Volcanic caves are filled with colorful microbial mats on the walls and ceilings. These volcanic caves are found worldwide, and studies are finding vast bacteria diversity within these caves. One group of bacteria that can be abundant in volcanic caves, as well as other caves, is Actinobacteria. As Actinobacteria are valued for their ability to produce a variety of secondary metabolites, rare and novel Actinobacteria are being sought in underexplored environments. The abundance of novel Actinobacteria in volcanic caves makes this environment an excellent location to study these bacteria. Scanning electron microscopy (SEM) from several volcanic caves worldwide revealed diversity in the morphologies present. Spores, coccoid, and filamentous cells, many with hair-like or knobby extensions, were some of the microbial structures observed within the microbial mat samples. In addition, the SEM study pointed out that these features figure prominently in both constructive and destructive mineral processes. To further investigate this diversity, we conducted both Sanger sequencing and 454 pyrosequencing of the Actinobacteria in volcanic caves from four locations, two islands in the Azores, Portugal, and Hawai'i and New Mexico, USA. This comparison represents one of the largest sequencing efforts of Actinobacteria in volcanic caves to date. The diversity was shown to be dominated by Actinomycetales, but also included several newly described orders, such as Euzebyales, and Gaiellales. Sixty-two percent of the clones from the four locations shared less than 97% similarity to known sequences, and nearly 71% of the clones were singletons, supporting the commonly held belief that volcanic caves are an untapped resource for novel and rare Actinobacteria. The amplicon libraries depicted a wider view of the microbial diversity in Azorean volcanic caves revealing three additional orders, Rubrobacterales, Solirubrobacterales, and Coriobacteriales. Studies of microbial ecology in volcanic caves are still very limited. To rectify this deficiency, the results from our study help fill in the gaps in our knowledge of actinobacterial diversity and their potential roles in the volcanic cave ecosystems.

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Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the ‘caves as laboratory’ paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well‐established methodology of ‘horizon scanning’ to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top‐priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis‐driven studies likely to resonate broadly from the traditional boundaries of this field.

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The terrestrial subsurface microbiome has gained considerable amount of interests in the recent years because of its rich potential resource for biomining novel genes coding for metabolites possessing antimicrobial activities. In our previous study, we identified two Streptomyces isolates, designated as ICC1 and ICC4, from the Iron Curtain Cave, Chilliwack, Canada that exhibited antagonistic activities against the multidrug resistant strains of Escherichia coli . In this study, the genomes of these two isolates were sequenced by Illumina MiSeq, assembled and annotated. The genes associated with secondary metabolite production were identified and annotated using the bioinformatics platforms antiSMASH and BAGEL. ICC1 and ICC4 were then cultivated and ICC1 metabolome characterized by UHPLC-ESI-HRMS. The Global Natural Products Social Molecular Networking was used to identify metabolites based on the MS/MS spectral data. ICC1 and ICC4 showed a high level of sequence identity with the terrestrial bacteria Streptomyces lavendulae ; however, they possess a greater secondary metabolite potential as estimated by the total number of identified biosynthetic gene clusters (BGCs). In particular, ICC1 and ICC4 had a greater number of polyketide and non-ribosomal peptide BGCs. The most frequently detected BGCs were those predicted to generate terpenes, small and low complexity dipeptides and lipids. Spectral analysis clearly identified a number of diketopiperazine products through matched reference spectra for cyclo (Leu-Pro), cyclo (Pro-Val) and cyclo [(4-hydroxyPro)-Leu]. One of the terpenes gene clusters predicted by antiSMASH possesses a seven-gene pathway consistent with diazepinomicin biosynthesis. This molecule contains a very rare core structure and its BGC, to date, has only been identified from a single bacterial genome. The tetrapeptide siderophore coelichelin BGC was unambiguously identified in the genome, however, the metabolite could not be identified from the culture extracts. Two type III polyketides, 2′, 5′ – dimethoxyflavone and nordentatin, were identified from the UHPLC-HRMS data of the aqueous and n -butanolic fractions of Streptomyces sp. ICC1, respectively. A BGC likely encoding these metabolites was predicted in both genomes. The predicted similarities in molecule production and genome shared by these two strains could be an indicative of a cooperative mode of living in extreme habitats instead of a competitive one. This secondary metabolite potential may contribute to the fitness of ICC1 and ICC4 in the Iron Curtain Cave.