Showing papers in "American Journal of Botany in 2019"

Tracking temporal shifts in area, biomes, and pollinators in the radiation of Salvia (sages) across continents: leveraging anchored hybrid enrichment and targeted sequence data.

Abstract: Premise of the study A key question in evolutionary biology is why some clades are more successful by being widespread geographically, biome diverse, or species-rich. To extend understanding of how shifts in area, biomes, and pollinators impact diversification in plants, we examined the relationships of these shifts to diversification across the mega-genus Salvia. Methods A chronogram was developed from a supermatrix of anchored hybrid enrichment genomic data and targeted sequence data for over 500 of the nearly 1000 Salvia species. Ancestral areas and biomes were reconstructed using BioGeoBEARS. Pollinator guilds were scored, ancestral pollinators determined, shifts in pollinator guilds identified, and rates of pollinator switches compared. Key results A well-resolved phylogenetic backbone of Salvia and updated subgeneric designations are presented. Salvia originated in Southwest Asia in the Oligocene and subsequently dispersed worldwide. Biome shifts are frequent from a likely ancestral lineage utilizing broadleaf and/or coniferous forests and/or arid shrublands. None of the four species diversification shifts are correlated to shifts in biomes. Shifts in pollination system are not correlated to species diversification shifts, except for one hummingbird shift that precedes a major shift in diversification near the crown of New World subgen. Calosphace. Multiple reversals back to bee pollination occurred within this hummingbird clade. Conclusions Salvia diversified extensively in different continents, biomes, and with both bee and bird pollinators. The lack of tight correlation of area, biome, and most pollinator shifts to the four documented species diversification shifts points to other important drivers of speciation in Salvia.

What makes a weed a weed? A large- scale evaluation of arable weeds through a functional lens

Abstract: PREMISE OF THE STUDY: Despite long-term research efforts, a comprehensive perspective on the ecological and functional properties determining plant weediness is still lacking. We investigated here key functional attributes of arable weeds compared to non-weed plants, at large spatial scale. METHODS: We used an intensive survey of plant communities in cultivated and non-cultivated habitats to define a pool of plants occurring in arable fields (weeds) and one of plants occurring only in open non-arable habitats (non-weeds) in France. We compared the two pools based on nine functional traits and three functional spaces (LHS, reproductive and resource requirement hypervolumes). Within the weed pool, we quantified the trait variation of weeds along a continuum of specialization to arable fields. KEY RESULTS: Weeds were mostly therophytes and had higher specific leaf area, earlier and longer flowering, and higher affinity for nutrient-rich, sunny and dry environments compared to non-weeds, although functional spaces of weeds and non-weeds largely overlapped. When fidelity to arable fields increased, the spectrum of weed ecological strategies decreased as did the overlap with non-weeds, especially for the resource requirement hypervolume. CONCLUSIONS: Arable weeds constitute a delimited pool defined by a trait syndrome providing tolerance to the ecological filters of arable fields (notably, regular soil disturbances and fertilization). The identification of such a syndrome is of great interest to predict the weedy potential of newly established alien plants. An important reservoir of plants may also become weeds after changes in agricultural practices, considering the large overlap between weeds and non-weeds.

Variation in the transcriptome of different ecotypes of Arabidopsis thaliana reveals signatures of oxidative stress in plant responses to spaceflight.

Abstract: Premise of the study Spaceflight provides a unique environment in which to dissect plant stress response behaviors and to reveal potentially novel pathways triggered in space. We therefore analyzed the transcriptomes of Arabidopsis thaliana plants grown on board the International Space Station to find the molecular fingerprints of these space-related response networks. Methods Four ecotypes (Col-0, Ws-2, Ler-0 and Cvi-0) were grown on orbit and then their patterns of transcript abundance compared to ground-based controls using RNA sequencing. Key results Transcripts from heat-shock proteins were upregulated in all ecotypes in spaceflight, whereas peroxidase transcripts were downregulated. Among the shared and ecotype-specific changes, gene classes related to oxidative stress and hypoxia were detected. These spaceflight transcriptional response signatures could be partly mimicked on Earth by a low oxygen environment and more fully by oxidative stress (H2 O2 ) treatments. Conclusions These results suggest that the spaceflight environment is associated with oxidative stress potentially triggered, in part, by hypoxic response. Further, a shared spaceflight response may be through the induction of molecular chaperones (such as heat shock proteins) that help protect cellular machinery from the effects of oxidative damage. In addition, this research emphasizes the importance of considering the effects of natural variation when designing and interpreting changes associated with spaceflight experiments.

RNA-seq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity.

Abstract: Premise Plants synthesize information from multiple environmental stimuli when determining their direction of growth. Gravity, being ubiquitous on Earth, plays a major role in determining the direction of growth and overall architecture of the plant. Here, we utilized the microgravity environment on board the International Space Station (ISS) to identify genes involved influencing growth and development of phototropically stimulated seedlings of Arabidopsis thaliana. Methods Seedlings were grown on the ISS, and RNA was extracted from 7 samples (pools of 10-15 plants) grown in microgravity (μg) or Earth gravity conditions (1-g). Transcriptomic analyses via RNA sequencing (RNA-seq) of differential gene expression was performed using the HISAT2-Stringtie-DESeq2 RNASeq pipeline. Differentially expressed genes were further characterized by using Pathway Analysis and enrichment for Gene Ontology classifications. Results For 296 genes that were found significantly differentially expressed between plants in microgravity compared to 1-g controls, Pathway Analysis identified eight molecular pathways that were significantly affected by reduced gravity conditions. Specifically, light-associated pathways (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) were significantly downregulated in microgravity. Conclusions Gene expression in A. thaliana seedlings grown in microgravity was significantly altered compared to that of the 1-g control. Understanding how plants grow in conditions of microgravity not only aids in our understanding of how plants grow and respond to the environment but will also help to efficiently grow plants during long-range space missions.

Autopolyploid lineage shows climatic niche expansion but not divergence in Arabidopsis arenosa

Abstract: PREMISE OF THE STUDY Successful establishment of neopolyploids, and therefore polyploid speciation, is thought to be contingent on environmental niche shifts from their progenitors. We explore this niche shift hypothesis in the obligate outcrosser Arabidopsis arenosa complex, which includes diploid and recently formed autotetraploid populations. METHODS To characterize the climatic niches for both cytotypes in Arabidopsis arenosa, we first gathered climatic data from localities with known ploidy types. We then estimated the climatic niches for diploids and autotetraploids and calculated niche overlap. Using this niche overlap statistic, we tested for niche equivalency and similarity. We explored differences in niches by estimating and comparing niche optimum and breadth and then calculated indices of niche expansion and unfilling. KEY RESULTS Climatic niche overlap between diploids and autotetraploids is substantial. Although the two niche models are not significantly divergent, they are not identical as they differ in both optimum and breadth along two environmental gradients. Autotetraploids fill nearly the entire niche space of diploids and have expanded into novel environments. CONCLUSIONS We find climatic niche expansion but not divergence, together with a moderate change in the niche optimum, in the autotetraploid lineage of Arabidopsis arenosa. These results indicate that the climatic niche shift hypothesis alone cannot explain the coexistence of tetraploid and diploid cytotypes.

The monocotyledonous underground: global climatic and phylogenetic patterns of geophyte diversity.

Abstract: Premise Geophytes-plants that typically possess a bulb, corm, tuber, and/or rhizome-have long captured the attention of hobbyists and researchers. However, despite the economic and evolutionary importance of these traits, the potential drivers of their morphological diversity remain unknown. Using a comprehensive phylogeny of monocots, we test for correlations between climate and geophyte growth form to better understand why we observe such a diversity of underground traits in geophytes. Understanding the evolutionary factors promoting independent origins of these potentially adaptive organs will lend insights into how plants adapt to environmental hardships. Methods Using a comprehensive phylogeny incorporated with global occurrence and climate data for the monocots, we investigated whether climatic patterns could explain differences between geophytes and non-geophytes, as well as differences among bulbous, cormous, tuberous, rhizomatous, and non-geophytic taxa. We used phylogenetically-informed ANOVAs, MANOVAs, and PCAs to test differences in climatic variables between the different growth forms. Results Geophytes inhabit cooler, drier, and more thermally variable climates compared to non-geophytes. Although some underground traits (i.e., bulb, corm, and tuber) appear to inhabit particular niches, a result supported by strong phylogenetic signal, our data has limited evidence for an overall role of climate in the evolution of these traits. However, temperature may be a driving force in rhizome evolution, as well as the evolution of taxa which we considered here as non-geophytic (e.g., trees, epiphytes, etc.). Conclusions While precipitation patterns have played a role in the evolution of geophytism, our results suggest that temperature should be more strongly considered as a contributing factor promoting the evolution of belowground bud placement, specifically in rhizomatous and non-geophytic taxa. Bulbous, cormous, and tuberous taxa need closer examination of other mechanisms, such as anatomical constraints or genetic controls, in order to begin to understand the causes behind the evolution of their underground morphology.

Phylogenomics in Cactaceae: A case study using the chollas sensu lato (Cylindropuntieae, Opuntioideae) reveals a common pattern out of the Chihuahuan and Sonoran deserts.

Abstract: Premise Although numerous phylogenetic studies have been conducted in Cactaceae, whole-plastome datasets have not been employed. We used the chollas to develop a plastome dataset for phylogeny reconstruction to test species relationships, biogeography, clade age, and morphological evolution. Methods We developed a plastome dataset for most known diploid members of the chollas (42 taxa) as well as for other members of Cylindropuntieae. Paired-end, raw reads from genome skimming were reference-mapped onto a de novo plastome assembly of one species of cholla, Cylindropuntia bigelovii, and were used to build our plastome dataset, which was analyzed using various methods. Results Our plastome dataset resolved the phylogeny of the chollas, including most interspecific and intraspecific relationships. Tribe Cylindropuntieae arose ~18 mya, during the early Miocene in southern South America, and is supported as sister to the South American clade Tephrocacteae. The (Micropuntia (Cylindropuntia + Grusonia)) clade most likely originated in the Chihuahuan Desert region around 16 mya and then migrated into other North American desert regions. Key morphological characters for recognizing traditional taxonomic series in Cylindropuntia (e.g., spiny fruit) are mostly homoplasious. Conclusions This study provides the first comprehensive plastome phylogeny for any clade within Cactaceae. Although the chollas s.l. are widespread throughout western North American deserts, their most recent common ancestor likely arose in the Chihuahuan Desert region during the mid-Miocene, with much of their species diversity arising in the early to mid-Pliocene, a pattern strikingly similar to those found in other western North American desert groups.

Seed freeze sensitivity and ex situ longevity of 295 species in the native Hawaiian flora.

Abstract: Premise Ex situ seed banking is critical for plant conservation globally, especially for threatened floras in tropical ecosystems like Hawai'i. Seed bank managers must maximize longevity, and species managers must plan restoration before seeds lose viability. Previous observations suggested some native Hawaiian seeds lost viability in frozen storage (-18°C). We investigated seed storage behavior in the Hawaiian flora to optimize storage conditions and recommend re-collection intervals (RCI) to maximize viability of stored seeds. Methods Using 20+ years of real-time seed storage viability data, we tested freeze sensitivity for 197 species and calculated RCIs for 295 species. Using paired tests of accessions stored >2 yr at 5°C and -18°C, we developed an index of relative performance to determine freeze sensitivity. We calculated RCIs at 70% of highest germination (P70). Results We identified four families (Campanulaceae, Cyperaceae, Rubiaceae, and Urticaceae) and four genera with seed freeze sensitivity and six additional genera with likely freeze sensitivity. Storage longevity was variable, but 195 species had viability >70% at the most recent tests (1 to 20+ yr), 123 species had RCIs >10 yr, and 45 species had RCIs Conclusions Freeze sensitive storage behavior is more widely observed in Hawai'i than any other regional flora, perhaps due to insufficient testing elsewhere. We present a new protocol to test seed freeze sensitivity, which is often not evident until 2-5 years of storage. Re-collection intervals will guide restoration practices in Hawai'i, and results inform seed conservation efforts globally, especially tropical and subtropical regions.

Rapid changes in eastern Himalayan alpine flora with climate change.

Abstract: Premise of the study With biodiversity and rates of climate change among the highest, the eastern Himalaya are critical for understanding the interaction of these two variables. However, there is a dearth of longitudinal data sets that address the effects of climate change on the exceptional alpine biodiversity of the Himalaya. Methods We established permanent alpine vegetation monitoring plots in three mountain chains of the Hengduan Mountains, the easternmost Himalaya, which have warmed 0.03-0.05°C yr-1 since 1985. Recently, we resampled plots (176 1-m2 quadrat plots and 88 sections of 11 summits in three Hengduan mountain chains) to measure changes in vegetation after 7 years. Key results Over 7 years, Tibetan alpine vegetation increased in number of species (+8 species/summit; +2.3 species/m2 ), in frequency (+47.8 plants/m2 ), and in diversity (+1.6 effective species/m2 ). Stepwise regressions indicated that warmer temperatures, southerly aspects, and higher elevations were associated with greater increases in these vegetation metrics. Unexpectedly, Himalayan endemic species increased (+1.4 species/m2 ; +8.5 plants/m2 ), especially on higher-elevation summits. In contrast, the increase in relative abundance of non-alpine species was greater at lower-elevation summits. Plants used by local Tibetans also increased (+1.3 species/m2 ; +32 plants/m2 ). Conclusions As in other alpine areas, biodiversity is increasing with climate change in the Himalaya. Unlike other areas, endemic species are proliferating at the highest summits and are indicators of change.

A taxonomically broad metagenomic survey of 339 species spanning 57 families suggests cystobasidiomycete yeasts are not ubiquitous across all lichens.

Abstract: PREMISE Lichens are fungi that enter into obligate symbioses with photosynthesizing organisms (algae, cyanobacteria). Traditional narratives of lichens as binary symbiont pairs have given way to their recognition as dynamic metacommunities. Basidiomycete yeasts, particularly of the genus Cyphobasidium, have been inferred to be widespread and important components of lichen metacommunities. Yet, the presence of basidiomycete yeasts across a wide diversity of lichen lineages has not previously been tested. METHODS We searched for lichen-associated cystobasidiomycete yeasts in newly generated metagenomic data from 413 samples of 339 lichen species spanning 57 families and 25 orders. The data set was generated as part of a large-scale project to study lichen biodiversity gradients in the southern Appalachian Mountains Biodiversity Hotspot of southeastern North America. RESULTS Our efforts detected cystobasidiomycete yeasts in nine taxa (Bryoria nadvornikiana, Heterodermia leucomelos, Lecidea roseotincta, Opegrapha vulgata, Parmotrema hypotropum, P. subsumptum, Usnea cornuta, U. strigosa, and U. subgracilis), representing 2.7% of all species sampled. Seven of these taxa (78%) are foliose (leaf-like) or fruticose (shrubby) lichens that belong to families where basidiomycete yeasts have been previously detected. In several of the nine cases, cystobasidiomycete rDNA coverage was comparable to, or greater than, that of the primary lichen fungus single-copy nuclear genomic rDNA, suggesting sampling artifacts are unlikely to account for our results. CONCLUSIONS Studies from diverse areas of the natural sciences have led to the need to reconceptualize lichens as dynamic metacommunities. However, our failure to detect cystobasidiomycetes in 97.3% (330 species) of the sampled species suggests that basidiomycete yeasts are not ubiquitous in lichens.

Repeated evolution of a morphological novelty: a phylogenetic analysis of the inflated fruiting calyx in the Physalideae tribe (Solanaceae)

Abstract: PREMISE OF THE STUDY The evolution of novel fruit morphologies has been integral to the success of angiosperms. The inflated fruiting calyx, in which the balloon-like calyx swells to completely surround the fruit, has evolved repeatedly across angiosperms and is postulated to aid in protection and dispersal. We investigated the evolution of this trait in the tomatillos and their allies (Physalideae, Solanaceae). METHODS The Physalideae phylogeny was estimated using four regions (ITS, LEAFY, trnL-F, waxy) with maximum likelihood (ML) and Bayesian inference. Under the best-fitting ML model of trait evolution, we estimated ancestral states along with the numbers of gains and losses of fruiting calyx accrescence and inflation with Bayesian stochastic mapping. Also, phylogenetic signal in calyx morphology was examined with two metrics (parsimony score and Fritz and Purvis's D). KEY RESULTS Based on our well-resolved and densely sampled phylogeny, we infer that calyx evolution has proceeded in a stepwise and directional fashion, from non-accrescent to accrescent to inflated. In total, we inferred 24 gains of accrescence, 24 subsequent transitions to a fully inflated calyx, and only two reversals. Despite this lability, fruiting calyx accrescence and inflation showed strong phylogenetic signal. CONCLUSIONS Our phylogeny greatly improves the resolution of Physalideae and highlights the need for taxonomic work. The comparative analyses reveal that the inflated fruiting calyx has evolved many times and that the trajectory toward this phenotype is generally stepwise and irreversible. These results provide a strong foundation for studying the genetic and developmental mechanisms responsible for the repeated origins of this charismatic fruit trait.

Adaptation and divergence in edaphic specialists and generalists: serpentine soil endemics in the California flora occur in barer serpentine habitats with lower soil calcium levels than serpentine tolerators.

Abstract: PREMISE Adaptation to harsh edaphic substrates has repeatedly led to the evolution of edaphic specialists and generalists. Yet, it is unclear what factors promote specialization versus generalization. Here, we search for habitat use patterns associated with serpentine endemics (specialists) and serpentine tolerators (generalists) to indirectly test the hypothesis that trade-offs associated with serpentine adaptation promote specialization. We predict that (1) endemics have adapted to chemically harsher and more bare serpentine habitats than tolerators, and (2) edaphic endemics show more habitat divergence from their sister species than tolerators do among on- and off-serpentine populations. METHODS We selected 8 serpentine endemic and 9 serpentine tolerator species representing independent adaptation to serpentine. We characterized soil chemistry and microhabitat bareness from one serpentine taxon of each species and from a paired nonserpentine sister taxon, resulting in 8 endemic and 9 tolerator sister-taxa pairs. RESULTS We find endemic serpentine taxa occur in serpentine habitats averaging twice as much bare ground as tolerator serpentine taxa and 25% less soil calcium, a limiting macronutrient in serpentine soils. We do not find strong evidence that habitat divergence between sister taxa of endemic pairs is greater than between sister taxa of tolerator pairs. CONCLUSIONS These results suggest serpentine endemism is associated with adaptation to chemically harsher and more bare serpentine habitats. It may be that this adaptation trades off with competitive ability, which would support the longstanding, but rarely tested, competitive trade-off hypothesis.

Fire cues trigger germination and stimulate seedling growth of grass species from Brazilian savannas

Abstract: Premise Although fire cues (high temperatures and smoke) influence seed germination in numerous species from fire-prone environments, their effects on seed germination of species from neotropical savannas are poorly understood. Methods We exposed seeds of eight grass species from the Cerrado, the Brazilian savanna to heat-shock (80°C or 110°C for 5 min) and/or smoke water, and then set them to germinate in light or dark, at either summer (28°C/18°C) or winter (27°C/14°C) temperature regimes in an incubator. In addition, we evaluated the effects of smoke water on seedling root and shoot growth for four of the species. Results Smoke interacted with the dark treatment to increase germination from 28% to 93% in Aristida recurvata and 77% to 95% in Aristida riparia. Smoke had no effect on germination of either of these species in the light. Heat-shock alone also promoted seed germination in A. recurvata. For Digitaria lehmanniana, smoke interacted with heat-shock to improve germination from 5% to 16%. In contrast, the fire treatments did not have any effect on the seed germination of the remaining five species. Smoke water stimulated root growth for A. riparia, A. recurvata, and Ctenium cirrosum but had no effect on their shoot growth. Conclusions The strong promotive effect of smoke on Aristida germination suggests that these species are fire-adapted. Aristida species have an active awn system, which facilitates seed burial, and the smoke and dark interaction would ensure buried seeds germinated post-fire. The species that showed no response to fire cues may either have adapted via alternative strategies or require different concentrations of smoke or levels of heat. This study is one of very few examples showing a positive germination and seedling growth response to smoke for species from neotropical savannas.

Phylogeography of a widespread eastern North American shrub, Viburnum lantanoides.

Abstract: Premise of the study There have been relatively few phylogeographic studies of eastern North American plants, especially of animal-dispersed shrubby species, and this leaves a significant gap in our understanding of how such species were affected by glacial events. Here, we analyzed the phylogeography of the widespread understory shrub Viburnum lantanoides. Methods We generated RADseq data and paleoclimatic species distribution models (SDMs) to identify the locations of refugia where V. lantanoides may have survived the Last Glacial Maximum (LGM) and how its range expanded as glaciers receded. Key results Genetic diversity falls off with increasing latitude and longitude, indicating that range expansion likely occurred via serial founder events from southern source populations. Samples from the southern Appalachians form a grade, while those from the north form a clade, suggesting that a single genetic lineage recolonized the north. SDMs indicate that V. lantanoides probably survived the LGM in refugia on the mid-Atlantic Coastal Plain and/or the interior Gulf Coastal Plain. Conclusions Our analyses indicate that V. lantanoides survived the LGM in refugia south of the glacier but north of the extensive refugium along the Gulf Coast. Following the LGM, a single population expanded northward along the Appalachian Mountains and eventually into eastern Canada. The patterns observed here suggest that range expansion occurred in a stepwise manner, similar to postglacial dynamics observed in a number of European plant species.

A meta-analysis of whole genome duplication and the effects on flowering traits in plants

Abstract: Premise of the study Polyploidy, or whole genome duplication (WGD), is common in plants despite theory suggesting that polyploid establishment is challenging and polyploids should be evolutionarily transitory. There is renewed interest in understanding the mechanisms that could facilitate polyploid establishment and explain their pervasiveness in nature. In particular, premating isolation from their diploid progenitors is suggested to be a crucial factor. To evaluate how changes in assortative mating occur, we need to understand the phenotypic effects of WGD on reproductive traits. Methods We used literature surveys and a meta-analysis to assess how WGD affects floral morphology, flowering phenology, and reproductive output in plants. We focused specifically on comparisons of newly generated polyploids (neopolyploids) and their parents to mitigate potential confounding effects of adaptation and drift that may be present in ancient polyploids. Key results The results indicated that across a broad representation of angiosperms, floral morphology traits increased in size, reproductive output decreased, and flowering phenology was unaffected by WGD. Additionally, we found that increased trait variation after WGD was uncommon for the phenotypic traits examined. Conclusions Our results suggest that the phenotypic effects on traits important to premating isolation of neopolyploids are small, in general. Changes in flowering phenology, reproductive output, and phenotypic variation resulting from WGD may be less critical in facilitating premating isolation and neopolyploid establishment. However, floral traits for which size is an important component of function (e.g., pollen transfer) could be strongly influenced by WGD.

Inflorescence evolution in Santalales: integrating morphological characters and molecular phylogenetics.

Abstract: PREMISE OF THE STUDY The sandalwood order (Santalales) includes members that present a diverse array of inflorescence types, some of which are unique among angiosperms. This diversity presents not only interpretational challenges but also opportunities to test fundamental concepts in plant morphology. Here we used modern phylogenetic approaches to address the evolution of inflorescences in the sandalwood order. METHODS Phylogenetic analyses of two nuclear and three chloroplast genes were conducted on representatives of 146 of the 163 genera in the order. A matrix was constructed that scored nine characters dealing with inflorescences. One character, "trios", that encompasses any grouping of three flowers (i.e., both dichasia and triads) was optimized on samples of the posterior distribution of trees from the Bayesian analysis using BayesTraits. Three nodes were examined: the most recent common ancestors of (A) all ingroup members, (B) Loranthaceae, and (C) Opiliaceae, Santalaceae s.l., and Viscaceae. KEY RESULTS The phylogenetic analysis resulted in many fully resolved nodes across Santalales with strong support for 18 clades previously named as families. The trios character was not supported for nodes A and C, whereas it was supported for node B where this partial inflorescence type is best described as a triad. CONCLUSIONS Essentially every major inflorescence type can be found in Santalales; however, the dichasium, a type of partial inflorescence, is rarely seen and is not plesiomorphic for the order. In the family Erythropalaceae, inflorescences are mostly in small, axillary fascicles or cymes. Successive families show both cymose and racemose types and compound systems (e.g., thyrses). Inflorescences in Amphorogynaceae and Viscaceae are not dichasial and in general are difficult to compare to "standard" inflorescences.

Differences in flowering time maintain species boundaries in a continental radiation of Viburnum

Abstract: Premise We take an integrative approach in assessing how introgression and Pleistocene climate fluctuations have shaped the diversification of the core Lentago clade of Viburnum, a group of five interfertile species with broad areas of sympatry. We specifically tested whether flowering time plays a role in maintaining species isolation. Methods RAD-seq data for 103 individuals were used to infer the species relationships and the genetic structure within each species. Flowering times were compared among species on the basis of historical flowering dates documented by herbarium specimens. Results Within each species, we found a strong relationship between flowering date and latitude, such that southern populations flower earlier than northern ones. In areas of sympatry, the species flower in sequence rather than simultaneously, with flowering dates offset by ≥9 d for all species pairs. In two cases it appears that the offset in flowering times is an incidental consequence of adaptation to differing climates, but in the recently diverged sister species V. prunifolium and V. rufidulum, we find evidence that reinforcement led to reproductive character displacement. Long-term trends suggest that the two northern-most species are flowering earlier in response to recent climate change. Conclusions We argue that speciation in the Lentago clade has primarily occurred through ecological divergence of allopatric populations, but differences in flowering time were essential to maintain separation of incipient species when they came into secondary contact. This combination of factors may underlie diversification in many other plant clades.

Model clades are vital for comparative biology, and ascertainment bias is not a problem in practice: a response to Beaulieu and O'Meara (2018).

Abstract: In several previous publications we have questioned the significance of largescale phylogenetic studies, highlighting instead the value of analyzing smaller, comprehensively sampled clades (Donoghue and Edwards, 2014; Edwards et al., 2015, 2017). In the recent issue of the American Journal of Botany on the Plant Tree of Life (2018, vol. 105, issue 3), Beaulieu and O’Meara (2018) explored an opposing concern, namely, that the study of selected small clades could lead to incorrect conclusions about evolutionary processes. Specifically, they performed a series of simulations designed to test whether studies of smaller clades present an “ascertainment bias”. The core idea is that comparative biologists tend to study clades that show variation in a trait of interest and, correspondingly, fail to focus sufficient attention on clades that are not so variable in that trait. Consequently, this form of taxonomic sampling could result in a biased inference about the rate of change in the trait of interest across the larger clade that includes both variable and nonvariable subclades. Surprisingly, their simulations showed that this was not generally the case. In fact, averaging across many smaller, variable subclades, there turned out not to be a significant deviation from the rates that were estimated for a more inclusive clade based on a random sample of a similar number of species. However, they did show that sampled subclades exhibited much higher variance around the estimated transition rates for the more inclusive clade. Beaulieu and O’Meara (2018) concluded that ascertainment bias is a major problem in comparative biology and argued that large, global phylogenetic analyses are needed to overcome this bias. In the same special issue, Folk et al. (2018) concurred with their assessment of the “risks” of relying on smaller studies. Many details of the Beaulieu and O’Meara study are worthy of careful examination, but here we reflect solely on the central conceptual issue that they raised for comparative biologists. Our main concern is that they implicitly misrepresent the motivation behind the study of smaller variable clades. As reflected in their simulations, their premise appears to be that we study small tractable subclades (e.g., Viburnum) to estimate transition probabilities for a more inclusive clade (e.g., Campanulidae). But this is typically not the goal at all. That is, most comparative biologists are not studying a particular subclade to generate such an estimate for a more inclusive clade. And, indeed, it would be a big risk to extend the inference for a subclade to more inclusive clades, not only because the sample of species is typically much smaller, but also because, as Beaulieu and O’Meara correctly pointed out, our estimate might then be highly biased by the particular subclade in question. If we were, in fact, attempting to estimate rates for a more inclusive clade, there would be an ascertainment bias problem. However, because those who study smaller clades typically are not intending to extend such estimates beyond the clade of interest, ascertainment bias, as described by Beaulieu and O’Meara, is simply not an issue in practice. One might then ask: Why bother studying a smaller variable clade if one is not interested in estimating more global transition rates (or speciation rates, or extinction rates, etc.)? Asked another way: What good are such targeted studies if not to render broader INVITED SPECIAL ARTICLE: COMMENTARY

Divergent gene expression levels between diploid and autotetraploid Tolmiea relative to the total transcriptome, the cell, and biomass

Abstract: PREMISE OF THE STUDY Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance. METHODS We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass. KEY RESULTS In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered, respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size. CONCLUSIONS While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.

Patterns and effects of heterospecific pollen transfer between an invasive and two native plant species: the importance of pollen arrival time to the stigma

Abstract: Premise Invasive plant species can integrate into native plant-pollinator communities, but the underlying mechanisms are poorly understood. Competitive interactions between invasive and native plants via heterospecific pollen (HP) and differential invasive HP effects depending on HP arrival time to the stigma may mediate invasion success, but these have been little studied. Methods We evaluated patterns and effects of HP receipt on pollen tube growth in two native and one invasive species in the field. We also used hand-pollination experiments to evaluate the effect of invasive HP pollen and its arrival time on native reproductive success. Results Native species receive smaller and less-diverse HP loads (5-7 species) compared to invasive species (10 species). The load size of HP had a negative effect on the proportion of pollen tubes in both native species but not in the invasive, suggesting higher HP tolerance in the latter. Invasive HP arrival time differentially affected pollen tube success in native species. Conclusions Our results highlight the need to study reciprocal HP effects between invasive and native species and the factors that determine differential responses to HP receipt to fully understand the mechanisms facilitating invasive species integration into native plant-pollinator communities.

Epigenetic variation for agronomic improvement: an opportunity for vegetatively propagated crops

Abstract: Crop breeding has been historically associated with genetic variation because genetic variants harboring traits with agronomic value can be artificially generated (e.g., hybrid crosses) and selected at the population (e.g., mass selection) or individual level (e.g., single seed descent) through classical crop breeding methods. Crop genetic improvement, however, has become increasingly difficult, and crop genetic diversity has been declining at an alarming rate, threatening world food security and sustainable development (Esquinas‐Alcázar, 2005). The stakes are high to find solutions. Cross breeding with closely related wild species, genomic selection informed by molecular markers and creation of genetically modified organisms are examples of ongoing research developments for crop improvement. Another potential research approach that is currently arising is epigenetics (Kenchanmane Raju and Niederhuth, 2018). Epigenetic variants in crops might represent an additional and timely resource for crop breeding. In this essay, we do not aim to comprehensively review the potential interest of epigenetics in agriculture but to outline the nature of some possible drawbacks, such as the instability of epigenetic marks, and opportunities, for example, clonal multiplication, associated with this potential. EPIGENETIC VARIANTS OF INTEREST FOR CROP BREEDING: WHERE ARE WE NOW?

A functional decomposition of sex inconstancy in the dioecious, colonizing plant Mercurialis annua.

Abstract: Plants with separate sexes often show "inconstant" or "leaky" sex expression, with females or males producing a few flowers of the opposite sex. The frequency and degree of such inconstancy may reflect residual hermaphroditic sex allocation after an evolutionary transition from combined to separate sexes. Sex inconstancy also represents a possible first step in the breakdown of dioecy back to hermaphroditism. In the Mercurialis annua (Euphorbiaceae) species complex, monoecy and androdioecy have evolved from dioecy in polyploid populations. Here, we characterize patterns of sex inconstancy in dioecious M. annua and discuss how sex inconstancy may have contributed to the breakdown of separate sexes in the genus. We measured sex inconstancy in three common gardens of M. annua over 2 years using a modification of Lloyd's phenotypic gender in terms of frequency and degree, with the degree calibrating inconstancy against the sex allocation of constant males and constant females, yielding a measure of gender that does not depend on the distribution of gender in the population. Unusually for dioecious plants, the frequency of sex inconstancy in M. annua was greater in females, but its degree was greater for males in the 2 years of study. We suggest that this pattern is consistent with the maintenance of inconstancy in dioecious M. annua by selection for reproductive assurance under mate limitation. Our study illustrates the utility of decomposing measures of sex inconstancy into its frequency and its degree and throws new light on the origin of variation in sexual systems in Mercurialis.

Anatomy solves the puzzle of explosive pollen release in wind-pollinated urticalean rosids

Abstract: Premise of the study This study details the unusual synorganization of the staminate flower in wind-pollinated urticalean rosids to add the missing pieces that complete the puzzle of the explosive mechanism of pollen release in this group. Methods Flower buds and flowers were analyzed using light and scanning electron microscopy. Key results The pistillode, stamens, and sepals form a floral apparatus that explosively releases pollen to be carried by the wind. The anthers dehisce when the stamens are still inflexed on the floral bud and are enveloped by the sepals and supported by an inflated pistillode. The distension of the filaments presses the pistillode, which decreases the pressure exerted on the anthers by releasing the air accumulated internally through its apical orifice. The extended filaments and the dehiscent free anthers move rapidly outward from the center of the flower. This movement of the filaments is then blocked by the robust basally united sepals, which causes a rapid inversion of the anther position, thus hurling the pollen grains far from the flower. The pollen grains are released grouped by the mucilage produced in high quantity in the cells found in all floral organs. Conclusions The anatomical structure of the pistillode and the finding of mucilaginous cells are the main features that help in the understanding the explosive mechanism of pollen release in urticalean rosids. The pistillode can be considered an exaptation because it was evolved later to provide a new role in the plant, optimizing male fitness.

Varieties of the highly dispersible and hypervariable tree, Metrosideros polymorpha, differ in response to mechanical stress and light across a sharp ecotone.

Abstract: Premise The drivers of isolation between sympatric populations of long-lived and highly dispersible conspecific plants are not well understood. In the Hawaiian Islands, the landscape-dominant tree, Metrosideros polymorpha, displays extraordinary phenotypic differences among sympatric varieties despite high dispersibility of its pollen and seeds, thereby presenting a unique opportunity to investigate how disruptive selection alone can maintain incipient forms. Stenophyllous M. polymorpha var. newellii is a recently evolved tree endemic to the waterways of eastern Hawai'i Island that shows striking neutral genetic differentiation from its ancestor, wet-forest M. polymorpha var. glaberrima, despite sympatry of these forms. We looked for evidence for, and drivers of, differential local adaptation of these varieties across the range of M. polymorpha var. newellii. Methods For paired populations of these varieties, we compared seedling performance under contrasting light conditions and a strong water current characteristic of the riparian zone. We also conducted a reciprocal transplant experiment and contrasted adult leaf anatomy. Results Results suggest that the riparian zone is harsh and that selection involving the mechanical stress of rushing water, and secondarily, light, led to significant reciprocal immigrant inviability in adjacent forest and riparian environments. The strongest adaptive divergence between varieties was seen in leaves and seedlings from the site with the sharpest ecotone, coincident with the strongest genetic isolation of M. polymorpha var. newellii observed previously. Conclusions These findings suggest that disruptive selection across a sharp ecotone contributes to the maintenance of an incipient riparian ecotype from within a continuous population of a long-lived and highly dispersible tree species.

The humidity inside leaves and why you should care: implications of unsaturation of leaf intercellular airspaces

Abstract: We are unaware of published measurements of smes in P. edulis, but smes varied from 14.4 to 40 m2 m-2 across 15 diverse species in a study by Tomas et al. (2013). We adopt smes = 10 m2 m-2 to ensure the resulting estimate of Pm is not an underestimate due to uncertainty in this parameter. Applying Eq. S6 to the data of Cernusak et al. (2018) indicates that, to sustain a symplastic water potential of -2 MPa while water flows across the cell membranes to an apoplastic space with water potential given by Eq. S1 would require Pm as low as 1.710-9 m s-1, or 0.0017 m s-1, for P. edulis, and as low as 9.810-8 m s-1, or 0.0098 m s-1, for J. monosperma (Appendix S3a). For P. edulis, 25% of measurements indicate Pm at least as small as 0.0083 m s-1; for J. monosperma, 25% of measurements indicated Pm < 0.099 m s. By comparison, mean values of Pm previously reported for mesophyll protoplasts are orders of magnitude larger (e.g., 5–25 m s-1; Shatil-Cohen et al., 2013).

Phylogenetic and geographic distribution of nickel hyperaccumulation in neotropical Psychotria.

Abstract: PREMISE Hyperaccumulation of heavy metals in plants has never been documented from Central America or Mexico. Psychotria grandis, P. costivenia, and P. glomerata (Rubiaceae) have been reported to hyperaccumulate nickel in the Greater Antilles, but they also occur widely across the neotropics. The goals of this research were to investigate the geographic distribution of hyperaccumulation in these species and explore the phylogenetic distribution of hyperaccumulation in this clade by testing related species. METHODS Portable x-ray fluorescence (XRF) spectroscopy was used to analyze 565 specimens representing eight species of Psychotria from the Missouri Botanical Garden herbarium. RESULTS Nickel hyperaccumulation was found in specimens of Psychotria costivenia ranging from Mexico to Costa Rica and in specimens of P. grandis from Guatemala to Ecuador and Venezuela. Among related species, nickel hyperaccumulation is reported for the first time in P. lorenciana and P. papantlensis, but no evidence of hyperaccumulation was found in P. clivorum, P. flava, or P. pleuropoda. Previous reports of hyperaccumulation in P. glomerata appear to be erroneous, resulting from taxonomic synonymy and specimen misidentification. CONCLUSIONS Hyperaccumulation of nickel by Psychotria is now known to occur widely from southern Mexico through Central America to northwestern South America, including some areas not known to have ultramafic soils. Novel aspects of this research include the successful prediction of new hyperaccumulator species based on molecular phylogeny, use of XRF technology to nondestructively obtain elemental data from herbarium specimens, and documentation of previously unknown areas of ultramafic or nickel-rich soil based on such data.

Evolution of RADIALIS and DIVARICATA gene lineages in flowering plants with an expanded sampling in non-core eudicots.

Abstract: Premise of the study Bilateral symmetry in core eudicot flowers is established by the differential expression of CYCLOIDEA (CYC), DICHOTOMA (DICH), and RADIALIS (RAD), which are restricted to the dorsal portion of the flower, and DIVARICATA (DIV), restricted to the ventral and lateral petals. Little is known regarding the evolution of these gene lineages in non-core eudicots, and there are no reports on gene expression that can be used to assess whether the network predates the diversification of core eudicots. Methods Homologs of the RAD and DIV lineages were isolated from available genomes and transcriptomes, including those of three selected non-core eudicot species, the magnoliid Aristolochia fimbriata and the monocots Cattleya trianae and Hypoxis decumbens. Phylogenetic analyses for each gene lineage were performed. RT-PCR was used to evaluate the expression and putative contribution to floral symmetry in dissected floral organs of the selected species. Key results RAD-like genes have undergone at least two duplication events before eudicot diversification, three before monocots and at least four in Orchidaceae. DIV-like genes also duplicated twice before eudicot diversification and underwent independent duplications specific to Orchidaceae. RAD-like and DIV-like genes have differential dorsiventral expression only in C. trianae, which contrasts with the homogeneous expression in the perianth of A. fimbriata. Conclusions Our results point to a common genetic regulatory network for floral symmetry in monocots and core eudicots, while alternative genetic mechanisms are likely driving the bilateral perianth symmetry in the early-diverging angiosperm Aristolochia.

Plant responses to climate warming: physiological adjustments and implications for plant functioning in a future, warmer world.

Abstract: American Journal of Botany 106(8): 1049–1051, 2019; http://www.wileyonlinelibrary.com/journal/AJB © 2019 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. ON THE NATURE OF THINGS: ESSAYS New Ideas and Directions in Botany