Grapevine bacterial communities display compartment-specific dynamics over space and time within the Central Valley of California.

BACKGROUND: Plant organs (compartments) host distinct microbiota which shift in response to variation in both development and climate. Grapevines are woody perennial crops that are clonally propagated and cultivated across vast geographic areas, and as such, their microbial communities may also reflect site-specific influences. These site-specific influences along with microbial differences across sites compose 'terroir', the environmental influence on wine produced in a given region. Commercial grapevines are typically composed of a genetically distinct root (rootstock) grafted to a shoot system (scion) which adds an additional layer of complexity via genome-to-genome interactions. RESULTS: To understand spatial and temporal patterns of bacterial diversity in grafted grapevines, we used 16S rRNA amplicon sequencing to quantify soil and compartment microbiota (berries, leaves, and roots) for grafted grapevines in commercial vineyards across three counties in the Central Valley of California over two successive growing seasons. Community composition revealed compartment-specific dynamics. Roots assembled site-specific bacterial communities that reflected rootstock genotype and environment influences, whereas bacterial communities of leaves and berries displayed associations with time. CONCLUSIONS: These results provide further evidence of a microbial terroir within the grapevine root systems but also reveal that the microbiota of above-ground compartments are only weakly associated with the local soil microbiome in the Central Valley of California.

X-ray imaging of 30 year old wine grape wood reveals cumulative impacts of rootstocks on scion secondary growth and Ravaz index.

Annual 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.

Increases in vein length compensate for leaf area lost to lobing in grapevine.

PREMISE: Leaf lobing and leaf size vary considerably across and within species, including among grapevines (Vitis spp.), some of the best-studied leaves. We examined the relationship between leaf lobing and leaf area across grapevine populations that varied in extent of leaf lobing. METHODS: We used homologous landmarking techniques to measure 2632 leaves across 2 years in 476 unique, genetically distinct grapevines from five biparental crosses that vary primarily in the extent of lobing. We determined to what extent leaf area explained variation in lobing, vein length, and vein to blade ratio. RESULTS: Although 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 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. CONCLUSIONS: For leaves with different extents of lobing but the same area, the 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.

Multifaceted DNA metabarcoding of guano to uncover multiple classes of ecological data in two different bat communities.

DNA contained in animal scat provides a wealth of information about the animal, and DNA metabarcoding of scat collections can provide key information about animal populations and communities. Next-generation DNA sequencing technologies and DNA metabarcoding provide an efficient means for obtaining information available in scat samples. We used multifaceted DNA metabarcoding (MDM) of noninvasively collected bat guano pellets from a Myotis lucifugus colony on Fort Drum Military Installation, New York, USA, and from two mixed-species bat roosts on Fort Huachuca Military Installation, Arizona, USA, to identify attributes such as bat species composition, sex ratios, diet, and the presence of pathogens and parasites. We successfully identified bat species for nearly 98% of samples from Fort Drum and 90% of samples from Fort Huachuca, and identified the sex for 84% and 67% of samples from these same locations, respectively. Species and sex identification matched expectations based on prior censuses of bat populations utilizing those roosts, though samples from some species were more or less common than anticipated within Fort Huachuca roosts. Nearly 62% of guano samples from Fort Drum contained DNA from Pseudogymnoascus destructans, where bats with wing damage from White-nose Syndrome were commonly observed. Putative dietary items were detected in a majority of samples from insectivorous bats on Fort Drum (81%) and Fort Huachuca (63%). A minority of guano samples identified as the nectarivorous Leptonycteris yerbabuenae (28%) provided DNA sequences from putative forage plant species. Finally, DNA sequences from both putative ecto- and endoparasite taxa were detected in 35% and 56% of samples from Fort Drum and Fort Huachuca, respectively. This study demonstrates that the combination of noninvasive sampling, DNA metabarcoding, and sample and locus multiplexing provide a wide array of data that are otherwise difficult to obtain.

Multi-dimensional leaf phenotypes reflect root system genotype in grafted grapevine over the growing season.

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.

Understanding how an amphicarpic species with a mixed mating system responds to fire: a population genetic approach.

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.

The phylogeographic history of a range disjunction in eastern North America: the role of post-glacial expansion into newly suitable 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.

Conservation genetics of the threatened plant species Physaria filiformis (Missouri bladderpod) reveals strong genetic structure and a possible cryptic species.

Understanding genetic diversity and structure in a rare species is critical for prioritizing both in situ and ex situ conservation efforts. One such rare species is Physaria 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 of P. filiformis is 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 of P. filiformis from across its geographical range and one population of Physaria gracilis for comparison and analyzed genetic diversity and structure. Populations of P. filiformis showed 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 of P. filiformis; future morphological analyses are needed to identify whether it merits recognition as a new, extremely rare species.

Grapevine Microbiota Reflect Diversity among Compartments and Complex Interactions within and among Root and Shoot Systems.

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 'Chambourcin', 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 '1103P' and 'SO4' 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.

Evaluating the efficacy of sample collection approaches and DNA metabarcoding for identifying the diversity of plants utilized by nectivorous bats.

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.

Multifaceted DNA metabarcoding: Validation of a noninvasive, next-generation approach to studying bat populations.

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.