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The global biodiversity and land degradation crises have brought about an urgent need and great demand for restoration actions. However, restoration outcomes are often less than ideal, indicating a need for improved restoration practices. Soil microbiota are extremely diverse and functionally important and should be further considered in restoration. However, despite their importance, there remains a gap in understanding of how soil microbiota respond following native plant revegetation. Several studies have used cross-sectional study designs of restoration chronosequences to infer that revegetation causes the recovery of soil microbiota, but it is near-impossible to determine cause and effect relationships with cross-sectional study designs. Here we used high-throughput amplicon sequencing of the bacterial 16s rRNA gene from soil samples collected at two timepoints, 6 years apart, at a revegetation chronosequence in South Australia. Our results show some indications of recovery but not the additional recovery in bacterial community composition toward the reference sites as expected after this 6-year period-a result that appears at odds to the expected patterns of revegetation causing recovery of soil microbiota. Spatially dependent factors (e.g. soil chemistry), biotic and abiotic barriers, seasonal differences in sampling, and variability among the ecological reference sites could each help explain this apparent lack of additional microbial recovery. More detailed longitudinal and/or experimental manipulation work is required to further examine the cause-effect relationships. Our study contributes important new information and highlights knowledge gaps in how soil microbiota respond to revegetation, and we urge caution when attempting to infer causation from cross-sectional chronosequence studies.

Environmental microbiota are becoming more conventional components of restoration ecology studies due to their functional importance in ecosystems. Studying these microbiota offers insight into how they respond to, and potentially drive, ecosystem restoration. However, microbes are everywhere and therefore they pose a risk to sample integrity via uncontrolled contamination, and many of these risks are introduced before entering a molecular facility. Field ecologists who have limited experience in microbial and/or molecular studies may lack the knowledge on how to mitigate microbial contamination risks and, accordingly, may find rigorous collection of microbial samples a daunting task. Here, we present a practical guide that builds on our previous paper to help manage the risks of microbial contamination when undertaking a microbiota restoration study prior to entering a molecular facility. We cover study design and planning, undertaking field sampling, and sample transport and storage. We hope to provide a useful and practical guide to restoration ecologists who wish to include a microbiota component in their studies. If done well, this inclusion offers improved research quality and ultimately enhanced restoration outcomes.

Mining activities modify both aboveground and belowground ecological communities, presenting substantial challenges for restoration. The soil microbiome is one of these impacted communities and performs important ecosystem functions but receives limited focus in restoration. Sequencing soil DNA enables accurate and cost‐effective assessment of soil microbiota, allowing for comparisons across land use, environmental, and temporal gradients. We used amplicon sequencing of the bacterial 16s rRNA gene extracted from soil samples across a 28‐year post‐mining rehabilitation chronosequence to assess soil bacterial composition and diversity following rehabilitation at a bauxite mine in Western Australia's jarrah forest. We show that while bacterial alpha diversity did not differ between reference and rehabilitated sites, bacterial community composition changed dramatically across the chronosequence, suggesting strong impacts by mining and rehabilitation activities. Bacterial communities generally became increasingly similar to unmined reference sites with time since rehabilitation. Soil from sites rehabilitated as recently as 14 years ago did not have significantly different communities to reference sites. Overall, our study provides evidence indicating the recovery of soil bacterial communities toward reference states following rehabilitation. Including several ecological reference sites revealed substantial natural variability in bacterial communities from within a single mine site. We urge future restoration chronosequence studies to sample reference sites that geographically span the restored sites and/or are spatially paired with restored sites to ensure this variability is captured and to improve any inferences on recovery.

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