ddRAD-seq reveals the genetic structure and detects signals of selection in Italian brown trout.
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
- ✓Any deviation was negligible
- 🟡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 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 -> clean 1:1. The authors deposited the exact STACKS genotype VCF (figshare doi:10.6084/m9.figshare.12999725, the central pipeline input) plus their R/shell repo (github.com/genomeud/GenSal @4f7ea79). Recomputing the clearly-specified downstream numbers directly from that VCF: 90 individuals (exact), 40 chromosomes (exact), 126,134 SNPs vs reported 126,124 (within-tol, diff 10 = 0.008%), and genome-wide FIS 0.3936 vs reported ~0.39 (exact, via the repo's own b11/vcftools --hardy logic). NOT attempted: (a) re-running STACKS from raw reads PRJNA663991 (upstream demux/catalog step is not in the deposited repo) since the deposited VCF makes it unnecessary for these claims; (b) the ZHp selection-region COUNT (17 regions) because a05_ZHp.r leaves the significance threshold commented out and never sets it, and the window->region merging is unspecified -- the Hp/Z values are computable but the count is not cleanly reproducible (the 20%, skipped). Note: VCF header says Stacks v2.4 while Methods say v2.0; BRIEF's data accession PRJEB32115 is the reference genome fSalTru1, not the reads.
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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 87assessed: 2026-06-15 ⛓ 0362c0cfdef2
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Provenance — full disclosure
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- 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
- 2026-08-05
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: opusWhat is the genetic structure of Italian brown trout populations, to what extent have stocking practices introduced admixture (particularly with the Atlantic lineage), and can genome-wide ddRAD-seq data detect signatures of selection distinguishing the lineages?
- ★ Italian brown trout populations are genetically differentiated but show strong admixture introduced by stocking, especially with the Atlantic lineage. finding
- ★ Most analysed populations show high levels of kinship and inbreeding, indicating eroded genetic diversity. finding
- ★ Regions putatively under selection with reduced heterozygosity across populations are enriched for genes involved in the response to viral infections. finding
- ★ ddRAD-seq generated more than 100,000 SNPs in 90 brown trout samples to characterize population structure and selection signatures. resource
- ★ Putative signatures of selection were detected using complementary approaches (ZHp pooled heterozygosity, HapFLK haplotype-based, IBD sliding-window kinship). method
- Candidate selected regions associated with resistance to infectious diseases constitute candidates for studying infection resistance in wild and farmed trout. finding
- The Mediterranea lineage was subdivided into Mediterranea Island and Mediterranea Mainland, yielding five lineages. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| ddRAD-seq (double-digest restriction-site associated DNA sequencing) genotyping | Salmo trutta (brown trout); fish farms and rivers in Italy, Corsica, Austria | none | >100,000 SNP genotypes | Illumina HiSeq2500, paired-end 2×125 bp, V4 chemistry; SphI/BstYI digestion; BluePippin size selection; MagAttract HMW DNA kit |
| Read alignment and variant calling | Salmo trutta v1.1 reference genome (NCBI PRJEB32115) | none | loci detection and genotyping | Stacks v2.0 (process_radtags, gstacks, populations); BWA-MEM |
| Population structure / admixture analysis | 90 brown trout samples (5 lineages) | none | ancestry probabilities Q, K populations | Admixture software |
| Hybrid characterization | subset of SNPs (high FST >0.95, low LD <0.2) | none | hybrid class assignment | NewHybrids |
| PCA and phylogenetic tree analysis | brown trout SNP dataset | none | principal components, ML trees with migration events | R SNPRelate; SNPhylo; iTOL; Treemix |
| Relatedness/IBD and inbreeding estimation | brown trout individuals within and between lineages | none | IBD, inbreeding coefficient FIS = 1-Ho/He, heterozygosity, nucleotide diversity π | R SNPRelate |
| Signatures of selection — pooled heterozygosity | five lineages (whole sample and per population; farmed vs wild) | none | ZHp Z-score in 1 Mb sliding windows (step 200 kb, ~67 SNPs) | — |
| Haplotype-based selection scan | studied brown trout populations | none | haplotype divergence p-values (windows of ≥2 consecutive SNPs) | HapFLK (K=20, 20 iterations) |
- – Stocking practices introduced strong admixture in endemic Italian trout, especially with the Atlantic lineage.
- ▲ Most analysed populations showed high levels of kinship and inbreeding.
- ▼ Regions under selection with reduced heterozygosity across all populations are enriched for genes involved in response to viral infections.
- – More than 100,000 SNPs were obtained from 90 brown trout samples via ddRAD-seq. >100,000 SNPs
- – Admixture analysis identified five ancestral populations/lineages (K=5): AT, CA, MA, MI, MM. K=5
- count >100,000 SNPs (SNPs genotyped across brown trout samples)
- count 96 fishes sampled (individuals sampled from Italy, Corsica, Austria)
- count 6 samples excluded (avg coverage <5×) (samples removed from analysis for low coverage)
- other ZHp threshold < −2.81 or > 2.81 (two-tail p ≤ 0.005) (significance threshold for pooled heterozygosity selection signal)
- pvalue nominal p ≤ 0.01 (HapFLK putative selected regions (≥2 consecutive SNPs))
- other maxQ > 0.95 (threshold defining admixed individuals)
- other kinship coefficient ≥ 0.05 (IBD sliding-window candidate regions (5 Mb window, 2.5 Mb step))
- other FST > 0.95, LD < 0.2 (SNP subset selection for NewHybrids analysis)
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 study used ddRAD-seq to genotype 90 brown trout individuals across five Italian lineages, employing ADMIXTURE, PCA, and maximum-likelihood phylogenetic methods (SNPhylo, TreeMix) to characterize population structure and gene flow. Relatedness and inbreeding were estimated with ML-IBD and F_IS via SNPRelate. Putative signatures of selection were detected with three complementary genome-scan approaches—ZHp sliding-window heterozygosity, HapFLK haplotype-based analysis, and sliding-window IBD kinship—each with permutation-based false-positive control (1,000 reshuffles). Candidate selected regions were functionally annotated using KEGG pathway enrichment.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| ADMIXTURE (ML-based ancestry proportion estimation, K determined by cross-validation) | Population structure across all five lineages / Fig. 1b | 90 | not stated |
| NewHybrids (Bayesian hybrid class assignment) | Hybrid structure characterization using SNPs with FST > 0.95 and LD < 0.2 | 90 | not stated |
| PCA (principal component analysis via SNPRelate) | Population structure visualization | 90 | na |
| Maximum-likelihood phylogenetic tree with bootstrap (SNPhylo) | Phylogenetic relationships among lineages | 90 | not stated |
| TreeMix ML phylogenetic network with migration edges (1–6 migration events evaluated) | Gene flow directions between lineages | 90 | not stated |
| ML estimator of pairwise IBD (SNPRelate); F_IS = 1 − Ho/He | Within- and between-lineage relatedness and inbreeding characterization | 90 | not stated |
| ZHp statistic (Z-transformed pooled heterozygosity), sliding window (1 Mb window, 200 kb step, ~67 SNPs/window), |ZHp| > 2.81 (two-tail p ≤ 0.005), ≥ 2 overlapping significant windows required; permutation resampling (1,000 reshuffles, region-level p < 0.05) | Genome-wide scan for enrichment/depletion of homozygosity as signatures of selection | 90 | not stated |
| HapFLK haplotype-based selection test (K = 20 local haplotype clusters, 20 iterations), nominal p ≤ 0.01 for ≥ 2 consecutive SNPs; permutation resampling (1,000 reshuffles, region-level p < 0.05) | Genome-wide scan for recent selective sweeps differentiating studied populations | 90 | not stated |
| Sliding-window IBD kinship estimation (SNPRelate; 5 Mb window, 2.5 Mb step), average pairwise kinship ≥ 0.05 across all population pairs in ≥ 2 overlapping windows; permutation resampling (1,000 reshuffles, region-level p < 0.05) | Identification of IBD-elevated regions shared between populations as a proxy for selection | 90 | not stated |
| KEGG pathway enrichment analysis (specific test not stated; p < 0.05, minimum 3 pathway occurrences required to test) | Functional annotation of putative selected regions | — | not stated |
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Population structure was inferred using ADMIXTURE with cross-validation to choose K; no second structural method was applied as a primary comparison↳ Could also: STRUCTURE (MCMC-based) or sNMF could also estimate ancestry proportions; DAPC (discriminant analysis of principal components) provides a model-free complement — Comparing results across two algorithms with different assumptions (EM vs. MCMC; parametric vs. model-free) can confirm that inferred K and Q-values are robust to methodological choices, which is informative given the complex admixture history of these lineages
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Signatures of selection were scanned with ZHp, HapFLK, and IBD kinship, each controlled by its own within-method permutation at region-level p < 0.05, with no joint FDR across the three methods↳ Could also: A single genome-wide Benjamini-Hochberg FDR correction applied jointly across all tested windows (or all SNPs) within each method would also be a standard approach; outlier methods such as PCAdapt or BayPass that model population structure as a nuisance covariate could additionally serve as comparators — A pooled FDR across all windows provides a directly interpretable false-discovery rate; methods that co-model structure reduce confounding between differentiation driven by demography and that driven by selection
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KEGG pathway enrichment was assessed at nominal p < 0.05 with a minimum term-count filter of 3 and no correction across pathways↳ Could also: Benjamini-Hochberg FDR correction across all tested pathways could also be applied; a hypergeometric or Fisher's exact test framework (e.g., clusterProfiler) with FDR adjustment is a widely used alternative — When many pathways are tested simultaneously, applying an FDR correction reduces the expected number of false-positive enriched categories and is commonly reported to aid interpretation
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Inbreeding was quantified as F_IS = 1 − Ho/He at the SNP level and via the ML-IBD estimator in SNPRelate↳ Could also: Runs-of-homozygosity (ROH)-based inbreeding coefficients (F_ROH) could also be estimated from the dense SNP dataset; KING-robust or GCTA GREML could provide additional genomic relatedness matrices — F_ROH captures autozygosity from extended IBD segments and can distinguish recent (long ROH) from ancestral (short ROH) inbreeding, providing a complementary perspective to the single-locus F_IS approach, especially relevant for the small population sizes noted in this study
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Recombination rates near putative selected regions were approximated by anchoring a Salmo salar linkage map to the S. trutta genome via BLAST, treating S. salar rates as proxies↳ Could also: Crossover rates could also be estimated directly from the ddRAD genotype data using LD-decay-based methods such as LDhat or LDhelmet within the S. trutta dataset itself — Direct within-species estimation avoids the assumption that recombination landscapes are conserved between S. salar and S. trutta, which may differ in locally rearranged regions, and would yield recombination estimates for all candidate loci rather than only those with anchored linkage-map markers
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Farmed and wild individuals were combined in primary analyses and stratified post hoc by repeating key analyses separately in each group↳ Could also: A mixed-model framework (e.g., GEMMA or SAIGE) that explicitly includes farm/wild origin as a fixed or random effect in the selection scan could also be applied — Explicit statistical modeling of farm/wild status as a covariate in the genome scan can formally partition domestication-associated signals from signals of natural local adaptation, complementing the stratified replication approach used here
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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Stocking practices introduced strong admixture into endemic Italian brown trout populations, predominantly from the Atlantic lineage.other salmo trutta up 2022×1papers★ This paper is the founder (earliest)
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Most analysed brown trout populations exhibited elevated kinship and inbreeding coefficients (FIS).other salmo trutta up 2022×1papers★ This paper is the founder (earliest)
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Admixture analysis of 90 brown trout individuals resolved five ancestral lineages (K=5): AT, CA, MA, MI, MM.other salmo trutta 2022×1papers★ This paper is the founder (earliest)
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Genomic windows under positive selection (reduced heterozygosity, low ZHp) across all brown trout populations are enriched for genes involved in response to viral infections.other salmo trutta down 2022×1papers★ This paper is the founder (earliest)
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Strong, near-1:1 reproduction: the authors deposited the exact STACKS genotype VCF and their own pipeline repo, and recomputing directly from that file matches the paper on individuals (90), chromosomes (40), SNP total (126,134 vs 126,124, Δ0.008%) and genome-wide FIS (0.3936 vs ~0.39). The one gap is the ZHp selection-region count (17), which is not derivable from the shared code because the significance threshold is commented out and the window→region merging is unspecified — an authors-side code incompleteness, not a fabrication signal. A minor provenance inconsistency (Stacks v2.4 in the VCF header vs v2.0 in Methods) is noted but immaterial. Overall yellow: solid descriptive reproduction with the selection-scan half of the central claim only partially confirmable.
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