Plasmid transmission dynamics and evolution of partner quality in a natural population of Rhizobium leguminosarum.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓The central claim held under reproduction
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough to reproduce the core pipeline outputs. The 62 deposited PacBio-HiFi complete genomes (BioProject PRJNA310138, GenBank GCA_052941675.1..GCA_052947415.1) are the exact FASTA input for the named third-party tools (Spine-Nucmer-SNPs + sourmash). REPRODUCED 1:1 (exact): the entire plasmid inventory from the deposited replicon structure -- 256 extrachromosomal elements, 1-6 per strain, 226 (88%) in Types I-IV, 16 (6%) in Types V-VIII, and Table 1 N column 56/56/56/58 (all C1-C5 exact). WITHIN-TOL: an INDEPENDENT sourmash rerun (scaled=10000,k=31, Jaccard>=0.1, connected components) re-derives 4 dominant plasmid clusters holding 232/256 = 90.6% of plasmids vs the paper's 226/88% -- same pipeline, same parameters, not trusting author labels (C7). PARTIAL: Spine core-genome length (Table 1) reproduces for the two types with a real conserved backbone -- Type I 0.62 vs 0.61 Mbp and pSym Type IV 0.05 vs 0.05 Mbp -- but Types II/III collapse to ~0 under Spine's default 100%-presence core, so the paper's 0.39/0.50 Mbp require a relaxed/curated core threshold (likely PIRATE) that Methods do not pin down (C8). PARTIAL: plasmid genome fraction 29.19% is not exactly recoverable from deposited metadata (35.0% counting all plasmids vs 21.6% excluding the ptI chromid; the paper's definition is unstated) (C6). NOT ATTEMPTED (the hard last ~20%): re-assembly from raw PacBio reads (Trycycler/Flye/Hifiasm/Raven), the GRF gene-tree discordance distributions (Fig 4), the pSym Chi-squared treatment test (Fig 5, P=0.042), and repABC operon annotation counts -- bespoke stats / annotation-dependent and not cleanly specified. Fabrication: none detected; the headline inventory and typing numbers are backed exactly by the public data. Spine bioconda package is broken (missing scripts/nucmer_multi.pl) -> fixed by git-cloning egonozer/Spine.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 86assessed: 2026-06-14 ⛓ 55bf62556629
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-14
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusHow do patterns of gene content, sequence diversity, size, and horizontal versus vertical transmission vary across the multiple coexisting plasmid types within a natural population of clover-associated Rhizobium leguminosarum, and does plasmid horizontal transmission underlie the long-term nitrogen fertilization-driven decline in symbiotic partner quality?
- ★ Of the four most frequent plasmid types, types II and III have more stable size, larger core genomes, and track the chromosomal phylogeny (more vertical transmission), while types I and IV (pSym) vary in size and gene content with phylogenies consistent with frequent horizontal transmission. finding
- ★ Differentiation in pSym (type IV) subtypes is driven by long-term nitrogen fertilization and implicates plasmid horizontal transmission in the rapid evolution of partner quality decline. finding
- ★ The symbiosis plasmid (pSym) is the most discordant with the chromosomal phylogeny, indicating the most horizontal transmission among plasmid types. finding
- ★ A k-mer-based clustering approach considering core and non-core plasmid regions, combined with phylogenomic GRF-distance distributions, is used to categorize plasmids and infer mode of inheritance. method
- ★ 62 novel reference-quality long-read genomes characterize 256 replicons (the plasmidome) of a natural Rhizobium population from fertilized vs unfertilized LTER plots. resource
- Canonical symbiosis genes (nif, fix, nod) are limited to type IV plasmids; four of 62 strains lacked the pSym entirely. finding
- Plasmid types track chromosomal genospecies (types I–III and V–VIII), except type IV which is found across both gsB and gsE. finding
- Plasmid gene content often, but not always, tracks the repABC-based Rh incompatibility group. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Long-read whole-genome sequencing and de novo assembly of closed reference-quality genomes | 62 clover-associated Rhizobium leguminosarum nodule isolates (natural population from LTER KBS, Michigan) | long-term nitrogen fertilization (28 fertilized, 34 unfertilized field plots) | complete genome assemblies; number/size of extrachromosomal replicons | — |
| k-mer signature clustering / network analysis | 256 plasmids/replicons of the Rhizobium plasmidome | none | Jaccard index similarity, plasmid type categorization (weighted undirected network) | — |
| Genome annotation and COG functional assignment | plasmidome of 62 isolates | none | gene presence-absence, orthologous gene clusters, COG groups | NCBI PGAP |
| Principal coordinate analysis (PCoA) | 256 plasmids | none | Jaccard distance of presence-absence of orthologous gene clusters | — |
| repABC operon detection / Rh typing | 256 replicons | none | presence of repABC operons, incompatibility Rh group assignment | — |
| Phylogenomic / phylogenetic tree building and tanglegram comparison | chromosomal core genes and homologous plasmid core genes (gsE and gsB) | none | tree topology, Generalized Robinson-Foulds (GRF) discordance distributions | — |
| Population genomic diversity statistics | plasmid types with >1 representative | none | nucleotide diversity (π), average nucleotide identity (ANI), core gene number/length | — |
| Whole-genome synteny alignment | gsB plasmid types VII and VIII aligned to gsE type III plasmid | none | syntenic aligned blocks / shared plasmid history | — |
- – 256 extrachromosomal elements identified across 62 strains; 255 carried at least one repABC operon and 17 had two distinct repABC operons 256 replicons
- – Plasmids account for ~29.19% of the genomes on average 29.19%
- – 226 of 256 plasmids (88%) fell into four main types (I–IV); 16 (6%) into types V–VIII 88% (226/256)
- – pSym (type IV) most discordant with chromosome; mean GRF distance between chromosome gene trees was 72.46 72.46 ± 5.40 sd
- – Type IV plasmids vary substantially in size and have the smallest core (0.05 Mbp core length, 49 core genes), versus more stable types II/III type IV size 0.26–0.46 Mbp, 49 core genes
- – Types I, II, III present in all 56 gsE strains; type IV found in 58/62 strains across both genospecies 58/62 strains
- – Four of 62 strains lacked the pSym, corroborating loss of this non-essential element in a natural population 4/62
- – Pairwise core gene content overlap: type I vs V = 56%, type II vs VI = 74%; 209 genes shared between accessory plasmids and pSyms 56%, 74%, 209 genes
- other ~29.19% (average proportion of genomes made up by plasmids)
- count 256 (total extrachromosomal elements identified in the population)
- count 226 (88%) (plasmids falling into the four main types I–IV)
- other 72.46 ± 5.40 sd (mean GRF distance between gene trees of the chromosome (gsE))
- other π = 0.015–0.021 (nucleotide diversity range across plasmid types I–IV)
- other ANI 95.25–97.94% (average nucleotide identity across plasmid types I–IV)
- other 56% and 74% (core gene content overlap type I vs V and type II vs VI)
- count 209 (genes shared between at least one accessory plasmid and one pSym)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This comparative genomics study assembled 62 closed reference-quality genomes of natural Rhizobium leguminosarum isolates (28 from long-term nitrogen-fertilized plots, 34 from unfertilized controls) and characterized 256 extrachromosomal replicons. Plasmid grouping used k-mer Jaccard similarity networks; gene content diversity was visualized by PCoA on binary presence-absence matrices of orthologous gene clusters; and vertical versus horizontal transmission propensity was inferred by comparing plasmid and chromosomal phylogenies via Generalized Robinson-Foulds (GRF) distance distributions from resampled gene trees. Nucleotide diversity (π) and ANI were reported as descriptive summaries; dispersion was expressed as standard deviation.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| All-vs-all Jaccard Index k-mer similarity network clustering (threshold > 0.1) | Grouping all 256 replicons into discrete plasmid types (Fig. 1) | 256 plasmids | not stated |
| Principal Coordinate Analysis (PCoA) on Jaccard distance from binary presence-absence matrix of orthologous gene clusters | Visualization of gene content variation across plasmid types (Fig. 3) | 256 plasmids | not stated |
| Generalized Robinson-Foulds (GRF) distance on resampled gene tree distributions | Quantifying phylogenetic discordance between each plasmid type and the chromosomal tree (Fig. 4; Fig. S3A–D) | 56 gsE strains for within-genospecies core comparison | not stated |
| Bootstrap support on maximum-likelihood concatenated core chromosomal phylogeny | Core chromosomal tree topology (Fig. 2); nodes with bootstrap > 85 marked | 62 strains | not stated |
| Nucleotide diversity (π) and Average Nucleotide Identity (ANI) as descriptive population-genetic statistics | Summary of within-type sequence diversity across plasmid types (Table 1) | Varies by type (e.g., n = 56 for types I–III; n = 4 for types V–VIII) | na |
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Plasmids were grouped via k-mer Jaccard similarity networks with a fixed threshold (> 0.1) to define plasmid types↳ Could also: Alignment-free distance tools such as MASH, or dedicated plasmid-typing frameworks such as MOB-suite or PopPUNK, could also define plasmid clusters — Established typing tools provide reference-anchored nomenclature and may offer data-driven or statistically motivated thresholds; comparing results across methods can confirm that cluster boundaries are robust rather than threshold-dependent
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Gene content variation was visualized by PCoA on binary (presence-absence) Jaccard distances among orthologous gene clusters↳ Could also: Non-metric multidimensional scaling (NMDS) or UMAP could also be applied to the same distance matrix — NMDS does not assume linearity of distances and can better preserve rank-order relationships in non-Euclidean spaces; reporting a stress value or goodness-of-fit statistic alongside ordination axes quantifies how faithfully the 2-D projection represents the full distance matrix
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Phylogenetic discordance was quantified using GRF distance distributions derived from gene tree subsampling↳ Could also: Gene concordance factors (gCF) or site concordance factors (sCF), as implemented in IQ-TREE2, could also quantify discordance per branch — Concordance factors summarize, for each internal branch, the fraction of loci or sites that support that clade, providing a per-branch complement to the global GRF distribution and not requiring a fixed reference topology for comparison
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Dispersion of GRF distances across gene tree subsamples was reported as mean ± standard deviation↳ Could also: Bootstrap 95% confidence intervals or full empirical distributions (violin or box plots) could also characterize spread — GRF distances are bounded at zero and may be skewed; CIs or distributional plots complement SD by communicating uncertainty around the mean and the shape of the distribution without assuming symmetry
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Differentiation of pSym subtypes between fertilized and unfertilized plots was described in terms of observed frequency patterns↳ Could also: A chi-squared test, Fisher's exact test, or a permutation/randomization test on subtype composition could formally assess whether subtype frequencies differ between fertilization treatments — Formal tests would attach a measure of uncertainty (p-value or CI) to the observed frequency differences, complementing the descriptive comparison and clarifying whether the pattern is consistent with sampling variation alone
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Nucleotide diversity (π) and ANI were used as descriptive per-type summaries without formal comparison across plasmid types↳ Could also: A non-parametric Kruskal-Wallis test with post-hoc Dunn correction, or a permutation ANOVA, on per-strain π or ANI values could also compare diversity levels across types — Formal tests would quantify whether observed differences in diversity metrics across plasmid types exceed those expected by chance, providing inferential complement to the descriptive summaries in Table 1
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41212030
Paper: Vereau Gorbitz et al. 2025, mBio. Plasmid transmission dynamics and evolution of partner quality in a natural population of Rhizobium leguminosarum. PMID 41212030 · PMCID PMC12691615 · DOI 10.1128/mbio.02497-25
Named code (brief): https://github.com/Alan-Collins/Spine-Nucmer-SNPs — a third-party SNP/core-genome pipeline (Spine → nucmer → SNP extraction → alignment → IQ-TREE). Applying this existing tool to the paper's own data is a valid reproduction (brief rule P16).
Data: BioProject PRJNA310138 / SRA SRP069749. The reproducible artifact is the set of 62 complete genome assemblies (PacBio HiFi, Trycycler) deposited as GenBank WGS GCA_052941675.1 … GCA_052947415.1 (chromosome + plasmids as labelled replicons). These FASTA assemblies are exactly the input the Spine-Nucmer-SNPs and sourmash pipelines consume. All 62 resolve publicly on NCBI Datasets.
Pipelines named in Methods and whether in scope
| Reported result | Pipeline | In scope? | Why |
|---|---|---|---|
| Replicon inventory: 256 extrachromosomal elements; 1–6 per strain | assembly structure (count replicons) | YES (primary) | directly derivable from deposited complete-genome replicon structure |
| Plasmid clustering: 226 (88%) in Types I–IV; 16 (6%) in V–VIII | sourmash (scaled=10000, k=31) + Jaccard≥0.1 graph components | YES | exact parameters given; rerun independently on plasmid FASTAs |
| Table 1: per-type core genome length + core gene count (Types I–IV) | SPINE-Nucmer pipeline (the named repo) + Mummer 3.2 | YES (stretch) | the named third-party tool; needs per-type grouping first |
| Table 1: per-type ANI (97.94 / 97.32 / 97.89 / 95.25 %) | Popgenome v2.7.2 | partial | derivable from core alignment; secondary |
| Chromosomal phylogeny: 56 gsE + 4 gsB + 2 outliers | SPINE-Nucmer core SNPs + IQ-TREE2 | stretch | the named tool on chromosomes; qualitative clade recovery |
| Plasmid genome fraction ~29.19% | assembly bp ratios | YES (reported, def. unclear) | computable but exact definition not recoverable → honest grade |
| Gene-tree discordance GRF distances (Fig 4); pSym Chi² P=0.042 (Fig 5) | custom R resampling | NO (out of scope) | bespoke 1000× resampling stats, the hard last-20% |
| Assembly itself (Trycycler/Flye/Hifiasm/Raven from raw PacBio) | assembly | NO | re-assembly of raw reads not attempted; we consume the deposited assemblies |
| repABC operon counts (255/256, 17 with two) | manual/annotation | NO | annotation-dependent, not cleanly specified |
Strategy (80/20)
- Replicon inventory from deposited assemblies — exact counts (DONE from metadata).
- sourmash clustering independently re-derives plasmid types — validates the author-deposited ptI–ptVIII labels rather than trusting them.
- Spine core genome per type → Table 1 core length / gene count (stretch). We do NOT attempt re-assembly, GRF discordance stats, or the Chi² treatment test.
Assessments & scoring basis
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An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
The headline plasmid inventory and typing (C1–C5 exact, C7 within-tolerance at 90.6% vs 88%) reproduce 1:1 from the deposited 62 complete genomes, with no fabrication signal — the central descriptive conclusion holds. The only deviations are C6 (the 29.19% genome fraction is definition-dependent and sits between 35.0% and 21.6%, with the paper's definition unstated) and C8 (Types II/III core lengths collapse to 0.0 under Spine's strict core because the relaxed threshold is not specified). These are explainable underspecification gaps on the authors'/method side, not core-logic errors, so the overall reproduction is solid-with-deviations rather than 1:1 or critical.
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Reproduction footprint
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