Molecular Phylogenetics and Evolution 132 (2019) 100–104

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Molecular Phylogenetics and Evolution

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Historical biogeography of the clade Rhinotermitinae (: T Isoptera) Menglin Wanga, Aleš Bučeka, Jan Šobotníkb, David Sillam-Dussèsc,d, Theodore A. Evanse,h, ⁎ Yves Roisinf, Nathan Log, Thomas Bourguignona,b, a Okinawa Institute of Science & Technology Graduate University, 1919–1 Tancha, Onna-son, Okinawa 904–0495, Japan b Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic c Institut de Recherche pour le Développement – Sorbonne Universités, iEES-Paris, U 242, Bondy, France d Université Paris 13 - Sorbonne Paris Cité, LEEC, EA 4443, Villetaneuse, France e School of Biological Sciences, University of Western Australia, Perth, WA 6009, Australia f Evolutionary Biology and Ecology, Université Libre de Bruxelles, Belgium g School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia h Department of Biological Sciences, National University of Singapore, Singapore, Singapore

ARTICLE INFO ABSTRACT

Keywords: are the principal decomposers in tropical and subtropical ecosystems around the world. Time-cali- Dolichorhinotermes brated molecular phylogenies show that some lineages of Neoisoptera diversified during the Oligocene and Isoptera Miocene, and acquired their pantropical distribution through transoceanic dispersal events, probably by rafting Parrhinotermes in wood. In this paper, we intend to resolve the historical biogeography of one of the earliest branching lineages Rhinotermes of Neoisoptera, the Rhinotermitinae. We used the mitochondrial genomes of 27 species of Rhinotermitinae to Schedorhinotermes build two robust time-calibrated phylogenetic trees that we used to reconstruct the ancestral distribution of the group. Our analyses support the monophyly of Rhinotermitinae and all genera of Rhinotermitinae. Our mole- cular clock trees provided time estimations that diverged by up to 15.6 million years depending on whether or not 3rd codon positions were included. Rhinotermitinae arose 50.4–64.6 Ma (41.7–74.5 Ma 95% HPD). We detected four disjunctions among biogeographic realms, the earliest of which occurred 41.0–56.6 Ma (33.0–65.8 Ma 95% HPD), and the latest of which occurred 20.3–34.2 Ma (15.9–40.4 Ma 95% HPD). These results show that the Rhinotermitinae acquired their distribution through a combination of transoceanic dis- persals and dispersals across land bridges.

1. Introduction the global distribution of earliest branching lineages. However, the termite lineages that currently dominate warm ecosystems evolved only Termites form a small group, comprising ∼3000 species ∼50 Ma (Bourguignon et al., 2015), and their distribution across the (Krishna et al., 2013). They primarily feed on wood or grass, but many tropics is best explained by transoceanic and land bridge dispersals. species of Termitidae evolved to feed on decomposed substrates such as Several tens of such dispersal events, each leading to termite lineages humus or soil (Abe, 1979). Termites reach their highest abundance in with modern representatives, took place during the Oligocene-Miocene tropical and subtropical terrestrial ecosystems, where they are the period, 34–6 Ma (Bourguignon et al., 2016, 2017). This outstanding principal decomposers of organic matter (Sugimoto et al., 2000). ability to disperse and colonise new continents is largely due to the Phylogenies based on mitochondrial genomes have shown that ex- wood feeding/nesting habit of many termite species, which favours tant termites descend from a common ancestor that lived ∼150 Ma dispersal by rafting across oceans in blown down trees and logs (Bourguignon et al., 2015), and their distribution includes all of the (Bourguignon et al., 2016, 2017). continents other than Antarctica. Vicariance, through plate tectonics, Several recent studies have intended to resolve the historical and dispersal, across oceans and land bridges, are the two processes biogeography of termites, primarily focusing on Neoisoptera that can explain how organisms acquired their current distributions (Bourguignon et al., 2016, 2017; Dedeine et al., 2016). However, one of across continents. In the case of termites, plate tectonics might explain the most basal lineages of Neoisoptera, the Rhinotermitinae, has been

⁎ Corresponding author at: Okinawa Institute of Science & Technology Graduate University, 1919–1 Tancha, Onna-son, Okinawa 904–0495, Japan. E-mail address: [email protected] (T. Bourguignon). https://doi.org/10.1016/j.ympev.2018.11.005 Received 22 June 2018; Received in revised form 5 November 2018; Accepted 10 November 2018 Available online 29 November 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved. M. Wang et al. Molecular Phylogenetics and Evolution 132 (2019) 100–104 ignored so far. The Rhinotermitinae includes six genera 2004), with default settings, implemented in MEGA 7.0.26 (Kumar (Krishna et al., 2013): Acorhinotermes, Schedorhinotermes, Rhinotermes, et al., 2016). Ribosomal RNAs and transfer RNAs were aligned as DNA, Dolichorhinotermes, Parrhinotermes, and the doubtful Macrorhinotermes, and protein-coding genes as codons. Alignments were concatenated a suspected synonym of Schedorhinotermes (Snyder, 1949). The group using FASconCAT (Kück and Meusemann, 2010). has living representatives in the Neotropical (13 species), Afrotropical We separated the dataset into five partitions (four partitions for (2 spp.), Oriental (35 spp.), New Guinean and Australian regions analyses without third codon position): one partition for the combined (16 spp.) (as defined by Holt et al., 2013), all of which feed on and tRNAs, one partition for the 12S and 16S, and one partition for each live in dead wood (Krishna et al., 2013). Previous molecular phyloge- codon position of protein-coding genes. The same partition scheme was netic trees unambiguously supported Rhinotermitinae as a used for all phylogenetic elaborations. We reconstructed phylogenies monophyletic group, and time-calibrated trees estimated the last using Bayesian and maximum likelihood methods. We examined each common ancestor of the group to be at 43 Ma (32–54 Ma 95% HPD) codon position of the protein-coding genes using the Xia’s method in (Lo et al., 2004; Bourguignon et al., 2015). Since the divergence of DAMBE (Xia and Lemey, 2009), and found no evidence of saturation at

Rhinotermitinae postdates the breakup of Gondwana, Rhinotermitinae the third codon positions (NumOTU = 32, ISS = 0.611, are expected to have acquired their geographic distribution through ISS.CAsym = 0.809). Therefore, all phylogenetic analyses were per- dispersal, presumably via wood rafting across oceans, or via crossing of formed twice, once with the third codon position included, and once land bridges. In this study, we reconstructed the historical biogeo- with the third codon position excluded. We implemented Bayesian in- graphy of Rhinotermitinae using phylogenetic trees inferred from the ferences in MrBayes version 3.2.3 (Ronquist et al., 2012), with unlinked full mitochondrial genomes of 27 species. We used 12 termite fossils to partitions. All analyses were performed twice, once using a GTR+G+I infer a time-calibrated tree of Rhinotermitinae and to determine the model of nucleotide substitution, and once using a GTR+G model of timing of dispersal events. nucleotide substitution. In all cases, we ran four MCMC chains (three hot and one cold) for 107 generations, and sampled the chain every 2. Methods 5000 generations to estimate the posterior distribution. We excluded as burn-in the first 106 generations, as determined by Tracer version 1.5 2.1. Mitochondrial genome sequencing (Rambaut and Drummond, 2007). We inferred maximum likelihood tree using RAxML v.8.2.4 (Stamatakis, 2014) and a GTRGAMMA model We sequenced specimens from 23 termite species and used an ad- of nucleotide substitution. Branch supports were calculated using 1000 ditional four samples of Rhinotermitinae sequenced in previous studies bootstrap replicates. (see Table S1). The outgroups comprise 71 non-Rhinotermitinae sam- ples used by Bourguignon et al. (2015). Termite specimens were pre- 2.4. Molecular dating served in RNAlater® and stored at -80 °C until DNA extraction. Whole genomic DNA was extracted from worker head and legs using the Molecular clock trees were computed in BEAST version 1.8.4 phenol-chloroform extraction procedure. The complete mitochondrial (Drummond and Rambaut, 2007). We used an uncorrelated lognormal genome was amplified in two long-PCR reactions with the TaKaRa LA relaxed clock to model rate variation among branches, with single Taq polymerase using primers previously designed for termites (see model for each partition, allowing different relative rate. A Yule spe- Table S2) (Bourguignon et al., 2015, 2016). The PCR conditions for the ciation model was used as tree prior. We used a GTR+G model of 10 kb-fragment were as followed: initial denaturation at 95 °C for 1 min, nucleotide substitution for each partition. MCMC chains were run for followed by 30 cycles of 98 °C for 10 s, 65 °C for 30 s, and 68 °C for 108 generations, from which the first 710 generations were discarded as 11 min, and a final extension at 72 °C for 10 min. The PCR conditions burn-in. The chain was sampled every 10,000 generations to estimate for the 6 kb-fragment were identical, except for the extension step the posterior distribution. We used 12 fossils as minimum age con- during the 30 cycles, which was set to 7 min at 68 °C instead of 11 min. straints (see Table S3). We determined soft maximum bounds using We measured the concentration of both long-PCR fragments with the phylogenetic bracketing (Ho and Phillips, 2009). Each calibration was Qubit 3.0 fluorometer and mixed them in equimolar concentration. implemented as exponential priors of node time. Each BEAST analysis, Sequencing of mixed long-PCR fragments was carried out through with and without third codon position, was executed thrice to ensure commercial service from BGI tech. Long-PCR fragments were frag- convergence of the chains. mented and inserts of 300–500 bp were multiplexed and 88 bp-paired- end sequenced using Illumina HiSeq2000. 2.5. Biogeographic analyses

2.2. Mitochondrial genome assembly and annotation We reconstructed the geographic range of termites using the ace function implemented in the R package APE version 5.0 (Paradis et al., We assembled 88 bp paired-end reads using the CLC suite of soft- 2004). We used the Maximum Likelihood model described by Pagel ware, as previously described by Bourguignon et al. (2015). For poly- (1994) and an equal-rates of transition. We also used the Bayesian morphic base pairs, we selected the most frequent base. The control Binary Method implemented in RASP version 4.0 (Yu et al. 2015), using region was systematically omitted from the final assembly because it both estimated and fixed model of state frequencies, and gamma and comprises repeated regions that were difficult to assemble accurately equal distribution rates across sites. Sampling locations were used to with short reads. assign each tip to one biogeographic realm. We used the MITOS webserver to annotate the two ribosomal RNAs, 22 transfer RNAs, and 13 protein-coding genes (Bernt et al., 2013). In 3. Results all cases, we ran MITOS using the invertebrate mitochondrial genetic code and default parameters. Annotated mitochondrial genomes were 3.1. Molecular phylogeny quality-checked against published termite mitochondrial genomes. Mitochondrial genomes were deposited in GenBank under accession Each of our analyses yielded identical tree topologies (Fig. 1), numbers provided in Table S1. consistently supporting the monophyly of Rhinotermitinae. Par- rhinotermes formed a monophyletic taxon, the sister group to all re- 2.3. Phylogenetic analyses maining Rhinotermitinae. Schedorhinotermes was also found to be monophyletic, and formed the sister group of a clade composed of We aligned each gene separately using the Muscle algorithm (Edgar, Dolichorhinotermes and Rhinotermes.

101 M. Wang et al. Molecular Phylogenetics and Evolution 132 (2019) 100–104

Fig. 1. Reconstruction of Rhinotermitinae lineage ancestral distributions, and estimates of divergence times within the group based on whole mitochondrial gen- omes, with third codon positions excluded. The map shows the biogeographic areas that were described in Holt et al. 2013. Phylogenetic tree was estimated in RAxML and Mrbayes. Branch support values indicate maximum-likelihood bootstrap support (percentage) and posterior probability in RaxML and MrBayes trees, respectively. Asterisks indicate 100% bootstrap support and 1.0 posterior probability. The bars at the nodes indicate the 95% HPD intervals for the ages. Pie charts indicate likelihoods of ancestral geographic range reconstructed with a maximum likelihood model.

3.2. Divergence dating analyses Dolichorhinotermes shared a last common ancestor at 24.0–35.7 Ma (16.8–45.3 Ma 95% HPD) and diverged from Schedorhinotermes The analysis with third codon positions yielded consistently older 41.0–56.6 Ma (33.0–65.7 Ma 95% HPD). Our biogeographic re- age estimates (Supplementary Fig. S1), up to 15.6 My older than the constructions suggested that they dispersed to the Neotropical realm, analysis without third codon positions (Fig. 1). We provide the results possibly from the Oriental region. In contrast, our Bayesian binary of both analyses conjointly. The divergence time between Par- models favour a Neotropical origin of the group, and a subsequent rhinotermes and other Rhinotermitinae was estimated at 50.4–64.6 Ma dispersal to the African realm. Schedorhinotermes consists of three (41.7–74.5 Ma 95% HPD). The most recent common ancestor of the lineages, each exclusively distributed in the African, Australian or Or- examined Parrhinotermes species was estimated at 29.7–42.4 Ma iental realms, respectively. The African Schedorhinotermes diverged (23.3–50.0 Ma 95% HPD). The clade composed of Dolichorhino- from other Schedorhinotermes 26.5–41.3 Ma (21.1–48.8 Ma 95% HPD), termes + Rhinotermes diverged from Schedorhinotermes 41.0–56.6 Ma and the Oriental Schedorhinotermes diverged from the Australian Sche- (33.0–65.8 Ma 95% HPD), and the most recent common ancestor of all dorhinotermes 20.3–34.2 Ma (15.9–40.4 Ma 95% HPD). The scenario Schedorhinotermes species was dated to 26.5–41.3 Ma (21.1–48.8 Ma favoured by the maximum likelihood model is an Oriental origin of 95% HPD). Schedorhinotermes and a subsequent dispersal to Africa and Australia. The scenario favoured by the Bayesian binary models is an African 3.3. Biogeographic reconstruction origin of Schedorhinotermes, followed by one dispersal to the Oriental realm, from where it dispersed once more to the Australian realm. Our ancestral range reconstruction analyses all yielded similar re- sults, and failed to precisely determine ancestral distributions. For 4. Discussion simplicity, we present here the results of the maximum likelihood model (Fig. 1), and the results obtained with the Bayesian binary model In this study, we conducted the most advanced phylogenetic with estimated state frequencies and gamma-distributed rates across analysis yet of the Rhinotermitinae. We found that the monophyletic sites (Supplementary Fig. S2). Our analyses revealed four disjunctions Parrhinotermes forms the sister group of all other Rhinotermitinae, and among biogeographic realms. Significant uncertainties in the ancestral that these two lineages diverged 50.4–64.6 Ma (41.7–74.5 Ma 95% range reconstruction prevent us from drawing definitive conclusions on HPD). Among the remaining Rhinotermitinae, Schedorhinotermes forms the direction of the dispersal. Within Parrhinotermes, the New Guinean the sister group of Dolichorhinotermes + Rhinotermes, from which it P. browni diverged from Oriental Parrhinotermes at 29.7–42.4 Ma diverged 41.0–56.6 Ma (33.0–65.8 Ma 95% HPD). The split between (23.3–50.0 Ma 95% HPD). The maximum likelihood model suggests Dolichorhinotermes and Rhinotermes was estimated at 24.0–35.7 Ma that Oriental Parrhinotermes dispersed to the New Guinean realm once, (16.8–45.3 Ma 95% HPD). Two previous studies agreed with this while the Bayesian binary models suggest that Oriental and New Guinea branching pattern (Austin et al., 2004; Bourguignon et al., 2015), origins are equally probable. The Neotropical Rhinotermes and while two other studies supported a sister relationship between

102 M. Wang et al. Molecular Phylogenetics and Evolution 132 (2019) 100–104

Schedorhinotermes and Parrhinotermes (Lo et al., 2004; Inward et al., Acknowledgements 2007). The position of Parrhinotermes as the earliest lineage in the tree, combined with the fact that it possesses monomorphic soldier and Financial support was provided by the Czech Science Foundation worker castes, suggests that the soldier dimorphism of other (project No. 15-07015Y), by the projects CIGA No. 20184306 and IGA Rhinotermitinae genera evolved once and was not lost since then, FLD No. A30/17 (Czech University of Life Sciences, Prague), and by the except perhaps in Acorhinotermes (Roisin, 2000). Alliance National University of Singapore-Université Sorbonne Paris Our molecular clock analysis with third codon positions yielded Cité. ages up to 15.6 My older than those without third codon positions. This large divergence is likely the result of the high substitution rate at the Appendix A. Supplementary material third codon position, which may have biased these estimations. Analyses with substitution-saturated sites are known to yield ex- Supplementary data to this article can be found online at https:// aggerated age estimations, especially for recent divergences, which doi.org/10.1016/j.ympev.2018.11.005. sometimes appear to be several times their actual age (Zheng et al., 2011). Therefore, our analysis without third codon positions is likely to References be more realistic (Fig. 1), and the actual divergence times within the Rhinotermitinae are probably similar to those of other termite lineages Abe, T., 1979. Studies on the distribution and ecological role of termites in a lowland rain analysed to date (Bourguignon et al., 2016, 2017). forest of west Malaysia. 2. Food and feeding habits of termites in Pasoh Forest Reserve. Jpn. J. Ecol. 29, 121–135. We found that the most recent common ancestor of modern Austin, J.W., Szalanski, A.L., Cabrera, B.J., 2004. 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104 Table S1. Rhinotermitinae samples used in this study, with details of collection locality, date of collection, sample code, collection holder, and 2 GenBank accession numbers. Collection holders: JŠ, Jan Šobotník; YR, Yves Roisin.

GenBank Sample Collection Species Collection Locality Date Reference Accession code holder Number Dolichorhinotermes longilabius Petit Saut, French Guiana 01-Feb-11 NA JŠ Bourguignon et al. 2015 Parrhinotermes aequalis near Kuala Belait, Brunei 13-Feb TBRU1.1b JŠ MK246849 Parrhinotermes browni 50 km from Nabire, West Papua, Indonesia 01-Jun-11 NG52 JŠ Bourguignon et al. 2015 Parrhinotermes microdentiformis near Kuala Belait, Brunei 13-Feb TBRU1.22c JŠ MK246851 Parrhinotermes sp. C Penang, Malaysia 13-Jul MAL29 JŠ MK246842 Parrhinotermes sp. A Khao Chong, Thailand 03-Nov-13 THAI023 JŠ MK246854 Parrhinotermes sp. B Bukit Batog, Singapore 13-Dec T6.9a JŠ MK246848 Rhinotermes hispidus Petit Saut, French Guiana 14-Oct G751 YR Bourguignon et al. 2016 Schedorhinotermes breinlia 19 km. Nth of Ayr, QLD, Australia 08-May-08 ISO314 Cameron et al. 2012 Schedorhinotermes breinlib near Gordonvale, Queensland, Australia 13-Feb-13 AUS87 JŠ MK246841 Schedorhinotermes lamanianus Yangambi, D.R. Congo 12-Jul-13 RDCT112 YR MK246844 Schedorhinotermes longirostris Mac Ritchie, Singapore 12-Dec T2.13c JŠ MK246846 Schedorhinotermes Mac Ritchie, Singapore 13-Dec-12 SING75 JŠ medioobscurus MK246845 Schedorhinotermes putorius Yangambi, D.R. Congo 05-Jul-13 RDCT003 YR MK246843 Schedorhinotermes sarawakensis near Kuala Belait, Brunei 13-Feb TBRU2.3A JŠ MK246852 Schedorhinotermes sp. near Nan, Thailand 12-Nov-13 THAI084 JŠ MK246857 Schedorhinotermes sp. 1 Khao Chong, Thailand 03-Nov-13 THAI030 JŠ MK246855 Schedorhinotermes sp. 2 Thung Chang, Thailand 13-Nov-13 THAI092 JŠ MK246858 Schedorhinotermes sp. 3 Khao Chong, Thailand 06-Nov-13 THAI063 JŠ MK246856 Schedorhinotermes sp. 4 Milla Milla, Queensland, Australia 22-Jul-14 AUS104 JŠ MK246837 Schedorhinotermes sp. 5 near Kuala Belait, Brunei 13-Feb TBRU1.20 JŠ MK246850 Schedorhinotermes sp. 6 Darwin, Australia 12-Jun AUS11 JŠ MK246838 Schedorhinotermes sp. 7 Darwin, Australia 09-Feb-14 AUS61 JŠ MK246839 Schedorhinotermes sp. 8 Cairns, Australia 16-Jul-14 AUS68 JŠ MK246840 Schedorhinotermes sp. 9 near Kuala Belait, Brunei 13-Feb TBRU5.4b JŠ MK246853 Schedorhinotermes sp. 10 Penang, Malaysia 13-Jul TP1.25B JŠ MK246859 Schedorhinotermes translucens Mac Ritchie, Singapore 12-Dec T2.1d JŠ MK246847

Table S2. PCR primers used in this study.

Primer name Gene Sequence (5’ - 3’) Direction Fragment size Source COIIF3150 COII TGG CAG ATA AGT GCR BTG GAT TTA AG Forward 10-kb fragment Bourguignon et al. 2016 16R13687 16S GAA GGG CCG CGG TAT TTT GAC C Reverse 10-kb fragment Bourguignon et al. 2016 16S13530F 16S TWA AAC TCT ATA GGG TCT TCT CGT CCC A Forward 6-kb fragment Bourguignon et al. 2015 COII3810R COII TTT GCY CCR CAR ATT TCT GAG CAT TG Reverse 6-kb fragment Bourguignon et al. 2015

Table S3. Fossils used as calibrations for estimating divergence times among Rhinotermitinae lineages

Age Soft maximum (Myo)/minimum Species Calibration group bound (97.5% Reference Comments on soft max. bound age constraint for probability) group

Mylacris Dictyoptera + First insect fossil (Engel and 315 407 Scudder, 1868 anthracophila Phasmatodea Grimaldi 2004)

Juramantis initialis 145 Dictyoptera 315.2 Vršanský, 2002 First cockroach fossils

Hodotermitidae + Krishna et al. 2013 Triassoblatta argentina, earliest Valditermes other Isoptera, 130 235 and references fossil of Mesoblattinidae brenanae excluding therein (Martins-Neto and Gallego 2005) Mastotermes Kalotermitidae + Cratokalotermes 112 Rhinotermitidae + 145 Grimaldi et al. 2008 Earliest fossil of termites santanensis Termitidae

Dolichorhinotermes Dolichorhinotermes Schlemmermeyer First fossil of Rhinotermitinae 16 94.3 dominicanus + Rhinotermes and Cancello 2000 (Krishna and Grimaldi 2009)

Reticulitermes + Reticulitermes Engel and Krishna First fossil of Rhinotermitinae 33.9 Coptotermes + 94.3 antiquus 2007 (Krishna and Grimaldi 2009) Heterotermes Coptotermes Coptotermes 16 33.9 Emerson 1971 First Heterotermes fossil sucineus +Heterotermes

Constrictotermes Constrictotermes + First fossil of Termitidae (Engel et 13.8 47.8 Krishna 1996 electroconstrictus Caetermes al. 2011)

All Apicotermitinae Anoplotermes sensu except Jugositermes Krishna and First fossil of Termitidae (Engel et 13.8 47.8 lato and Grimaldi 2009 al. 2011) Duplidentitermes Microcerotermes Microcerotermes + Krishna and First fossil of Termitidae (Engel et 13.8 47.8 insularis Syntermitinae Grimaldi 2009 al. 2011) Termitidae + Coptotermes + First fossil of Rhinotermitinae Nanotermes 47.8 94.3 Engel et al. 2011 Heterotermes + (Krishna and Grimaldi 2009) Reticulitermes Macrotermes + Macrotermes First fossil of Termitidae (Engel et 16 Odontotermes + 47.8 Charpentier 1843 pristinus al. 2011) Synacanthotermes

BRA31_Serritermes_serrifer

* Schedorhinotermes sp. 1 * Schedorhinotermes translucens * Schedorhinotermes sp. 9 * Schedorhinotermes medioobscurus * Schedorhinotermes sp. 5 * Schedorhinotermes longirostris * Schedorhinotermes sp. 2 * * Schedorhinotermes sp. * Schedorhinotermes sp. 3 Schedorhinotermes sp. 10 Schedorhinotermes sarawakensis Neotropical realm * * Schedorhinotermes breinli a Afrotropical realm * Schedorhinotermes breinli b Oriental realm * Schedorhinotermes sp. 7 * Australian realm * * Schedorhinotermes sp. 8 Oceanian realm (New Guinea) Schedorhinotermes sp. 4 Schedorhinotermes sp. 6 * * Schedorhinotermes lamanianus Schedorhinotermes putorius * Rhinotermes hispidus Dolichorhinotermes longilabius * * Parrhinotermes sp. A * Parrhinotermes sp. C * * Parrhinotermes aequalis Parrhinotermes sp. B * Parrhinotermes microdentiformis Parrhinotermes browni

6 0 5 0 4 0 3 0 2 0 1 0 Ma

Supplementary Fig. S1. Reconstruction of Rhinotermitinae ancestral distribution and estimates of cladogenesis time based on whole mitochondrial genome, including third codon positions. The map shows the biogeographic areas that were described in Holt et al. 2013. Phylogenetic tree was estimated in BEAST v1.8.4. Branch support values indicate posterior probability. Asterisks indicate 1.0 posterior probability. The bars at the nodes indicate the 95% HPD intervals for the ages. Pie charts indicate likelihoods of ancestral geographic range reconstruction.

98/0.51 Schedorhinotermes sp. 1 * Schedorhinotermes translucens * Schedorhinotermes sp. 9 * Schedorhinotermes medioobscurus * Schedorhinotermes sp. 5 * Schedorhinotermes longirostris * Schedorhinotermes sp. 2 * * Schedorhinotermes sp. * Schedorhinotermes sp. 3 Schedorhinotermes sp. 10 Schedorhinotermes sarawakensis Neotropical realm * * Schedorhinotermes breinli a Afrotropical realm * Schedorhinotermes breinli b Oriental realm 99/1 Schedorhinotermes sp. 7 Australian realm * * * Schedorhinotermes sp. 8 Oceanian realm (New Guinea) Schedorhinotermes sp. 4 96/1 Schedorhinotermes sp. 6 * Schedorhinotermes lamanianus Schedorhinotermes putorius * Rhinotermes hispidus Dolichorhinotermes longilabius * * Parrhinotermes sp. A * Parrhinotermes sp. C * * Parrhinotermes aequalis Parrhinotermes sp. B * Parrhinotermes microdentiformis Parrhinotermes browni

6 0 5 0 4 0 3 0 2 0 1 0 Ma

Supplementary Fig. S2. Reconstruction of Rhinotermitinae ancestral distribution and estimates of cladogenesis time based on whole mitochondrial genome, with third codon positions excluded. The map shows the biogeographic areas that were described in Holt et al. 2013. Phylogenetic tree was estimated in RAxML and MrBayes. Branch support values indicate maximum-likelihood bootstrap support (percentage) and posterior probability in RaxML and MrBayes trees, respectively. Asterisks indicate 100% bootstrap support and 1.0 posterior probability. The bars at the nodes indicate the 95% HPD intervals for the ages. Pie charts indicate likelihoods of ancestral geographic range reconstructed with a Bayesian binary model with estimated state frequencies and gamma-distributed rates across sites.

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