Biodiversity assessment underpins our understanding of ecosystems and determines environmental management decisions on resource use and conservation priorities. Recently, a new discipline – environmental or ecological genomics (ecogenomics) – has emerged from major advances in sequencing technologies, such as pyrosequencing (a technique based on the detection of pyrophosphate during nucleotide incorporation), and enabled extraordinary progress in the way biodiversity can be assessed. Since 2008, numerous high‐impact microbial metagenomic sequencing studies, which have relied on both classical and next‐generation sequencing, have been published. As a result, many previously unrecognized taxa and biota have been identified, but none of these studies explored eukaryote diversity. Here, we illustrate the power of applying next‐generation pyrosequencing to identify and enumerate eukaryote species assemblages in the context of assessing the impacts of human activity on ecosystems.
Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species.
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.
The complete length of the Asia I member of the Bemisia tabaci species complex mitochondrial DNA genome (mitogenome) is 15,210 bp (GenBank accession no. KJ778614) with an A-T biased nucleotide composition (A: 32.7%; T: 42.4%; G: 14.0%; C: 10.8%). The mitogenome consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNA (rRNAs) and a 467 bp putative control region which also includes the A+T rich repeat region. All PCGs have an ATA (n = 8) or ATG (n = 5) start codon. Gene synteny of Asia I is overall similar to B. afer and two other members of the B. tabaci species complex Mediterranean and New World 1, and contains the tRNA-Ser2 located between the Cytb and ND1 genes found in Mediterranean and New World 1, but which is absent in B. afer. The orientation of the tRNA-Arg in Asia I is on the "plus" strand and differed from Mediterranean which is found on the "minus" strand. The Asia I mitogenome size is currently ranked the second smallest after B. afer (14,968 bp) followed by New World 1 (15,322 bp) and Mediterranean (15,632 bp).
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.
Here we show how the 10 genes of the alpha esterase cluster of Drosophila melanogaster have diverged substantially in their expression profiles. Together with previously described sequence divergence this suggests substantial functional diversification. By peptide mass fingerprinting and in vitro gene expression we have also shown that two of the genes encode the isozymes EST9 (formerly ESTC) and EST23. EST9 is the major 'alpha staining' esterase in zymograms of gut tissues in feeding stages while orthologues of EST23 confer resistance to organophosphorus insecticides in other higher Diptera. The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters. However, many of the other genes in the cluster show developmental or tissue-specific expression that seems inconsistent with such roles. Furthermore, there is generally poor correspondence between the mRNA expression patterns of the remaining eight genes and isozymes previously characterized by standard techniques of electrophoresis and staining, suggesting that the alpha cluster might only account for a small minority of the esterase isozyme profile.
Museum specimens represent valuable genomic resources for understanding host-endosymbiont/parasitoid evolutionary relationships, resolving species complexes and nomenclatural problems. However, museum collections suffer DNA degradation, making them challenging for molecular-based studies. Here, the mitogenomes of a single 1912 Sri Lankan Bemisia emiliae cotype puparium, and of a 1942 Japanese Bemisia puparium are characterised using a Next-Generation Sequencing approach. Whiteflies are small sap-sucking insects including B. tabaci pest species complex. Bemisia emiliae’s draft mitogenome showed a high degree of homology with published B. tabaci mitogenomes, and exhibited 98–100% partial mitochondrial DNA Cytochrome Oxidase I (mtCOI) gene identity with the B. tabaci species known as Asia II-7. The partial mtCOI gene of the Japanese specimen shared 99% sequence identity with the Bemisia ‘JpL’ genetic group. Metagenomic analysis identified bacterial sequences in both Bemisia specimens, while hymenopteran sequences were also identified in the Japanese Bemisia puparium, including complete mtCOI and rRNA genes, and various partial mtDNA genes. At 88–90% mtCOI sequence identity to Aphelinidae wasps, we concluded that the 1942 Bemisia nymph was parasitized by an Eretmocerus parasitoid wasp. Our approach enables the characterisation of genomes and associated metagenomic communities of museum specimens using 1.5 ng gDNA, and to infer historical tritrophic relationships in Bemisia whiteflies.
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