Amphistomy: stomata patterning inferred from 13C content and leaf-side specific deposition of epicuticular wax.

BACKGROUND AND AIMS: The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax (EW) on the opposite leaf sides is studied. METHODS: We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from (i) wax synthesis being located exclusively in epidermal cells and (ii) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL, fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of EW, i.e. the difference in abaxial and adaxial δ(δab-δad), was used to calculate the leaf dorsi-ventral CO2 gradient. Leaf-side specific EW deposition, amphiwaxy level (AWL), was estimated and related to ASL. KEY RESULTS: In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one independently of light conditions. In high-light and low-light grown AS leaves, the isotopic polarity and CO2 gradient varied in parallel with ASL. AS leaves grown under high light increased ASL compared to low light, and δab-δad approached near-zero values. Changes in ASL occurred concomitantly with changes in AWL. CONCLUSIONS: The leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.

Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian 'ilima (Sida fallax).

PREMISE: The adaptive significance of amphistomy (stomata on both upper and lower leaf surfaces) is unresolved. A widespread association between amphistomy and open, sunny habitats suggests the adaptive benefit of amphistomy may be greatest in these contexts, but this hypothesis has not been tested experimentally. Understanding amphistomy informs its potential as a target for crop improvement and paleoenvironment reconstruction. METHODS: We developed a method to quantify "amphistomy advantage" ( AA $\text{AA}$ ) as the log-ratio of photosynthesis in an amphistomatous leaf to that of the same leaf but with gas exchange blocked through the upper surface (pseudohypostomy). Humidity modulated stomatal conductance and thus enabled comparing photosynthesis at the same total stomatal conductance. We estimated AA $\text{AA}$ and leaf traits in six coastal (open, sunny) and six montane (closed, shaded) populations of the indigenous Hawaiian species 'ilima (Sida fallax). RESULTS: Coastal 'ilima leaves benefit 4.04 times more from amphistomy than montane leaves. Evidence was equivocal with respect to two hypotheses: (1) that coastal leaves benefit more because they are thicker and have lower CO2 conductance through the internal airspace and (2) that they benefit more because they have similar conductance on each surface, as opposed to most conductance being through the lower surface. CONCLUSIONS: This is the first direct experimental evidence that amphistomy increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase CO2 supply to chloroplasts. The prevalence of amphistomatous leaves in open, sunny habitats can partially be explained by the increased benefit of amphistomy in "sun" leaves, but the mechanistic basis remains uncertain.

How Important Are Functional and Developmental Constraints on Phenotypic Evolution? An Empirical Test with the Stomatal Anatomy of Flowering Plants.

AbstractQuantifying the relative contribution of functional and developmental constraints on phenotypic variation is a long-standing goal of macroevolution, but it is often difficult to distinguish different types of constraints. Alternatively, selection can limit phenotypic (co)variation if some trait combinations are generally maladaptive. The anatomy of leaves with stomata on both surfaces (amphistomatous) present a unique opportunity to test the importance of functional and developmental constraints on phenotypic evolution. The key insight is that stomata on each leaf surface encounter the same functional and developmental constraints but potentially different selective pressures because of leaf asymmetry in light capture, gas exchange, and other features. Independent evolution of stomatal traits on each surface imply that functional and developmental constraints alone likely do not explain trait covariance. Packing limits on how many stomata can fit into a finite epidermis and cell size-mediated developmental integration are hypothesized to constrain variation in stomatal anatomy. The simple geometry of the planar leaf surface and knowledge of stomatal development make it possible to derive equations for phenotypic (co)variance caused by these constraints and compare them with data. We analyzed evolutionary covariance between stomatal density and length in amphistomatous leaves from 236 phylogenetically independent contrasts using a robust Bayesian model. Stomatal anatomy on each surface diverges partially independently, meaning that packing limits and developmental integration are not sufficient to explain phenotypic (co)variation. Hence, (co)variation in ecologically important traits like stomata arises in part because there is a limited range of evolutionary optima. We show how it is possible to evaluate the contribution of different constraints by deriving expected patterns of (co)variance and testing them using similar but separate tissues, organs, or sexes.

Technical comment on "Negative-assortative mating for color in wolves".

Hedrick et al. (2016) (Negative-assortative mating for color in wolves. Evolution, 70, 757-766) reported on "negative-assortative mating for color in wolves" from Yellowstone National Park, the "first documented case of significant negative-assortative mating in mammals." Here I report a logical inconsistency in their population genetic model that effectively imposes selection against some assortatively mating genotype. After pointing out this inconsistency, I derive new expressions for the frequency of different matings and the equilibrium allele frequencies. Compared to Hedrick et al. (2016) (Negative-assortative mating for color in wolves. Evolution, 70, 757-766), the system rapidly approaches an equilibrium based on observed levels of negative-assortative mating in this population. Hence, the revised model may strengthen the case that assortative mating can maintain polymorphism and provides logically consistent results to inform the study of nonrandom mating in other organisms.

Selection on early survival does not explain germination rate clines in Mimulus cardinalis.

PREMISE: Many traits covary with environmental gradients to form phenotypic clines. While local adaptation to the environment can generate phenotypic clines, other nonadaptive processes may also. If local adaptation causes phenotypic clines, then the direction of genotypic selection on traits should shift from one end of the cline to the other. Traditionally, genotypic selection on non-Gaussian traits like germination rate have been hampered because it is challenging to measure their genetic variance. METHODS: Here we used quantitative genetics and reciprocal transplants to test whether a previously discovered cline in germination rate showed additional signatures of adaptation in the scarlet monkeyflower (Mimulus cardinalis). We measured genotypic and population level covariation between germination rate and early survival, a component of fitness. We developed a novel discrete log-normal model to estimate genetic variance in germination rate. RESULTS: Contrary to our adaptive hypothesis, we found no evidence that genetic variation in germination rate contributed to variation in early survival. Across populations, southern populations in both gardens germinated earlier and survived more. CONCLUSIONS: Southern populations have higher early survival but it is not caused by faster germination. This pattern is consistent with nonadaptive forces driving the phenotypic cline in germination rate, but future work will need to assess whether there is selection at other life stages. This statistical framework should help expand quantitative genetic analyses for other waiting-time traits.

Core arbuscular mycorrhizal fungi are predicted by their high abundance-occupancy relationship while host-specific taxa are rare and geographically structured.

Habitat restoration may depend on the recovery of plant microbial symbionts such as arbuscular mycorrhizal (AM) fungi, but this requires a better understanding of the rules that govern their community assembly. We examined the interactions of soil and host-associated AM fungal communities between remnant and restored patches of subtropical montane forests. While AM fungal richness did not differ between habitat types, community membership did and was influenced by geography, habitat and host. These differences were largely driven by rare host-specific AM fungi that displayed near-complete turnover between forest types, while core AM fungal taxa were highly abundant and ubiquitous. The bipartite networks in the remnant forest were more specialized and hosts more specific than in the restored forest. Host-associated AM fungal communities nested within soil communities in both habitats, but only significantly so in the restored forest. Our results provide evidence that restored and remnant forests harbour the same core fungal symbionts, while rare host-specific taxa differ, and that geography, host identity and taxonomic resolution strongly affect the observed distribution patterns of these fungi. We suggest that host-specific interactions with AM fungi, as well as spatial processes, should be explicitly considered to effectively re-establish target host and symbiont communities.

Principles of resilient coding for plant ecophysiologists.

Plant ecophysiology is founded on a rich body of physical and chemical theory, but it is challenging to connect theory with data in unambiguous, analytically rigorous and reproducible ways. Custom scripts written in computer programming languages (coding) enable plant ecophysiologists to model plant processes and fit models to data reproducibly using advanced statistical techniques. Since many ecophysiologists lack formal programming education, we have yet to adopt a unified set of coding principles and standards that could make coding easier to learn, use and modify. We identify eight principles to help in plant ecophysiologists without much programming experience to write resilient code: (i) standardized nomenclature, (ii) consistency in style, (iii) increased modularity/extensibility for easier editing and understanding, (iv) code scalability for application to large data sets, (v) documented contingencies for code maintenance, (vi) documentation to facilitate user understanding; (vii) extensive tutorials and (viii) unit testing and benchmarking. We illustrate these principles using a new R package, {photosynthesis}, which provides a set of analytical and simulation tools for plant ecophysiology. Our goal with these principles is to advance scientific discovery in plant ecophysiology by making it easier to use code for simulation and data analysis, reproduce results and rapidly incorporate new biological understanding and analytical tools.

Phylogenetic history of vascular plant metabolism revealed using a macroevolutionary common garden.

While the fundamental biophysics of C3 photosynthesis is highly conserved across plants, substantial leaf structural and enzymatic variation translates into variability in rates of carbon assimilation. Although this variation is well documented, it remains poorly understood how photosynthetic rates evolve, and whether macroevolutionary changes are related to the evolution of leaf morphology and biochemistry. A substantial challenge in large-scale comparative studies is disentangling evolutionary adaptation from environmental acclimation. We overcome this by using a 'macroevolutionary common garden' approach in which we measured metabolic traits (Jmax and Vcmax) from 111 phylogenetically diverse species in a shared environment. We find substantial phylogenetic signal in these traits at moderate phylogenetic timescales, but this signal dissipates quickly at deeper scales. Morphological traits exhibit phylogenetic signal over much deeper timescales, suggesting that these are less evolutionarily constrained than metabolic traits. Furthermore, while morphological and biochemical traits (LMA, Narea and Carea) are weakly predictive of Jmax and Vcmax, evolutionary changes in these traits are mostly decoupled from changes in metabolic traits. This lack of tight evolutionary coupling implies that it may be incorrect to use changes in these functional traits in response to global change to infer that photosynthetic strategy is also evolving.

Quantitative trait locus mapping reveals an independent genetic basis for joint divergence in leaf function, life-history, and floral traits between scarlet monkeyflower (Mimulus cardinalis) populations.

PREMISE: Across taxa, vegetative and floral traits that vary along a fast-slow life-history axis are often correlated with leaf functional traits arrayed along the leaf economics spectrum, suggesting a constrained set of adaptive trait combinations. Such broad-scale convergence may arise from genetic constraints imposed by pleiotropy (or tight linkage) within species, or from natural selection alone. Understanding the genetic basis of trait syndromes and their components is key to distinguishing these alternatives and predicting evolution in novel environments. METHODS: We used a line-cross approach and quantitative trait locus (QTL) mapping to characterize the genetic basis of twenty leaf functional/physiological, life history, and floral traits in hybrids between annualized and perennial populations of scarlet monkeyflower (Mimulus cardinalis). RESULTS: We mapped both single and multi-trait QTLs for life history, leaf function and reproductive traits, but found no evidence of genetic co-ordination across categories. A major QTL for three leaf functional traits (thickness, photosynthetic rate, and stomatal resistance) suggests that a simple shift in leaf anatomy may be key to adaptation to seasonally dry habitats. CONCLUSIONS: Our results suggest that the co-ordination of resource-acquisitive leaf physiological traits with a fast life-history and more selfing mating system results from environmental selection rather than functional or genetic constraint. Independent assortment of distinct trait modules, as well as a simple genetic basis to leaf physiological traits associated with drought escape, may facilitate adaptation to changing climates.

Adaptation across geographic ranges is consistent with strong selection in marginal climates and legacies of range expansion.

Every species experiences limits to its geographic distribution. Some evolutionary models predict that populations at range edges are less well adapted to their local environments due to drift, expansion load, or swamping gene flow from the range interior. Alternatively, populations near range edges might be uniquely adapted to marginal environments. In this study, we use a database of transplant studies that quantify performance at broad geographic scales to test how local adaptation, site quality, and population quality change from spatial and climatic range centers toward edges. We find that populations from poleward edges perform relatively poorly, both on average across all sites (15% lower population quality) and when compared to other populations at home (31% relative fitness disadvantage), consistent with these populations harboring high genetic load. Populations from equatorial edges also perform poorly on average (18% lower population quality) but, in contrast, outperform foreign populations (16% relative fitness advantage), suggesting that populations from equatorial edges have strongly adapted to unique environments. Finally, we find that populations from sites that are thermally extreme relative to the species' niche demonstrate strong local adaptation, regardless of their geographic position. Our findings indicate that both nonadaptive processes and adaptive evolution contribute to variation in adaptation across species' ranges.

Developmental changes in the reflectance spectra of temperate deciduous tree leaves and implications for thermal emissivity and leaf temperature.

Leaf optical properties impact leaf energy balance and thus leaf temperature. The effect of leaf development on mid-infrared (MIR) reflectance, and hence thermal emissivity, has not been investigated in detail. We measured a suite of morphological characteristics, as well as directional-hemispherical reflectance from ultraviolet to thermal infrared wavelengths (250 nm to 20 µm) of leaves from five temperate deciduous tree species over the 8 wk following spring leaf emergence. By contrast to reflectance at shorter wavelengths, the shape and magnitude of MIR reflectance spectra changed markedly with development. MIR spectral differences among species became more pronounced and unique as leaves matured. Comparison of reflectance spectra of intact vs dried and ground leaves points to cuticular development - and not internal structural or biochemical changes - as the main driving factor. Accompanying the observed spectral changes was a drop in thermal emissivity from about 0.99 to 0.95 over the 8 wk following leaf emergence. Emissivity changes were not large enough to substantially influence leaf temperature, but they could potentially lead to a bias in radiometrically measured temperatures of up to 3 K. Our results also pointed to the potential for using MIR spectroscopy to better understand species-level differences in cuticular development and composition.

A Stomatal Model of Anatomical Tradeoffs Between Gas Exchange and Pathogen Colonization.

Stomatal pores control leaf gas exchange and are one route for infection of internal plant tissues by many foliar pathogens, setting up the potential for tradeoffs between photosynthesis and pathogen colonization. Anatomical shifts to lower stomatal density and/or size may also limit pathogen colonization, but such developmental changes could permanently reduce the gas exchange capacity for the life of the leaf. I developed and analyzed a spatially explicit model of pathogen colonization on the leaf as a function of stomatal size and density, anatomical traits which partially determine maximum rates of gas exchange. The model predicts greater stomatal size or density increases the probability of colonization, but the effect is most pronounced when the fraction of leaf surface covered by stomata is low. I also derived scaling relationships between stomatal size and density that preserves a given probability of colonization. These scaling relationships set up a potential anatomical conflict between limiting pathogen colonization and minimizing the fraction of leaf surface covered by stomata. Although a connection between gas exchange and pathogen defense has been suggested empirically, this is the first mathematical model connecting gas exchange and pathogen defense via stomatal anatomy. A limitation of the model is that it does not include variation in innate immunity and stomatal closure in response to pathogens. Nevertheless, the model makes predictions that can be tested with experiments and may explain variation in stomatal size and density among plants. The model is generalizable to many types of pathogens, but lacks significant biological realism that may be needed for precise predictions.

tealeaves: an R package for modelling leaf temperature using energy budgets.

Plants must regulate leaf temperature to optimize photosynthesis, control water loss and prevent damage caused by overheating or freezing. Physical models of leaf energy budgets calculate the energy fluxes and leaf temperatures for a given set leaf and environmental parameters. These models can provide deep insight into the variation in leaf form and function, but there are few computational tools available to use these models. Here I introduce a new R package called tealeaves to make complex leaf energy budget models accessible to a broader array of plant scientists. This package enables novice users to start modelling leaf energy budgets quickly while allowing experts to customize their parameter settings. The code is open source, freely available and readily integrates with other R tools for scientific computing. This paper describes the current functionality of tealeaves, but new features will be added in future releases. This software tool will advance new research on leaf thermal physiology to advance our understanding of basic and applied plant science.

Is Amphistomy an Adaptation to High Light? Optimality Models of Stomatal Traits along Light Gradients.

Stomata regulate the supply of CO2 for photosynthesis and the rate of water loss out of the leaf. The presence of stomata on both leaf surfaces, termed amphistomy, increases photosynthetic rate, is common in plants from high light habitats, and rare otherwise. In this study I use optimality models based on leaf energy budget and photosynthetic models to ask why amphistomy is common in high light habitats. I developed an R package leafoptimizer to solve for stomatal traits that optimally balance carbon gain with water loss in a given environment. The model predicts that amphistomy is common in high light because its marginal effect on carbon gain is greater than in the shade, but only if the costs of amphistomy are also lower under high light than in the shade. More generally, covariation between costs and benefits may explain why stomatal and other traits form discrete phenotypic clusters.

Geographic variation in reproductive assurance of Clarkia pulchella.

Climate can affect plant populations through direct effects on physiology and fitness, and through indirect effects on their relationships with pollinating mutualists. We therefore expect that geographic variation in climate might lead to variation in plant mating systems. Biogeographic processes, such as range expansion, can also contribute to geographic patterns in mating system traits. We manipulated pollinator access to plants in eight sites spanning the geographic range of Clarkia pulchella to investigate geographic and climatic drivers of fruit production and seed set in the absence of pollinators (reproductive assurance). We examined how reproductive assurance and fruit production varied with the position of sites within the range of the species and with temperature and precipitation. We found that reproductive assurance in C. pulchella was greatest in populations in the northern part of the species' range and was not well explained by any of the climate variables that we considered. In the absence of pollinators, some populations of C. pulchella have the capacity to increase fruit production, perhaps through resource reallocation, but this response is climate dependent. Pollinators are important for reproduction in this species, and recruitment is sensitive to seed input. The degree of autonomous self-pollination that is possible in populations of this mixed-mating species may be shaped by historic biogeographic processes or variation in plant and pollinator community composition rather than variation in climate.

Light and growth form interact to shape stomatal ratio among British angiosperms.

In most plants, stomata are located only on the abaxial leaf surface (hypostomy), but many plants have stomata on both surfaces (amphistomy). High light and herbaceous growth form have been hypothesized to favor amphistomy, but these hypotheses have not been rigorously tested together using phylogenetic comparative methods. I leveraged a large dataset including stomatal ratio, Ellenberg light indicator value, growth form and phylogenetic relationships for 372 species of British angiosperms. I used phylogenetic comparative methods to test how light and/or growth form influence stomatal ratio and density. High light and herbaceous growth form are correlated with amphistomy, as predicted, but they also interact; the effect of light is pronounced in therophytes (annuals) and perennial herbs, but muted in phanerophytes (shrubs and trees). Furthermore, amphistomy and stomatal density evolve together in response to light. Comparative analyses of British angiosperms reveal two major insights. First, light and growth form interact to shape stomatal ratio; amphistomy is common under high light, but mostly for herbs. Second, coordinated evolution of adaxial stomatal density and light tolerance indicates that amphistomy helps to optimally balance light acquisition with gas exchange. Stomatal ratio may have potential as a functional trait for paleoecology and crop improvement.

Pervasive antagonistic interactions among hybrid incompatibility loci.

Species barriers, expressed as hybrid inviability and sterility, are often due to epistatic interactions between divergent loci from two lineages. Theoretical models indicate that the strength, direction, and complexity of these genetic interactions can strongly affect the expression of interspecific reproductive isolation and the rates at which new species evolve. Nonetheless, empirical analyses have not quantified the frequency with which loci are involved in interactions affecting hybrid fitness, and whether these loci predominantly interact synergistically or antagonistically, or preferentially involve loci that have strong individual effects on hybrid fitness. We systematically examined the prevalence of interactions between pairs of short chromosomal regions from one species (Solanum habrochaites) co-introgressed into a heterospecific genetic background (Solanum lycopersicum), using lines containing pairwise combinations of 15 chromosomal segments from S. habrochaites in the background of S. lycopersicum (i.e., 95 double introgression lines). We compared the strength of hybrid incompatibility (either pollen sterility or seed sterility) expressed in each double introgression line to the expected additive effect of its two component single introgressions. We found that epistasis was common among co-introgressed regions. Interactions for hybrid dysfunction were substantially more prevalent in pollen fertility compared to seed fertility phenotypes, and were overwhelmingly antagonistic (i.e., double hybrids were less unfit than expected from additive single introgression effects). This pervasive antagonism is expected to attenuate the rate at which hybrid infertility accumulates among lineages over time (i.e., giving diminishing returns as more reproductive isolation loci accumulate), as well as decouple patterns of accumulation of sterility loci and hybrid incompatibility phenotypes. This decoupling effect might explain observed differences between pollen and seed fertility in their fit to theoretical predictions of the accumulation of isolation loci, including the 'snowball' effect.

Weak coordination between leaf structure and function among closely related tomato species.

Theory predicts that natural selection should favor coordination between leaf physiology, biochemistry and anatomical structure along a functional trait spectrum from fast, resource-acquisitive syndromes to slow, resource-conservative syndromes. However, the coordination hypothesis has rarely been tested at a phylogenetic scale most relevant for understanding rapid adaptation in the recent past or for the prediction of evolutionary trajectories in response to climate change. We used a common garden to examine genetically based coordination between leaf traits across 19 wild and cultivated tomato taxa. We found weak integration between leaf structure (e.g. leaf mass per area) and physiological function (photosynthetic rate, biochemical capacity and CO2 diffusion), even though all were arrayed in the predicted direction along a 'fast-slow' spectrum. This suggests considerable scope for unique trait combinations to evolve in response to new environments or in crop breeding. In particular, we found that partially independent variation in stomatal and mesophyll conductance may allow a plant to improve water-use efficiency without necessarily sacrificing maximum photosynthetic rates. Our study does not imply that functional trait spectra, such as the leaf economics spectrum, are unimportant, but that many important axes of variation within a taxonomic group may be unique and not generalizable to other taxa.

Constraint around Quarter-Power Allometric Scaling in Wild Tomatoes (Solanum sect. Lycopersicon; Solanaceae).

The West-Brown-Enquist (WBE) metabolic scaling theory posits that many organismal features scale predictably with body size because of selection to minimize transport costs in resource distribution networks. Many scaling exponents are quarter-powers, as predicted by WBE, but there are also biologically significant deviations that could reflect adaptation to different environments. A central but untested prediction of the WBE model is that wide deviation from optimal scaling is penalized, leading to a pattern of constraint on scaling exponents. Here, we demonstrate, using phylogenetic comparative methods, that variation in allometric scaling between mass and leaf area across 17 wild tomato taxa is constrained around a value indistinguishable from that predicted by WBE but significantly greater than 2/3 (geometric-similarity model). The allometric-scaling exponent was highly correlated with fecundity, water use, and drought response, suggesting that it is functionally significant and therefore could be under selective constraints. However, scaling was not strictly log-log linear but rather declined during ontogeny in all species, as has been observed in many plant species. We caution that although our results supported one prediction of the WBE model, it did not strongly test the model in other important respects. Nevertheless, phylogenetic comparative methods such as those used here are powerful but underutilized tools for metabolic ecology that complement existing methods to adjudicate between models.

Making pore choices: repeated regime shifts in stomatal ratio.

Ecologically important traits do not evolve without limits. Instead, evolution is constrained by the set of available and viable phenotypes. In particular, natural selection may only favour a narrow range of adaptive optima constrained within selective regimes. Here, I integrate data with theory to test whether selection explains phenotypic constraint. A global database of 599 plant species from 94 families shows that stomatal ratio, a trait affecting photosynthesis and defence against pathogens, is highly constrained. Most plants have their stomata on the lower leaf surface (hypostomy), but species with half their stomata on each surface (amphistomy) form a distinct mode in the trait distribution. A model based on a trade-off between maximizing photosynthesis and a fitness cost of upper stomata predicts a limited number of adaptive solutions, leading to a multimodal trait distribution. Phylogenetic comparisons show that amphistomy is the most common among fast-growing species, supporting the view that CO2 diffusion is under strong selection. These results indicate that selective optima stay within a relatively stable set of selective regimes over macroevolutionary time.