Phylogenetic patterns of Cytochrome B variation
We analysed 71 P. imperialis wasps from 26 different F. rubiginosa trees, representing 18 sites in Australia and one introduced population in the USA (Table 1, Fig. 1). We found 44 unique cytb haplotypes, 444 bp in length, of which 139 (31.1%) nucleotide sites were polymorphic and 107 (24.1%) parsimony-informative.
Forty-four P. imperialis sequences were used in phylogenetic analyses, together with sequences from 14 other Pleistodontes species [33] and four Ceratosolen fig-pollinating wasp species [34] as outgroups. The MP (not shown) and Bayesian phylogenies (Fig. 2) had very similar topologies, both supporting the monophyly of P. imperialis, but dividing the species into four deep mitochondrial clades. The (GTR) pairwise genetic distances between taxa fell into two distinct groups: 1) distances within each of the four P. imperialis clades (0–7%); and 2) distances between P. imperialis clades (9–17%) and between morphologically distinguishable Pleistodontes species (10–26%). Consequently, the large genetic distances between the four P. imperialis clades are similar to those between morphologically distinct Pleistodontes and indicate cryptic species. There was one divergent P. imperialis sample, from which three wasps grouped only weakly with the rest of clade 1 (Figure 2). Based upon the commonly used mitochondrial DNA (mtDNA) clock rate of 2.3% pairwise divergence/Myr [35], clades 1 and 2 diverged from clades 3 and 4 at least 7.1 million years ago (MYA), clades 3 and 4 split 5.6 MYA and clades 1 and 2 split 4.4 MYA
Geographic distribution of Cytochrome B clades
The four cytb clades show different geographic distributions (Fig. 1). Clade 1 was found throughout the natural range (Queensland (except Townsville) and New South Wales (NSW)) of P. imperialis and was also the only clade found outside the native range (in S. Australia, Victoria and USA). Clade 2 was found only in the Townsville area of Northern Queensland and contained all the yellow wasps that are also restricted to this area. Clade 3 wasps were found only in N. Queensland, while clade 4 was found in N. and S. Queensland, but not in NSW. Overall, clade diversity appears to decrease from North to South (Fig. 1).
There is not, however, simple geographic replacement of clades, as most sites in Queensland have two or more clades present. For example, wasps collected from just five trees near Townsville represent three of the four clades (Table 1). In most cases, all individuals from one fig tree belonged to the same clade. However, in three cases individuals from the same tree belonged to two different clades (Table 1). Given that only 3–5 wasps were sampled per fig tree, this suggests the frequent occurrence of wasps from two or more clades in a single crop of fig fruits.
Wolbachia infections
All 71 wasps harboured Wolbachia, but some carried one strain and others two. We obtained wsp sequences from 27 insects, revealing 6 with single and 21 with double infections (Table 1). The wsp sequences all belong to the Wolbachia A-clade, and revealed three strains (W1, W2 and W3). W1 and W2 strains differed by a single synonymous A/G substitution at position 268, while there was 8.5% nucleotide divergence, including a 3 bp and a 21 bp indel between W2 and W3. There was a highly consistent pattern between Wolbachia infection status and cytb clades. Wasps in clade 3 had a single infection with W1, while wasps from the other three clades all had both the W2 and W3 infections. The single exception was one clade 2 wasp that harboured only strain W2.
Nuclear sequence variation
We sequenced nuclear 28S (1033 bp) and wg (433 bp) DNA fragments for 22 wasps representing the four cytb clades (Table 1). The 28S data revealed two weakly supported clades (28S.I and 28S.II) that differed from each other by 4–8 nucleotide substitutions, while there was a maximum of 2 base differences between wasps within the two clades (Fig. 3). 28S.II contains individuals from cytb clade 4 only, while 28S.I individuals fall into cytb clades 1–3 (Table 2). Wg also divided the wasps into two clades (wgA and wgB) and these differed from each other by a single nucleotide substitution at a synonymous site (position 175) (0.23% nucleotide divergence). Group wgA contained individuals from cytochrome b clade 3 only, while wgB individuals belonged to clades 1, 2 and 4 (Table 2). To place this in context, we also sequenced wg from eight outgroup taxa: four other Pleistodontes species and four Ceratosolen species. Nucleotide divergence ranged from 1.16% to 3.23% between morphologically distinguishable Pleistodontes species and up to 16% between genera. While there is no clear association between 28S variation and Wolbachia infection, there is an association of Wolbachia with genetic divergence of wg between clade 3 (W1 infection; wgA) and clades 1, 2 and 4 (W2 and W3 infections; wgB). The overall correspondence of different markers is summarised in Table 2.
Answers to all your Biology Questions
