Anchored Phylogenomics, Evolution and Systematics of Elateridae: Are All Bioluminescent Elateroidea Derived Click Beetles?

Click-beetles (Coleoptera: Elateridae) are an abundant, diverse, and economically important beetle family that includes bioluminescent species. To date, molecular phylogenies have sampled relatively few taxa and genes, incompletely resolving subfamily level relationships. We present a novel probe set for anchored hybrid enrichment of 2260 single-copy orthologous genes in Elateroidea. Using these probes, we undertook the largest phylogenomic study of Elateroidea to date (99 Elateroidea, including 86 Elateridae, plus 5 non-elateroid outgroups). We sequenced specimens from 88 taxa to test the monophyly of families, subfamilies and tribes. Maximum likelihood and coalescent phylogenetic analyses produced well-resolved topologies. Notably, the included non-elaterid bioluminescent families (Lampyridae + Phengodidae + Rhagophthalmidae) form a clade within the otherwise monophyletic Elateridae, and Sinopyrophoridae may not warrant recognition as a family. All analyses recovered the elaterid subfamilies Elaterinae, Agrypninae, Cardiophorinae, Negastriinae, Pityobiinae, and Tetralobinae as monophyletic. Our results were conflicting on whether the hypnoidines are sister to Dendrometrinae or Cardiophorinae + Negastriinae. Moreover, we show that fossils with the eucnemid-type frons and elongate cylindrical shape may belong to Eucnemidae, Elateridae: Thylacosterninae, ancestral hard-bodied cantharoids or related extinct groups. Proposed taxonomic changes include recognition of Plastocerini as a tribe in Dendrometrinae and Hypnoidinae stat. nov. as a subfamily within Elateridae.

Aquatic Bacterial Communities Associated With Land Use and Environmental Factors in Agricultural Landscapes Using a Metabarcoding Approach.

This study applied a 16S rRNA gene metabarcoding approach to characterize bacterial community compositional and functional attributes for surface water samples collected within, primarily, agriculturally dominated watersheds in Ontario and Québec, Canada. Compositional heterogeneity was best explained by stream order, season, and watercourse discharge. Generally, community diversity was higher at agriculturally dominated lower order streams, compared to larger stream order systems such as small to large rivers. However, during times of lower relative water flow and cumulative 2-day rainfall, modestly higher relative diversity was found in the larger watercourses. Bacterial community assemblages were more sensitive to environmental/land use changes in the smaller watercourses, relative to small-to-large river systems, where the proximity of the sampled water column to bacteria reservoirs in the sediments and adjacent terrestrial environment was greater. Stream discharge was the environmental variable most significantly correlated (all positive) with bacterial functional groups, such as C/N cycling and plant pathogens. Comparison of the community structural similarity via network analyses helped to discriminate sources of bacteria in freshwater derived from, for example, wastewater treatment plant effluent and intensity and type of agricultural land uses (e.g., intensive swine production vs. dairy dominated cash/livestock cropping systems). When using metabarcoding approaches, bacterial community composition and coexisting pattern rather than individual taxonomic lineages, were better indicators of environmental/land use conditions (e.g., upstream land use) and bacterial sources in watershed settings. Overall, monitoring changes and differences in aquatic microbial communities at regional and local watershed scales has promise for enhancing environmental footprinting and for better understanding nutrient cycling and ecological function of aquatic systems impacted by a multitude of stressors and land uses.

Assessing Performance of Spore Samplers in Monitoring Aeromycobiota and Fungal Plant Pathogen Diversity in Canada.

Spore samplers are widely used in pathogen surveillance but not so much for monitoring the composition of aeromycobiota. In Canada, a nationwide spore-sampling network (AeroNet) was established as a pilot project to assess fungal community composition in air and rain samples collected using three different spore samplers in the summers of 2010 and 2011. Metabarcodes of the internal transcribed spacer (ITS) were exhaustively characterized for three of the network sites, in British Columbia (BC), Québec (QC), and Prince Edward Island (PEI), to compare performance of the samplers. Sampler type accounted for ca. 20% of the total explainable variance in aeromycobiota compositional heterogeneity, with air samplers recovering more Ascomycota and rain samplers recovering more Basidiomycota. Spore samplers showed different abilities to collect 27 fungal genera that are plant pathogens. For instance, Cladosporium spp., Drechslera spp., and Entyloma spp. were collected mainly by air samplers, while Fusarium spp., Microdochium spp., and Ustilago spp. were recovered more frequently with rain samplers. The diversity and abundance of some fungi were significantly affected by sampling location and time (e.g., Alternaria and Bipolaris) and weather conditions (e.g., Mycocentrospora and Leptosphaeria), and depended on using ITS1 or ITS2 as the barcoding region (e.g., Epicoccum and Botrytis). The observation that Canada's aeromycobiota diversity correlates with cooler, wetter conditions and northward wind requires support from more long-term data sets. Our vision of the AeroNet network, combined with high-throughput sequencing (HTS) and well-designed sampling strategies, may contribute significantly to a national biovigilance network for protecting plants of agricultural and economic importance in Canada.IMPORTANCE The current study compared the performance of spore samplers for collecting broad-spectrum air- and rain-borne fungal pathogens using a metabarcoding approach. The results provided a thorough characterization of the aeromycobiota in the coastal regions of Canada in relation to the influence of climatic factors. This study lays the methodological basis to eventually develop knowledge-based guidance on pest surveillance by assisting in the selection of appropriate spore samplers.

Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil.

Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and negative soil samples was dually demonstrated for both the SSU and ITS-1 assays.

Comparative mapping of the oat Dw6/dw6 dwarfing locus using NILs and association with vacuolar proton ATPase subunit H.

Seven pairs of oat near-isogenic lines (NILs) (Kibite in Crop Sci 41:277-278, 2001) contrasting for the Dw6 dwarfing gene were used to test for correlation between tall/dwarf phenotype and polymorphic genotype using restriction fragment length polymorphism (RFLP) and other molecular markers selected from the Kanota × Ogle (K×O) (Wight et al. in Genome 46:28-47, 2003) and Terra × Marion (De Koeyer et al. in Theor Appl Genet 108:1285-1298, 2004) recombination maps. This strategy located the Dw6/dw6 locus to a small chromosomal region on K×O linkage group (LG) KO33, near or at a putative RFLP locus aco245z. Aco245z and other tightly linked flanking markers have potential for use in marker-assisted selection (MAS), and PCR-based markers were developed from several of these. RFLP genotyping of the Dw6 NILs indicated that 13 of the 14 individual lines were homogeneously maternal or paternal for a large genomic region near Dw6/dw6, an unexpected result for NILs. The cDNA clone aco245 codes for a vacuolar proton ATPase subunit H, a potential candidate gene for Dw6. Vacuolar proton ATPase enzymes have a central role in plant growth and development and a mutation in subunit C is responsible for the det3 dwarfing mutation in Arabidopsis thaliana (Schumacher et al. in Genes Dev 13:3259-3270, 1999). Aco245 affords the potential of designing highly precise diagnostic markers for MAS for Dw6. The Dw6 NILs have potential utility to investigate the role of vacuolar proton ATPases in growth and development in plants.

Loci affecting flowering time in oat under short-day conditions.

Flowering time (or days to heading) is an important characteristic in crop plants that affects adaptation to cropping cycles and growing seasons. The objectives of this study were to identify molecular markers associated with flowering time in 3 oat populations developed from Brazilian oat varieties, and to compare their map locations with those of other loci that might influence flowering time. Flowering time was studied in recombinant inbred lines from 3 hexaploid oat populations: UFRGS 8 x Pc68/5*Starter; UFRGS 881971 x Pc68/5*Starter; and UFRGS 8 x UFRGS 930605. Bulked segregant analysis, using amplified fragment length polymorphism, was followed by selective mapping in each population and in a reference population, 'Kanota' x 'Ogle' (KxO). One quantitative trait locus (QTL) with major effects on flowering time was identified in each cross. Comparative mapping showed that a major QTL, with earliness alleles originating from UFRGS 8 and UFRGS 881971, is in a region with close homology to KxO linkage group 17 and to a locus that reportedly confers day-length insensitivity in oat (Di1). This is the first report to identify the map location of the Di1 locus, and putatively confirm the presence of Di1 alleles in new germplasm. Further comparative mapping and the alignment of mapped oat markers with the sequenced rice genome suggest that this QTL and (or) Di1 is orthologous to the Hd1 locus in rice and the CONSTANS gene in Arabidopsis and other species. A different QTL with major effects segregated in the UFRGS 8 x UFRGS 930605 cross, where the early-flowering allele for Di1 was probably fixed. Two additional QTLs with smaller effects were identified in the UFRGS 8 x Pc68/5*Starter population. These results suggest that the Brazilian oat line UFRGS 8 contains an optimal set of alleles conditioning earliness under the short-day conditions of the Brazilian winter growing season, and that molecular selection could be used to introgress these alleles into other breeding material.