SummaryCyanobacteria 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.

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.

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.

Abstract. Endolithic microbial communities are prominent features of intertidal marine habitats, where they colonize a variety of substrates, contributing to their erosion. Almost 2 centuries worth of naturalistic studies focused on a few true-boring (euendolithic) phototrophs, but substrate preference has received little attention. The Isla de Mona (Puerto Rico) intertidal zone offers a unique setting to investigate substrate specificity of endolithic communities since various phosphate rock, limestone and dolostone outcrops occur there. High-throughput 16S rDNA genetic sampling, enhanced by targeted cultivation, revealed that, while euendolithic cyanobacteria were dominant operational taxonomic units (OTUs), the communities were invariably of high diversity, well beyond that reported in traditional studies and implying an unexpected metabolic complexity potentially contributed by secondary colonizers. While the overall community composition did not show differences traceable to the nature of the mineral substrate, we detected specialization among particular euendolithic cyanobacterial clades towards the type of substrate they excavate but only at the OTU phylogenetic level, implying that close relatives have specialized recurrently into particular substrates. The cationic mineral component was determinant in this preference, suggesting the existence in nature of alternatives to the boring mechanism described in culture that is based exclusively on transcellular calcium transport.

Phylogenetic placement of query samples on an existing phylogeny is increasingly used in microbiome analyses and other biological studies. As the size of available reference trees used in microbiome analyses continues to grow, there is a growing need for methods that place sequences on ultra-large trees with high accuracy.In this paper, we build on our previous work and introduce APPLES-2, a distance-based phylogenetic placement method. In extensive studies, we show that APPLES-2 is more accurate and much more scalable than APPLES 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 dataset 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. As a proof of concept, we also tested APPLES-2 for placing scaffolds produced by metagenomic assembly. It was able to place 3318 scaffolds on the backbone tree with 10575 taxa in less than 3 hours. The accuracy of these placement was high as long as a scaffold included tens of marker genes.

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 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.

<p><strong>Abstract.</strong> Endolithic microbial communities are prominent features of intertidal marine habitats, where they colonize a variety of substrates, contributing to their erosion. Almost two centuries worth of naturalistic studies focused on a few true-boring (euendolithic) phototrophs, but substrate preference has received little attention. The Isla de Mona (Puerto Rico) intertidal zone offers a unique setting to investigate substrate specificity of endolithic communities since various phosphate rock, limestone, and dolostone outcrops occur there. High-throughput 16S rDNA genetic sampling, enhanced by targeted cultivation, revealed that, while euendolithic cyanobacteria were dominant, the communities were invariably of high diversity, well beyond that reported in traditional studies, and implying an unexpected metabolic complexity, potentially contributed by secondary colonizers. While the overall community composition did not show differences traceable to the nature of the mineral substrate, we detected specialization among particular euendolithic cyanobacterial clades towards the type of substrate they excavate, but only at the OTU phylogenetic level, implying that close relatives have specialized recurrently into particular substrates. The cationic mineral component was determinant in this preference, calling for the existence in nature of alternatives to the boring mechanism described in culture that is based exclusively on transcellular calcium transport.</p>