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Home , Kalotermes flavicollis, Mastotermes darwiniensis, Neotermes castaneus, Prorhinotermes simplex, Rhinotermitidae

1 Supporting Information for

2 Evidence for reduced immune gene diversity and activity during the evolution of

3

4 Shulin He1,2,3, Thorben Sieksmeyer1,2, Yanli Che4, M. Alejandra Esparza Mora1,2, Petr Stiblik3, 5 Ronald Banasiak2, Mark C. Harrison5, Jan Šobotník6, Zongqing Wang4, Paul R. Johnston1,7,8,†, 6 Dino P. McMahon1,2,*†

7 8 1 Institute of Biology, Freie Universität Berlin, Schwendenerstr. 1, 14195 Berlin, Germany. 9 2 BAM Federal Institute for Materials Research and Testing, Department for Materials and 10 Environment, Unter den Eichen 87, 12205 Berlin, Germany. 11 3 Faculty of Forestry and Wood Science, Czech University of Life Science Prague, Kamýcká 129, 12 16500 Prague, Czech Republic. 13 4 College of Plant Protection, Southwest University, Tiansheng 2, 400175 Chongqing, China. 14 5 Institute for Evolution and Biodiversity, University of Münster, Münster, Germany. 15 6 Faculty of Tropical AgriSciences, Czech University of Life Science Prague, Kamýcká 129, 16500 16 Prague, Czech Republic. 17 7 Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 310, 18 12587 Berlin, Germany. 19 8 Berlin Center for Genomics in Biodiversity Research, Königin-Luise-Str. 6-8, 14195 Berlin, 20 Germany. 21 † These authors contributed equally.

22 *Correspondence to: D. P. M. ([email protected]).

23 Supplementary Text

24 Phylogenetic analysis 25 In order to construct a comprehensive phylogeny, we analyzed 30 transcriptomes and genomes, 26 of which 1 genome and 10 available raw data sets were included alongside the 19 27 assemblies from our study (Tab. S11 and Tab. S12). To facilitate phylogenetic inference, we 28 removed raw reads derived from rRNA and mitochondrial DNA in 19 sequenced using 29 Botwie2(Langmead and Salzberg 2012) with converted indices built from related sequences of 30 , termites and protists from NCBI. Retained reads were assembled by Trinity (version 31 v2.5.1) (Grabherr, et al. 2011)with default parameters (Kmer length: 25) and trimmomatic to clean 32 low-quality reads. After assembling, gene expression was quantified by using Kallisto(Bray, et al. 33 2016) for each assembly. To reduce redundancy, the highest expressed isoform for each gene 34 was selected with a script in Trinity. Redundancy was further reduced in each assembly by CD- 35 HIT-EST(Fu, et al. 2012) implementing a 95% similarity cut-off. The assemblies were further 36 filtered by Botwie2 to remove rRNA and mitochondrial DNA as we had done previously to the raw 37 reads. Subsequently, the final assemblies were translated into proteins by Transdecoder (version 38 5.0.1) with a minimum length of 60 amino acids. The raw sequence reads were downloaded from 39 the SRA database in NCBI and the details are listed in Supplementary Table S12. For assembling, 40 we applied the same procedures for raw Illumina sequence reads and assembled the Raw 454 41 sequence reads using Newbler v2.7 (454 Life Sciences/ Roche).The translated protein sets were 42 used for ortholog analysis by OrthoFinder (version v2.0.0), which is an all-to-all and gene length 43 balanced method to find ortholog groups, suitable for transcriptome data(Emms and Kelly 2015). 44 For the ortholog analysis, we also included an official gene set Macrotermes natalensis 45 (http://gigadb.org/dataset/100057).

46 After ortholog prediction, the single ortholog groups that met the following criteria were selected 47 for matrix building. To mitigate representation bias per orthogroup, we selected orthogroups 48 that included at least one representative of each of the following taxa: 1) , 2) 49 Zootermopsis and Hodotermopsis, 3) (, , Cryptotermes), 4) 50 , 5) , 6) . The longest sequence from each selected 51 orthogroup was queried against the ncbi nr database using blast to check for bacterial and protist 52 contamination. Subsequently, these orthogroups were aligned using MAFFT(Katoh and Standley 53 2013) with the L-INS-i alignment algorithm. To minimize alignment ambiguities, each aligned 54 orthogroup was masked by trimAI v1.2(Capella-Gutiérrez, et al. 2009) with the gappyout function. 55 Orthogroups were then concatenated with Phyutility(Smith and Dunn 2008). An amino acid data 56 matrix with an average of 82.06% gene occupancy per species was assembled from predicted 57 orthogroups. The resulting matrix comprised 152 orthogroups with 22898 amino acid positions 58 and 17.00% missing data. 59 We employed two different approaches to constructing the phylogeny: maximum likelihood with 60 RAxML (v8.2.12) (Stamatakis 2014)and Bayesian inference with ExaBayes (v1.4.1)(Aberer, et al. 61 2014). In RAxML, 1000 rapid bootstrap replicates were calculated by employing the 62 PROTGAMMAAUTO model. The parsimony random seed (-p) and bootstrap random seed (-x) 63 were set to 12345. For ExaBayes, two runs were performed and each with four chains. The 64 starting seed (-s) was set to 258. Analyses were run until both runs had average standard 65 deviation of split frequencies (asdsf) below 1% for at least 106 generations. The phylogenetic 66 trees obtained from two different methods, RaxML and ExaBayes, have identical topologies (Fig. 67 1, Fig. S1). Cryptocercidae and Isoptera are sister groups and form a that is closely related 68 to . is the family of the termites and comprises a sister lineage to 69 all other groups. is located between Mastotermitidae and Kalotermitidae. 70 Kalotermitidae is a monophyletic grouping in the phylogeny. is a polyphyletic

71 group, comprised of the monophyletic Rhinotermitinae, (consisting of 72 Coptotermes and Reticulitermes), and Psammotermitinae (consisting of , 73 Prorhinotermes, Termitogeton) and Stylotermitinae. is monophyletic and a sister group 74 to Rhinotermitinae. 75 To estimate the divergence times for termites, a molecular clock analysis was performed with 76 PhyloBayes (v4.1) (Lartillot and Philippe 2004). The topology of the phylogenetic tree was 77 constrained to the consensus tree obtained from ExaBayes. An uncorrelated relaxed clock model, 78 using uncorrelated gamma multipliers (-ugam), was applied in our analysis under a birth death 79 prior (-bd) with soft bounds (-sb). Four independent chains were run with 5 calibration points. 80 The following age constraints were employed in this study: all cockroaches and Isoptera: 145.5- 81 315.2 mya (representing the age of the root) (Vršanský 2002), and Isoptera: 130- 82 235 mya (Krishna, et al. 2013), Kalotermitidae and Rhinotermitidae plus Termitidae: 94.3-235 83 mya (Krishna and Grimaldi 2003), Termitidae and Coptotermes plus Reticulitermes: 47.8-94.3 84 mya (Engel, et al. 2011), Reticulitermes and Coptotermes: 33.9-94.3 mya (Engel, et al. 2007). 85 We assessed burn-in, convergence among runs, and run performance by examining parameter 86 files with the program TRACER v1.6.0 (Suchard, et al. 2018). Each chain was run for over 10000 87 cycles, sampling posterior rates and dates with an initial burn in of 20%. Posterior estimation of 88 divergence times was computed from the chain with the highest ESS. As illustrated in the time 89 calibrated phylogenetic tree (Fig. 1), the most recent common ancestor (MRCA) of Cryptocercus 90 and termites can be dated to the lower Jurassic, 179.436± 24.1544 (133.939-225.204, 95% 91 confidence interval (CI)) million years ago (mya), which diverged from the Blattidae in the upper 92 Triassic, around 216.657±28.6003 (160.664-267.785, 95% CI) mya. The root of termites is 93 estimated to be 155.341±21.3062 (115.826-195.454,95% CI) million years old from the upper 94 Jurassic. The MRCA of the higher termites, Termitidae, is estimated to be around 95 58.9309±8.74701 (42.2055-74.7823, 95%CI) million years old from the upper Paleocene and 96 diverged from lower termites around 76.6184±10.5918 (55.8417-93.9591, 95%CI) mya in upper 97 Cretaceous. Although the estimated ages in our study are generally older those derived from 98 mitochondrial or phenotypic data(Engel, et al. 2009; Bourguignon, et al. 2015)and a recent 99 phylogenetic study of evolution(Evangelista, et al. 2019), our date estimates are in line 100 with a multiple-fossil calibration analysis(Ware, et al. 2010)and a comprehensive recent study of 101 termite evolution(Bucek, et al. 2019).

102 Expansion and contraction of immune gene families 103 We sequenced 15 termite, 2 Cryptocercus, and an additional 2 cockroach transcriptomes. After 104 quality control and assembling, each assembly per species contained 120- 210 thousand 105 transcripts with 82.7%-97.7% complete BUSCOs (except Pericapritermes sp. with 69.0% BUSCO 106 completeness, which was excluded for further analysis) (Tab. S13). 107 Immune related genes from 50 families were categorized as either receptor, effector or signaling 108 molecules. Using a combined identification of hmmsearch and trinotate annotation, every gene 109 family was represented by each cockroach and termite species (Fig. 2), except drosomycin, a 110 family of effectors that has been lost in termites and wood roaches. 111 In the phylosignal analysis, we found no evidence of phylogenetic signal among species for 112 BUSCO scores (Cmean = 0.058, p-value=0.178; Moran’s I= -0.059, p-value=0.467; K=0.371, p- 113 value=0.365; K*=0.489, p-value=0.286; λ<0.0001, p-value=1.0). Conversely, we detected a 114 strong pattern of total immune gene diversity loss during the evolution of termites (Cmean = 0.449, 115 p-value=0.002; Moran’s I=0.055, p-value=0.023; K=1.391, p-value=0.002; K*=0.869, p- 116 value=0.008; λ=0.830, p-value=0.008) with significant positive autocorrelation among species 117 (Fig S2).

118 The following structures (M1-M25) were tested in our CAFE analysis and each structure was 119 repeated 5 times to check for convergence: 120 M1: 5 λs in solitary cockroaches, subsocial cockroaches, lower termites (except Rhinotermes), 121 Rhinotermes, and higher termites 122 M2: 4 λs in solitary cockroaches, subsocial cockroaches, lower termites, and higher termites 123 M3: 3 λs in solitary cockroaches, subsocial cockroaches, all termites 124 M4: 2 λs in solitary cockroaches, all subsocial cockroaches and termites 125 M5: 3 λs in solitary cockroaches, subsocial cockroaches and lower termites (except Rhinotermes), 126 Rhinotermes and higher termites 127 M6: 2 λs in all cockroaches, and all termites 128 M7: a common global λ in all species 129 M8: all nodes have different λ rates 130 M9 – M25: different λ rates based on kmeans clusters (k from 2 to 17) 131 132 After testing all structures, we found that two λ rates, based on with a solitary and sub- or 133 social system (structure M4), represented the best fitting model. After applying an error correction, 134 we found the global evolutionary rate of immune gene families in solitary cockroaches (birth/death 135 rate[λ]=0.0037) to be higher than that of subsocial cockroaches and termites (λ=0.0016). Among 136 effector genes, we found that the thioredoxin peroxidase (TPX) gene family had undergone a 137 contraction in the Termitidae crown group, while we find an antimicrobial peptide family, defensin, 138 to have undergone an expansion in the same group. Aside from these immune genes, lysozyme 139 (LYS) also showed expansion in an internal node of higher termites (Fig. S3). In the receptors, 140 we found that C-type lectins (CTL) underwent two contraction events during the evolution of 141 termite sociality (Fig. 1, Fig. S3), once in the MRCA of subsocial wood roaches + social termites, 142 and once in the MRCA of Rhinotermitidae + Termitidae. Interestingly, CTLs appear to have also 143 undergone a re-expansion in higher termites, coinciding with the expansion of lysozymes in this 144 group. We also detected evidence of GNBP undergoing an expansion in the common ancestor of 145 the subsocial cockroaches and contractions of CLIP (serine protease) and autophage related 146 genes (ATG) in the MRCA of Rhinotermitidae and Termitidae.

147 Individual experiment: transcriptional responses of immune-challenged individuals 148 To explore the relationship between immune response and division of labour in termites, we 149 quantified the number of immune-related genes which were differentially expressed in response 150 to a common immune challenge in three termite castes, a subsocial cockroach and a solitary 151 cockroach. As in the first experiment, we compared individuals that were injected with heat-killed 152 microbes or an equivalent Ringer’s control solution, treated were kept individually in order 153 to investigate the individual immune response. 154 In the solitary cockroach Blatta orientalis, we detected 263 and 165 significantly down- and 155 upregulated genes in immune-challenged individuals respectively. Upregulated genes were again 156 represented by significantly enriched immune related GO terms (Tab. S1). Among total 157 differentially expressed genes, 25 and 10 represented up- and downregulated immune related 158 genes, respectively (Fig. S4). In an equivalent experiment in the subsocial cockroach C. 159 meridianus, we found a similar pattern to B. orientalis with 248 and 382 genes to be significantly 160 downregulated and upregulated (log2FoldChange>2, p<0.01), respectively. The upregulated 161 genes represented a comprehensive immune response and were significantly enriched in immune

162 related GO terms (Tab. S2). Among total differentially expressed genes, 24 and 19 represented 163 up- and downregulated immune related genes, respectively (Fig. S5). 164 In N. castaneus, only 3 genes were found to be significantly upregulated in immune-challenged 165 individuals of all three castes (log2FoldChange>2, p<0.01). These were a Jerky protein homolog- 166 like, a peroxidase and an uncharacterized gene. One gene (poly [ADP-ribose] polymerase 12- 167 like) was significantly upregulated in both false workers and reproductives, while 7 genes were 168 upregulated in both false workers and soldiers (log2FoldChange>2, p<0.01). Interestingly, the two 169 castes representing terminal moults: soldiers and reproductive, shared 84 upregulated genes in 170 immune-challenged individuals (log2FoldChange>2, p<0.01). Significantly upregulated genes in 171 false workers (N=30) were not significantly enriched for any GO terms (Tab. S3), while 172 upregulated genes in soldiers (N=161) were significantly enriched in immune-related and 173 transport as well as metabolic process GO terms (Supplementary Table S4). Upregulated genes 174 in reproductives (N=220) were significantly enriched in positive regulation of antifungal peptide 175 production (GO:0002804) and phenol-containing compound biosynthetic processes 176 (GO:0046189) (Supplementary Table S5). Five, 11 and 9 immune related genes were significantly 177 upregulated in false workers, soldiers and reproductives, respectively (Fig. S5). Among these 178 gene, a peroxidase was significantly upregulated in all castes. 179 In a comparison across the 3 species, we found that the immune responses of the 2 cockroach 180 species were similarly comprehensive, with evidence of significant upregulation of receptor genes, 181 signalling components, and effectors. By contrast, termite reproductives and soldiers displayed a 182 similar but relatively reduced pattern of immune gene expression following immune-challenge. 183 The response of false workers was comparatively even weaker, with differential expression limited 184 to a serpin and 3 effector genes: attacin, lysozyme and peroxidase (Fig. 3).

185 Caste-specific immunity in the termite N. castaneus 186 We also compared overall caste-specific genes expression patterns by grouping treatments and 187 comparing differentially expressed genes between castes. We found genes to be significantly 188 grouped by caste (Fig. S9, Fig. S10, Fig. S11). Compared with both false workers and soldiers, 189 reproductives harboured highly expressed genes that were significantly enriched for reproductive 190 and developmental processes as well as in pheromone synthesis, whereas lowly expressed 191 genes were significantly enriched for oxidative phosphorylation (GO:0006119) 192 (log2FoldChange>2, p<0.01) (Tab. S6). In false workers, highly expressed genes were 193 significantly enriched in carboxylic acid biosynthetic processes (GO:0016053) whereas lowly 194 expressed genes were enriched for multi-multicellular organism processes (GO:0044706) 195 compared to both soldiers and reproductive (Tab. S7). Compared to both false workers and 196 reproductive, soldiers harboured highly expressed genes that were enriched, but not significantly, 197 for muscle related activity and development related GO terms, whereas lowly expressed gene 198 were significantly enriched for developmental processes (GO:0032502), proteolysis 199 (GO:0006508), defecation rhythm (GO:0035882) and macromolecule catabolic processes 200 (GO:0009057) (Tab. S8). 201 Interestingly, we found that expression of immune related genes could be effectively categorized 202 by caste in a principle component analysis (Fig. 2). Reproductives displayed the highest levels 203 of immune-related gene expression, when compared with both soldiers and false workers (Fig. 204 S7). 205 Social experiment: transcriptional responses of nestmates exposed to immune-challenged 206 individuals

207 In this experiment, we studied the social immune responses of different N. castaneus castes 208 following exposure to immune-challenged or Ringer’s solution-injected individuals. In order to 209 record transcriptional responses, we challenged two false workers and then returned them to a 210 mini-colony composed of 6 members, comprised of 2 (non-challenged) individuals of each caste, 211 i.e. 2 false workers, 2 soldiers, and 2 reproductives (king and queen). 212 We investigated gene expression responses of different castes between treatments, following 24 213 hours of exposure to 2 immune-challenged or Ringer-injected false workers (Fig 4). Among 214 reproductives, we detected 1 significantly upregulated (Fatty-acid amide hydrolase 2 like) and 215 one significantly downregulated gene (Glucose dehydrogenase [FAD, quinone]) between 216 treatments (log2FoldChange>1, p<0.05). Among soldiers, 1 gene was significantly upregulated 217 (Guanylate cyclase 32E like) while no genes were downregulated (log2FoldChange>1, p<0.05). 218 By contrast, among false workers, 12 genes were significantly upregulated while 96 genes were 219 significantly downregulated(log2FoldChange>1, p<0.05). Upregulated genes included Fatty acid 220 synthase-1, Trypsin-1-like, Thiamine transporter 2, Zinc finger protein like, probable cytochrome 221 P450, and a gustatory and odorant receptor 24-like isoform X2, mite allergen Der f 3-like, and 5 222 uncharacterized genes. The downregulated genes (Tab. S9) mainly comprised transport-related, 223 oxidation-related and protease related genes, and included 6 immune-related genes (5 serine 224 proteases and 1 Chorion peroxidase). 225 In the equivalent experiment using the solitary cockroach B. orientalis, conspecific individuals 226 between treatments exposed to immune-challenged or Ringer’s solution-injected showed 227 significant upregulation and downregulation of 9 and 7 genes, respectively (log2FoldChange>1, 228 p<0.05). Upregulated genes in conspecifics included 2 serine proteases, a trypsin-4, an Ankyrin 229 repeat and fibronectin type-III domain-containing protein 1 as well as 5 other uncharacterized 230 genes. Upregulated genes were enriched in the “serine-type peptidase activity” molecular 231 functional (MF) GO term (Tab. S10). Downregulated genes contained a Hemolymph 232 lipopolysaccharide-binding protein, a troponin T, a Protein obstructor-E and 4 other 233 uncharacterized genes.

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234 0.0 235 Figure S1. Phylogeny of termites based on RAxML and Exabayes. The number on each node 236 represents support of boostrap values from RAxML/likelihood score from Exabayes. Different 237 colors of lines indicate traditional classification of termites and cockroaches.

238

239 Figure S2. Phylosignal analysis. (A) Time-calibrated phylogeny and corresponding phylogenetic 240 signal of two trait values associated with each tip (species). Left: associated BUSCO scores as a 241 control for the effect of transcriptome assembly quality. Right: total predicted immune genes 242 derived from each assembly. (B) Phylogenetic correlograms displaying the extent of trait 243 autocorrelation among related tips, revealing the pattern and location in the of any 244 detected phylogenetic signal. Higher and lower levels of autocorrelation than expected by chance 245 are depicted in red and blue, respectively.

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246 247 Figure S3. Gene family names in grey and black on the phylogeny indicate significant 248 contractions and expansions of individual gene families, respectively. The gene family evolution 249 analysis was conducted in CAFE. Significance levels of 0.05 (*) and 0.01 (**) are shown. The 250 gene number of contraction or expansion in each family were indicated in square brackets. The 251 contractions and expansion in the right panel indicate the significant changes in gene families in 252 individual tested species.

253

254 Figure S4. Differentially expressed immune genes after injection (left panel, subsocial 255 cockroach, C. meridianus; right panel, solitary cockroach, B. orientalis).

256 257 258 Figure S5. Differentially expressed immune genes in different castes after injection in individual 259 experiment. Red squares indicate upregulated immune gene expression in caste after injected 260 with heat killed microorganisms, and blue squares indicate downregulated immune gene 261 expression in caste after injected with heat killed microorganisms.

262

Upregulated Downregulated Species/caste non- non- immune immune immune immune Reproductives 211 9 384 4 Soldiers 150 11 130 1 N. castaneus False Workers 25 5 68 3 Total 313 20 485 6

C. meridianus 358 24 229 19

B. orientalis 238 25 155 10 263 264 Figure S6. Summary numbers of differentially expressed genes (immune genes and non-immune 265 genes) in 3 castes of the termite N. castaneus, subsocial cockroach C. meridianus, and solitary 266 cockroach B. orientalis injected with heat-killed microbes versus Ringer’s solution (individual 267 experiment). The numbers of total differentially expressed genes are different from sum up of 268 individual castes because of the shared genes among different castes.

269 270 271 Figure S7. Heatmap of total identified immune gene expression in 3 N. castaneus termite 272 castes following exposure to nestmate focal individuals injected with heat-killed microbes 273 (treatment) or Ringer’s solution (control) (social experiment). Library legend: CW1-3: false 274 workers in control-exposed replicates, TW1-3: false workers in treatment-exposed replicates, 275 CS1-3: soldiers in control-exposed replicates, TS1-3: soldiers in treatment-exposed replicates, 276 CR1-3: reproductives in control-exposed replicates, TR1-3: reproductives in treatment-exposed 277 replicates.

278

Species/caste Upregulated Downregulated Reproductives 1 1 Neotermes Soldiers 1 0 castaneus False Workers 12 96 Blatta orientalis 9 7 279

280 Figure S8. Number of differentially regulated genes in 3 castes of the termite N. castaneus or 281 conspecifics of the cockroach B. orientalis following exposure to nestmate or conspecific focal 282 individuals that had been injected with heat-killed microbes versus Ringer’s solution (social 283 experiment).

284 285 Figure S9. PCA analysis of immune gene expression in 3 different castes of the termite N. 286 castaneus following exposure to nestmate focal individuals injected with heat-killed microbes 287 (treatment, blue) versus Ringer’s solution (control, red) (social experiment).

288

289 290 Figure S10. MA plots of total differential gene expression comparisons between the 3 different 291 castes of the termite N. castaneus, regardless of social treatment exposure (combined 292 treatment and control-exposure replicates from the social experiment). From left to right: false 293 workers vs reproductives; soldiers vs reproductives; soldiers vs false workers.

Only false Only soldiers Soldier & False Worker workers Reproductives-up 601 770 165 Reproductives-down 421 28 436

Reproductives & False Only false Only reproductives Worker workers Soldiers-up 327 122 90 Soldiers-down 1275 96 321

Reproductives & False Only reproductives Workers Only soldiers Workers-up 275 189 238 Workers-down 912 23 189 294 295 Figure S11. Number of differentially expressed genes between the 3 different castes of the 296 termite N. castaneus, regardless of social exposure (combined treatment- and control-exposure 297 replicates from the social experiment) (|log2FoldChange| > 2 and p-value<0.01).

298 References

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