Environmental conditions play a key role in controlling the composition and diversity of Colombian biocrust microbiomes.

Drylands soils worldwide are naturally colonized by microbial communities known as biocrusts. These soil microbiomes render important ecosystem services associated with soil fertility, water holding capacity, and stability to the areas they cover. Because of the importance of biocrusts in the global cycling of nutrients, there is a growing interest in describing the many microbial configurations these communities display worldwide. However, comprehensive 16S rRNA genes surveys of biocrust communities do not exist for much of the planet: for example, in the continents of South America and the northern part of Africa. The absence of a global understanding of biocrust biodiversity has lead us to assign a general importance to community members that may, in fact, be regional. Here we report for the first time the presence of biocrusts in Colombia (South America) through 16S rRNA genes surveys across an arid, a semi-arid and a dry subtropical region within the country. Our results constitute the first glance of the Bacterial/Archaeal communities associated with South American biocrust microbiomes. Communities where cyanobacteria other than Microcoleus vaginatus prevail, despite the latter being considered a key species elsewhere, illustrate differentiable results in these surveys. We also find that the coastal biocrust communities in Colombia include halo-tolerant and halophilic species, and that niche preference of some nitrogen fixing organisms deviate from previously described global trends. In addition, we identified a high proportion (ranging from 5 to 70%, in average) of cyanobacterial sequences that did not match any formally described cyanobacterial species. Our investigation of Colombian biocrusts points to highly diverse communities with climatic regions controlling taxonomic configurations. They also highlight an extensive local diversity to be discovered which is central to better design management and restoration strategies for drylands soils currently undergoing disturbances due to land use and global warming. Finally, this field study highlights the need for an improved mechanistic understanding of the response of key biocrust community members to changes in moisture and temperature.

Spatial self-segregation of pioneer cyanobacterial species drives microbiome organization in biocrusts.

Microbial communities are typically characterized by some degree of self-organization. In biological soil crust (biocrust) communities, vertical organization of resident populations at the mm scale is driven by organismal adaptations to physicochemical microniches. However, the extent of horizontal organization and its driving processes are unknown. Using a combination of observational and genetic mapping, we provide evidence for a highly defined, horizontal self-organization (patchiness) at the mm to cm scale in a successionally early biocrust community dominated by the pioneer cyanobacteria, Microcoleus vaginatus (Microcoleaceae) and Parifilum sp. (Coleofasciculaceae). Experiments with representative isolates of each species demonstrate that the phenomenon is driven by active spatial segregation based on cross-species sensing through the exometabolome acted upon with motility responses. Further, we show that both species share the ability to enrich for specialized cyanospheres of heterotrophic bacteria at smaller scales, and that these cyanospheres are characterized by compositional host-specificity, thus expanding the reach of spatial patchiness beyond primary producers. Our results highlight the importance of specific microbial interactions in the emergence of microbiome compositional architecture and the enhancement of microbial diversity.

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.

A symbiotic nutrient exchange within the cyanosphere microbiome of the biocrust cyanobacterium, Microcoleus vaginatus.

Microcoleus vaginatus plays a prominent role as both primary producer and pioneer in biocrust communities from dryland soils. And yet, it cannot fix dinitrogen, essential in often nitrogen-limited drylands. But a diazotroph-rich "cyanosphere" has been described in M. vaginatus, hinting that there exists a C for N exchange between the photoautotroph and heterotrophic diazotrophs. We provide evidence for this by establishing such a symbiosis in culture and by showing that it is selective and dependent on nitrogen availability. In natural populations, provision of nitrogen resulted in loss of diazotrophs from the cyanosphere of M. vaginatus compared to controls, but provision of phosphorus did not. Co-culturing of pedigreed cyanosphere diazotroph isolates with axenic M. vaginatus resulted in copious growth in C and N-free medium, but co-culture with non-cyanosphere diazotrophs or other heterotrophs did not. Unexpectedly, bundle formation in M. vaginatus, diacritical to the genus but not seen in axenic culture, was restored in vitro by imposed nitrogen limitation or, even more strongly, by co-culture with diazotrophic partners, implicating this trait in the symbiosis. Our findings provide direct evidence for a symbiotic relationship between M. vaginatus and its cyanosphere and help explain how it can be a global pioneer in spite of its genetic shortcomings.

What Could Explain δ13C Signatures in Biocrust Cyanobacteria of Drylands?

Dryland ecosystems are increasing in geographic extent and contribute greatly to interannual variability in global carbon dynamics. Disentangling interactions among dominant primary producers, including plants and autotrophic microbes, can help partition their contributions to dryland C dynamics. We measured the δ13C signatures of biological soil crust cyanobacteria and dominant plant species (C3 and C4) across a regional scale in the southwestern USA to determine if biocrust cyanobacteria were coupled to plant productivity (using plant-derived C mixotrophically), or independent of plant activity (and therefore purely autotrophic). Cyanobacterial assemblages located next to all C3 plants and one C4 species had consistently more negative δ13C (by 2‰) than the cyanobacteria collected from plant interspaces or adjacent to two C4 Bouteloua grass species. The differences among cyanobacterial assemblages in δ13C could not be explained by cyanobacterial community composition, photosynthetic capacity, or any measured leaf or root characteristics (all slopes not different from zero). Thus, microsite differences in abiotic conditions near plants, rather than biotic interactions, remain a likely mechanism underlying the observed δ13C patterns to be tested experimentally.

A Fog-Irrigated Soil Substrate System Unifies and Optimizes Cyanobacterial Biocrust Inoculum Production.

Biological soil crusts (biocrusts) are globally important microbial communities inhabiting the top layer of soils. They provide multiple services to dryland ecosystems but are particularly vulnerable to anthropogenic disturbance from which they naturally recover only slowly. Assisted inoculation with cyanobacteria is held as a promising approach to promote biocrust regeneration. Two different methodologies have been developed for this purpose: mass cultivation of biocrust pioneer species (such as the cyanobacteria Microcoleus spp.) on cellulose supports, and polymicrobial cultivation of biocrusts in soils within greenhouse settings. Here, we aimed to test a novel method to grow cyanobacterial biocrust inoculum based on fog irrigation of soil substrates (FISS) that can be used with either culture-based or mixed-community approaches. We found that the FISS system presents clear advantages over previous inoculum production methodologies; overall, FISS eliminates the need for specialized facilities and decreases user effort. Specifically, there were increased microbial yields and simplification of design compared to those of the culture-based and mixed-community approaches, respectively. Its testing also allows us to make recommendations on underexplored aspects of biocrust restoration: (i) field inoculation levels should be equal to or greater than the biomass found in the substrate and (ii) practices regarding evaluation of cyanobacterial biomass should, under certain circumstances, include proxies additional to chlorophyll aIMPORTANCE Biocrust inoculum production for use in dryland rehabilitation is a powerful tool in combating the degradation of dryland ecosystems. However, the facilities and effort required to produce high-quality inoculum are often a barrier to effective large-scale implementation by land managers. By unifying and optimizing the two foremost methods for cyanobacterial biocrust inoculum production, our work improves on the ease and cost with which biocrust restoration technology can be translated to practical widespread implementation.

Niche Partitioning with Temperature among Heterocystous Cyanobacteria (Scytonema spp., Nostoc spp., and Tolypothrix spp.) from Biological Soil Crusts.

Heterocystous cyanobacteria of biocrusts are key players for biological fixation in drylands, where nitrogen is only second to water as a limiting resource. We studied the niche partitioning among the three most common biocrust heterocystous cyanobacteria sts using enrichment cultivation and the determination of growth responses to temperature in 30 representative isolates. Isolates of Scytonema spp. were most thermotolerant, typically growing up to 40 °C, whereas only those of Tolypothrix spp. grew at 4 °C. Nostoc spp. strains responded well at intermediate temperatures. We could trace the heat sensitivity in Nostoc spp. and Tolypothrix spp. to N2-fixation itself, because the upper temperature for growth increased under nitrogen replete conditions. This may involve an inability to develop heterocysts (specialized N2-fixing cells) at high temperatures. We then used a meta-analysis of biocrust molecular surveys spanning four continents to test the relevance of this apparent niche partitioning in nature. Indeed, the geographic distribution of the three types was clearly constrained by the mean local temperature, particularly during the growth season. This allows us to predict a potential shift in dominance in many locales as a result of global warming, to the benefit of Scytonema spp. populations.

Optimizing the Production of Nursery-Based Biological Soil Crusts for Restoration of Arid Land Soils.

Biological soil crusts (biocrusts) are topsoil communities formed by cyanobacteria or other microbial primary producers and are typical of arid and semiarid environments. Biocrusts promote a range of ecosystem services, such as erosion resistance and soil fertility, but their degradation by often anthropogenic disturbance brings about the loss of these services. This has prompted interest in developing restoration techniques. One approach is to source biocrust remnants from the area of interest for scale-up cultivation in a microbial "nursery" that produces large quantities of high-quality inoculum for field deployment. However, growth dynamics and the ability to reuse the produced inoculum for continued production have not been assessed. To optimize production, we followed nursery growth dynamics of biocrusts from cold (Great Basin) and hot (Chihuahuan) deserts. Peak phototrophic biomass was attained between 3 and 7 weeks in cold desert biocrusts and at 12 weeks in those from hot deserts. We also reused the resultant biocrust inoculum to seed successive incubations, tracking both phototroph biomass and cyanobacterial community structure using 16S rRNA gene amplicon sequencing. Hot desert biocrusts showed little to no viability upon reinoculation, while cold desert biocrusts continued to grow, but at the expense of progressive shifts in species composition. This leads us to discourage the reuse of nursery-grown inoculum. Surprisingly, growth was highly variable among replicates, and overall yields were low, a fact that we attribute to the demonstrable presence of virulent and stochastically distributed but hitherto unknown cyanobacterial pathogens. We provide recommendations to avoid pathogen incidence in the process.IMPORTANCE Biocrust communities provide important ecosystem services for arid land soils, such as soil surface stabilization promoting erosion resistance and contributing to overall soil fertility. Anthropogenic degradation to biocrust communities (through livestock grazing, agriculture, urban sprawl, and trampling) is common and significant, resulting in a loss of those ecosystem services. Losses impact both the health of the native ecosystem and the public health of local populations due to enhanced dust emissions. Because of this, approaches for biocrust restoration are being developed worldwide. Here, we present optimization of a nursery-based approach to scaling up the production of biocrust inoculum for field restoration with respect to temporal dynamics and reuse of biological materials. Unexpectedly, we also report on complex population dynamics, significant spatial variability, and lower than expected yields that we ascribe to the demonstrable presence of cyanobacterial pathogens, the spread of which may be enhanced by some of the nursery production standard practices.

Genomics, Biology and Phylogeny Aurantiochytrium acetophilum sp. nov. (Thraustrochytriaceae), Including First Evidence of Sexual Reproduction.

Strain HS-399 was isolated from a mangrove swamp in Biscayne Bay (Florida, USA) and selected for its capacity to accumulate lipids (84.0±1.0% DW), particularly docosahexaenoic acid (DHA; 22:6 n-3) (28.3±0.1% DW). Molecular phylogenetic analysis demonstrated that the new organism belonged to the genus Aurantiochytrium, and when the whole nuclear genome was blasted against the type species (and only described species), A. limacinum SR21, there was a 5.38% difference at the protein level. We described our new organism as Aurantiochytrium acetophilum sp. nov. (Thraustochytriaceae, Thraustochytriales) using light microscopy, electron microscopy, substrate assimilation, biochemical composition and nuclear genomic data. We found some characteristics of biotechnological relevance that were not previously described in this family. First, strain HS-399 of A. acetophilum was extremely tolerant to acetate toxicity, and it used this substrate as a sole carbon source. Second, we observed putative gametes that fused together to form a zygote. Zygote fate and the life stage with meiosis were not determined; however, we found several meiosis genes in the genome, further supporting the possibility of breeding for these industrially relevant organisms.

Spatial segregation of the biological soil crust microbiome around its foundational cyanobacterium, Microcoleus vaginatus, and the formation of a nitrogen-fixing cyanosphere.

BACKGROUND: Biological soil crusts (biocrusts) are a key component of arid land ecosystems, where they render critical services such as soil surface stabilization and nutrient fertilization. The bundle-forming, filamentous, non-nitrogen-fixing cyanobacterium Microcoleus vaginatus is a pioneer primary producer, often the dominant member of the biocrust microbiome, and the main source of leaked organic carbon. We hypothesized that, by analogy to the rhizosphere of plant roots, M. vaginatus may shape the microbial populations of heterotrophs around it, forming a specialized cyanosphere. RESULTS: By physically isolating bundles of M. vaginatus from biocrusts, we were able to study the composition of the microbial populations attached to it, in comparison to the bulk soil crust microbiome by means of high-throughput 16S rRNA sequencing. We did this in two M. vaginatus-dominated biocrust from distinct desert biomes. We found that a small, selected subset of OTUs was significantly enriched in close proximity to M. vaginatus. Furthermore, we also found that a majority of bacteria (corresponding to some two thirds of the reads) were significantly more abundant away from this cyanobacterium. Phylogenetic placements suggest that all typical members of the cyanosphere were copiotrophs and that many were diazotrophs (Additional file 1: Tables S2 and S3). Nitrogen fixation genes were in fact orders of magnitude more abundant in this cyanosphere than in the bulk biocrust soil as assessed by qPCR. By contrary, competition for light, CO2, and low organic carbon concentrations defined at least a part of the OTUs segregating from the cyanobacterium. CONCLUSIONS: We showed that M. vaginatus acts as a significant spatial organizer of the biocrust microbiome. On the one hand, it possesses a compositionally differentiated cyanosphere that concentrates the nitrogen-fixing function. We propose that a mutualism based on C for N exchange between M. vaginatus and copiotrophic diazotrophs helps sustains this cyanosphere and that this consortium constitutes the true pioneer community enabling the colonization of nitrogen-poor soils. On the other hand, a large number of biocrust community members segregate away from the vicinity of M. vaginatus, potentially through competition for light or CO2, or because of a preference for oligotrophy.

Microbial Nursery Production of High-Quality Biological Soil Crust Biomass for Restoration of Degraded Dryland Soils.

Biological soil crusts (biocrusts) are slow-growing, phototroph-based microbial assemblages that develop on the topsoils of drylands. Biocrusts help maintain soil fertility and reduce erosion. Because their loss through human activities has negative ecological and environmental health consequences, biocrust restoration is of interest. Active soil inoculation with biocrust microorganisms can be an important tool in this endeavor. We present a culture-independent, two-step process to grow multispecies biocrusts in open greenhouse nursery facilities, based on the inoculation of local soils with local biocrust remnants and incubation under seminatural conditions that maintain the essence of the habitat but lessen its harshness. In each of four U.S. Southwest sites, we tested and deployed combinations of factors that maximized growth (gauged as chlorophyll a content) while minimizing microbial community shifts (assessed by 16S rRNA sequencing and bioinformatics), particularly for crust-forming cyanobacteria. Generally, doubling the frequency of natural wetting events, a 60% reduction in sunlight, and inoculation by slurry were optimal. Nutrient addition effects were site specific. In 4 months, our approach yielded crusts of high inoculum quality reared on local soil exposed to locally matched climates, acclimated to desiccation, and containing communities minimally shifted in composition from local ones. Our inoculum contained abundant crust-forming cyanobacteria and no significant numbers of allochthonous phototrophs, and it was sufficient to treat ca. 6,000 m2 of degraded dryland soils at 1 to 5% of the typical crust biomass concentration, having started from a natural crust remnant as small as 6 to 30 cm2 IMPORTANCE: Soil surface crusts can protect dryland soils from erosion, but they are often negatively impacted by human activities. Their degradation causes a loss of fertility, increased production of fugitive dust and intensity of dust storms with associated traffic problems, and provokes general public health hazards. Our results constitute an advance in the quest to actively restore biological soil covers by providing a means to obtain high-quality inoculum within a reasonable time (a few months), thereby allowing land managers to recover essential, but damaged, ecosystem services in a sustainable, self-perpetuating way as provided by biocrust communities.

Differential Responses of Dinitrogen Fixation, Diazotrophic Cyanobacteria and Ammonia Oxidation Reveal a Potential Warming-Induced Imbalance of the N-Cycle in Biological Soil Crusts.

N2 fixation and ammonia oxidation (AO) are the two most important processes in the nitrogen (N) cycle of biological soil crusts (BSCs). We studied the short-term response of acetylene reduction assay (ARA) rates, an indicator of potential N2 fixation, and AO rates to temperature (T, -5°C to 35°C) in BSC of different successional stages along the BSC ecological succession and geographic origin (hot Chihuahuan and cooler Great Basin deserts). ARA in all BSCs increased with T until saturation occurred between 15 and 20°C, and declined at 30-35°C. Culture studies using cyanobacteria isolated from these crusts indicated that the saturating effect was traceable to their inability to grow well diazotrophically within the high temperature range. Below saturation, temperature response was exponential, with Q10 significantly different in the two areas (~ 5 for Great Basin BSCs; 2-3 for Chihuahuan BSCs), but similar between the two successional stages. However, in contrast to ARA, AO showed a steady increase to 30-35°C in Great Basin, and Chihuhuan BSCs showed no inhibition at any tested temperature. The T response of AO also differed significantly between Great Basin (Q10 of 4.5-4.8) and Chihuahuan (Q10 of 2.4-2.6) BSCs, but not between successional stages. Response of ARA rates to T did not differ from that of AO in either desert. Thus, while both processes scaled to T in unison until 20°C, they separated to an increasing degree at higher temperature. As future warming is likely to occur in the regions where BSCs are often the dominant living cover, this predicted decoupling is expected to result in higher proportion of nitrates in soil relative to ammonium. As nitrate is more easily lost as leachate or to be reduced to gaseous forms, this could mean a depletion of soil N over large landscapes globally.