| 05 Jan 2026
Contrasting isotopic responses of dryland and wetland plants to a century of global anthropogenic changes in nutrient cycling
Abstract. Anthropogenic emissions of carbon dioxide and reactive nitrogen in various forms disrupt the functioning of ecosystems around the world. In Europe, many valuable habitats, particularly wetlands and semi-natural dry grasslands, are under threat from ongoing eutrophication. However, due to contrasting water regimes, the uptake of anthropogenic nitrogen by plants in these ecosystems is different and is also interrelated with an increase in trophic level in both habitats.
In our study, we measured the δ15N and δ13C values, as well as the total nitrogen content (TN), of 99 pairs of foliar samples collected from seven species of vascular plants in both dry grasslands and wetlands in Poland. Each pair consisted of a historical sample collected from a herbarium voucher dating from before 1939 (i.e. before artificial fertilisers were widely used in agriculture) and a contemporary sample collected in 2024 from the same species in a similar location.
We performed t-tests to determine whether there were significant differences in the means of δ15N, TN and δ13C between samples from the two different habitats. Next, we calculated the differences in δ15N, TN and δ13C between the contemporary and historical samples for each pair. We then tested whether the difference for each species and habitat type was significantly different from zero using 90 % confidence intervals. Using multiple linear regression, we analysed the relationships between differences in δ15N and TN over time and the following factors: habitat type, the proportion of farmland in the surrounding landscape, the consumption of synthetic nitrogen fertiliser, and NOx deposition.
δ15N and TN values were lower in dry grassland species than in wetland species, in both the contemporary and historical subsets. For dry grassland species, the mean δ15N value was lower in contemporary samples than in historical ones. For wetland species, the opposite was true. The difference in δ15N values between pairs of samples was positively related to the amount of farmland in the surrounding landscape. The mean TN was higher in contemporary than in historical wetland samples, but not in dry grassland plants. The mean δ13C value, corrected for the Suess effect, was lower in contemporary samples than in historical ones. The mean difference was −0.51 ‰ for dry grassland and −3.85 ‰ for wetland species.
Our study revealed that the century of fossil fuel-derived carbon emissions, increased nitrogen input into the environment, and dominance of artificial fertilisers and combustion-derived nitrogen over biological nitrogen sources have not led to consistent responses across habitats and species. While the isotopic composition of nitrogen and carbon in plant tissues in Central Europe has undoubtedly changed, this change is highly context-dependent. Its magnitude and direction are impacted by the type of habitat and the identity and/or ecology of the species. As expected, man-made alterations appear to be more pronounced in wetland environments than in dryland habitats. Furthermore, the source of disruption may differ between the two habitat types. Specifically, wetlands are exposed to a multitude of anthropogenic nitrogen and carbon sources, whereas dry grasslands seem to be predominantly affected by changes in the composition of the atmosphere.
The manuscript by Dembicz et al. addresses a timely and important topic, focusing on how accelerated anthropogenic CO2 emissions and eutrophication have been recorded in plant tissues over more than a century using an isotopic and stoichiometric approach. In the Introduction, the discussion is largely limited to artificial fertilizers, with little attention given to organic manure, including both solid and liquid forms. Considering their well-known contribution to nutrient enrichment, I recommend incorporating this aspect to provide a more comprehensive background.
The study is thorough, based on a large number of sites and well-executed methods. However, in the Methods section, I have raised a few questions and suggestions that should be addressed. The results are clearly presented, but in Figures 3, 4, and B1, I recommend indicating statistically significant differences more explicitly. While values clearly different from zero may be considered significant, adding markers (e.g., asterisks) would improve the clarity and reliability of these figures. The discussion is sound and well-written.
The specific comments line-by-line with some editorial suggestions are listed below:
Line 11 and the whole manuscript: I strongly recommend using italics for small delta, as it is a common use in the stable isotopic-oriented literature.
Line 25: I strongly recommend briefly explaining the Suess effect in the abstract for clarity.
Line 66-72: What about organic fertilizers, particularly liquid forms? I suggest also providing information on their relevance at both the EU and Poland scales.
Line 85 and the whole manuscript: A different font type was used than in the previous description of the nitrogen isotopic signature; please unify the style throughout the entire manuscript.
Line 103 – Please change "organic" to "inorganic".
Line 106 - In the reference list, the 2024 publication has only one author (Pronin), so “et al.” should not be used.
Line 119: Since the authors described this above, I suggest replacing "below" with "above".
Line 146: The species name is used here instead of the Latin family name, unlike in other cases. Please clarify this inconsistency.
Line 190: Did the authors test whether the samples contained CaCO3 that could influence the carbon isotope results? Carbonates may be present, particularly in lake samples. Please clarify how this potential issue was addressed.
Line 194-195: The names and isotopic values of the standards used for calibration should be reported.
Line 225-226: Please provide the name of the software package used, along with the appropriate citation.
Line 240: Figure 2 Caption: What do the whiskers in these boxplots represent? Please clarify.
Line 318: The upper index in the nitrogen signature is missing.
Line 320-321: The upper index in the nitrogen signature is missing.
Line 323: The upper index in the carbon signature is missing.
Line 325: It would be beneficial to include, in the introduction, relevant information on this issue in Poland in recent years, particularly after 2022, when the prices of artificial fertilizers increased dramatically due to the escalation of the war in Ukraine.
Line 374: The lower index of 3 in nitrite is missing
Line 440 Figure B2: The equation of the regression lines, as well as the R2 and P values, should be presented.