Geographic variation in bacterial assemblages on cane toad skin is influenced more by local environments than by evolved changes in host traits.

Bacterial assemblages on amphibian skin may play an important role in protecting hosts against infection. In hosts that occur over a range of environments, geographic variation in composition of bacterial assemblages might be due to direct effects of local factors and/or to evolved characteristics of the host. Invasive cane toads (Rhinella marina) are an ideal candidate to evaluate environmental and genetic mechanisms, because toads have evolved major shifts in physiology, morphology, and behavior during their brief history in Australia. We used samples from free-ranging toads to quantify site-level differences in bacterial assemblages and a common-garden experiment to see if those differences disappeared when toads were raised under standardised conditions at one site. The large differences in bacterial communities on toads from different regions were not seen in offspring raised in a common environment. Relaxing bacterial clustering to operational taxonomic units in place of amplicon sequence variants likewise revealed high similarity among bacterial assemblages on toads in the common-garden study, and with free-ranging toads captured nearby. Thus, the marked geographic divergence in bacterial assemblages on wild-caught cane toads across their Australian invasion appears to result primarily from local environmental effects rather than evolved shifts in the host.

Microbial communities associated with mounds of the Orange-footed scrubfowl Megapodius reinwardt.

Megapodius reinwardt, the orange-footed scrubfowl, belongs to a small family of birds that inhabits the Indo-Australian region. Megapodes are unique in incubating their eggs in mounds using heat from microbial decomposition of organic materials and solar radiation. Little is known about the microorganisms involved in the decomposition of organic matter in mounds. To determine the source of microbes in the mounds, we used 16S and 18S rRNA gene sequencing to characterize the microbial communities of mound soil, adjacent soil and scrubfowl faeces. We found that the microbial communities of scrubfowl faeces were substantially different from those of the mounds and surrounding soils, suggesting that scrubfowls probably do not use their faeces to inoculate their mounds although a few microbial sequence variants were present in both faeces and mound samples. Further, the mound microbial community structure was significantly different to the adjacent soils. For example, mounds had a high relative abundance of sequence variants belonging to Thermomonosporaceae, a thermophilic soil bacteria family able to degrade cellulose from plant residues. It is not clear whether members of Thermomonosporaceae disproportionately contribute to the generation of heat in the mound, or whether they simply thrive in the warm mound environment created by the metabolic activity of the mound microbial community. The lack of clarity in the literature between designations of heat-producing (thermogenic) and heat-thriving (thermophilic) microbes poses a challenge to understanding the role of specific bacteria and fungi in incubation.

Mitochondrial phylogeny within the Yellow Chat (Epthianura crocea) does not support subspecific designation of endangered Alligator Rivers population.

The delineation of subspecies is important in the evaluation and protection of biodiversity. Subspecies delineation is hampered by inconsistently applied criteria and a lack of agreement and shifting standards on how a subspecies should be defined. The Australian endemic Yellow Chat (Epthianura crocea) is split into three subspecies (E. c. crocea, E. c. tunneyi, and E. c. macgregori) based on minor plumage differences and geographical isolation. Both E. c. tunneyi (Endangered) and E. c. macgregori (Critically Endangered) are recognized under Australian legislation as threatened and are the subject of significant conservation effort. We used mitochondrial DNA to evaluate the phylogeny of the Yellow Chat and determine how much genetic variation is present in each of the three subspecies. We found no significant difference in the cytochrome b sequences (833 base pairs) of E. c. crocea and E. c. tunneyi, but approximately 0.70% or 5.83 bp difference between E. c macgregori and both E. c. crocea and E. c. tunneyi. This analysis supports the delineation of E. c. macgregori as a valid subspecies but does not support separation of E. c. crocea from E. c. tunneyi. We also found very low levels of genetic variation within the Yellow Chat, suggesting it may be vulnerable to environmental change. Our results cast doubt upon the geographic isolation of E. c. crocea from E. c. tunneyi, but more advanced genetic sequencing and a robust comparison of plumage are needed to fully resolve taxonomy.

Opportunistic pathogens and large microbial diversity detected in source-to-distribution drinking water of three remote communities in Northern Australia.

In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.

Sources and drivers of contamination along an urban tropical river (Ciliwung, Indonesia): Insights from microbial DNA, isotopes and water chemistry.

Wastewater treatment infrastructure is lacking in many developing countries, often resulting in high loads of contaminants discharged to urban rivers. In these countries, targeted pollution mitigation requires an understanding of where, how and when contaminants enter water bodies. Here we report on contamination of the Ciliwung River, a dynamic, tropical system flowing through the Jakarta metropolitan area (Indonesia). We measured a set of isotopic, chemical and microbial tracers in representative water and contamination sources, as well as longitudinally within the river, to assess the spatial and temporal variations in contaminant levels in and pathways to the river. In the dry season, we observed a tight coupling between locally recharged groundwater sources and the river, whereas in the wet season, one single water source originating from the fractured headwaters predominantly contributed to river flow. Yet, the flushing of upstream waters in the wet season did not always lead to the dilution of contaminants downstream. We delineated several contamination hotspots along the river, particularly active during the wet season due to higher hydrological connectivity between sources and the river. These hotspots may originate from septic tank leakage, as supported by metal ratios and dominant microbial communities, although we could not rule out other potential sources such as urban runoff or sediment resuspension. Bayesian source tracking on the whole microbial community proved useful in outlining processes that conventional tracers did not capture, such as the occurrence of a localised domestic contamination in the upper catchment, and the inflow of agricultural runoff all along the river profile during the wet season. Our study emphasises the role of rivers as biogeochemical reactors that constantly process and transform contaminants and microbial communities. We also demonstrate the value of using isotopic, chemical and microbial tools together to trace the movement of water and contaminants through urban rivers.

Ecological patterns in the skin microbiota of frogs from tropical Australia.

The microbiota of frog skin can play an important role in protecting against diseases and parasites. The frog skin microbial community represents a complex mix of microbes that are promoted by the chemical environment of the frog skin and influenced by the animal's immediate past environment. The microbial communities of six species of frogs sampled from the campus of Charles Darwin University (CDU) were more similar within species than between species. The microbiota of the introduced cane toad (Rhinella marina) was most dissimilar among the species. Pairwise comparisons showed that the microbial communities of each species were different, except for the terrestrial Litoria nasuta and the arboreal L. rothii. The microbial communities of the six species were not related to ecological habit (arboreal or terrestrial), and neither was the alpha diversity of the microbes. The core microbes (defined as being on ≥90% of individuals of a species or group) were significantly different among all species, although 89 microbial operational taxonomic units (OTUs) were core microbes for all six species at CDU. Two species, Rhinella marina and Litoria rothii, were sampled at additional sites approximately 10 and 30 km from CDU. The microbial communities and the core OTU composition were different among the sites, but there were nevertheless 194 (R. marina) and 181 (L. rothii) core OTUs present at all three sites. Thus, the core microbiota varied with respect to geographic range and sample size.

Differences in structure of northern Australian hypolithic communities according to location, rock type, and gross morphology.

Hypolithic communities (under translucent rocks) were compared between a semi-arid site (Wave Hill) and a site with considerably higher rainfall (Lake Bennett) to test the hypothesis that the communities at the higher rainfall site would be more diverse. A total of 153 cyanobacteria operational taxonomic units (OTUs) were identified, and only 50 of those were found at both sites. Of these, only two were core OTUs, as defined as being present in ≥90% of samples, highlighting the extreme differences in the cyanobacterial communities at the two sites. At Wave Hill, we compared the composition of the cyanobacterial components under two different rock types (quartz and prehnite) to determine if the different minerals would result in different hypolithic communities, but no differences were found. Of the 42 core OTUs found at Wave Hill, 22 (52%) were shared between the two rock types. As hypothesised, the diversity of both cyanobacteria and eukaryotes in the hypolithic communities was significantly higher at Lake Bennett. Some hypolithic communities were thin and tightly adhered to the rock surface, but others were thicker and could be peeled off the rock in sheets. However, the two types were not significantly different in OTU composition. Metazoans, primarily nematodes, were ubiquitous, raising the possibility that nematodes may act as vectors to transport the components of hypolithic communities from rock to rock as a mechanism of colonization.

Molecular screening of microbial communities for candidate indicators of multiple metal impacts in marine sediments from northern Australia.

Coastal sediments accumulate metals from anthropogenic sources and as a consequence industry is required to monitor sediment health. The total concentration of a metal does not necessarily reflect its potential toxicity or biological impact, so biological assessment tools are useful for monitoring. Rapid biological assessment tools sensitive enough to detect relatively small increases in metal concentrations would provide early warning of future ecosystem impact. The authors investigated in situ populations of Archaea and Bacteria as potential tools for rapid biological assessment in sediment at 4 northern Australian coastal locations over 2 yr, in both wet and dry seasons. The 1 M HCl-extractable concentrations of metals in sediment were measured, and Archaeal and Bacterial community profiles were obtained by next-generation sequencing of sediment deoxyribonucleic acid (DNA). Species response curves were used to identify several taxonomic groups with potential as biological indicators of metal impact. Spatial variation, sediment grain size, water depth, and dissolved oxygen also correlated with microbial population shifts. Seasonal variation was less important than geographic location. Metal-challenge culture trials supported the identification of metal-resistant and -sensitive taxa. In situ Archaea and Bacteria are potentially sensitive indicators for changes in bioavailable concentrations of metals; however, the complexity of the system suggests it is important to identify metal-specific functional genes that may be informed by these sequencing surveys, and thus provide a useful addition to identity-based assays.