Phylogenomics reveals accelerated late Cretaceous diversification of bee flies (Diptera: Bombyliidae).

Bombyliidae is a very species-rich and widespread family of parasitoid flies with more than 250 genera classified into 17 extant subfamilies. However, little is known about their evolutionary history or how their present-day diversity was shaped. Transcriptomes of 15 species and anchored hybrid enrichment (AHE) sequence captures of 86 species, representing 94 bee fly species and 14 subfamilies, were used to reconstruct the phylogeny of Bombyliidae. We integrated data from transcriptomes across each of the main lineages in our AHE tree to build a data set with more genes (550 loci versus 216 loci) and higher support levels. Our overall results show strong congruence with the current classification of the family, with 11 out of 14 included subfamilies recovered as monophyletic. Heterotropinae and Mythicomyiinae are successive sister groups to the remainder of the family. We examined the evolution of key morphological characters through our phylogenetic hypotheses and show that neither the "sand chamber subfamilies" nor the "Tomophthalmae" are monophyletic in our phylogenomic analyses. Based on our results, we reinstate two tribes at the subfamily level (Phthiriinae stat. rev. and Ecliminae stat. rev.) and we include the genus Sericosoma Macquart (previously incertae sedis) in the subfamily Oniromyiinae, bringing the total number of bee fly subfamilies to 19. Our dating analyses indicate a Jurassic origin of the family (165-194 Ma), with the sand chamber evolving early in bee fly evolution, in the late Jurassic or mid-Cretaceous (100-165 Ma). We hypothesize that the angiosperm radiation and the hothouse climate established during the late Cretaceous accelerated the diversification of bee flies, by providing an expanded range of resources for the parasitoid larvae and nectarivorous adults.

Molecular phylogeny reveals food plasticity in the evolution of true ladybird beetles (Coleoptera: Coccinellidae: Coccinellini).

BACKGROUND: The tribe Coccinellini is a group of relatively large ladybird beetles that exhibits remarkable morphological and biological diversity. Many species are aphidophagous, feeding as larvae and adults on aphids, but some species also feed on other hemipterous insects (i.e., heteropterans, psyllids, whiteflies), beetle and moth larvae, pollen, fungal spores, and even plant tissue. Several species are biological control agents or widespread invasive species (e.g., Harmonia axyridis (Pallas)). Despite the ecological importance of this tribe, relatively little is known about the phylogenetic relationships within it. The generic concepts within the tribe Coccinellini are unstable and do not reflect a natural classification, being largely based on regional revisions. This impedes the phylogenetic study of important traits of Coccinellidae at a global scale (e.g. the evolution of food preferences and biogeography). RESULTS: We present the most comprehensive phylogenetic analysis of Coccinellini to date, based on three nuclear and one mitochondrial gene sequences of 38 taxa, which represent all major Coccinellini lineages. The phylogenetic reconstruction supports the monophyly of Coccinellini and its sister group relationship to Chilocorini. Within Coccinellini, three major clades were recovered that do not correspond to any previously recognised divisions, questioning the traditional differentiation between Halyziini, Discotomini, Tytthaspidini, and Singhikaliini. Ancestral state reconstructions of food preferences and morphological characters support the idea of aphidophagy being the ancestral state in Coccinellini. This indicates a transition from putative obligate scale feeders, as seen in the closely related Chilocorini, to more agile general predators. CONCLUSIONS: Our results suggest that the classification of Coccinellini has been misled by convergence in morphological traits. The evolutionary history of Coccinellini has been very dynamic in respect to changes in host preferences, involving multiple independent host switches from different insect orders to fungal spores and plants tissues. General predation on ephemeral aphids might have created an opportunity to easily adapt to mixed or specialised diets (e.g. obligate mycophagy, herbivory, predation on various hemipteroids or larvae of leaf beetles (Chrysomelidae)). The generally long-lived adults of Coccinellini can consume pollen and floral nectars, thereby surviving periods of low prey frequency. This capacity might have played a central role in the diversification history of Coccinellini.

Prediction of Potato Tuber Damage by Root-Knot Nematodes using Quantitative DNA Assay of Soil.

Root-knot nematodes (Meloidogyne fallax and M. hapla) cause significant reductions in potato yield by reducing tuber quality. Concentrations of M. fallax and M. hapla DNA in soil were determined by quantitative polymerase chain reaction following sampling at planting and harvest within 78 fields across 3 years in Australia. Meloidogyne spp. were also detected using a tomato bioassay. M. fallax was more prevalent than M. hapla and DNA concentrations of M. fallax in soil were significantly higher in samples collected at harvest compared with those at planting. In contrast, M. hapla DNA in soil did not significantly change from planting to harvest. Using receiver operating characteristic curve analysis, M. fallax DNA in soil at planting and harvest was a highly accurate predictor of tuber damage at harvest and galling on tomato. Prediction accuracy for tuber damage was highest for M. fallax DNA compared with M. hapla or M. fallax + M. hapla. Both Meloidogyne spp. were detected in the peel of asymptomatic certified seed. For M. fallax, the addition of seedborne inoculum did not improve tuber damage predictions. This suggested that soilborne M. fallax populations contributed most substantially to tuber damage. These findings highlight the utility of this approach for predicting risk of crop damage from nematodes. The use of this technique as a practical management tool is discussed.

Pratylenchus quasitereoides n. sp. from cereals in Western Australia.

Pratylenchus quasitereoides n. sp. is described from Western Australia. It is characterized by 2 external incisures in the head cuticle, 4 lateral incisures at mid body, stylet length 17 µm to 19 µm, V greater than 75%, PUS less than 2 body diameters long and crenate tail terminus. Molecular data confirm the separation of the new species from morphologically similar and sympatric congeners. The host range also differs from P. teres as well as the sympatric P. neglectus, P. thornei and P. penetrans. Reproduction rates on oat and lupin differed between the new species and P. neglectus. The species was originally described as P. teres, but the species concept of P. teres now encompasses a considerable range of different attributes spread over two described subspecies and three variant populations. The new species differs from all these subspecies and populations in at least two characters. It differs from all populations of P. teres teres most notably in having four rather than 6 lateral lines and a more posterior vulva. It differs from P. teres vandebergae in having a longer stylet and longer overlap of the intestine by the oesophageal glands. Characters which can be used under low magnification to separate the new species from the closest sympatric congeners (P. thornei and P. crenatus) are discussed.

DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees.

Understanding the root distribution of trees by soil coring is time -: consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m(-2)) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23-28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R(2) = 0.9307, P < 0.001) with the dry matter (g m(-2)) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g.

A DNA-based method for studying root responses to drought in field-grown wheat genotypes.

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Improved inference of taxonomic richness from environmental DNA.

Accurate estimation of biological diversity in environmental DNA samples using high-throughput amplicon pyrosequencing must account for errors generated by PCR and sequencing. We describe a novel approach to distinguish the underlying sequence diversity in environmental DNA samples from errors that uses information on the abundance distribution of similar sequences across independent samples, as well as the frequency and diversity of sequences within individual samples. We have further refined this approach into a bioinformatics pipeline, Amplicon Pyrosequence Denoising Program (APDP) that is able to process raw sequence datasets into a set of validated sequences in formats compatible with commonly used downstream analyses packages. We demonstrate, by sequencing complex environmental samples and mock communities, that APDP is effective for removing errors from deeply sequenced datasets comprising biological and technical replicates, and can efficiently denoise single-sample datasets. APDP provides more conservative diversity estimates for complex datasets than other approaches; however, for some applications this may provide a more accurate and appropriate level of resolution, and result in greater confidence that returned sequences reflect the diversity of the underlying sample.

Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils.

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi-arid floodplain would transition towards comprising mainly dry-soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post-flood) were dominated by persistent rather than either aquatic or dry-specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post-flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post-flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi-arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.

Predicting Take-All Severity in Second-Year Wheat Using Soil DNA Concentrations of Gaeumannomyces graminis var. tritici Determined with qPCR.

The lack of accurate detection of Gaeumannomyces graminis var. tritici inoculum in soil has hampered efforts to predict the risk of severe take-all for wheat growers. The current study used a molecular method to quantify soil G. graminis var. tritici concentrations in commercial wheat fields in New Zealand and to compare them with the proportion of crops surpassing the thresholds for visible and moderate to severe take-all over three growing seasons. The study evaluated a soil G. graminis var. tritici DNA-based take-all prediction system developed in Australia, with four take-all risk categories. These categories were found to be useful for predicting disease severity in second wheat but did not clearly separate risk between fields in medium- and high-risk categories. A sigmoidal relationship was identified between inoculum concentration and the proportion of fields exceeding the two disease thresholds. A logistic response curve was used to further examine this relationship and evaluate the boundaries between take-all risk categories. G. graminis var. tritici boundaries between medium- and high-risk categories were clustered near or within the upper plateau of the relationship. Alternative G. graminis var. tritici boundaries for a three-category system were identified that provided better separation of take-all risk between categories. This information could improve prediction of the risk of severe take-all.

Carbon source accounting for fish using combined DNA and stable isotope analyses in a regulated lowland river weir pool.

Determining the source and flow of carbon, energy and nutrients through food webs is essential for understanding ecological connectivity and thus determining the impact of management practices on biodiversity. We combined DNA sequencing, microarrays and stable isotope analyses to test whether this approach would allow us to resolve the carbon flows through food webs in a weir pool on the lower Murray River, a highly impacted, complex and regulated ecosystem in southern Australia. We demonstrate that small fish in the Murray River consume a wide range of food items, but that a significant component of carbon and nitrogen entering the food web during dry periods in summer, but not spring, is derived from nonconventional sources other than in-channel primary producers. This study also showed that isotopic analyses alone cannot distinguish food sources and that a combined approach is better able to elucidate food-consumer dynamics. Our results highlight that a major river ecosystem, stressed by reduced environmental flows, can rapidly undergo significant and previously undetected changes that impact on the ecology of the system as a whole.

Putative natural hybrid between Puccinia lagenophorae and an unknown rust fungus on Senecio madagascariensis in KwaZulu-Natal, South Africa.

Several specimens of an aecial rust fungus were collected on Senecio madagascariensis during a field survey carried out in KwaZulu-Natal, South Africa. As telia were not present in the specimens collected, DNA sequence analyses were undertaken to determine the identity of the rust species. ITS and beta-tub1 sequencing confirmed that one of the isolates recovered is Puccinia lagenophorae sensu lato. On the other hand, sequencing and RFLP analysis revealed the presence of two divergent copies of ITS and beta-tub1 in all the other six isolates investigated. In both phylogenetic trees, one copy of the gene region grouped within a well supported clade with sequences of P. lagenophorae accessions from different geographical origins and hosts, and the Australian rusts Puccinia saccardoi and Puccinia stylidii. The other copy of these gene regions grouped within a separate clade comprising European accessions of Puccinia dioicae (ITS) and Uromyces sommerfeltii (beta-tub1) that occur on Asteraceae hosts. Multiple copies of these gene regions were not observed in Australian isolates of P. lagenophorae. Our study provides some evidence that an interspecific hybrid rust fungus, with P. lagenophorae as one of its parents, may occur on S. madagascariensis in South Africa. The identity of the other parent remains unknown.

Matching the origin of an invasive weed for selection of a herbivore haplotype for a biological control programme.

The Florida Everglades have been invaded by an exotic weed fern, Lygodium microphyllum. Across its native distribution in the Old World tropics from Africa to Australasia it was found to have multiple location-specific haplotypes. Within this distribution, the climbing fern is attacked by a phytophagous mite, Floracarus perrepae, also with multiple haplotypes. The genetic relationship between mite and fern haplotypes was matched by an overarching geographical relationship between the two. Further, mites that occur in the same location as a particular fern haplotype were better able to utilize the fern than mites from more distant locations. From a biological control context, we are able to show that the weed fern in the Everglades most likely originated in northern Queensland, Australia/Papua New Guinea and that the mite from northern Queensland offers the greatest prospect for control.