Data used in manuscript, "Context-dependent concordance between physiological divergence and phenotypic selection in sister taxa with contrasting phenology and mating systems"

PREMISE: The study of phenotypic divergence of, and selection on, functional traits in closely related taxa provides the opportunity to detect the role of natural selection in driving diversification. Where selection in field populations differs between taxa in a pattern that is consistent with the phenotypic difference between them, this provides evidence that natural selection reinforces the divergence. Few studies have sought evidence for such concordance for physiological traits. METHODS: Herbarium specimen records were used to detect phenological differences between sister taxa independent of the effects on flowering time of long-term variation in the climate across collection sites. In the field, physiological divergence in photosynthetic rate, transpiration rate, and instantaneous water use efficiency were recorded during vegetative growth and flowering in 13 field populations of two taxon pairs of Clarkia, each comprised of a self-pollinating and a outcrossing taxon. RESULTS: Historically, each selfing taxon flowered earlier than its outcrossing sister taxon, independent of the effects of local long-term climatic conditions. Sister taxa differed in all focal traits, but the degree and (in one case) the direction of divergence depended on life stage. In general, self-pollinating taxa exhibited higher gas exchange rates, consistent with their earlier maturation. In 6 of 18 comparisons, patterns of selection were concordant with the phenotypic divergence (or lack thereof) between sister taxa. CONCLUSIONS: Patterns of selection on physiological traits measured in heterogeneous conditions do not reliably reflect divergence between sister taxa, underscoring the need for replicated studies of the direction of selection within and among taxa.This README.txt file was generated on 2022-02-01 by Susan Mazer and David Hunter GENERAL INFORMATION 1. Title of Dataset: 2. Author Information A. Principal Investigator Contact Information Name: Susan J. Mazer Institution: University of California, Santa Barbara Address: Department of Ecology, Evolution & Marine Biology, UCSB, Santa Barbara, CA, 93106 Email: sjmazer@ucsb.edu B. Associate or Co-investigator Contact Information Name: David J. Hunter Institution: Westmont College, Santa Barbara, California, 93108 Address: Department of Mathematics and Computer Science Email: dhunter@westmont.edu C. Alternate Contact Information Name: Alisa A. Hove Institution: Warren Wilson College Address: Biology Department, P.O. Box 9000, Asheville, North Carolina, 28815 Email: ahove@warren-wilson.edu C. Alternate Contact Information Name: Leah Dudley Institution: East Central University Address: Department of Biological and Environmental Sciences, East Central University, Ada, Oklahoma, 74820 Email: ldudley@ecok.edu 3. Date of data collection (single date, range, approximate date) : Field data were collected from 2010-04-01 to 2010-06-15, with subsequent specimen processing (to measure above-ground stem biomass and to estimate fruit production) occurring approximately from 2010-07-01 to 2010-12-01 4. Geographic location of data collection : Names and GPS coordinates of populations surveyed for physiological traits and total above-ground stem biomass in 2010. Locality Name Taxon Elevation (m) Latitude (N) Longitude (W) Willow Springs exilis 365 35.670 -118.902 Stark Creek exilis 457 35.474 -118.726 Stark Creek unguiculata 457 35.474 -118.726 Live Oak unguiculata 430 35.479 -118.749 Democrat Springs Campground unguiculata 590 35.531 -118.663 Democrat Springs Campground xantiana 590 35.531 -118.663 China Gardens unguiculata 641 35.533 -118.646 Borel Road xantiana 707 35.584 -118.522 Camp 3 xantiana 896 35.812 -118.453 Fay Ranch Road parviflora 927 35.700 -118.304 Sawmill Road parviflora 1003 35.678 -118.481 Sawmill Road xantiana 1217 35.675 -118.510 Long Valley parviflora 1628 35.815 -118.091 5. Information about funding sources that supported the collection of the data: Funding for this research was awarded by the National Science Foundation (award IOS-0718227) to L.S.D. and S.J.M. A.A.H. received funding from the Luce Foundation, the Ralph M. Parsons Foundation, and the California Native Plant Society. SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: None 2. Links to publications that cite or use the data: Manuscript under review in the American Journal of Botany 3. Links to other publicly accessible locations of the data: N/A 4. Links/relationships to ancillary data sets: N/A 5. Was data derived from another source? No A. If yes, list source(s): 6. Recommended citation for this dataset: Mazer, S.J., D. J. Hunter, A. A. Hove, and L. S. Dudley. 2022. Data used in manuscript, "Context-dependent concordance between physiological divergence and phenotypic selection in sister taxa with contrasting phenology and mating systems" (in review in the American Journal of Botany" DATA & FILE OVERVIEW 1. File List: Three data sets are available. 1) CUCE_herbarium_Normals_1951_1980_na.rm.csv: These are the occurrence records and climatic parameters associated with the herbarium specimens of Clarkia unguiculata and Clarkia exilis analyzed in the current manuscript. 2) CXXCXP_herbarium_Normals_1951_1980_na.rm.csv: These are the occurrence records and climatic parameters associated with the herbarium specimens of Clarkia xantiana ssp. xantiana and Clarkia xantiana ssp. parviflora analyzed in the current manuscript. The R code used to analyze these data sets and to create the figures included in the manuscript is presented, along with the output, in the following html file (also uploaded into Dryad): 2022-01-17-Analyses_of_Herbarium_Records-Dryad 3) ClarkiaFieldData2010.csv: This data set contains all of the information for the field-collected data recorded in the wild populations of all four taxa (Clarkia unguiculata, C. exilis, C. xantiana ssp. xantiana and C. xantiana ssp. parviflora). These data were used in the analyses of total and direct phenotypic selection on photosynthetic rate, transpiration rate, and instantaneous water use efficiency presented in the manuscript. In addition, these data were used to detect physiological divergence between sister taxa. The R code used to analyze this data set and to create the figures included in the manuscript is presented, along with the output, in the following html files (also uploaded into Dryad): Gas_Exchange_Least_Squares_Means_Table.html Gas_Exchange_Confidence_Intervals.html Gas_Exchange_Summary.html Gas_Exchange_Means_Figure.html Total_Selection_Gradient_Models.html Total_Selection_Figure.html Direct_Selection_Gradient_Models.html Direct_Selection_Figure.html loadClarkiaData.R 2. Relationship between files, if important: The file loadClarkiaData.R is a helper script used by all of the other scripts. 3. Additional related data collected that was not included in the current data package: N/A 4. Are there multiple versions of the dataset? No A. If yes, name of file(s) that was updated: i. Why was the file updated? ii. When was the file updated? METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: Study species—Clarkia unguiculata Lindley and C. xantiana ssp. xantiana A.Gray are self-compatible winter annuals that flower in late spring, are endemic to California, and occupy oak and pine woodlands, grasslands, and roadside habitats, California. While C. xantiana ssp. xantiana is restricted to the southern Sierra Nevada and Transverse ranges to the south, the geographic range of C. unguiculata is much more widespread, occupying both the Sierra Nevada and the Coastal and Transverse Ranges (Lewis and Lewis, 1955). In the Lake Isabella region of the southern Sierra Nevada (Kern and Tulare counties), both taxa may occur alone or in sympatry with their predominantly selfing sister taxa (C. exilis and C. xantiana ssp. parviflora, respectively). Where sister taxa co-occur, reproductive isolation is reinforced by differences between sister taxa in flowering phenology and floral morphology, with selfers flowering earlier and showing greater levels of herkogamy and dichogamy than their bee-pollinated outcrossing counterparts (Eckhart and Geber, 1999; Dudley et al., 2007). In the southern Sierra Nevada, the sister taxa studied here differ with respect to habitat. Although C. exilis is sometimes encountered at the same sites as C. unguiculata, when these species are in sympatry, C. unguiculata occupies a broader range of habitats, growing in woodlands and encroaching into grasslands. By contrast, C. exilis has a more patchy distribution; it is generally restricted to relatively cool canyons, stream beds, and ravines, or is found near boulders or rocky outcrops that provide some shade and/or soil moisture (Vasek, 1958). The join distributions of C. xantiana ssp. xantiana and ssp. parviflora follow a west to east gradient, with xantiana occurring in populations to the west and parviflora occurring towards the east, nearly reaching the Mojave desert. Although there is a narrow central zone of contact between these subspecies (Eckhart et al., 2004; Geber and Eckhart, 2005), sites occupied solely by ssp. parviflora receive less precipitation than those occupied solely by ssp. xantiana. Historical climatic and phenological differences between sister taxa: herbarium-based study—Occurrence data and the GPS coordinates of all specimen records available for each of the focal taxa were downloaded from the Consortium of California Herbarium portal (https://cch2.org) on July 31, 2021 (Fig.1). The date of specimen collection was converted to day-of-year (DOY: 1-365; 366 for leap years). The GPS coordinates of each collection site were then used to extract from the climate database, ClimateNA v.7.01 (Wang et al., 2016) the mean values of 23 annual climatic variables estimated from 1951-1980, the 30-year period preceding the point at which climate warming is considered to have begun to accelerate (IPCC, 2013). The climate variables estimated are defined in Appendix S1 (see Supplemental Data with this article). To estimate climatic parameters, ClimateNA uses interpolated values from the PRISM climate database (PRISM Climate Group, Oregon State University, http://prism.oregonstate.edu) to generate scale-free estimates of local climatic conditions at a resolution of 4km x 4km. The DOY of specimen collection does not provide a precise record of the flowering date of a specimen because an individual plant collected in flower may have been collected at any time after flowering has begun, and the duration of an individual's flowering period may be several weeks or longer. Nevertheless, the date of specimen collection has been found to be a reliable proxy for flowering time (Davis et al., 2015), and has been used in many studies to detect the factors influencing flowering phenology (Davis et al., 2015; Matthews and Mazer, 2016; Willis et al., 2017; Ferris and Willis, 2018; Park and Mazer, 2018, 2019; Ellwood et al., 2019; Love et al., 2019; Park et al., 2019; Mazer et al., 2021; Park, Breckheimer, et al., 2021; Park, Ramirez‐Parada, et al., 2021; Pearson et al., 2021). Among the focal taxa of this study, images were available from CCH2 on 25 December 2021 for 26, 679, 10, and 33 specimens of C. exilis, C. unguiculata, C. xantiana ssp. parviflora, and C. xantiana ssp. xantiana, respectively. Each image was visually inspected to determine whether the individual plant(s) on each sheet bore one or more open flowers. Of these, 24 (92%), 659 (97%), 8 (80%), and 30 (91%) specimens bore one or more open flowers, indicating that the DOY of specimen collection for these taxa generally reflects the specimen's flowering time at a given site. Statistical analysis of herbarium-derived data and climate—To assess whether sister taxa differ with respect to mean flowering time independent of the effects of local climatic conditions on flowering phenology due to evolutionary and/or short-term plastic responses to climate, we controlled statistically for climatic variation among collection sites when testing for differences between taxa in mean flowering time. To characterize each collection site using a multivariate index of its climate, principal components (PCs) for the 24 annual climatic variables were estimated using the pooled data for each pair of sister taxa. In both pairs of taxa, the first two principal components (PC1 and PC2) for the 24 climatic parameters together account for >75% of the variance in climate (Appendix S2). In the pooled data for Clarkia unguiculata and C. exilis, PC1 and PC2 account for 46% and 31% of the variance, respectively; in Clarkia xantiana ssp. xantiana and ssp. parviflora, PC1 and PC2 explain 62% and 21% of the variance, respectively (Appendix S2). In both taxon pairs, across their geographic distribution, the loadings of PC1 indicate that it represents an axis dominated by temperature, while PC2 represents an axis dominated by humidity (Appendix S3). For each data set, the first two PCs were used to determine whether sister taxa differ with respect to their flowering phenology (controlling for site-specific climatic conditions) and in the chronic climatic conditions to which they are exposed. The function prcomp() was used to estimate PCs, following centering and scaling of the data to unit variance; the function fviz_pca_var() in the package factoextra (v.1.0.7) was used to visualize the contributions of each variable in each PC. These and all subsequent analyses were conducted in RStudio (v. 1.4.1717) with R v. 4.1.1. Phenological differences between sister taxa—To determine whether sister taxa differ in their mean flowering date while controlling for the effects of geographic variation in local chronic climatic conditions (which influence DOY; see Results), we conducted four linear regressions on each sister pair. In all of these models, the day of year of specimen collection (DOY) was the response variable (Table 1). In neither taxon pair were either of the two-way interactions (Taxon x PC1 or Taxon x PC2) statistically significant. Below, we present the results for the model with the lowest AIC value. Type III sums of squares were used to detect a significant difference between taxa in their mean date of collection, controlling for variation among collection sites with respect to the covariates. The lm function with the default setting for contrasts and the lme4 package (v. 1.1-27.1) were used. Climatic differences between sister taxa —To determine whether sister taxa differ with respect to the chronic climatic conditions at their respective herbarium specimen collection sites, we used linear models to detect statistically significant differences between taxa with respect to the mean values of the first and the second PCs. In these analyses, either PC1 or PC2 was the response variable, and taxon was the predictor variable. The lm function (using the default setting for contrasts: c[contr.treatment, contr.poly]) in the lme4 package (v. 1.1-27.1) was used. Contemporary differences between sister taxa: phenotypic divergence in physiological traits—Sampling of extant populations: sample sizes and life stages—Naturally occurring field populations were surveyed in the region of Lake Isabella, California in the southern Sierra Nevada, where we sampled populations at elevations ranging from 457 to 1628 m. In spring 2010, 13 sites were identified that, at the onset of spring, contained a population of one or more of our focal taxa with a sufficient number of healthy juvenile plants that we judged we would be able to survey at least 90 individuals for their physiological performance during both vegetative and flowering stages (Fig.2; see Appendices S4 and S5 for a summary of each population's GPS coordinates, elevation, sampling dates, and sample sizes). Each population was surveyed once during vegetative growth and once during flowering in spring 2010; a different group of individuals was sampled at each life stage. For each survey, 90 individuals were selected at random positions along one or more transects traversing the population, with the restriction that a surveyed plant had to bear at least one leaf long enough to sample with a LiCoR-6400 gas exchange analyzer (leaves that were too short could not cross the leaf chamber when it was closed). Sampled plants were typically at least 1m apart from one another. In total, we measured the physiological performance of >1800 individuals. We analyzed and compared these traits while controlling statistically for leaf position on the primary stem and for leaf temperature at the time of measurement, both of which can influence measures of physiological performance (Mazer et al., 2010). Physiological survey—Gas exchange measurements were recorded on leaves produced by the primary branch on each plant. Measured leaves remained attached to the stem while using a portable infrared gas exchange analyzer (IRGA; LI-COR 6400, Lincoln, Nebraska, USA). We recorded photosynthetic rate (A; μmol CO2 m-2 s-1), transpiration rate (E; mol H2O m-2 s-1), and the derived parameter, A/E, instantaneous water use efficiency (WUEi; μmolCO2 molH2O-1 x 0.0001). In addition, the position of each measured leaf, recorded as the number of the node that bore it, was recorded; the node just above the cotyledons was referred to as node 1, with subsequently produced nodes counted in sequence. The temperature of the leaf (°C) at the time of measurement was also recorded. Node number and leaf temperature were included as covariates in our statistical models in order to control for their potential effects on physiological rates, as observed by Mazer et al. (2010). The following LI-COR settings were used: light source, 6400-40 fluorometer; stability, values of CO2 and H2O remained stable for at least 15 s with a change in slope of