Environmental contributions to the evolution of trait differences in Geum triflorum: implications for restoration

Premise of the Study Understanding how environment influences the distribution of trait variation across a species’ range has important implications for seed transfer during restoration. Heritable genetic differences associated with environment could impact fitness when transferred into new environments. Here, we test the degree to which the environment shapes the evolution and distribution of genetic effects for traits important to adaptation. Methods In a common garden experiment, we quantified trait differentiation for populations of Geum triflorum sourced from three distinct ecoregions and evaluated the ability of climate to predict trait variation. Populations were sourced from alvar ecoregions which experience predictable extremes in seasonal water availability and the prairie ecoregion which exhibits unpredictable changes in water availability. Key Results Plants sourced from alvar ecoregions exhibited smaller but more numerous stomata and greater intrinsic water use efficiency relative to prairie plant populations supporting the evolution of ecotypic differences. Estimates of standing genetic variance and heritable genetic variation for quantitative traits suggest alvar populations have greater adaptive potential. However, reduced evolvability suggest all populations of G. triflorum may have limited capacity to evolve in response to environmental change. Conclusions These results point towards the importance of understanding the role of environment in shaping the distribution and evolution of genetic differences across seed populations and how these data may inform recommendations for seed transfer across novel environments and our expectations of populations’ adaptive potential.


INTRODUCTION
Understanding how the environment influences trait variation is essential, particularly 48 within the context of restoration (Wang et al., 2010). The evolution of ecotypic differences for 49 vegetative, physiological, or reproductive life history traits can lead to differential success  The history of selection, particularly the degree to which environmental heterogeneity has 77 been predictable or unpredictable across a species' range, may impact the distribution of genetic 78 variation underlying traits and consequently their capacity to adapt. Here, we define 79 environmental predictability as repeatable seasonal cues associated with a given climate variable 80 (Reed et al., 2010). Theory suggests that where populations have experienced predictable 81 environmental cues, heritable genetic variance for phenotypic traits will increase as the total 82 phenotypic variance is reduced ( Fig. 1 ). Thus, we suggest the alvar ecoregion reflects a 'predictable' history of selection, whereas 115 the prairie ecoregion reflects an 'unpredictable' history of selection in response to changing 116 water availability. These ecoregions provide an ideal system to evaluate the role predictability of 117 the environment may play in influencing the amount and distribution of genetic variance for 118 phenotypic traits. Physiological traits, including stomatal size and density along with water-use 119 efficiency (WUE) are expected to vary between prairie and alvar ecoregions. Given the 120 importance of stomatal traits and WUE to plant persistence, examining how environment of 121 origin has influenced variation in these traits will inform seed transfer recommendations.

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Using a common garden experiment of maternal seed families for G. triflorum sourced 123 from both prairie and alvar ecoregions, we evaluated the role source environment has had on the 124 distribution of physiological trait variation linked to plant water use. We quantified ecoregional 125 differentiation for each trait and tested for correlations between functional traits and climate of 126 origin for all sampled populations. Lastly, we quantified standing genetic variance for stomatal 127 traits, including estimates of heritability and evolvability. Specifically, we ask 1) do 128 physiological traits exhibit ecoregional differences, 2) is there a relationship between 129 physiological trait variation and source climate, and 3) does the history of selection associated 130 with seed source environment structure the distribution of additive genetic variance and the 131 heritability or evolvability of physiological traits? We predict alvar ecoregions will exhibit 132 smaller, but more numerous stomata in addition to greater WUE relative to prairie populations. 133 We also expect populations from the alvar ecoregions will have greater heritability for 134 quantitative traits relative to prairie populations due to the history of selection associated with 135 predictable changes in water availability. An understanding of how differentiation in 136 physiological traits evolve and the role selection may play in shaping the distribution of heritable 137 7 trait variation will be valuable for predicting the response of seeds to restored environments and 138 estimating their longer-term evolutionary potential.    Table 1).

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To test whether differences in stomatal traits and WUE could be explained by climatic 206 variation summarized as PC1 and PC2, we fit linear regressions using the lm function in R (R

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Core Team, 2018). We tested three models using PC1 and PC2 as predictor variables. Two Broad-sense heritability (H 2 ) was calculated as follows using variance components from the 228 mixed model: The variance components associated with the kth family within the ith population and the block x ) interaction effect was subsequently used to estimate narrow-sense heritability 232 and the evolvability. Narrow-sense heritability (h 2 ) for leaf surface traits was estimated using the 233 following equation:  294 Additive genetic variance (V A ) provides an estimate of the amount of genetic variation 295 available for selection to act upon (Falconer and Mackay, 1996). Ecoregion-specific estimates 296 for additive genetic variance (V A ) of stomatal traits were quantified using the half-sibling design 297 ( providing an understanding of potential response to selection. There was substantial variability in 321 narrow sense heritabilities estimated for physiological traits across ecoregions (Table 2). On 322 average, individuals from the GLA ecoregion exhibited greater h 2 for all traits, excluding 323 stomatal area index, which was greatest for individuals sourced from the PRA ecoregion (Table   324 2). Narrow-sense heritability was similar between GLA (0.   341 To determine whether the adaptive capacity of stomatal traits differs across ecoregions 342 we calculated evolvability. While alvar and prairie ecoregions exhibited eco-region differences 343 in V A , evolvability did not vary by ecoregion (Table 2). This suggests, that while the amount of 344 additive genetic variance is greater for alvar ecoregions, the per-generation change expected due 345 to any given selection coefficient is similar across ecoregions. Although evolvability did not vary 346 by ecoregion it did vary across traits (Table 2) suggesting that the per generation change will be greater for stomatal area index traits than for 349 guard cell length. Overall, evolvabilities for all traits were low ranging from 0-0.18 indicating 350 that the expected per generation change in these traits is likely limited (Table 2).  407 Using climate associated with population origin we performed a PCA to identify those 408 climate variables that structure population variation across the range of G. triflorum. We found 409 ecoregions were differentiated primarily by temperature (PC1) and water availability (PC2).

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While PC1 explained the most variation between ecoregions, it did not predict physiological trait 411 variation. However, we did observe a relationship between PC2 and physiological trait variation.

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Using the PC2 axis, we observed greater annual climate moisture deficit (CMD) and annual heat Heritability of stomatal traits 441 Quantifying heritability provides insight into the degree to which trait variation is largely 442 mediated by genetic or environmental effects. We predicted that heritable trait variation would 443 be greater within alvar environments as they experience environmental heterogeneity that is 444 predictable. Interestingly, broad-sense heritabilities for stomatal traits ranged from 0.09-0.9 and 445 narrow-sense heritability estimates ranged from 0.1-0.5 and were similar across ecoregions 446 (Table 2). This suggests that while the genetic effects for some traits is substantial, there is also a 447 substantial proportion of variance attributable to environmental variance. Indeed, unpredictable, diminish over time (Donohue, 2009). However, given these caveats, our estimates of both broad 457 and narrow-sense heritability likely represent upper bounds (Falconer and Mackay, 1996). noted that all estimates were close to zero with little to no differences across ecoregions (Table   476 2). This suggests that populations used in this experiment may have limited capacity to respond 477 to selection. This is a concern in the context of restoration, which will require seed transferred to