Eucharitidae are parasitoids of ants; adults deposit their eggs in or on vegetation and the active first-instar larvae are responsible for gaining access to the ant host (Heraty, 1994, 2002). Eucharitids are known to attack five subfamilies of ants, Myrmicinae, Formicinae, Ponerinae and Myrmeciinae, with one, probably erro- neous, record from Ecitoninae (Heraty, 2000). Because of a large number of informative morphological characters, the proposed phylogenies of Eucharitidae are fairly well resolved and concordant in different studies with different charac- ters and taxa (Heraty, 1994, 2000, 2002). The most recent analysis of morpho- logical data encompasses all of the genera (Heraty, 2002), and supports a monophyletic Oraseminae that is a sister group to the Eucharitinae, which is comprised of the monophyletic Psilocharitini and Eucharitini (Fig. 3.4).
Importantly, the genera Gollumiellaand Anorasema are placed basally within the tribe Eucharitini, and another genus,Tricoryna, is included with Pseudometageain a group basal to the major radiation of genera within the tribe.
The results from a molecular analysis of the 28S-D2, 28S-D3 and 18S-E23 regions (Fig. 3.4) are almost identical to the morphological analyses (Heraty and Hawks, unpublished), but with three important differences found in both inde- pendent and combined analyses of the three gene regions: (i) Gollumiella and Anorasemaare highly supported (bootstrap values of 100%) as the sister group of Oraseminae and Eucharitinae; (ii) Psilocharitini are a paraphyletic group (boot- strap support of 80%); and (iii) Tricoryna, which are parasitoids of Rhytidoponera (large Ponerinae in Australia), is consistently placed as the sister group of Austeucharis, which are parasitoids of Myrmecia (Myrmeciinae; in Australia) and nested within a group that are all parasitic on large ponerine ants of the tribes Ectatommini or Ponerini, or Myrmeciinae (morphologically and behaviourally similar to large Ponerinae) (Heraty, 2002). A re-evaluation of morphological data led to the discovery of a unique ovipositor bulb in Tricorynaand Austeucharis, which places the two as sister taxa, but inclusion of this character in the morphological analyses did not change the position of this group. Morphological features were found that supported the monophyly of Gollumiellaand Anorasema, but not their exclusion from Eucharitinae. There is no morphological support for paraphyly of Psilocharitini.
Why are these differences relevant? Gollumiella and the eucharitine genus, Pseudometagea, which is placed unequivocally within Eucharitinae in both analyses (Fig. 3.4), are both parasitoids of members of the tribe Lasiini within the ant sub- family Formicinae. This places Lasiini as the potential ancestral host for Eucharitidae. Under the morphological hypothesis, the ancestral host could be Myrmicinae, Ponerinae (host for Neolosbanus and other derived Eucharitini) or Formicinae. Furthermore, internal parasitism of the host ant larva by the
54 J. Heraty
Molecular Systematics55
Fig. 3.4.Comparative phylogenies of Eucharitidae based on morphology (Heraty, 2002) and a parsimony analysis of the 28S-D2, -D3 and 18S-E23 gene regions (Heraty and Hawks, unpublished). Bootstrap values are indicated above branches. Ant hosts that are Formicinae (open boxes) or Ponerinae (black boxes) are indicated.
first-instar eucharitid larva occurs in Gollumiella, Oraseminae and Pseudometagea, but not Neolosbanusor other Eucharitini in which all larval stages are external par- asites. The molecular hypothesis supports internal parasitism as an ancestral behaviour, whereas in the morphological hypothesis it is equivocal and either internal or external parasitism is possible.
With regard to the Psilocharitini, if the group is monophyletic, as suggested by the morphological hypothesis, then parasitism of small Ponerinae and exter- nal parasitism of the host ant larvae, as found in Neolosbanus, would be predicted for Psilocharis. However, no such assumptions can be made under the molecular hypothesis for Psilocharis. Finally, the new placement of Tricoryna in the group which is parasitic on large Ponerinae makes greater biological sense and leads to a single host shift in the common ancestor of this group, as compared with two independent acquisitions of a large ponerine host. When strongly supported by multiple genes, different analytical procedures, adequate taxonomic sampling, and after comparison with traditional morphology-based phylogenies, molecular hypotheses can be used to examine evolutionary change in a new perspective. It cannot, however, be done without first adopting a pessimistic view in which the molecular data are assumed to be wrong.
Encarsia: Unchallenged Trees and the Interpretation of Change
Species of Encarsia(Aphelinidae) are a diverse group of minute wasps, less than 2 mm in size. Immature stages usually develop as endoparasitoids of whiteflies and armoured scales, and perhaps less commonly in immatures of Hormaphididae, themselves, or eggs of Lepidoptera (Williams and Polaszek, 1996; Hunter and Woolley, 2001).Encarsiais one of the most important parasitic groups being exploited in biological control, and various species are currently being collected as part of foreign exploration efforts to search for biological control agents (Noyes and Hayat, 1994). Many species have attributes that allow them to be placed into discrete groups. These species groups are often defined by combinations of characters, many of which are characteristic of one or more species placed in other species groups. Importantly, these loosely defined groups are our first approximation of the phylogenetic relationships of species. However, species grouped arbitrarily on the basis of overall similarity can lead to miscon- ceptions about behaviour and host associations. If we can prove that their defin- ing features and behavioural traits have an evolutionary basis, then we can enhance our ability to place, and rapidly evaluate, the potential effectiveness of new species for use in biological control programmes.
Within Encarsia, 25 species groups are currently recognized, with 60 of the 273 described species unplaced (Heraty and Woolley, 2002). Because of their small size and general reduction or loss of morphological features, these groups are difficult to characterize on morphological data alone (Babcock et al., 2001).
56 J. Heraty
What appear to be obvious group characteristics can be found in unrelated groups of species; for example, the close placement of scutellar sensilla, which were considered diagnostic of the strenua group, are now known to be conver- gent and found in several very unrelated species groups (Heraty and Polaszek, 2000). Even the obvious characteristic of a reduction in number of tarsomeres from five to four was regarded as a poor character for defining species groups by Hayat (1998). Various analyses of morphological characters have led to differing opinions regarding the relationships, composition and placement of species into groups of Encarsia(Hayat, 1998; Huang and Polaszek, 1998). Trying to analyse these morphological traits within a phylogenetic framework yields little resolution of relationships (Babcock et al., 2001).
The relationships of species within Encarsia were analysed in two papers using 28S-D2 rDNA (Babcock et al., 2001; Manzari et al., 2002). The species in the two data sets were re-analysed along with new sequence data for a total of 31 species of Encarsia and two outgroup genera (Fig. 3.5; Heraty et al., 2003).
Parsimony analysis resulted in only three competing tree topologies, with support for the inaron, luteola and strenua species groups, but not the parvella species group. The relationships between groups is not highly supported, and one of the outgroup genera,Encarsiella, occurs within Encarsia. The inclusion of this genus within Encarsiais strongly supported, with only an extra 3 steps required to force Encarsiato be monophyletic. These results were supported in the earlier studies (Babcock et al., 2001; Manzari et al., 2002). This odd generic placement could be an artefact of the 28S-D2 gene region (Fig. 3.5); however, studies with other genes (ITS2, COI) also support these conclusions. Quite possibly, the minor mor- phological differences between groups of Encarsia are representative of deeper phylogenetic differences that have not been recognized by taxonomists. Clearly, with only 31 of 273 described species represented in the molecular analyses, it is too early to make any formal conclusions from the data.
Each of the three studies reaches the same general conclusions, and if we have faith in the strongly supported species groups, then we can use these results to examine certain morphological and behavioural features for their ability to define monophyletic groups. The close placement of scutellar sensillae is present in all members of the strenua group and its putative sister group,Encarsia querci- cola(Se; Fig. 3.5). A reduction to a four-segmented tarsus is found in all members of the luteola group, and separately in Encarsia nigricephalaof the cubensis group (4; Fig. 3.5). Based on whitefly parasitism, which is characteristic of the outgroup taxa, a shift to parasitism of Diaspididae was derived, possibly as many as three times (D; Fig. 3.5). There does not appear to be any phylogenetic component to the parasitism of various whitefly genera (Bemisia [B], Trialeurodes [T], other whitefly genera [Ot]; Fig. 3.5). The association of thelytoky (Th) with sex-ratio- distorting bacteria (Encarsiabacterium [EB] and Wolbachia[W]; Fig. 3.5) also does not have a phylogenetic component, although Wolbachia is known only in E.
formosa (Zchori-Fein et al., 2001). In these cases, the lack of phylogenetic con- straint affects how we approach our understanding of the results. For example, if the EB bacteria are associated with different lineages of Encarsia, are the EB
Molecular Systematics 57
58 J. Heraty
Fig. 3.5.Strict consensus of three trees of 844 steps recovered from a parsimony analysis of 28S-D2 rDNA from 31 species of Encarsiaand two closely related genera (Coccophagoidesand Encarsiella) (Heraty et al., 2003). Data were analysed with PAUP4.0*b9 using 100 random addition sequences and TBR branch swapping. Bootstrap proportions greater than 50% are shown above branches.
The same results were obtained when additional populations, as identified by the numbers in parentheses, were added for a total of 80 terminal taxa (sequences from Babcock et al. (2001) and Manzari et al. (2002); some names corrected from Schmidt et al. (2001)), but with five trees of 893 steps. These were identical to the three trees after pruning out the extra populations. Both analyses were stable to successive approximations character weighting (Babcock et al., 2001).
Behavioural attributes indicated: Se = scutellar sensillae closely placed; 4 = mid- tarsus four-segmented; D = Diaspididae host; B = Bemisiahost; T = Trialeurodes vaporariorumhost; Ot = other whitefly host; S = symbiotic association (EB = Encarsiabacterium (eb is variable within species); W = Wolbachia; x = tested but none found (Zchori-Fein et al., 2001)); Th = parthenogenetic species.
lineages concordant, indicating coevolution, or random, indicating horizontal transfer?