{"id":"f86f1904-be24-4613-9070-36d2da78bb47","arxiv_id":"2507.07761","paper_version":1,"verdict":"CONDITIONAL","confidence":"MODERATE","novelty_score":6.0,"correctness_risk":"medium","formal_verification":"none","parameter_count":4,"one_line_summary":"RIFinder detects 622 remote gene transfers between major grass lineages, with functional enrichment in stress-response genes and two detailed adaptive cases.","lead":"This paper introduces a computational method, RIFinder, that scans grass genomes for DNA segments swapped between distantly related subfamilies, and reports 622 such events. It highlights a drought-related gene cluster acquired by a desert grass and traces a defensive chemical's gene cluster to remote transfers.","discovery_kind":"new_method","skeptic_critique":{"model":"deepseek-v4-flash","headline":"The mBLT that separates the 622 RI events from ILS is never benchmarked on ILS-only simulations, and its stated null contradicts standard coalescent expectations; the count of true events is unsupported without that control.","rationale":"The reader's weakest assumption identifies exactly the load-bearing gap: the modified branch-length test is not benchmarked against ILS. My reading of the Methods confirms that the simulation benchmark never includes an ILS-only condition, so precision/recall numbers from the existing benchmark cannot constrain the false-positive rate in real grass genomes where ILS is expected to be common across an 80-Myr split. This is the most load-bearing concern because the headline '622 events' is the quantitative foundation for the functional enrichment and adaptive-introgression narrative; if a substantial fraction of those 622 are ILS artifacts, the prevalence conclusion weakens regardless of the individual case studies. The two deep-dive case studies (the Cleistogenes segment and gramine clusters) are supported by additional evidence—synteny, read coverage, expression, and topology tests—and may survive even if the genome-wide count is revised. The paper also contains an internal inconsistency: the null says coalescence times should not differ between topologies, but the text immediately states ILS leads to older coalescence times; under the multispecies coalescent, discordant topologies do have older coalescence times, so the null distribution is mis-specified. This reinforces the need for an explicit ILS-only control. Because the central claim is potentially right but currently under-supported, the verdict should remain CONDITIONAL: the paper should be accepted only if the authors supply an ILS-only simulation benchmark and show that the mBLT false-positive rate is negligible at the observed ILS levels. No change from the reader's verdict is needed; the concern strengthens the condition but does not by itself warrant rejection, given prior independent evidence for some inter-subfamily transfers and the availability of code and data for re-analysis.","tokens_in":34958,"tokens_out":5933,"duration_ms":76495,"concrete_test":"Simulate 10,000 gene trees under the multispecies coalescent with no introgression, using the same 122-tip grass species tree, divergence times, and sampling as Supplementary Table 1, with population sizes chosen to generate ILS discordance comparable to the real data (e.g., msprime or SimPhy). Run the released RIFinder pipeline unchanged, including RI scoring, mBLT, and Benjamini-Hochberg FDR. Count how many 'RI events' pass. If pure-ILS simulations yield a nonzero number of significant calls approaching or exceeding the 5% FDR level, the mBLT is not discriminating and the reported 622 events require downward revision.","verdict_should_be":"UNCHANGED","load_bearing_attack":"The central claim of '622 true remote introgression events' depends on the modified Branch-Length Test (Methods, 'Modified Branch-Length testing') being able to exclude incomplete lineage sorting. The paper states the null as 'in the absence of introgression, coalescence times should not differ significantly between topologies,' yet the same paragraph says ILS 'typically leads to older coalescence time.' Under the multispecies coalescent, a discordant topology arises from deeper coalescence, so the donor-acceptor distance is expected to be longer, not equal, than the donor-sister distance. The implemented test is a two-tailed independent t-test, and it is not described how a longer donor-acceptor distance is handled; if only significant t-tests are retained regardless of direction, ILS itself could generate exactly the kind of significant difference the test is meant to exclude. Critically, the simulation benchmark (Methods, 'Simulation') only injects RI at known rates and reports precision/recall on those datasets; no ILS-only simulation without any gene flow is run through RIFinder. Therefore, the false-positive rate of RIFinder under pure ILS is unknown, and the 622-event count may be substantially inflated. The arbitrary Cayley-formula scoring threshold (n(n-2), with the exponent missing in the text) adds a second uncalibrated filter, reinforcing the need for an explicit null benchmark.","agreement_with_reader":"agree"},"referee_report":{"model":"deepseek-v4-flash","summary":"The paper introduces RIFinder, a phylogeny-based pipeline for detecting remote introgression (RI) between deeply diverged lineages, and applies it to 122 haploid grass genomes. The authors report 622 RI events between the PACMAD and BOP clades, functional enrichment in stress-response genes, a Triticeae-derived drought-associated segment in Cleistogenes songorica, and RI-mediated assembly of gramine biosynthetic gene clusters. The manuscript also describes simulation benchmarks, detailed case studies, expression analyses, synteny, and ecological niche comparisons.","tokens_in":35294,"tokens_out":4646,"duration_ms":57647,"significance":"If the main claim holds, the paper establishes that inter-subfamily gene transfer is widespread and adaptively important in grasses, a major expansion beyond documented cases of HGT and close-range introgression. The study is valuable for its large, openly described dataset, the public availability of RIFinder code and results, and the multi-pronged case studies. The C. songorica segment and gramine cluster examples are supported by independent evidence beyond the genome scan, including raw-read coverage, synteny, phylogenetic topology tests, expression, and metabolomics. The central quantitative claim, however, currently rests on a detection method whose ability to exclude incomplete lineage sorting has not been demonstrated, so the 622-event count should be treated as provisional until a proper null benchmark is supplied.","major_comments":[{"comment":"The central claim of 622 true RI events depends on mBLT excluding ILS, but mBLT is never benchmarked on an ILS-only simulation. The Simulation section only injects RI at rates 0.1%–2% and reports precision/recall; it does not run pure-ILS gene trees through RIFinder, so the false-positive rate under ILS is unknown. In addition, the mBLT null is stated as 'coalescence times should not differ significantly between topologies' while the same paragraph notes that ILS 'typically leads to older coalescence time.' Under the multispecies coalescent, the discordant topology labeled as introgression has a deeper coalescence, so d(Donor, Accepted) is expected to be longer, not equal, than d(Donor, Sister). Since the implemented test is a two-tailed independent t-test, the authors do not specify whether only the one-sided direction supporting introgression is retained; if significant differences are kept regardless of direction, ILS itself could generate exactly the signal the test is meant to exclude. Please add a no-gene-flow null simulation, report the false-positive rate at the same taxonomic sampling, and clarify the directional decision rule.","section":"Methods, 'Modified Branch-Length testing' and 'Simulation'"},{"comment":"The RI score threshold is described as determined by Cayley's formula n(n-2), where n is the number of taxa. The text appears to intend n^(n-2), but Cayley's formula counts the number of labeled trees on n labeled vertices and is not a distribution of discordance scores expected under ILS; applying it as a significance threshold is uncalibrated and unexplained. Because this threshold is one of the filters that converts candidate signals into '622 RI events,' its derivation needs to be replaced by an explicit null model or by calibration on simulations that include ILS. As written, the threshold is an ad hoc parameter.","section":"Methods, 'RI scoring'"},{"comment":"The performance evaluation is partly circular. The observed RI rate (0.03%–0.3%) is produced by RIFinder, and the simulation study then selects transfer rates (0.04% and 0.1%) at which RIFinder reports high precision and recall. Such a loop does not independently validate the observed rate. Please provide external positive controls, such as the previously documented Bx, momilactone, and Panicum-derived segments, alongside a negative control with no gene flow, in addition to the current simulation.","section":"Results, 'Inferring remote introgression events using RIFinder'"}],"minor_comments":[{"comment":"The pseudocode and surrounding text contain a literal placeholder for the minimum taxonomic representation threshold, written as 'default: ?'; this must be replaced with the actual value or formula used in the analyses.","section":"Methods, 'Compressing and splitting gene trees'"},{"comment":"The notation is inconsistent: the text says distances to 'all donor leaves d(Donor, Accepted) and all sister clade leaves d(Donor, Sister),' but the second distance should presumably be d(Acceptor, Sister) or d(Donor, Sister) depending on which pairwise comparison is intended; please clarify the definitions of the three clades and the measured distances.","section":"Methods, 'Modified Branch-Length testing'"},{"comment":"The x-axis label says 'Transfer proportion' in the figure but the Methods text specifies transfer rates per lineage-million-years; the units should be made consistent.","section":"Figure 1b and Methods, 'Simulation'"},{"comment":"The text says 'Primes used for constructs' but should read 'Primers'; also, the internal control genes are listed without a corresponding reference for the Z. latifolia 18S primer.","section":"Methods, 'Gramine-related gene expression'"}],"recommendation":"major_revision","confidential_remarks":"I agree with the stress-test assessment: the mBLT/ILS issue is the main risk and is load-bearing for the 622-event count. The authors should be required to run an ILS-only simulation and report the false-positive rate, and to specify the one-sided versus two-sided decision rule. The threshold issue and the circularity of the simulation rates are also fixable in revision. The case studies are strong enough that the paper is not beyond repair."},"author_rebuttal":null,"desk_editor":{"model":"deepseek-v4-flash","letter":"Best to start with the punchline: this is a substantial empirical paper with real strengths, but the headline number of 622 remote introgression events is not yet trustworthy because the filter that separates RI from incomplete lineage sorting is never properly validated.\n\nWhat's actually new: a genome-wide systematic scan for inter-subfamily transfers in grasses, with code and data released, and two carefully investigated case studies. The Cleistogenes segment is well-supported by synteny, read-depth checks, topology tests, and geographic evidence; the gramine cluster is more interpretive but still uses decent phylogenetic and expression data. RIFinder recalls previously known transfers (Bx, momilactone, the Panicum→Hordeum segment), which is a good sanity check. The simulation benchmark shows good precision/recall when the simulated data matches the method's assumptions.\n\nThe weak spot is the modified Branch-Length Test. The stated null is that under ILS, coalescence times should not differ between topologies, but standard coalescent theory says the opposite: a discordant topology arises from deeper coalescence, so the donor–acceptor distance is expected to be longer under ILS, not equal. The test is a two-tailed t-test; it's not clear whether a longer donor–acceptor distance is treated as evidence against introgression or just as a significant difference. More importantly, the simulation study never runs an ILS-only null through RIFinder. Without that, we don't know the false-positive rate under pure ILS, and the 622 count could be substantially inflated. The Cayley-formula threshold is also arbitrary; the formula counts labeled trees, not unrooted phylogenies, and the text has a typo (n(n-2) vs n^(n-2)). It's an ad hoc cutoff, not a principled calibration.\n\nThe validation loop—estimating the RI rate from the data and then using that rate in simulations—is partially self-referential, but not fatally; it's a reasonable way to pick simulation parameters if the method were otherwise trustworthy.\n\nIf I were refereeing, I'd ask for an ILS-only simulation benchmark and a clarification of the mBLT's directional logic before the 622 count is accepted. The case studies and the overall framing likely survive that test.\n\nThis paper deserves a serious referee. The dataset and the two deep dives are valuable. The count needs strengthening, but the work is not a desk-reject.","headline":"The two case studies and the dataset are solid, but the 622-event count rests on a branch-length filter that has never been shown to remove ILS.","tokens_in":35832,"tokens_out":2657,"would_cite":true,"duration_ms":29521,"reading_group":"yes","serious_thinker":"yes","would_accept_peer_review":true},"rs_alignment":null,"lean_confirmation":null,"pith_extraction":{"msc":[],"pacs":[],"model":"deepseek-v4-flash","headline":"The paper reports 622 remote introgression events between the two deep grass lineages, detected by a new pipeline called RIFinder.","keywords":["remote introgression","grass phylogenomics","incomplete lineage sorting","modified branch-length test","stress-response genes","biosynthetic gene clusters","drought tolerance","RIFinder"],"falsifier":"Simulate the grass PACMAD/BOP species tree under a realistic deep coalescent model with zero transfer, run RIFinder on the simulated gene trees, and count how many RI events it reports; if pure incomplete lineage sorting routinely triggers its branch-length criterion, the 622-event claim collapses. A second check is direct: remap long-read assemblies of Cleistogenes songorica and Achnatherum splendens and confirm the 30-kb segment's boundaries and absence from Cson-B; if the segment is present across broad Chloridoideae diversity or shows genealogies that intermix with multiple outgroup species, the recent-transfer interpretation is undermined.","tokens_in":34780,"feed_emoji":"🌾","tokens_out":9959,"duration_ms":103627,"temperature":0.7,"pith_summary":"The paper proposes a distinct category of genetic exchange, remote introgression (RI), meaning DNA transfer between species whose lineages have been separate for tens of millions of years, and it builds RIFinder, a phylogeny-based pipeline to detect such events. RIFinder works by comparing individual gene trees to the reference grass tree, looking for genes of one deep lineage (PACMAD) nested inside the other (BOP), then filtering candidates with a modified branch-length test aimed at ruling out incomplete lineage sorting. Applied to 122 haploid grass genomes, the pipeline reports 622 RI events from 543 genes, with introgressed genes enriched in stress-response functions; the paper's two worked examples are a Triticeae-derived 30-kb segment in the drought-tolerant Cleistogenes songorica and the gramine biosynthetic gene cluster, which appears to have been assembled partly through such transfers. The reason to care is that if this is right, foreign DNA from very distant relatives is a regular and adaptively relevant force in grass genomes, not an occasional anomaly.","feed_headline":"622 gene transfers found between distant grass clades","feed_subtitle":"The new screen shows grasses exchanged stress genes across an 80-million-year-old split.","key_machinery":"The central object is RIFinder, a phylogeny-based remote-introgression detection pipeline. It works by clustering proteins into homology groups, inferring gene trees, collapsing monophyletic single-clade branches, splitting multi-copy trees into ortholog-like subtrees, then scoring each leaf's topological incongruence against a reference two-clade species tree (PACMAD vs. BOP); candidate events must survive a modified branch-length test that compares donor-to-acceptor distances with donor-to-sister distances, on the logic that introgression makes the donor-acceptor pair look younger than incomplete lineage sorting would. The threshold for a significant RI signal uses Cayley's formula for the number of possible unrooted topologies on a given set of tips.","core_discovery":"On the paper's own terms, the discovery is that DNA transfer between subfamilies of grasses that diverged more than eighty million years ago is widespread: 622 candidate remote introgression events, traced to 543 distinct homologous genes, with the Pooideae receiving the most introgressed genes and the Bambusoideae the fewest. The introgressed copies show signatures of post-transfer local adaptation, with significant enrichment in stress-response protein domains. Two cases are worked out in detail: a roughly 30-kilobase Triticeae-derived segment in Cleistogenes songorica that is absent from its other subgenome and from other Chloridoideae, and whose genes respond to drought and heat stress; and the gramine biosynthetic gene cluster, whose AMIS and NMT components show discordant phylogenies placing Panicoideae and Chloridoideae copies inside the Oryzoideae lineage, implying that remote introgression contributed to the cluster's origin and diversification. The paper also reports that RIFinder recovers previously documented transfers, such as the Bx benzoxazinoid genes in Triticeae and the momilactone cluster in rice, and that simulated benchmarks yield high precision and recall.","pith_inferences":["The reported 622 events are a lower bound by the paper's own conservative filters, so denser sampling and k-mer or pangenome-based detection should raise the count; re-running the pipeline on the additional published grass genomes would be a direct test.","If the branch-length assumption survives deep-coalescent benchmarking, the same workflow could be applied to other large plant families or animal radiations, potentially making remote introgression a general feature of eukaryotic genome evolution rather than a grass quirk.","The sympatry of Achnatherum splendens and Cleistogenes songorica suggests an ecological predictor: deeply divergent species with overlapping ranges should show elevated RI enrichment; a systematic comparison of sympatric versus allopatric pairs across the 122 genomes could test this.","The rice pangenome shows the gramine cluster is nearly restricted to one cultivated haplotype group with selection signals, implying RI-derived defense clusters may be subject to fast presence/absence evolution; searching other pangenomes for recently introgressed metabolic clusters would test that."],"forward_implications":["Gene trees in grass phylogenomic data sets will need to be treated as potentially composite: a gene's history can include a foreign branch from the opposite deep lineage even when the species tree is uncontroversial.","Functional screens for stress-tolerance genes in grasses should check for discordant phylogenetic placement, because the enriched stress-response signal means candidate adaptive genes may be introgressed rather than native.","The asymmetry between donor and acceptor counts points to life history, geographic range, and reproductive timing as predictors of how much inter-lineage DNA a plant family acquires, which is a testable comparative hypothesis.","The documented assembly of gramine biosynthetic gene clusters by remote introgression means the presence of a metabolic cluster in a species does not by itself establish vertical ancestry with other cluster-bearing species.","If the Cleistogenes segment's drought stress response is causal, then multigenic stress modules can be acquired ready-made from sympatric distant relatives, which is an evolutionary shortcut unavailable under a purely vertical-gene model."],"supporting_citations":[{"why":"Documents the Panicum-derived stress-gene segment in Hordeum that RIFinder is required to recall, supplying a real previously known case of inter-subfamily transfer.","marker":"Mahelka et al., 2021"},{"why":"Documents the transfer of Bx benzoxazinoid biosynthetic genes from ancestral Panicoideae to Triticeae, the case RIFinder recovers and extends with a newly found Bx6-like clade.","marker":"Wu et al., 2022b"},{"why":"Documents lateral transfer of momilactone biosynthetic genes from wheat to rice, providing the gene-cluster precedent that underlies the gramine cluster argument.","marker":"Wu et al., 2022a"},{"why":"Supplies the branch-length logic that the modified branch-length test adapts to separate introgression from incomplete lineage sorting.","marker":"Suvorov et al., 2022"},{"why":"Provides the D3 statistic, the conceptual predecessor of the branch-length comparison used to test each candidate RI event.","marker":"Hahn and Hibbins, 2019"},{"why":"Reports multiple horizontal transfers of nuclear ribosomal genes between distantly related grass lineages, extending the prior evidence that remote transfer happens.","marker":"Mahelka et al., 2017"},{"why":"Establishes the well-supported Poaceae nuclear phylogeny that defines the PACMAD and BOP clades used as the reference tree for incongruence scanning.","marker":"Huang et al., 2022"},{"why":"Provides the nuclear phylogenomic grass tree used to interpret gene-tree discordance and to date the inferred gramine cluster introgression.","marker":"GrassPhylogenyWorkingGroupIII, 2024"}],"fun_headline_variants":["Remote introgression: 622 genes crossed 80-million-year grass gap","Grasses swapped stress genes across distant clades, 622 times","RIFinder exposes 622 cross-clade gene jumps in grass genomes","Distant grass lineages exchanged 622 genes, study finds","Grass survival genes jumped between clades 80 million years apart"],"cache_read_input_tokens":3200,"weakest_assumption_plain":"The count assumes that ordinary sorting of ancestral genetic variation cannot by itself make a donor and acceptor look closer in time than the sister lineage; if it can, many of the 622 'transfers' could be artifacts of that sorting.","fun_headline_variants_meta":{"raw":{"variants":["Remote introgression: 622 genes crossed 80-million-year grass gap","Grasses swapped stress genes across distant clades, 622 times","RIFinder exposes 622 cross-clade gene jumps in grass genomes","Distant grass lineages exchanged 622 genes, study finds","Grass survival genes jumped between clades 80 million years apart"]},"model":"deepseek-v4-flash","effort":"low","cost_usd":0.0006,"raw_usage":{"total_tokens":2842,"prompt_tokens":1020,"completion_tokens":1822,"prompt_tokens_details":{"cached_tokens":384},"prompt_cache_hit_tokens":384,"prompt_cache_miss_tokens":636,"completion_tokens_details":{"reasoning_tokens":1730}},"tokens_in":636,"tokens_out":1822,"duration_ms":13763,"temperature":1.0,"reasoning_tokens":1730,"cache_read_input_tokens":384,"cache_creation_input_tokens":0},"cache_creation_input_tokens":0},"created_at":"2026-08-06T18:33:01.854728+00:00","model_set":{"reader":"deepseek-v4-flash"},"falsifier":"Simulate the grass PACMAD/BOP species tree under a realistic deep coalescent model with zero transfer, run RIFinder on the simulated gene trees, and count how many RI events it reports; if pure incomplete lineage sorting routinely triggers its branch-length criterion, the 622-event claim collapses. A second check is direct: remap long-read assemblies of Cleistogenes songorica and Achnatherum splendens and confirm the 30-kb segment's boundaries and absence from Cson-B; if the segment is present across broad Chloridoideae diversity or shows genealogies that intermix with multiple outgroup species, the recent-transfer interpretation is undermined.","supporting_citations":[],"review_version":1}