Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape.

The cistrome is the complete set of transcription factor (TF) binding sites (cis-elements) in an organism, while an epicistrome incorporates tissue-specific DNA chemical modifications and TF-specific chemical sensitivities into these binding profiles. Robust methods to construct comprehensive cistrome and epicistrome maps are critical for elucidating complex transcriptional networks that underlie growth, behavior, and disease. Here, we describe DNA affinity purification sequencing (DAP-seq), a high-throughput TF binding site discovery method that interrogates genomic DNA with in-vitro-expressed TFs. Using DAP-seq, we defined the Arabidopsis cistrome by resolving motifs and peaks for 529 TFs. Because genomic DNA used in DAP-seq retains 5-methylcytosines, we determined that >75% (248/327) of Arabidopsis TFs surveyed were methylation sensitive, a property that strongly impacts the epicistrome landscape. DAP-seq datasets also yielded insight into the biology and binding site architecture of numerous TFs, demonstrating the value of DAP-seq for cost-effective cistromic and epicistromic annotation in any organism.

Epigenomic Diversity in a Global Collection of Arabidopsis thaliana Accessions.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Persistence and plasticity in bacterial gene regulation.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Transcriptome and DNA methylome divergence of inflorescence development between 2 ecotypes in Panicum hallii.

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation. Hall's panicgrass (Panicum hallii, P. hallii) is a wild perennial grass that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the 2 major ecotypes in P. hallii, the upland ecotype (P. hallii var hallii, HAL2 genotype) with compact inflorescence and large seed and the lowland ecotype (P. hallii var filipes, FIL2 genotype) with an open inflorescence and small seed. Here we conducted a comparative analysis of the transcriptome and DNA methylome, an epigenetic mark that influences gene expression regulation, across different stages of inflorescence development using genomic references for each ecotype. Global transcriptome analysis of differentially expressed genes (DEGs) and co-expression modules underlying the inflorescence divergence revealed the potential role of cytokinin signaling in heterochronic changes. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of P. hallii inflorescence. We found that a large proportion of differentially methylated regions (DMRs) were located in the flanking regulatory regions of genes. Intriguingly, we observed a substantial bias of CHH hypermethylation in the promoters of FIL2 genes. The integration of DEGs, DMRs, and Ka/Ks ratio results characterized the evolutionary features of DMR-associated DEGs that contribute to the divergence of the P. hallii inflorescence. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a genomic resource for perennial grass biology.

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus.

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle.

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. We interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

High-throughput identification of novel heat tolerance genes via genome-wide pooled mutant screens in the model green alga Chlamydomonas reinhardtii.

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome-wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat-sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high-confidence Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.

Highly integrated single-base resolution maps of the epigenome in Arabidopsis.

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

SECONDARY WALL ASSOCIATED MYB1 is a positive regulator of secondary cell wall thickening in Brachypodium distachyon and is not found in the Brassicaceae.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon.

Expression and regulatory asymmetry of retained Arabidopsis thaliana transcription factor genes derived from whole genome duplication.

BACKGROUND: Transcription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after whole genome duplication (WGD) event, transcription factors are retained at a significantly higher rate. Little is known about how TF duplicates have diverged in their expression and regulation, the answer to which may contribute to a better understanding of the elevated retention rate among TFs. RESULTS: Here we assessed what features may explain differences in the retention of TF duplicates and other genes using Arabidopsis thaliana as a model. We integrated 34 expression, sequence, and conservation features to build a linear model for predicting the extent of duplicate retention following WGD events among TFs and 19 groups of genes with other functions. We found that TFs was the least well predicted, demonstrating the features of TFs are substantially deviated from duplicate genes in other function groups. Consistent with this, the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates: one "ancestral" TF duplicate retains most ancestral expression and cis-regulatory sites, while the "non-ancestral" duplicate is enriched for novel regulatory sites. By modeling the retention of ancestral expression and cis-regulatory states in duplicate pairs using a system of differential equations, we found that TF duplicate pairs in a partitioned state are preferentially maintained. CONCLUSIONS: These TF duplicates with asymmetrically partitioned ancestral states are likely maintained because one copy retains ancestral functions while the other, at least in some cases, acquires novel cis-regulatory sites that may be important for novel, adaptive traits.

Shoring up the base: the development and regulation of cortical sclerenchyma in grass nodal roots.

Plants depend on the combined action of a shoot-root-soil system to maintain their anchorage to the soil. Mechanical failure of any component of this system results in lodging, a permanent and irreversible inability to maintain vertical orientation. Models of anchorage in grass crops identify the compressive strength of roots near the soil surface as key determinant of resistance to lodging. Indeed, studies of disparate grasses report a ring of thickened, sclerenchyma cells surrounding the root cortex, present only at the base of nodal roots. Here, in the investigation of the development and regulation of this agronomically important trait, we show that development of these cells is uncoupled from the maturation of other secondary cell wall-fortified cells, and that cortical sclerenchyma wall thickening is stimulated by mechanical forces transduced from the shoot to the root. We also show that exogenous application of gibberellic acid stimulates thickening of lignified cell types in the root, including cortical sclerenchyma, but is not sufficient to establish sclerenchyma identity in cortex cells. Leveraging the ability to manipulate cortex development via mechanical stimulus, we show that cortical sclerenchyma development alters root mechanical properties and improves resistance to lodging. We describe transcriptome changes associated with cortical sclerenchyma development under both ambient and mechanically stimulated conditions and identify SECONDARY WALL NAC7 as a putative regulator of mechanically responsive cortex cell wall development at the root base.

Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

An optimized ChIP-Seq framework for profiling histone modifications in Chromochloris zofingiensis.

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high-quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein cross-linking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

Linking genotype to phenotype using the Arabidopsis unimutant collection.

The large collections of Arabidopsis thaliana sequence-indexed T-DNA insertion mutants are among the most important resources to emerge from the sequencing of the genome. Several laboratories around the world have used the Arabidopsis reference genome sequence to map T-DNA flanking sequence tags (FST) for over 325,000 T-DNA insertion lines. Over the past decade, phenotypes identified with T-DNA-induced mutants have played a critical role in advancing both basic and applied plant research. These widely used mutants are an invaluable tool for direct interrogation of gene function. However, most lines are hemizygous for the insertion, necessitating a genotyping step to identify homozygous plants for the quantification of phenotypes. This situation has limited the application of these collections for genome-wide screens. Isolating multiple homozygous insert lines for every gene in the genome would make it possible to systematically test the phenotypic consequence of gene loss under a wide variety of conditions. One major obstacle to achieving this goal is that 12% of genes have no insertion and 8% are only represented by a single allele. Generation of additional mutations to achieve full genome coverage has been slow and expensive since each insertion is sequenced one at a time. Recent advances in high-throughput sequencing technology open up a potentially faster and cost-effective means to create new, very large insertion mutant populations for plants or animals. With the combination of new tools for genome-wide studies and emerging phenotyping platforms, these sequence-indexed mutant collections are poised to have a larger impact on our understanding of gene function.

Aspects of the Neurospora crassa Sulfur Starvation Response Are Revealed by Transcriptional Profiling and DNA Affinity Purification Sequencing.

Accurate nutrient sensing is important for rapid fungal growth and exploitation of available resources. Sulfur is an important nutrient source found in a number of biological macromolecules, including proteins and lipids. The model filamentous fungus Neurospora crassa is capable of utilizing sulfur found in a variety of sources from amino acids to sulfate. During sulfur starvation, the transcription factor CYS-3 is responsible for upregulation of genes involved in sulfur uptake and assimilation. Using a combination of RNA sequencing and DNA affinity purification sequencing, we performed a global survey of the N. crassa sulfur starvation response and the role of CYS-3 in regulating sulfur-responsive genes. The CYS-3 transcription factor bound the promoters and regulated genes involved in sulfur metabolism. Additionally, CYS-3 directly activated the expression of a number of uncharacterized transporter genes, suggesting that regulation of sulfur import is an important aspect of regulation by CYS-3. CYS-3 also directly regulated the expression of genes involved in mitochondrial electron transfer. During sulfur starvation, genes involved in nitrogen metabolism, such as amino acid and nucleic acid metabolic pathways, along with genes encoding proteases and nucleases that are necessary for scavenging nitrogen, were activated. Sulfur starvation also caused changes in the expression of genes involved in carbohydrate metabolism, such as those encoding glycosyl hydrolases. Thus, our data suggest a connection between sulfur metabolism and other aspects of cellular metabolism. IMPORTANCE Identification of nutrients present in the environment is a challenge common to all organisms. Sulfur is an important nutrient source found in proteins, lipids, and electron carriers that are required for the survival of filamentous fungi such as Neurospora crassa. Here, we transcriptionally profiled the response of N. crassa to characterize the global response to sulfur starvation. We also used DNA affinity purification sequencing to identify the direct downstream targets of the transcription factor responsible for regulating genes involved in sulfur uptake and assimilation. Along with genes involved in sulfur metabolism, this transcription factor regulated a number of uncharacterized transporter genes and genes involved in mitochondrial electron transfer. Our data also suggest a connection between sulfur, nitrogen, and carbon metabolism, indicating that the regulation of a number of metabolic pathways is intertwined.

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics.

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.