Earlham Institute

Biological soil crusts (BSCs) are formed by phototrophic organisms at the soil surface and play a key role in structuring microbial communities and biogeochemical processes in dryland ecosystems. Similar surface associated communities occur in temperate agricultural soils, but their development, assembly dynamics and functional significance remain poorly understood. We investigated the temporal development of the soil surface microbiome following tillage and during subsequent growth of a winter wheat crop, integrating multi-kingdom amplicon sequencing with metagenomic analysis to track phototrophic, bacterial, fungal and protist communities, together with co-amplified macroscopic phototrophs. Distinct surface communities of phototrophs, bacteria and protists established rapidly, within 4 weeks of tillage, and underwent marked succession from early dominance by yellow-green algae (Xanthophyceae) to cyanobacteria, charophytes and ultimately mosses. Across all taxonomic groups, community assembly at the soil surface increasingly shifted towards dispersal limitation over time, whereas bulk soil communities were predominantly shaped by ecological drift. By the end of the growing season, the soil surface had developed a functional profile distinct from bulk soil, characterised by a greater representation of photosynthetic processes, largely due to eukaryotic algal carbon fixation, alongside increased genetic potential for heterotrophic carbon, nitrogen, phosphorus and sulphur cycling. These functional shifts were associated with enrichment of Actinobacteria, Bacteroidetes and Proteobacteria. Our results demonstrate that the soil surface of temperate agricultural systems represents a dynamic and functionally differentiated microbial habitat, which shares key biological features with dryland BSCs while exhibiting distinct functional and successional trajectories, revealing an overlooked component of managed temperate ecosystems • Soil surface communities establish rapidly in temperate agricultural soil • Dispersal limitation structures surface communities, while drift dominates in bulk soil • Surface soils show a distinct functional signature from phototrophs and heterotrophs

Biodiversity genomics is converging from historically separate approaches — DNA barcoding and reference genome sequencing — into an integrated digital ecosystem driven by shared data stewardship principles: transparent provenance, persistent identifiers and interoperable repositories. We demonstrate how these workflows can operate within a unified informatics architecture spanning data generation, validation, publication and reuse. We describe coordinated infrastructure, including the European BOLD mirror, ERGA Genome Tracking Console and metadata platforms COPO and PlutoF. These systems employ harmonised validation pipelines, shared metadata standards that bridge the Darwin Core and Genomic Standards Consortium vocabularies and automated data exchange amongst ENA, UNITE and GBIF. Workflows in Galaxy, Nextflow and Snakemake are registered in WorkflowHub as Research Object Crates (RO-Crates), ensuring reproducibility and complete provenance. Key outcomes include comprehensive data flow documentation, automated quality control using BUSCO and ERGA Assembly Reports and robust specimen-to-data linkage. We identify challenges in metadata harmonisation, distributed tracking, collaborative attribution and infrastructure sustainability and provide recommendations for addressing them through existing platforms and emerging RO-Crate standards. This work establishes practical foundations for treating biodiversity molecular data as a continuum, demonstrating how FAIR principles can scale to continental initiatives.

Abstract Background The contribution of cytotoxic T cells to the pathogenesis and treatment outcomes of ulcerative colitis (UC) remains poorly understood. Methods We integrated in vitro functional assays, multiparameter flow cytometry, bulk transcriptomics, single-cell RNA sequencing (scRNAseq), and single-cell spatial transcriptomics analyses to characterise cytotoxic T-cell responses in UC and evaluate their association with clinical and endoscopic outcomes. Results In vitro stimulation of lamina propria mononuclear cells (LPMCs) from UC patients with agonistic anti-CD3 antibodies upregulated cytotoxic gene programs, including GZMB, indicating enhanced T-cell cytotoxicity. Bulk transcriptomics analysis using the TaMMA resource1 confirmed upregulation of these cytotoxic genes in the UC colonic mucosa compared to non-IBD controls and Crohn’s disease. Multiparameter flow cytometry of 42 UC patients and 22 controls showed increased GZMB+ LPMCs in UC. Surprisingly, CD4+ T cells were a major source of GZMB in UC patients and were significantly more abundant than in controls. These GZMB+ CD4+ T cells exhibited a “hyper-inflammatory” polyfunctional phenotype, co-expressing IFN-γ, IL-13, IL-17, IL-22, and TNFα, with cells producing ≥3 pro-inflammatory cytokines significantly increased in UC compared to controls (p = 0.004). Analysis of UC scRNAseq data2 confirmed that GZMB+ CD4+ T cells co-expressed cytotoxic genes (GZMA, PRF1) and pro-inflammatory mediators (IFNG, IL26, OSM) compared to GZMB- CD4+ T cells. A cytotoxic CD4+ T-cell gene signature derived from these data was strongly enriched in UC compared to controls in a large independent cohort (n = 525)3, increasing with endoscopic disease severity and stratifying UC patients by degree of cytotoxic enrichment (Figure 1). Independent single-cell spatial transcriptomics analysis further validated enrichment of this cytotoxic CD4+ T-cell signature in UC versus non-IBD colonic tissues. This signature was also detected in multiple murine models of colitis, with strongest upregulation observed in T-cell transfer colitis. Notably, higher cytotoxic CD4+ T-cell signature enrichment predicted poor response to ustekinumab at week 8 in the UNIFI trial4 (n = 384) for mucosal healing (OR 0.11, p-value = 0.042), even after adjusting for CRP, faecal calprotectin, Total Mayo Score, disease duration, and prior anti-TNF failure. Similarly, the signature was upregulated (p-value = 0.013) in baseline biopsies from UC patients who failed to achieve mucosal healing to infliximab compared with responders in an independent cohort. Conclusion Pro-inflammatory cytotoxic GZMB+ CD4+ T cells are increased in the colonic mucosa of UC patients, correlate with endoscopic disease severity, and predict resistance to biologic therapies.

Gramene (gramene.org) is a comprehensive reference database for comparative plant genomics and pathway analysis, integrating functional annotations, evidence-based curated pathways and their projections, and multi-omics datasets. Since our last report, Gramene has added crop-specific pan-genome portals for maize, sorghum, rice, and grapevine. These pan-genome portals host population-scale datasets and multiple assembled genomes per species, all anchored by shared reference genomes. Importantly, these portals now adopt standardized rsIDs for genetic variants, advancing FAIR data principles and enabling cross-database interoperability. The main site is now Gramene Plants, emphasizing its broad genome coverage. Release 69 features 233 reference genomes, curated pathways for 139 species, expression data from 1026 studies across 27 species, and genetic variation data mapped to 27 genomes from 19 species. Key updates to the integrated search functionality include embedded expression viewers from the Bio-Analytic Resource for Plant Biology and EMBL-EBI Expression Atlas, a literature-curated catalog of gene functions, and a new Germplasm tab linking accessions with loss-of-function alleles to seed repositories. These advances reinforce Gramene as a comprehensive platform for exploring plant genomic diversity, gene function, and evolutionary conservation across the Green Tree of Life and within key agricultural species.

Abstract Current long-read single-nucleotide variant callers were designed primarily for genomic data—particularly human genomes. While some have been used on metagenomic data, their underlying assumptions and training procedures fail to account for the inherent complexity of metagenomic samples. To date, no long-read variant caller has been purpose-built for metagenomic applications. To address this gap, we present SNooPy, a SNP-calling tool that implements a new statistical framework tailored to long-read metagenomic data. Unlike previous genomic methods, our approach makes no assumptions about the number of haplotypes present, their evolutionary relationships, or their sequence divergence. We demonstrate that SNooPy outperforms both traditional statistical and deep learning–based SNP callers. Our results suggest that future integration of this framework with deep learning approaches could further enhance variant calling performance. SNooPy is freely available on github.com/rolandfaure/snoopy.

Abstract Lichens are symbiotic associations between filamentous fungi and photosynthetic micro-organisms, such as algae or cyanobacteria, that result in a single anatomically-complex structure that can thrive in environments inhospitable to most organisms, including arctic tundra, high mountains, and deserts. Recent evidence suggests that lichens may be even more complex than previously appreciated, containing multiple microbial constituents that operate as mini-ecosystems, but how genomes of the principal fungal symbiont (which provides the majority of biomass in lichen tissue) have been shaped during evolution is largely unexplored. Recently, giant transposable elements called Starships have been found in many genomes of filamentous fungi, but to which extent they occur in lichen-forming fungi is not known. In this report, we describe a Starship -like element from the lichen fungus Xanthoria parietina . This element, named Tangerine , contains several genes that have signatures of horizontal gene transfer from non-lichen-forming fungi, most likely from black yeasts of the Chaetothyriales, that are often lichen-associated. Furthermore, the “captain” gene responsible for transposition of the Starship defines a small lichen-specific clade of tyrosine recombinases within clade 1 of the tyrosine recombinase typology, suggesting a longstanding association of these elements with lichen-forming fungi. Internal repeats within Tangerine , and other sites in Xanthoria genomes, are affected by repeat-induced point mutation (RIP), a mechanism of genome defense against transposable elements, consistent with fungal sexual reproduction which always precedes new lichen formation by Xanthoria . We conclude that Starships may have played a significant, yet hitherto unrecognized role, in lichen genome evolution.

The emergence of nanopore sequencing technology has the potential to transform metagenomics by offering low-cost, portable, and long-read sequencing capabilities. Furthermore, these platforms enable real-time data generation, which could significantly reduce the time from sample collection to result, a crucial factor for point-of-care diagnostics and biosurveillance. However, the full potential of real-time metagenomics remains largely unfulfilled due to a lack of accessible, open-source bioinformatic tools. We present Metagenomic Analysis in Real-Time (MARTi), an innovative open-source software designed for the real-time analysis, visualization, and exploration of metagenomic data. MARTi supports various classification methods, including BLAST, Centrifuge, and Kraken2, letting users customize parameters and utilize their own databases for taxonomic classification and antimicrobial resistance analysis. With a user-friendly, browser-based graphical interface, MARTi provides dynamic, real-time updates on community composition and AMR gene identification. MARTi's architecture and operational flexibility make it suitable for diverse research applications, ranging from in-field analysis to large-scale metagenomic studies. Using both simulated and real-world data, we demonstrate MARTi's performance in read classification, taxon detection, and relative abundance estimation. By bridging the gap between sequencing and actionable insights, MARTi marks a significant advance in the accessibility and functionality of real-time metagenomic analysis.

Abstract The radiations of cichlid fishes in East African Lakes Victoria, Malawi, and Tanganyika showcase a remarkable example of rapid adaptive speciation, with over 2000 species evolving diverse morphological and ecological adaptations within the last few million years. Understanding the molecular basis of this phenotypic diversity remains a key challenge. Building on prior evidence of gene regulatory network (GRN) rewiring underpinning adaptive traits, we profiled chromatin accessibility (ATAC-seq) and matched transcriptomes across brain, eye, liver, and testis tissues in five representative cichlid species using optimised protocols. We show extensive divergence in chromatin accessibility corresponding to phylogenetic lineages and tissue identity, with many regulatory regions exhibiting accelerated nucleotide evolution. Transcription factor binding site (TFBS) variation correlates with both chromatin accessibility and differential gene expression, particularly in genes linked to sensory systems. By integrating TF footprinting with regulatory motif turnover analyses, we demonstrate that dynamic nucleotide changes drive GRN rewiring, concordant with ecological niche and lineage-specific adaptations. Our findings highlight regulatory variation at conserved and novel TFBSs as critical drivers of phenotypic innovation across radiating and non-radiating East African cichlids. This study provides foundational epigenomic evidence establishing GRN divergence as a key mechanism facilitating rapid adaptive diversification in this iconic vertebrate radiation.

Abstract The surface of dryland soils is typically covered by biological soil crusts (BSC) dominated by cyanobacteria, lichens or moss which contribute key ecosystem functions. However, while photosynthetic biota have been observed on the surface of temperate agricultural soils, understanding of the development, biodiversity and functional significance of these communities is extremely limited. We investigated the temporal development of the soil surface community following tillage and during subsequent growth of a wheat crop. Amplicon analysis showed that establishment of the soil surface microbiome was rapid over the winter period, with distinct communities of phototrophs, bacteria and protists detected 60 days after tillage. There was rapid succession with yellow-green algae (Xanthophyceae) dominating early stages of development, followed by Cyanobacteria and Charophytes and finally moss. Temporal dynamics of fungal and protist communities was influenced by eDNA deposition from plant pathogens inhabiting the crop canopy and wild animal parasites. For all groups, assembly processes in the surface soil became increasingly dominated by dispersal limitation over time while assembly of bulk soil communities became predominantly influenced by drift. Metagenome analysis showed that the soil surface had developed a distinct functional profile relative to bulk soil after 9 months, enriched in photosynthetic processes which were largely from eukaryotic algae rather than cyanobacteria. Additionally the 3-Hydroxypropionate bicycle and reductive TCA carbon fixation pathways were enriched in surface soil. The abundance of genes involved in heterotrophic carbon, nitrogen, phosphorus and sulphur cycling processes were also enriched in surface soil. This included reads of carbohydrate active enzymes, nitrification, denitrification and sulphate reduction genes, largely attributed to the enrichment of Actinobacteria, Bacteroidetes and Proteobacteria in the soil surface. Overall, we show overlap in dominant phototrophic taxa between temperate agricultural and BSC in arid soils, but with notable differences, including abundance of Xanthphycae and Charophytes. Furthermore, the functional signatures of the soil surface communities indicate that they may play an important role in the delivery of ecosystem functions in temperate agricultural systems.