ELECTRONIC APPENDIX. This is the Electronic Appendix to the article

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1 ELECTRONIC APPENDIX This is the Electronic Appendix to the article Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic by Charles E. Cook, Qiaoyun Yue, Michael Akam Proc. R. Soc. B (doi:1.198/rspb ) Electronic appendices are refereed with the text; however, no attempt is made to impose a uniform editorial style on the electronic appendices.

2 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p.1 Supplement Table A1. List of taxa used to construct the data sets used for phylogenetic analysis. Taxa newly reported in this paper are in bold. Taxon Accession number Taxonomy Crustacea Artemia franciscana NC_162 Branchiopoda; Anostraca Daphnia pulex NC_844 Branchiopoda; Cladocera Triops cancriformis NC_4465 Branchiopoda; Notostraca Triops longicaudatus AY Branchiopoda; Notostraca Hutchinsoniella macracantha AY Cephalocarida Parhyale hawaiiensis AY Malacostraca; Amphipoda Pagurus longicarpus AF15756 Malacostraca; Decapoda Panulirus japonicus NC_4251 Malacostraca; Decapoda Penaeus monodon NC_2184 Malacostraca; Decapoda Portunus trituberculatus NC_537 Malacostraca; Decapoda Squilla mantis AY Malacostraca; Hoplocarida Argulus americus AY Maxilliopoda; Branchiura Pollicipes polymerus AY Maxilliopoda; Cirripedia Tigriopus japnoicus NC_3979 Maxillopoda; Copepoda Vargula hilgendorfii NC_536 Maxillopoda; Ostracoda Armillifer armillatus AY Pentastomida Speleonectes tulumensis AY45619 Remipedia Hexapoda Gomphiocephalus hodgsoni NC_5438 Collembola Onychiurus orientalis AY Collembola Podura aquatica AY Collembola Tetrodontophora bielanensis NC_2735 Collembola Insecta Crioceris duodecimpunctata NC_3372 Coleoptera Pyrocoelia rufa NC_397 Coleoptera Tribolium castaneum NC_381 Coleoptera Chrysomya putoria NC_2697 Diptera; Muscomorpha Cochliomyia hominivorax NC_266 Diptera; Muscomorpha Drosophila melanogaster NC_179 Diptera; Muscomorpha Drosophila yakuba NC_1322 Diptera; Muscomorpha Ceratitis capitata NC_857 Diptera; Muscomorpha Anopheles gambiae NC_284 Diptera; Nematocera Anopheles quadrimaculatus NC_875 Diptera; Nematocera Triatoma dimidiata NC_269 Hemiptera Apis mellifera NC_1566 Hymenoptera Bombyx mandarina NC_3395 Lepidoptera Bombyx mori NC_2355 Lepidoptera Ostrinia furnicalis NC_3368 Lepidoptera Ostrinia nubilalis NC_3367 Lepidoptera Melipona bicolor NC_4529 Neoptera; Hymenoptera Antheraea pernyi NC_4622 Neoptera; Lepidoptera lepidopsocid RS-21 NC_4816 Neoptera; Psocoptera Thrips imaginis NC_4371 Neoptera; Thysanoptera Locusta migratoria NC_1712 Orthoptera

3 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p.2 Heterodoxus macrupus NC_2651 Phthiraptera Tricholepidion gertschi NC_5437 Thysanura Thermobia domestica AY Thysanura Chelicerata Ixodes hexagonus NC_21 Arachnida; Acari; Parasitiformes Ixodes persulcatus NC_437 Arachnida; Acari; Parasitiformes Ornithodoros moubata NC_4357 Arachnida; Acari; Parasitiformes Rhipicephalus sanguineus NC_274 Arachnida; Acari; Parasitiformes Varroa destructor NC_4454 Arachnida; Acari; Parasitiformes Limulus polyphemus AF21623 Merostomata; Xiphosura Myriapoda Lithobius forficatus NC_2629 Chilopoda Narceus annularus NC_3343 Diplopoda Thyropygus sp. NC_3344 Diplopoda

4 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p.3 Supplement Table A2. Percentage of each gene retained for phylogenetic analysis after elimination of poorly aligned regions using Gblocks. Note that the second column, total aa, refers to the number of amino acid positions in the complete alignment. Because there are gaps in some alignments this figure is greater than the total number of amino acid residues in a gene for any single taxon. Similarly, column five, percentage retained, is calculated based upon the total number of amino acid residues in the complete alignment. Gene Total aa aa retained Nucleotides retained Percentage retained atp atp8 58 cox cox cob nad nad nad nad nad4l nad nad Total

5 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p.4 Supplement Table A3. Results of composition-bias test from Tree-Puzzle for each of the 3 taxa used in the analysis, for each of four different data sets: nucleotide first and second positions only, all nucleotide positions, nucleotide data set in which first and third positions were RYencoded, nucleotide data set in which third positions were RY-encoded, and amino acids. Note that when third positions are RY-encoded no taxa fail the composition-bias test. Taxon 3taxa 3taxa 1st2nd all 3taxa 1st3rdRY 3taxa Amino 3rdRY acid Argulus americus f f f p f Armillifer armillatus f f f p f Artemia franciscana f f p p p Bombyx mandarina f f f p f Ceratitis capitata f f p p p Daphnia pulex f f p p f Gomphiocephalus hodgsoni p f p p p Hutchinsoniella macracantha f f p p p lepidopsocid RS-21 f f p p f Limulus polyphemus p f p p p Lithobius forficatus f f p p p Locusta migratoria p f p p p Narceus annularus f f p p p Onychiurus orientalis p f p p p Pagurus longicarpus p f p p p Parhyale hawaiiensis f f p p p Penaeus monodon p f p p p Podura aquatica p f p p p Pollicipes polymerus p f p p p Speleonectes tulumensis p f p p p Squilla mantis p f p p f Tetrodontophora bielanensis f f p p p Thermobia domestica p f p p p Thyropygus sp. p f p p p Tigriopus japnoicus f f p p p Triatoma dimidiata p f p p p Tribolium castaneum f f f p f Tricholepidion gertschi p f p p p Triops longicaudatus p f p p p Vargula hilgendorfii f f f p f number passing

6 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p.5 (a) First positions (b) First positions RY encoded (c) Second positions (d) First and second positions p-distances (e) Third positions (f) Third positions RY encoded (g) First, second + third RY (h) All positions GTR+I+G distances GTR+I+G distances Supplement Figure A1. Saturation testing. X-axis: maximum likelihood distances calculated using the best-fit model (GTR+I+G8). Y-axis: uncorrected p-distances. Distances were calculated with PAUP* [Swofford, 1998 #212] for the 3-taxon data set using various combinations of 1st, 2nd, and 3rd positions, including those data sets in which 1st and/or 3rd positions were RY encoded as indicated by each label. For each subset parameters were re-optimized before ML distances were calculated. If there is no saturation in the nucleotide substition process the points will fall on the line of Y=X. As saturation increases the line deviates. All axes have been normalized for comparison, except the X-axis for the third position data set. This data set shows near complete saturation when all four nucleotides are used to calculate distances (e), but reveals some signal when nucleotides are RY-encoded (f).

7 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p % 28.6% 21.9% 3.% 3.3% 3.8% 3.% 7.3% 3.4% 5.4% 3.8% 22.4% 27.7% 3.1% 28.5% 28.2% 3.5% 27.5% 22.4% 3.6% 22.2% 1st positions: 83.6% 2nd positions: 84.3% 3rd positions: 66.5% 31.6% 3% 17.3% 1.4% 2.3% 5.3% 1.2% 1.% 1.8% 1.7% 4.5% 32.% 31.6% 1.3% 32.% All positions: 95.2% 3% 1.7% 31.5% 1st&2nd positions: 92.5% 18.1% 5.3% 17.5% 3rd positions RY: 52.9% 31.6% 28.7% 32.3% 1.5% 2.8% 1.3% 1.3% 1.1% 3.% 4.2% 1.4% % 32.% 1.2% 31.3% 1st2nd3rdRY: 94.9% 29.3% 3.2% 28.9% 1stRY2nd3rdYR: 86.9% 31.6% 1.5% 31.4% Amino acids: 95.3% Supplement Figure A2. Likelihood maps for 3 taxa nucleotide data sets using various combinations of 1st, 2nd, and 3rd positions, including those data sets in which 1st and/or 3rd positions were RY encoded. Corners represent well-resolved quartets. The percentage of quartets within each region is indicated, and the total for all three corners is shown. The higher the total the more phylogenetic signal there is in that data set.

8 Cook, Yue & Akam: Mitochondria suggest paraphyly of hexapods and crustaceans Supplement p Vargula hilgendorfii Maxillopoda Ostracoda Artemia franciscana Branchiopoda Tigriopus japonicus Maxillopoda Copepoda Armillifer armillatus Maxillopoda Branchiura Argulus americus Maxillopoda Pentastomida Hutchinsoniella macracantha Maxillopoda Cephalocarida Parhyale hawaiiensis Malacostraca Gomphiocephalus hodgsoni Podura aquatica Collembola Onychiurus orientalis Tetrodontophora bielanensis Bombyx mandarina Tribolium castaneum lepidopsocid RS-21 Triatoma dimidiata Ceratitis capitata Locusta migratoria Thermobia domestica Tricholepidion gertschi Daphnia pulex Triops longicaudatus Squilla mantis Pagurus longicarpus Penaeus monodon Pollicipes polymerus Maxillopoda Cirripedia Speleonectes tulumensis Remmipedia Narceus annularis Thyropygus sp. Lithobius forficatus Insecta Branchiopoda Malacostraca Myriapoda Limulus polyphemus Chelicerata Supplement Figure A3. Estimates of arthropod phylogeny using an amino acid data set with 3 taxa. This is the consensus tree generated by MrBayes. Numbers indicate posterior probabilities for that branch of the tree in the Bayesian analysis. The positions of four taxa; A. franciscana, H. macracantha, P. hawaiiensis, and S. tulumensis, are different from the positions of these taxa in Figure 1.

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