Asymmetric introgression and thermal advantage jointly drive climate-mediated lineage turnover in a mixed-ploidy reed

Reviewer #1 (Public review):

Summary:

The article is testing the relative advantages of plant lineages with differing ploidy and admixture across environmental gradients. The results show that intraspecific variation in ploidy and admixture between lineages impacts plant traits that may enable persistence and range expansion.

Strengths:

Suitable marker panel size and convincing results that include attempts to analyse mixed ploidy level data, which is a challenge.

Weaknesses:

(1) Inadequate explanation of allele dosage for ploidy levels, some of which do not match the allele counts expected for genome copy number.

(2) The setup and sample sizes of the common garden experiments are very unclear. The numbers implied are extremely low to draw robust conclusions.

(3) Unclear how allele dosage is determined. Given it's so central to many analyses, it would be useful to see how this is done rather than use a citation.

Reviewer #2 (Public review):

Summary:

This manuscript describes a combination of species distribution mapping experimental data from common garden and physiological experiments to project the future distribution of genetic subgroups with the widespread grass Phragmites australis. Overall, the sample sizes seem appropriate for the questions being asked, and the key results regarding projected change in distribution of the focal lineages are well supported. However, at this point, it is difficult to evaluate the broader impact of the work on the field or the utility of the data for the broader community outside of those studying the focal species, P. austrina.

Strengths:

A key strength of the paper is the use of common garden and physiological experiments in conjunction with species distribution modeling. The experiments provide a mechanistic basis for the correlations between interspecific lineage and climatic data, suggesting that the distributional patterns are more likely to result from genetic differences rather than limited dispersal among regions. I would, in fact, emphasize the experimental validation of modeling efforts even more in the introduction.

Weaknesses:

I see two weaknesses with the framing of the ms and the presentation of the results. First, no data support the claims that polyploidy has any causal effect. The ploidy levels are, in fact, completely confounded with other genetic differences, so it is not possible to eliminate genetic variation, independent of ploidy, as the causative factor. As the authors note, ploidy was not manipulated in the reported experiments. Thus, the focus on polyploidy in the introduction and elsewhere distracts from the novel and informative experiments that were conducted. Second, the manuscript indicates that intraspecific variation is critical for the evolutionary potential of a species to respond to environmental change, but intraspecific variation is seldom considered in species distribution models. To me, an assessment of evolutionary potential requires estimates of heritable genetic variation and responses to selection. The sample sizes presented here are modest to estimate heritabilities, but the manuscript could be framed with this perspective in mind. However, instead, the manuscript performs species distribution modeling on a small number of sub-specific lineages, essentially treating them as homogeneous "species" - thus the analysis commits the same oversimplification that the manuscript highlights, but does so at a finer evolutionary scale than species. Not acknowledging this simplification (or better, examining phenotypic variation within the genetically defined lineages) hinders what would otherwise be a strength of the manuscript.

The title suggests that asymmetric introgression and thermal tolerance are the most important findings of the work. However, the introduction contains no explanation of the potential importance of gene flow (other than to say that asymmetric gene flow was suggested by some preliminary analyses), and the discussion offers only a limited explanation of either the potential mechanisms underlying the asymmetric gene flow or its importance for the long-term evolution of the species. Similarly, the novelty of combining experiments and species distribution modeling is scarcely mentioned, and there is no exploration of the connection between tolerance alleles and gene flow. Could introgression of heat tolerance alleles alter the spread of the hybridizing lineages, for example? A greater emphasis on these general population genetic parameters could potentially highlight the broader impact of this work.

Author response:

We sincerely thank the editors and reviewers for the positive assessment of our work and for the constructive and insightful feedback.

We fully agree with the major points raised in the public reviews and outline below our planned revisions to address them.

Reviewer #1 raised two important concerns regarding our methodology. First, the determination of allele dosage is insufficiently explained, which is central to our ploidy assignment and downstream analyses. Second, the setup and sample sizes of the common garden experiments are unclear, raising questions about the robustness of our conclusions. We accept these criticisms and will address them as follows.

Regarding allele dosage, we will add a detailed step-by-step description of our calling pipeline in the Methods section, including the criteria for peak height ratios and thresholds used to assign copy numbers. We will also clarify a crucial biological detail: the common reed (Phragmites australis) is an allotetraploid in its origin. As a consequence, many molecular markers, including the widely used SSR markers in previous studies, behave as disomic markers (i.e., two homeologous copies inherited in a Mendelian manner). Therefore, observing more than two alleles at a locus is indeed indicative of higher-level ploidy (hexaploidy or octoploidy) in this system. We will explicitly state this to resolve any confusion about why tetraploids in our dataset are treated as having a maximum of two alleles, while hexaploids and octoploids can carry more.

Regarding the common garden experiment, we will explicitly report the replication number for each lineage-by-treatment combination and clarify the experimental design. We will also discuss the statistical approaches used given the sample sizes, while acknowledging that the consistency between experimental results and distributional patterns lends additional support to our conclusions.

Reviewer #2 raised three substantive framing issues. First, ploidy is completely confounded with genetic background, yet our manuscript places undue emphasis on polyploidy as a causal factor. Second, our species distribution models treat each lineage as a homogeneous entity, failing to capture within-lineage variation and thus repeating the oversimplification we criticize. Third, we insufficiently explore the evolutionary significance of asymmetric introgression, gene flow, and the novelty of combining SDM with experiments. We fully agree with these points and will revise accordingly.

To address the confounding issue, we will substantially reframe the manuscript to de-emphasize claims about polyploidy as a causal driver, and instead focus on the adaptive differentiation among distinct genetic lineages that happen to differ in ploidy. The Discussion will explicitly state that dissecting ploidy effects from background genetic effects will require future experimental approaches.

To address the simplification in SDMs, we will add a clear acknowledgment of this limitation, discussing how it may affect predictive accuracy and suggesting that future studies incorporating population-level genomic data could more directly assess evolutionary potential.

To address the insufficient exploration of introgression and the novelty of our approach, we will expand the Introduction to better highlight the value of coupling controlled experiments with SDMs at the intraspecific level. In the Discussion, we will elaborate on the evolutionary significance of asymmetric introgression, including testable hypotheses about how gene flow might mediate the spread of heat-tolerance alleles and influence lineage geographical limits under climate change.
We also thank the reviewer for the suggestion to emphasize the experimental validation of SDM efforts, which we will incorporate into a revised Introduction.

Looking beyond the present study, we envision three complementary directions that build upon our current findings. Expanding common garden experiments to include admixed individuals would test whether introgressed genomic blocks confer fitness advantages under thermal stress. Leveraging the population genomic framework established here, we will transition to whole-genome resequencing for selection scans and genotype-environment association analyses to pinpoint adaptive loci and reveal whether heat-tolerance alleles are preferentially transferred via asymmetric introgression. We will also integrate transcriptomic profiling with phenotypic measurements to identify candidate genes whose expression correlates with thermal performance and introgressed ancestry, helping to disentangle ploidy effects from genetic background. Together, these directions span expanded phenotyping, whole-genome resequencing, and transcriptome-guided discovery, forming an integrated framework that moves from the correlative patterns reported here toward mechanistic understanding. These perspectives are briefly outlined in our Discussion, and we hope the present study will serve as a foundation for these future investigations, which we plan to pursue in subsequent work.

We believe these revisions will substantially strengthen the manuscript.