DOI: 10.25349/d9mw4m
title: Data from: Genotype x environment interaction obscures genetic sources of variation in seed size in Dithyrea californica but provides the opportunity for selection on phenotypic plasticity

description: Premise: Seed size is a functional trait that influences individual
 fitness but the genetic basis of variation in individual seed size in wild
 species remains still largely unknown. The evolutionary trajectory of seed
 size depends, in part, on whether seed size variation is determined by
 additive, maternal, or non-additive genetic variance. The expression of
 these sources of variance in seed size may be environment-dependent,
 reflecting genetic variation in phenotypic plasticity. In this study, we
 used a quantitative genetic approach to reveal the genetic basis of seed
 size and its plastic response to drought stress in
 Dithyrea californica. Methods: We used a diallel mating design to
 estimate genetic and environmental variance components for seed size in
 each of three greenhouse-raised populations sampled from California and
 northern Mexico. We replicated diallels in two watering treatments to
 examine genetic parameters and genotype x environment interactions
 affecting seed size. We estimated general combining ability (GCA),
 specific combining ability (SCA), reciprocal effects (RGCA and RSCA), and
 their interactions with water availability. We examined norms of reaction
 for maternal and paternal families and for each maternal/paternal pair to
 reveal the magnitude of genetic variation in phenotypic plasticity for
 seed size in each population. Results: Seed size in the sampled
 populations of D. californica is determined primarily by the combination
 of watering treatment, GCA and RGCA; parental identity alone does not
 consistently affect seed size. Across genotypes, water availability did
 not have a significant effect on seed size. Conclusion: Genetic variation
 in phenotypic plasticity of seed size is greater than variation among
 genotypes.
description: <strong><em>Study
 species</em></strong><em>—Dithyrea californica </em>(Harvey)<em> </em>is a self-incompatible
 winter annual native to sand dune habitats in the Mohave and Sonoran
 Deserts. <em>D. californica</em> germinates in response to
 late-fall or early-winter rains. Once germinated, it grows as a basal
 rosette for approximately two months, flowering and producing seeds in
 late February to early April, depending on the timing of precipitation.
 Flowers are hermaphroditic and protogynous, and if unpollinated, they
 typically remain open for 3-4 days. The pistil becomes receptive before
 the petals open, after which the anthers dehisce simultaneously with petal
 opening. Anthers protrude slightly from the distal end of the corolla
 while pistils extend to a height that is no more than one quarter of the
 length of the petals, making the flowers easy to emasculate. Fruits
 consist of two subunits called “mericarps”, each of which contains one
 seed. The
 geographic distribution of <em>D. californica</em> is broad,
 occurring in most arid sand dune systems in western North America
 (Northern Mexico and southwest USA). <em>Dithyrea
 californica</em> is an ideal species with which to investigate the
 selective dynamics of seed size in the field due to a special feature of
 the germinated seed. When a seed inside a mericarp germinates, the rim of
 the mericarp (the diameter of which provides a measure of seed size)
 remains attached to the root until plants become adults. This special
 feature has allowed us to track individual plants in the field and to
 relate seed size to both survival and fecundity, the main components of
 individual fitness (Larios et al., 2014; Larios and Venable,
 2018). <em>D. californica</em> is
 also a suitable species in which to investigate quantitative genetic
 variation because it is easy to emasculate and is self-incompatible,
 preventing accidental pollinations when conducting controlled crosses to
 create pedigreed seeds. <em> </em> <strong><em>Source
 populations</em></strong><em>—</em>During the spring of 2017, seeds of <em>D.
 californica</em> were collected in three locations. The northernmost
 population is 800 km away from the southernmost location. A northern
 population located in the Mohave desert was collected from the Kelso dunes
 at the Mohave National Preserve in California, USA (34.88° N, -115.77° W,
 altitude: 676 m). A central population was located in the Sonoran Desert
 on a coastal dune near the town of Puerto Lobos, Sonora, Mexico (30.35° N,
 -112.81° W, altitude: 70 m). The most southern population is also located
 in the Sonoran Desert and was collected on a coastal dune in the town of
 Desemboque de los Seris in Hermosillo, Sonora, Mexico (29.51° N, -112.41°
 W, altitude: 10 m). Seeds were stored in an outdoor shed (protected from
 rainfall) but exposed to ambient temperature for two years in paper bags
 until the spring of 2019; this exposure to natural year-round conditions
 facilitated the loss of dormancy. <em> </em> <strong><em>Breeding
 design</em></strong><em>—</em>The
 experiment was performed in a greenhouse located at the University of
 California, Santa Barbara campus in Goleta, California, USA. In the
 greenhouse, we planted eight seeds representing each maternal line, one
 seed per pot, and selected two plants of each maternal line to be grown in
 two alternative watering treatments (one individual/treatment). Once
 plants became reproductive, we conducted controlled pollinations using 5 x
 5 diallel breeding designs, performing reciprocal crosses and excluding
 self-pollinations. Because individuals of <em>D.
 californica</em> are cosexual, this breeding design allows us to
 estimate each individual’s effect on seed size as both a maternal
 (seed-bearing) and paternal (pollen-donating) plant.  We created three
 diallels from the “Kelso” population, 3 diallels from the “Seri”
 population and only one diallel from the “Lobos” population. The number of
 diallels per population was determined by the number of field-collected
 maternal families from which seeds successfully germinated in the
 greenhouse. Pollinations from a given pollen donor were performed on a
 pollen recipient’s single reproductive stalk so we could have  better
 control of the pedigree of resulting seeds. Pollinations were conducted by
 collecting entire dehiscent stamens and rubbing them against a receptive
 stigma using alcohol-sterilized forceps. We sterilized the forceps with
 rubbing alcohol after each flower was pollinated. To avoid self-pollen
 contamination, we emasculated all flowers prior to anther dehiscence by
 slicing off their tips prior to the flower bud opening (the short style
 prevented damage to the stigma). Each
 diallel was replicated in two watering treatments (Fig. 1); here,
 replication meant that each of the five individuals in a given diallel had
 a maternal sibling in a paired diallel exposed to the other treatment. All
 pots were watered using a nutrient solution comprised of MiracleGro
 (Marysville, Ohio, USA); we diluted one tablespoon of fertilizer in 3.78
 liters of deionized water, as suggested by the manufacturer.  Wet
 conditions were achieved by watering the pots every three days, which kept
 the soil saturated for the duration of the reproductive stage. In the wet
 treatment, plants were maintained at full turgidity throughout the
 experiment. Dry conditions were achieved by watering the pots each week
 with the nutrient solution and allowing the soil to dry until we observed
 the onset of wilting, but plants retained the capacity to return to
 turgidity after watering. Pedigreed seeds resulting from these controlled
 pollinations were identified by color coding each seed with small dots of
 acrylic paint to identify their maternal and paternal origin. Seeds were
 allowed to mature on the plant and were harvested when they became easy to
 detach. <em> </em> <strong><em>Data
 analysis</em></strong><em>—</em>The
 linear dimensions of each harvested seed were measured using the imaging
 software <em>ImageJ</em> (Schneider et al., 2012).
 <em>D. californica</em> seeds are shaped as flat ellipses; we
 estimated seed size as the area of one face of the ellipse, using the two
 major axes of each seed and the following equation:
   where H is the length of the longest axis and W the length
 of the longest axis perpendicular to H. To
 estimate the variance components of seed size we analyzed variation in
 ellipse area produced by crosses using the random diallel analysis that
 includes reciprocal crosses and no self-pollinations (diallel type III sensu Hayman, 1954; Griffing,
 1956). From this analysis we estimated the
 general combining ability (GCA), the specific combining ability (SCA), the
 reciprocal general combining ability (RGCA), the reciprocal specific
 combining ability (RSCA), and the interactions of each of these variance
 components with the watering treatment, for each population separately.
 GCA refers to additive effects of genes (nuclear effects) and it is
 represented as the mean deviation between the mean seed size produced by
 both parents and the diallel’s grand mean (Hayman, 1954; Griffing, 1956).
 SCA refers to non-additive effects of nuclear genes (dominance and
 epistatic effects) and it is represented as the deviation of mean seed
 size of specific parental combinations from the parental seed size means.
 Reciprocal effects are deviations in mean seed size due to the direction
 of a cross between two parents and due to the effects of the parents’
 functional gender on offspring genotype. In the Hayman (1954)
 model, reciprocal effects are partitioned into reciprocal general
 combining ability (RGCA), which is represented by the deviation in mean
 seed size produced by a parental genotype functioning as a male vs. as a
 female. The reciprocal specific combining ability (RSCA) is represented by
 the deviation in mean seed size between a given cross (between two
 parents) and its reciprocal. This model is defined by the following
 equation:   where <em>Y<sub>ijk</sub></em> are
 the individual seed sizes produced by the combination of dam<em>
 j</em> and sire <em>k</em>,
 <em>m</em> is
 the grand mean of seed size within a diallel,
 <em>G<sub>j</sub></em> and
 <em>G<sub>k</sub></em> are the GCAs of the dam
 <em>j</em> and sire <em>k</em>,
 <em>S<sub>jk</sub></em> is the SCA of the
 <em>jth</em> and <em>kth</em> parent combination,
 <em>RG<sub>j</sub></em> and
 <em>RG<sub>k</sub></em> are the RGCAs of the
 <em>jth</em> and the <em>kth</em> parents,
 <em>RS<sub>jk</sub></em> is the RSCA of the
 <em>jth</em> and <em>kth</em> parent combination,
 and <em>E<sub>ijk</sub></em> is the residual
 variance. We
 analyzed the data using the R package ‘<em>lmDiallel’</em>
 (Onofri et al., 2021)
 along with the ‘<em>sommer’</em> package to estimate random
 effects of variance components (Covarrubias-Pazaran, 2016). We
 first constructed design matrices that indicated the identity of dams and
 sires for each pedigreed seed with the <em>lmDiallel</em>
 package functions <em>GCA</em>, <em>SCA</em>,
 <em>RGCA</em> and <em>RSCA</em>. We fitted
 generalized linear mixed-effects models where we used ellipse area (as a
 measure of seed size) as the response variable, watering treatment as a
 fixed explanatory variable, and <em>GCA</em>,
 <em>SCA</em>, <em>RGCA</em>,
 <em>RSCA</em>, <em>GCA x water</em>, <em>SCA
 x water</em>, <em>RGCA x water</em>, and <em>RSCA
 x water</em> as random effects. We tested the statistical
 significance of each random effect with likelihood-ratio tests comparing a
 model that contained all random predictor variables (the full model) with
 models from which we excluded the random variable to be tested. We tested
 for a change in mean seed size between watering treatments per population
 with likelihood-ratio tests between the full models that contained the
 watering treatment as a fixed factor and sub-models that did not contain
 the watering treatment as the fixed factor. Additionally, we performed the diallel analysis on each
 population and watering treatment separately in order to estimate
 environment-specific variance components of seed size and the significance
 of maternal and paternal genetic contributions to seed size in each
 treatment. We
 estimated narrow-sense heritability per population and per watering
 treatment as the additive genetic variance
 divided by total phenotypic variance
 (Falconer and Mackay, 2003).             We also estimated broad-sense heritability
 as total genetic variance
  divided by total phenotypic
 variance. We
 estimated norms of reaction of seed size using the residual variation of
 ellipse area within each diallel cross. Residual variation was estimated
 for the dams and sires separately by subtracting the diallel mean seed
 size of each dam or sire from the mean seed size produced by each of the
 dams or sires within a diallel. We pooled all dam and sire residuals by
 population and represented the norms of reaction by population rather than
 by individual diallel. By examining the norms of reaction for each
 population, we can observe the environmentally induced effect of watering
 treatment on the mean seed size and phenotypic rank of each maternal and
 paternal genotype. We tested for a change in variance in seed size between
 watering treatments with Levene’s tests of homogeneity of variance per
 population where we compared the within-population variance among dams or
 sires in ellipse area of dry and wet treatments.
 We examined the sex-specific performance of,
 and correlations between, cosexual individuals with respect to the size of
 seeds produced in each watering treatment. To analyze the correlation
 between sexes, we conducted general linear models to examine the bivariate
 relationship between the residual mean ellipse area produced by
 individuals as pollen donors (sires) and as pollen recipients (dams).
 Finally, to analyze the cross-environment correlation of sex-specific
 residual seed size, we conducted general linear models to examine the
 bivariate relationship between the residual mean ellipse area produced by
 individuals as pollen donors (sires) in the dry and wet treatments, and as
 pollen recipients (dams) in the dry and wet treatments. For each diallel
 matrix, we used the diallel mean ellipse area of all sires and dams to
 calculate sire- and dam-specific residuals. Then, all residuals for a
 given population were pooled prior to plotting the bivariate relationships
 presented here.