Supplementary materials

Transcription

1 1 Supplementary materials Mitochondrial diversity. The mtdna sequences used to compare nucleotide diversity between Culicidae species were available on GenBank: Ae. albopictus (cytb: AJ AJ9702, AY072044; COI: AF253022, AY072044, AY , AY AY101854, DQ181451, DQ181457, DQ181458, DQ DQ397912), Ae. aegypti (cytb: AJ AJ970958; ND4: AF AF203366, AF AF334859, AF AF334865), Ae. caspius (COI: FJ FJ210908; COII: DQ DQ300499), Ae. vexans (COI: AY AY645247; COII: AY AY645309, GU229896); C. pipiens (ND4: AY AY793693, EF028084, EF030092, EF033661; COI: AJ557889, AJ557891, AJ557892, AJ AJ633086, AY33086, GQ GQ255651, GQ GQ25564, GQ255666; COII: EU014281, EU014282, L344351); Culex sp. (ND4: AY AY ), C. tarsalis (nad4: EF EF125862), An. aconitus (COI: AY423055, DQ DQ000264; COII: AJ AJ194451, AJ AJ547369, AY AY626978), An. funestus (cytb: AF AF062511), An. gambiae (COI: AF020967, AF020968, AF020970, AF020971, AF020973, AF020980, AF020988, AF020989, AF AF02093, AF020998, AF020999, AF022, AF023, AF AF021023) and An. maculipennis (COI: AF AF342722, AF AF491736). 1

2 19 20 Gene Putative product Locus tag in wpip(pel) Primer (5'-3') Size (bp) Number of alleles found in this study (accession numbers) References gatb Glutamyl-tRNA(Gln) amidotransferase, WPa_0087 gatb_f1-gakttaaaycgygcaggbgtt (Baldo et al. 2006) subunit B gatb_r1-tggyaaytcrggyaaagatga coxa Cytochrome c oxidase, subunit I WPa_0082 coxa_f1-ttggrgcratyaactttatag (Baldo et al. 2006) coxa_r1-ctaaagactttkacrccagt hcpa Conserved hypothetical protein WPa_1214 hcpa_f1-gaaatarcagttgctgcaaa (Baldo et al. 2006) hcpa_r1-gaaagtyragcaagytctg ftsz Cell division protein WPa_0577 ftsz_f1-atyatggarcatataaargatag (Baldo et al. 2006) ftsz_r1-tcragyaatggattrgatat fbpa Fructose-bisphosphate aldolase WPa_1081 fbpa_f1-gctgctccrcttggywtgat (Baldo et al. 2006) fbpa_r1-ccrccagaraaaayyactattc wsp Surface protein WPa_ F-TGGTCCAATAAGTGATGAAGAAAC (Braig et al. 1998) 691R-AAAAATTAAACGCTACTCCA MutL DNA mismatch repair protein WPa_0278 F- ACTTCATTGCCCTTCCAGCT 0-1,063 6 (HQ HQ709394) This study R -GGCATCAAATTAAGGGACA ank2 Ankyrin domain protein WPa_0652 F-CTTCTTCTGTGAGTGTACGT (AM AM397072) (Duron et al. 2007) R2-TCCATATCGATCTACTGCGT pk1 Ankyrin domain protein WPa_0256 (1) F-CCACTACATTGCGCTATAGA 1,334-1,349 5 (AM AM397079) (Sinkins et al. 2005) WPa_0313 (2) R-ACAGTAGAACTACACTCCTCCA (Duron et al. 2007) WPa_1306 (3) pk2 Ankyrin domain protein WPa_0299 (1) F-ATTATGATAAAGCTTGGTAAGAA (AM AM397073; DQ DQ000472) (Sinkins et al. 2005) WPa_0413 (2) R-TTAGCCCTTCATAAATAGCTT (Duron et al. 2007) GP12 Phage related DNA methylase-like protein WPa_0258 (1) F-ATGAATTTAGCAATCCACTACT 1,215-1,302 7 (GU GU827987; HQ HQ709398) (Atyame et al. in press) WPa_0317 (2) R-TTACTAAATAACAGACATATTGCT WPa_1310 (3) WPa_0429 (4) GP15 (=vrlc) Phage related probable secretory protein WPa_1322 F1-ACCATTACAGAACTTGAGGA 1,511-1,538 7 (GU GU827991; HQ HQ709401) (Duron, Fort, and Weill 2006) R1-TAGACGTTCATAGGCAACCA (Atyame et al. in press) F2-ACCTGACTCTGCAGTACTTGA R2-ACTGCTTCTCTCATAAATTCA RepA Phage related replication protein WPa_1312 Tr1e-F1-ACTTTAGAGGGGTGCTTTCT 583-1,501 2 (AJ ; AJ646887) (Duron et al. 2005) Tr1e-R2- ACAAACAACGGCACAGATT Table S1. List of primers and characteristics of genes used to examine the Wolbachia polymorphism. 2

3 Mitochondrial forward primers (5'-3') Mitochondrial reverse primers (5'-3') 1F AATGAATTGCCTGATAAAAAGGA 417R TGAAGAGGCAAAAGCTTGAGT 161F a GCTATTGGGTTCATACCCCAC 773R a GCTATTAATATTCAACCTAAG 286F TGGCTTGGTGCTTGAATAGGGT 1442R AATGGCTGAAGTTTAGGCGAT 1254F ACTAATAGCCTTCAAAGCTGA 2123R TGGATCTCCTCCTCCAATTGGA 2045F AGCTGGTGCTATTACTATGT 3921R AGTTAATCATCTAATAGGGGCT 2768F TCCAGATAGTTACTTAGCATGA 4798R AGCTCCAATAGCTCCTGT 3738F TTCATTAGATGACTGAAAGCA 5968R TTAGGTCGAAACTAATTGCA 4781F ACAGGAGCTATTGGAGCT 7002R CTTTTTTAGCAGGGTTTTATTC 5949F TGCAATTAGTTTCGACCTAA 7723R b GGGTGGGATGGATTAGGATTGG 6290F CATCTTCAGTGTCATGCTCT 8112R b GATTTGTGGTGTCAATGATA 6981F b GAATAAAACCCTGCTAAAAAAG 8871R TGATTACCTAAGGCTCATGT 7702F b CCAATCCTAATCCATCCCACCC 9259R AGCAAGAGAAAGAGTTGTACGA 7940F TGAAACAATTTCCCATTCA 99R AATAAAACTAATATTCCTCCT 8636F TGAGCAACAGAAGAATAAGCA 11217R c ACTAAAGGATTAGCAGGAATGA 8781F GTAATAATCCATATCCTCCT 12178R TACGAGCGGTTGCTCAAACA 9239F CGTACAACTCTTTCTCTTGCT 12409R TACTAAGGAACAAACTTATCCT 9851F AGAAATCTCTTTGTCACTAACT 13182R TGAATGAGATATATACTGTCT 10366F c CTTTATTAGTAACTGTAAAAATTAC 13587R TATTTTAAGGGATTAGCTTTAA 10912F ACAATGGATTTGAGGAGGA 13706R TAATTAGAAATGAAATGTTAATCG 11985F AGGAGTACGATTAGTTTCAGCT 14067R TTAAAGCTTAATTAGTAAAGTA 12387F AGGATAAGTTTGTTCCTTAGTAA 14998R AGCAATGGGAAGGCTTACACT 12856F TCCAACATCGAGGTCGCAATC 13338F GCCGAATTCCTTATTTAAACCTTTC 13566F TTAAAGCTAATCCCTTAAAATA 13802F ACCCTGATACACAAGGTACA 14793F AATTCACACAAAAATTTACATGT Table S2. List of primers used to examine the Culex pipiens mitochondrial polymorphism. The name of the primers indicates their position in the mitochondrial genome. a,b,c, primers used to amplified fragments of the ND2, ND5 and cytb genes, respectively. GenBank accession numbers: 3

4 25 26 ND2 (HQ HQ709413), ND5 (HQ HQ724613), cytb (HQ HQ709409), complete mitochondrial genomes (HQ HQ724617). 4

5 Gene No. of alleles a Fragment size % of VI b π b G+C content (%) b Ka/Ks b Intragenic recombination (Sawyer's test) a MutL , Yes (P<10-4 ) ank No (P=0.33) pk1 5 1,292-1, Yes (P<10-4 ) pk Yes (P<10-4 ) GP12 7 1,193-1, Yes (P<10-4 ) GP15 8 c 1,470-1, Yes (P<10-4 ) RepA , not reliable Table S3. Genetic characteristics of the seven polymorphic genes used for wpip characterization. VI; number of variable sites; π : pairwise nucleotide diversity based on the average of all pairwise comparisons; a Characteristics estimated considering indels in sequence alignments; b characteristics assessed excluding indels; c including the null GP15 wpip(jhb) allele. Note that primer regions were not considered in these analyses. 5

6 Genes MutL ank2 pk1 pk2 GP12 GP15 RepA MutL 0.000*** 0.000*** *** 0.000*** ank *** *** 0.000*** pk *** 0.000*** pk GP *** GP RepA Table S4. Linkage disequilibrium (LD) measures and tests of association between the wpip genes. The upper half shows probabilities based on the null hypothesis of random association of allelic diversity between loci. The lower half shows LD measures (D values). ***, the null hypothesis is rejected at α = taking into account a Bonferonni s adjustment for 21 comparisons. 6

7 Gene Position, Direction of transcription trna anticodon/position Start codon End codon t-rna Ile 2-69, CW GAU/31-33 t-rna Gln , CCW UUG/ t-rna Met , CW CAU/ ND , CW _ ATC (Ile) TAA trna Trp , CW UCA/ trna Cys , CCW GCA/ trna Tyr , CCW GUA/ COI , CW _ TCG (Ser) T trna Leu , CW UAA/ COII , CW _ ATG (Met) T trna Lys , CW CUU/ trna Asp , CW GUC/ ATPase , CW _ ATT (Ile) TAA ATPase , CW _ ATG (Met) TAA COIII , CW _ ATG (Met) TAA trna Gly , CW UCC/ ND , CW _ ATT (Ile) TAA trna Arg , CW UCG/ trna Ala , CW UGC/ trna Asn , CW GUU/ trna Ser , CW GCA/ trna Glu , CW UUC/ trna Phe , CCW GAA/ ND , CCW _ GTG (Val) TAA trna His , CCW GUG/ ND , CCW _ ATG (Met) TAA ND4L , CCW _ ATG (Met) TAA trna Thr , CW UGU/ trna Pro , CCW UGG/ ND , CW _ ATA (Met) TAA CytB , CW _ ATG (Met) TAA trna Ser , CW UGA/ ND , CCW _ TTG (Phe) TAA trna Leu , CCW UAG/ Large rrna , CCW _ trna Val , CCW UAC/ Small rrna , CCW _ A + T rich region _ Table S5. Summary of the Culex pipiens mitochondrial genome. Position: expressed in nucleotides based on the Pel sequence. Direction of transcription: CW, clockwise; CCW, counterclockwise. 7

8 Gene, position Mitotype ND2 ND5 cytb Mosquito line ,061 7,106 7,280 7,341 7,345 7,571 7,824 7,826 7,927 10,502 10,554 10,758 10,887 10,918 10,943 10,952 11,118 pi1 A T C T T T A T G C G A C A A A G G G C A A Pel pi2 G G G Cot-A, Cot-B, Ma-B pi3 G T - G G Ep-A, Ep-B pi4 G - - C - A G G - A Ko, Tn pi5 G - - C G G - A Bf-A pi6 G A - G G T - - G G Au pi7 G A G T - - G G Lv pi8 G C - - A - - C G T G - G G Ke-A pi9 G C - - A G T - - G G Ke-B pi10 G A G T - G G G Bf-B, Mc pi11 G A G T - G G G G Sl pi12 G - G - A A - A G T - - G - - A Is pi13 G A G T - - G A - - T - - Ka-C pi14 G A G T - - G T - - Ma-A Table S6: Nucleotide polymorphism in the ND2, ND5 and cytb mitochondrial genes of Culex pipiens. Mosquito lines are listed according to mitotype (pi1 to pi14). Only polymorphic site are indicated, and a dash indicates similarity with the top sequence. Position: expressed in nucleotides based on the complete mitochondrial sequence of the Pel C. pipiens line. 8

9 48 Supplementary figures Figure S1. Wolbachia phylogeny constructed using Bayesian inferences on concatenated sequences of the five MLST genes gatb, coxa, hcpa, ftsz and hcpa. Wolbachia of major supergroups (A, B, D, F and H) were included in the analysis to delineate the wpip group (highlighted). Host species of Wolbachia are reported, followed by the name of the Wolbachia strain. The scale bar is in units of substitutions/site Figure S2. Examples of recombination breakpoints along the pk1 (A, B), pk2 (C, D, E) and GP12 (F) sequences. For each alignment, only polymorphic sites around the breakpoints are shown. Polymorphisms shared with the underlined sequence are highlighted in grey. Arrows indicated the significant breakpoints and the nucleotide position detected by Sawyer s procedure Figure S3. Mapping of the 13 genes examined in this study on the wpip(pel) genome and on the five major contigs of the wpip(jhb) genome. Black boxes designate prophage genes, or genes inserted in phage regions. Lines connect orthologous genes. The wpip(jhb) genome description corresponds to the current situation and could change when the assembling is achieved Figure S4. Wolbachia phylogenies constructed with six wpip polymorphic genes. A: MutL; B: ank2; C: pk1; D: pk2; E: GP12; F: GP15. The phylogeny of the RepA gene was not performed because the polymorphism with this gene is only based on the presence or the absence of the transposon Tr1. The scale bar is in units of substitutions/site. 9

10 Figure S5. Map of the Culex pipiens mitochondrial genome. The map has been linearized and nucleotide 1 is arbitrary allocated to trna Ile transcription start. All genes are indicated as boxes above (transcription from left to right) or below (transcription from right to left) the baseline. trnas are represented by the single-letter code for the cognate amino acid. Sites found polymorphic between the five C. pipiens mtdna genomes (without the A+T rich region) are indicated by stars. 10

11 Figure S1 11

12 82 A wpip(pel) T T T A G A A G C G C G T G C A T A G G A C T G A T A A A T T G T G G A T G T C G A C G T T C T T C G A C T G G G T G C C wpip(jhb) wpip(is) C A C T A G C T C T A G A T G A T C G T A G T C A A A C A T T wpip(lv) A A A G A T C A A A A T C A T G C G A A T T C A G C G G G G C A C T A G C T C T A G A T G A T C G T A G T C A A A C A T T B wpip(pel) C T G G T A G T T C G C T A C G T A A G T T A A T G G A G C G T A C C G T T T A T T C C A C C C A A A A A C A wpip(jhb) wpip(ka-c) G A C A G G C A T C A A A C G A G A A G C G A C G T C A A T T G G G G A G wpip(sl) C T T C G T G A T A T C G T A C G. A C A G G C A T C A A A C G A G. A G C G A C G T C A A T T G G G. A G 2 wpip(pel) A T C C G T A A T A C G G G A T G A C G G A A C G A A A T G G T G A G C A C G C T A A G T A wpip(jhb) wpip(ep-a) A C A G A T G T T A G G T A G G G C A A A C G A G G T A A C G G A C G wpip(bf-b)g G T T A A G C C G T A C A G A T G T T A G G T A G G G C A A A C. A G G T A A C G G A C G D E F 159 wpip(pel) A T C C G T A A T A C G G G A T G A C G G A A C G A A A T G G T G G C A C G C T A A G T A wpip(jhb) wpip(sl) C A G G T A A C G G A C G wpip(bf-b) G G T T A A G C C G T A C A G A T G T T A G G T A G G G C A A A C A G G T A A C G G A C G wpip(pel) G G G A T G A C G G A A C G A A A T G G T G A G C A C G C T A A G T A wpip(jhb) wpip(sl) C. A G G T A A C G G A C G wpip(ep-a) A C A G A T G T T A G G T A G G G C A A A C G A G G T A A C G G A C G wpip(pel) G C A G C C C C A G G A G C C G G A A A A A T G C G A T T A G C A C A C C G G A C G G A A C T T T G C G wpip(jhb) wpip(ka-c) A T G T T T A T G A A G A T T A A T T C G T A A T A C C C T A T G T T A T wpip(ma-a) A T G T T T A T G A A G A T T A A T T C G T A A T A C C C T A T G T T A T A A C A A T C C T A G C A G A Figure S2 12

13 w Pip(JHB) contig 1299 (478,325bp) w Pip(JHB) contig 1298 (316,943bp) w Pip(JHB) contig 1302 (42,565bp) w Pip(JHB) contig 1301 (126,623bp) w Pip(JHB) contig 1300 (466,173bp) w Pip(Pel) genome (1,482,355bp) gatb coxa coxa gatb // GP12 wsp fbpa GP12 RepA ftsz ank2 pk2? pk2 pk1 GP12 MutL ank2 hcpa pk1 GP12 MutL pk2 pk1 GP12 pk2 GP12 // ftsz ank2 wsp fbpa hcpa pk1 GP12 RepA GP15 // // // Figure S3 13 / // /

14 14 90 wpip(pel) wpip(jhb) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(lv) wpip(ke-a) wpip(ke-b) wpip(au) wpip(is) wpip(ka-c) wpip(ma-a) wpip(ep-a) wpip(ep-b) wpip(bf-b) wpip(mc) wpip(sl) 0.1 wpip(pel) wpip(jhb) wpip(ep-a) wpip(ep-b) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(lv) wpip(mc) wpip(ke-a) wpip(ke-b) wpip(is) wpip(sl) wpip(bf-b) wpip(ka-c) wpip(ma-a) wpip(au) wpip(pel) wpip(jhb) wpip(ep-a) wpip(ep-b) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(lv) wpip(ke-b) wpip(au) wpip(bf-b) wpip(mc) wpip(sl) wpip(ke-a) wpip-(is) wpip(ma-a) wpip(ka-c) wpip(pel) wpip(jhb) wpip(ep-a) wpip(ep-b) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(ka-c) wpip(ma-a) wpip(lv) wpip(ke-a) wpip(ke-b) wpip(au) wpip(bf-b) wpip(mc) wpip(sl) wpip(is) wpip(pel) wpip(ep-a) wpip(ep-b) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(is) wpip(ke-a) wpip(ke-b) wpip(lv) wpip(au) wpip(bf-b) wpip(mc) wpip(sl) wpip(ka-c) wpip(ma-a) A B C D E F WP0652-Pel WP0652-Ep-A WP0652-Ep-B WP0652-Cot-A WP0652-Cot-B WP0652-Ma-B WP0652-Bf-A WP0652-Ko WP0652-Tn WP0652-Lv WP0652-Ke-A WP0652-Ke-B WP0652-Au WP0652-Bf-B WP0652-Mc WP0652-Sl WP0652-Is WP0652-Ka-C WP0652-Ma-A 0.05 wpip(pel) wpip(jhb) wpip(cot-a) wpip(cot-b) wpip(ma-b) wpip(bf-a) wpip(ko) wpip(tn) wpip(ep-a) wpip(ep-b) wpip(lv) wpip(ke-a) wpip(ke-b) wpip(au) wpip(mc) wpip(is) wpip(sl) wpip(bf-b) wpip(ka-c) wpip(ma-a) Figure S4 92

15 I M Q ND4L ND2 T P W CY ND6 cytb ATPase8 L K D G R A N S E COI COII ATPase6 COIII ND3 F S ND1 Large rrna Small rrna L V Figure S5 ND5 A+T rich region H ND4 15

16 Literature Cited Atyame, C., O. Duron, P. Tortosa, N. Pasteur, P. Fort, and M. Weill Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations. Mol Ecol (in press). Baldo, L., J. C. Dunning Hotopp, K. A. Jolley, S. R. Bordenstein, S. A. Biber, R. R. Choudhury, C. Hayashi, M. C. Maiden, H. Tettelin, and J. H. Werren Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol 72 : Braig, H. R., W. Zhou, S. L. Dobson, and S. L. O'Neill Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 180 : Duron, O., A. Boureux, P. Echaubard, A. Berthomieu, C. Berticat, P. Fort, and M. Weill Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens. J Bacteriol 189 : Duron, O., P. Fort, and M. Weill Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the mosquito Culex pipiens. Proc Biol Sci 273 : Duron, O., J. Lagnel, M. Raymond, K. Bourtzis, P. Fort, and M. Weill Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination. Mol Ecol 14 : Sinkins, S. P., T. Walker, A. R. Lynd, A. R. Steven, B. L. Makepeace, H. C. Godfray, and J. Parkhill Wolbachia variability and host effects on crossing type in Culex mosquitoes. Nature 436 :