Large-Scale Phylogenomics of the Lactobacillus casei Group Highlights Taxonomic Inconsistencies and Reveals Novel Clade-Associated Features.
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 are derivable from the shared data
- ✓Any deviation was negligible
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡The central claim did not (fully) hold under reproduction
- 🟡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 light, clearly-specified pipeline outputs 1:1. Method: downloaded the exact 183 NCBI assemblies listed in supplementary Table S1 (datasets CLI, on a «our HPC» compute node, «infra»), recomputed GC% per genome, and ran fastANI all-vs-all on the same genomes. GENOME SET + GC CONTENT (stage 04b) reproduce essentially exactly: per-species mean GC L. paracasei 46.30 (reported 46.3), L. rhamnosus 46.67 (46.7), L. zeae 47.75 (47.8), and crucially the L. casei GC is bimodal with EXACTLY 5 high-GC genomes (incl. type strain ATCC393) as the paper reports; original NCBI annotation counts 92/36/38/2 match exactly. ANI three-clade species separation (stage 05; paper used pyANI ANIb) was reproduced with fastANI (a faster third-party tool on the paper's identical data, explicitly valid per the brief): within-clade mins 97.6/94.1/96.5 and between-clade max 82.2 reproduce the same structure and the same clade-B-borderline signal, though absolute values differ because fastANI(mapping) != ANIb(BLAST). Authentic pyANI ANIb was launched («job») then cancelled in the makeblastdb stage as the hard 20% (all-vs-all BLAST on 183 ~3Mb genomes ~ 12h node time for a proxy->authentic refinement). NOT ATTEMPTED (deliberately, the heavy downstream chain): SPAdes assembly of AMBR2, Prokka annotation, Roary pangenome / 776 marker genes, RAxML phylogeny (Fig 2), OrthoFinder (5915 orthogroups / 521567 genes), and the interest-driven catalase / SecA2-SecY2 / glycosyltransferase analyses; the headline biological claims (only 10 L. casei after reclassification, catalase-positivity, 6/10 SecA2/SecY2) depend on that chain and are recorded as reported-only, not reproduced. No fabrication signals: every reproduced value is derivable from the shipped Table S1 accession list and matches closely. Grades are provisional; a human auditor decides (see AUDIT.md).
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
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v1 current initial assessment Score 94assessed: 2026-06-16 ⛓ 148c5b2d0b43
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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
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no 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: opusThe taxonomy of the Lactobacillus casei group (L. casei, L. paracasei, L. rhamnosus) is inconsistent; large-scale comparative phylogenomics of 184 genomes can resolve the group's structure, correct misclassifications, and reveal clade-associated functional features.
- ★ The L. casei group resolves into three distinct clades (A, B, C) supported by phylogeny, GC content, ANI, and TETRA, and many strains are misclassified relative to their nearest type strain. finding
- ★ Reclassifying genomes to their most closely related type strain leaves only 10 genomes as L. casei sensu stricto (clade B), the smallest clade. finding
- ★ All 10 L. casei sensu stricto (clade B) strains carry a heme-dependent catalase gene and are catalase positive, making L. casei the first described catalase-positive species in the Lactobacillus genus. finding
- ★ L. casei AMBR2 was isolated from the human upper respiratory tract, the first L. casei strain from this niche. resource
- ★ 6 of 10 L. casei genomes contain a SecA2/SecY2 gene cluster encoding two putative glycosylated surface adhesins. finding
- ★ L. casei and L. paracasei are paraphyletic taxa under current annotations, being completely intermixed in clade A, whereas L. rhamnosus is monophyletic (clade C). finding
- A high-quality maximum-likelihood phylogenetic tree was built from 776 conserved single-copy marker genes with L. nasuensis JCM 17158 as outgroup. method
- Superoxide dismutase (SOD)-encoding genes are present only in clade A strains (69 of 70 genomes), restricting SOD to a single species rather than two. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Whole-genome sequencing and assembly | L. casei AMBR2, human upper respiratory tract isolate | none | genome assembly / sequence | — |
| Comparative genomics / phylogenomics (maximum-likelihood tree) | 184 L. casei group genome assemblies plus L. nasuensis JCM 17158 outgroup | none | phylogenetic tree from 776 single-copy marker genes | roary |
| Pairwise genome comparison (ANIb and TETRA) | all L. casei group genomes | none | ANI and tetranucleotide frequency distances / species delimitation | BLAST-based ANIb |
| Orthogroup / gene content analysis | 184 L. casei group genomes | none | core and accessory orthogroups, genes per genome | OrthoFinder |
| Functional category mapping and PCoA | all orthogroups of L. casei group genomes | none | predicted functional capacity across 18 categories | eggNOG database v4.5 |
| Gene/protein domain identification (catalase, SOD) | L. casei group genomes (clade B catalase; clade A SOD) | none | presence of catalase (Pfam PF00199, PF05067) and SOD genes | HMMER, Pfam |
| 16S rRNA gene screening | unclassified Lactobacillus sp. NCBI genomes | none | identification of additional L. casei group members | RDP database v11 |
| Catalase activity microbiology test | 12 selected strains (4 per clade) grown in Weissella medium broth under anaerobic/aerobic conditions ± hemin/menaquinone | H2O2 exposure; respiration-promoting supplementation | bubble/froth formation indicating catalase activity | 3% H2O2 standard lab test |
- – After reclassification, only 10 genomes remain as L. casei sensu stricto (clade B), the smallest clade 10 genomes
- – Heme-dependent catalase gene found in all 10 clade B genomes; manganese-dependent catalase in 7 of 10 10/10 and 7/10
- – Only clade B strains tested (particularly AMBR2) were experimentally catalase positive na
- – 6 of 10 L. casei genomes contain a SecA2/SecY2 cluster with two putative glycosylated surface adhesins 6/10
- – SOD-encoding genes present only in clade A strains (69 of 70 genomes) 69/70
- – Inter-clade ANI values ≤85.1% while intra-clade ANI ≥96.1% (A), ≥93.6% (B), ≥96.3% (C), supporting three-clade separation ≤85.1% vs ≥93.6-96.3%
- ▲ Clade B genomes show elevated GC content (47.74-47.76%) similar to L. zeae versus ~46.1-46.6% in L. paracasei-range 47.74-47.76% vs 46.10-46.60%
- – At least 17 assemblies show very high similarity to L. rhamnosus GG, suggesting repeated sequencing of the same strain 17 assemblies
- count 184 L. casei group strains studied (183 public + AMBR2) (total genomes analyzed)
- count 776 single-copy marker genes (phylogenetic tree construction)
- correlation ANI inter-clade ≤85.1%; intra-clade ≥96.1% (A), ≥93.6% (B), ≥96.3% (C) (ANIb species delimitation)
- other TETRA ≥0.9941 (A), ≥0.9872 (B), ≥0.9882 (C) (tetranucleotide frequency within clades)
- count 521,567 genes total; 2,827 ± 141 genes/genome average (gene content; clustered into 5,915 orthogroups (1,814 core, 4,101 accessory))
- count GC contents: L. paracasei 46.3%, L. rhamnosus 46.7%, L. zeae 47.8% (average GC content by species)
- count 2,662 orthogroups mapped to category S (function unknown) (eggNOG functional annotation)
- other heme-dependent catalase 487 amino acids; manganese-dependent catalase 269 amino acids (two catalase types identified in clade B)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This is a comparative/phylogenomics study of 184 Lactobacillus casei group genomes rather than a hypothesis-testing experimental study. The overall approach is descriptive: a maximum-likelihood phylogenetic tree built from 776 single-copy marker genes (with bootstrap support thresholds), pairwise genome-distance metrics (ANIb and TETRA) compared against published species-delimitation cutoffs, orthogroup/gene-content tabulation, and ordination (PCoA) of eggNOG functional categories. Results are reported as counts, ranges, threshold comparisons, and means ± SD, with one experimental catalase assay summarized qualitatively in a supplemental table; no formal inferential significance tests or P values are reported.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Maximum-likelihood phylogenetic inference with bootstrap support (clades indicated at bootstrap >70) | Fig. 2A-D, whole group and clade subtrees based on 776 single-copy marker genes | 184 genomes (plus L. nasuensis outgroup); 776 marker genes | not stated |
| Average nucleotide identity (ANIb) compared to published species cutoffs (95-96%) | Fig. 3, pairwise within- and between-clade comparisons | all pairwise comparisons among L. casei group genomes | na |
| Tetranucleotide frequency correlation (TETRA) | Fig. 3, within-clade pairwise values | all pairwise comparisons among L. casei group genomes | na |
| Principal-coordinate analysis (PCoA) of functional-category gene content | Fig. 4, clustering of clades by eggNOG functional category | orthogroups mapped per genome across 184 genomes | na |
| Orthogroup clustering / core vs accessory gene-content tabulation | Table 1, gene content per clade | 521,567 genes; 5,915 orthogroups across 184 genomes | na |
| Qualitative catalase activity assay (bubble/froth on H2O2 exposure) | Table S2, 12 strains (4 per clade) under 4 growth conditions | 12 strains tested | na |
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Species boundaries were assessed by comparing ANIb and TETRA values against previously published fixed cutoffs (e.g., 95-96% ANI).↳ Could also: A clustering or gap-statistic / threshold-optimization approach (or model-based delineation such as those in GGDC or autoMLST) could also be applied to the pairwise distance matrix. — Such approaches would let the species boundary emerge from the data distribution itself and could quantify how robust the clade assignments are to the chosen threshold, which is useful when within-clade values fall near the cutoff (as noted for clade B).
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Clade separation in functional capacity was shown via PCoA visualization of eggNOG category profiles.↳ Could also: A complementary permutational test of group separation (e.g., PERMANOVA/adonis) or ANOSIM on the same distance matrix could also be reported. — This would attach a quantitative measure of between-clade versus within-clade variation to the visual clustering, giving readers an explicit summary statistic alongside the ordination plot.
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Per-genome gene and orthogroup counts were summarized as mean ± SD per clade in Table 1.↳ Could also: A 95% confidence interval or the full range/IQR could also accompany the mean. — For unequal and sometimes small group sizes (e.g., clade B with 10 genomes), a CI or range conveys uncertainty in the mean directly and is often preferred for comparing groups of differing n.
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Branch support was conveyed by marking clades with bootstrap support >70.↳ Could also: Reporting the full bootstrap values (or alternative supports such as SH-aLRT or Bayesian posterior probabilities) could also be presented. — Showing the continuous support values, rather than a single threshold dichotomy, lets readers gauge the strength of each split and compare relative confidence across nodes.
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The catalase phenotype was assessed qualitatively (presence/absence of bubble formation) for 12 selected strains.↳ Could also: A semi-quantitative or replicated readout with a defined scoring scheme could also be used. — Replicated, scaled measurements would allow the phenotype to be related to genotype (e.g., catalase gene copy presence) with an explicit comparison and would document measurement reproducibility.
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The analysis used the full available set of genomes, including apparently redundant assemblies (e.g., ~17 highly similar to L. rhamnosus GG, duplicate L. zeae sequencing).↳ Could also: A dereplication or down-weighting step (e.g., collapsing near-identical genomes) could also be applied before summary statistics. — Accounting for sampling redundancy would help ensure per-clade averages and ordination patterns are not influenced by repeatedly sequenced strains, which the authors already flag as a feature of the dataset.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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Only clade B (L. casei sensu stricto) strains show catalase enzymatic activity experimentally, confirming clade-specific catalase function.other lactobacillus casei 2017×1papers★ This paper is the founder (earliest)
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Inter-clade ANI ≤85.1% versus intra-clade ANI ≥93.6–96.3% across L. casei group genomes, supporting three species-level clades (A, B, C).WGS lactobacillus casei group 2017×1papers★ This paper is the founder (earliest)
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Phylogenomic reclassification restricts L. casei sensu stricto to 10 genomes (clade B), the smallest of three distinct clades.WGS lactobacillus casei group 2017×1papers★ This paper is the founder (earliest)
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At least 17 public L. casei group assemblies are highly similar to L. rhamnosus GG, indicating substantial database redundancy from repeated sequencing of the same strain.WGS lactobacillus casei group 2017×1papers★ This paper is the founder (earliest)
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Heme-dependent catalase gene is present in all 10 clade B (L. casei sensu stricto) genomes and manganese-dependent catalase in 7/10, absent from other clades.WGS lactobacillus casei 2017×1papers★ This paper is the founder (earliest)
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Clade B (L. casei sensu stricto) genomes have elevated GC content (47.74–47.76%) relative to clade A/C genomes (~46.1–46.6%).WGS lactobacillus casei up 2017×1papers★ This paper is the founder (earliest)
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A SecA2/SecY2 secretion cluster encoding two putative glycosylated surface adhesins is present in 6/10 L. casei sensu stricto (clade B) genomes.WGS lactobacillus casei 2017×1papers★ This paper is the founder (earliest)
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Superoxide dismutase (SOD)-encoding genes are present in 69/70 clade A (L. paracasei) genomes and absent from clades B and C.WGS lactobacillus paracasei 2017×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
No assessed neighbours yet — the network grows as more papers are assessed.
Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Reproduction scope — pmid-28845461
Paper: Wuyts et al. 2017, mSystems 2:e00061-17. "Large-Scale Phylogenomics of
the Lactobacillus casei Group..." PMID 28845461 / PMC5566788.
Code: https://github.com/LebeerLab/caseiGroup_mSystems_pipeline @ commit
78f013f861d28080d1ed11c4ceb236752381cafe (public, master, last push 2018-05-14).
A collection of stage-numbered shell/R scripts (authors' own pipeline, P16-valid).
Data: 183 public NCBI genome assemblies (exact accessions in supplementary
Table S1 = sys004172127st1.csv) + 1 isolate (L. casei AMBR2, PRJEB21025).
Pipeline stages (from repo) and reproduction status
| stage | tool | output | in scope? |
|---|---|---|---|
| 01 assembly | SPAdes | AMBR2 assembly from reads | NO — single isolate, raw-read assembly, hard 20% |
| 02 download genomes | wget NCBI | the 183 .fna set | YES (we download the exact Table S1 accessions) |
| 04a quality control | QUAST | N75/length/#contigs | partial — QC thresholds (N75>10kb) are a selection rule, not re-run |
| 04b GC content | custom (per-genome GC%) | per-species GC averages | YES — PRIMARY, light compute |
| 05 ANI | pyani ANIb/TETRA | within/between-clade ANI ranges | YES — STRETCH, heavy all-vs-all BLAST |
| 06 gene prediction | Prokka | .gff annotations | NO — heavy, feeds 07/09 |
| 07 core alignment | Roary | 776 marker genes | NO — heavy, depends on 06 |
| 08 phylogeny | RAxML | tree (Fig 2) | NO — heavy, depends on 07 |
| 09 orthofinder | OrthoFinder | 5915 orthogroups, 521567 genes | NO — heavy, depends on 06 |
| 10–11 gene content | R / HMMER / BLAST | catalase, SecA2/SecY2, GTs | NO — interest-driven, downstream |
In-scope targets (clearly-specified, pipeline-derived)
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Genome-set composition (Results, para 1): 183 public genomes; original NCBI annotation = 92 L. rhamnosus, 36 L. casei, 38 L. paracasei, 2 L. zeae (= 168) + 15 Lactobacillus sp. Audited directly against Table S1 (metadata), no compute. Provisional grade only — human confirms.
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GC content per species (Results "GC content"; Fig 1; methods stage 04b): reported averages L. paracasei 46.3 %, L. rhamnosus 46.7 %, L. zeae 47.8 %; L. casei splits into a large group (46.10–46.60 %) and a small group of 5 genomes (47.74–47.76 %). Reproduce by downloading the exact 183 Table S1 assemblies and recomputing GC% per genome. This is the clean 1:1 (≈183 data points → 4 per-species averages).
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ANIb three-clade separation (Results; Fig 3; methods "ANIb and TETRA", tool = pyani): within-clade ANIb min ≥96.1 % (A), ≥93.6 % (B), ≥96.3 % (C); between-clade ANIb ≤85.1 %. STRETCH — pyani ANIb all-vs-all on 183 genomes is a heavy BLAST job; attempted on «our HPC» only if budget allows.
Out of scope (and why)
Assembly, Prokka, Roary, RAxML, OrthoFinder, HMMER/eggNOG, the interest-driven catalase/SecA2/SecY2/glycosyltransferase analyses (stages 06–11): each is heavy, multi-tool, and chained off de-novo annotation — the hard last 20 %. The headline biological claims (only 10 L. casei after reclassification; all catalase-positive; 6/10 with SecA2/SecY2) depend on this full chain and are NOT attempted; they are recorded as reported-only.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
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.
What we could check reproduces cleanly: genome-set composition and GC content (C1–C6) match the paper essentially 1:1 from the shipped Table S1 accession list, including the diagnostic 5-genome high-GC L. casei split with type strain ATCC393 — no fabrication signal. The ANI three-clade species separation (C7–C8) was reproduced with fastANI as a proxy for the paper's pyANI ANIb, so absolute values differ (82.21 vs ≤85.1) on our methodological choice while the biological conclusion holds. The deviation is on our side (tool substitution) and benign, not authors'. Overall yellow because the paper's headline biological conclusions depend on a heavy downstream chain that was deliberately not run, leaving the central claim only partially confirmed.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.