DOI: 10.5061/dryad.x3ffbg7th
title: Regional differences in leaf evolution facilitate photosynthesis following severe drought

description: Characterizing physiological and anatomical changes that underlie rapid
 evolution following climatic perturbation can broaden our understanding of
 how climate change is affecting biodiversity. It can also provide evidence
 of cryptic adaptation despite stasis at higher levels of biological
 organization. Here we compared evolutionary changes in populations
 of Mimulus cardinalis from historically different climates in the
 north and south of the species’ range following an exceptional drought. We
 grew seeds produced from pre-drought ancestral plants alongside
 peak-drought descendants in a common greenhouse and exposed them to wet
 and dry conditions. Prior to the drought, northern ancestral populations
 expressed traits contributing to drought escape, while southern ancestral
 populations expressed drought avoidance. Following the drought, both
 regions evolved to reduce water loss and maintain photosynthesis in dry
 treatments (drought avoidance), but via different anatomical alterations
 in stomata, trichomes, and palisade mesophyll. Additionally, southern
 populations lost the ability to take advantage of wet conditions. These
 results reveal rapid evolution towards drought avoidance at an anatomical
 level following an exceptional drought, but suggest that differences in
 the mechanisms between regions incur different trade-offs. This sheds
 light on the importance of characterizing underlying mechanisms for
 downstream life-history and macromorphological traits.
description: Climate data was collected from ClimateWNA. Seeds
 of <em>Mimulus cardinalis </em>(scarlet monkeyflower) were
 originally field collected prior to a severe drought (2010 and 2011) and
 during the peak drought (2014 and 2015) that occurred throughout the range
 . Following a refresher generation, the seeds were grown in a common
 garden experiment and exposed to either wet or dry treatments. Plants were
 monitored to maintain a similar drought across all plants. Leaves were
 sampled with a licor 6800 photosynthetic instrument for physiological
 traits. The leaves were collected and sectioned to examin leaf cell layers
 and epidermal peels were made using nail polish to examine leaf surface
 architecture. Data was directly input into a spreadsheet for anatomical
 traits. Data from Licor instrument was imported into Excel and cleaned for
 just the variables of interest using R software.  
 <strong> </strong>
description: # Regional differences in leaf evolution facilitate photosynthesis
 following severe drought
 [https://doi.org/10.5061/dryad.x3ffbg7th](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-](http://...) 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