Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis.
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 and reproduced 1:1. The paper's central pipeline-derived result is a per-population list of fixed (>=90%) non-synonymous adaptive mutations from pooled-population WGS vs ancestor (S-Table, Fig 3B/3C). We reproduced this for 4 of 15 generation-375 populations (P4,P10,P13,P15; SRA E3-P*), chosen to span every named functional module, using the koschwanez/mutantanalysis logic (bwa->samtools->VarScan somatic vs ancestor E0-11) with current builds + snpEff R64-1-1 annotation (legacy Python2/bwa-aln/GATK3 binaries substituted; reference Ensembl R64-1-1 == S288C). RESULT: 21/21 reported SNPs reproduced EXACTLY (identical ref>alt and frequency), incl. cohesin SMC1/SMC3 (missense), separase ESP1 (missense), and Cdk8/mediator SSN2/SRB8/BOI2 recovered as stop_gained -- directly confirming the paper's 'early stop codons in the Cdk8 module'. 2/2 indels reproduced as the correct frameshift/gene (ALT repr differs due to Excel-mangled table + left-alignment). A systematic +1 coordinate offset (paper 0-based vs VCF 1-based) is present; allele identity and frequency match. ONE flagged discrepancy for human review: the recurrent TIR1 chrV:175658 indel reported at ~96% in 6 populations is only ~5-7% in the raw reads (present in ancestor too), so not called somatic -- possibly an artifact of the paper's GATK indel-realignment (which we omitted) in a repetitive subtelomeric gene; flagged, not asserted as fabrication. NOT ATTEMPTED (out of scope / optional 20%): the remaining 11 populations and the gen-1750 set, wet-lab phenotypes, ChIP-seq, backcross-segregant linkage, plasmid-borne REC8 E148Q (plasmid absent from genome ref), and exact rerun of the legacy GATK3/HTML classifier.
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 90assessed: 2026-06-15 ⛓ 7e2ccc98b2f7
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
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
- 2026-06-15
- 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
- 2026-09-19
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: sonnetThe paper asks how cells adapt when a protein is forced to perform a different biological function, using the budding yeast meiotic kleisin Rec8 substituted for its mitotic paralog Scc1 to test whether experimental evolution can repair the resulting mitotic defects and to identify which genes/modules must change.
- ★ Replacing Scc1 with Rec8 in mitosis impairs sister chromosome cohesion, advances the timing of genome replication, and reduces reproductive fitness by 45% finding
- ★ Experimental evolution of 15 parallel Rec8-expressing populations for 1,750 generations substantially increased their fitness finding
- ★ Adaptive mutations arose mainly in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce the G1-to-S transition, rather than in REC8 itself (only one population had a REC8 mutation) finding
- ★ The recovered adaptive mutations improve sister chromosome cohesion and delay genome replication timing back toward the wild-type pattern in Rec8-expressing cells finding
- ★ Rec8 instability in mitosis is caused by residual separase (Esp1) activity outside of anaphase mechanism
- Rec8 shows reduced protein levels and reduced binding to mitotic chromosomes compared to Scc1 finding
- ★ Engineered mutations that reduce replication origin firing or slow replication forks improve the fitness of Rec8-expressing cells, revealing a link between genome replication and sister chromosome cohesion finding
- A PGAL1-SCC1 conditional expression system allows acute, single-cell-cycle analysis of kleisin function without confounding chronic aneuploidy method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Competitive growth fitness assay | Saccharomyces cerevisiae (Rec8-expressing vs wild-type strain) | Rec8 substituted for Scc1 (PSCC1-REC8 vs PSCC1-SCC1) | relative fitness (ratio change of PACT1-mCitrine-marked strain vs wild type over generations) | — |
| Live-cell fluorescence microscopy (sister chromatid cohesion assay) | S. cerevisiae, Chromosome 5 tagged with GFP-tetR/tetO array | Rec8 vs Scc1 vs scc1Δ, with PGAL1-SCC1 shut-off | fraction of budded cells with one vs two GFP dots (cohesion loss) | fluorescence microscopy |
| Flow cytometry (DNA content analysis) | S. cerevisiae, synchronized G1-released cultures | Rec8 vs Scc1 expression; mad2Δ (spindle checkpoint removal) | cell cycle profile / DNA content (1C, 2C) over time | flow cytometer |
| Western blot (protein level and stability) | S. cerevisiae, Scc1-3xHA / Rec8-3xHA tagged strains | cycloheximide chase; esp1-1 temperature-sensitive separase mutation | kleisin protein abundance, cleavage product, half-life | anti-HA antibody western blot; Hxk1 loading control |
| Chromatin immunoprecipitation (ChIP) | S. cerevisiae, HA-tagged Scc1/Rec8, benomyl-arrested mitotic cells | Rec8 vs Scc1 expression | chromosome-bound kleisin at known cohesin binding sites (DNA associated with immunoprecipitated protein) | anti-HA immunoprecipitation |
| Experimental evolution (serial passaging) | S. cerevisiae, 15 parallel Rec8-expressing populations | long-term propagation for 1,750 generations | fitness change over generations | — |
| Whole-genome sequencing (WGS) | S. cerevisiae, evolved populations | none (post-evolution genotyping) | identification of adaptive mutations | deposited in NCBI GEO (GSE141598) and BioProject (PRJNA594153) |
- ▼ Rec8-expressing strain fitness is 55% that of wild type 55% of WT (45% fitness reduction)
- ▲ Rec8-expressing cells show two GFP dots (loss of cohesion) in 10% of cells during S phase, rising to 50% in mitosis 10% to 50%
- ▼ Rec8 protein level is 4-fold lower than Scc1 during G1 and S phase 4-fold
- ▼ Rec8 half-life (58 minutes) is much shorter than Scc1 half-life (200 minutes) in mitotically arrested cells 58 min vs 200 min
- ▲ Reducing separase activity via esp1-1 increases Rec8 half-life to 190 minutes 58 to 190 min
- – Rec8-expressing cells show more cells with 2C DNA content earlier (30 minutes) and a prolonged 2C/mitotic period (75-180 minutes) compared to wild type
- ▲ Removing Mad2 (spindle checkpoint) from Rec8-expressing cells increases the fraction of cells reaching 1C DNA content and restores S-phase kinetics similar to wild type
- ▲ 15 evolved populations increased fitness after 1,750 generations, with adaptive mutations concentrated in mediator complex, cohesin-related genes, and cell cycle (G1-to-S) regulators rather than REC8
- other fitness = 55% of wild type (competitive growth assay, Rec8- vs wild-type strain, p < 0.01)
- other 45% fitness reduction (overall reproductive fitness cost of Rec8 substitution)
- other half-life: Scc1 = 200 min, Rec8 = 58 min (protein stability in benomyl-arrested mitotic cells, p < 0.01)
- other Rec8 half-life increased to 190 min in esp1-1 (reduced separase activity stabilizes Rec8, p < 0.01)
- fold_change 4-fold less Rec8 than Scc1 (protein levels during G1 and S phase, p < 0.01)
- other two-GFP-dot frequency: 10% (S phase) to 50% (mitosis) (sister chromosome cohesion loss in Rec8-expressing strain, at least 100 cells imaged per time point)
- count 15 parallel evolved populations, 1,750 generations (experimental evolution of Rec8-expressing yeast)
- count 1 population with a Rec8 mutation (out of 15 evolved populations, most adaptive mutations elsewhere)
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.
The paper uses an experimental-evolution design in budding yeast, expressing the meiotic kleisin Rec8 in place of mitotic Scc1 and evolving 15 parallel populations for 1,750 generations. The primary phenotypic comparisons (fitness, protein levels, protein stability, and sister-cohesion frequencies) are made between two-strain pairings using two-tailed Student's t-tests, with results expressed as means ± SD from three or more biological replicates. Statistical significance is indicated with a threshold annotation (**p < 0.01) rather than exact p-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed Student's t-test | Fitness of Rec8-expressing strain relative to wild type (Fig 1B) | 3 biological replicates | not stated |
| two-tailed Student's t-test | Rec8 vs. Scc1 protein levels across synchronous cell cycle time points (Fig 2A) | 3 biological replicates | not stated |
| two-tailed Student's t-test | Protein stability / half-life comparison of Scc1 vs. Rec8, and effect of esp1-1 mutation (Fig 2B) | ≥3 biological replicates | not stated |
| not stated (visual/descriptive comparison of proportion with two GFP dots) | Sister chromosome cohesion assay over time in mitosis (Fig 1C) | ≥100 cells per time point, 3 biological repeats | na |
| flow cytometry (descriptive cell-cycle profiles, no explicit test stated) | S-phase and G2/M progression comparison across strains (Fig 1D) | not stated | na |
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Proportions of cells with two vs. one GFP dot were compared descriptively across time points and strains (Fig 1C)↳ Could also: A chi-square test or Fisher's exact test (for small expected counts) on the counts of cells in each cohesion category at each time point could also be used — Proportion data with a fixed denominator (≥100 cells) follow a binomial distribution; a test designed for count/proportion data would directly quantify whether the difference in proportions between strains exceeds chance variation and yield a p-value tied to the specific comparison
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Two-tailed Student's t-tests were used with n = 3 biological replicates for continuous measurements such as protein levels and fitness↳ Could also: A Mann-Whitney U (Wilcoxon rank-sum) test could also be used for pairwise comparisons with very small n — With only three replicates per group, the normality assumption of the t-test cannot be verified empirically; a non-parametric alternative makes no distributional assumption and is often recommended when n is too small to assess normality
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Multiple independent t-tests were conducted across several figures and comparisons without a stated multiplicity correction↳ Could also: A single ANOVA (or repeated-measures ANOVA for the time-course data) followed by a post-hoc correction such as Tukey HSD or Benjamini-Hochberg FDR could also be applied across the family of comparisons — Conducting many pairwise t-tests increases the nominal type-I error rate across the experiment; a unified model with a post-hoc correction would control the family-wise error rate or false discovery rate while still identifying which specific pairs differ
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Results were summarized using the mean ± one standard deviation↳ Could also: A 95% confidence interval around the mean could also be reported alongside or instead of SD — With n = 3, SD describes spread of the sample but a CI directly conveys uncertainty about the population mean and is often easier to interpret for readers assessing practical significance; some journals and reporting guidelines (e.g., Nature Methods) now recommend CIs over SD for small n
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Statistical significance was reported only as a threshold annotation (**p < 0.01)↳ Could also: Exact p-values (e.g., p = 0.003) and a standardized effect size such as Cohen's d could also be reported — Exact p-values let readers judge the strength of evidence more precisely and are required for meta-analyses; effect sizes convey the magnitude of difference independently of sample size, which is informative when n is very small
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Cell-cycle progression was characterized by flow cytometry profiles displayed as visual traces at selected time points (Fig 1D) without a stated statistical test↳ Could also: A quantitative summary such as the mean time to reach 50% of cells in G2/M (T50) with a t-test or Kaplan-Meier–style analysis of cell-cycle exit could also be applied — Reducing continuous time-course data to a single derived quantity (e.g., T50 or area under the curve) enables formal statistical comparison across strains and replicates, complementing the visual impression from gel-lane profiles
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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MAD2 deletion in Rec8-expressing cells relieves spindle assembly checkpoint-dependent mitotic delay, increasing the 1C fraction.flow-cytometry saccharomyces cerevisiae mixed 2020×1papers★ This paper is the founder (earliest)
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Rec8-expressing cells show accelerated S-phase progression, with more 2C-content cells at 30 min post-G1 release compared to wild type.flow-cytometry saccharomyces cerevisiae up 2020×1papers★ This paper is the founder (earliest)
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Rec8-expressing cells lose sister chromatid cohesion in mitosis, with the two-GFP-dot fraction rising from 10% in S phase to 50% in mitosis.imaging saccharomyces cerevisiae down 2020×1papers★ This paper is the founder (earliest)
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S. cerevisiae cells expressing Rec8 in place of Scc1 have 55% relative fitness compared to wild-type Scc1 cells.other saccharomyces cerevisiae down 2020×1papers★ This paper is the founder (earliest)
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Experimental evolution for 1,750 generations in 15 parallel Rec8-expressing populations substantially increased fitness.other saccharomyces cerevisiae up 2020×1papers★ This paper is the founder (earliest)
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Rec8 protein half-life (58 min) is shorter than Scc1 (200 min) due to premature separase-mediated cleavage; esp1-1 separase mutation restores Rec8 stability to ~190 min.western-blot saccharomyces cerevisiae down 2020×1papers★ This paper is the founder (earliest)
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Evolved Rec8-expressing populations accumulated recurrent adaptive mutations in mediator complex, cohesin-related, and S-phase-inducing genes; only 1 of 15 populations carried a mutation in REC8 itself.WGS saccharomyces cerevisiae 2020×1papers★ 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.
Scope — pmid-32155147
Paper: Hsieh YP, Makrantoni V, Robertson D, Marston AL, Murray AW (2020). Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis. PLoS Biol 18(3):e3000635.
System: S. cerevisiae in which the only cohesin kleisin is the meiotic Rec8 (mitotic Scc1/Mcd1 deleted). Founder is sick; 15 populations were evolved (~1,750 generations) and recovered fitness. The paper asks which mutations drive the adaptation.
Pipeline-derived results (IN SCOPE)
The central computational result is whole-genome variant calling of the evolved populations against the ancestor, yielding the list of adaptive mutations (non-synonymous, frequency ≥90%) reported in:
- S1 Table — mutations at generation 375
- S2 Table — mutations at generation 1,750
- Summarised in Fig 3B/3C (functional modules hit).
Reported high-level findings to check against:
- Cdk8 / mediator module (SSN2/MED13, SSN3/CDK8, SSN8/CYC8?, SRB8/MED12): hit in 12 of 15 populations; 9 produced early stop codons.
- Cohesin: SMC3 in 7 populations, SMC1 in 2, ESP1 in 4.
- G1→S regulators (MBP1, CLN2, SWI4, SWI6): 6 populations total.
- Chr IX (chromosome 9) aneuploidy in several populations.
- One population (P12) carries REC8 E148Q on the plasmid copy.
Pipeline / methods named
- Methods: "WGS data were processed as described [ref 61]".
- Cited analysis code: github.com/koschwanez/mutantanalysis (J. Koschwanez).
Pipeline =
bwa(aln/samse/sampe) →samtoolssort/index → GATK IndelRealigner → VarScan (variant calling, ancestor vs sample) →mutantanalysis.pyclassifies SNP/indel as synonymous / non-syn / stop and reports per gene. Reference genome shipped in repo: S. cerevisiae S288C (SGD) with SGD_features annotation.
Data
- SRA BioProject PRJNA594153 (GEO GSE141598). 120 paired-end runs:
E0-xx(5 ancestral clones: 03,05,09,11,13) — the founders.E3-P1..P15(15 populations, earlier timepoint).E14-P1..P15(15 populations, later timepoint, ~gen 1,750).YPH.../YPH-II...— backcross segregant pools (out of primary scope).
Reproduction strategy (80/20)
Map the koschwanez tool onto the paper's pooled-population data: treat an
E0 ancestral clone as ancestor and an evolved population pool as clone
(segregation/pool args are optional in mutantanalysis.py). For a few CLEAR
data points:
- Align ancestor + selected evolved pools to S288C with
bwa+samtools. - Call variants with VarScan (the tool's own caller) — sample vs ancestor.
- Annotate (snpEff S288C R64 / SGD features) → non-syn, stop, frequency.
- Filter to non-syn ≥90% and check whether the specific genes/mutations the paper reports for those populations are recovered (gene, type, AA, freq).
Faithfulness note: the tool is Python2 + bwa aln + GATK3 + legacy samtools
(env-fragile). The substance — VarScan calling against the ancestor — is
reproduced with current builds of the same caller; the GATK3 indel-realign step
(deprecated, optional refinement) and the HTML classifier are not required to
recover the reported mutations. This is recorded honestly, not as a 1:1 rerun of
the exact legacy binaries.
OUT OF SCOPE (not attempted)
- Wet-lab phenotypes (growth/fitness assays, microscopy, tetrad dissection).
- ChIP-seq analysis (separate repo PhoebeHsieh-yuying; different result class).
- Backcross-segregant linkage (YPH pools) — secondary, large, optional 20%.
- Exact reproduction of legacy GATK3 indel realignment / HTML output.
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.
Strong reproduction. All 21 reported non-synonymous SNPs across the four sampled gen-375 populations were recovered from the raw SRA reads with identical ref>alt and matching allele frequency, including every headline module mutation (cohesin SMC1/SMC3, separase ESP1, and Cdk8/mediator SSN2/SRB8/BOI2 confirmed as stop_gained), so the paper's central conclusion holds 1:1. The only systematic difference is a cosmetic +1 coordinate offset (0-based paper vs 1-based VCF). One genuine flag remains for human review: the recurrent TIR1 chrV:175658 indel reported at 91-96% in 6/15 populations is only ~5-7% in the raw reads of both ancestor and evolved pool — plausibly an artifact of the paper's omitted GATK indel-realignment, but not derivable from the deposited data and biologically suspicious as an identical-position recurrent indel. Overall a solid reproduction with explainable deviations.
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.