Intragenomic internal transcribed spacer 2 variation in a genus of parasitoid wasps (Hymenoptera: Braconidae): implications for accurate species delimitation and phylogenetic analysis.

A recent DNA barcoding study of Australian microgastrines (Hymenoptera: Braconidae) sought to use next-generation sequencing of the cytochrome c oxidase subunit 1 (COI) barcoding gene region, the wingless (WG) gene and the internal transcribed spacer 2 (ITS2) to delimit molecular species in a highly diverse group of parasitic wasps. Large intragenomic distances between ITS2 variants, often larger than the average interspecific variation, caused difficulties in using ITS2 for species delimitation in both threshold and tree-based approaches, and the gene was not included in the reported results of the previous DNA barcoding study. We here report on the intragenomic, and the intra- and interspecies, variation in ITS2in the microgastrine genus Diolcogasterto further investigate the value of ITS2as a marker for species delimitation and phylogenetics of the Microgastrinae. Distinctive intragenomic variant patterns were found in different species of Diolcogaster, with some species possessing a single major variant, and others possessing many divergent variants. Characterizing intragenomic variation of ITS2is critical as it is a widely used marker in hymenopteran phylogenetics and species delimitation, and large intragenomic distances such as those found in this study may obscure phylogenetic signal.

Response to host density by the parasitoid Dolichogenidea tasmanica (Hymenoptera: Braconidae) and the influence of grapevine variety.

Natural enemies that respond to prey in a density-dependent manner may be able to quickly suppress pest populations before they reach economically damaging levels. Although it is primarily the combination of a natural enemy's functional response and a population numerical response that will influence the maximum number of pests attacked, other factors may influence a density-dependent response. We conducted large-scale field experiments, both artificially inoculating grapevines with larvae and using naturally occurring populations, to quantify and characterize the response of a parasitoid, Dolichogenidea tasmanica (Cameron) (Hymenoptera: Braconidae) to different densities of its host, the pest of grapevines, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). We showed that the response of D. tasmanica to the density of E. postvittana was inversely density-dependent, and that the degree of parasitism was consistently and significantly higher in the grape variety Cabernet Sauvignon compared with Chardonnay. While the significant effect of variety on the degree of parasitism may provide an option for increasing the parasitism of E. postvittana by D. tasmanica, it also highlights how differences in host plant can influence trophic interactions.

Extraordinary micro-endemism in Australian desert spring amphipods.

Increasing pressure for water in the Australian arid zone is placing enormous stress on the diverse endemic communities inhabiting desert springs. Detailed information about the evolutionary processes occurring within and between individual endemic species will help to develop effective and biologically relevant management strategies this fragile ecosystem. To help determine conservation priorities, we documented the genetic structure of the endemic freshwater amphipod populations in springs fed by the Great Artesian Basin in central Australia. Phylogenetic and phylogeographic history and genetic diversity measures were examined using nuclear and mitochondrial DNA from approximately 500 chiltoniid amphipods across an entire group of springs. Pronounced genetic diversity was identified, demonstrating that levels of endemism have been grossly underestimated in these amphipods. Using the GMYC model, 13 genetically divergent lineages were recognized as Evolutionarily Significant Units (ESUs), all of which could be considered as separate species. The results show that due to the highly fragmented ecosystem, these taxa have highly restricted distributions. Many of the identified ESUs are endemic to a very small number of already degraded springs, with the rarest existing in single springs. Despite their extraordinarily small ranges, most ESUs showed relative demographic stability and high levels of genetic diversity, and genetic diversity was not directly linked to habitat extent. The relatively robust genetic health of ESUs does not preclude them from endangerment, as their limited distributions ensure they will be highly vulnerable to future water extraction.

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment.

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km(2) region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.

What lies beneath: molecular phylogenetics and ancestral state reconstruction of the ancient subterranean Australian Parabathynellidae (Syncarida, Crustacea).

The crustacean family Parabathynellidae is an ancient and significant faunal component of subterranean ecosystems. Molecular data were generated in order to examine phylogenetic relationships amongst Australian genera and assess the species diversity of this group within Australia. We also used the resultant phylogenetic framework, in combination with an ancestral state reconstruction (ASR) analysis, to explore the evolution of two key morphological characters (number of segments of the first and second antennae), previously used to define genera, and assess the oligomerization principle (i.e. serial appendage reduction over time), which is commonly invoked in crustacean systematics. The ASR approach also allowed an assessment of whether there has been convergent evolution of appendage numbers during the evolution of Australian parabathynellids. Sequence data from the mtDNA COI and nDNA 18S rRNA genes were obtained from 32 parabathynellid species (100% of described genera and ~25% of described species) from key groundwater regions across Australia. Phylogenetic analyses revealed that species of each known genus, defined by traditional morphological methods, were monophyletic, suggesting that the commonly used generic characters are robust for defining distinct evolutionary lineages. Additionally, ancestral state reconstruction analysis provided evidence for multiple cases of convergent evolution for the two morphological characters evaluated, suggesting that caution needs to be shown when using these characters for elucidating phylogenetic relationships, particularly when there are few morphological characters available for reconstructing relationships. The ancestral state analysis contradicted the conventional view of parabathynellid evolution, which assumes that more simplified taxa (i.e. those with fewer-segmented appendages and setae) are derived and more complex taxa are primitive.

Evidence for population fragmentation within a subterranean aquatic habitat in the Western Australian desert.

The evolution of subterranean animals following multiple colonisation events from the surface has been well documented, but few studies have investigated the potential for species diversification within cavernicolous habitats. Isolated calcrete (carbonate) aquifers in central Western Australia have been shown to contain diverse assemblages of aquatic subterranean invertebrate species (stygofauna) and to offer a unique model system for exploring the mechanisms of speciation in subterranean ecosystems. In this paper, we investigated the hypothesis that microallopatric speciation processes (fragmentation and isolation by distance (IBD)) occur within calcretes using a comparative phylogeographic study of three stygobiontic diving beetle species, one amphipod species and a lineage of isopods. Specimens were sequenced for the mitochondrial cytochrome c oxidase 1 gene from three main sites: Quandong Well, Shady Well (SW) and Mt. Windarra (MW), spanning a 15 km region of the Laverton Downs Calcrete. Phylogenetic and haplotype network analyses revealed that each species possessed a single divergent clade of haplotypes that were present only at the southern MW site, despite the existence of other haplotypes at MW that were shared with SW. IBD between MW and SW was evident, but the common phylogeographic pattern most likely resulted from fragmentation, possibly by a salt lake adjacent to MW. These findings suggest that microallopatric speciation within calcretes may be a significant diversifying force, although the proportion of stygofauna species that may have resulted from in situ speciation in this system remains to be determined.

Fine-scale comparative phylogeography of a sympatric sister species triplet of subterranean diving beetles from a single calcrete aquifer in Western Australia.

Calcrete aquifers in the arid Yilgarn region of central Western Australia are a biodiversity hotspot for stygofauna. A distinct pattern of interspecific size class variation among subterranean dytiscid beetle species has been observed in 29 of these aquifers where either two or three small, medium and/or large sympatric species are found that are in some cases sister species. We used a 3.5 km(2) grid of bores to sample dytiscids on a fine-scale and employed a comparative phylogeographical and population genetic approach to investigate the origins of a sympatric sister species triplet of diving beetles from a single aquifer. Mitochondrial DNA sequence data from the Cytochrome oxidase c subunit I gene revealed that all three species have high levels of haplotype diversity with ancient (approximately 1 million years ago) intra-specific coalescence of haplotypes, but low levels of nucleotide diversity. Population analyses provide evidence for multiple expansion events within each species. There was spatial heterogeneity in the distribution of genetic variation and abundance both within and among the three taxa. Population analyses revealed significant fine-scale differentiation with isolation by distance for Paroster macrosturtensis and P. mesosturtensis, but not the smallest species P. microsturtensis. Haplotype network analyses provided limited or no evidence for past population fragmentation within the large and small species, but substantial historical divergence was observed in P. mesosturtensis that was not spatially structured. A patchy population structure with contemporaneous and historical isolation by distance in the three species is likely to have been a significant isolating and diversifying force, preventing us from ruling out a potential role for allopatric divergence during speciation of this beetle sister triplet.

Estimating ancestral geographical distributions: a Gondwanan origin for aphid parasitoids?

We tested the published hypothesis of a Gondwanan origin for the overwhelmingly northern hemisphere aphid parasitoids (Aphidiinae) as follows: (i) finding their sister group by a phylogenetic analysis of the entire Braconidae (Insecta: Hymenopterai using sequence data from approximately 500 bp fragments of both the nuclear 28S (D2 region) and mitochondrial 16S rDNA genes, (ii) using this sister-group relationship and the more informative 28S D2 gene to estimate the phylogeny of the Aphidiinae and (iii) estimating the ancestral distribution for the Aphidiinae using maximum-likelihood and maximum-parsimony methods. Both methods indicated a Gondwanan origin.

Seed viability in declining populations of Caladenia rigida (Orchidaceae): are small populations doomed?

Despite comparatively good rates of pollination and seed production, some populations of the endangered terrestrial orchid Caladenia rigida continue to decline. To determine whether seed quality may be limiting reproductive potential, we assessed seed viability among declining populations of C. rigida (in the southern part of its distribution) and among populations that are regarded as stable (in the northern part of its distribution). We also compared differences in seed viability to plant traits, population size and habitat characteristics (soil properties, canopy cover, presence of proximate vegetation). Seed capsules from southern populations were significantly smaller, with only 9% of seeds being viable, compared to 36% in capsules from northern populations. Soil phosphorus concentrations differed between regions, but other habitat characteristics did not correlate with seed viability. Using calculations based on seedling recruitment data from other Caladenia species, we predict that seed output is insufficient to ensure the long-term persistence of the smallest C. rigida populations.

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone.

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.

Systematics, evolution, and biology of scelionid and platygastrid wasps.

The Platygastroidea comprises two families of parasitoids, Scelionidae and Platygastridae, and nearly 4500 described species. They parasitize a diverse array of insects as well as spiders. Idiobiont endoparasitism of eggs is the putative ground plan biology, as reflected by all scelionids, but most Platygastridae are koinobiont endoparasitoids of immature Auchenorrhyncha, Sternorrhyncha, and Cecidomyiidae. The superfamily is demonstrably monophyletic but its phylogenetic position remains uncertain. Relationships within the Platygastroidea are also poorly known and the group is in need of comprehensive phylogenetic study. Significant information is available on host relationships and biology, although much of this is biased to a few genera of Telenominae that are employed as biocontrol agents. Hosts for many genera are unknown, in particular those that inhabit leaf litter or parasitize solitary host eggs. The Trissolcus basalis-Nezara viridula parasitoid-host association has become a favored model system in ecological, behavioral, and physiological research on insects.

A study on the level of knowledge of health matters among patients in a rural practice.

How much do patients know about everyday matters that can affect their health? A study of 200 patients attending a rural G.P. surgery was undertaken to find out. It revealed some ignorance about the link between smoking and heart disease especially among female smokers (22% for this group). It showed a great deal of ignorance about alcohol in relation to health. Patients had a poor understanding of the relative alcohol content of different beverages, diseases related to excessive drinking and safe limits of alcohol consumption.

Evidence for AT-transversion bias in wasp (Hymenoptera: Symphyta) mitochondrial genes and its implications for the origin of parasitism.

We inferred the incidence of nucleotide conversions in the COI and 16S rRNA mitochondrial genes of members of the Symphyta and basal Apocrita (Hymenoptera). Character-state reconstructions in both genes suggested that conversions between A and T (AT transversions) occurred much more frequently than any other type of change, although we cannot wholly discount an underlying transition bias. Parsimony analysis of COI nucleotide characters did not recover phylogeny; e.g., neither the Tenthredinoidea nor Apocrita were recovered as monophyletic. However, analysis of COI amino acid characters did recover these relationships, as well as others based on fossil and morphological evidence. Analysis of 16S rRNA characters also recovered these relationships providing conversions between A and T were down-weighted. Analysis of the combined data sets gave relatively strong support for various relationships, suggesting that both data sets supported similar topographies. These data sets, both separately and combined, suggested that the phytophagous Siricidae were more closely related to the predominantly parasitic Apocrita than were the ectoparasitic Orussoidea. This suggests that the wasp parasitic lifestyle did not have a single origin, unless the Siricidae have more recently reverted to phytophagy. Alternatively, parasitism evolved twice independently, once in the Orussoidea and again in the Apocrita. The latter scenario is supported by the observation that the evolution of parasitism was accompanied by a tendency for the larvae to develop inside plant tissues. Adaptations that accompanied the movement of wasps into a confined, wood-boring habitat may have preadapted them to becoming ectoparasitic.

Molecular phylogeny of the insect order Hymenoptera: apocritan relationships.

Phylogenetic relationships among the major groups of hymenopteran insects were investigated by using comparative sequence information from the mitochondrial 16S rRNA gene. The placement of the ectoparasitic Stephanidae as the sister group to the remaining Apocrita confirmed ectoparasitism as the ground plan biology for the Apocrita. Endoparasitism evolved at least eight times within the Apocrita, and the consequent association with polydnaviruses and virus-like particles evolved at least three times. The Evaniomorpha were consistently placed as basal to the remaining Apocrita but were not resolved as monophyletic. The Gasteruptiidae were resolved as the sister group to the Evaniidae, but the relationship between the Trigonalyoidea and the Evanioidea was unclear. The Proctotrupomorpha (sensu Rasnitsyn) was resolved by topology-dependent permutation tail probability (T-PTP) testing as monophyletic, with strong evidence for a sister group relationship between the Platygastroidea and the Chalcidoidea. Strong evidence was found for the monophyly of the Ichneumonomorpha (Ichneumonidae + Braconidae) and the sister-group relationship between the Aculeata (Vespomorpha) and the Ichneumonomorpha.

Increased genetic diversity in mitochondrial genes is correlated with the evolution of parasitism in the Hymenoptera.

A higher AT content and rate of mtDNA sequence divergence was found in parasitic wasps (Apocrita) compared with nonparasitic wasps (Symphyta). The compositional bias was reflected in extreme codon bias for a cytochrome oxidase I protein coding gene fragment as well as in the types of amino acid substitutions that have occurred during the evolution of this gene fragment. In some instances, compositional bias influenced the definition of a conservative amino acid change. The increased rate of mtDNA sequence evolution probably arose during the early Jurassic, coincident with the first appearance of parasitic wasps in the fossil record. Our results suggest a causal link between the rate of sequence divergence and the parasitic lifestyle.

Evolutionary dynamics of a mitochondrial rearrangement "hot spot" in the Hymenoptera.

The arrangement of tRNA genes at the junction of the cytochrome oxidase II and ATPase 8 genes was examined across a broad range of Hymenoptera. Seven distinct arrangements of tRNA genes were identified among a group of wasps that have diverged over the last 180 Myr (suborder Apocrita); many of the rearrangements represent evolutionarily independent events. Approximately equal proportions of local rearrangements, inversions, and translocations were observed, in contrast to vertebrate mitochondria, in which local rearrangements predominate. Surprisingly, homoplasy was evident among certain types of rearrangement; a reversal of the plesiomorphic gene order has arisen on three separate occasions in the Insecta, while the tRNA(H) gene has been translocated to this locus on two separate occasions. Phylogenetic analysis indicates that this gene translocation is real and is not an artifactual translocation resulting from the duplication of a resident tRNA gene followed by mutation of the anticodon. The nature of the intergenic sequences surrounding this region does not indicate that it should be especially prone to rearrangement; it does not generally have the tandem or inverted repeats that might facilitate this plasticity. Intriguingly, these findings are consistent with the view that during the evolution of the Hymenoptera, rearrangements increased at the same time that the rate of point mutations and compositional bias also increased. This association may direct investigations into mitochondrial genome plasticity in other invertebrate lineages.

Phylogenetic relationships among the microgastroid wasps (Hymenoptera: braconidae): combined analysis of 16S and 28S rDNA genes and morphological data.

Relationships among the microgastroid complex of braconid wasps were investigated using sequence data from the 16S mitochondrial rDNA and 28S (D2 expansion region) nuclear rDNA genes, as well as morphological data. Parsimony analysis of these gene fragments, both separately and combined, indicated that Neoneurus (Neoneurinae) and Ichneutes (Ichneutinae) were no more closely related to the microgastroids than were a range of helconoid taxa. Combined parsimony analysis of the microgastroids indicated the relationships ((Cardiochilinae + Microgastrinae) + Miracinae) + Cheloninae, with Adeliinae falling inside the Cheloninae. Bootstrap proportions for each of these nodes were greater than 70%. Character reweighting (sensu Farris), using the rescaled consistency index, also recovered these relationships. Mapping of lifestyle traits onto this relatively well supported phylogeny indicated that solitary endoparasitism is ancestral for the microgastroids, with a single origin for egg-larval endoparasitism in the Cheloninae + Adeliinae. Mapping of the radiation of the microgastroids into lepidopteran hosts was less clear, due to the specialized biology of the most basal microgastroid clade, the Cheloninae + Adeliinae. Our data are consistent with attack of concealed lepidopteran hosts as the plesiomorphic lifestyle, at least for the Miracinae + Cardiochilinae + Microgastrinae, with radiation into more exposed hosts in the Cardiochilinae + Microgastrinae.

Contrasting rates of mitochondrial molecular evolution in parasitic Diptera and Hymenoptera.

We investigated the putative association between the parasitic lifestyle and an accelerated rate of mt genetic divergence, compositional bias, and gene rearrangement, employing a range of parasitic and nonparasitic Diptera and Hymenoptera. Sequences were obtained for the cox1, cox2, 16S, 28S genes, the regions between the cox2 and atp8 genes, and between the nad3 and nad5 genes. Relative rate tests indicated generally that the parasitic lifestyle was not associated with an increased rate of genetic divergence in the Diptera but reaffirmed that it was in the Hymenoptera. Similarly, a departure from compositional stationarity was not associated with parasitic Diptera but was in parasitic Hymenoptera. Finally, mitochondrial (mt) gene rearrangements were not observed in any of the dipteran species examined. The results indicate that these genetic phenomena are not accelerated in parasitic Diptera compared with nonparasitic Diptera. A possible explanation for the differences in the rate of mt molecular evolution in parasitic Diptera and Hymenoptera is the extraordinary level of radiation that has occurred within the parasitic Hymenoptera but not in any of the dipteran parasitic lineages. If speciation events in the parasitic Hymenoptera are associated with founder events, a faster rate of molecular evolution is expected. Alternatively, biological differences between endoparasitic Hymenoptera and endoparasitic Diptera may also account for the differences observed in molecular evolution.

Evolutionary relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA gene sequences.

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA gene sequence data. Analyses recovered the few well-supported relationships evident in this family from morphological analyses, viz the monophyly of the microgastroid complex of subfamilies, the monophyly of the cyclostome complex of subfamilies (= braconoids), a sister-group relationship between the Alysiinae and Opiinae, and a close relationship between the Helconinae and Blacinae. With respect to the braconoid complex of subfamilies, a sister-group relationship was recovered between Aphidiinae and Mesostoinae, and a clade composed of Gnamptodontinae + Histeromerinae + Rhyssalinae + Aphidiinae + Mesostoinae was also recovered. The Doryctinae and Rogadinae sensu lato (s.l.) were generally not resolved as monophyletic. With respect to the helconoid complex of subfamilies, a sister-group relationship was recovered between Sigalphinae and Agathidinae, whereas Neoneurinae fell out among other helconoid subfamilies. Other relationships among the helconoid subfamilies were unclear from these analyses. With respect to the microgastroid complex of subfamilies, our data conform to morphological estimates, recovering ((Microgastrinae + Miracinae) + Cardiochilinae) + Cheloninae. The topology of our trees suggests that the cyclostome subfamilies are a natural derived group, inferring that endoparasitism (not ectoparasitism) is the ancestral state for the Braconidae, unless all of the ectoparasitic ancestors of the helconoid + microgastroid subfamilies are now extinct.