Electronic supplementary material Evolutionary rates are correlated between cockroach symbiont and mitochondrial genomes Daej A. Arab1, Thomas Bourguignon1,2,3, Zongqing Wang4, Simon Y. W. Ho1, & Nathan Lo1 1School of Life and Environmental Sciences, University of Sydney, Sydney, Australia 2Okinawa Institute of Science and Technology Graduate University, Tancha, Onna-son, Okinawa, Japan 3Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic 4College of Plant Protection, Southwest University, Chongqing, China Sampling, DNA extraction, and sequencing A list of samples and collection data for each cockroach and outgroup examined is provided in table S1. For the majority of taxa examined in this study, partial Blattabacterium genomic data were available from a previous study of cockroach mitochondrial genomes [1]. In some cases, new genomic data were obtained from fat bodies of individual cockroaches, as follows. DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s protocol. Individual DNA samples were tagged with unique barcode combinations, mixed in equimolar concentration, and 150 bp paired-end-reads-sequenced with an Illumina HiSeq4000, following the methods described in ref. [1]. All specimens examined in this study are stored at the Okinawa Institute of Science and Technology, Japan. The genome sequences of Blattabacterium outgroups were obtained from GenBank and included three strains of Sulcia muelleri (accession numbers CP002163, AP013293, and CP010828), one Flavobacterium gilvum (CP017479), one Lutibacter sp. (CP017478), one Tenacibaculum dicentrachi (CP013671), and one Polaribacter sp. (LT629752). In total, we obtained 104 orthologous genes consistently present across the taxa that we examined. Phylogenetic analysis The 104 genes selected for analysis were found in 95% of all taxa; genes that were missing more than two taxa were excluded. All taxa had over 90% of the selected genes, except for Aeluropoda insignis which only had ~80% of all genes. Missing genes were presumed to be because of uneven sequencing coverage of samples and the relatively low sequencing coverage used, rather than the actual absence of these genes from their genomes. All genes were concatenated into a 107,187 bp data set. We aligned the protein-coding Blattabacterium genes at the amino acid level using MAFFT v7.305b [2], then back- translated them into nucleotide sequences using PAL2NAL [3]. The same alignment methods were used for the cockroach mtDNA genome analyses. Outgroups for the cockroach mtDNA genome analyses were as described in ref. [1]. The same phylogenetic methods were used for both the cockroach mtDNA and Blattabacterium data sets. Tests in DAMBE indicated that rd 3 codon positions in the mitochondrial dataset were saturated (NumOTU = 32, ISS = 0.804, ISS.CAsym =0.809). Although the Blattabacterium sequences were not significantly saturated at 3rd codon sites (NumOTU = 32, ISS = 0.649, ISS.CAsym =0.819), we did not use it for our phylogenetic analyses because the test statistic was close to the critical value. Therefore, we performed phylogenetic analyses using alignments of 1st and 2nd codon sites only. First, we inferred the phylogeny using maximum likelihood in RAxML v8.2 [4], using 1000 bootstrap replicates to estimate node support. We used the GTR+G substitution model for Blattabacterium combined 1st and 2nd codon sites. The cockroach mtDNA data set was partitioned into 1st+2nd codon sites and 12S+16S+tRNAs, with each subset assigned a separate GTR+G substitution model. Tests for congruence between host and symbiont datasets We used ParaFit in R 3.5.1 [5] to quantify congruence between host and symbiont topologies. We first created matrices of patristic distances calculated from maximum-likelihood host and symbiont phylogenies and a host-symbiont association matrix, then implemented a global test with 999 permutations, using the ParafitGlobal value and a p-value threshold of 0.05 to determine significance. Molecular dating The molecular clock was calibrated using minimum age constraints based on the fossil record, implemented as uniform priors on node ages. Soft maximum bounds were determined using phylogenetic bracketing [6]. Altogether we used four fossils to calibrate four nodes (table S2). We selected fossils following suggested criteria for justifying fossil calibrations [7]. A birth-death process was used for the tree prior. A single Markov chain Monte Carlo (MCMC) analysis was performed, with the tree and parameter values sampled every 104 MCMC steps over a total of 108 steps. The initial 10% of trees were discarded as burn-in. A maximum-clade-credibility tree was obtained using TreeAnnotator in the BEAST software package [8]. Acceptable sample sizes and convergence to the stationary distribution were checked using Tracer 1.7 [8]. Simulations We simulated the evolution of sequences from both host and symbiont along the Blattabacterium tree topology inferred using RAxML, with evolutionary parameters obtained from our separate RAxML analyses of the original data. Tree lengths for host and symbiont were rescaled according to their relative rates, but the relative branch rates were maintained between the two trees. We performed comparisons of evolutionary rates inferred from these synthetic data, using the methods described above. These analyses were carried out a total of three times. Supplementary Table S1. A list of samples and collection data for each cockroach examined. Species Family Sample ID Accession Collecting locality Collector Date number Aeluropoda insignis Blaberidae B002 TBA Breeding colony of Kyle Kandilian N/A N/A Allacta australiensis Ectobiidae AUS Allacta James Cook University, Rainforest site, Queensland, David Rentz 22-Jun-15 TBA Australia Amazonina sp. Ectobiidae Z256E TBA Ecuador, Bosque Protector del Alto Nangaritza Frantisek Juna Apr-2016 Anallacta methanoides Ectobiidae B057 TBA Breeding colony of Kyle Kandilian N/A N/A Anaplecta calosoma Anaplectidae Cockroach contig Kuranda, Queensland, Australia David Rentz 17-Nov- TBA 1688 15 Anaplecta omei Anaplectidae Anaplecta omei TBA Mt. Emeishan, Sichuan Province, China Zongqing Wang 01-Jul-13 Balta sp. Ectobiidae Balta_sp. Cairns, Australia David Rentz 18-Dec- TBA 15 Beybienkoa kurandanensis Ectobiidae Cairns, Australia David Rentz 18-Dec- Beybienkoa_karandanensis TBA 15 Blaberus giganteus Blaberidae BGIGA CP003535- GenBank N/A N/A CP003536 Blaptica dubia Blaberidae B056 TBA Breeding colony of Kyle Kandilian N/A N/A Blatta orientalis Blattidae BOR CP003605- GenBank N/A N/A CP003606 Blattella germanica Ectobiidae BGE CP001487 GenBank N/A N/A Carbrunneria paramaxi Ectobiidae Carbru TBA Cairns, Australia David Rentz 05-Oct-15 Chorisoserrata sp. Ectobiidae CHORI TBA Yunnan Province, China Zongqing Wang Cosmozosteria sp. Blattidae B117 TBA Cape Upstart, Australia James Walker 13-Oct-15 Cryptocercus hirtus Cryptocercidae HIR TBA Mt.Taibaishan, Shaanxi Province,China N/A 06-Oct-13 Cryptocercus punctulatus Cryptocercidae CPU CP003015- GenBank N/A N/A CP003016 Deropeltis paulinoi Blattidae B069 TBA Breeding colony of Kyle Kandilian N/A N/A Ectobius sp. Ectobiidae Z254C TBA Slovenia Frantisek Juna Apr-2016 Ectoneura hanitschi Ectobiidae Ectoneura_hanitschi James Cook University, Rainforest site, Queensland, David Rentz 18-Dec- TBA Australia 15 Epilampra maya Blaberidae B095 TBA Arcadia, Florida, USA Kyle Kandilian 07-Jul-09 Eublaberus distanti Blaberidae B025 TBA Breeding colony of Kyle Kandilian N/A Euphyllodromia sp. Ectobiidae Z257 TBA Ecuador, Podocarpus National Park Frantisek Juna Apr-2016 Eupolyphaga sinensis Corydiidae B081 TBA Breeding colony of Kyle Kandilian N/A N/A Eurycotis decipiens Blattidae B071 TBA Breeding colony of Kyle Kandilian N/A N/A Galiblatta cribrosa Blaberidae Z98 TBA Nouragues, French Guiana N/A 14-Jun-15 Gromphadorhina Blaberidae B030 Breeding colony of Kyle Kandilian N/A N/A TBA grandidieri Gyna capucina Blaberidae Z139GY TBA Ebogo, Cameroon Frantisek Juna 08-Sep-15 Ischnoptera Ectobiidae B083 Torreya State Park, Bristol, Florida, USA 07-Jul-09 deropeltiformis Lamproblatta sp. Lamproblattidae LA male TBA Petit saut, French Guiana Frantisek Juna 08-Jul-09 Laxta sp. Blaberidae AUS2 Olney State Forest, New South, Wales, Australia Nathan Lo and 25-Aug- TBA Thomas 15 Bourguignon Macropanesthia Blaberidae B092 Breeding colony of Kyle Kandilian N/A N/A TBA rhinoceros Mastotermes darwiniensis Isoptera MADAR CP003000, GenBank N/A N/A CP003095 Megaloblatta sp. Ectobiidae ECMD1 TBA Podocarpus National Park, Ecuador Frantisek Juna Apr-2016 Melanozosteria sp. Blattidae Melanozosteria_sp. Cairns, Australia David Rentz 18-Dec- TBA 15 Methana sp. Blattidae AUS1 North Manly, New South Wales, Australia Nathan Lo 01-Aug- TBA 15 Nauphoeta cinerea Blaberidae BNCIN CP005488- GenBank - Kambhampati et al. 2013 N/A N/A CP005489 Neolaxta mackerrasae Blaberidae B107 TBA Paluma Range, Australia David Rentz 15-Oct-15 Opisthoplatia orientalis Blaberidae Z15100 TBA Breeding colony of J. Hromádka N/A N/A Panchlora nivea Blaberidae B044 TBA Breeding colony of Kyle Kandilian N/A N/A Panesthia angustipennis Blaberidae Z138 Breeding colony in Czech Republic., orig Vietnam N/A 01-Aug- TBA 15 Panesthia sp. Blaberidae Panesthia_sp TBA Bubeng, province Yunnan, China N/A 08-Jul-09 Paranauphoeta Blaberidae PARA N/A N/A N/A TBA circumdata Paratemnopteryx Ectobiidae B061 TBA Breeding colony of Kyle Kandilian N/A N/A Parcoblatta virginica Ectobiidae B102 TBA Breeding colony of Kyle Kandilian