https://doi.org/10.5061/dryad.x3ffbg7th

We use Mimulus cardinalis (scarlet monkeyflower) to ask the following questions:

1. Are there differences in ancestral (2010-2011) leaf trait means and plasticity between regions with distinct historical climates?
2. Are these differences maintained at the peak of the drought (2014-2015) or do populations from different historical climates evolve to drought stress differently?
3. If so, via what type of leaf changes (those that promote escape or those that promote avoidance)? 

We conducted this experiment on plants from six populations (three from north and three from the southern part of the range) with different climates. To obtain the differences in climate between the north and south, we first obtained 30-year-averages for mean annual precipitation and mean annual temperature from ClimateWNA. The precipitation seasonality was obtained from the biovars function from 'dismo' package in R.  Seeds were collected from the field during 2010 and 2011, which was prior to a severe drought, and then were also collected from the same populations during the peak of the drought. All seeds were then refreshed in a common greenhouse. These refreshed seeds (maternal lines) were then planted in wet and dry treatments to conduct the experiment. Data from running A/Ci curves with the LI-6800 were cleaned to obtain the carboxylation rate, RuBP-regeneration rate, CO2 concentration when Vcmax switches to Jmax (Ci transition point), stomatal conductance, leaf temperature. The anatomical traits were gathered by making nail polish peels to count stomata and trichome densities and cross-sections for mesophyll layers and leaf thickness. All traits measured were run against a correlation and those with greater than 0.60 were removed as traits to continue to examine. This only excluded Jmax and the Ci transition point. 

Description of the data and file structure

Data:

aci_data_all

               vcmax = carboxylation rate (µmol per m-squared per second) values obtained from calculations in the
               plantecophys R package from raw data from LICOR 6800 photosynthetic instrument.

               Jmax = RuBP regeneration rate (µmol per m-squared per second) values obtained from calculations in the
               plantecophys R package from raw data from LICOR 6800 photosynthetic instrument.  

               Rd = respiration rate (µmol per m-squared per second) values obtained from calculations in the
               plantecophys R package from raw data from LICOR 6800 photosynthetic instrument.

               Ci.transition = the point where vcmax and jmax flip (ppm)            
               Treatment = wet or dry treatment experienced by plants
               Year = year of collection
               Site = ID for the location where the seeds were collected
               Rep = temporal replicate number
               ID = Individual maternal line name
               PrePeak = the time that the seeds were collected relative to the drought event
               Region = north or south

all_stom_trich

                  Rep = temporal replicate number
                  Placement = ab for above leaf and be for below leaf
                  Stomata = total number of stomata counted from microscope images
                  Trichome = total number of trichomes counted from microscope images
                  Name = the site name
                  Site = the ID of the site location
                  Year = year of collection
                  ID = Individual maternal line name
                  Treatment = wet or dry treatment experienced by plants
                  PrePeak = the time that the seeds were collected relative to the drought event (pre- or peak- drought)
                  Region = north or south
                  Trich_mm = number of trichomes standardized for mm-squared
                  Stom_mm = number of stomata standardized for mm-squared

chl_fl_all

                  Treatment = wet and dry
                  Rep = temporal replicate number
                  Timeline = name of maternal plant
                  Fs = steady state fluorescence
                  Fm’ = maximal F, dark-adapted
                  PhiPS2 = 1-Fs/Fm’
                  ETR = electron transport rate in micromole per second (µmol/s)
                  A = assimilation rate (µmol per m-squared per second)
                  Year = year of collection
                  PrePeak = the time that the seeds were collected relative to the drought event (pre- or peak- drought)
                  Site = ID for the location where the seeds were collected
                  Fv.Fm. = 1-Fo(minimal F, dark adapted)/Fm’
                  Region = north or south

Climate

                  Region = north or south
                  Site = the site name
                  MAP = mean annual precipitation obtained from climateWNA
                  MAT = mean annual temperature obtained from climateWNA
                  CVI = coefficient of covariance for precipitation seasonality obtained from the biovars package using
                  climateNA in R

gsw_50

                  A = carbon assimilation rate value obtained from LICOR-6800 (µmol per m-squared per second)
                  Ca = ambient carbon dioxide concentration value obtained from LICOR-6800 (ppm)
                  Ci = intercellular carbon dioxide concentration value obtained from LICOR-6800 (ppm)
                  gsw = gas exchange value obtained from LICOR-6800 (µmol per m-squared per second)
                  Tleaf  = leaf temperature value obtained from LICOR-6800 (celsius)
                  ID = Individual maternal line name
                  Treatment = wet or dry treatment experienced by plants
                  Rep = temporal replicate number
                  Year = year of collection
                  Site = ID for the location where the seeds were collected
                  PrePeak = the time that the seeds were collected relative to the drought event (pre- or peak- drought)
                  Region = north or south

 
gsw_400

                  A = carbon assimilation rate value obtained from LICOR-6800 (µmol per m-squared per second)
                  Ca = ambient carbon dioxide concentration value obtained from LICOR-6800 (ppm)
                  Ci = intercellular carbon dioxide concentration value obtained from LICOR-6800 (ppm)
                  gsw = gas exchange value obtained from LICOR-6800 (µmol per m-squared per second)
                  Tleaf  = leaf temperature value obtained from LICOR-6800 (celsius)
                  ID = Individual maternal line name
                  Treatment = wet or dry treatment experienced by plants
                  Rep = temporal replicate number
                  Year = year of collection
                  Site = ID for the location where the seeds were collected
                  PrePeak = the time that the seeds were collected relative to the drought event (pre- or peak- drought)
                  Region = north or south

hbPopulations

                  Site = name of the location where populations are
                  Group = letter ID for each location
                  ID = site numerical ID
                  Lat = latitude
                  Lon = longitude

Mesophyll_proportions_all

                  Rep = temporal replicate number
                  Treatment = wet or dry treatment experienced by plants
                  ID =  maternal seed line identification number
                  PrePeak = the time that the seeds were collected relative to the drought event (pre- or peak- drought)
                  Region = north or south
                  Site = ID for the location where the seeds were collected
                  Length 1 = measure (of mesophyll thickness total (including palisade and spongy) (µm)
                  Length 2 = 2nd measure of mesophyll thickness total (including palisade and spongy) (µm)
                  Length 3 = 3rd measure of mesophyll thickness total (including palisade and spongy) (µm)
                  Palisade 1 = measure of palisade thickness (µm)
                  Palisade 2 = 2nd measure of palisade thickness (µm)
                  Palisade 3 = 3rd measure of palisade thickness (µm)
                  Ratio 1 = proportion between total length and first palisade thickness
                  Ratio 2 = proportion between total length and second palisade thickness
                  Ratio 3 = proportion between total length and third palisade thickness
                  Average_prop = average ratio number
                  Thickness 1 = thickness of leaf including epidermal pavement cells (not trichomes) (µm)
                  Thickness 2 = 2nd thickness of leaf including epidermal pavement cells (not trichomes) (µm)
                  Thickness 3 = 3rd thickness of leaf including epidermal pavement cells (not trichomes) (µm)
                  Average_thick = average leaf thickness (µm)
                  Palisade_length = average palisade thickness (µm)

RandomID

                  Name = population site location
                  Site = numerical site name
                  Year = year of collection
                  Timelines = name of maternal plant
                  ID = new ID made for each maternal plant for randomization
                  Treatment = wet or dry treatments
                  Unique = combination of ‘Site’, ‘Timeline’, ‘Year’, and ‘Treatment’
                  PrePeak = collection year relative to drought timing

sites

                  ID = numerical name for each site
                  Name = population site name
                  Latitude = latitude
                  Longitude = longitude
                  Elevation = elevation
                  Region = South, Centre, and North

timeSer3

                  Year = the year the weather data is from climateNA
                  ID1 = name of site
                  ID2 = each site by year combination has 1000 weather points within a 62km radius
                  Latitude = latitude location of weather site near the ID1 population location
                  Longitude = longitude location of weather site near the ID1 population location
                  Elevation = elevation location of weather site near the ID1 population location
                  Tmax = maximum temperature (kelvin)

                                    01-12 = month of the year

                  Tmin = minimum temperature (kelvin)

                                    01-12 = month of the year

                  Tave = average temperature (kelvin)

                                    01-12 = month of the year

                  PPT = precipitation amount (mm)

                                    01-12 = amount of precipitation within each month

                  Columns BC-ET = parameters generated by climateNA that are not used

                  CMD = climate moisture deficit

                                    01-12 = month of the year

                  RH = relative humidity

                                    01-12 = month of the year

                  Bio1 = mean annual temperature (kelvin)
                  Bio2 = mean diurnal range (mean of max temp – min temp)
                  Bio3 = Isothermality (bio2/bio7)*(100)
                  Bio4 = temperature seasonality
                  Bio5 = Max temperature of warmest month
                  Bio6 = min temperature of coldest month
                  Bio7 = temperature annual range (bio5-bio6)
                  Bio8 = mean temperature of wettest quarter
                  Bio9 = mean temperature of driest quarter
                  Bio10 = mean temperature of warmest quarter
                  Bio11 = mean temperature of coldest quarter
                  Bio12 = total annual precipitation
                  Bio13 = precipitation of wettest month
                  Bio14 = precipitation of driest month
                  Bio15 = precipitation seasonality (coefficient of variation)
                  Bio16 = precipitation of wettest quarter
                  Bio17 = precipitation of driest quarter
                  Bio18 = precipitation of warmest quarter
                  Bio19 = Precipitation of coldest quarter      
                  PopID = site number

Script file structure:

0.1_map.R = generate a map of Oregon and California with the six focal populations. 

0.5_bio15.R = first grab precipitation seasonality. This is obtained from calculating the weather averages for precipitation and temperatures for each year across the 30 years of interest and using the* biovars* function from 'dismo'.

1.0_clim.R = Graphical representation of the 30 year climate averages for the two regions.

2.0_aci_fit.R = using the 'plantecophys' package to generate the ACi curve fitting and grab all variables of interest to create new dataframes of the ACi variables for each replicate. Merge all files together.

3.0_vcmax.R = run a mixed-effects model with a type 3 ANOVA using the 'lme4' package. The effect sizes are generated using* omega_squared* from the 'effectsize' spackage and following Field (2013). To compare the contrasts of interest, use 'emmeans' and specify the 12 contrasts wished to examine and posthoc with a Benjamini Hochberg analysis to reduce type-2 error. Generate a graphical representation of the residuals of the data using 'ggplot2'. 

4.0_Assim.R = same as 3.0 but with assimilation data for max assimilation rate and net assimilation rate at 400ppm CO2 concentration. 

5.0_gsw.R = same as 3.0 but with max stomatal conductance at 50 ppm CO2 concentration and net stomatal conductance at 400ppm CO2 concentration. 

6.0_mesophyll.R = same as 3.0 but with average proportion of the palisade mesophyll layer relative to the spongy mesophyll layer. 

6.5_thickness.R = same as 3.0 but with leaf thickness. 

7.0_stomtrich.R = same as 3.0 but with stomatal and trichome densities. Each model is made for the above and below densities. 

Sharing/Access information

Links to other publicly accessible locations of the data:

• https://github.com/haleybranch/Regional-differences-in-leaf-evolution-

Data was derived from the following sources:

• ClimateNA, ClimateWNA, biovars, seed collections, demography data, LI-6800 portable photosynthetic instruments, microscopy 

Code/Software

R version 4.0.4

Packages required in alphabetical order:

car
cowplot
dismo
dplyr
effectsize
emmeans
ggplot2
Hmisc
lme4
lmerTest
multtest
MuMIn
plantecophys
tidyverse
visreg