Genetic canalization of nutrient resorption: evidence from a widespread grass under effective salt stress

Reviewer #1 (Public review):

Summary:

This study demonstrates that nutrient resorption efficiency (NuRE) in Phragmites australis is genetically canalized rather than plastic to salt stress. Using 110 genotypes in a common garden, the authors show that intraspecific variation in NuRE is explained by phylogeographic lineage, ecotype, and latitude, not by effective salinity. Element-specific regulatory strategies further reveal how N, P, and K resorption are differentially controlled. At the population level, this is an important study that fundamentally advances our understanding of plant functional trait evolution and its implications for ecosystem nutrient dynamics under global change.

Strengths:

This study is the first to demonstrate genetic determination of a key nutrient conservation trait under effective salt stress in a widespread macrophyte, directly testing the 'plastic acclimation versus inherent conservatism' paradigm in a non-nutrient stress context. The experimental design is rigorous: each genotype was paired across control and salt treatments, and multilevel stress effectiveness (metabolomics, biomass, Na accumulation) was confirmed before evaluating NuRE. The large sample size of a macrophyte and dual classification (phylogeography + ecotype) allow robust disentangling of genetic versus plastic sources of variation.

The analysis comprehensively tests three resorption control hypotheses using appropriate SMA regression, revealing element-specific and condition-dependent patterns. The latitudinal gradient and variation partitioning provide strong evidence that genetic origin and geographic context outweigh short-term plasticity, with important implications for predicting ecosystem nutrient cycling under global change. This study provides a clear empirical demonstration that a key nutrient conservation trait can remain homeostatic under non-nutrient stress, and that intraspecific variation is primarily a product of population differentiation rather than short-term plasticity.

Weaknesses:

First, the salinity treatment spanned only one growing season. The conclusion of genetic canalization therefore specifically refers to the absence of plasticity to an acute salt shock. Whether long-term, multigenerational chronic salinity could act as a selective agent or induce transgenerational plasticity remains an open and interesting question for further research. Likewise, the physiological mechanisms underlying the observed lack of plastic increase in NuRE (for example, phloem loading or senescence gene expression) are not directly resolved, leaving some inference about trade-offs versus true unresponsiveness. These points do not weaken the study's main conclusion. Instead, they suggest productive future directions, such as longer-term field manipulations and targeted molecular investigations.

Second, the test of nutrient limitation control relies on resorbed N:P and N:K ratios as proxies, an established but indirect approach. Direct nutrient addition experiments would provide stronger causal evidence. Also, the metabolomic analysis is used primarily to validate stress effectiveness; deeper integration of specific metabolites with NuRE variation across genotypes could have offered mechanistic insights but was not pursued. Additionally, the potential collinearity between ecotype and phylogeographic lineage among Chinese populations is not quantitatively addressed. None of these considerations undermines the main finding, which is supported by a robust experimental design and widely accepted analytical approaches.

Reviewer #2 (Public review):

Summary:

The study finds that nutrient resorption efficiency in Phragmites australis shows no plastic response to salinity stress but is canalized by phylogeographic lineage, ecotype, and latitude. In a common garden with 110 genotypes, salinity induced stress, yet no plastic change occurred for N, P, or K resorption. The authors conclude that intraspecific variation is historical and geographic; thus, predictions of wetland nutrient cycling need to account for phylogeographic composition.

Strengths:

The core finding that NuRE shows no plastic response to salinity, but is instead evolutionarily canalized by lineage and latitude, challenges a key assumption of broad trait plasticity. This conclusion is firmly supported by a robust common garden design with 110 genotypes, rigorous multi-level stress validation, and element-specific resorption analyses. The work provides compelling evidence that intraspecific variation in this critical nutrient cycling trait is shaped by phylogeographic history rather than short-term acclimation. The implications for predicting wetland responses to salinization are significant, as ecosystem-level nutrient dynamics may be constrained by the genetic composition of plant populations.

Weaknesses:

The experiment covers only one growing season, with salinity applied in June and measurements in December. While the stress is clearly effective, longer-term or multi-year stress might reveal acclimation or epigenetic effects that are not captured. Given the author team's expertise in parental and transgenerational effects in clonal plants, this limitation is particularly relevant and warrants more thorough discussion in the manuscript.

The salinity treatment uses a single moderate level of 10 ppt, which does not allow assessment of whether more extreme stress might trigger a plastic response. A dose-response design across a gradient would have provided stronger inference about the threshold at which NuRE canalization might be overcome. Additionally, the ecotype analysis in Figure 4 applies only to Chinese populations, as classification was not available for non-Chinese populations, which should be stated more explicitly in the Results.

The variation partitioning shows latitude as a significant predictor, but the R² values are relatively low, indicating that much variance remains unexplained. The manuscript should avoid overinterpreting latitude's explanatory power and more openly acknowledge the role of unmeasured factors. The interpretation of slopes greater than 1 for the resorbed N:P versus green N:P relationship, labeled as "inverted limitation", also needs further explanation regarding its functional significance.

Author response:

We sincerely thank the editors and reviewers for the positive assessment of our work and for the constructive and insightful feedback. We are grateful that the experimental design and the evidence for genetically canalized nutrient resorption efficiency (NuRE) were recognized as compelling and important, with clear implications for predicting wetland nutrient cycling under salinization. We fully agree with the major points raised in the public reviews and outline below our planned revisions to address them, with particular attention to the weaknesses noted.

Reviewer #1 raised two important concerns regarding the scope and generality of our conclusions. First, the salinity treatment spanned only one growing season, so the genetic canalization we document specifically refers to the absence of a plastic response to an acute salt shock; whether long-term, chronic or multigenerational salinity could act as a selective agent or induce transgenerational plasticity remains an open and interesting question. Second, the test of nutrient limitation control relies on resorbed N:P and N: K ratios as proxies rather than direct nutrient manipulation, and the metabolomic analysis is used primarily to validate stress effectiveness. We accept these criticisms and will address them as follows.

Regarding the temporal scope of the treatment, we will explicitly state in the Discussion that our conclusion of canalization pertains to short-term acclimation to an acute salt shock, and we will discuss the scenarios under which chronic, more severe or multigenerational exposure could trigger plastic, acclimatory or transgenerational responses. Given our team’s prior work on parental and transgenerational effects in clonal plants, we will frame these as testable hypotheses for future research. We will also acknowledge that the physiological mechanisms underlying the lack of a plastic increase in NuRE (for example, phloem loading or senescence-associated gene expression) are not directly resolved in the present study and will propose targeted molecular investigations as a natural next step.

Regarding the nutrient limitation tests, we will clearly acknowledge in the Discussion that the resorbed N:P and N:K ratios provide an established but indirect proxy for nutrient limitation, and we will discuss how direct nutrient addition experiments could provide stronger causal evidence, while deepening the integration of the metabolomic profiles with genotype-level NuRE variation where feasible. We will also quantitatively assess the potential collinearity between ecotype and phylogeographic lineage among the Chinese populations.

Reviewer #2 raised three substantive issues regarding the interpretation and presentation of our results. First, although latitude emerged as a significant predictor in the variation partitioning, the relatively low R² values indicate that much variance remains unexplained, and our interpretation should be more cautious; in addition, the functional significance of the “inverted” nutrient limitation (slopes greater than 1 for resorbed versus green N:P) needs further explanation. Second, the single moderate salinity level (10 ppt) does not allow assessment of whether more extreme stress might trigger a plastic response. Third, the ecotype analysis applies only to the Chinese populations, as ecotype classification was not available for non-Chinese populations, and this should be stated explicitly in the Results. We fully agree with these points and will revise accordingly.

To address the concern about latitude, we will temper the language in the Results and Discussion, explicitly noting the limited proportion of variance explained by latitude and acknowledging the role of unmeasured factors, while retaining the study’s central message that genetic and phylogeographic origin outweigh short-term plasticity in shaping NuRE. We will also expand the Discussion to explain the functional significance of the “inverted” nutrient limitation, that is, why P and K are resorbed more completely relative to N, whether as a strategy to maintain optimal N:P:K ratios or a reflection of the higher costs and lower availability of N.

To address the salinity gradient concern, we will acknowledge in the Discussion that the single moderate salinity level may not have been severe enough to trigger a plastic response and will justify future dose-response experiments to identify the threshold at which canalization of NuRE might be overcome.

To address the ecotype limitation, we will explicitly state in the Results that the ecotype analysis in Figure 4 is based only on the Chinese populations, for which ecotype classification was available.