Development and comparative analysis of initiation ability in large-scale Heuchera propagation using tissue culture versus cuttings.

The Heuchera genus, a member of the Saxifragaceae family, encompasses a wide array of varieties and hybrids, serving both traditional medicinal and ornamental purposes. However, a significant knowledge gap persists in achieving efficient mass propagation of diverse Heuchera cultivars creating a substantial market void. To address this, our study focuses on expedited seedling regeneration by investigating leaf cutting and tissue culture techniques to offer novel insights to cultivators. Herein, we successfully rooted thirteen distinct cultivars from the Heuchera and Heucherella (Heuchera × Tiarella) genera through cutting. Moreover, in vitro culture experiments led to the successful induction of calli and shoots from petiole samples. Notably, variations in measured parameters were observed across cultivars in both cutting and tissue culture methodologies. When petiole explants were exposed to cytokinin 6-benzylaminopurine (BA) at concentrations of 0.5, 1.0, and 2.0 mg/L along with auxin α-naphthaleneacetic acid (NAA) at 0.5 mg/L, shoots were produced either directly or indirectly during the primary culture. Exposure to darkness and the application of 2,4-dichlorophenoxyacetic acid (2,4-D) did not promote shoot formation but were beneficial for callus stimulation. Interestingly, a negative correlation was observed between the ease of initiating cutting recovery and inducting tissue culture regeneration, suggesting that cultivars that easily regenerate through cutting might encounter difficulties during induction by tissue culture. In light of these findings, we devised a streamlined and effective protocol for rapid Heuchera propagation. This protocol involves micropropagation, directly acquiring adventitious shoots from primary cultures supplemented by cutting-based propagation methods.

Neopolyploidy causes increased nutrient requirements and a shift in plant growth strategy in Heuchera cylindrica.

Functional traits fall along a continuum from resource conservative to acquisitive and are powerful predictors of the ecological settings necessary for a species to persist and establish. As a consequence, a major problem that functional trait analysis could address is understanding the ecological contexts necessary for the persistence of polyploid plants, because early generation polyploids, or "neopolyploids," are at a high extinction risk. Because neopolyploidy could increase nutrient limitation, growth strategies should shift to accommodate the increased need for resources, but this prediction is untested. To address this gap, we compared the functional trait responses of diploids, synthetic neotetraploids, and naturally occurring tetraploids of Heuchera cylindrica, an herbaceous perennial plant, to nutrient manipulations in a greenhouse experiment. We found strong support for the hypothesis that neotetraploidy increases nutrient requirements, as evidenced by reduced productivity and increased tissue concentrations of nitrogen and phosphorus in neotetraploids. We also found that the repeated formation of independent origins of neotetraploidy led to differing responses to nutrient supply, but neotetraploidy generally shifted functional traits to be more resource acquisitive and inefficient. Taken together, our results suggest that shifts in functional trait responses may constrain the ability of neopolyploids to establish in nutrient-poor habitats.

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells.

Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast.

The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast.

The plant defensin HsAFP1 is characterized by broad-spectrum antifungal activity and induces apoptosis in Candida albicans. In this study, we performed a transcriptome analysis on C. albicans cultures treated with HsAFP1 to gain further insight in the antifungal mode of action of HsAFP1. Various genes coding for cell surface proteins, like glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in cation homeostasis, autophagy and in cell cycle were differentially expressed upon HsAFP1 treatment. The biological validation of these findings was performed in the model yeast Saccharomyces cerevisiae. To discriminate between events linked to HsAFP1's antifungal activity and those that are not, we additionally used an inactive HsAFP1 mutant. We demonstrated that (i) HsAFP1-resistent S. cerevisiae mutants that are characterized by a defect in processing GPI-anchors are unable to internalize HsAFP1, and (ii) moderate doses (FC50, fungicidal concentration resulting in 50% killing) of HsAFP1 induce autophagy in S. cerevisiae, while high HsAFP1 doses result in vacuolar dysfunction. Vacuolar function is an important determinant of replicative lifespan (RLS) under dietary restriction (DR). In line, HsAFP1 specifically reduces RLS under DR. Lastly, (iii) HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. However, the latter HsAFP1-induced event is not linked to its antifungal activity, as the inactive HsAFP1 mutant also impairs the G2/M phase. In conclusion, we demonstrated that GPI-anchored proteins are involved in HsAFP1's internalization, and that HsAFP1 induces autophagy, vacuolar dysfunction and impairment of the cell cycle. Collectively, all these data provide novel insights in the mode of action of HsAFP1 as well as in S. cerevisiae tolerance mechanisms against this peptide.

Intraspecific polyploidy correlates with colonization by arbuscular mycorrhizal fungi in Heuchera cylindrica.

PREMISE: Polyploidy is known to cause physiological changes in plants which, in turn, can affect species interactions. One major physiological change predicted in polyploid plants is a heightened demand for growth-limiting nutrients. Consequently, we expect polyploidy to cause an increased reliance on the belowground mutualists that supply these growth-limiting nutrients. An important first step in investigating how polyploidy affects nutritional mutualisms in plants, then, is to characterize differences in the rate at which diploids and polyploids interact with belowground mutualists. METHODS: We used Heuchera cylindrica (Saxifragaceae) to test how polyploidy influences interactions with arbuscular mycorrhizal fungi (AMF). Here we first confirmed the presence of AMF in H. cylindrica, and then we used field-collected specimens to quantify and compare the presence of AMF structures while controlling for site-specific variation. RESULTS: Tetraploids had higher colonization rates as measured by total, hyphal, and nutritional-exchange structures; however, we found that diploids and tetraploids did not differ in vesicle colonization rates. CONCLUSIONS: The results suggest that polyploidy may alter belowground nutritional mutualisms with plants. Because colonization by nutritional-exchange structures was higher in polyploids but vesicle colonization was not, polyploids might form stronger associations with their AMF partners. Controlled experiments are necessary to test whether this pattern is driven by the direct effect of polyploidy on AMF colonization.

Geographic Range Dynamics Drove Ancient Hybridization in a Lineage of Angiosperms.

Elucidating the dynamic distribution of organismal lineages has been central to biology since the nineteenth century, yet the difficulty of combining biogeographic methods with shifts in habitat suitability remains a limitation. This integration, however, is critical to understanding geographic distributions, present and past, as well as the time-extended trajectories of lineages. Here, we link previous advances in phyloclimatic modeling to develop a framework that overcomes existing methodological gaps by predicting potential ecological and geographic overlap directly from estimated ancestral trait distributions. We show the utility of this framework by focusing on a clade in the montane angiosperm genus Heuchera, which is noteworthy in that it experienced ancient introgression from circumboreally distributed species of Mitella, lineages now ~1,300 km disjunct. Using this system, we demonstrate an application of ancestral state reconstruction to assess geographic range dynamics in a lineage lacking a fossil record. We test hypotheses regarding inferred past geographic distributions and examine the potential for ancient geographic contact. Application of this multifaceted approach suggests potential past contact between species of Heuchera and Mitella in western North America during cooler periods of the Pleistocene. Integration of niche models and phylogenetic estimates suggests that climatic cooling may have promoted range contact and gene flow between currently highly disjunct species. Our approach has wide applicability for testing hypotheses concerning organismal co-occurrences in deep time.

Phylogenetic relationships and character evolution in Heuchera (Saxifragaceae) on the basis of multiple nuclear loci.

UNLABELLED: • PREMISE OF THE STUDY: The use of multiple genetic regions from the nuclear genome, including low-copy markers, has long been recognized as essential to robust phylogenetic construction, addressing gene tree incongruence, and allowing increased resolution to test current taxonomy and resolve basic hypotheses about character evolution, biogeography, and other organismal traits of interest to biologists. Heuchera, the largest genus of Saxifragaceae endemic to North America, has presented an unusually difficult case for systematists with limited sampling in previous molecular studies. We used morphological and multilocus molecular phylogenetic data to test the monophyly of Heuchera, better resolve hypotheses of relationships, and test hypotheses of character evolution, biogeography, and diversification rates.• METHODS: Phylogenetic relationships were inferred using sequences from six nuclear loci and 39 morphological characters using concatenation and coalescent analysis. Ancestral state reconstruction, diversification, and correlated evolution of morphological traits were performed using parsimony, BiSSE, and Pagel's method, respectively.• KEY RESULTS: The concatenation and species tree analyses gave confident support to the monophyly of Heuchera and suggested several well-supported subclades. The addition of morphological data significantly improved support numbers. Ancestral character reconstruction suggested frequent homoplasy and reversal of floral characters and a complex biogeographical history.• CONCLUSIONS: Heuchera is a natural genus; however, the current subgeneric classification is artificial and requires revision. Biogeographic reconstructions suggest a Pacific Northwest origin for the genus; morphological shifts in stamen exsertion and hypanthium length are hypothesized to have driven diversification in Heuchera in concert with possible pollinator shifts.

Polyploidization in Heuchera cylindrica (Saxifragaceae) did not result in a shift in climatic requirements.

PREMISE OF THE STUDY: Polyploidization is a key factor involved in the diversification of plants. Although polyploids are commonly found, there remains controversy on the mechanisms that lead to their successful establishment. One major problem that has been identified is that newly formed polyploids lack mates of the appropriate ploidy level and may experience severely reduced fertility due to nonproductive intercytotype crosses. Niche differentiation has been proposed as a primary mechanism that can alleviate this reproductive disadvantage and facilitate polyploid establishment. Here we test whether the establishment of tetraploid cytotypes of Heuchera cylindrica (Saxifragaceae) is consistent with climatic niche differentiation. • METHODS: We use a combination of field surveys, flow cytometry and species distribution models to: (1) examine the distribution of diploid and tetraploid cytotypes; and (2) determine whether tetraploid Heuchera cylindrica occupy climates that differ from those of its diploid progenitors. • KEY RESULTS: The geographic distributions of diploid and tetraploid cytotypes are largely allopatric as an extensive survey of 636 plants from 43 locations failed to detect any populations with both cytotypes. Although diploids and tetraploids occur in different geographic areas, polyploid Heuchera cylindrica occur almost exclusively in environments that are predicted to be suitable to diploid populations. • CONCLUSIONS: Climatic niche differentiation does not explain the geographic distribution of tetraploid Heuchera cylindrica. We propose instead that tetraploid lineages were able to establish by taking advantage of glacial retreat and expanding into previously unoccupied sites.

Entangling ancient allotetraploidization in Asian Mitella: an integrated approach for multilocus combinations.

The reconstruction of an ancient polyploidization history is often challenging, although it is a crucial step in clarifying the mechanisms underlying the contemporary success and diversity of polyploids. Phylogenetic relationships of duplicated gene pairs of polyploids, with respect to their orthologs in related diploids, have been used to address this problem, but they often result in conflicting topologies among different genes. Asimitellaria is an East Asian endemic tetraploid lineage of perennials (genus Mitella; Saxifragaceae) that has diversified in riparian habitats. Phylogenetic analyses of four nuclear-encoded, single-copy (per haploid) genes GBSSI-A, GBSSI-B, GS-II, and PepCK all supported a single allopolyploid origin of Asimitellaria, but they did not lead to a consensus about which diploid lineage gave rise to each of the Asimitellaria subgenomes. To address this issue, we used an integrated approach, whereby the four gene data sets and an additional nuclear ribosomal external transcribed spacer and internal transcribed spacer (including a 5.8S ribosomal DNA) data set were concatenated in all possible combinations, and the most probable data combination was determined together with the phylogenetic inference. This resulted in relatively robust support for the two closely related North American diploid species as the ancestral lineages of the Asimitellaria subgenomes, suggesting ancient intercontinental migration of the diploid or tetraploid lineages and subsequent tetraploid diversification in the Japanese Archipelago. The present approach enabled sorting out the duplicated genes into their original combinations in their preduplication ancestors under a maximum-likelihood framework, and its extension toward genome sequencing data may help in the reconstruction of ancestral, preduplicated, whole-genome structures.

A unified model of autopolyploid establishment and evolution.

The prevalence of polyploidy among flowering plants is surprising given the hurdles impeding the establishment and persistence of novel polyploid lineages. In the absence of strong assortative mating, reproductive assurance, or large intrinsic fitness advantages, new polyploid lineages face almost certain extinction through minority cytotype exclusion. Consequently, much work has focused on a search for adaptive advantages associated with polyploidy such as increased competitive ability, enhanced ecological tolerances, and increased resistance to pathogens. Yet, no consistent adaptive advantages of polyploidy have been identified. Here, to investigate the potential for autopolyploid establishment and persistence in the absence of any intrinsic fitness advantages, we develop a simulation model of a diploid population that sporadically gives rise to novel autopolyploids. The autopolyploids have only very small levels of initial assortative mating or niche differentiation, generated entirely by dosage effects of genome duplication, and they have realistic levels of reproductive assurance. Our results show that by allowing assortative mating and competitive interactions to evolve, establishment of novel autopolyploid lineages becomes common. Additional scenarios where adaptive optima change over time reveal that rapid environmental change promotes the replacement of diploid lineages by their autopolyploid descendants. These results help to explain recent empirical findings that suggest that many contemporary polyploid lineages arose during the Cretaceous-Tertiary extinction, without invoking adaptive advantages of polyploidy.

Neopolyploidy and diversification in Heuchera grossulariifolia.

Newly formed polyploid lineages must contend with several obstacles to avoid extinction, including minority cytotype exclusion, competition, and inbreeding depression. If polyploidization results in immediate divergence of phenotypic characters these hurdles may be reduced and establishment made more likely. In addition, if polyploidization alters the phenotypic and genotypic associations between traits, that is, the P and G matrices, polyploids may be able to explore novel evolutionary paths, facilitating their divergence and successful establishment. Here, we report results from a study of the perennial plant Heuchera grossulariifolia in which the phenotypic divergence and changes in phenotypic and genotypic covariance matrices caused by neopolyploidization have been estimated. Our results reveal that polyploidization causes immediate divergence for traits relevant to establishment and results in significant changes in the structure of the phenotypic covariance matrix. In contrast, our results do not provide evidence that polyploidization results in immediate and substantial shifts in the genetic covariance matrix.

Evolution of polyploidy and the diversification of plant-pollinator interactions.

One of the major mechanisms of plant diversification has been the evolution of polyploid populations that differ from their diploid progenitors in morphology, physiology, and environmental tolerances. Recent studies have indicated that polyploidy may also have major effects on ecological interactions with herbivores and pollinators. We evaluated pollination of sympatric diploid and tetraploid plants of the rhizomatous herb Heuchera grossulariifolia (Saxifragaceae) along the Selway and Salmon Rivers of northern Idaho, USA, during four consecutive years. Previous molecular and ecological analyses had indicated that the tetraploid populations along these two river systems are independently derived and differ from each other in multiple traits. In each region, we evaluated floral visitation rate by all insect visitors, pollination efficacy of all major visitors, and relative contribution of all major pollinators to seed set. In both regions, diploid and tetraploid plants attracted different suites of floral visitors. Most pollination was attributable to several bee species and the moth Greya politella. Lasioglossum bees preferentially visited diploid plants at Lower Salmon but not at Upper Selway, queen Bombus centralis preferentially visited tetraploids at both sites, and worker B. centralis differed between sites in their cytotype preference. Hence, diploid and autotetraploid H. grossulariifolia plants act essentially as separate ecological species and may experience partial reproductive isolation through differential visitation and pollination by their major floral visitors. Overall the results, together with recent results from other studies, suggest that the repeated evolution of polyploidy in plants may contribute importantly to the structure and diversification of ecological interactions in terrestrial communities.

The contribution of parasitism to selection on floral traits in Heuchera grossulariifolia.

Parasites are ubiquitous and have well-documented ecological consequences. In contrast, the extent to which parasites drive phenotypic evolution in hosts remains obscure. We use a recently developed statistical technique--selective source analysis--to analyse the strength of phenotypic selection acting on floral traits in the plant Heuchera grossulariifolia attributable to attack by the seed-parasitic moth, Greya politella. This analysis spanned 3 years and included two sympatric populations of the host plant H. grossulariifolia that differ in ploidy. Our analyses revealed that attack by G. politella contributed to phenotypic selection for flowering time and floral display size, favouring earlier flowering in the polyploid population, later flowering in the diploid population and increased floral display size in the polyploid population. Although selection imposed by parasite attack was generally quite weak, in one of the 3 years parasites generated a modestly strong selection gradient (beta = -0.059) that explained 38.6% of total observed phenotypic selection for earlier flowering in the polyploid population. Together, our results demonstrate parasites can generate significant phenotypic selection, but that such selection may be sporadic across populations and time.

Selection for phenotypic divergence between diploid and autotetraploid Heuchera grossulariifolia.

Much of the diversity of flowering plants is associated with genomic duplication through polyploidy. Little is known, however, about the evolutionary mechanisms responsible for the diversification of novel polyploid lineages. We evaluated the possibility that divergence is driven by natural selection by estimating the strength of phenotypic selection acting on three floral traits in sympatric populations of diploid and autotetraploid Heuchera grossulariifolia over three years. Our results demonstrate consistent directional selection for increasing scape length and floral display in both diploid and tetraploid populations. In contrast, selection acting on flowering phenology varied across year and ploidy. Specifically, selection was found to favor late-flowering diploids in 2001 and 2002 but early-flowering tetraploids in 2003. We investigated the mechanistic basis of divergent selection for flowering phenology in 2003 by estimating the relationship between plant flowering phenology and the probability of intercytotype pollinator movement. The results demonstrated that less divergent tetraploids were significantly more likely to experience intercytotype flights than were more divergent tetraploids. This result is consistent with the pattern of phenotypic selection observed. Taken together, our results suggest that divergence of polyploids and their diploid progenitors may be driven by a process analogous to reinforcement whereby selection favors phenotypes that reduce the probability of intercytotype matings with reduced fertility.