The study of ancient DNA (aDNA) from sediments (sedaDNA) offers great potential for paleoclimate interpretation, and has recently been applied as a tool to characterise past marine life and environments from deep ocean sediments over geological timescales. Using sedaDNA, palaeo-communities have been detected, including prokaryotes and eukaryotes that do not fossilise, thereby revolutionising the scope of marine micropalaeontological research. However, many studies to date have not reported on the measures taken to prove the authenticity of sedaDNA-derived data from which conclusions are drawn. aDNA is highly fragmented and degraded and extremely sensitive to contamination by non-target environmental DNA. Contamination risks are particularly high on research vessels, drilling ships and platforms, where logistics and facilities do not yet allow for sterile sediment coring, and due consideration needs to be given to sample processing and analysis following aDNA guidelines. This review clarifies the use of aDNA terminology, discusses common pitfalls and highlights the urgency behind adopting new standards for marine sedaDNA research, with a focus on sampling optimisation to facilitate the incorporation of routine sedaDNA research into International Ocean Discovery Program (IODP) operations. Currently available installations aboard drilling ships and platforms are reviewed, improvements suggested, analytical approaches detailed, and the controls and documentation necessary to support the authenticity of aDNA retrieved from deep-sea sediment cores is outlined. Beyond practical considerations, concepts relevant to the study of past marine biodiversity based on aDNA, and the applicability of the new guidelines to the study of other contamination-susceptible environments (permafrost and outer space) are discussed.

Bundera sinkhole, located in north-western Australia, is the only known continental anchialine system in the Southern Hemisphere. Anchialine environments are characterised by stratified water columns with complex physicochemical profiles spanning hypoxic and anoxic regions, often displaying high levels of endemism. Research on these systems has focused on eukaryotic fauna, however interest in the microbial diversity of these environments is growing, enabled by next-generation DNA sequencing. Here we report detailed analyses of the microbial communities across a depth profile within Bundera sinkhole (from 2 to 28 m), involving parallel physicochemical measurements, cell population counts and 16S rRNA amplicon analyses. We observed clear shifts in microbial cell counts, community diversity, structure and membership across the depth profile, reflecting changing levels of light, organic and inorganic energy sources as well as shifts in pH and salinity. While Proteobacteria were the most abundant phylum found, there was a high degree of taxonomic novelty within these microbial communities, with 13,028 unique amplicon sequence variants (ASVs) identified, belonging to 67 identifiable bacterial and archaeal phyla. Of these ~4,600, more than one third of the total, were unclassified below family level. A small number of ASVs were highly abundant at select depths, all of which were part of the set not classified below family level. The 2 m and 6 m samples had in common two highly abundant ASVs, belonging to the Ectothiorhodospiraceae and Thiotrichaceae families, while the 8 m community contained a single predominant ASV belonging to family Thioglobaceae. At lower depths a different Ectothiorhodospiraceae ASV comprised up to 68% relative abundance, peaking at 26 and 28 m. Canonical correspondence analyses indicated that community structure was strongly influenced by differences in key physicochemical parameters, particularly salinity, dissolved organic and inorganic carbon, phosphate and sulphate concentrations. This work highlights the potential for anchialine systems to house considerable microbial novelty, potentially driven by adaptations to the specific physicochemical makeup of their local environment. As only a small number of anchialine systems have been examined via microbial community studies to date, this work is particularly valuable, contributing new insight regarding the microbial residents of these important and sensitive environments.

Background Each year, approximately 9.5 million metric tons of plastic waste enter the ocean with the potential to adversely impact all trophic levels. Until now, our understanding of the impact of plastic pollution on marine microorganisms has been largely restricted to the microbial assemblages that colonize plastic particles. However, plastic debris also leaches considerable amounts of chemical additives into the water, and this has the potential to impact key groups of planktonic marine microbes, not just those organisms attached to plastic surfaces. Results To investigate this, we explored the population and genetic level responses of a marine microbial community following exposure to leachate from a common plastic (polyvinyl chloride) or zinc, a specific plastic additive. Both the full mix of substances leached from polyvinyl chloride (PVC) and zinc alone had profound impacts on the taxonomic and functional diversity of our natural planktonic community. Microbial primary producers, both prokaryotic and eukaryotic, which comprise the base of the marine food web, were strongly impaired by exposure to plastic leachates, showing significant declines in photosynthetic efficiency, diversity, and abundance. Key heterotrophic taxa, such as SAR11, which are the most abundant planktonic organisms in the ocean, also exhibited significant declines in relative abundance when exposed to higher levels of PVC leachate. In contrast, many copiotrophic bacteria, including members of the Alteromonadales, dramatically increased in relative abundance under both exposure treatments. Moreover, functional gene and genome analyses, derived from metagenomes, revealed that PVC leachate exposure selects for fast-adapting, motile organisms, along with enrichment in genes usually associated with pathogenicity and an increased capacity to metabolize organic compounds leached from PVC. Conclusions This study shows that substances leached from plastics can restructure marine microbial communities with the potential for significant impacts on trophodynamics and biogeochemical cycling. These findings substantially expand our understanding of the ways by which plastic pollution impact life in our oceans, knowledge which is particularly important given that the burden of plastic pollution in the marine environment is predicted to continue to rise.

Investigating the composition and metabolic capacity of aquatic microbial assemblages usually requires the filtration of multi-litre samples, which are up to 1 million-fold larger than the microenvironments within which microbes are predicted to be spatially organised. To determine if community profiles can be reliably generated from microlitre volumes, we sampled seawater at a coastal and an oceanic site, filtered and homogenised them, and extracted DNA from bulk samples (2 L) and microvolumes (100, 10 and 1 μL) using two new approaches. These microvolume DNA extraction methods involve either physical or chemical lysis (through pH/thermal shock and lytic enzymes/surfactants, respectively), directly followed by the capture of DNA on magnetic beads. Downstream analysis of extracted DNA using both amplicon sequencing and metagenomics, revealed strong correlation with standard large volume approaches, demonstrating the fidelity of taxonomic and functional profiles of microbial communities in as little as 1 μL of seawater. This volume is six orders of magnitude smaller than most standard operating procedures for marine metagenomics, which will allow precise sampling of the heterogenous landscape that microbes inhabit.

a b s t r a c tThe microbiome present in individuals of Talitrus saltator belonging to seven populations distributed along the Tuscan coast (Italy) was assessed by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Talitrus saltator is one of the key species of the damp band of European sandy beaches and despite of the large interest on animal-associated bacteria, only a few and preliminary data were present. Results showed a high diversity of the microbiome, composed mainly by members of Alphaproteobacteria, Gammaproteobacteria, Bacillales and Clostridiales classes. The microbiome fingerprints were highly variable among individuals, even from the same populations, the inter-individual differences accounting for 88.7% of total fingerprint variance. However, statistically significant population-specific microbiome signatures were detected, and accounted for the remaining 11.3% of total fingerprint variance. These population-specific differences were mainly attributed to sequences from members of known host-associated bacteria such as Gammaproteobacteria and Betaproteobacteria, Cytophagia and Spirochaetia. This study showed the high complexity of the microbiome associated with an amphipod species and on the inter-individual microbiome variation with potential importance for understanding amphipod trophic and ecologic processes.