segments III+) in dorsal 2498 view. — Observed in various Camponotini, particularly Polyrhachis (Bolton 1994). 2499 This state also applies to groups which were not sampled in the present study, such as 2500 various Myrmicinae. 2501 Metasoma, posterior to segment III. 2502 100. Cinctus of abdominal segment IV defining presclerites (correlated with formation of 2503 postpetiole) present. — In many Formicidae, a given tergum or sternum may be divided 2504 into pre- and post-sclerites by a transverse sulcus (cinctus) (Bolton 1990). 2505 101. Abdominal segment IV tergosternal fusion present (whether partial or complete). — A 2506 probable synapomorphy of the poneroid clade, with reversal in Adetomyrma, tabulated 2507 by Bolton in Fisher & Bolton (2016); see also Bolton (2003). 2508 102. Spiracles of abdominal segments V–VII exposed (if false, then concealed by preceding 2509 tergite). — An autapomorphy of the Dorylinae (Bolton 1990, 1994, 2003). 2510 103. Stridulitrum of abdominal segment IV tergum present. — Stridulitra occur, with some 2511 frequency, on the fifth abdominal tergum throughout the Formicidae.

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2512 104. Stridulitrum of abdominal segment IV sternum present. — The stridulitrum of the fifth 2513 abdominal sternum is a unique derivation of Nothomyrmecia. 2514 105. Abdominal terga VI and VII with dense, uniform layer of short and strongly curved 2515 setae. 2516 106. Sting present. — A functional sting has been reduced in various Formicidae (Kugler 2517 1978, 1979), including the Attini (sensu Ward et al. 2015) and Crematogastrini (sensu 2518 Blaimer et al. 2018). The sting is also absent in the Formicinae and Dolichoderinae, as 2519 has been recognized since the classical era of ant taxonomy (Mayr 1868, 1869; Forel 2520 1878). 2521 107. Acidopore and coronula present. — The seventh abdominal sternum in Formicinae is 2522 characteristically curved, forming the acidopore, and is usually surmounted by a well- 2523 defined ring of setae, the coronula. See Hung & Brown (1966) and Keller (2011). 2524 Larval phenotype. 2525 108. Larval food pocket (trophothylax) present. — Recorded as an autapomorphy of the 2526 Pseudomyrmecinae by Bolton (2003). 2527 Polyphenism. 2528 109. Major (soldier) caste present. — Pronounced allometry has evolved many times in the 2529 Formicidae. Here, majors were recorded for Aneuretus, Pheidole, most Camponotini, 2530 Gesomyrmex, most Melophorini, and Pseudolasius. 2531 110. At least some workers with phragmotic face. — Among sampled formicoids, 2532 phragmotic faces occur only in Colobopsis (see Ward et al. 2016 for distinguishing 2533 features of Colobopsis). 2534 2535 Characters used in previous studies 2536 Most setational characters of Maruyama et al. (2008; 18–20, 23–29, 34, 35, 64–66, 102) not 2537 included, except 26 which relates to presence of cranial setae. Additionally, body color, used to 2538 define the fuliginosus group (M08: 1, 2) not included. 2539 2540 A note on Santschiella 2541 Bolton (pers. comm.) recently dissected a specimen of the bizarre formicine Santschiella and 2542 noted that the definition of this genus in Fisher & Bolton (2016) needs to be updated (character 2543 states quoted verbatim): (1) Mandible has 7 teeth, with tooth 3 reduced (smaller than 4). (2) 2544 Maxillary palpi are very long and extend back almost to the occipital foramen. (3) Metacoxae are 2545 closely approximated. (4) Propodeal foramen is short. (5) Tergite of helcium is entire, as in 2546 Echinopla and most Polyrhachis—the characteristic formicine notch is absent. (6) Post-helcial 2547 sulcus is present on abdominal segment III. 2548 2549 2550 References for Appendix S2 2551 Agosti, D. (1990) What makes the Formicini the Formicini. Insects Sociaux, 6, 295–303. 2552 Agosti, D. & Bolton, B. (1990) New characters to differentiate the ant genera Lasius F. and 2553 Formica L. (Hymenoptera: Formicidae). Entomologist’s Gazette, 41, 149–156. 2554 Barden, P. & Grimaldi, D. A. (2016) Adaptive radiation in socially advanced stem-group ants 2555 from the Cretaceous. Current Biology, 26, 1–7.

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2556 Baroni Urbani, C. (2000) Rediscovery of the Baltic amber ant genus Prionomyrmex 2557 (Hymenoptera, Formicidae) and its taxonomic consequences. Eclogae Geologicae Helvetiae, 2558 93, 471–480. 2559 Blaimer, B.B., Ward, P.S., Schultz, T.R., Fisher, B.L. & Brady, S.G. (2018). Paleotropical 2560 diversification dominates the evolution of the hyperdiverse ant tribe Crematogastrini 2561 (Hymenoptera: Formicidae). Insect Systematics and Diversity, 2(5), 1–14. 2562 Bolton, B. (1990a) Abdominal characters and status of the cerapachyine ants (Hymenoptera, 2563 Formicidae). Journal of Natural History, 24, 53–68. 2564 Bolton, B. (1990b) Army ants reassessed: the phylogeny and classification of the doryline 2565 section (Hymenoptera, Formicidae). Journal of Natural History, 24, 1339–1364. 2566 Bolton, B. (1994) Identification Guide to the Ant Genera of the World. Harvard University Press, 2567 Cambridge, Mass. 2568 Bolton, B. (1999) Ant genera of the tribe Dacetonini (Hymenoptera: Formicidae). Journal of 2569 Natural History, 33, 1639–1689. 2570 Bolton, B. (2003) Synopsis and classification of Formicidae. Memoirs of the American 2571 Entomological Institute, 71, 1–370. 2572 Boudinot, B.E., Probst, R.S., Brandão, C.R.F., Feitosa, R.M. & Ward, P.S. (2016) Out of the 2573 Neotropics: newly discovered relictual species sheds light on the biogeographical history of 2574 spider ants (Leptomyrmex, Dolichoderinae, Formicinae). Systematic Entomology, 41, 658– 2575 671. 2576 Brothers, D. J. (1975) Phylogeny and classification of aculeate Hymenoptera, with special 2577 reference to Mutillidae. University of Kansas Science Bulletin, 50, 483–648. 2578 Buren, W.F., Hermann, H.R. & Blum, M.S. (1970) The widespread existence of mandibular 2579 grooves of the aculeate Hymenoptera. Journal of the Georgia Entomological Society, 5. 2580 Ettershank, G. (1966) A generic revision of the worl Myrmicinae related to Solenopsis and 2581 Pheidologeton (Hymenoptera: Formicidae). Australian Journal of Zoology, 14, 73–171. 2582 Fisher, B. L. (2016) Ants of Africa and Madagascar: A guide to the genera. University of 2583 California Press, Oakland. 2584 Forel, A. (1874) Les fourmis de la Suisse. Systématique, notices anatomiques et physiologiques, 2585 architecture, distribution géographique, nouvelles expériences et observations de moeurs. 2586 Neue Denkschriften der Allgemeinen Schweizerischen Gesellschaft für die Gesammten 2587 Naturwissenschaften, 26, 1–452. 2588 Gotwald, W.H., Jr. (1969) Comparative morphological studies of the ants, with particular 2589 reference to the mouthparts (Hymenoptera: Formicidae). Cornell University Agricultural 2590 Experiment Station Memoirs, 408, 1–150. 2591 Hermann, H.R., Hunt, A.N. & Buren, W.F. (1971) Manibular gland and mandibular groove in 2592 Polistes annularis (L.) an Vespula maculata (L.) (Hymenoptera: Formicidae). International 2593 Journal of Insect Morphology & Embryology, 1, 43–49. 2594 Hung, A.C.F. & Brown, W.L., Jr. (1966) Structure of the gastric apex as a subfamily character of 2595 the Formicinae (Hymenoptera: Formicidae). New York Entomological Society, 74, 198–200. 2596 Janda, M., Folková, D. & Zrzavy, J. (2004) Phylogeny of Lasius ants based on mitochondrial 2597 DNA and morphology, and the evolution of social parisitism. Molecular Phylogenetics & 2598 Evolution, 33, 595–614. 2599 Keller, R. (2011) A phylogenetic analysis of ant morphology (Hymenoptera: Formicidae) with 2600 special reference to the poneromorph subfamilies. Bulletin of the American Museum of 2601 Natural History, 355, 1–90.

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2602 Kugler, C. (1978) A comparative study of the mymicine sting apparatus (Hymenoptera, 2603 Formicidae). Studia Entomologia, 20, 413–548. 2604 Kugler, C. (1979) Evolution of the sting apparatus in the myrmicine ants. Evolution, 33, 117– 2605 130. 2606 Maruyama, M., Steiner, F.M., Stauffer, C., Akino, T., Crozier, R.H. & Schlick-Steiner, B.C. 2607 (2008) A DNA and morphology based phylogenetic framework of the ant genus Lasius with 2608 hypotheses for the evolution of social parasitism and fungiculture. BMC Evolutionary 2609 Biology, 8, 237. 2610 Mayr, G. (1868) Die Ameisen des baltischen Bernsteins. Beiträge zur Naturkunde Preussens, 1, 2611 1–102. 2612 Mayr, G. (1869) Die Ameisen des baltischen Bernsteis. Neues Jahrbuch für Mineralogie, 2613 Geologie und Paläontologie, 1869, 620–625. 2614 Peeters, C. & Ito, F. (2015) Wingless and dwarf workers underlie the ecological success of ants 2615 (Hymenoptera: Formicidae. Myrmecological News, 21, 117–130. 2616 Serna, F. & Mackay, W. (2010) A descriptive morphology of the ant genus Procryptocerus 2617 (Hymenoptera: Formicidae). Journal of Insect Science, 10, 1–36. 2618 Serna, F., Bolton, B. & Mackay, W. (2011) On the morphology of Procryptocerus 2619 (Hymenoptera: Formicidae). Zootaxa, 2923, 67–68. 2620 Shattuck, S.O. (1992) Generic revision of the ant subfamily Dolichoderinae (Hymenoptera: 2621 Formicidae). Sociobiology, 21, 1–181. 2622 Shattuck, S.O. (1995) Generic-level relationships within the ant subfamily Dolichoderinae 2623 (Hymenoptera: Formicidae). Systematic Entomology, 20, 217–228. 2624 Taylor, R. W. (1978) Nothomyrmecia macrops: A living-fossil ant rediscovered. Science, 201, 2625 979–985. 2626 Ward, P.S. & Brady, S.G. (2003) Phylogeny and biogeography of the ant subfamily 2627 Myrmeciinae (Hymenoptera: Formicidae). Invertebrate Systematics, 17, 361–386. 2628 Ward, P.S., Brady, S.G., Fisher, B.L., & Schultz, T.R. (2015) The evolution of myrmicine ants: 2629 phylogeny and biogeography of a hyperdiverse ant clade (Hymenoptera: Formicidae). 2630 Systematic Entomology, 40, 61–81. 2631 Ward, P.S., Blaimer, B.B. & Fisher, B.L. (2016) A revised phylogenetic classification of the ant 2632 subfamily Formicidae (Hymenoptera: Formicidae), with resurrection of the genera 2633 Colobopsis and Dinomyrmex. Zootaxa, 4072, 343–357. 2634 Wilson, E.O. (1955) A monographic revision of the ant genus Lasius. Bulletin of the Museum of 2635 Comparative Zoology, 113, 1–201. 2636 Wilson, E.O., Carpenter, F.M. & Brown, W.L., Jr. (1967a) The first Mesozoic ants, with the 2637 description of a new subfamily. Psyche, 74, 1–19. 2638 Wilson, E.O., Carpenter, F.M. & Brown, W.L., Jr. (1967b) The first Mesozoic ants. Science, 2639 157, 1038–1040. 2640 2641 2642 Figure legends for supplementary files 2643 2644 Fig. S1. Schematics of the constraints used in the topology tests for Bayes factor analysis of the 2645 Lasiini_55t dataset. (A) H0: XXX and Lasius genus group monophyletic; H1: XXX and Prenolepis 2646 genus group monophyletic (B) H0: Lasius s. str. monophyletic; H1: Lasius s. str. paraphyletic (C)

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2647 H0: Chthonolasius monophyletic; H1: Chthonolasius polyphyletic (D) H0: Cautolasius 2648 monophyletic; H1: Cautolasius paraphyletic. 2649 2650 Fig. S2. Results of the concordance factor analysis inferred from the Lasius_55t dataset in 2651 IQTREE. Node support values are reported as gene concordance factor (gCF) vs. site 2652 concordance factor (sCF). 2653 2654 Fig. S3. Results of analysis of the Lasiini_w_outgroups_w_fossils_morphology_154t dataset in 2655 MrBayes. Node support values are reported as Bayesian posterior probabilities (PP). 2656 2657 Fig. S4. Results of analysis of the Lasiini_w_outgroups_135t dataset in MrBayes. Node support 2658 values are reported as Bayesian posterior probabilities (PP). 2659 2660 Fig. S5. Results of analysis of the Lasiini_w_outgroups_135t dataset in IQTREE. Node support 2661 values are reported as maximum likelihood bootstrap (BS). 2662 2663 Fig. S6. Results of analysis of the Lasiini_w_outgroups_w_fossils_154t dataset in MrBayes 2664 without a clock model. Node support values are reported as Bayesian posterior probabilities 2665 (PP). 2666 2667 Fig. S7. Results of analysis of the Lasiini_w_outgroups_w_fossils_154t dataset in MrBayes with 2668 a uniform branch length prior and IGR clock rate prior. Node support values are reported as 2669 Bayesian posterior probabilities (PP). Horizontal blue bars at nodes are 95% highest posterior 2670 density (HPD) intervals. 2671 2672 Fig. S8. Results of analysis of the Lasiini_w_outgroups_w_fossils_154t dataset in MrBayes with 2673 a uniform branch length prior and TK02 clock rate prior. Node support values are reported as 2674 Bayesian posterior probabilities (PP). Horizontal blue bars at nodes are 95% highest posterior 2675 density (HPD) intervals. 2676 2677 Fig. S9. Results of analysis of the Lasiini_w_outgroups_w_fossils_154t dataset in MrBayes with 2678 a FBD branch length prior and TK02 clock rate prior. Node support values are reported as 2679 Bayesian posterior probabilities (PP). Horizontal blue bars at nodes are 95% highest posterior 2680 density (HPD) intervals. 2681 2682 Fig. S10. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2683 compound eye location under the equal rates assumption. In the pie charts, red = eyes anteriorly 2684 set and blue = eyes set at or posterior to head midlength. 2685 2686 Fig. S11. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2687 coxal separation under the equal rates assumption. In the pie charts, red = close-set and blue = 2688 wide-set. 2689 2690 Fig. S12. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2691 sulcus presence posteroventral to the helcium. In the pie charts, red = sulcus absent and blue = 2692 sulcus present.

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2693 2694 Fig. S13. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2695 propodeal spiracle shape. In the pie charts, red = spiracle circular to elliptical and blue = spiracle 2696 slit-shaped. 2697 2698 Fig. S14. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2699 proventricular form. In the pie charts, red = asepalous, blue = sepalous. 2700 2701 Fig. S15. Ancestral state estimation (ace) results from analysis of a 135-taxon chronogram for 2702 tergosternal suture conformation of abdominal segment III. In the pie charts, pink = without 2703 narrow shoulder, blue = low narrow shoulder, red = high narrow shoulder. 2704 2705 Fig. S16. Ancestral state estimation (ace) results from analysis of a 21-taxon chronogram 2706 representing Lasius for temporary social parasitism. In the pie charts, red = absence, blue = 2707 presence. 2708 2709 Fig. S17. Ancestral state estimation (ace) results from analysis of a 21-taxon chronogram 2710 representing Lasius for fungiculture. In the pie charts, red = absence, blue = presence. 2711 2712 Fig. S18. Dot plot of longitudinal distance from anterolateral corners of clypeus to posterior 2713 margins of compound eyes (ED2) by maximum head length (HL2) contrasting the two primary 2714 clades of Lasius with the genera XXX and †XXX, and demonstrating that the compound eyes of 2715 the latter two taxa are clearly set more-anteriorly than those of Lasius. 2716 2717 Fig. S19. Box and whisker plots of values for six eye position indices, showing differentiation 2718 among Lasius, XXX, and †XXX. 2719 2720 Fig. S20. Box and whisker plot of relative eye size (ESI), the index of average eye length (ELx) 2721 proportional to maximum head length (HL2), contrasting the two primary clades of Lasius with 2722 the genera XXX and †XXX.

61 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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 Fig. 1 available under aCC-BY-NC-ND 4.0 International license. A B Acanthomyops Lasius sensu stricto Chthonolasius Lasius s.s. brunneus Lasius s.s pallitarsis Dendrolasius Cautolasius Cautolasius Chthonolasius Lasius sensu stricto Dendrolasius Lasius s.s. brunneus C D Acanthomyops Cautolasius Lasius s.s. brunneus Dendrolasius Lasius sensu stricto Lasius sensu stricto Dendrolasius L. (Cautolasius) nearcticus Lasius s.s. brunneus Lasius s.s pallitarsis Lasius s.s pallitarsis L. (Chth.) atopus Chthonolasius L. (Cautolasius) flavus Austrolasius Chthonolasius Acanthomyops Austrolasius Acanthomyops bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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 Fig. 2 available under aCC-BY-NC-ND 4.0 International license. Cladomyrma_petalae Myrmecocystus_mexicanus Myrmecocystus_flaviceps Myrmecocystus_tenuinodis Lasius_brunneus 1/95 brunneus grp. Lasius_turcicus 1 Lasius_fuliginosus fuliginosus grp. Lasius_spathepus niger Lasius_emarginatus clade Lasius_platythorax Lasius genus group 1/99 Lasius_psammophilus niger grp. Lasius_niger 0.96/87 0.73/67 Lasius_nr_americanus 0.98/86 Lasius_nearcticus nearcticus grp. Lasius_sonobei Lasius_brevicornis 1/98Lasius_flavus flavus grp. 1/87 Lasius_myops Lasius_nr_atopus atopus grp. flavus 0.95/56 core 0.97/46 Lasius_pallitarsis pallitarsis grp. clade Lasiini Lasius_nr_subumbratus 1/95 umbratus grp. 0.96/50 Lasius_sabularum Lasius_carniolicus carniolicus grp. 1/91 Lasius_californicus 1/98 latipes grp. Lasius_latipes XXX_myrmidon XXX Zatania_gloriosa Zatania_albimaculata 1/79 Zatania_cisipa 0.84/40 Paraparatrechina_glabra Paraparatrechina_oceanica 0.99/54 Prenolepis_sp_PHL02 Prenolepis_melanogaster Prenolepis_imparis 0.43/78 Prenolepis_nitens Pseudolasius_australis Pseudolasius_sp_PHL01

Pseudolasius_typhlops Prenolepis genus group Euprenolepis_procera Euprenolepis_wittei 1/97 Paratrechina_longicornis Paratrechina_antsingy 1/95 1/72 Paratrechina_zanjensis Nylanderia_vitiensis Nylanderia_emmae Nylanderia_hystrix Nylanderia_phantasma 1/98 Nylanderia_vividula 0.83/63 Nylanderia_cf_madagascarensis 1/96 Nylanderia_sp_GUY03 1/84 Nylanderia_sp_PER01 Nylanderia_amblyops 1/* Nylanderia_sp_MAD01 Nylanderia_cf_vaga 0.99/100 0.63/* Nylanderia_dodo

0.008 substitutions per site bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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 Fig. 3 available under aCC-BY-NC-ND 4.0 International license. Tetraponera_rufonigra Dolichoderus_pustulatus Aneuretus_simoni Nothomyrmecia_macrops Myrmecia_pyriformis Manica_bradleyi Acanthoponera_minor Rhytidoponera_chalybaea Myrmelachista_flavocotea Brachymyrmex_sp_BR01 Brachymyrmex_depilis Cladomyrma_petalae Myrmecocystus_flaviceps 0.95 Lasius_californicus Lasius genus group Lasius_niger

Prenolepis_imparis Prenolepis genus group Zatania_albimaculata Paraparatrechina_oceanica Paraparatrechina_glabra Nylanderia_dodo Nylanderia_sp_MAD01 Nylanderia_hystrix Nylanderia_emmae Pseudolasius_australis Euprenolepis_procera Paratrechina_longicornis Paratrechina_antsingy Paratrechina_zanjensis Stigmacros_cf_clivispina Melophorus_sp_AU01 Prolasus_convexus Teratomyrmex_greavesi 0.97 Notoncus_capitatus Pseudonotoncus_hirsutus 0.75 Myrmecorhynchus_emeryi 0.34 Notostigma_carazzii Lasiophanes_atriventris Santschiella_kohli Gigantiops_destructor Myrmoteras_iriodum 0.46 Colobopsis_sp_BCA01 0.64 Colobopsis_saundersi Colobopsis_conithorax Colobopsis_vitiensis Opisthopsis_sp_PG01 Opisthopsis_respiciens Dinomyrmex_gigas Polyrhachis_sp_Hagio01 Polyrhachis_decumbens Echinopla_australis Echinopla_nr_striata Calomyrmex_albertisi Calomyrmex_laevissimus Camponotus_maritimus Camponotus_hyatti 0.94 Camponotus_gibbinotus 0.7 Camponotus_sp_MG089 Camponotus_sp_ZA01 0.29 Camponotus_phillipinensis Camponotus_cf_bedoti 0.68 Camponotus_claviscapus Camponotus_sp_MG001 Camponotus_sp_MG131 Bajcaridris_theryi Proformica_mongolica 0.92 Rossomyrmex_anatolicus Cataglyphis_cursor Polyergus_breviceps Iberoformica_subrufa Formica_moki Formica_neogagates 0.6 Gesomyrmex_sp_TH01 0.82 Gesomyrmex_sp_KH01 Oecophylla_smaragdina Oecophylla_longinoda 0.76 Anoplolepis_gracilipes Anoplolepis_custodiens Tapinolepis_sp_MG01 Tapinolepis_sp_ZA01 Petalomyrmex_phylax Aphomomyrmex_afer Lepisiota_sp_AFRC_LIM_03 Lepisiota_canescens 0.8 Plagiolepis_sp_MG05 Plagiolepis_alluaudi Acropyga_sp_CF01 Acropyga_acutiventris Agraulomyrmex_sp_TZ01 Formicinae_sp_Genus_01_ZA02 1 coalescent unit Formicinae_sp_Genus_01_ZA03 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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 Fig. 4 available under aCC-BY-NC-ND 4.0 International license. Jurassic Cretaceous Paleogene Neogene Q. Late Jurassic Early Cretaceous Late Cretaceous Paleoc. Eocene Oligo. Miocene Pl. P/H

Lioponera_larvata 0.96 †Procerapachys_annosus 0.87 Vicinopone_conciliatrix 0.87 Simopone_oculata Tanipone_zona 0.99 Aneuretus_simoni 0.72 †Chronomyrmex_medicinehatensis Dolichoderus_pustulatus 0.98 †Dolichoderus_tertiarius Myrmecia_pyriformis 0.96 Nothomyrmecia_macrops †Prionomyrmex_janzeni 0.45 †Tetraponera_groehni Tetraponera_rufonigra Myrcidris_epicharis 0.99 Pseudomyrmex_osurus †Pseudomyrmex_vicinus Rhytidoponera_chalybaea 0.73 Acanthoponera_minor †Gnamptogenys_levinates Manica_bradleyi Pogonomyrmex_vermiculatus Veromessor_andrei Terataner_bottegoi 0.75 Rogeria_stigmatica Pheidole_clydei †Pheidole_primagenia Myrmelachista_flavocotea Brachymyrmex_depilis 0.99 Brachymyrmex_sp_BR01 †Kyromyrma_neffi Cladomyrma_petalae †Lasius_schiefferdeckeri Myrmecocystus_mexicanus Myrmecocystus_flaviceps 0.89 Myrmecocystus_tenuinodis Lasius_brunneus Lasius_turcicus Lasius_fuliginosus 0.99 Lasius_spathepus Lasius_emarginatus Lasius_niger 0.77 Lasius_nr_americanus Lasius_platythorax 0.64 Lasius_psammophilus Lasius_nearcticus Lasius_sonobei 0.94 Lasius_brevicornis Lasius_flavus 0.9 Lasius_myops Lasius_nr_atopus 0.65 Lasius_pallitarsis Lasius_nr_subumbratus Lasius_sabularum Lasius_carniolicus Lasius_californicus Lasius_latipes 0.94 †XXX_pumilus XXX_myrmidon 0.99 †Prenolepis_henschei Paraparatrechina_glabra 0.91 Paraparatrechina_oceanica 0.99 Prenolepis_sp_PHL02 Prenolepis_melanogaster Prenolepis_imparis 0.41 Prenolepis_nitens 0.47 Zatania_gloriosa 0.95 Zatania_albimaculata Zatania_cisipa Pseudolasius_australis 0.5 Pseudolasius_sp_PHL01 Pseudolasius_typhlops 0.92 Euprenolepis_procera Euprenolepis_wittei Paratrechina_longicornis 0.98 Paratrechina_antsingy Paratrechina_zanjensis Nylanderia_vitiensis Nylanderia_emmae Nylanderia_hystrix Nylanderia_phantasma 0.8 Nylanderia_vividula Nylanderia_cf_madagascarensis Nylanderia_sp_GUY03 Nylanderia_sp_PER01 Nylanderia_amblyops 0.94 Nylanderia_sp_MAD01 Nylanderia_cf_vaga 0.76 Nylanderia_dodo Anoplolepis_custodiens Anoplolepis_gracilipes Tapinolepis_sp_MG01 0.99 0.75 Tapinolepis_sp_ZA01 Aphomomyrmex_afer 0.99 Petalomyrmex_phylax Lepisiota_sp_AFRC_LIM_03 0.84 Lepisiota_canescens Plagiolepis_alluaudi Plagiolepis_sp_MG05 Acropyga_acutiventris Acropyga_sp_CF01 Agraulomyrmex_sp_TZ01 Formicinae_sp_Genus_01_ZA02 Formicinae_sp_Genus_01_ZA03 Paratrechina_kohli 0.4 0.82 Melophorus_sp_AU01 0.99 Stigmacros_cf_clivispina 0.66 0.57 Myrmecorhynchus_emeryi 0.86 Lasiophanes_atriventris 0.54 Notostigma_carazzii 0.96 Notoncus_capitatus 0.21 0.92 Pseudonotoncus_hirsutus Prolasius_convexus Teratomyrmex_greavesi †Gesomyrmex_hoernesi 0.92 Gesomyrmex_sp_TH01 0.49 Myrmoteras_iriodum 0.33 Santschiella_kohli †Glaphyromyrmex_oligocenicus 0.65 0.97 Polyergus_breviceps 0.97 Iberoformica_subrufa 0.41 Formica_moki Formica_neogagates 0.36 †Formica_gustawi 0.83 Bajcaridris_theryi 0.99 Proformica_mongolica Cataglyphis_cursor Rossomyrmex_anatolicus †Oecophylla_brischkei Oecophylla_longinoda 0.13 †XXX_boreus 0.48 Gigantiops_destructor †Camponotus_mengei 0.37 Dinomyrmex_gigas 0.43 0.74 Colobopsis_conithorax Colobopsis_vitiensis Colobopsis_saundersi Colobopsis_sp_BCA01 Polyrhachis_decumbens Polyrhachis_sp_Hagio01 Calomyrmex_albertisi 0.85 Calomyrmex_laevissimus Echinopla_australis Echinopla_nr_striata Camponotus_cf_bedoti 0.51 Camponotus_cf_philippinensis Camponotus_hyatti Camponotus_maritimus Camponotus_sp_MG089 0.45 Camponotus_sp_ZA01 Camponotus_gibbinotus Camponotus_claviscapus Camponotus_sp_MG001 Camponotus_sp_MG131 Late Jurassic Early Cretaceous Late Cretaceous Paleoc. Eocene Oligo. Miocene Pl. P/H

150 125 100 7 5 5 0 2 5 0 Mya bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. Fig. 5

Cretaceous Paleogene Neogene Q. parasitism fungiculture Late Cretaceous Paleoc. Eocene Oligo. Miocene Pl. P/H region Cladomyrma petalae Myrmecocystus mexicanus Myrmecocystus flaviceps Myrmecocystus tenuinodis Lasius brunneus Lasius turcicus Lasius fuliginosus crown Lasius Lasius spathepus Lasius emarginatus genus grp. Lasius niger Lasius sp. nr. americanus Lasius platythorax Lasius psammophilus Lasius nearcticus Lasius sonobei Lasius brevicornis Lasius flavus core Lasius myops Lasiini Lasius sp. nr. atopus Lasius pallitarsis Lasius sp. nr. subumbratus Lasius sabularum Lasius carniolicus Lasius californicus Lasius latipes XXX myrmidon Paraparatrechina glabra Paraparatrechina oceanica Prenolepis sp. PHL02 Prenolepis melanogaster Prenolepis imparis Prenolepis nitens Zatania gloriosa Zatania albimaculata Prenolepis Zatania cisipa genus grp. Pseudolasius australis Pseudolasius sp. PHL01 Regions Pseudolasius typhlops Euprenolepis procera Neotropical Euprenolepis wittei Nearctic Paratrechina longicornis Palearctic Paratrechina antsingy Paratrechina zanjensis Afrotropic Nylanderia vitiensis Indomalayan Nylanderia emmae Nylanderia hystrix Australasian Nylanderia phantasma Traits Nylanderia vividula parasitism Nylanderia cf. madagascarensis Nylanderia sp. GUY03 fungiculture Nylanderia sp. PER01 Nylanderia amblyops Nylanderia sp. MAD01 Nylanderia cf. vaga Nylanderia dodo

Hot Earth, 1

Late Cretaceous: ~94 Mya Early Eocene: ~50 Mya Mid Miocene: ~15 Mya Hot Earth, I K/Pg Extinction Eocene Optimum Hot Earth, II Antarctic Ice Sheet Arctic Ice Sheet Late Cretaceous Paleoc. Eocene Oligo. Miocene Pl. P/H 80 70 60 50 40 30 20 10 0 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 1 Question Data Program Analysis Phylogenetic Analyses UCE: summary coalescence 89 taxa, 959 UCE locus alignments UCEs: ASTRAL-II 135 taxa, 11 Sanger loci: Bayesian tree inference, & 1. Is Lasius monophyletic? 135 taxa, 11 Sanger loci Sanger: MrBayes maximum likelihood bootstrap 55 taxa, 11 Sanger loci 3.2.6, IQ-TREE 55 taxa, 11 Sanger loci: stepping stone, concordance factor Bayesian: Bayesian tree inference, stepping stone 2. Are the subgenera of Lasius MrBayes 3.2.6, 55 taxa, 11 Sanger loci ML: maximum likelihood bootstrap, concordance monophyletic? IQ-TREE factor 154 taxa: 11 Sanger loci for 135 extant 3. What are the relationships of fossil taxa, and 110 binary morphological Tree search: (clock vs. non-clock; morphology-only MrBayes 3.2.6 Formicinae? characters for 136 extant and 18 extinct vs. combined data) taxa Evolutionary History 4. Under what paleoclimatological 135 extant taxon, 11 Sanger loci conditions and where biogeographically BioGeoBEARS: several models (with and without chronogram trimmed from 154 taxon tree BioGeoBEARS did the Lasiini and its internal clades +J) generated in Question 4 originate? 135 taxon chronogram from Question 5, 5. What was the evolutionary sequence of 5 binary morphological characters and 1 transformation for taxonomically ace with ER (equal rates) vs. SYM (symmetrical ternary morphological character; 21 R (ape) important characters and life history rates) vs. ARD (all rates different) models taxon chronogram trimmed from traits? Question 5, 2 binary life history traits Taxonomy Qualitative: N/a 6. What are the morphological states that Qualitative comparisons (various) Qualitative: Key + Tabulation Quantitative: R define the Lasiini and focal clades therein? Morphometrics (104 specimens, 56 spp.) Quantitative: Plotting (ggplot) bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 2 Species Specimen code Locality (year) Collector Lasius americanus_nr CASENT0732004 CA, U.S.A. (2014) Borowiec, M.L. Lasius atopus _nr CASENT0234858 CA, U.S.A. (2014) Boudinot, B.E., et al. Lasius brevicornis CASENT0234864 CA, U.S.A. (2012) Borowiec, M.L. Lasius brunneus CASENT0234862 Dolny Śląsk, Poland (2010) Borowiec, M.L. Lasius carniolicus CASENT0731085 Dolny Śląsk, Poland (2012) Salata, S. Lasius emarginatus CASENT0234865 Dolny Śląsk, Poland (2010) Borowiec, M.L. Lasius flavus CASENT0234860 Dolny Śląsk, Poland (2010) Borowiec, M.L. Lasius fuliginosus CASENT0234861 Dolny Śląsk, Poland (2010) Borowiec, M.L. Lasius latipes CASENT0234866 CA, U.S.A. (2007) Ward, P.S. Lasius myops CASENT0732040 N Calabria, Italy (2014) Borowiec, L. Lasius nearcticus CASENT0732003 MA, U.S.A. (2008) Alpert, G.D. & Borowiec, M.L. Lasius pallitarsis CASENT0234859 CA, U.S.A. (2014) Borowiec, M.L. Lasius platythorax CASENT0732002 N. Dalmatia, Croatia (2000) Borowiec, M.L. & Poprawska, M. Lasius psammophlus CASENT0732000 Dolny Śląsk, Poland (2009) Borowiec, M.L. Lasius sabularum CASENT0234863 Dolny Śląsk, Poland (2010) Borowiec, M.L. Lasius sonobei CASENT0730575 Hokkaido, Japan (2002) Ward, P.S. Lasius spathepus CASENT0732006 Hokkaido, Japan (2002) Ward, P.S. Lasius subumbratus _nr CASENT0731084 CA, U.S.A. (2014) Ward, P.S. Lasius turcicus CASENT0732001 Rhodes, Greece (2008) Borowiec, M.L. XXX myrmidon CASENT0811544 Korinthia, Greece (2013) Borowiec, L. Myrmecocystus mexicanus CASENT0234867 CA, U.S.A. (2014) Borowiec, M.L. Myrmecocystus tenuinodis CASENT0234868 CA, U.S.A. (2004) Ward, P.S. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 3 dataset name purpose of analyses data type taxa clock type Lasiini_w_outgroups_w_fossils_morphology_154t fossil placement comparison morphology extant & fossil no clock Lasiini_w_outgroups_136t relaxed clock prior estimation combined extant taxa no clock vs. strict clock fossil placement comparison, relaxed clock: IGR vs. TK02; Lasiini_w_outgroups_w_fossils_154t combined extant & fossil divergence dating uniform clock: IGR vs.TK02 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 4

SS Run Marg. Log Likelihood H0 Marg. Log Likelihood H1 2lne(B10) XXX + Lasius g. g. XXX + Prenolepis g. g. 1 -29156.21 -29092.2 2 -29153.15 -29094.4 mean -29153.79 -29092.79 -122 Lasius monophyletic Lasius paraphyletic 1 -29068.73 -29072.46 2 -29068.86 -29072.04 mean -29068.79 -29072.23 6.88 Lasius s. str. monophyletic Lasius s. str. paraphyletic 1 -29092.4 -29057.37 2 -29094.02 -29058.11 mean -29092.91 -29057.67 -70.48 Cautolasius monophyletic Cautolasius paraphyletic 1 -29063.91 -29054.38 2 -29064.12 -29054.21 mean -29064.01 -29054.29 -19.44 Chthonolasius monophyletic Chthonolasius polyphyletic 1 -29100.32 -29047.78 2 -29099.17 -29046.75 mean -29099.59 -29047.13 -104.92 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 5 divergence dating biogeography reconstruction DEC (lnL DIVALIKE (lnL BAYAREALIKE FBD IGR clock FBD TK02 clock uniform IGR clock uniform TK02 clock −136.77) −132.24) (lnL −150.98) node mean age (95% HPD) PP mean age (95% HPD) PP mean age (95% HPD) PP mean age (95% HPD) PP range likelihood range likelihood range likelihood Lasiini 70.0 (57.6-82.7) 0.99 97.2 (89.6-106.0) 0.91 80.8 (68.6-91.8) 0.96 100.0 (90.9-112.3) 0.94 PI 0.33 PI 0.37 PI 0.43 core Lasiini 62.3 (51.2-72.9) 0.94 88.6 (79.8-97.8) 0.64 72.5 (59.0-85.4) 0.94 92.0 (83.5-102.2) 0.49 P 0.42 P 0.56 PI 0.43 XXX + Prenolepis g. grp. 51.1 (42.2-60.2) 0.99 71.0 (60.8-81.9) 1.00 59.9 (48.7-72.8) 0.99 75.6 (65.1-86.5) 1.00 P 0.40 P 0.53 PI 0.48 Prenolepis g. grp. 39.0 (36.5-50.9 0.47 53.1 (43.6-62.6) 1.00 50.9 (40.8-61.3) 1.00 57.0 (48.2-66.5) 1.00 PI 0.42 PI 0.50 PI 0.64 Lasius g. grp. 24.9 (18.0-33.0) 1.00 67.9 (54.9-81.6) 0.99 31.1 (22.4-40.5) 0.99 73.7 (61.9-88.3) 0.99 NaP 0.53 Na 0.55 NaP 0.84 crown Lasius 21.9 (15.3-28.6) 0.77 63.7 (52.4-75.1) 0.63 27.3 (19.4-35.6) 0.73 64.3 (58.4-82.2) 0.71 P 0.72 P 0.71 NaP 0.87 niger clade 15.8 (10.0-21.6) 1.00 53.2 (41.2-65.3) 0.70 20.0 (12.7-27.7) 1.00 60.6 (46.1-73.0) 0.56 P 0.87 P 0.99 NaP 0.62 flavus clade 15.7 (10.4-21.0) 1.00 49.1 (36.7-61.8) 1.00 19.7 (13.2-26.8) 1.00 55.9 (41.1-69.4) 1.00 Na 0.71 Na 0.90 NaP 0.91 Myrmecocystus 14.6 (7.3-21.5) 1.00 47.9 (33.5-63.1) 1.00 17.8 (8.9-26.9) 1.00 55.8 (40.0-69.8) 1.00 Na 0.87 Na 0.97 Na 0.53 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 6 model Log lik. Df Df change Resid. Dev Pr(>|Chi|) Eyes_ER -49.43 1 NA NA NA Eyes_ARD -49.12 2 1 0.63 0.43 Coxae_ER -26.97 1 NA NA NA Coxae_ARD -26.24 2 1 1.48 0.22 Prov_ER -27.56 1 NA NA NA Prov_ARD -27.29 2 1 0.53 0.47 Spiracle_ER -28.83 1 NA NA NA Spiracle_ARD -27.64 2 1 2.38 0.12 Sulcus_ER -12.74 1 NA NA NA Sulcus_ARD -12.25 2 1 0.97 0.32 AIII_ER -42.53 1 NA NA NA AIII_SYM -39.71 3 2 5.66 0.06 AIII_ARD -38.36 6 3 2.69 0.44 Parasitism_ER -7.74 1 NA NA NA Parasitism_ARD -7.39 2 1 0.71 0.40 Fungiculture_ER -7.75 1 NA NA NA Fungiculture_ARD -7.73 2 1 0.03 0.87 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.14.452383; this version posted July 15, 2021. 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-ND 4.0 International license.

Table 7 propodeal eyes metacoxae AIII shoulder proventriculus sulcus spiracle absent/ low, high, circle/ post. ant. close wide slit asepal. sepal. absent present node broad narrow narrow ellipse doryloformicoids (root) 0.02 0.98 1.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 myrmecioformicoids 0.00 1.00 1.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 Formicinae 0.02 0.98 0.08 0.92 0.72 0.06 0.22 1.00 0.00 0.33 0.67 1.00 0.00 Myrmelachistini 0.02 0.99 0.01 0.99 0.06 0.01 0.93 1.00 0.00 0.33 0.67 1.00 0.00 Lasiini + core Formicinae 0.05 0.95 0.07 0.93 0.69 0.11 0.19 1.00 0.00 0.11 0.89 1.00 0.00 Lasiini 0.05 0.95 0.00 1.00 0.04 0.86 0.10 1.00 0.00 0.01 0.99 1.00 0.00 core Lasiini 0.07 0.93 0.00 1.00 0.01 0.89 0.10 1.00 0.00 0.00 1.00 1.00 0.00 XXX + Prenolepis g. grp. 0.03 0.97 0.00 1.00 0.01 0.85 0.14 1.00 0.00 0.00 1.00 1.00 0.00 Prenolepis g. grp. 0.03 0.97 0.00 1.00 0.00 0.00 1.00 1.00 0.00 0.00 1.00 1.00 0.00 core Formicinae 0.13 0.87 0.09 0.91 0.71 0.09 0.20 1.00 0.00 0.11 0.89 1.00 0.00 Melophorini 0.98 0.02 1.00 0.00 1.00 0.00 0.00 1.00 0.00 0.90 0.10 0.00 1.00 formicoform radiation 0.30 0.70 0.99 0.01 1.00 0.00 0.00 1.00 0.00 0.02 0.98 0.93 0.07 Formicini 0.98 0.02 1.00 0.00 1.00 0.00 0.00 0.03 0.97 0.00 1.00 0.00 1.00 Plagiolepidini 0.11 0.89 0.00 1.00 0.28 0.16 0.56 1.00 0.00 0.95 0.05 1.00 0.00 Camponotini 0.01 0.99 1.00 0.00 1.00 0.00 0.00 0.01 0.99 0.00 1.00 0.00 1.00