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Background: Plant hormones influence phenology, development, and function of above and belowground plant structures. In seedlings, auxin influences the initiation and development of lateral roots and root systems. How auxin-related genes influence root initiation at early life stages has been investigated from numerous perspectives. There is a gap in our understanding of how these genes influence root size through the life cycle and in mature plants. Across development, the influence of a particular gene on plant phenotypes is partly regulated by the addition of a poly-A tail to mRNA transcripts via alternative polyadenylation (APA). Auxin related genes have documented variation in APA, with auxin itself contributing to APA site switches. Studies of the influence of exogenous auxin on natural plant accessions and mutants of auxin pathway gene families exhibiting variation in APA are required for a more complete understanding of genotype by development by hormone interactions in whole plant and fitness traits., Methods: We studied Arabidopsis thaliana homozygous mutant lines with inserts in auxin-related genes previously identified to exhibit variation in number of APA sites. Our growth chamber experiment included wildtype Col-0 controls, mutant lines, and natural accession phytometers. We applied exogenous auxin through the life cycle. We quantified belowground and aboveground phenotypes in 14 day old, 21 day old seedlings and plants at reproductive maturity. We contrasted root, rosette and flowering phenotypes across wildtype, auxin mutant, and natural accession lines, APA groups, hormone treatments, and life stages using general linear models., Results: The root systems and rosettes of mutant lines in auxin related genes varied in response to auxin applications across life stages and varied between genotypes within life stages. In seedlings, exposure to auxin decreased size, but increased lateral root density, whereas at reproductive maturity, plants displayed greater aboveground mass and total root length. These differences may in part be due to a shift which delayed the reproductive stage when plants were treated with auxin. Root traits of auxin related mutants depended on the number of APA sites of mutant genes and the plant's developmental stage. Mutants with inserts in genes with many APA sites exhibited lower early seedling belowground biomass than those with few APA sites but only when exposed to exogenous auxin. As we observed different responses to exogenous auxin across the life cycle, we advocate for further studies of belowground traits and hormones at reproductive maturity. Studying phenotypic variation of genotypes across life stages and hormone environments will uncover additional shared patterns across traits, assisting efforts to potentially reach breeding targets and enhance our understanding of variation of genotypes in natural systems., Competing Interests: The authors declare that they have no competing interests., (© 2024 Sydow and Murren.)

Phenology, or seasonal variation in life cycle events, is poorly described for many macroalgal species. We describe the phenology of a non-native population of Gracilaria vermiculophylla whose thalli are free-living or anchored by decorating polychaetes to tube caps. At a site in South Carolina, USA, we sampled 100 thalli approximately every month from January 2014 to January 2015. We assessed the reproductive state and measured thallus size based on wet weight, thallus length, and thallus surface area from herbarium mounts. Because life cycle stage cannot be assigned using morphology, we implemented a PCR assay to determine the life cycle stage-tetrasporophyte, female gametophyte, or male gametophyte-of each thallus. Tetrasporophytes dominated throughout the year, making up 81%-100% of thalli sampled per month. Reproductive tetrasporophytes varied between 0% and 65% of monthly samples and were most common in warm summer months (July through September) when thalli also tended to be larger. The vast majority of the reproductive thalli were worm-anchored and not fixed to hard substratum via a holdfast. Thus, free-living thalli can be reproductive and potentially seed new non-native populations. Given G. vermiculophylla reproduction seems tied closely to temperature, our work suggests phenology may change with climate-related changes in seawater temperatures. We also highlight the importance of understanding the natural history of macroalgae to better understand the consequence of range expansions on population dynamics., (© 2023 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

In-person undergraduate research experiences (UREs) promote students' integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration and whether they might perceive doing research less favorably (i.e., not beneficial or too costly). To address these questions, we examined indicators of scientific integration and perceptions of the benefits and costs of doing research among students who participated in remote life science URE programs in Summer 2020. We found that students experienced gains in scientific self-efficacy pre- to post-URE, similar to results reported for in-person UREs. We also found that students experienced gains in scientific identity, graduate and career intentions, and perceptions of the benefits of doing research only if they started their remote UREs at lower levels on these variables. Collectively, students did not change in their perceptions of the costs of doing research despite the challenges of working remotely. Yet students who started with low cost perceptions increased in these perceptions. These findings indicate that remote UREs can support students' self-efficacy development, but may otherwise be limited in their potential to promote scientific integration.

Selection leaves signatures in the DNA sequence of genes, with many test statistics devised to detect its action. While these statistics are frequently used to support hypotheses about the adaptive significance of particular genes, the effect these genes have on reproductive fitness is rarely quantified experimentally. Consequently, it is unclear how gene-level signatures of selection are associated with empirical estimates of gene effect on fitness. Eukaryotic data sets that permit this comparison are very limited. Using the model plant Arabidopsis thaliana, for which these resources are available, we calculated seven gene-level substitution and polymorphism-based statistics commonly used to infer selection (dN/dS, NI, DOS, Tajima's D, Fu and Li's D*, Fay and Wu's H, and Zeng's E) and, using knockout lines, compared these to gene-level estimates of effect on fitness. We found that consistent with expectations, essential genes were more likely to be classified as negatively selected. By contrast, using 379 Arabidopsis genes for which data was available, we found no evidence that genes predicted to be positively selected had a significantly different effect on fitness than genes evolving more neutrally. We discuss these results in the context of the analytic challenges posed by Arabidopsis, one of the only systems in which this study could be conducted, and advocate for examination in additional systems. These results are relevant to the evaluation of genome-wide studies across species where experimental fitness data is unavailable, as well as highlighting an increasing need for the latter., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The COVID-19 pandemic shut down undergraduate research programs across the United States. A group of 23 colleges, universities, and research institutes hosted remote undergraduate research programs in the life sciences during Summer 2020. Given the unprecedented offering of remote programs, we carried out a study to describe and evaluate them. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from students' perspectives. Strengths included the quality of mentorship, opportunities for learning and professional development, and a feeling of connection with a larger community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote Research Experiences for Undergraduates (REUs) that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

Stomata are essential pores flanked by guard cells that control gas exchange in plants. We can utilize stomatal size and density measurements as a proxy for a plant's capacity for gas exchange. While stomatal responses to stressful environments are well studied; data are lacking in the responses across mutant genotypes of the same species in these trait and treatment interactions or genetic variation in phenotypic plasticity. We evaluated the effects of soil nutrient variation on macroscopic and stomatal traits of Arabidopsis thaliana T-DNA insertion mutants for which prior performance in a single benign growing condition were available. Nutrient-induced stress significantly impacted traits including plant biomass, height, fruit number, and leaf number which we denote as macroscopic traits. We found evidence that genotype by environment effects exist for macroscopic traits, yet total stomatal area variation, or "microscopic variation" across environments was modest. Divergence from the wildtype line varied by mutant background and these responses were variable among traits. These findings suggest that Arabidopsis employs a strategy of physiological compensation, sacrificing morphological traits to maintain stomatal production., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The genomic variation of an invasive species may be affected by complex demographic histories and evolutionary changes during the invasion. Here, we describe the relative influence of bottlenecks, clonality, and population expansion in determining genomic variability of the widespread red macroalga Agarophyton vermiculophyllum. Its introduction from mainland Japan to the estuaries of North America and Europe coincided with shifts from predominantly sexual to partially clonal reproduction and rapid adaptive evolution. A survey of 62,285 SNPs for 351 individuals from 35 populations, aligned to 24 chromosome-length scaffolds indicate that linkage disequilibrium (LD), observed heterozygosity (H o ), Tajima's D, and nucleotide diversity (Pi) were greater among non-native than native populations. Evolutionary simulations indicate LD and Tajima's D were consistent with a severe population bottleneck. Also, the increased rate of clonal reproduction in the non-native range could not have produced the observed patterns by itself but may have magnified the bottleneck effect on LD. Elevated marker diversity in the genetic source populations could have contributed to the increased H o and Pi observed in the non-native range. We refined the previous invasion source region to a ~50 km section of northeastern Honshu Island. Outlier detection methods failed to reveal any consistently differentiated loci shared among invaded regions, probably because of the complex A. vermiculophyllum demographic history. Our results reinforce the importance of demographic history, specifically founder effects, in driving genomic variation of invasive populations, even when localized adaptive evolution and reproductive system shifts are observed., (© 2021 John Wiley & Sons Ltd.)

Undergraduates phenotyping Arabidopsis knockouts (unPAK) is a biology research network that has provided undergraduate research experiences (URE) since 2010. In 2019, unPAK expanded to include a summer URE that engaged undergraduate researchers from across the network in an intensive collaborative program. In response to the COVID-19 pandemic in 2020, unPAK rapidly shifted to provide the summer URE program remotely. This article describes (i) the instructional and communication processes of unPAK in the remote URE; and (ii) a summative assessment of the outcomes associated with the remote summer program as compared with the 2019 in-person program. We conclude by offering timely recommendations for educators in biology that emerged from the 2020 remote summer research experience, which may be applicable to other remote UREs and course-based research experiences (CUREs)., (©2021 Author(s). Published by the American Society for Microbiology.)

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double-digest restriction site-associated DNA sequencing (ddRAD-seq) to identify sex-linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD-seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non-native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female-linked and 19 putatively male-linked sequences. Four female- and eight male-linked markers amplified in all three life cycle stages. Using one female- and one male-linked marker that were sex-specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non-native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD-seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex-linked markers in other haplodiplontic macroalgae for which genomes are lacking., (© 2020 Phycological Society of America.)

Biology research is becoming increasingly dependent on large-scale, "big data," networked research initiatives. At the same time, there has been a corresponding effort to expand undergraduate participation in research to benefit student learning and persistence in science. This essay examines the confluence of this trend through eight years of a collaboration within a successful biology research network that explicitly incorporates undergraduates into large-scale scientific research. We draw upon interviews with faculty in this network to consider the interplay of scientific and pedagogical objectives at the heart of this undergraduate-focused network research project. We identify ways that this network has expanded and diversified access to scientific knowledge production for faculty and students and examine a goal conflict that emerged around the dual objectives of mentoring emerging scientists while producing high-quality scientific data for the larger biology community. Based on lessons learned within this network, we provide three recommendations that can support institutions and faculty engaging in networked research projects with undergraduates: (1) establish rigorous protocols to ensure data and database quality, (2) protect personnel time to coordinate network and scientific processes, and (3) select appropriate partners and establish explicit expectations for specific collaborations.

Premise: In the complex soil nutrient environments of wild populations of annual plants, in general, low nutrient availability restricts growth and alters root-shoot relationships. However, our knowledge of natural selection on roots in field settings is limited. We sought to determine whether selection acts directly on root traits and to identify which components of the soil environment were potential agents of selection., Methods: We studied wild native populations of Arabidopsis thaliana across 4 years, measuring aboveground and belowground traits and analyzing soil nutrients. Using multivariate methods, we examined patterns of natural selection and identified soil attributes that contributed to whole-plant form. In a common garden experiment at two field sites with contrasting soil texture, we examined patterns of selection on root and shoot traits., Results: In wild populations, we uncovered selection for above- and belowground size and architectural traits. We detected variation through time and identified soil components that influenced fruit production. In the garden experiment, we detected a distinct positive selection for total root length at the site with greater water-holding capacity and negative selection for measures of root architecture at the field site with reduced nutrient availability and water holding capacity., Conclusions: Patterns of natural selection on belowground traits varied through time, across field sites and experimental gardens. Simultaneous investigations of above- and belowground traits reveal trait functional relationships on which natural selection can act, highlighting the influence of edaphic features on evolutionary processes in wild annual plant populations., (© 2020 Botanical Society of America.)

Determining how genes are associated with traits in plants and other organisms is a major challenge in modern biology. The unPAK project - undergraduates phenotyping Arabidopsis knockouts - has generated phenotype data for thousands of non-lethal insertion mutation lines within a single Arabidopsis thaliana genomic background. The focal phenotypes examined by unPAK are complex macroscopic fitness-related traits, which have ecological, evolutionary and agricultural importance. These phenotypes are placed in the context of the wild-type and also natural accessions (phytometers), and standardized for environmental differences between assays. Data from the unPAK project are used to describe broad patterns in the phenotypic consequences of insertion mutation, and to identify individual mutant lines with distinct phenotypes as candidates for further study. Inclusion of undergraduate researchers is at the core of unPAK activities, and an important broader impact of the project is providing students an opportunity to obtain research experience., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Premise: Determining how species perform in novel climatic environments is essential for understanding (1) responses to climate change and (2) evolutionary consequences of biological invasions. For the vast majority of species, the number of population characteristics that will predict performance and patterns of natural selection in novel locations in the wild remains limited., Methods: We evaluated phenological, vegetative, architectural, and fitness-related traits in experimental gardens in contrasting climates (Ontario, Canada, and South Carolina, USA) in the North American non-native distribution of Arabidopsis thaliana. We assessed the effects of climatic distance, geographic distance, and genetic features of history on performance and patterns of natural selection in the novel garden settings., Results: We found that plants had greater survivorship, flowered earlier, were larger, and produced more fruit in the south, and that genotype-by-environment interactions were significant between gardens. However, our analyses revealed similar patterns of natural selection between gardens in distinct climate zones. After accounting for genetic ancestry, we also detected that population climatic distance best predicted performance within gardens., Conclusions: These data suggest that colonization success in novel, non-native environments is determined by a combination of climate and genetic history. When performance at novel sites was assessed with seed sources from geographically and genetically disparate, established non-native populations, proximity to the garden alone was insufficient to predict performance. Our study highlights the need to evaluate seed sources from diverse origins to describe comprehensively phenotypic responses to novel environments, particularly for taxa in which many source populations may contribute to colonization., (© 2019 Botanical Society of America.)

We present a curriculum description, an initial student outcome investigation, and sample scientific results for a representative Course-Based Undergraduate Research Experience (CURE) that is part of the "Undergraduates Phenotyping Arabidopsis Knockouts" (unPAK) network. CUREs in the unPAK network characterize quantitative phenotypes of the model plant Arabidopsis from across environments to uncover connections between genotype and phenotype. Students in unPAK CUREs grow plants in a replicated block design and make quantitative measurements throughout the semester. This CURE enables students to answer plant science questions that draw from fields such as environmental science, genetics, ecology, and evolution. Findings indicate that this experience provides students with opportunities to make relevant scientific discoveries. Eighty percent of student datasets produced from the CURE met criteria for inclusion in the project database, indicative of student learning in data collection and analysis of quantitative plant traits. Student datasets uncovered novel effects of mutation on plant form. In addition, students' science self-efficacy increased as a result of course participation, and faculty feedback on course implementation was positive. We present unPAK as a new network that supports CUREs and research experiences focused on collecting biological data made publicly available to the scientific community. The unPAK CUREs can be tailored to address instructor interests or pedagogical needs while involving students in research investigating quantitative plant phenotypes.

Organisms are faced with variable environments and one of the most common solutions to cope with such variability is phenotypic plasticity, a modification of the phenotype to the environment. These modifications are commonly modelled in evolutionary theories as adaptive, influencing ecological and evolutionary processes. If plasticity is adaptive, we would predict that the closer to fitness a trait is, the less plastic it would be. To test this hypothesis, we conducted a meta-analysis of 213 studies and measured the plasticity of each reported trait as a coefficient of variation. Traits were categorized as closer to fitness-life-history traits including reproduction and survival related traits, and farther from fitness-non-life-history traits including traits related to development, metabolism and physiology, morphology and behaviour. Our results showed, unexpectedly, that although traits differed in their amounts of plasticity, trait plasticity was not related to its proximity to fitness. These findings were independent of taxonomic groups or environmental types assessed. We caution against general expectations that plasticity is adaptive, as assumed by many models of its evolution. More studies are needed that test the adaptive nature of plasticity, and additional theoretical explorations on adaptive and non-adaptive plasticity are encouraged.

Theory predicts that the maintenance of haplodiplontic life cycles requires ecological differences between the haploid gametophytes and diploid sporophytes, yet evidence of such differences remain scarce. The haplodiplontic red seaweed Gracilaria vermiculophylla has invaded the temperate estuaries of the Northern Hemisphere, where it commonly modifies detrital and trophic pathways. In native populations, abundant hard substratum enables spore settlement, and gametophyte:tetrasporophyte ratios are ~40:60. In contrast, many non-native populations persist in soft-sediment habitats without abundant hard substratum, and can be 90%-100% tetrasporophytic. To test for ecologically relevant phenotypic differences, we measured thallus morphology, protein content, organic content, "debranching resistance" (i.e., tensile force required to remove a branch from its main axis node), and material properties between male gametophytes, female gametophytes, and tetrasporophytes from a single, nonnative site in Charleston Harbor, South Carolina, USA in 2015 and 2016. Thallus length and surface area to volume ratio differed between years, but were not significantly different between ploidies. Tetrasporophytes had lower protein content than gametophytes, suggesting the latter may be more attractive to consumers. More force was required to pull a branch from the main axis of tetrasporophytes relative to gametophytes. A difference in debranching resistance may help to maintain tetrasporophyte thallus durability relative to gametophytes, providing a potential advantage in free-floating populations. These data may shed light on the invasion ecology of an important ecosystem engineer, and may advance our understanding of life cycle evolution and the maintenance of life cycle diversity., (© 2018 Phycological Society of America.)

The rapid evolution of non-native species can facilitate invasion success, but recent reviews indicate that such microevolution rarely yields expansion of the climatic niche in the introduced habitats. However, because some invasions originate from a geographically restricted portion of the native species range and its climatic niche, it is possible that the frequency, direction, and magnitude of phenotypic evolution during invasion have been underestimated. We explored the utility of niche shift analyses in the red seaweed Gracilaria vermiculophylla , which expanded its range from the northeastern coastline of Japan to North America, Europe, and northwestern Africa within the last 100 years. A genetically informed climatic niche shift analysis indicates that native source populations occur in colder and highly seasonal habitats, while most non-native populations typically occur in warmer, less seasonal habitats. This climatic niche expansion predicts that non-native populations evolved greater tolerance for elevated heat conditions relative to native source populations. We assayed 935 field-collected and 325 common-garden thalli from 40 locations, and as predicted, non-native populations had greater tolerance for ecologically relevant extreme heat (40°C) than did Japanese source populations. Non-native populations also had greater tolerance for cold and low-salinity stresses relative to source populations. The importance of local adaptation to warm temperatures during invasion was reinforced by evolution of parallel clines: Populations from warmer, lower-latitude estuaries had greater heat tolerance than did populations from colder, higher-latitude estuaries in both Japan and eastern North America. We conclude that rapid evolution plays an important role in facilitating the invasion success of this and perhaps other non-native marine species. Genetically informed ecological niche analyses readily generate clear predictions of phenotypic shifts during invasions and may help to resolve debate over the frequency of niche conservatism versus rapid adaptation during invasion.

Premise of the Study: Studies on phenotypic plasticity and plasticity of integration have uncovered functionally linked modules of aboveground traits and seedlings of Arabidopsis thaliana , but we lack details about belowground variation in adult plants. Functional modules can be comprised of additional suites of traits that respond to environmental variation. We assessed whether shoot and root responses to nutrient environments in adult A. thaliana were predictable from seedling traits or population-specific geologic soil characteristics at the site of origin., Methods: We compared 17 natural accessions from across the native range of A. thaliana using 14-day-old seedlings grown on agar or sand and plants grown to maturity across nutrient treatments in sand. We measured aboveground size, reproduction, timing traits, root length, and root diameter. Edaphic characteristics were obtained from a global-scale dataset and related to field data., Key Results: We detected significant among-population variation in root traits of seedlings and adults and in plasticity in aboveground and belowground traits of adult plants. Phenotypic integration of roots and shoots varied by population and environment. Relative integration was greater in roots than in shoots, and integration was predicted by edaphic soil history, particularly organic carbon content, whereas seedling traits did not predict later ontogenetic stages., Conclusions: Soil environment of origin has significant effects on phenotypic plasticity in response to nutrients, and on phenotypic integration of root modules and shoot modules. Root traits varied among populations in reproductively mature individuals, indicating potential for adaptive and integrated functional responses of root systems in annuals., (© 2017 Botanical Society of America.)

Screens of organisms with disruptive mutations in a single gene often fail to detect phenotypic consequences for the majority of mutants. One explanation for this phenomenon is that the presence of paralogous loci provides genetic redundancy. However, it is also possible that the assayed traits are affected by few loci, that effects could be subtle or that phenotypic effects are restricted to certain environments. We assayed a set of T-DNA insertion mutant lines of Arabidopsis thaliana to determine the frequency with which mutation affected fitness-related phenotypes. We found that between 8% and 42% of the assayed lines had altered fitness from the wild type. Furthermore, many of these lines exhibited fitness greater than the wild type. In a second experiment, we grew a subset of the lines in multiple environments and found whether a T-DNA insert increased or decreased fitness traits depended on the assay environment. Overall, our evidence contradicts the hypothesis that genetic redundancy is a common phenomenon in A. thaliana for fitness traits. Evidence for redundancy from prior screens of knockout mutants may often be an artefact of the design of the phenotypic assays which have focused on less complex phenotypes than fitness and have used single environments. Finally, our study adds to evidence that beneficial mutations may represent a significant component of the mutational spectrum of A. thaliana., (© 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.)

Baker's Law predicts uniparental reproduction will facilitate colonization success in novel habitats. While evidence supports this prediction among colonizing plants and animals, few studies have investigated shifts in reproductive mode in haplo-diplontic species in which both prolonged haploid and diploid stages separate meiosis and fertilization in time and space. Due to this separation, asexual reproduction can yield the dominance of one of the ploidy stages in colonizing populations. We tested for shifts in ploidy and reproductive mode across native and introduced populations of the red seaweed Gracilaria vermiculophylla. Native populations in the northwest Pacific Ocean were nearly always attached by holdfasts to hard substrata and, as is characteristic of the genus, haploid-diploid ratios were slightly diploid-biased. In contrast, along North American and European coastlines, introduced populations nearly always floated atop soft-sediment mudflats and were overwhelmingly dominated by diploid thalli without holdfasts. Introduced populations exhibited population genetic signals consistent with extensive vegetative fragmentation, while native populations did not. Thus, the ecological shift from attached to unattached thalli, ostensibly necessitated by the invasion of soft-sediment habitats, correlated with shifts from sexual to asexual reproduction and slight to strong diploid bias. We extend Baker's Law by predicting other colonizing haplo-diplontic species will show similar increases in asexuality that correlate with the dominance of one ploidy stage. Labile mating systems likely facilitate colonization success and subsequent range expansion, but for haplo-diplontic species, the long-term eco-evolutionary impacts will depend on which ploidy stage is lost and the degree to which asexual reproduction is canalized., (© 2016 John Wiley & Sons Ltd.)

Phenotypic plasticity is ubiquitous and generally regarded as a key mechanism for enabling organisms to survive in the face of environmental change. Because no organism is infinitely or ideally plastic, theory suggests that there must be limits (for example, the lack of ability to produce an optimal trait) to the evolution of phenotypic plasticity, or that plasticity may have inherent significant costs. Yet numerous experimental studies have not detected widespread costs. Explicitly differentiating plasticity costs from phenotype costs, we re-evaluate fundamental questions of the limits to the evolution of plasticity and of generalists vs specialists. We advocate for the view that relaxed selection and variable selection intensities are likely more important constraints to the evolution of plasticity than the costs of plasticity. Some forms of plasticity, such as learning, may be inherently costly. In addition, we examine opportunities to offset costs of phenotypes through ontogeny, amelioration of phenotypic costs across environments, and the condition-dependent hypothesis. We propose avenues of further inquiry in the limits of plasticity using new and classic methods of ecological parameterization, phylogenetics and omics in the context of answering questions on the constraints of plasticity. Given plasticity's key role in coping with environmental change, approaches spanning the spectrum from applied to basic will greatly enrich our understanding of the evolution of plasticity and resolve our understanding of limits.

Understanding the evolution of reaction norms remains a major challenge in ecology and evolution. Investigating evolutionary divergence in reaction norm shapes between populations and closely related species is one approach to providing insights. Here we use a meta-analytic approach to compare divergence in reaction norms of closely related species or populations of animals and plants across types of traits and environments. We quantified mean-standardized differences in overall trait means (Offset) and reaction norm shape (including both Slope and Curvature). These analyses revealed that differences in shape (Slope and Curvature together) were generally greater than differences in Offset. Additionally, differences in Curvature were generally greater than differences in Slope. The type of taxon contrast (species vs. population), trait, organism, and the type and novelty of environments all contributed to the best-fitting models, especially for Offset, Curvature, and the total differences (Total) between reaction norms. Congeneric species had greater differences in reaction norms than populations, and novel environmental conditions increased the differences in reaction norms between populations or species. These results show that evolutionary divergence of curvature is common and should be considered an important aspect of plasticity, together with slope. Biological details about traits and environments, including cryptic variation expressed in novel environmental conditions, may be critical to understanding how reaction norms evolve in novel and rapidly changing environments.

Proper functioning of complex phenotypes requires that multiple traits work together. Examination of relationships among traits within and between complex characters and how they interact to function as a whole organism is critical to advancing our understanding of evolutionary developmental plasticity. Phenotypic integration refers to the relationships among multiple characters of a complex phenotype, and their relationships with other functional units (modules) in an organism. In this review, I summarize a brief history of the concept of phenotypic integration in plant and animal biology. Following an introduction of concepts, including modularity, I use an empirical case-study approach to highlight recent advance in clarifying the developmental and genomic basis of integration. I end by highlighting some novel approaches to genomic and epigenetic perturbations that offer promise in further addressing the role of phenotypic integration in evolutionary diversification. In the age of the phenotype, studies that examine the genomic and developmental changes in relationships of traits across environments will shape the next chapter in our quest for understanding the evolution of complex characters.

Introduced species frequently show geographic differentiation, and when differentiation mirrors the ancestral range, it is often taken as evidence of adaptive evolution. The mouse-ear cress (Arabidopsis thaliana) was introduced to North America from Eurasia 150-200 years ago, providing an opportunity to study parallel adaptation in a genetic model organism. Here, we test for clinal variation in flowering time using 199 North American (NA) accessions of A. thaliana, and evaluate the contributions of major flowering time genes FRI, FLC, and PHYC as well as potential ecological mechanisms underlying differentiation. We find evidence for substantial within population genetic variation in quantitative traits and flowering time, and putatively adaptive longitudinal differentiation, despite low levels of variation at FRI, FLC, and PHYC and genome-wide reductions in population structure relative to Eurasian (EA) samples. The observed longitudinal cline in flowering time in North America is parallel to an EA cline, robust to the effects of population structure, and associated with geographic variation in winter precipitation and temperature. We detected major effects of FRI on quantitative traits associated with reproductive fitness, although the haplotype associated with higher fitness remains rare in North America. Collectively, our results suggest the evolution of parallel flowering time clines through novel genetic mechanisms.

Background and Aims: Since the early 1990s, research on genetic variation of phenotypic plasticity has expanded and empirical research has emphasized the role of the environment on the expression of inbreeding depression. An emerging question is how these two evolutionary ecology mechanisms interact in novel environments. Interest in this area has grown with the need to understand the establishment of populations in response to climate change, and to human-assisted transport to novel environments., Methods: We compare performance in the field of outcrossed (O) and inbred lines (S1, S2) from 20 maternal families from each of two native populations of Mimulus guttatus. The experiment was planted in California in each population's home site, in the other populations's home site, in a novel site within the native range of M. guttatus, and in a novel site within the non-native range in North America. The experiment included nearly 6500 individuals. Survival, sexual reproduction and above-ground biomass were examined in order to evaluate inbreeding depression, and stem diameter and plant height were examined in order to evaluate phenotypic plasticity., Key Results: Across all field sites, approx. 36 % of plants survived to flowering. Inbreeding depression differed among sites and outcrossed offspring generally outperformed selfed offspring. However, in the native-novel site, self-progeny performed better or equally well as outcross progeny. Significant phenotypic plasticity and genetic variation in plasticity was detected in the two architectural traits measured. The absolute value of plasticity showed the most marked difference between home and non-native novel site or non-native-novel site. Evidence was detected for an interaction between inbreeding and plasticity for stem diameter., Conclusions: The results demonstrate that during initial population establishment, both inbreeding depression and phenotypic plasticity vary among field sites, and may be an important response to environments outside a species' currently occupied range. However, the interaction between inbreeding and plasticity may be limited and environment-dependent.

To better understand invasion dynamics, it is essential to determine the influence of genetics and ecology in species persistence in both native and nonnative habitats. One approach is to assess patterns of selection on floral and growth traits of individuals in both habitats. Mimulus guttatus (Phrymaceae) has a mixed mating system and grows under variable water conditions across its native and nonnative range in North America. Field investigations of patterns of selection of floral and plant size traits were conducted in two native and two nonnative populations. Field-collected seed was grown and crossed in the glasshouse using a paternal half-sib design. The resulting offspring were grown in saturated and dry-down low-water conditions and the same traits were measured in both environments. Patterns of selection varied across years in the native range. Nonnative populations exhibited selection for increased floral size, consistent with the hypothesis that selection favors larger size in nonnative habitats. In the glasshouse, we detected genetic variation for traits across population/treatment combinations. However, size hierarchy in the glasshouse was dependent on water conditions. Our results suggest that both variable selection pressures and local adaptation probably influence the persistence of both native and nonnative populations.

Low Ca/Mg ratios (a defining component of serpentine soils) and low water environmental conditions often co-occur in nature and are thought to exert strong selection pressures on natural populations. However, few studies test the individual and combined effects of these environmental factors. We investigated the effects of low Ca/Mg ratio and low water availability on plant leaf, stem, stolon, and floral traits of Mimulus guttatus, a bodenvag species, i.e., a species that occurs in serpentine and non-serpentine areas. We quantified genetic variation and genetic variation for plasticity for these leaf, stem, stolon, and floral traits at three hierarchical levels: field-habitat type, population, and family, and we evaluated the relative importance of local adaptation and plasticity. We chose two populations and 10 families per population from four distinct field "habitat types" in northern California: high Ca/Mg ratio (non-serpentine) and season-long water availability, high Ca/Mg ratio and seasonally drying, low Ca/Mg ratio (serpentine) and season-long water availability, and low Ca/Mg ratio and seasonally drying. Seedlings were planted into greenhouse treatments that mimicked the four field conditions. We only detected genetic variation for stem diameter and length of longest leaf at the field-habitat level, but we detected genetic variation at the family level for nearly all traits. Soil chemistry and water availability had strong phenotypic effects, alone and in combination. Our hypothesis of an association between responses to low water levels and low Ca/Mg ratio was upheld for length of longest leaf, stem diameter, corolla width, and total number of reproductive units, whereas for other traits, responses to Ca/Mg ratio and low water were clearly independent. Our results suggest that traits may evolve independently from Ca/Mg ratios and water availability and that our focal traits were not simple alternative measures of vigor. We found genetic variation for plasticity both at the field-habitat type and family levels for half of the traits studied. Phenotypic plasticity and genetic variation for plasticity appear to be more important than local adaptation in the success of these M. guttatus populations found across a heterogeneous landscape in northern California. Phenotypic plasticity is an important mechanism maintaining the broad ecological breadth of native populations of M. guttatus.

In addition to considerable debate in the recent evolutionary literature about the limits of the Modern Synthesis of the 1930s and 1940s, there has also been theoretical and empirical interest in a variety of new and not so new concepts such as phenotypic plasticity, genetic assimilation and phenotypic accommodation. Here we consider examples of the arguments and counter-arguments that have shaped this discussion. We suggest that much of the controversy hinges on several misunderstandings, including unwarranted fears of a general attempt at overthrowing the Modern Synthesis paradigm, and some fundamental conceptual confusion about the proper roles of phenotypic plasticity and natural selection within evolutionary theory.

Thigmomorphogenesis refers to the widespread ability of sessile organisms to modify their morphology in response to a variety of mechanical stimulations, from direct contact with the stem by insects or other plants to flexure caused by wind, water, or snow. In this paper we investigated the differences in the reaction norms to wind exposure of seven species of the Brassicaceae that constitute a well-studied complex of known phylogenetic relationships. The goals included the characterization of differences between allopolyploids and their parental species and the comparison of wild and fast-cycling accessions within each species. We found statistically significant variation for plasticity among species or accessions for several characters, but the majority of the phenotypic variance was accounted for by overall (across-environment) differences among species and accessions and not by variation in plasticity. Allopolyploids displayed an array of behaviors when compared to their parents, from co-dominance to complete dominance to exceeding both parental means. Furthermore, fast-cycling plants showed distinct features from their wild relatives, suggesting that wild populations should be included with artificially selected lines in ecological studies. We proposed further steps to gain a more comprehensive understanding of thigmomorphogenetic responses, by integrating current research on the molecular bases of thigmomorphogenesis with insights into the ecology and evolution of plants exposed to wind.

The idea of genetic assimilation, that environmentally induced phenotypes may become genetically fixed and no longer require the original environmental stimulus, has had varied success through time in evolutionary biology research. Proposed by Waddington in the 1940s, it became an area of active empirical research mostly thanks to the efforts of its inventor and his collaborators. It was then attacked as of minor importance during the "hardening" of the neo-Darwinian synthesis and was relegated to a secondary role for decades. Recently, several papers have appeared, mostly independently of each other, to explore the likelihood of genetic assimilation as a biological phenomenon and its potential importance to our understanding of evolution. In this article we briefly trace the history of the concept and then discuss theoretical models that have newly employed genetic assimilation in a variety of contexts. We propose a typical scenario of evolution of genetic assimilation via an intermediate stage of phenotypic plasticity and present potential examples of the same. We also discuss a conceptual map of current and future lines of research aimed at exploring the actual relevance of genetic assimilation for evolutionary biology.

Spatial and temporal genetic structures were examined across sites on islands and mainland (continuous forest) populations of an epiphytic orchid, Catasetum viridiflavum, using 17 polymorphic allozyme loci. I tested whether patches on islands or at mainland sites comprised small local populations or a large population. Low among population differentiation was observed across the landscape suggesting that the species-specific pollinator and tiny wind-dispersed seeds maintain interconnections among distant patches. Temporal genetic structure among stage classes, and among breeding individuals are important components of the maintenance of genetic variation in this orchid. The natural history of this species including small breeding populations, probable high frequency of mating among relatives, and the high rates of seed movement among sites contribute to the high FIS. These data show that physically isolated patches in this epiphytic orchid comprise a single larger genetic population, which is independent of the physical distances among sites. Although quite different in ecological and life history characteristics, the genetic structure of this orchid demonstrates a pattern similar to temperate and tropical trees in fragmented landscapes.

Phenotypic integration is a necessary characteristic of living organisms that results from genetic, developmental, and functional relationships among traits. The nature of these relationships can be influenced by the environment. We examined patterns of phenotypic integration of six species of rapid cycling Brassica and of Raphanus sativus within a phylogenetic context. Specifically, we tested the hypothesis that hybrid species show intermediate levels of integration in morphological and life-history characters compared to their putative parentals. We used matrix correlation tests to examine if cytogenetic relationships or ecological similarities among species partially explained the patterns of phenotypic integration. There was a significant negative relationship between the ecological and cytogenetic matrices, suggesting that more closely related species were ecologically dissimilar. However, neither ecological nor cytogenetic matrices significantly explained differences among species in the pattern of their phenotypic correlations. Set correlation analysis indicated that important traits within the modules and the strength of the correlations within modules differed across species. We also found that there were a greater number of significant correlations between modules than within modules. Hybrid species were more integrated (had greater number of significant trait correlations) than either of their parents, both within and between modules. However, univariate analyses of character means of the hybrid species were not significantly different from the combined mean of their putative parents for 5, 6, or 7 of the 11 phenotypic characters (for Brassica napus, B. juncea and B. carinata, respectively); for the remaining characters, the hybrids were more similar to one of the parents.