Liana T. Burghardt

Genetic and physiological bases for phenological responses to current and predicted climates.

We are now reaching the stage at which specific genetic factors with known physiological effects can be tied directly and quantitatively to variation in phenology. With such a mechanistic understanding, scientists can better predict phenological responses to novel seasonal climates. Using the widespread model species Arabidopsis thaliana, we explore how variation in different genetic pathways can be linked to phenology and life-history variation across geographical regions and seasons. We show that the expression of phenological traits including flowering depends critically on the growth season, and we outline an integrated life-history approach to phenology in which the timing of later life-history events can be contingent on the environmental cues regulating earlier life stages. As flowering time in many plants is determined by the integration of multiple environmentally sensitive gene pathways, the novel combinations of important seasonal cues in projected future climates will alter how phenology responds to variation in the flowering time gene network with important consequences for plant life history. We discuss how phenology models in other systems—both natural and agricultural—could employ a similar framework to explore the potential contribution of genetic variation to the physiological integration of cues determining phenology.

Modeling the Influence of Genetic and Environmental Variation on the Expression of Plant Life Cycles across Landscapes

Organisms develop through multiple life stages that differ in environmental tolerances. The seasonal timing, or phenology, of life-stage transitions determines the environmental conditions to which each life stage is exposed and the length of time required to complete a generation. Both environmental and genetic factors contribute to phenological variation, yet predicting their combined effect on life cycles across a geographic range remains a challenge. We linked submodels of the plasticity of individual life stages to create an integrated model that predicts life-cycle phenology in complex environments. We parameterized the model for Arabidopsis thaliana and simulated life cycles in four locations. We compared multiple “genotypes” by varying two parameters associated with natural genetic variation in phenology: seed dormancy and floral repression. The model predicted variation in life cycles across locations that qualitatively matches observed natural phenology. Seed dormancy had larger effects on life-cycle length than floral repression, and results suggest that a genetic cline in dormancy maintains a life-cycle length of 1 year across the geographic range of this species. By integrating across life stages, this approach demonstrates how genetic variation in one transition can influence subsequent transitions and the geographic distribution of life cycles more generally.

Applying developmental threshold models to evolutionary ecology

Process-based models of development predict developmental rates and phenology as a function of physiological responses to multiple dynamic environmental factors. These models can be adapted to analyze diverse processes in evolutionary ecology. By linking models across life stages, they can predict life cycles and generation times. By incorporating fitness, they can identify environmental and physiological factors that limit species distributions. By incorporating population variance, they can investigate mechanisms of intraspecific variation or synchronization. By incorporating genetics, they can predict genotype-specific phenology under diverse climatic scenarios and examine causes and consequences of pleiotropy across life stages. With further development, they have the potential to predict genotype-specific ranges and identify key genes involved in determining phenology and fitness in variable and changing environments.

Multiple paths to similar germination behavior in Arabidopsis thaliana.

Germination timing influences plant fitness, and its sensitivity to temperature may cause it to change as climate shifts. These changes are likely to be complex because temperatures that occur during seed maturation and temperatures that occur post-dispersal interact to define germination timing. We used the model organism Arabidopsis thaliana to determine how flowering time (which defines seed-maturation temperature) and post-dispersal temperature influence germination and the expression of genetic variation for germination. Germination responses to temperature (germination envelopes) changed as seeds aged, or after-ripened, and these germination trajectories depended on seed-maturation temperature and genotype. Different combinations of genotype, seed-maturation temperature, and after-ripening produced similar germination envelopes. Likewise, different genotypes and seed-maturation temperatures combined to produce similar germination trajectories. Differences between genotypes were most likely to be observed at high and low germination temperatures. The germination behavior of some genotypes responds weakly to maternal temperature but others are highly plastic. We hypothesize that weak dormancy induction could synchronize germination of seeds dispersed at different times. By contrast, we hypothesize that strongly responsive genotypes may spread offspring germination over several possible germination windows. Considering germination responses to temperature is important for predicting phenology expression and evolution in future climates.

Secondary dormancy dynamics depends on primary dormancy status in Arabidopsis thaliana

Seed dormancy can prevent germination under unfavourable conditions that reduce the chances of seedling survival. Freshly harvested seeds often have strong primary dormancy that depends on the temperature experienced by the maternal plant and which is gradually released through afterripening. However, seeds can be induced into secondary dormancy if they experience conditions or cues of future unfavourable conditions. Whether this secondary dormancy induction is influenced by seed-maturation conditions and primary dormancy has not been explored in depth. In this study, we examined secondary dormancy induction in seeds of Arabidopsis thaliana matured under different temperatures and with different levels of afterripening. We found that low water potential and a range of temperatures, from 8°C to 35°C, induced secondary dormancy. Secondary dormancy induction was affected by the state of primary dormancy of the seeds. Specifically, afterripening had a non-monotonic effect on the ability to be induced into secondary dormancy by stratification; first increasing in sensitivity as afterripening proceeded, then declining in sensitivity after 5 months of afterripening, finally increasing again by 18 months of afterripening. Seed-maturation temperature sometimes had effects that were independent of expressed primary dormancy, such that seeds that had matured at low temperature, but which had comparable germination proportions as seeds matured at warmer temperatures, were more easily induced into secondary dormancy. Because seed-maturation temperature is a cue of when seeds were matured and dispersed, these results suggest that the interaction of seed-maturation temperature, afterripening and post-dispersal conditions all combine to regulate the time of year of seed germination.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

GWA studies combined with multiple mutagenesis technologies, including CRISPR/Cas9, were used to functionally validate novel candidate genes contributing to phenotypic variation in symbiosis between legume plants and rhizobial bacteria. Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.

The community‐level effect of light on germination timing in relation to seed mass: a source of regeneration niche differentiation

Within a community, species may germinate at different times so as to mitigate competition and to take advantage of different aspects of the seasonal environment (temporal niche differentiation). We illustrated a hypothesis of the combined effects of abiotic and biotic competitive factors on germination timing and the subsequent upscale effects on community assembly. We estimated the germination timing (GT) for 476 angiosperm species of the eastern Tibetan Plateau grasslands under two light treatments in the field: high (i.e. natural) light and low light. We also measured the shift in germination timing (SGT) across treatments for all species. Furthermore, we used phylogenetic comparative methods to test if GT and SGT were associated with seed mass, an important factor in competitive interactions. We found a significant positive correlation between GT and seed mass in both light treatments. Additionally, small seeds (early germinating seeds) tended to germinate later and large seeds (late germinating seeds) tended to germinate earlier under low light vs high light conditions. Low light availability can reduce temporal niche differentiation by increasing the overlap in germination time between small and large seeds. In turn, reduced temporal niche differentiation may increase competition in the process of community assembly.

Avoiding the crowds: the evolution of plastic responses to seasonal cues in a density-dependent world

Summary Across a range of organisms, life-history transitions can be triggered by specific levels of seasonal cues, such as day length or temperature. This acts to regulate the life cycle relative to seasonal fluctuations, so that, for example, reproduction occurs when resources are most abundant. At an environmentally optimal cue level, individuals might be selected to respond in a similar way, and consequently abruptly increase population density through, for example, the production of offspring. However, if density dependence is operating, crowding will reduce individual fitness, and selection will then favour life-history transitions at different cue levels. We explore the eco-evolutionary dynamics that result from the interaction between density dependence and phenology using a life-history model of an annual plant and focus on the timing of seed germination within the year. We characterize the adaptive dynamics of the mean and variance across the year of germination timing. Our results indicate that, even in predictably varying environments, mean timing of germination is not evolutionarily stable, but may experience evolutionary branching if variance in germination timing is sufficiently constrained. Unpredictably varying environments extend contexts where this is the case. Synthesis. Even if there is a restricted time period in the year during which environmental conditions are most appropriate for germination, observed timing of germination might be very diverse. Density dependence selects for persistence of traits conferring variance in timing across the year and additionally can lead to evolutionary branching of mean germination timing, resulting in multiple coexisting genotypes corresponding to different mean germination timings.

Transcriptomic basis of genome by genome variation in a legume-rhizobia mutualism

In the legume‐rhizobia mutualism, the benefit each partner derives from the other depends on the genetic identity of both host and rhizobial symbiont. To gain insight into the extent of genome × genome interactions on hosts at the molecular level and to identify potential mechanisms responsible for the variation, we examined host gene expression within nodules (the plant organ where the symbiosis occurs) of four genotypes of Medicago truncatula grown with either Ensifer meliloti or E. medicae symbionts. These host × symbiont combinations show significant variation in nodule and biomass phenotypes. Likewise, combinations differ in their transcriptomes: host, symbiont and host × symbiont affected the expression of 70%, 27% and 21%, respectively, of the approximately 27,000 host genes expressed in nodules. Genes with the highest levels of expression often varied between hosts and/or symbiont strain and include leghemoglobins that modulate oxygen availability and hundreds of Nodule Cysteine‐Rich (NCR) peptides involved in symbiont differentiation and viability in nodules. Genes with host × symbiont‐dependent expression were enriched for functions related to resource exchange between partners (sulphate/iron/amino acid transport and dicarboxylate/amino acid synthesis). These enrichments suggest mechanisms for host control of the currencies of the mutualism. The transcriptome of M. truncatula accession HM101 (A17), the reference genome used for most molecular research, was less affected by symbiont identity than the other hosts. These findings underscore the importance of assessing the molecular basis of variation in ecologically important traits, particularly those involved in biotic interactions, in multiple genetic contexts.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments

Significance We describe an empirical approach to measure the outcomes of selection and competition in bacterial populations. This approach differs from others in that it examines selection acting on naturally occurring variation rather than new mutations. We demonstrate this method by examining selection on rhizobial bacteria living both in symbiosis with leguminous plants and independently in the soil. We identify fitness correlations across environments that could affect the maintenance of the mutualism and natural genomic variants underlying bacterial fitness. Identifying selection inside and outside of hosts may lead to future manipulation of the mutualism to increase agricultural yields. Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the “evolve and resequence” approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti. We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.