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PremiseThere is considerable variation in leaf lobing and leaf size, including among grapevines, some of the most well-studied leaves. We examined the relationship between leaf lobing and leaf size across grapevine populations which varied in extent of leaf lobing.MethodsWe used homologous landmarking techniques to measure 2,632 leaves across two years in 476 unique, genetically distinct grapevines from 5 biparental crosses which vary primarily in the extent of lobing. We determined to what extent leaf area could explain variation in lobing, vein length, and vein to blade ratio.ResultsAlthough lobing was the primary source of variation in shape across the leaves we measured, leaf area varied only slightly as a function of lobing. Rather, leaf area increases as a function of total major vein length, total branching vein length, and decreases as a function of vein to blade ratio. These relationships are stronger for more highly lobed leaves, with the residuals for each model differing as a function of distal lobing.ConclusionsFor a given leaf area, more highly lobed leaves have longer veins and higher vein to blade ratios, allowing them to maintain similar leaf areas despite increased lobing. These findings show how more highly lobed leaves may compensate for what would otherwise result in a reduced leaf area, allowing for increased photosynthetic capacity through similar leaf size.

As the climate changes, plants and their associated microbiomes face greater water limitation and increased frequency of drought. Historical precipitation patterns can leave a legacy effect on soil and root-associated microbiomes, but the impact of this conditioning on future drought performance is poorly understood. Moreover, agricultural practices, like tilling and fertilization, may override precipitation legacies in natural systems. Precipitation gradients provide a means to assess these legacy effects. We collected six soil microbiomes across a Kansas precipitation gradient from both agricultural fields and native prairies. Seedlings of twoZea maysgenotypes were inoculated with each soil microbiome in a factorial drought experiment. Droughted plants exhibited decreased shoot mass accumulation rates and greater root mass relative to shoot mass. Restructuring of the bacterial root-associated microbiome was apparent, with depletion observed in Pseudomonadota and enrichment in Actinomycetota, while the fungal microbiome was largely unaffected by drought. A historical precipitation legacy effect on soil microbiomes interacted with plants during drought treatment, but only among prairie soils. Prairie soils from historically wetter locations increased maize shoot biomass under drought more so than agricultural or historically drier prairie soils. We demonstrate links between legacy effects and drought performance, suggesting that future drying climates may condition soils to negatively impact plant performance.

SummaryAnnual rings from 30 year old vines in a California rootstock trial were measured to determine the effects of 15 different rootstocks on Chardonnay and Cabernet Sauvignon scions. Viticultural traits measuring vegetative growth, yield, berry quality, and nutrient uptake were collected at the beginning (1995 to 1999) and end (2017 to 2020) of the lifetime of a vineyard initially planted in 1991 and removed in 2021. X-ray Computed Tomography (CT) was used to measure ring widths in 103 vines. Ring width was modeled as a function of ring number using a negative exponential model. Early and late wood ring widths, cambium width, and scion trunk radius were correlated with 27 traits. Modeling of annual ring width shows that scions alter the width of the first rings but that rootstocks alter the decay of later rings, consistently shortening ring width throughout the lifetime of the vine. Ravaz index, juice pH, photosynthetic assimilation and transpiration rates, and instantaneous water use efficiency are correlated with scion trunk radius. Ultimately, our research indicates that rootstocks modulate secondary growth over years, altering physiology and agronomic traits. Rootstocks act in similar but distinct ways from climate to modulate ring width, which borrowing techniques from dendrochronology, can be used to monitor both genetic and environmental effects in woody perennial crop species.

PremiseGrapevine leaves have diverse shapes and sizes. Their shape and size is known to be influenced by many factors including genetics, vine phytosanitary status, environment, leaf and vine age, and node position on the shoot. In order to determine the importance of grapevine leaf shape and size to canopy temperature, we examined the relationship in five seedling populations grown in a vineyard in California, USA.MethodsAll of the populations had one parent with compound leaves of the Vitis piasezkii type and each population had a different second parent with non-compound leaves. In previous work, we measured leaf shape and size using 21 homologous landmarks. Here, we paired these morphology data with measurements taken using an infrared thermometer to measure the temperature of the canopy. By recording time of sampling and canopy temperature, we were able to determine which vines were cooler or hotter than expected, using a linear model.ResultsWe established a relationship between leaf size and canopy temperature: vines with larger leaves were cooler than expected. In contrast, leaf shape was not strongly correlated with variation in temperature.ConclusionsUltimately, these findings indicate that vines with larger leaves may contribute to the reduction of overall vine canopy temperature, but further work is needed to determine if this is due to variation in leaf size, differences in the openness of the canopy, or other related traits.

SummaryAnnual rings from 30 year old vines in a California rootstock trial were measured to determine the effects of 15 different rootstocks on Chardonnay and Cabernet Sauvignon scions. Viticultural traits measuring vegetative growth, yield, berry quality, and nutrient uptake were collected at the beginning and end of the lifetime of the vineyard.X-ray Computed Tomography (CT) was used to measure ring widths in 103 vines. Ring width was modeled as a function of ring number using a negative exponential model. Early and late wood ring widths, cambium width, and scion trunk radius were correlated with 27 traits.Modeling of annual ring width shows that scions alter the width of the first rings but that rootstocks alter the decay thereafter, consistently shortening ring width throughout the lifetime of the vine. The ratio of yield to vegetative growth, juice pH, photosynthetic assimilation and transpiration rates, and stomatal conductance are correlated with scion trunk radius.Rootstocks modulate secondary growth over years, altering hydraulic conductance, physiology, and agronomic traits. Rootstocks act in similar but distinct ways from climate to modulate ring width, which borrowing techniques from dendrochronology, can be used to monitor both genetic and environmental effects in woody perennial crop species.

Background Modern biological approaches generate volumes of multi-dimensional data, offering unprecedented opportunities to address biological questions previously beyond reach owing to small or subtle effects. A fundamental question in plant biology is the extent to which below-ground activity in the root system influences above-ground phenotypes expressed in the shoot system. Grafting, an ancient horticultural practice that fuses the root system of one individual (the rootstock) with the shoot system of a second, genetically distinct individual (the scion), is a powerful experimental system to understand below-ground effects on above-ground phenotypes. Previous studies on grafted grapevines have detected rootstock influence on scion phenotypes including physiology and berry chemistry. However, the extent of the rootstock's influence on leaves, the photosynthetic engines of the vine, and how those effects change over the course of a growing season, are still largely unknown. Results Here, we investigate associations between rootstock genotype and shoot system phenotypes using 5 multi-dimensional leaf phenotyping modalities measured in a common grafted scion: ionomics, metabolomics, transcriptomics, morphometrics, and physiology. Rootstock influence is ubiquitous but subtle across modalities, with the strongest signature of rootstock observed in the leaf ionome. Moreover, we find that the extent of rootstock influence on scion phenotypes and patterns of phenomic covariation are highly dynamic across the season. Conclusions These findings substantially expand previously identified patterns to demonstrate that rootstock influence on scion phenotypes is complex and dynamic and underscore that broad understanding necessitates volumes of multi-dimensional data previously unmet.

Amphicarpic plants produce both above-ground and below-ground seeds. Because below-ground seeds are protected in the soil and may maintain viability when above-ground conditions are stressful, they were proposed as an adaptation to recolonize a site after disturbance. However, whether below-ground seeds are the main colonizers after a disturbance remains unknown. Our goal was to understand whether recolonization by an amphicarpic species after fire was accomplished primarily through germination of seeds produced above-ground or below-ground. We investigated Polygala lewtonii, an amphicarpic, perennial species endemic to fire-prone Florida sandhill and scrub, where fire kills plants but subsequently increases recruitment and population sizes. Polygala lewtonii produces three flower types: above-ground chasmogamous flowers and above-ground and below-ground cleistogamous flowers, with previous research demonstrating chasmogamous flowers produce a much greater proportion of seeds than cleistogamous flowers. We quantified outcrossing in seeds produced by chasmogamous flowers to determine whether it differed from the 100 % self-fertilized below-ground seeds. Approximately 25 % of seeds from chasmogamous flowers showed evidence of cross-pollination. Assuming that chasmogamous flowers produce the majority of the above-ground seeds, as was shown previously, this indicates it is possible to differentiate between germination by above-ground versus below-ground seeds in post-fire colonization. We next compared genetic diversity, admixture, inbreeding and population genetic structure pre- and post-fire. If fire promoted germination of chasmogamous seeds, heterozygosity and admixture would increase, and genetic structure and inbreeding would decrease. Instead, inbreeding and genetic structure increased and admixture decreased, suggesting that the below-ground selfed seeds (with limited dispersal ability) increased their contribution to the population after fire, possibly because fire reduced above-ground seed viability. Additionally, new alleles not found previously in range-wide analyses emerged from the seed bank post-fire. These results suggest that amphicarpy is a powerful adaptation to preserve genetic variation, maintain adaptive potential and promote rapid post-fire colonization., We examined the mating system’s response to fire in Polygala lewtonii, a species endemic to fire-prone Florida sandhill and scrub. Polygala lewtonii has three flower types: above-ground chasmogamous (open-pollinated) and cleistogamous (closed, obligately self-fertilizing) flowers and below-ground cleistogamous flowers, with fruits produced both above-ground and below-ground. We used a genetic approach to test whether post-fire recolonization was accomplished primarily through germination of above-ground or below-ground seed. The below-ground selfed seeds showed an increased contribution to the population and new alleles emerged from the seed bank post-fire. These results suggest that amphicarpy is an adaptation to promote rapid post-fire colonization and preserve genetic variation and adaptive potential.

In this study, we evaluated the efficacy of sample collection approaches and DNA metabarcoding to identify plants utilized by nectivorous bats. Samples included guano collected from beneath bat roosts and pollen-swabs from bat fur, both of which were subjected to DNA metabarcoding and visual identification of pollen (microscopy) to measure plant diversity. Our objectives were to determine whether DNA metabarcoding could detect likely food plants of nectivorous bats, whether sample types would produce different estimates of plant diversity, and to compare results of DNA metabarcoding to visual identification. Visual identification found that 99% of pollen was from Agave, which is thought to be the bats’ main food source. The dominant taxon found by metabarcoding was also Agavoideae, but a broader diversity of plant species was also detected, many of which are likely “by-catch” from the broader environment. Metabarcoding outcomes differed between sample types, likely because pollen-swabs measured the plant species visited by bats and guano samples measured all items consumed in the bat’s diet, even those that were not pollen or nectar. Overall, metabarcoding is a powerful, high-throughput tool to understand bat ecology and species interactions, but careful analysis of results is necessary to derive accurate ecological conclusions.

Understanding genetic diversity and structure in a rare species is critical for prioritizing bothin situandex situconservation efforts. One such rare species isPhysaria filiformis(Brassicaceae), a threatened, winter annual plant species. The species has a naturally fragmented distribution, occupying three different soil types spread across four disjunct geographical locations in Missouri and Arkansas. The goals of this study were to understand: (1) whether factors associated with fragmentation and small population size (i.e., inbreeding, genetic drift or genetic bottlenecks) have reduced levels of genetic diversity, (2) how genetic variation is structured and which factors have influenced genetic structure, and (3) how much extant genetic variation ofP.filiformisis currently publicly protected and the implications for the development of conservation strategies to protect its genetic diversity. Using 16 microsatellite markers, we genotyped individuals from 20 populations ofP.filiformisfrom across its geographical range and one population ofPhysaria gracilisfor comparison and analyzed genetic diversity and structure. Populations ofP.filiformisshowed comparable levels of genetic diversity to its congener, except a single population in northwest Arkansas showed evidence of a genetic bottleneck and two populations in the Ouachita Mountains of Arkansas showed lower genetic variation, consistent with genetic drift. Populations showed isolation by distance, indicating that migration is geographically limited, and analyses of genetic structure grouped individuals into seven geographically structured genetic clusters, with geographic location/spatial separation showing a strong influence on genetic structure. At least one population is protected for all genetic clusters except one in north-central Arkansas, which should therefore be prioritized for protection. Populations in the Ouachita Mountains were genetically divergent from the rest ofP.filiformis; future morphological analyses are needed to identify whether it merits recognition as a new, extremely rare species.

Grafting connects root and shoot systems of distinct individuals, bringing microbial communities of different genotypes together in a single plant. How do root system and shoot system genotypes influence plant microbiota in grafted grapevines? To address this, we utilized clonal replicates of the grapevine &lsquo, Chambourcin&rsquo, growing ungrafted and grafted to three different rootstocks in three irrigation treatments. Our objectives were to (1) characterize the microbiota (bacteria and fungi) of below-ground compartments (roots, adjacent soil) and above-ground compartments (leaves, berries), (2) determine how rootstock genotype, irrigation, and their interaction influences grapevine microbiota in different compartments, and (3) investigate abundance of microorganisms implicated in the late-season grapevine disease sour rot (Acetobacterales and Saccharomycetes). We found that plant compartment had the largest influence on microbial diversity. Neither rootstock genotype nor irrigation significantly influenced microbial diversity or composition. However, differential abundance of bacterial and fungal taxa varied as a function of rootstock and irrigation treatment, in particular, Acetobacterales and Saccharomycetes displayed higher relative abundance in berries of grapevines grafted to &lsquo, 1103P&rsquo, and &lsquo, SO4&rsquo, rootstocks and varied across irrigation treatments. This study demonstrates that grapevine compartments retain distinct microbiota and identifies associations between rootstock genotypes, irrigation treatment, and the relative abundance of agriculturally relevant microorganisms in the berries.

SummaryIn many perennial crops, grafting the root system of one individual to the shoot system of another individual has become an integral part of propagation performed at industrial scales to enhance pest, disease, and stress tolerance and to regulate yield and vigor. Grafted plants offer important experimental systems for understanding the extent and seasonality of root system effects on shoot system biology.Using an experimental vineyard where a common scion ‘Chambourcin’ is growing ungrafted and grafted to three different rootstocks, we explore associations between root system genotype and leaf phenotypes in grafted grapevines across a growing season. We quantified five high-dimensional leaf phenotyping modalities: ionomics, metabolomics, transcriptomics, morphometrics, and physiology and show that rootstock influence is subtle but ubiquitous across modalities.We find strong signatures of rootstock influence on the leaf ionome, with unique signatures detected at each phenological stage. Moreover, all phenotypes and patterns of phenotypic covariation were highly dynamic across the season.These findings expand upon previously identified patterns to suggest that the influence of root system on shoot system phenotypes is complex and broad understanding necessitates volumes of high-dimensional, multi-scale data previously unmet.

BackgroundWithin an individual plant, different compartments (e.g. roots, leaves, fruits) host distinct communities of microorganisms due to variation in structural characteristics and resource availability. Grafting, which joins the root system of one individual with the shoot system of a second genetically distinct individual, has the potential to bring the microbial communities of different genotypes together. An important question is the extent to which unique root system and shoot system genotypes, when grafted together, influence the microbiota of the graft partner. Our study sought to answer this question by utilizing an experimental vineyard composed of ‘Chambourcin’ vines growing ungrafted and grafted to three different rootstocks, replicated across three irrigation treatments. We characterized bacterial and fungal communities in roots, leaves, and berries, as well as surrounding soil. Our objectives were to (1) characterize the microbiota of compartments within the root system (roots and adjacent soil) and the shoot system (leaves and berries), (2) determine the influence of rootstock genotypes, irrigation, and their interaction on the microbiota of aboveground and belowground compartments, and (3) investigate the distribution of microorganisms implicated in the late-season grapevine bunch rot disease sour rot (Acetobacterales and Saccharomycetes).ResultsCompartments were significantly differentiated in bacterial and fungal richness and composition. Abundance-based machine learning accurately predicted the compartment and differential abundance analysis showed a large portion of taxa differed significantly across compartments. Rootstock genotypes did not differ significantly in microbial community richness or composition; however, individual microbial taxa exhibited significant differences in abundance based on rootstock and irrigation treatment. The relative abundance of Acetobacterales and Saccharomycetes in the berry was influenced by complex interactions among rootstock genotype and irrigation.ConclusionOur results indicate that grapevine compartments retain distinct core microbiota regardless of the rootstock to which they are grafted. While rootstock genotype generally had a subtle impact on global patterns of microbial diversity, we found associations between rootstock genotypes and specific groups of microorganisms. Further experimental validation is needed in order to understand how associations with these microorganisms impacts a vine’s susceptibility to sour rot upon damage and whether the characteristics of wine are impacted.

Amphicarphic species produce both aboveground and belowground seeds; the belowground seeds have been proposed to be an adaptation to disturbed sites because they are protected belowground, enabling them to persist and recolonize a site after disturbance. However, it is unknown whether such seeds indeed serve as the main colonizers after a disturbance. The amphicarpic species Polygala lewtonii is endemic to fire-prone Florida sandhill and scrub and is among only a few species with three flower types (aboveground chasmogamous flowers and both above and belowground cleistogamous flowers). The goal of this study was to understand whether recolonization of sites by P. lewtonii was accomplished primarily through germination of belowground seed. First, we quantified the outcrossing rate in seeds produced by aboveground chasmogamous flowers to determine whether we could detect differences in colonization of between seeds produced aboveground vs. belowground. Approximately 25% of seeds from aboveground chasmogamous flowers showed evidence of cross pollination and the seeds showed greater heterozygosity and lower inbreeding coefficients than pure selfing, indicating that it is possible to differentiate between selfed and non-selfed seed types in postfire colonization. Second, we analyzed genetic diversity, inbreeding, and genetic structure of the populations before and after a prescribed fire. If heterozygosity and admixture increased, and spatial population genetic structure and inbreeding decreased, this would indicate that fire promoted germination of outcrossed seed from aboveground flowers. However, inbreeding increased and spatial genetic structure and admixture decreased after fire, suggesting that selfed seed produced by belowground flowers predominantly recolonized the site after fire. Thus, amphicarpy is a powerful adaptation to fire-maintained environments by producing seeds that are well suited to the range of conditions presented by a highly variable, disturbance prone habitat.

Premise A disjunct distribution, where a species' geographic range is discontinuous, can occur through vicariance or long-distance dispersal. Approximately 75 North American plant species exhibit a ~650 km disjunction between the Ozark and Appalachian regions. This disjunction is attributed to biogeographic forces including: (1) Eocene-Oligocene vicariance by the formation of the Mississippi embayment; (2) Pleistocene vicariance from interglacial flooding; (3) post-Pleistocene northward colonization from separate glacial refugia; (4) Hypsithermal vicariance due to climate fluctuations; and (5) recent long-distance dispersal. We investigated which of these pathways most likely gave rise to the Appalachian-Ozark disjunction in Delphinium exaltatum. Methods We genotyped populations of D. exaltatum from five Ozark and seven Appalachian localities, analyzed genetic structure, tested the order and timing of divergences using DIYABC, and conducted niche reconstructions up to 21,000 years before present (YBP). Results Populations fell into five main genetic clusters, i.e., a group in the central Appalachians, and four "lowland" groups. DIYABC analyses showed the central Appalachian and lowland lineages diverging 11,300 to17,000 YBP, and the lowland groups diverging 6800 to 10,900 YBP. Niche reconstructions showed that suitable climate for the central Appalachian lineage experienced large spatial discontinuity starting 14,000 YBP, such that divergence and persistence before this period is less plausible than divergence thereafter. Conclusions Our results did not fully support any of the original hypotheses. Rather, the oldest divergence likely occurred after 13,500 YBP through expansion into newly opened habitat in the Appalachians. The Appalachian-Ozark disjunction likely resulted from northward dispersal of the lowland lineage as climate warmed during the Holocene.

As multiple species of bats are currently experiencing dramatic declines in populations due to white‐nose syndrome (WNS) and other factors, conservation managers have an urgent need for data on the ecology and overall status of populations of once‐common bat species. Standard approaches to obtain data on bat populations often involve capture and handling, requiring extensive expertise and unavoidably resulting in stress to the bats. New methods to rapidly obtain critical data are needed that minimize both the stress on bats and the spread of WNS. Guano provides a noninvasive source of DNA that includes information from the bat, but also dietary items, parasites, and pathogens. DNA metabarcoding is a high‐throughput, DNA‐based identification technique to assess the biodiversity of environmental or fecal samples. We investigated the use of multifaceted DNA metabarcoding (MDM), a technique combining next‐generation DNA sequencing (NGS), DNA barcodes, and bioinformatic analysis, to simultaneously collect data on multiple parameters of interest (bat species composition, individual genotype, sex ratios, diet, parasites, and presence of WNS) from fecal samples using a single NGS run. We tested the accuracy of each MDM assay using samples in which these parameters were previously determined using conventional approaches. We found that assays for bat species identification, insect diet, parasite diversity, and genotype were both sensitive and accurate, the assay to detect WNS was highly sensitive but requires careful sample processing steps to ensure the reliability of results, while assays for nectivorous diet and sex showed lower sensitivity. MDM was able to quantify multiple data classes from fecal samples simultaneously, and results were consistent whether we included assays for a single data class or multiple data classes. Overall, MDM is a useful approach that employs noninvasive sampling and a customizable suite of assays to gain important and largely accurate information on bat ecology and population dynamics.

Polygala lewtonii is a federally endangered, amphicarpic plant with a mixed mating system and three types of flowers: (1) aboveground, chasmogamous flowers (i.e., open-pollinated; CH), (2) aboveground, cleistogamous flowers (i.e., closed, selfing; CL) and (3) CL flowers on belowground stems (amphicarpy). Aboveground seeds are ant-dispersed, whereas belowground seeds are spaced across the length of the rhizome. Here, we collected individuals of P. lewtonii at both range-wide and fine geographic scales and genotyped them at 11 microsatellite loci. We analyzed patterns of genetic diversity and structure to understand: (1) the predominant mating system (selfing or outcrossing), (2) the movement of pollen and seeds across the landscape, and (3) the optimal strategy to conserve the full range of genetic variation. P. lewtonii reproduces predominantly by selfing or bi-parental inbreeding, but reproduction occurred through each of the three flower types. Some individuals produced by selfing/inbreeding were tightly clustered spatially, and were likely produced either by belowground flowers or by aboveground flowers with limited seed dispersal. Other selfed/inbred individuals were spatially separated (maximum of 15 m), and were likely produced by aboveground flowers followed by seed dispersal by ants. Fine-scale patterns of genetic structure indicate that some gene flow is occurring among aboveground CH flowers but both pollen and outcrossed seeds are moving limited distances (maximum of 0.5 km). Because genetic variation is structured at a fine spatial scale, protecting many populations is necessary to fully conserve the genetic variation in P. lewtonii. Conservation seed banking, if accompanied by research on seed germination requirements, may also contribute to the effective protection of genetic variation in P. lewtonii.