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Metagenomic analysis of the effects of European bison Bison bonasus (Linnaeus, 1758) presence on soil community structure and function in West Blean and Thornden Woods, Kent

徐崇翰iD, Hester J. van SchalkwykiD, Owain Powell, Carla GustaveiD, Lawrence BalliD, Kora Ross, Edward Murray, Heidi Aguirregoicoa, Hannah Mackins, Kirsty Swinnerton, Thomas J. CreedyiD, Laura SivessiD, Jumayma Jones, Kath Castillo, Rosie Bleet, Silvia SalatinoiD, Yuen-Ting Chan, Raju MisraiD, Darren Chooneea, Pedro Lebre, Hermine V. MkrtchyaniD, Piotr CuberiD

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Abstract

The reintroduction of extinct or endangered species to restore ecosystem function is an essential aspect of rewilding. The Wilder Blean Project at West Blean and Thornden Woods in Canterbury, UK, is committed to rewilding natural processes and enhancing biodiversity in one of England's oldest and largest areas of ancient woodland. The introduction of European bison (Bison bonasus) is an important part of the project. However, how the reintroduction of large herbivores influences local biodiversity and ecosystem functions during the early stages of rewilding remains poorly understood. Soil samples were collected from the same sampling sites before and two years after bison were reintroduced and profiled by metagenomic sequencing using Oxford Nanopore Technologies sequencing platforms. The results showed that the alpha diversity of soil organisms did not change significantly before and after the introduction of European bison, while beta diversity showed modest shifts in community composition. The relative abundance of some nitrogen-fixing and photosynthetic microbial genera showed declines in the 2024 Bison Area, while the mycorrhizal fungus genus Rhizophagus was significantly less abundant than in the 2024 Control Area. Despite relatively stable taxonomic diversity, functional composition differed significantly between the 2022 and 2024 Bison areas and among the 2024 rewilding treatments, revealing a decoupling between taxonomic diversity and functional composition. Amino acid synthesis pathways and carbon metabolism pathways were significantly enriched. These findings highlight the potential of long-read Oxford Nanopore metagenomics to reveal functional shifts that may not be apparent from taxonomic diversity alone. Although these early-stage responses cannot yet predict long-term rewilding trajectories, continued longitudinal monitoring integrating microbial, soil physicochemical, and ecosystem-level measurements will be essential to determine the persistence and ecological significance of these functional shifts.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (15 Sept 2026) · CC BY 4.0

Nanopore shotgun metagenomics of 56 Kent woodland soils finds stable alpha diversity but significantly shifted KEGG functional composition two years after European bison release.

Most evidence on rewilding outcomes comes from vegetation and vertebrates, so little is known about how soil microbes respond when large herbivores are reintroduced. The study compares soils before and after the first UK woodland release of European bison across three management areas, and tests whether soil functional potential shifts sooner than taxonomic diversity.

Key results

  1. ShownTwo years after release, the functional potential (KEGG pathway composition) of Bison Area soils differed from the same area in 2022 and from both the 2024 Control and 2024 Grazer areas.

    PERMANOVA on Bray–Curtis distances of KEGG pathway profiles, 2024 Bison vs 2022 Bison: R² = 0.35, F = 7.53, P = 0.001; vs 2024 Control: R² = 0.26, F = 5.63, P = 0.008; vs 2024 Grazer: R² = 0.33, F = 7.75, P = 0.002. In PCoA, 2024 Bison samples separated from the other groups. · 3.6 Functional beta diversity of KEGG pathways; Tables; 2.5 Genome assembly and KEGG pathway annotation

  2. ShownTaxonomic alpha diversity stayed high and showed no significant change across years or areas. By contrast, Bison Area community composition shifted between 2022 and 2024, and its 2024 samples were more spread out.

    Median Simpson index above 0.9 in every group; all five comparisons non-significant (Benjamini-Hochberg adjusted p>0.05). ANOSIM: 2024 vs 2022 Bison R=0.37, p<0.05; three 2024 areas R = 0.20, P < 0.05, but no pairwise difference stayed significant after correction. · 3.2 Alpha diversity; 3.3 Beta diversity; 4.1 Effect of the presence of European bison on…

  3. SuggestedDifferential-abundance and enrichment analysis found carbon metabolism and amino acid biosynthesis enriched in 2024 Bison soils in all three comparisons. Fatty acid metabolism was enriched vs 2022 Bison, pyruvate metabolism vs 2024 Control, and quorum sensing uniquely vs 2024 Grazer.

    Carbon metabolism and amino acid biosynthesis enriched in 2024 Bison vs each of 2022 Bison, 2024 Control and 2024 Grazer; fatty acid metabolism vs 2022 Bison, pyruvate metabolism vs 2024 Control (Discussion also cites Grazer), quorum sensing uniquely vs 2024 Grazer; no enrichment statistics reported in text. · 3.7 KEGG pathway enrichment analysis; 4.2 Changes in soil functional diversity…; 4.3 Functional shifts as early indicators of…

  4. SuggestedThe nitrogen-fixing genus Azorhizobium fell significantly in the Bison Area by 2024. Most other rhizobial and photosynthetic genera had lower medians there, but these differences were not significant.

    Azorhizobium lower (p<0.05) in 2024 vs 2022 Bison Area and vs both other 2024 areas; Mesorhizobium, Neorhizobium, Rhizobium and Sinorhizobium also had lower 2024 Bison medians; ten of eleven photosynthetic genera were below 2022 Bison levels; none of these trends significant. · 3.4 Spatial and temporal difference of rhizobia…

  5. ShownThe arbuscular mycorrhizal genus Rhizophagus was less abundant in the 2024 Grazer and Bison areas than in the 2024 Control area. Diversity of plant taxa detected by metagenomics did not differ between groups.

    Rhizophagus higher in 2024 than 2022 Control Area and lower in 2024 Grazer and 2024 Bison than 2024 Control (p<0.05); 3652 plant taxa screened via World Flora Online showed no significant Simpson index differences across five comparisons (p>0.05). · 3.5 Spatial and temporal difference of…; 2.4 Normalization and relative abundance…

Methods, data and reproducibility

Soil cores from the Control, Grazer and Bison areas, sampled in 2022 (pre-release) and 2024, were sequenced by Oxford Nanopore (ONT) shotgun metagenomics. Centrifuge classification against NCBI nt fed TPM (transcripts per million) normalised diversity and genus tests. MEGAHIT assemblies annotated by eggNOG-mapper fed KEGG PERMANOVA and DESeq2 enrichment.

  • 2022 pre-introduction soil cores (30 cm depth) from Control, Grazer and Bison areas of West Blean and Thornden Woods, Kent, UK; 56 of 140 collected cores were sequenced overall · mixed soil biota (bacteria, fungi, plant and animal DNA) · n = 28 (10 Control, 10 Grazer, 8 Bison)
  • 2024 soil cores from the same sites two years after bison release; Bison Area cored by Kent Wildlife Trust, other areas by Natural History Museum · mixed soil biota (bacteria, fungi, plant and animal DNA) · n = 28 (10 Control, 10 Grazer, 8 Bison)
  • Triplicate DNA extracts per sample, ONT-sequenced and merged per sample, plus molecular-grade water extraction blanks (no taxa detected) · n = 168 extracts; 12 negative controls (6 per year)
  • ONT shotgun reads: 2022 run 25.7 million reads (41.17 Gb); 2024 run 27.29 million reads (40.77 Gb); reads mostly 600 bp to 1200 bp · mixed soil biota · n = 8,689,711 classified reads; 108 phylum-level taxa
  • NCBI nucleotide non-redundant (nt) database, last updated 16 May 2024, built into a local Centrifuge index · multiple species
  • World Flora Online plant species backbone used to screen plant taxa in the abundance matrix · plants · n = 3652 plant taxa detected
Methods & tools
DNeasy PowerSoil Pro and PowerClean Pro Cleanup kits (Qiagen), ONT Rapid Barcoding 96 V14 kit (SQK-RBK114.96) on PromethION flow cells, nf-core/taxprofiler v1.1.8, FastQC, Porechop, NanoFilt (Q10 filter), Centrifuge (local NCBI nt index), Custom Python script for TPM normalisation, vegan 2.6.6.1 (Simpson, Bray–Curtis, ANOSIM), WorldFlora R package 1.14.4, MEGAHIT v1.2.9, QUAST v5.2.0, eggNOG-mapper v2.1.12 (KEGG annotation), DESeq2 1.40.2
Reagents & environment
nf-core/taxprofiler v1.1.8; R 4.3.3 with vegan 2.6.6.1, WorldFlora 1.14.4, DESeq2 1.40.2; MEGAHIT v1.2.9 (k-mers 35 to 255); QUAST v5.2.0; eggNOG-mapper v2.1.12; Centrifuge index from NCBI nt dated 16 May 2024; ArcGIS Pro 3.0.0.
Reproducibility gaps
No code repository, read accession or data URL is given; the TPM normalisation script and abundance tables are only referenced as supplementary files (SI_1, SI_2, SI_5).
Limitations (6)
  • Sampling rounds are only two years apart, and pairwise taxonomic contrasts among 2024 areas were non-significant. The authors therefore cannot attribute the shifts to bison rather than climate or other environmental factors, or treat them as long-term trends. · stated by the authors
  • DNA concentrations from 2024 Bison Area soils were much lower than from other areas, so sequencing coverage of that group may be incomplete despite normalisation before abundance and KEGG analyses. · stated by the authors
  • Shotgun metagenomes measure the content and relative abundance of functional genes, not their expression or activity. KEGG enrichment therefore cannot show that pathways are operating; transcriptomics would be required. · stated by the authors
  • No soil chemistry (total carbon and nitrogen, C:N ratio, pH, moisture) or qPCR data were collected, so the proposed explanations based on nitrogen inputs and carbon processing remain untested. · stated by the authors
  • Sequencing batch is confounded with year (libraries were pooled by year on separate PromethION flow cells). The 2024 Bison cores were also collected by a different team from the other 2024 cores, so technical effects could mimic Bison-specific differences. · noted in this summary
  • Groups are small (8 Bison cores per year) and 2024 Bison samples were more dispersed, but no dispersion test is reported, so PERMANOVA and ANOSIM results may partly reflect spread. Sections 3.7 and 4.2 also disagree on which comparisons showed pyruvate enrichment. · noted in this summary

Read this if you monitor soil or microbial outcomes of rewilding or large-herbivore reintroduction and want a worked example of Nanopore metagenomics comparing functional and taxonomic responses.

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