Deep mtDNA divergences within "P. imperialis"
We have demonstrated that P. imperialis wasps fall into four major mtDNA clades. These differ from each other by 9–17% nucleotides and are supported strongly by both MP and Bayesian phylogenetic analyses (Fig. 2). These deep divergences are very similar to those (10–26%) found between morphologically distinct members of the same genus [33]. The mtDNA genetic distances between our clades are also slightly higher than those reported between cryptic species of Pegoscapus fig-pollinating wasps in Panama [14]. These deep divergences suggest strongly the existence of four cryptic species within the morphologically defined P. imperialis.
We also showed that all wasps harboured Wolbachia bacteria and that clade 3 wasps had a different infection to all other wasps. Wolbachia can influence both the diversity and evolution of mtDNA and can maintain mtDNA divergences within or between populations of a species [36]. The best-studied example is Drosophila simulans which has three distinct haplotypes [37] and is infected by at least five strains of Wolbachia [38-43]. However, in this and other cases where populations are polymorphic for Wolbachia infection (e.g. the gall wasp Biorhiza pallida [44]) the genetic distances between different haplotypes (clades) are very much lower than we report here. Nevertheless, given the very high incidence of Wolbachia infections in fig-pollinating wasps [26,27], these endosymbionts have probably played a role in host mtDNA evolution and reduce confidence in the applicability of a general mtDNA clock.
Evidence against a role for host shifts
Two wasp species may co-occur on a single fig species if one of them has shifted from another host fig. Indeed, some mismatches between figs and wasps at deep phylogenetic levels suggest that host shifts have occurred at times during their coevolutionary history [9,15]. In addition, there are cases of extant co-pollinators that are not closely related species, suggesting more recent host shifts (e.g. [14,34]. However, despite high genetic distances between the four cytb clades, all three wasp genes sequenced support the monophyly of the P. imperialis complex relative to other Pleistodontes species. Consequently, it seems unlikely that any of our four clades has shifted to F. rubiginosa from another Ficus species. In addition, we have not recorded P. imperialis from another fig species, nor detected mitochondrial haplotype groups shared by Pleistodontes wasps from different fig species ([33] and further unpublished data).
Evidence against parallel divergences of F. rubiginosa and P. imperialis
Alternatively, the F. rubiginosa/P. imperialis species pair could be in the process of cospeciation, with the partner species diverging together. However, our existing data do not support this notion and it is also difficult to envisage how the fig species might split simultaneously into four genetic units. We found that in most areas, and even some individual trees, two or more wasp clades were present (Table 1), despite limited within-site sampling. Furthermore, as part of a long-term survey in Townsville, we have found that most F. rubiginosa syconia are entered by more than one foundress and that yellow (clade 2) and black wasps (clades 3 or 4 at this site) co-enter about 20% of syconia (53/198 figs from 10 different trees over 2 years). These results suggest that there is no simple segregation of wasp clades between different host trees and that they co-occur regularly in the same trees and even fruits, as reported in Panama for cryptic species of Pegoscapus fig-pollinating wasps [14,17].
One possibility is that the different wasp clades predominate in different habitats and that the fig species is diverging into habitat-specific races. For example, in West Africa Ficus ottonifolia occurs in a mosaic of forest and open habitats and has two pollinators. These co-occur locally, but one predominates in open habitat patches and the other in forest patches [12,45]. F. rubiginosa is also found in both forest and open habitats and further sampling might reveal a similar pattern. However, we believe that this is unlikely as, for logistical reasons, almost all of our samples are from open habitats.
A further possibility is that the fig is diverging into two or more races that are not habitat-specific. Indeed, Dixon et al. (2001) recognised two forms of F. rubiginosa (see background). Most of our sampling was performed before the description of these two forms, so we cannot yet compare pollinators between the different forms. However, most of our samples came from form rubiginosa, arguing against a simple split by form. In addition, the only taxonomic character that separates the two forms is the presence of hairs on the leaves, which may well be a simple polymorphism that has no connection with the pollinators. Finally, even if there is segregation by fig form, this can only provide a partial answer since there are four wasp clades, but only two fig forms. Clearly, genetic studies of the figs are needed to test ideas further. However, we note that recent genetic studies of wasps have revealed cryptic species [14], but genetic studies of the corresponding figs have not [8,15].
Wasp divergence without direct involvement of host plant
We argue above that there is no good evidence for either parallel divergence of F. rubiginosa and P. imperialis, or for recent host shifts by wasps. Fig wasp divergence might instead occur following the development of spatial or temporal barriers within a single wasp species. For example, temporary geographic isolation of wasp (and fig) populations could occur for periods of time that allow the evolution of reproductive isolation in the wasps, but not the figs. Selection and/or genetic drift may be involved and the approximately 100 times faster generation time of the insects may facilitate their population divergences [11]. Many scenarios are possible, but we suggest that the role of Wolbachia deserves further study, since we have detected infection differences and the acquisition of different Wolbachia infections in isolated populations can facilitate or even cause speciation [23-25].
Correspondence of other markers with mtDNA clades
There is very little variation in the slower-evolving nuclear genes studied. However, 28S sequences split the wasps into two clades that correspond to cytb clades 1–3 and clade 4, while wg also splits them into two groups, but corresponding to cytb clade 3 and clades 1, 2 and 4. Cytb clade 3 stands out further by differing in its Wolbachia infection status. Consequently, three markers support isolation of clade 3 from the others, and clade 4 differs from all others on the basis of variation in 28S. Clades 1 and 2 differ strongly in mtDNA, but not in the other genes studied. However, clade 2 contains only (and all) the yellow wasps sampled, while all other wasps are black and this effectively provides a nuclear marker supporting isolation of clade 2.
Further resolution of gene flow in the "P. imperialis complex" now requires data from from substantial numbers of wasps representing the different clades. Given the limited variability of the nuclear sequences studied here, a population genetic approach, using microsatellites, may be most appropriate.
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