Data from: A field experiment assessing the roles of drought, herbivory, and local climate on cyanogenesis cline formation and local adaptation in Trifolium repens
Data files
Mar 10, 2026 version files 16.24 GB
-
Albano_et_al_2026_JEB_Cline_Data_Means.csv
2.89 KB
-
Albano_et_al_2026_JEB_Cline_Locations.csv
1.02 KB
-
Albano_et_al_2026_JEB_Script.R
39.03 KB
-
Albano_et_al_2026_JEB_Soil_Moisture.csv
164.84 KB
-
Albano_et_al_2026_JEB_Trifolium_Manipulative_Experiment_Data.csv
101.32 KB
-
Albano_et_al_JEB_2026_PlantAreaPhotos.zip
16.24 GB
-
README.md
13.11 KB
Abstract
Projecting how a population will adapt to environmental changes requires a mechanistic understanding of the specific biotic and abiotic factors that impose selection on that population. In white clover (Trifolium repens), clines in an antiherbivore defense mechanism, hydrogen cyanide (HCN), form via variation in selection imposed by the environment. However, the specific environmental factors that select for or against chemical defense phenotypes in white clover remain unresolved. We performed a field experiment in high and low-latitude study sites, with a factorial manipulation of precipitation and herbivory at each site. These factors are hypothesized to be important in driving HCN cline formation, so we investigated their effects on the fitness of a white clover F3 recombinant population, segregating for the alleles underlying the HCN chemical defense phenotype. Surprisingly, we found that precipitation and herbivory either did not drive differential selection on HCN or its metabolic components, or did not impose selection in a manner consistent with the maintenance of observed HCN clines. Instead, we find that the production of only one of the metabolic components of HCN, cyanogenic glycosides, resulted in a fitness advantage, even when lacking the ability to produce HCN. This was most prominent at the northern study site, which is again contrary to expectations. These results suggest additional physiological roles that cyanogenic glycosides may play in adaptation and the evolutionary ecology of white clover. This study highlights the importance of experimental manipulations of environmental factors to understand how selection acts on genes underlying important phenotypic traits, often in unexpected ways.
Readme file associated with the paper Albano et al., published in the Journal of Evolutionary Biology in February 2026. https://doi.org/10.1093/jeb/voag008
Title: A field experiment assessing the roles of drought, herbivory, and local climate on cyanogenesis cline formation and local adaptation in Trifolium repens
In this paper, we perform a field experiment in high (King Township, ON, Canada) and low (Lafayette, LA, USA) latitude study sites, with a factorial manipulation of precipitation and herbivory at each site. Precipitation and herbivory factors are hypothesized to be important in driving HCN latitudinal cline formation, so we investigated their effects on the fitness of a T. repens F3 recombinant population segregating for the alleles underlying the HCN chemical defense phenotype. This experiment addressed four research questions: (Q1) Is there a cline in herbivory and HCN frequency across a latitudinal gradient in the introduced North American range of T. repens? (Q2) Is selection for or against cyanogenesis or its metabolic components influenced by the manipulation of precipitation or herbivory? (Q3) Is selection on cyanogenesis or its metabolic components by precipitation or herbivory also dependent on the latitude of the study site? (Q4) Is the outcome of any observed selection on cyanogenesis or its metabolic components consistent with the maintenance of the cyanogenesis polymorphism within populations or the formation of cyanogenesis clines across latitudinal gradients? Any NA in the data files represent data not available.
In order to address these research questions, we provide four main data files, an R script file, and a folder of photos:
- Datafile: Albano_et_al_2026_JEB_Cline_Locations.csv
- Datafile: Albano_et_al_2026_JEB_Cline_Data_Means.csv
- Datafile: Albano_et_al_2026_JEB_Soil_Moisture.csv
- Datafile: Albano_et_al_2026_JEB_Trifolium_Manipulative_Experiment_Data.csv
- R script file: Albano_et_al_2026_JEB_Script.R
- Folder: Albano_et_al_JEB_2026_PlantAreaPhotos.zip
The folder contains photos used to determine maximum plant area using the EasyLeafArea software (taken facing downwards in order to keep the entire lateral area of the plant within frame), with each experimental site containing separate folders for each month photos were taken within the growing season of each year. The R script file was originally run in R version 4.4.1 and requires the following packages: tidyverse, glmmTMB, car, DHARMa, emmeans, plotrix, and lme4. The R script contains the necessary code to run the analysis using data from all four of the main data files, which are each described in further detail below:
- In order to assess the presence of a latitudinal cline in the presence of HCN in North American T. repens, we sampled 50 T. repens populations from across a latitudinal gradient, extending from Lafayette, LA, USA, to Moosonee, ON, Canada. We extracted historical mean annual temperature data for each of these populations from WorldClim and determined their level of herbivory and proportion of cyanogenic plants. These data can be found in Albano_et_al_2026_JEB_Cline_Data_Means.csv, which contains the following columns:
- Site: The unique site ID, labelled from 1 through 50
- Latitude: Latitude of origin of each population. Units = decimal degrees
- Longitude: Longitude of origin of each population. Units = decimal degrees
- MAT: Mean annual temperature, extracted as the bio1 variable from WorldClim. Units = degrees Celsius
- AvgHerb: The % of leaf area consumed by herbivores, averaged across 50 individuals per population (1 trifoliate leaf per individual).
- HCN: The proportion of cyanogenic (HCN+ vs HCN, or AcLi vs Acli, acLi, and acli) individuals per population.
- Ac: The proportion of individuals that can produce cyanogenic glucosides (Ac vs ac) per population.
- Li: The proportion of individuals that can produce cyanogenic glucosides (Li vs li) per population.
- Individuals: The number of individuals contributing to the above three proportions in each population. Min = 1, Max = 5
- Cells containing NA represent populations for which not enough individuals germinated to determine the proportion of HCN, Ac, or Li.
2. The latitude and longitude used to extract data for each location from which T. repens individuals were sampled can be found in Albano_et_al_2026_JEB_Cline_Locations.csv. Populations are in order from 1 through 50, but there is no column to identify population ID for ease of raster extraction.
- Longitude: Longitude of origin of each population. Units = decimal degrees
- Latitude: Latitude of origin of each population. Units = decimal degrees.s
3. In order to determine the effectiveness of the precipitation manipulation, we periodically measured soil moisture in each split plot at each study site. These data can be found in Albano_et_al_2026_JEB_Soil_Moisture.csv, which contains the following columns:
- Garden: The location of the experimental site (Ontario or Louisiana)
- Date: The date of the data collection in format MM_DD
- Date2: The date of the data collection in format DD_MMM
- Year: The year of the data collection (2021 or 2022)
- WholePlotID: The unique ID of the whole plots in the experiment, each containing 8 plants (numbered 1 through 48)
- SplitPlotID: The unique ID of the split plots within each whole plot, each containing 4 plants (numbered 1 or 2)
- Rep: The replicate number of the soil moisture measurement within each split plot (3 or 4 reps per split plot)
- Treatment: The precipitation reduction treatment (Control or Reduced)
- PercentVol: One measure of soil moisture conducted at each study site. Units = % volume
- mV: The other measure of soil moisture was conducted at each study site. Units = millivolts
- Cells containing NA represent samples for which soil moisture data in mV are unavailable or samples that failed in reading soil moisture altogether.
4. All identifying, genotyping, and fitness data for all individual T. repens plants in each study site for the field manipulative experiment can be found in Albano_et_al_2026_Trifolium_Manipulative_Experiment_Data.csv, which contains the following columns:
- Garden: The location of the experimental site (Ontario or Louisiana)
- WholePlotID: The unique ID of the whole plots in the experiment, each containing 8 plants (numbered 1 through 48)
- SplitPlotID: The unique ID of the split plots within each whole plot, each containing 4 plants (numbered 1 or 2)
- PlantID: The unique ID of each plant with each split plot (numbered 1 through 4)
- Cyanotype: Describes 4 T. repens phenotypes (AcLi, Acli, acLi, or acli) with respect to HCN and its underlying loci based on the presence/absence of a dominant allele at each of two loci (Ac/ac or Li/li)
- Cyanogenesis: Describes whether or not a plant can produce HCN (AcLi = HCN+, Acli/acLi/acli = HCN-)
- Ac_ac: Describes whether or not a plant contains at least one dominant allele at the Ac/ac locus (Ac = yes, ac = no)
- Li_li: Describes whether or not a plant contains at least one dominant allele at the Li/li locus (Li = yes, li = no)
- Precipitation: The precipitation reduction treatment (Control or Reduced)
- Herbivores: The herbivore reduction treatment (Control or Reduced)
- Survived21: Whether or not each plant survived the first growing season (2021). Survived = 1, Did not survive = 0.
- Flowered21: Whether or not each plant flowered in the first growing season (2021). Flowered = 1, Did not flower = 0.
- Seeded21: Whether or not each plant flowered in the first growing season (2021). Flowered = 1, Did not flower = 0.
- FlowerHeadNumber21: The total number of flower heads produced by each plant in the first growing season (2021)
- SeedSetMass21: The total seed set mass produced by the flower heads collected from each plant in the first growing season (2021). Units = grams
- MaxArea21: The maximum lateral area taken up by each plant throughout the monthly measurements of the first growing season (2021). Units = cm^2
- GrowthRate21: Growth rate of each plant during the first growing season (2021), measured as ln(maximum lateral area) minus the ln(initial lateral area) divided by the # of days between those timepoints. Units = cm^2/day
- Herbivory21: The % of leaf area consumed by herbivores, averaged across 5 trifoliate leaves per plant, in July of the first growing season (2021).
- SurvivedWinter: Whether or not each plant survived the winter between the 2021 and 2022 growing seasons. Survived = 1, Did not survive = 0.
- Survived22: Whether or not each plant survived the second growing season (2022). Survived = 1, Did not survive = 0.
- Flowered22: Whether or not each plant flowered in the second growing season (2022). Flowered = 1, Did not flower = 0.
- Seeded22: Whether or not each plant flowered in the second growing season (2022). Flowered = 1, Did not flower = 0.
- FlowerHeadNumber22: The total number of flower heads produced by each plant in the second growing season (2022)
- SeedSetMass22: The total seed set mass produced by the flower heads collected from each plant in the second growing season (2022). Units = grams
- MaxArea22: The maximum lateral area taken up by each plant throughout the monthly measurements of the second growing season (2022). Units = cm^2
- GrowthRate22: Growth rate of each plant during the second growing season (2022), measured as ln(maximum lateral area) minus the ln(initial lateral area) divided by the # of days between those timepoints. Units = cm^2/day
- Herbivory22: The % of leaf area consumed by herbivores, averaged across 5 trifoliate leaves per plant, in the second growing season (2022).
- For FlowerHeadNumber21, SeedSetMass21, MaxArea21, GrowthRate21, and Herbivory21: Cells containing NA represent individuals for which accurate data was unable to be successfully collected.
- For SurvivedWinter: In Ontario, cells containing NA represent individuals that already died during the 2021 growing season prior to the winter season in question. In Louisiana, all cells contain NA because Louisiana does not experience the winter conditions being tested by this variable.
- For Survived 22, Flowered 22, Seeded 22, FlowerHeadNumber22, SeedSetMass22, MaxArea22, GrowthRate22, and Herbivory22: Cells containing NA represent individuals that already died prior to the 2022 growing season or individuals for which accurate data was unable to be successfully collected.
Albano_et_al_2026_JEB_Trifolium_Manipulative_Experiment_Data:
-
Garden: Location or site where the plants are grown (e.g., Ontario).
-
WholePlotID: Identifier for the whole plot or main experimental block.
-
SplitPlotID: Identifier for a smaller sub-division within the whole plot.
-
PlantID: Unique identifier for each plant.
-
Cyanotype: Plant type based on cyanogenesis (whether the plant produces cyanogenic compounds). Values like AcLi, Acli, etc., indicate different cyanotype genotypes.
-
Cyanogenesis: Whether the plant is cyanogenic or not (produces cyanide compounds) — e.g., Cyanogenic or Acyanogenic.
-
Ac_ac and Li_li: Specific alleles/genetic markers related to cyanogenesis genes. Different capitalization indicates allele variants.
-
Precipitation: Experimental treatment for water — e.g., reduced or controlled precipitation.
-
Herbivores: Herbivory pressure treatment — e.g., reduced or Control (normal) herbivore presence.
-
Survived21: Binary (0/1) indicating whether the plant survived in year 21.
-
Flowered21: Binary (0/1) indicating whether the plant flowered in year 21.
-
Seeded21: Binary (0/1) indicating whether the plant produced seeds in year 21.
-
FlowerHeadNumber21: Number of flower heads produced by the plant in year 21.
-
SeedSetMass21: Mass (likely grams or mg) of seeds set in year 21.
-
MaxArea21: Maximum area (likely leaf or plant canopy area) measured in year 21.
-
GrowthRate21: Growth rate of the plant during year 21 (units unclear, possibly relative or absolute growth).
-
Herbivory21: Measure of herbivore damage or intensity on the plant in year 21.
-
SurvivedWinter: Binary (0/1) indicating whether the plant survived over the winter period.
-
Survived22: Binary (0/1) indicating survival during year 22.
-
Flowered22: Binary (0/1) indicating flowering status in year 22.
-
Seeded22: Binary (0/1) indicating seed production in year 22.
-
FlowerHeadNumber22: Number of flower heads produced in year 22.
-
SeedSetMass22: Mass of seeds set in year 22.
-
MaxArea22: Maximum area measured in year 22.
-
GrowthRate22: Growth rate during year 22.
-
Herbivory22: Herbivore damage measure during year 22.
Albano_et_al_2026_JEB_Script:
-
This R script analyzes experimental data from Trifolium repens (white clover) to test how drought (precipitation), herbivory, and plant cyanotype affect plant survival, growth, reproduction, and herbivore damage. It loads datasets, prepares and transforms variables, then uses mixed-effects models (glmmTMB/lmer) and ANOVA/post-hoc tests to evaluate treatment effects across two sites (Ontario and Louisiana) and two years (2021–2022).
