DOI: 10.5061/dryad.k0p2ngfm3
title: Data from: Repeated polyploidization shapes divergence in floral morphology in Lithophragma bolanderi (Saxifragaceae)

description: Polyploidization is an important driver of evolution and diversification
 in flowering plants. Here, we assess how repeated polyploidization may
 have shaped diversification of floral morphology in Lithophragma bolanderi
 (Saxifragaceae). This species comprises multiple cytotypes and varies
 geographically in its interactions with specialized pollinating moths in
 the genus Greya (Prodoxidae). Past studies have shown that coevolution
 with these moths has favored particular suites of floral characters but
 does not fully explain local and regional floral diversification. We
 combined phenotypic and genomic data from more than 1800 individuals from
 40 L. bolanderi populations spread across its entire range.
 Flow-cytometric analyses revealed a geographic mosaic of populations
 comprising one to four of three dominant (diploid, tetraploid, hexaploid)
 and three rare (triploid, pentaploid, octoploid) cytotypes. Whole-genome
 resequencing of a subset of populations suggested that polyploids arose
 from multiple autopolyploidization events, rather than a single event
 and/or through hybridization, albeit with some signals consistent with low
 levels introgression from the congener Lithophragma glabrum.
 Quantification of flower traits from plants grown in a common garden
 showed that cytotype explained more than 15% of the variation in floral
 morphology, with polyploids showing more variability than diploids.
 Experimental induction of neopolyploids directly induced phenotypic
 changes but also indicated that local selection may have favored
 subsequent convergence in floral morphology among cytotypes in natural
 populations. Collectively, this comprehensive and integrative approach
 provides novel insights into how variability generating processes, such as
 polyploidization integrates with selection from species interactions to
 shape local floral diversification.
description: In 29 natural populations of the plant <em>Lithophragma
 bolanderi</em> in the Sierra Nevada, California, USA, seeds were
 collected. A subset of these seeds was grown in a greenhouse
 common-garden. When the plants were well established, leaf material was
 collected and analyzed using flow cytometry (conducted by one of the
 authors with <em>Brassica napus</em> as internal standard) to
 assess plant ploidy level, flower diameter and corolla-opening diameter
 were measured of fresh flowers using digital calipers, and flowers were
 collected, stored in 70% ethanol, dissected, and photographed to quantify
 additional floral morphological traits based on the photos and to assess
 the shape of the petal edge. As the flowers were apportioned among three
 persons to conduct the measurements, one of these persons remeasured a
 subset of the flowers measured by the other two persons. In cases where
 the measurements were not highly correlated, this one person remeasured
 these traits for all samples. This one person also visually checked for
 “outliers” separately for each population and cytotype, double-checked
 these measures, and remeasured them if necessary. The morphological floral
 traits with all pairwise correlations &lt; 0.7 were included in a
 principal coordinate analysis (PCA) and the principal components (PCs)
 used for further analyses were also included in the dataset.
 In a subset of the natural populations of the plant <em>L.
 bolanderi</em>, in which seeds were collected, flower diameter and
 corolla-opening diameter were measured of fresh flowers using digital
 calipers and compared to the flower diameter and corolla-opening diameter
 measured on a randomly selected subset of the plants from the same
 populations grown in the greenhouse common-garden (see above).
 In an addition 11 natural populations of the plant <em>L.
 bolanderi</em>, leaf material was collected and analyzed using flow
 cytometry (conducted by Plant Cytometry Service [https://www.plantcytometry.nl/] with <em>Allium schoenoprasum</em> as internal standard) to assess plant ploidy level. Some <em>L. bolanderi</em> plants were grown from seeds (derived from crosses between individuals of the same cytotype and from the same population but from different seed families of plants grown from root bulbils of the greenhouse common-garden plants) in a greenhouse common-garden. When the plants were well established, leaf material was collected and analyzed using flow cytometry (conducted by Plant Cytomerty Service once with<em> B. napus</em> and once with <em>A. schoenoprasum</em> as internal standard). The sample to internal standard rations were compared between the two internal standards and with those of the parental seed families quantified by one of the authors with <em>B. napus</em> as internal standard. An additional subset of the seeds collected in one natural <em>L. bolanderi</em> population, in which both diploids and tetraploids grow, was grown in a greenhouse common-garden. A subset of the diploid seedlings was treated with colchicine to induce polyploidization. When plants were well established, leaf material was collected and analyzed using flow cytometry to verify plant ploidy level. When these plants were flowering, hand-pollination crossings were conducted among colchicine treated plants that were at least partially polyploid, among diploid control plants, and among tetraploid control plants. These crosses were done to mitigate side effects of colchicine. The seeds resulting from these crossings were used to grow F1 plants. When the F1 plants were well established, leaf material of all colchicine-treated plants and a subset of the diploid and tetraploid control plants was collected and analyzed using flow cytometry (conducted by Plant Cytomerty Service with <em>A. schoenoprasum</em> as internal standard) to verify plant ploidy level, flower diameter and corolla-opening diameter were measured of fresh flowers using digital calipers, and flowers were collected, stored in 70% ethanol, dissected, and photographed to quantify the additional floral morphological traits based on the photos (see above). The same morphological floral traits as for the data of the <em>L. bolanderi</em> plants from the multi-population greenhouse common-garden were included in a PCA and the PCs used for further analyses were also included in the dataset.   For details, see: Karin Grossa, Homa Papoli Yazdi, Elisabeth Schlager, Jodie Lilley, Andrés Romero-Bravo, Anna Runemark, John N Thompson, and Magne Friberg. Repeated polyploidization shapes divergence in floral morphology in <em>Lithophragma bolanderi</em> (Saxifragaceae). Proceedings of the National Academy of Sciences.
description: # Data from: Repeated polyploidization shapes divergence in floral
 morphology in Lithophragma bolanderi (Saxifragaceae) --- This dataset
 contains all morphological and flow cytometry data newly collected for the
 following article: Karin Grossa, Homa Papoli Yazdi, Elisabeth Schlager,
 Jodie Lilley, Andrés Romero-Bravo, Anna Runemark, John N Thompson, and
 Magne Friberg. Repeated polyploidization shapes divergence in floral
 morphology in Lithophragma bolanderi (Saxifragaceae). Proceedings of the
 National Academy of Sciences. Details concerning the methodology and
 analyses of all data are described in the main text and the SI Appendix
 file of the article above. This dataset consists of the following six data
 files (file type: tab-delimited .txt files): 1.
 01_FlowCytometry_Data_Gross_etal_2025.txt 2.
 02_FloralMorphology27PopulationCommonGarden_Data_Gross_etal_2025.txt 3.
 03_FloralMorphologySyntheticPolyploidization_Data_Gross_etal_2025.txt 4.
 04_FloralMorphologyFieldCommonGarden_Data_Gross_etal_2025.txt 5.
 05_FlowCytometryMethods_Data_Gross_etal_2025.txt 6.
 06_TraitMeasuringComparability_Data_Gross_etal_2025.txt ## Description of
 the data and file structure ### 1.
 01_FlowCytometry_Data_Gross_etal_2025.txt These data were used to assess
 the presence, abundance, and geographical distribution of Lithophragma
 bolanderi cytotypes. These data were also the source for all analyses in
 which ploidy level was used as a factor. The file contains the following
 columns: * FlowCytometryID: Individual ID used for flow cytometry
 analyses. Those starting with “UU_” were analyzed by one of the authors at
 Uppsala University and those starting with “PCS_” were analyzed by Plant
 Cytometry Service
 ([https://www.plantcytometry.nl/](https://www.plantcytometry.nl/)). *
 Population: Three-letter abbreviation of the population name. * Latitude:
 Latitude in °N on the population level. * Longitude: Longitude in °E on
 the population level. * SeedFamily: Seed family (seeds collected from a
 single plant being half- and/or full-sibs) identity. Note that as this is
 not applicable to our field-collected samples, “NA” is listed for these
 samples. * PlantID: Individual code for each plant. Note that for the
 field-collected samples (each sample is from a different individual), the
 “FlowCytometryID” and the “PlantID” are the same. * Year: Year when the
 plants were grown and/or collected and analyzed. “2017” for the plants
 from the first greenhouse common-garden cohort (sowed in September/October
 2016), “2018” for the plants from the second greenhouse common-garden
 cohort (sowed in August/September 2017), and “2019” for the samples
 collected in the field in spring 2019. *
 Place_PlantGrowing_SampleCollection: Information whether leaf material was
 collected from plants grown in the greenhouse common-garden at Uppsala
 University (“GreenhouseCommonGarden_UU”) or from plants in the natural
 populations in California (“Field_CA”). * AnalysisPlace: Information where
 and by whom flow cytometry analyses were run. “UppsalaUniversity” for the
 samples analyzed by one of the authors at BIOVIS (Biolgical Visualisation)
 at Uppsala University, Sweden
 ([https://biovis.uu.se/](https://biovis.uu.se/)), and
 “PlantCytometryService” for the samples analyzed by Plant Cytometry
 Service
 ([https://www.plantcytometry.nl/](https://www.plantcytometry.nl/)). * IS:
 The species used as internal standard (IS) for the flow cytometry
 analyses. * SampleISRatio: The sample:IS ratio calculated by dividing the
 median of the L. bolanderi peak by the median of the IS peak. *
 PloidyLevel: Plant ploidy level determined based on discontinuities in the
 sample:IS ratios, and the information on the known ploidy levels from
 karyological counts by Taylor (1965) [R. L. Taylor, The genus Lithophragma
 (Saxifragaceae). University of California Publications in Botany 37, 1–122
 (1965).]. “2x”: diploid, “3x”: triploid, “4x”: tetraploid, “5x”:
 pentaploid, “6x”: hexaploid, “8x”: octoploid. ### 2.
 02_FloralMorphology27PopulationCommonGarden_Data_Gross_etal_2025.txt These
 data, or a subset thereof, were used to assess differences in floral
 morphology among Lithophragma bolanderi cytotypes both in univariate and
 multivariate analyses. For multivariate analyses, only traits with all
 pairwise correlations &lt; 0.7 were included. These data were also used to
 estimate population means separately for diploids, tetraploids, and
 hexaploids using linear mixed-effect models (for details, see the main
 text and SI Appendix of the paper from which these data are), and these
 estimates, or a subset thereof, were used for additional analyses of
 differences among cytotypes that could only be conducted at the population
 level, such as the assessments of the effect of geographic distance on
 floral morphology differences and the relative contribution of plant
 ploidy level and Greya moth community on floral morphology variation. For
 details on how the traits were measured, see Fig. 2 and SI Appendix, Text
 S4, of the paper from which these data are. The file contains the
 following variables: * Population: Three-letter abbreviation of the
 population name. * SeedFamily: Seed family (seeds collected from a single
 plant being half- and/or full-sibs) identity. * PlantID: Individual code
 for each plant. * Latitude: Latitude in °N on the population level. *
 Longitude: Longitude in °E on the population level. * Year: Year when the
 plants were grown and/or collected and analyzed. “Year1” for the plants
 from the first greenhouse common-garden cohort (sowed in September/October
 2016), “Year2” for the plants from the second greenhouse common-garden
 cohort (sowed in August/September 2017). * PloidyLevel: Plant ploidy
 level. “2x”: diploid, “4x”: tetraploid, “6x”: hexaploid. *
 PloidyLevelMethod: Information whether relative ploidy level was measured
 using flow cytometry (“measured”), or whether it was set to be the same as
 the relative ploidy level measured using flow cytometry on another plant
 from the same seed family in populations where all analyzed samples had
 the same ploidy level (“inferred”). * PloidyPop: A factor combining the
 information on plant ploidy level from column “PloidyLevel” and the
 population identity from column “Population”. This factor was used to
 estimate population means separately for diploids, tetraploids, and
 hexaploids using linear mixed-effect models (for details, see the main
 text and SI Appendix of the paper from which these data are) for further
 analyses. * FlDi: Flower diameter in mm. * CorOpDi: Corolla-opening
 diameter in mm. * PetLen: Petal length in mm. * PetWid: Petal width in mm.
 * CorGap: Corolla gap in mm. * FlLen: Overall flower length in mm. *
 IntFlLen: Internal floral length in mm. * OvDep: Ovary depth in mm. *
 FlFlair: Floral flair in mm. * LgAng: Long floral angle in mm. * FlWid:
 Floral width in mm. * NectLen: Nectary disc length in mm. * LobeDi:
 Diameter of the largest stigmatic lobe in mm. * LobeC: Stigma
 circumference in mm. * StyleHt: Pistil height above the nectary disk in
 mm. * FrillCode: Binary trait for the shape of the petal edge with “1”
 representing a lobed petal edge and “0” a whole petal edge. “NA” indicates
 that the assessment of the shape of the petal edge was unfortunately not
 possible. * PC1: Score of the principal component 1 of a principal
 component analysis including the nine traits (“CorOpDi”, “PetLen”,
 “PetWid”, “CorGap”, “IntFlLen”, “OvDep”, “FlWid”, “LobeDi”, “StyleHt”)
 that had all pairwise correlations &lt; 0.7. * PC2: Score of the principal
 component 2 of a principal component analysis described in the description
 of the column “PC1”. ### 3.
 03_FloralMorphologySyntheticPolyploidization_Data_Gross_etal_2025.txt
 These data were used to assess the direct effects of polyploidization on
 floral morphology by comparing synthetically generated neopolyploids with
 established diploids and established tetraploids and colchicine-treated
 plants that remained diploid. With these data univariate and multivariate
 analyses were conducted. For multivariate analyses, the same raits as for
 the data of the Lithophragma bolanderi plants from the multi-population
 greenhouse common-garden were included. For details on how the traits were
 measured, see Fig. 2 and SI Appendix, Text S5.5, of the paper from which
 these data are. The file contains the following variables: * Population:
 Three-letter abbreviation of the population name. * SeedFamily: Seed
 family (seeds from a single cross) identity. * IndividualID: Individual
 code for each plant within a seed family. * Donor_Seed_Family: Seed family
 identity of the pollen donor of the cross. * Donor_ID: Individual identity
 of the pollen donor of the cross. * Receiver_Seed_Family: Seed family
 identity of the pollen receiver of the cross. * Receiver_ID: Individual
 identity of the pollen receiver of the cross. *
 ParentalSeedFamily_Donor_Receiver: Factor combining the information of the
 column “Donor_Seed_Family” (number in front of “*”) and the column
 “Receiver_Seed_Family” (number after “*”). * Year: Year when the plants
 were collected and analyzed. “2021” for the plants from the first cohort
 (sowed in March 2021), “2022” for the plants from the second cohort (sowed
 in December 2021). * FlowCytometryID: Individual ID used for flow
 cytometry analyses. “NA”: no flow cytometry analyses was conducted for
 this control plant. * SampleISRatio: The sample:internal-standard (IS)
 ratio calculated by dividing the median of the L. bolanderi peak by the
 median of the IS peak. “NA”: no flow cytometry analyses was conducted for
 this control plant. * PloidyLevel: Plant ploidy level determined based the
 sample:IS ratios and the ploidy level information previously determined
 (see “1. FlowCytometry_Data_Gross_etal_2025.txt”). “2x”: diploid, “3x”:
 triploid, “4x”: tetraploid. * Treatment: Information whether the parental
 plants were treated with colchicine (“treatment”) or not “control”). *
 Treatment_Ploidy: Factor combining the information from column “Treatment”
 and “PloidyLevel”. * FlDi: Flower diameter in mm. * CorOpDi:
 Corolla-opening diameter in mm. * PetLen: Petal length in mm. * PetWid:
 Petal width in mm. * CorGap: Corolla gap in mm. * IntFlLen: Internal
 floral length in mm. * FlFlair: Floral flair in mm. * LgAng: Long floral
 angle in mm. * FlLen: Overall flower length in mm. * OvDep: Ovary depth in
 mm. * FlWid: Floral width in mm. * NectLen: Nectary disc length in mm. *
 LobeDi: Diameter of the largest stigmatic lobe in mm. * LobeC: Stigma
 circumference in mm. * StyleHt: Pistil height above the nectary disk in
 mm. * PC1: Score of the principal component 1 of a principal component
 analysis including the nine traits (“CorOpDi”, “PetLen”, “PetWid”,
 “CorGap”, “IntFlLen”, “OvDep”, “FlWid”, “LobeDi”, “StyleHt”) also included
 in principal component analysis run on the traits in the data file “2.
 FloralMorphology27PopulationCommonGarden_Data_Gross_etal_2025.txt”. * PC2:
 Score of the principal component 2 of a principal component analysis
 described in the description of the column “PC1”. ### 4.
 04_FloralMorphologyFieldCommonGarden_Data_Gross_etal_2025.txt These data
 were used to compare the floral morphology between plants growing in
 natural populations and plants grown in the multi-population greenhouse
 common-garden from seeds collected in these natural populations for a
 subset of the populations included in the data file “2.
 FloralMorphology27PopulationCommonGarden_Data_Gross_etal_2025.txt”. The
 file contains the following variables: * Population: Three-letter
 abbreviation of the population name. * PlantID: Individual code for each
 plant. Note that the numerical part of the “PlantID” for the
 greenhouse-grown plants is the seed family (seeds collected from a single
 plant being half- and/or full-sibs) identity and the letter code part of
 the “PlantID” for plants in the natural population is the three-letter
 abbreviation of the name of the population. * Latitude: Latitude in °N on
 the population level. * Longitude: Longitude in °E on the population
 level. * Origin: Information whether the measurements were taken from
 plants in the natural populations (“Field”) or from plants grown in the
 greenhouse common-garden (“Greenhouse”). * FlDi: Flower diameter in mm. *
 CorOpDi: Corolla-opening diameter in mm. ### 5.
 05_FlowCytometryMethods_Data_Gross_etal_2025.txt These data were used to
 assess the comparability of the flow-cytometry protocol by Plant Cytometry
 Service ([https://www.plantcytometry.nl/](https://www.plantcytometry.nl/))
 with the one used by the authors at BIOVIS (Biolgical Visualisation) at
 Uppsala University, Sweden
 ([https://biovis.uu.se/](https://biovis.uu.se/)). The file contains the
 following variables: * FlowCytometryID: Individual ID used for the flow
 cytometry analyses of the samples analyzed by Plant Cytometry Service. *
 SeedFamily: Seed family (seeds from a single cross) identity. * PlantID:
 Individual code for each plant. * Population: Three-letter abbreviation of
 the population name. * DonorID: Individual identity of the pollen donor of
 the cross. Note that the three letters at the beginning of the code are
 the three-letter abbreviation of the population name, and the numerical
 part is the seed family (seeds collected from a single plant being half-
 and/or full-sibs) identity. * ReceiverID: Individual identity of the
 pollen receiver of the cross. Note that the three letters at the beginning
 of the code are the three-letter abbreviation of the population name, and
 the numerical part is the seed family (seeds collected from a single plant
 being half- and/or full-sibs) identity. * PloidyLevel: Plant ploidy level.
 “2x”: diploid, “4x”: tetraploid, “6x”: hexaploid. *
 Donor_SampleBnRatio_UU: The sample:internal-standard (IS) ratio of the
 pollen-donor plant calculated by dividing the median of the Lithophragma
 bolanderi peak by the median of the IS peak (Brassica napus was used as
 IS). The flow cytometry analyses to quantify these sample:IS rations were
 conducted by one of the authors at Uppsala University. For the plant with
 the “DonorID” “MIN11450A” flow cytometry analysis was unfortunately not
 possible and the “Donor_SampleBnRatio_UU” was calculated as the mean
 sample:IS ratio of the other individuals of that plant’s seed family for
 which quantification of the sample:IS ratio was possible (these sample:IS
 ratios were 0.83423624 for MIN11450H, 0.82111261 for MIN11450I, 0.83191792
 for MIN11450J, 0.83046457 for MIN11450K, and 0.83886178 for MIN11450L). *
 Receiver_SampleBnRatio_UU: The sample:IS ratio of the pollen-donor plant
 calculated by dividing the median of the L. bolanderi peak by the median
 of the IS peak (B. napus was used as IS). The flow cytometry analyses to
 quantify these sample:IS rations were done by one of the authors at
 Uppsala University. * SampleIBnRatio_UU: The sample:IS ratio quantified
 based on the flow cytometry analyses conducted by one of the authors at
 Uppsala University and calculated as the mean of “Donor_SampleBnRatio_UU”
 and “Receiver_SampleBnRatio_UU”. * SampleBnRatio_PCS: The sample:IS ratio
 quantified by Plant Cytometry service using B. napus as IS in the flow
 cytometry analyses. * SampleAsRatio_PCS: The sample:IS ratio quantified by
 Plant Cytometry service using Allium schoenoprasum as IS in the flow
 cytometry analyses. ### 6.
 06_TraitMeasuringComparability_Data_Gross_etal_2025.txt These data were
 used to ensure that the measuring of the floral morphological traits were
 repeatable, because the flowers were apportioned among three persons to
 conduct the measurements. In particular, these data were used to compute
 the correlation of one person remeasuring a subset of the flowers measured
 by the two other persons. Note that the measures of the flowers in this
 data file might slightly differ from the measures for the flowers with the
 same plant ID in the file “2.
 FloralMorphology27PopulationCommonGarden_Data_Gross_etal_2025.txt”,
 because this one person remeasured some traits for all flowers and took
 some additional measures to ensure the accuracy of the measures (such as
 visually checking for “outliers” separately for each population and
 cytotype, double-checking these measures, and remeasuring them if
 necessary) after the assessment of the correlation of measurements between
 two different persons. For details on how the traits were measured, see
 Fig. 2 and SI Appendix, Text S4, of the paper from which these data are.
 The file contains the following variables: * Person: Person who measured
 the floral morphological traits based on the photos taken of the flowers.
 “P1”: person 1, “P2”: person 2, “P3”: person 3, who was the person
 remeasuring a subset of the flowers measured by P1 and P2. * Measuring:
 Information whether the measurements were initial measurements conducted
 by P1 or P2 (“1stMeasuring”) or remeasurements conducted by P3
 (“Remeasuring”). * Population: Three-letter abbreviation of the population
 name. * PlantID: Individual code for each plant. The numerical part of the
 “PlantID” is the seed family (seeds collected from a single plant being
 half- and/or full-sibs) identity. * PetLen1: Length of the first randomly
 selected petal in mm. * PetWid1: Width of the first randomly selected
 petal in mm. * PetLen2: Length of the second randomly selected,
 non-adjacent petal in mm. * PetWid2: Width of the second randomly
 selected, non-adjacent petal in mm. * CorGap: Corolla gap in mm. * FlLen:
 Overall flower length in mm. * IntFlLen: Internal floral length in mm. *
 FlFlair: Floral flair in mm. * LgAng1: Long floral angle measured in the
 first direction in mm. * LgAng2: Long floral angle measured in the second
 direction in mm. * FlWid: Floral width in mm. * NectLen: Nectary disc
 length in mm. * LobeDi: Diameter of the largest stigmatic lobe in mm. *
 LobeC1: Outer distance between stigmatic lobe 1 and 2 in mm. * LobeC2:
 Outer distance between stigmatic lobe 2 and 3 in mm. “NA”: this
 measurement was not possible for this flower because it had only two
 stigmatic lobes. * LobeC3: Outer distance between stigmatic lobe 3 and 1
 in mm or between stigmatic lobe 3 and 4 for flowers that had 4 stigmatic
 lobes. “NA”: this measurement was not possible (see explanation to
 “LobeC2”). * LobeC4: Outer distance between stigmatic lobe 4 and 1 in mm
 in case there were four stigmatic lobes. “NA”: this measure was not
 possible, because there were only two or three (this is the case by far
 the most flowers) stigmatic lobes in the flower. * StyleHt: Pistil height
 above the nectary disk in mm. ## Sharing/Access information These data are
 not accessible at another location. These are original data and not
 derived from another source.