Global distribution and diversity of marine euendolithic cyanobacteria.

Euendolithic, or true-boring, cyanobacteria actively erode carbonate-containing substrata in a wide range of environments and pose significant risks to calcareous marine fauna. Their boring activities cause structural damage and increase susceptibility to disease and are projected to only intensify with global climate change. Most research has, however, focused on tropical coral systems, and limited information exists on the global distribution, diversity, and substratum specificity of euendoliths. This metastudy aimed to collate existing 16S rRNA gene surveys along with novel data from the south coast of South Africa to investigate the global distribution and genetic diversity of endoliths to identify a "core endolithic cyanobacterial microbiome" and assess global diversification of euendolithic cyanobacteria. The cyanobacterial families Phormidesmiaceae, Nodosilineaceae, Nostocaceae, and Xenococcaceae were the most prevalent, found in >92% of categories surveyed. All four known euendolith clusters were detected in both intertidal and subtidal habitats, in the North Atlantic, Mediterranean, and South Pacific oceans, across temperate latitudes, and within rock, travertine tiles, coral, shell, and coralline algae substrata. Analysis of the genetic variation within clusters revealed many organisms to be unique to substratum type and location, suggesting high diversity and niche specificity. Euendoliths are known to have important effects on their hosts. This is particularly important when hosts are globally significant ecological engineers or habitat-forming species. The findings of this study indicate high ubiquity and diversity of euendolithic cyanobacteria, suggesting high adaptability, which may lead to increased community and ecosystem-level effects with changing climatic conditions favoring the biochemical mechanisms of cyanobacterial bioerosion.

Swapping Metagenomics Preprocessing Pipeline Components Offers Speed and Sensitivity Increases.

Increasing data volumes on high-throughput sequencing instruments such as the NovaSeq 6000 leads to long computational bottlenecks for common metagenomics data preprocessing tasks such as adaptor and primer trimming and host removal. Here, we test whether faster recently developed computational tools (Fastp and Minimap2) can replace widely used choices (Atropos and Bowtie2), obtaining dramatic accelerations with additional sensitivity and minimal loss of specificity for these tasks. Furthermore, the taxonomic tables resulting from downstream processing provide biologically comparable results. However, we demonstrate that for taxonomic assignment, Bowtie2's specificity is still required. We suggest that periodic reevaluation of pipeline components, together with improvements to standardized APIs to chain them together, will greatly enhance the efficiency of common bioinformatics tasks while also facilitating incorporation of further optimized steps running on GPUs, FPGAs, or other architectures. We also note that a detailed exploration of available algorithms and pipeline components is an important step that should be taken before optimization of less efficient algorithms on advanced or nonstandard hardware. IMPORTANCE In shotgun metagenomics studies that seek to relate changes in microbial DNA across samples, processing the data on a computer often takes longer than obtaining the data from the sequencing instrument. Recently developed software packages that perform individual steps in the pipeline of data processing in principle offer speed advantages, but in practice they may contain pitfalls that prevent their use, for example, they may make approximations that introduce unacceptable errors in the data. Here, we show that differences in choices of these components can speed up overall data processing by 5-fold or more on the same hardware while maintaining a high degree of correctness, greatly reducing the time taken to interpret results. This is an important step for using the data in clinical settings, where the time taken to obtain the results may be critical for guiding treatment.

Fast and accurate distance-based phylogenetic placement using divide and conquer.

Phylogenetic placement of query samples on an existing phylogeny is increasingly used in molecular ecology, including sample identification and microbiome environmental sampling. As the size of available reference trees used in these analyses continues to grow, there is a growing need for methods that place sequences on ultra-large trees with high accuracy. Distance-based placement methods have recently emerged as a path to provide such scalability while allowing flexibility to analyse both assembled and unassembled environmental samples. In this study, we introduce a distance-based phylogenetic placement method, APPLES-2, that is more accurate and scalable than existing distance-based methods and even some of the leading maximum-likelihood methods. This scalability is owed to a divide-and-conquer technique that limits distance calculation and phylogenetic placement to parts of the tree most relevant to each query. The increased scalability and accuracy enables us to study the effectiveness of APPLES-2 for placing microbial genomes on a data set of 10,575 microbial species using subsets of 381 marker genes. APPLES-2 has very high accuracy in this setting, placing 97% of query genomes within three branches of the optimal position in the species tree using 50 marker genes. Our proof-of-concept results show that APPLES-2 can quickly place metagenomic scaffolds on ultra-large backbone trees with high accuracy as long as a scaffold includes tens of marker genes. These results pave the path for a more scalable and widespread use of distance-based placement in various areas of molecular ecology.

Cydrasil 3, a curated 16S rRNA gene reference package and web app for cyanobacterial phylogenetic placement.

Cyanobacteria are a widespread and important bacterial phylum, responsible for a significant portion of global carbon and nitrogen fixation. Unfortunately, reliable and accurate automated classification of cyanobacterial 16S rRNA gene sequences is muddled by conflicting systematic frameworks, inconsistent taxonomic definitions (including the phylum itself), and database errors. To address this, we introduce Cydrasil 3 ( https://www.cydrasil.org ), a curated 16S rRNA gene reference package, database, and web application designed to provide a full phylogenetic perspective for cyanobacterial systematics and routine identification. Cydrasil 3 contains over 1300 manually curated sequences longer than 1100 base pairs and can be used for phylogenetic placement or as a reference sequence set for de novo phylogenetic reconstructions. The web application (utilizing PaPaRA and EPA-ng) can place thousands of sequences into the reference tree and has detailed instructions on how to analyze results. While the Cydrasil web application offers no taxonomic assignments, it instead provides phylogenetic placement, as well as a searchable database with curation notes and metadata, and a mechanism for community feedback.

Coleofasciculaceae, a Monophyletic Home for the Microcoleus steenstrupii Complex and Other Desiccation-tolerant Filamentous Cyanobacteria.

Cyanobacteria classified as Microcoleus steenstrupii play a significant role as pioneers of biological soil crusts (biocrusts), but this taxon is recognized to constitute a diverse complex of strains and field populations. With the aim of clarifying its systematics, we conducted a polyphasic characterization of this and allied taxa. A 16S ribosomal gene meta-analysis of published environmental sequences showed that the complex encompasses a variety of well supported genus-level clades with clade-specific environmental preferences, indicating significant niche differentiation. Fifteen strains in the M. steenstrupii complex were selected as representative of naturally occurring clades and studied using 16S rRNA gene phylogeny, morphology, and niche delineation with respect to temperature and rainfall. Bayesian phylogenetic reconstructions within a comprehensive, curated database of long 16S rRNA cyanobacterial sequences (1,000 base pairs or more) showed that they all belonged in a monophyletic, family-level clade (91.4% similarity) that included some other known genera of desiccation-resistant, largely terrestrial, filamentous, nonheterocystous cyanobacteria, including Coleofasciculus, the type genus for the family Coleofasciculaceae. To accommodate this biodiversity, we redescribe the Coleofasciculaceae, now composed of 11 genera, among which six are newly described herein (Funiculus, Parifilum, Arizonema, Crassifilum, Crustifilum, and Allocoleopsis), and five were previously recognized (Porphyrosiphon, Coleofasciculus, Pycnacronema, Potamolinea, and Wilmottia). We provide an evaluation of their respective niches and global distributions within biocrusts based on published molecular data. This new systematics treatment should help simplify and improve our understanding of the biology of terrestrial cyanobacteria.

Succession and Colonization Dynamics of Endolithic Phototrophs within Intertidal Carbonates.

Photosynthetic endolithic communities are common in shallow marine carbonates, contributing significantly to their bioerosion. Cyanobacteria are well known from these settings, where a few are euendoliths, actively boring into the virgin substrate. Recently, anoxygenic phototrophs were reported as significant inhabitants of endolithic communities, but it is unknown if they are euendoliths or simply colonize available pore spaces secondarily. To answer this and to establish the dynamics of colonization, nonporous travertine tiles were anchored onto intertidal beach rock in Isla de Mona, Puerto Rico, and developing endolithic communities were examined with time, both molecularly and with photopigment biomarkers. By 9 months, while cyanobacterial biomass and diversity reached levels indistinguishable from those of nearby climax communities, anoxygenic phototrophs remained marginal, suggesting that they are secondary colonizers. Early in the colonization, a novel group of cyanobacteria (unknown boring cluster, UBC) without cultivated representatives, emerged as the most common euendolith, but by 6 months, canonical euendoliths such as Plectonema (Leptolyngbya) sp., Mastigocoleus sp., and Pleurocapsalean clades displaced UBC in dominance. Later, the proportion of euendolithic cyanobacterial biomass decreased, as nonboring endoliths outcompeted pioneers within the already excavated substrate. Our findings demonstrate that endolithic cyanobacterial succession within hard carbonates is complex but can attain maturity within a year's time.

The Compositionally Distinct Cyanobacterial Biocrusts From Brazilian Savanna and Their Environmental Drivers of Community Diversity.

The last decade was marked by efforts to define and identify the main cyanobacterial players in biological crusts around the world. However, not much is known about biocrusts in Brazil's tropical savanna (cerrado), despite the existence of environments favorable to their development and ecological relevance. We examined the community composition of cyanobacteria in biocrusts from six sites distributed in the Southeast of the country using high throughput sequencing of 16S rRNA and phylogenetic placement in the wider context of biocrusts from deserts. Sequences ascribable to 22 genera of cyanobacteria were identified. Although a significant proportion of sequences did not match those of known cyanobacteria, several clades of Leptolyngbya and Porphyrosiphon were found to be the most abundant. We identified significant differences in dominance and overall composition among the cerrado sites, much larger than within-site variability. The composition of cerrado cyanobacterial communities was distinct from those known in biocrusts from North American deserts. Among several environmental drivers considered, the opposing trend of annual precipitation and mean annual temperature best explained the variability in community composition within Brazilian biocrusts. Their compositional uniqueness speaks of the need for dedicated efforts to study the ecophysiology of tropical savanna biocrust and their roles in ecosystem function for management and preservation.

Exposure to predicted precipitation patterns decreases population size and alters community structure of cyanobacteria in biological soil crusts from the Chihuahuan Desert.

Cyanobacteria typically colonize the surface of arid soils, building biological soil crust (biocrusts) that provide a variety of ecosystem benefits, ranging from fertilization to stabilization against erosion. We investigated how future scenarios in precipitation anticipated for the Northern Chihuahuan Desert affected abundance and composition of biocrust cyanobacteria in two grassland ecosystems. Scenarios included a decrease in precipitation and a delay of monsoon rainfall. After three years, both treatments negatively affected cyanobacteria, although the effects of monsoon delay were milder than those of decreased precipitation. Mature biocrusts in black grama grassland suffered severe losses in cyanobacterial biomass and diversity, but compositionally simpler biocrusts in blue grama-dominated grassland maintained biomass, only suffering diversity losses. This could be partially explained by the differential sensitivity of cyanobacterial taxa: nitrogen-fixing Scytonema spp. were the most sensitive, followed by phylotypes in the Microcoleus steenstrupii complex. Microcoleus vaginatus was the least affected in all cases, but is known to be very sensitive to warming. We predict that altered precipitation will tend to prevent biocrusts from reaching successional maturity, selecting for M. vaginatus over competing M. steenstrupii, among pioneer biocrust-formers. A shift towards heat-sensitive M. vaginatus could ultimately destabilize biocrusts when precipitation changes are combined with global warming.

A New Niche for Anoxygenic Phototrophs as Endoliths.

Anoxygenic phototrophic bacteria (APBs) occur in a wide range of aquatic habitats, from hot springs to freshwater lakes and intertidal microbial mats. Here, we report the discovery of a novel niche for APBs: endoliths within marine littoral carbonates. In a study of 40 locations around Isla de Mona, Puerto Rico, and Menorca, Spain, 16S rRNA high-throughput sequencing of endolithic community DNA revealed the presence of abundant phylotypes potentially belonging to well-known APB clades. An ad hoc phylogenetic classification of these sequences enabled us to refine the assignments more stringently. Even then, all locations contained such putative APBs, often reaching a significant proportion of all phototrophic sequences. In fact, in some 20% of samples, their contribution exceeded that of oxygenic phototrophs, previously regarded as the major type of endolithic microbe in carbonates. The communities contained representatives of APBs in the Chloroflexales, various proteobacterial groups, and Chlorobi The most abundant phylotypes varied with geography: on Isla de Mona, Roseiflexus and Chlorothrix-related phylotypes dominated, whereas those related to Erythrobacter were the most common in Menorca. The presence of active populations of APBs was corroborated through an analysis of photopigments: bacteriochlorophylls were detected in all samples, bacteriochlorophyll c and a being most abundant. We discuss the potential metabolism and geomicrobial roles of endolithic APBs. Phylogenetic inference suggests that APBs may be playing a role as photoheterotrophs, adding biogeochemical complexity to our understanding of such communities. Given the global extent of coastal carbonate platforms, they likely represent a very large and unexplored habitat for APBs.IMPORTANCE Endolithic microbial communities from carbonates, which have been explored for over 2 centuries in predominantly naturalistic studies, were thought to be primarily composed of eukaryotic algae and cyanobacteria. Our report represents a paradigm shift in this regard, at least for the marine environment, demonstrating the presence of ubiquitous and abundant populations of APBs in this habitat. It raises questions about the role of these organisms in the geological dynamics of coastal carbonates, including coral reefs.