Field sampling
Between 1998 and 2003 ripe syconia were collected from F. rubiginosa trees growing at many disparate locations throughout its natural range. Wasps were allowed to emerge naturally from their syconia and were then stored in 95% ethanol at -20C. A single collection tube comprised wasps from syconia from the same tree on the same day. We later conducted genetic analyses on 1–5 female wasps from each sample tube.
The head from each insect was removed and retained as a voucher specimen, and DNA was extracted from the remaining body parts using a simple Chelex extraction procedure [46]. We amplified a 444 bp fragment of mitochondrial cytochrome b (cytb) for all wasps using the primers CB1 and CB2 [47]. For selected individuals (see results), we also amplified a region of the nuclear 28S rRNA gene (28S), using primers D1F and D3R [33,48,49] as well as the wingless (wg) gene, using primers LepWG1 and LepWG2 [50]. All wasps were also screened for Wolbachia infection by PCR, employing the primers wsp81F and wsp 691R, which amplify part of the Wolbachia surface protein gene (wsp) [51].
Amplification of cytb was performed using a GeneAmp 2400 machine (Perkin-Elmer Cetus) with 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 1 min at 45°C, 1 min 30 s at 72°C, and a final elongation step of 7 min at 72°C. We increased the annealing temperature for the other gene fragments as follows: 55°C for wsp, 50°C for 28S and 58°C for wg. The sizes of PCR products were 444 bp (cytb), 564–588 bp (wsp), ~1,060 bp (28S), and 433 bp (wg). Ten microlitres of each PCR product was electrophoresed through a 1% agarose gel to determine amplicon size, and the gel band was excised for purification using a GFX DNA Purification Kit (Amersham Pharmacia Biotech Inc). We then sequenced fig wasp genes (cytb, 28S and wg) directly using the same primers employed in PCR.
For wsp, PCR products were first sequenced directly and, if direct sequencing failed three times (or repeatedly generated sequences with multiple peaks), the PCR product was cloned and 6–10 different clones were sequenced to test for the presence of multiple Wolbachia strains. We ligated each PCR product into a T-tailed vector (pGEM-T Easy Vector system, Promega Ltd.) and transformed into E. coli JM109. Positive colonies were selected and plasmid DNA was purified using a GFX Micro Plasmid Prep Kit (Amersham Pharmacia Biotech Inc) and the wsp inserts were sequenced using M13 vector primers. In all cases, we sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Perkin Elmer Inc.) and an ABI PRISM 3700 DNA Analyzer (Perkin Elmer Inc). All isolates were sequenced fully in both directions, and sequences have been deposited in GenBank: cytb [GenBank: AY567594–AY567638], 28S [GenBank: AY567639–AY567660] and wg [GenBank: DQ539361–DQ539391].
Sequence alignment and phylogenetic analysis
Sequences were edited and aligned using Sequencher™ (Gene Codes Corporation), and final adjustments to 28S and wsp alignments were made by eye, following previous alignments (wsp: Shoemaker et al. 2002; 28S: Lopez-Vaamonde et al. 2001).
Cytb phylogenies were estimated using Maximum Parsimony (MP) in PAUP* version 4.0b10 [52], and Bayesian methods in MRBAYES version 3.0 [53]. For MP analyses, we conducted an initial heuristic search with 10,000 random additions and TBR branch swapping, holding one tree per replicate. Trees generated by the initial search were then used as starting trees for a second heuristic search, in which multiple trees were saved. We assessed clade support using 1000 bootstrap replicates. For Bayesian analyses, the most appropriate model of nucleotide substitution was determined, using MrModeltest v2.2 [54], to be the general time reversible model (nst = 6). The analyses were run for 106 generations, with one tree retained every 100 generations. Likelihood stationarity occurred after 2.5 × 105 generations, and this "burn-in" period was excluded before creating a 50% majority-rule consensus tree in PAUP*.
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