Details of the multiple sequence alignment (MSA) methods used and maximum likelihood phylogenies estimated from them.

<p>We define the core length as the number of character positions from the first to the last of the characteristic cysteine residues (C1–C6) of the OBPs. The log-likelihoods (LnL), parsimony informative characters, tree size, and average approximate likelihood-ratio tests (aLRT) are derived from the ML analyses. Robinson-Foulds tree distances (RF distance) are calculated by comparing the ant and bee MAP trees to the ML trees derived under the other MSA methods. Best scores of the MSAs compared to the BAli-Phy MAP are in highlighted in bold italics.</p

Details of the <i>Solenopsis invicta</i> odorant binding proteins.

<p>*This single Sanger read appears to be partially unspliced and frameshifted.</p><p>**The total number of 454 reads contributing to this SiOBP3/<i>Gp-9</i> contig is unclear, because it strangely assembled in several different non-overlapping contigs.</p><p>The columns are: Gene – number we are assigning; cDNA – length of cDNA in base pairs, excluding polyA tail; TotAA – conceptual precursor protein length including signal sequence; MatAA – mature secreted protein length excluding signal sequence according to PSORTII; 454 – number of 454 reads contributing to contig; Sanger – number of Sanger reads contributing to contig; C – number of conserved cysteines; PolyA – presence of single or multiple poly-adenylation sites.</p

Maximum a-posteriori (MAP) phylogeny and alignment of the <i>Solenopsis invicta</i> (SiOBP) odorant binding proteins.

<p>A. The <i>S. invicta</i> MAP phylogeny. The branch in grey is collapsed in the 50% consensus tree. Branch support is posterior probabilities derived from 3241 samples taken after the burn-in was discarded. Even though the node support in the conserved ortholog clade is relatively poor, the exact same topology of the orthologs was recovered in the honey bee MAP tree (not shown), suggesting that the branching pattern is accurate. B. The <i>S. invicta</i> MAP-AU plot. The quality of the alignment is indicated through a heat map. Red (warm colors) indicates areas of high quality alignment, blue (cold colors) signifies areas of low certainty. Note that there are considerable high quality alignment blocks in the N-terminal signal peptide and the C-terminal protein tail.</p

Maximum likelihood phylogenies of the fire ant OBPs (SiOBPs) and honey bee OBPs (AmOBPs).

<p>The phylogenies are based on the two best alignments (top: MUSCLE, bottom: PRANK). All trees are midpoint rooted in the absence of a suitable outgroup. Branch support is SH-like aLRT derived from PhyML analyses.</p

Results of the selection analyses for the best alignment method (PRANK), and two others (CLUSTAL and MUSCLE) and the MAP dataset of <i>Solenopsis</i>.

<p>Given are the log-likelihoods of the null hypotheses (H₀), which assume no selection, and alternative hypotheses (H_a), which allow for positive selection. Likelihood-ratio tests (LRT) of positive selection are conducted to compare the two hypotheses. Levels of significance are 3.84 at 5% and 6.63 at 1% for the site model and 3.84 at 5% and 5.99 at 1% for the branch and branch-site models, following the χ₁² distribution to guide against violations of model assumptions. Statistically significant LRTs for positive selection are indicated by italics and *** for p≪0.01. Note that inference of positive selection greatly depends on the alignment method used.</p

Genomic organization of MdBV proviral segment loci.

<p>The upper part of the figure presents the eight proviral loci identified (L1–L8) and the corresponding <i>M. demolitor</i> genome scaffolds where they are located. Only the portion of the scaffold where the proviral locus resides is shown. For each locus, the upper scale bar in kilobases (k) names the scaffold(s). Below the scale bar, colored bars indicate the segments identified by deep sequencing DNA from MdBV virions (Encapsidated segments) and their corresponding orientation and location in a given locus as a proviral segment in the <i>M. demolitor</i> genome. Average coverage in thousands of reads is indicated above each segment in brackets, while below each segment is shown read coverage per nucleotide relative to the scale indicated to the right of the graph. Gaps in read coverage indicate regions flanking individual proviral segments that are not amplified, excised and packaged into virions. The gap seen in segment S (L6) is due to a region of N's in the reference sequence. Below each MdBV proviral segment is shown predicted genes in forward (black) and reverse (white) orientation. Individual introns, exons and untranslated regions are not shown. The lower part of the figure shows each scaffold containing MdBV proviral loci in their entirety. Scaffolds are drawn to scale and organized from largest (L3) to smallest (L5). Scale bar is in megabases (M).</p

Wasp Integration Motifs (WIMs) for all MdBV proviral segments.

<p>(A) Alignment of 200 nucleotides (nt) surrounding the 5′ WIM site of each segment with similarity for each site colored in shades of blue. Red box highlights the tetramer AGCT. 31 nt are conserved in each proviral segment following the AGCT motif (red box), while the 5′ flanking region upstream of this motif is AT rich and not well conserved. (B) Alignment of 200 nt surrounding the 3′ WIM site for proviral segments shows that the flanking region preceding the WIM site is AT rich but not conserved, whereas the first 100 nt of each proviral segment downstream of the WIM site shows high conservation. Maximum likelihood analyses indicated that the relationships between segments for the WIM sites (A) and 3′ flanking regions (B) cannot be resolved.</p

Distribution of nudivirus-like genes in loosely associated clusters in the <i>M. demolitor</i> genome.

<p>Five scaffolds containing nudivirus-like genes arranged from largest (1.6 Mb) to smallest (660 Kb) are shown in in gray. Named nudivirus-like genes (red) are separated by large stretches of DNA containing predicted <i>M. demolitor</i> genes (white). Wasp genes are depicted without indicating introns, exons, and untranslated regions.</p

Relatedness of Host Integration Motif (HIM) sequences for all MdBV proviral segments.

<p>Alignment of HIM sequences with similarity for each site colored in shades of blue. Maximum likelihood analysis shows that three segment groups contain HIMs that form monophyletic groups (segments N and J; segments P, K1 and K; segments A and B) but other relationships could not be resolved.</p

Two genomic regions flanking proviral segments are conserved among BV-carrying wasps.

<p>(A) The area surrounding segments N and J in <i>M. demolitor</i> Locus 3 is homologous to the regions surrounding segment 25 in Locus 5 of <i>G. flavicoxis</i> and <i>G. indiensis</i> and segment 1 in Locus 5 of <i>C. congregata.</i><i>M. demolitor</i> loci are oriented to match the orientation of sequence scaffolds and similarity between flanking regions reported in prior studies. Although the genes in the proviral segments themselves are usually not recognizably homologous (black background with genes in white), the regions flanking these proviral segments (gray background with genes in white) are conserved as indicated by regions of synteny shaded in light blue. To the right of the figure is shown the phylogenetic relationships between these wasp species and estimated divergence times. (B) The downstream region flanking segment P in <i>M. demolitor</i> Locus 1 is similar to the region between Locus 1 and 2 in <i>G. flavicoxis, G. indiensis, C. congregata</i> and <i>C. sesamiae Kitale</i>. Genes and segments are depicted as in (A) except for <i>odv-e66-like1</i> homologs are indicated in red in the <i>Glyptapanteles</i> and <i>Cotesia</i> genomes. Although the <i>odv-e66</i> homologs are located in an area of synteny, no <i>odv-e66</i> homologs exist in this region for <i>M. demolitor</i>. Accession numbers for sequences are MdL3 (KK043340), GiL5 (EF710656), GfL5 (EF710650), CcL5 (HF586476), MdL1 (KK044729), GfL1 (EF710644), GiL1 (EF710657), GiL2 (AC191960), CcL2 (HF586473), CcL1 (HF586472), CsL2 (EF710635), CsL1 (EF710629).</p