Terpenes are a major class of secondary metabolites present in all plants, and long hypothesized to have diversified in response to specific plant-herbivore interactions. Herbivory is a major biotic interaction that plays out across broad temporal and spatial scales that vary dramatically in temperature regimes, both due to climatic variation across geographic locations as well as the effect of seasonality. In addition, there is an emerging understanding that global climate change will continue to alter the temperature regimes of nearly every habitat on Earth over the coming centuries. Regardless of source, variation in temperature may influence herbivory, in particular via changes in the efficacy and impacts of plant defensive chemistry. This study aims to characterize temperature-driven variation in toxicological effects across several structural classes of terpenes in the model herbivore Vanessa cardui , the painted lady butterfly. We observed a general increase in monoterpene toxicity to larvae, pupa, and adults at higher temperatures, as well as an increase in development time as terpene concentration increased. Results obtained from this study yield insights into possible drivers of seasonal variation in plant terpene production as well as inform effects of rising global temperatures on plant-insect interactions. In the context of other known effects of climate change on plant-herbivore interactions like carbon fertilization and compensatory feeding, temperature-driven changes in plant chemical defense efficacy may further complicate the prediction of climate change impacts on the fundamental ecological process of herbivory.

Abstract Cultivated sunflower (Helianthus annuus) is not typically considered to have a pleasant floral fragrance. In field production, seed yield is often limited by pollination services, particularly in the production of hybrid seed. Improved floral fragrance, as determined by volatile organic compounds, may be a route to improving pollinator attraction for oilseed and confectionary production and could also add value to ornamental sunflowers. Wild relatives of H. annuus have a long history of being used to breed improved traits into cultivated varieties, yet it is unknown whether favorable scents are present in wild Helianthus species and thus an available resource for fragrance breeding. In order to assess the diversity of floral fragrance available in crop wild relatives, 30 diverse accessions of wild Helianthus as well as seven varieties of H. annuus spanning a domestication gradient were grown in greenhouse experiments and variation in floral volatiles was analyzed by solid phase microextraction-gas chromatography-mass spectrometry. While alpha-pinene made up a significant portion of the volatiles emitted for most taxa, there was substantial diversity present across the genus as well as within H. annuus. Most volatiles emitted were monoterpenoids with a significant share of sesquiterpenoids. The diversity identified here will inform further targeted study of which compounds affect pollinator attraction and health. Several wild accessions such as Helianthus debilis subsp. tardiflorus and Helianthus praecox subsp. praecox as well as open-pollinated domesticated accessions of H. annuus show promise for breeding for improved floral fragrance due to high volatile abundance and likely favorable compound compositions.

Terrestrial mammals span seven orders of magnitude in body size, ranging from the <2-g Etruscan pygmy shrew (Suncus etruscus) to the >3,900-kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in functional immune defenses changes with body size across species is unknown. Here, we (1) developed a novel 12-point dilution curve approach to describe and compare antibacterial capacity against three bacterial species among >160 terrestrial species of mammals and (2) tested published predictions about the scaling of immune defenses. Our study focused on the safety factor hypothesis, which predicts that broad, early-acting immune defenses should scale hypermetrically with body mass. However, our three statistical approaches demonstrated that antibacterial activity in sera across mammals exhibits isometry; killing capacity did not change with body size across species. Intriguingly, this result indicates that the serum of a large mammal is less hospitable to bacteria than would be predicted by its metabolic rates. In other words, if metabolic rates underlie the rates of physiological reactions as postulated by the metabolic theory of ecology, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of pathogens.

C 4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations in spite of sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C 4 , but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C 4 +CAM. It has been hypothesized that C 4 +CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of P. amilis and sampled mRNA from P. amilis and P. oleracea during CAM induction. Gene co-expression network analyses identified C 4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase (PEPC) to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C 4 and CAM mostly used mutually exclusive genes for primary carbon fixation and it is likely that nocturnal CAM malate stores are shuttled into diurnal C 4 decarboxylation pathways, but we find evidence that metabolite cycling may occur at low levels. C 4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C 4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.

PremiseAs plant lineages diversify across environmental gradients, species are predicted to encounter divergent biotic pressures. This study investigated the evolution of volatile secondary metabolism across species of Helianthus. MethodsLeaves and petals of 40 species of wild Helianthus were analyzed via gas chromatography-mass spectrometry to determine volatile secondary metabolite profiles. ResultsAcross all species, 500 compounds were identified; 40% were sesquiterpenes, 18% monoterpenes, 3% diterpenes, 4% fatty acid derivatives, and 35% other compounds such as phenolics and small organic molecules. Qualitatively, annuals and species from more arid western climates had leaf compositions with a higher proportion of total monoterpenes, while erect perennials and species from more mesic eastern habitats contained a higher proportion of total sesquiterpenes. Among species, mass-based leaf monoterpene and sesquiterpene abundance were identified as largely orthogonal axes of variation by principal component analysis. Profiles for leaves were not strongly correlated with those of petals. ConclusionsVolatile metabolites were highly diverse among wild Helianthus, indicating the value of this genus as a model system and rich genetic resource. The independence of leaf and petal volatile profiles indicates a low level of phenotypic integration between vegetative and reproductive structures, implying vegetative defense and reproductive defense or pollinator attraction functions mediated by terpene profiles in these two organs can evolve without major trade-offs. The major biosynthetic pathways for the major terpenes in wild Helianthus are already well described, providing a road map to deeper inquiry into the drivers of this diversity.

Terrestrial mammals span 7 orders of magnitude in body size, ranging from the < 2 g pygmy shrew (Suncus etruscus) to the > 3900 kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in immune defenses changes with body size across species is unknown. Comparative immunology studies are often limited because of reagents or because assays are calibrated for a specific species. We developed a novel 12‐point dilution‐curve approach to compare complement‐mediated antibacterial capacity against 3 diverse bacterial species (Escherichia coli, Salmonella enterica, Micrococcus luteus) among >160 terrestrial species of mammals. We tested published predictions about the scaling of immune defenses, focusing on the Safety Factor Hypothesis, which predicts that broad, early‐acting immune defense should scale hypermetrically with body mass. Multivariate linear mixed models using Markov chain Monte Carlo techniques demonstrate that antibacterial activity across mammals exhibited isometry (scaling coefficient = 0), regardless of the bacterial species used in the challenge. For example, the antibacterial ability of a specific dilution scaled isometrically when serum was tested against E. coli(b95% Credible Intervals =‐0.144 : 0.044), S. enterica (b = ‐0.102 : 0.061), and M, luteus (b = ‐0.401 : 0.177). Therefore, body size was unrelated to killing capacity across species. Intriguingly, these results indicate that the serum of large mammals was less hospitable to bacteria than would be predicted by their metabolic rates. That is, if metabolic rates restrict the rate of organismal reactions as postulated by the Rate of Metabolism Hypothesis, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of zoonotic pathogens. This work was supported by the National Science Foundation (award numbers IOS 1656551 to C.J.D. and IOS 1656618 to L.B.M), and the authors have no competing interests to declare.

Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0–70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense.

Pathogen host range and pathogen severity are dependent on interactions with their hosts and are hypothesized to have evolved as products of a coevolutionary arms race. An understanding of the factors that affect host range and pathogen severity is especially crucial in introduced pathogens that infect evolutionarily naïve hosts and cause substantial damage to ecosystems. Powdery mildews are detrimental pathogens found worldwide in managed and natural systems. Golovinomyces latisporus is a powdery mildew species that is especially damaging to plants within Asteraceae and to plants within the genus Helianthus in particular. In this study, we evaluated 126 species within Asteraceae to measure the role of host plant morphophysiological traits and evolutionary history on susceptibility to G. latisporus and disease severity. We observed phylogenetic signal in both susceptibility and severity within and among major clades of the Asteraceae. In general, there was a major phylogenetic structure of host severity to G. latisporus; however, there was some fine-scale phylogenetic variability. Phylogenetic statistical methods showed that chlorophyll content, biomass, stomatal index, and trichome density were not associated with disease severity, thus providing evidence that phylogenetic structure, rather than observed plant morphophysiological traits, is the most reliable predictor of pathogen severity. This work sheds light on the role that evolutionary history plays in plant susceptibility and severity to disease and underscores the relative unimportance of commonly assessed host plant traits in powdery mildew severity.

Aim The road network is increasing globally but the consequences of roadkill on the viability of wildlife populations are largely unknown. We provide a framework that allows us to estimate how risk of extinction of local populations increases due to roadkill and to generate a global assessment that identifies which mammalian species are most vulnerable to roadkill and the areas where they occur. Location Global. Time period 1995-2015. Major taxa studied Terrestrial mammals. Methods We introduce a framework to quantify the effect of roadkill on terrestrial mammals worldwide that includes three steps: (a) compilation of roadkill rates to estimate the fraction of a local population killed on the roads, (b) prediction of population risk of extinction based on observed roadkill rates (for a target group of species of conservation concern and non-threatened species with high roadkill rates), and (c) global assessment of vulnerability to roadkill for 4,677 terrestrial mammalian species estimated using phylogenetic regression models that link extinction risk to demographic parameters. Results We identified four populations among the 70 species in the target group that could become extinct in 50 years if observed roadkill levels persist in the study areas: maned wolf Chrysocyon brachyurus (Brazil), little spotted cat Leopardus tigrinus (Brazil), brown hyena Hyaena brunnea (Southern Africa) and leopard Panthera pardus (North India). The global assessment revealed roadkill as an added risk for 2.7% (n = 124) terrestrial mammals, including 83 species Threatened or Near Threatened. We identified regions of concern that have species vulnerable to roadkill with high road densities in areas of South Africa, central and Southeast Asia, and the Andes. Main conclusions Our framework revealed populations of threatened species that require special attention and can be incorporated into management and planning strategies informing road managers and conservation agencies.

Premise of research. Phylogenetic comparative methods provide a powerful approach for exploring the macroevolution of plant functional traits. Such approaches can uncover trait-trait correlations through evolutionary time as well as provide evidence of the role of traits in adaptation across environmental gradients. For continuous traits, most phylogenetic comparative approaches to date employ a single trait value per species-often a mean of sampled individuals-or alternatively incorporate intraspecific variation as a distribution around such a mean. It has been known for quite some time that many of the most physiologically and ecologically important plant traits are actually highly plastic, changing dynamically across a growing season, with whole-plant development or in response to environmental conditions. Here we demonstrate one possible approach to assessing the evolution of such dynamic traits: the use of function-valued phylogenetic comparative methods. Methodology. Leaf traits were sampled across 25 taxa in the genus Cornus at six time points throughout the growing season in a common-garden context, followed by contrasting sets of alternative analyses to demonstrate the consequences of researcher decisions on study conclusions. Pivotal results. The vast majority of assessed traits exhibit substantial seasonal shifts. These shifts cause traditional macroevolutionary correlations assessed at different sampling dates to yield conflicting results. Function-valued approaches indicate that seasonal shifts in many traits are evolutionarily correlated, with implications for the origin of trait-trait trade-offs. Seasonal trait plasticity is also evolutionarily correlated with native habitat environmental gradients across Cornus. Conclusions. Because a very large number of plant functional traits are not fixed but vary dynamically over time or with environmental conditions, stronger insights into the evolution of plant functional traits can emerge when this dynamism is explicitly incorporated into phylogenetic comparative approaches. We encourage the adoption of such approaches, as well as the development of better tools for doing so.

Terrestrial mammals span 7 orders of magnitude in body size, ranging from the < 2 g pygmy shrew (Suncus etruscus) to the > 3900 kg African elephant (Loxodonta africana). Although body size has profound effects on the behavior, physiology, ecology, and evolution of animals, how body mass affects immune functions is unknown. Here, using data from >160 terrestrial species of mammals, we show that the size-scaling of antibacterial activity in serum exhibits extreme hyperallometry. Compared to small mammals, the serum of large mammals is remarkably inhospitable to bacteria. Such hypermetric scaling of immune defenses provides novel perspectives on the ecology of host-pathogen interactions, and on their co-evolutionary dynamics. These results also have implications for effectively modeling human immunity and for identifying reservoirs of zoonotic pathogens.

Premise Defense investment in plant reproductive structures is relatively understudied compared to the defense of vegetative organs. Here the evolution of chemical defenses in reproductive structures is examined in light of the optimal defense, apparency, and resource availability hypotheses within the genusCornususing a phylogenetic comparative approach in relation to phenology and native habitat environmental data. Methods Individuals representing 25Cornusspecies were tracked for reproductive phenology over a full growing season at the Arnold Arboretum of Harvard University. Floral, fruit, and leaf tissue was sampled to quantify defensive chemistry as well as fruit nutritional traits relevant to bird dispersal. Native habitat environmental characteristics were estimated using locality data from digitized herbarium records coupled with global soil and climate data sets. Results The evolution of later flowering was correlated with increased floral tannins, and the evolution of later fruiting was correlated with increased total phenolics. Leaves were found to contain the highest tannin activity, while inflorescences contained the highest total flavonoids. Multiple aspects of fruit defensive chemistry were correlated with fruit nutritional traits. Floral and fruit defensive chemistry were evolutionarily correlated with aspects of native habitat temperature, precipitation, and soil characteristics. Conclusions Results provide tentative support for the apparency hypothesis with respect to both flower and fruit phenology, while relative concentrations of secondary metabolites across organs provide mixed support for the optimal defense hypothesis. The evolution of reproductive defense with native habitat provides, at best, mixed support for the resource availability hypothesis.

Hybrid enrichment is an increasingly popular approach for obtaining hundreds of loci for phylogenetic analysis across many taxa quickly and cheaply. The genes targeted for sequencing are typically single-copy loci, which facilitate a more straightforward sequence assembly and homology assignment process. However, this approach limits the inclusion of most genes of functional interest, which often belong to multi-gene families. Here, we demonstrate the feasibility of including large gene families in hybrid enrichment protocols for phylogeny reconstruction and subsequent analyses of molecular evolution, using a new set of bait sequences designed for the "portullugo" (Caryophyllales), a moderately sized lineage of flowering plants (~ 2200 species) that includes the cacti and harbors many evolutionary transitions to C$_{\mathrm{4}}$ and CAM photosynthesis. Including multi-gene families allowed us to simultaneously infer a robust phylogeny and construct a dense sampling of sequences for a major enzyme of C$_{\mathrm{4}}$ and CAM photosynthesis, which revealed the accumulation of adaptive amino acid substitutions associated with C$_{\mathrm{4}}$ and CAM origins in particular paralogs. Our final set of matrices for phylogenetic analyses included 75-218 loci across 74 taxa, with ~ 50% matrix completeness across data sets. Phylogenetic resolution was greatly improved across the tree, at both shallow and deep levels. Concatenation and coalescent-based approaches both resolve the sister lineage of the cacti with strong support: Anacampserotaceae $+$ Portulacaceae, two lineages of mostly diminutive succulent herbs of warm, arid regions. In spite of this congruence, BUCKy concordance analyses demonstrated strong and conflicting signals across gene trees. Our results add to the growing number of examples illustrating the complexity of phylogenetic signals in genomic-scale data.

Conservation management for environmental sustainability is now ubiquitous. The ecological effects of these actions are well-intentioned and well-known. Although conservation biologists and managers increasingly incorporate evolutionary considerations into management plans, the evolutionary consequences of management strategies have remained relatively unexplored and unconsidered. But what are the evolutionary consequences? Here, we advocate a new research agenda focused on identifying, predicting, and countering the evolutionary consequences of conservation management. We showcase the examples of park creation and invasive species management, and speculate further on five other major methods of management. Park creation may cause selection for altered dispersal and behavior that utilizes human foods and structures. Management of invasive species may favor the evolution of resistance to or tolerance of control methods. In these and other cases, evolution may cause deviations from the predicted consequences of management strategies optimized without considering evolution, particularly when management results in or coincides with major environmental change, if population size change strongly, or if life histories are short enough to allow more rapid evolution. We call for research focused on: (1) experimental predictions and tests of evolution under particular management strategies, (2) widespread monitoring of managed populations and communities, and (3) meta-analysis and theoretical study aimed at simplifying the process of evolutionary prediction, particularly at systematizing a means of identifying traits likely to evolve due to likely existing genetic variance or high mutation rates. Ultimately, conservation biologists should incorporate evolutionary prediction into management planning to prevent the evolutionary domestication of the species that they are trying to protect.

PREMISE OF THE STUDY Next-generation sequencing facilitates rapid production of well-sampled phylogenies built from very large genetic data sets, which can then be subsequently exploited to examine the molecular evolution of the genes themselves. We present an evolutionary analysis of 83 gene families (19 containing carbon-concentrating mechanism (CCM) genes, 64 containing non-CCM genes) in the portullugo clade (Caryophyllales), a diverse lineage of mostly arid-adapted plants that contains multiple evolutionary origins of all known photosynthesis types in land plants (C3 , C4 , CAM, C4 -CAM, and various intermediates). METHODS We inferred a phylogeny of 197 individuals from 167 taxa using coalescent-based approaches and individual gene family trees using maximum likelihood. Positive selection analyses were conducted on individual gene family trees with a mixed effects model of evolution (MEME). We devised new indices to compare levels of convergence and prevalence of particular residues between CCM and non-CCM genes and between species with different photosynthetic pathways. KEY RESULTS Contrary to expectations, there were no significant differences in the levels of positive selection detected in CCM versus non-CCM genes. However, we documented a significantly higher level of convergent amino acid substitutions in CCM genes, especially in C4 taxa. CONCLUSIONS Our analyses reveal a new suite of amino acid residues putatively important for C4 and CAM function. We discuss both the advantages and challenges of using targeted enrichment sequence data for exploratory studies of molecular evolution.

Assessing the importance of different taxa for inferring evolutionary history is a critical, but underutilized, aspect of systematics. Quantifying the importance of all taxa within a dataset provides an empirical measurement that can establish a ranking of extant taxa for ecological study and/or quantify the relative importance of newly announced or redescribed specimens to enable the disentangling of novelty and inferential influence. Here, we illustrate the use of taxon influence indices through analysis of both molecular and morphological datasets, introducing a modified Bayesian approach to the taxon influence index that accounts for model and topological uncertainty. Quantification of taxon influence using the Bayesian approach produced clear rankings for both dataset types. Bayesian taxon rankings differed from maximum likelihood (ML)-derived rankings from a mitogenomic dataset, and the highest ranking taxa exhibited the largest interquartile range in influence estimate, suggesting variance in the estimate must be taken into account when the ranking of taxa is the feature of interest. Application of the Bayesian taxon influence index to a recent morphological analysis of the Tully Monster (Tullimonstrum) reveals that it exhibits consistently low inferential importance across two recent treatments of the taxon with alternative character codings. These results lend support to the idea that taxon influence indices may be robust to character coding and therefore effective for morphological analyses. These results underscore a need for the development of approaches to, and application of, taxon influence analyses both for the purpose of establishing robust rankings for future inquiry and for explicitly quantifying the importance of individual taxa. Quantifying the importance of individual taxa refocuses debates in morphological studies from questions of character choice/significance and taxon sampling to explicitly analytical techniques, and guides discussion of the context of new discoveries.

Summary Over the past several years, phylogenetic comparative studies have increasingly approached trait evolution in a multivariate context, with a number of taxa that continues to rise dramatically. Recent methods for phylogenetic comparative studies have provided ways to incorporate measurement error and to address computational challenges. However, missing data remain a particularly common problem, in which data are unavailable for some but not all traits of interest for a given species (or individual), leaving researchers with the choice between omitting observations or utilizing imputation‐based approaches. Here, we introduce an r implementation of PhyloPars, a tool for phylogenetic imputation of missing data and estimation of trait covariance across species (phylogenetic covariance) and within species (phenotypic covariance). Rphylopars provides expanded capabilities over the original PhyloPars interface including a fast linear‐time algorithm, thus allowing for extremely large data sets (which were previously computationally infeasible) to be analysed in seconds or minutes rather than hours. In addition to providing fast and computationally efficient implementations, we introduce in Rphylopars methods to estimate macroevolutionary parameters under alternative evolutionary models (e.g. Early‐Burst, multivariate Ornstein‐Uhlenbeck). By providing fast and computationally efficient methods with flexible options for various phylogenetic comparative approaches, Rphylopars expands the possibilities for researchers to analyse large and complex data with missing observations, within‐species variation and deviations from Brownian motion.

AbstractAncestral state reconstruction is a method used to study the evolutionary trajectories of quantitative characters on phylogenies. Although efficient methods for univariate ancestral state reconstruction under a Brownian motion model have been described for at least 25 years, to date no generalization has been described to allow more complex evolutionary models, such as multivariate trait evolution, non‐Brownian models, missing data, and within‐species variation. Furthermore, even for simple univariate Brownian motion models, most phylogenetic comparative R packages compute ancestral states via inefficient tree rerooting and full tree traversals at each tree node, making ancestral state reconstruction extremely time‐consuming for large phylogenies. Here, a computationally efficient method for fast maximum likelihood ancestral state reconstruction of continuous characters is described. The algorithm has linear complexity relative to the number of species and outperforms the fastest existing R implementations by several orders of magnitude. The described algorithm is capable of performing ancestral state reconstruction on a 1,000,000‐species phylogeny in fewer than 2 s using a standard laptop, whereas the next fastest R implementation would take several days to complete. The method is generalizable to more complex evolutionary models, such as phylogenetic regression, within‐species variation, non‐Brownian evolutionary models, and multivariate trait evolution. Because this method enables fast repeated computations on phylogenies of virtually any size, implementation of the described algorithm can drastically alleviate the computational burden of many otherwise prohibitively time‐consuming tasks requiring reconstruction of ancestral states, such as phylogenetic imputation of missing data, bootstrapping procedures, Expectation‐Maximization algorithms, and Bayesian estimation. The described ancestral state reconstruction algorithm is implemented in the Rphylopars functions anc.recon and phylopars.