Matthew J. Morgan

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.

Metabarcoding of benthic eukaryote communities predicts the ecological condition of estuaries

DNA-derived measurements of biological composition have the potential to produce data covering all of life, and provide a tantalizing proposition for researchers and managers. We used metabarcoding to compare benthic eukaryote composition from five estuaries of varying condition. In contrast to traditional studies, we found biotic richness was greatest in the most disturbed estuary, with this being due to the large volume of extraneous material (i.e. run-off from aquaculture, agriculture and other catchment activities) being deposited in the system. In addition, we found strong correlations between composition and a number of environmental variables, including nutrients, pH and turbidity. A wide range of taxa responded to these environmental gradients, providing new insights into their sensitivities to natural and anthropogenic stressors. Metabarcoding has the capacity to bolster current monitoring techniques, enabling the decisions regarding ecological condition to be based on a more holistic view of biodiversity.

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.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database

BackgroundMicrobial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The ‘Biomes of Australian Soil Environments’ (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function.FindingsBASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project’s data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the ‘Atlas of Living Australia’.ConclusionsDeveloped within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services.

DNA barcoding to support conservation: species identification, genetic structure and biogeography of fishes in the Murray)Darling River Basin, Australia

Freshwater fish stocks worldwide are under increasing threat of overfishing, disease, pollution and competition from introduced species. In the Murray—Darling Basin (MDB), the largest river system of Australia, more than half the native species are listed as rare or endangered. Active management is required to counteract reduction in population sizes, prevent local extinctions and to maintain genetic diversity. We describe the first comprehensive set of DNA barcodes able to discriminate between all 58 native and introduced species of freshwater fish recorded in the MDB. These barcodes also distinguish populations from those in adjacent basins, with estimated separation times as short as 0.1 million years ago. We demonstrate the feasibility of using DNA fingerprinting of ribosomal RNA (12S and 18S rRNA) genes and mitochondrial DNA control region (mtDNA CR) sequences to identify species from eggs, larvae, tissues and predator gut contents as well as differentiate populations, morphologically cryptic species and hybrids. The DNA barcode resource will enhance capacity in many areas of fish conservation biology that can benefit from improved knowledge of genetic provenance. These include captive breeding and restocking programs, life history studies and ecological research into the interactions between populations of native and exotic species.

Characterization of a Drinking Water Distribution Pipeline Terminally Colonized by Naegleria fowleri

Free-living amoebae, such as Naegleria fowleri, Acanthamoeba spp., and Vermamoeba spp., have been identified as organisms of concern due to their role as hosts for pathogenic bacteria and as agents of human disease. In particular, N. fowleri is known to cause the disease primary amoebic meningoencephalitis (PAM) and can be found in drinking water systems in many countries. Understanding the temporal dynamics in relation to environmental and biological factors is vital for developing management tools for mitigating the risks of PAM. Characterizing drinking water systems in Western Australia with a combination of physical, chemical and biological measurements over the course of a year showed a close association of N. fowleri with free chlorine and distance from treatment over the course of a year. This information can be used to help design optimal management strategies for the control of N. fowleri in drinking-water-distribution systems.

Molecular homology and multiple-sequence alignment: an analysis of concepts and practice

Abstract. Sequence alignment is just as much a part of phylogenetics as is tree building, although it is often viewed solely as a necessary tool to construct trees. However, alignment for the purpose of phylogenetic inference is primarily about homology, as it is the procedure that expresses homology relationships among the characters, rather than the historical relationships of the taxa. Molecular homology is rather vaguely defined and understood, despite its importance in the molecular age. Indeed, homology has rarely been evaluated with respect to nucleotide sequence alignments, in spite of the fact that nucleotides are the only data that directly represent genotype. All other molecular data represent phenotype, just as do morphology and anatomy. Thus, efforts to improve sequence alignment for phylogenetic purposes should involve a more refined use of the homology concept at a molecular level. To this end, we present examples of molecular-data levels at which homology might be considered, and arrange them in a hierarchy. The concept that we propose has many levels, which link directly to the developmental and morphological components of homology. Of note, there is no simple relationship between gene homology and nucleotide homology. We also propose terminology with which to better describe and discuss molecular homology at these levels. Our over-arching conceptual framework is then used to shed light on the multitude of automated procedures that have been created for multiple-sequence alignment. Sequence alignment needs to be based on aligning homologous nucleotides, without necessary reference to homology at any other level of the hierarchy. In particular, inference of nucleotide homology involves deriving a plausible scenario for molecular change among the set of sequences. Our clarifications should allow the development of a procedure that specifically addresses homology, which is required when performing alignment for phylogenetic purposes, but which does not yet exist.

Comparison of biofilm ecology supporting growth of individual Naegleria species in a drinking water distribution system

Free-living amoebae (FLA) are common components of microbial communities in drinking water distribution systems (DWDS). FLA are of clinical importance both as pathogens and as reservoirs for bacterial pathogens, so identifying the conditions promoting amoebae colonisation of DWDSs is an important public health concern for water utilities. We used high-throughput amplicon sequencing to compare eukaryotic and bacterial communities associated with DWDS biofilms supporting distinct FLA species (Naegleria fowleri, N. lovaniensis or Vermamoeba sp.) at sites with similar physical/chemical conditions. Eukaryote and bacterial communities were characteristics of different FLA species presence, and biofilms supporting Naegleria growth had higher bacterial richness and higher abundance of Proteobacteria, Bacteroidetes (bacteria), Nematoda and Rotifera (eukaryota). The eukaryotic community in the biofilms had the greatest difference in relation to the presence of N. fowleri, while the bacterial community identified individual bacterial families associated with the presence of different Naegleria species. Our results demonstrate that ecogenomics data provide a powerful tool for studying the microbial and meiobiotal content of biofilms, and, in these samples can effectively discriminate biofilm communities supporting pathogenic N. fowleri. The identification of microbial species associated with N. fowleri could further be used in the management and control of N. fowleri in DWDS.

The complete mitochondrial DNA genomes for two lineages of Aedes notoscriptus (Diptera: Culicidae)

Abstract The complete mitochondrial genomes for two deeply divergent lineages of the urban adapted mosquito Aedes notoscriptus Skuse (Diptera: Culicidae) in Australia were sequenced using a combination of next generation Illumina and traditional Sanger sequencing. The 15,846 and 15,851 bp circular genomes share 95.0% nucleotide identity. They both have the full complement of 37 metazoan genes and identical gene arrangements to previously published Culicidae species with the one non-coding A + T rich control region present between rns and tRNA-Ile. All protein initiation codons are ATN apart from COX1 (TCG). Eight protein coding genes encode full TAA stop codons, one uses an incomplete TA and four use T. Typical cloverleaf structures containing DHU and TΨC stem and loops can be inferred for all 22 tRNAs.

Discrimination of Plant-Parasitic Nematodes from Complex Soil Communities Using Ecometagenetics

Many plant pathogens are microscopic, cryptic, and difficult to diagnose. The new approach of ecometagenetics, involving ultrasequencing, bioinformatics, and biostatistics, has the potential to improve diagnoses of plant pathogens such as nematodes from the complex mixtures found in many agricultural and biosecurity situations. We tested this approach on a gradient of complexity ranging from a few individuals from a few species of known nematode pathogens in a relatively defined substrate to a complex and poorly known suite of nematode pathogens in a complex forest soil, including its associated biota of unknown protists, fungi, and other microscopic eukaryotes. We added three known but contrasting species (Pratylenchus neglectus, the closely related P. thornei, and Heterodera avenae) to half the set of substrates, leaving the other half without them. We then tested whether all nematode pathogens-known and unknown, indigenous, and experimentally added-were detected consistently present or absent. We always detected the Pratylenchus spp. correctly and with the number of sequence reads proportional to the numbers added. However, a single cyst of H. avenae was only identified approximately half the time it was present. Other plant-parasitic nematodes and nematodes from other trophic groups were detected well but other eukaryotes were detected less consistently. DNA sampling errors or informatic errors or both were involved in misidentification of H. avenae; however, the proportions of each varied in the different bioinformatic pipelines and with different parameters used. To a large extent, false-positive and false-negative errors were complementary: pipelines and parameters with the highest false-positive rates had the lowest false-negative rates and vice versa. Sources of error identified included assumptions in the bioinformatic pipelines, slight differences in primer regions, the number of sequence reads regarded as the minimum threshold for inclusion in analysis, and inaccessible DNA in resistant life stages. Identification of the sources of error allows us to suggest ways to improve identification using ecometagenetics.