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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Integrating Systems and Synthetic Biology to Understand and Engineer Microbiomes

Microbiomes are complex and ubiquitous networks of microorganisms whose seemingly limitless chemical transformations could be harnessed to benefit agriculture, medicine, and biotechnology. Here, the spatial and temporal changes in microbiome composition and function are influenced by a multitude of molecular and ecological factors. This complexity yields both versatility and challenges in designing synthetic microbiomes and perturbing natural microbiomes in controlled, predictable ways. In this review, we describe factors that give rise to emergent spatial and temporal microbiome properties and the meta-omics and computational modeling tools that can be used to understand microbiomes at the cellular and system levels. We also describe strategies for designing and engineering microbiomes to enhance or build novel functions. Throughout the review,we discuss key knowledge and technology gaps for elucidating the networks and deciphering key control points for microbiome engineering, and highlight examples where multiple omics and modeling approaches can be integrated to address these gaps.

42 ENGINEERING↗
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2025

Enhancing Hydrogen Production from Bioenergy Crops via Photoreforming

Photoreforming perennial bioenergy crops (willow, Miscanthus , and poplar) has the potential to produce H 2 with reduced environmental impacts. To understand the compositional effects of the biomass on the average rate of H 2 production over the first 30 min of reaction ( r H 2 ), the r H 2 values of model biomass component (i.e., cellulose, hemicellulose, and lignin) mixtures were compared with those from the raw biomass. The higher cellulose or hemicellulose content in multicomponent mixtures resulted in higher r H 2 , whereas lignin reduced the hydrogen production rate. However, with raw biomass, the ratio of biomass components alone did not determine the r H 2 via photoreforming, with rates of hydrogen production for different varieties of willow ranging between 1.9 μmol h -1 and 12.3 μmol h -1 , 11.8 μmol h -1 for a poplar, and 6.8 μmol h -1 for a miscanthus biomass. In addition, comparable r H 2 values of raw poplar and its extracted cellulose via an IonoSolv treatment indicated the possibility of using raw biomass materials without delignification for generating H 2 via photoreforming. Importantly, r H 2 was positively correlated with the interaction between water and the biomass, as assessed by NMR relaxation via an examination of the T 1 /T 2 ratio. A stronger water-biomass interaction resulted in a higher r H 2 . Genetic modification of biomass has been suggested as a putative way to improve the r H 2 of biomass with an enhanced interaction with water. This research enhances the understanding of factors influencing H 2 production from lignocellulosic biomass by photoreforming and supports the breeding and management of perennial biomass crops to maximize H 2 yields while minimizing land area requirements.

biomass↗
DOE OSTI2024

Diverse fates of ancient horizontal gene transfers in extremophilic red algae

Abstract Horizontal genetic transfer (HGT) is a common phenomenon in eukaryotic genomes. However, the mechanisms by which HGT‐derived genes persist and integrate into other pathways remain unclear. This topic is of significant interest because, over time, the stressors that initially favoured the fixation of HGT may diminish or disappear. Despite this, the foreign genes may continue to exist if they become part of a broader stress response or other pathways. The conventional model suggests that the acquisition of HGT equates to adaptation. However, this model may evolve into more complex interactions between gene products, a concept we refer to as the ‘Integrated HGT Model’ (IHM). To explore this concept further, we studied specialized HGT‐derived genes that encode heavy metal detoxification functions. The recruitment of these genes into other pathways could provide clear examples of IHM. In our study, we exposed two anciently diverged species of polyextremophilic red algae from the Galdieria genus to arsenic and mercury stress in laboratory cultures. We then analysed the transcriptome data using differential and coexpression analysis. Our findings revealed that mercury detoxification follows a ‘one gene‐one function’ model, resulting in an indivisible response. In contrast, the ars H gene in the arsenite response pathway demonstrated a complex pattern of duplication, divergence and potential neofunctionalization, consistent with the IHM. Our research sheds light on the fate and integration of ancient HGTs, providing a novel perspective on the ecology of extremophiles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Nonparallel transcriptional divergence during parallel adaptation

Abstract How underlying mechanisms bias evolution toward predictable outcomes remains an area of active debate. In this study, we leveraged phenotypic plasticity and parallel adaptation across independent lineages of Trinidadian guppies ( Poecilia reticulata ) to assess the predictability of gene expression evolution during parallel adaptation. Trinidadian guppies have repeatedly and independently adapted to high‐ and low‐predation environments in the wild. We combined this natural experiment with a laboratory breeding design to attribute transcriptional variation to the genetic influences of population of origin and developmental plasticity in response to rearing with or without predators. We observed substantial gene expression plasticity, as well as the evolution of expression plasticity itself, across populations. Genes exhibiting expression plasticity within populations were more likely to also differ in expression between populations, with the direction of population differences more likely to be opposite those of plasticity. While we found more overlap than expected by chance in genes differentially expressed between high‐ and low‐predation populations from distinct evolutionary lineages, the majority of differentially expressed genes were not shared between lineages. Our data suggest alternative transcriptional configurations associated with shared phenotypes, highlighting a role for transcriptional flexibility in the parallel phenotypic evolution of a species known for rapid adaptation.

Fischer, Eva K.↗
DOE OSTI2024

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA). Final report

The goal of the Stahl lab is to use a combination of isotopic, genomic, and activity-based analyses in the field, of selected field isolates, and defined synthetic communities to identify sources and sinks of different nitrogen species in the field, focusing on the FRC at the ORNL. The most general research objective is to characterize variants in the pathway for denitrification among field isolates and synthetic microbial assemblies as a foundation for predicting the biotic and abiotic controls of nitrogen and carbon transformations in the field. Specifically, we have focused on characterizing environmental controls of nitrous oxide emissions, an important greenhouse gas and ozone depleting molecule. Studies have mapped the flux of nitrous oxide and controlling genetic elements within the FRC subsurface, indicating the importance of different enzyme variants in nitrous oxide production and consumption in controlling surface emissions. We have developed methods to quantify and catalog the isotopic signatures imparted on nitrous oxide by different nitrous oxide reductase variants in isolated organisms and model synthetic communities. We have developed methods to quantify and catalog the nitrous oxide affinity of field relevant isolates and their biomass yield when respiring nitrous oxide on different carbon substrates. We continue to expand the collection of field-relevant microbial isolates within ENIGMA by focused enrichment and isolation from field material. These objectives have been facilitated by the development of new technologies, including: 1) the development of a multiplexed automated optical density measurement system for quantifying growth kinetics of microorganisms dependent on gaseous substrates in monoculture or gas exchange in synthetic communities and 2) the development of a microcalorimetric analysis format to quantify the impact of environmental stress on microbial maintenance energy. Since field relevant processes are mediated by assemblies of organisms interacting with each other and their local environment, we have also collaborated with other ENIGMA investigators on the assembly and characterization of synthetic communities. These studies have evaluated the role of organisms lacking a complete pathway for denitrification in the control of field processes, measuring process rates of assemblies of organisms that individually lack the full denitrification pathway but mediate the complete denitrification when growing together. These efforts have so far resulted in the publication of 18 peer reviewed papers ranging from ecological and physiological drivers of pathway optimization to field-scale process characterization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Mitochondrial Genomes of the United States Distribution of Gray Fox (Urocyon cinereoargenteus) Reveal a Major Phylogeographic Break at the Great Plains Suture Zone

We examined phylogeographic structure in gray fox (Urocyon cinereoargenteus) across the United States to identify the location of secondary contact zone(s) between eastern and western lineages and investigate the possibility of additional cryptic intraspecific divergences. We generated and analyzed complete mitochondrial genome sequence data from 75 samples and partial control region mitochondrial DNA sequences from 378 samples to investigate levels of genetic diversity and structure through population- and individual-based analyses including estimates of divergence (FST and SAMOVA), median joining networks, and phylogenies. We used complete mitochondrial genomes to infer phylogenetic relationships and date divergence times of major lineages of Urocyon in the United States. Despite broad-scale sampling, we did not recover additional major lineages of Urocyon within the United States, but identified a deep east-west split (~0.8 million years) with secondary contact at the Great Plains Suture Zone and confirmed the Channel Island fox (Urocyon littoralis) is nested within U. cinereoargenteus. Genetic diversity declined at northern latitudes in the eastern United States, a pattern concordant with post-glacial recolonization and range expansion. Beyond the east-west divergence, morphologically-based subspecies did not form monophyletic groups, though unique haplotypes were often geographically limited. Gray foxes in the United States displayed a deep, cryptic divergence suggesting taxonomic revision is needed. Secondary contact at a common phylogeographic break, the Great Plains Suture Zone, where environmental variables show a sharp cline, suggests ongoing evolutionary processes may reinforce this divergence. Follow-up study with nuclear markers should investigate whether hybridization is occurring along the suture zone and characterize contemporary population structure to help identify conservation units. Comparative work on other wide-ranging carnivores in the region should test whether similar evolutionary patterns and processes are occurring.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Expanding standards in viromics: in silico evaluation of dsDNA viral genome identification, classification, and auxiliary metabolic gene curation

Viruses influence global patterns of microbial diversity and nutrient cycles. Though viral metagenomics (viromics), specifically targeting dsDNA viruses, has been critical for revealing viral roles across diverse ecosystems, its analyses differ in many ways from those used for microbes. To date, viromics benchmarking has covered read pre-processing, assembly, relative abundance, read mapping thresholds and diversity estimation, but other steps would benefit from benchmarking and standardization. Here we use in silico-generated datasets and an extensive literature survey to evaluate and highlight how dataset composition (i.e., viromes vs bulk metagenomes) and assembly fragmentation impact (i) viral contig identification tool, (ii) virus taxonomic classification, and (iii) identification and curation of auxiliary metabolic genes (AMGs). The in silico benchmarking of five commonly used virus identification tools show that gene-content-based tools consistently performed well for long (≥3 kbp) contigs, while k -mer- and blast-based tools were uniquely able to detect viruses from short (≤3 kbp) contigs. Notably, however, the performance increase of k -mer- and blast-based tools for short contigs was obtained at the cost of increased false positives (sometimes up to ~5% for virome and ~75% bulk samples), particularly when eukaryotic or mobile genetic element sequences were included in the test datasets. Furthermore, for viral classification, variously sized genome fragments were assessed using gene-sharing network analytics to quantify drop-offs in taxonomic assignments, which revealed correct assignations ranging from ~95% (whole genomes) down to ~80% (3 kbp sized genome fragments). A similar trend was also observed for other viral classification tools such as VPF-class, ViPTree and VIRIDIC, suggesting that caution is warranted when classifying short genome fragments and not full genomes. Finally, we highlight how fragmented assemblies can lead to erroneous identification of AMGs and outline a best-practices workflow to curate candidate AMGs in viral genomes assembled from metagenomes. Together, these benchmarking experiments and annotation guidelines should aid researchers seeking to best detect, classify, and characterize the myriad viruses ‘hidden’ in diverse sequence datasets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Winter connectivity and leapfrog migration in a migratory passerine

Abstract Technological advances in migratory tracking tools have revealed a remarkable diversity in migratory patterns. One such pattern is leapfrog migration, where individuals that breed further north migrate to locations further south. Here, we analyzed migration patterns in the Painted Bunting ( Passerina ciris ) using a genetic‐based approach. We started by mapping patterns of genetic variation across geographic space (called a genoscape) using 386 individuals from 25 populations across the breeding range. We then genotyped an additional 230 samples from 31 migration stopover locations and 178 samples from 16 wintering locations to map patterns of migratory connectivity. Our analyses of genetic variation across the breeding range show the existence of four genetically distinct groups within the species: Eastern, Southwestern, Louisiana, and Central groups. Subsequent assignment of migrating and wintering birds to genetic groups illustrated that birds from the Central group migrated during the fall via western Mexico or southern Texas, spent the winter from northeastern Mexico to Panama, and migrated north via the Gulf Coast of Mexico. While Louisiana birds overlapped with Central birds on their spring migratory routes along the Gulf Coast, we found that Louisiana birds had a more restricted wintering distribution in the Yucatan Peninsula and Central America. Further estimation of the straight‐line distance from the predicted breeding location to the wintering location revealed that individuals sampled at lower winter latitudes traveled to greater distances (i.e., the predicted breeding area was further north; p > .001), confirming that these species exhibit a leapfrog migration pattern. Overall, these results demonstrate the utility of a genoscape‐based approach for identifying range‐wide patterns of migratory connectivity such as leapfrog migration with a high degree of clarity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Comparative genome analyses suggest a hemibiotrophic lifestyle and virulence differences for the beech bark disease fungal pathogens Neonectria faginata and Neonectria coccinea

Abstract Neonectria faginata and Neonectria coccinea are the causal agents of the insect-fungus disease complex known as beech bark disease (BBD), known to cause mortality in beech forest stands in North America and Europe. These fungal species have been the focus of extensive ecological and disease management studies, yet less progress has been made toward generating genomic resources for both micro- and macro-evolutionary studies. Here, we report a 42.1 and 42.7 mb highly contiguous genome assemblies of N. faginata and N. coccinea, respectively, obtained using Illumina technology. These species share similar gene number counts (12,941 and 12,991) and percentages of predicted genes with assigned functional categories (64 and 65%). Approximately 32% of the predicted proteomes of both species are homologous to proteins involved in pathogenicity, yet N. coccinea shows a higher number of predicted mitogen-activated protein kinase genes, virulence determinants possibly contributing to differences in disease severity between N. faginata and N. coccinea. A wide range of genes encoding for carbohydrate-active enzymes capable of degradation of complex plant polysaccharides and a small number of predicted secretory effector proteins, secondary metabolite biosynthesis clusters and cytochrome oxidase P450 genes were also found. This arsenal of enzymes and effectors correlates with, and reflects, the hemibiotrophic lifestyle of these two fungal pathogens. Phylogenomic analysis and timetree estimations indicated that the N. faginata and N. coccinea species divergence may have occurred at ∼4.1 million years ago. Differences were also observed in the annotated mitochondrial genomes as they were found to be 81.7 kb (N. faginata) and 43.2 kb (N. coccinea) in size. The mitochondrial DNA expansion observed in N. faginata is attributed to the invasion of introns into diverse intra- and intergenic locations. These first draft genomes of N. faginata and N. coccinea serve as valuable tools to increase our understanding of basic genetics, evolutionary mechanisms and molecular physiology of these two nectriaceous plant pathogenic species.

Salgado-Salazar, Catalina↗
DOE OSTI2021

Natural soil microbiome variation affects spring foliar phenology with consequences for plant productivity and climate-driven range shifts

Identifying the potential for natural soil microbial communities to predictably affect complex plant traits is an important frontier in climate change research. Plant phenology varies with environmental and genetic factors, but few studies have examined if the soil microbiome interacts with plant population differentiation to affect phenology and ecosystem function. We compared soil microbial variation in a widespread tree species (Populus angustifolia) with different soil inoculum treatments in a common garden environment to test how the soil microbiome affects spring foliar phenology and subsequent biomass growth. We hypothesized and show that: 1) soil bacterial and fungal communities vary with tree conditioning from different populations and elevations, 2) this soil community variation influences patterns of foliar phenology and plant growth across populations and elevation gradients, and 3) transferring lower elevation plant genotypes to higher elevation soil communities delayed foliar phenology, thereby shortening the growing season and reducing annual biomass production. Our findings show the importance of plant-soil interactions that help shape the timing of tree foliar phenology and productivity. Here, these geographic patterns in plant population x microbiome interactions also broaden our understanding of how soil communities impact plant phenotypic variation across key climate change gradients, with consequences for ecosystem functioning.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Life strategies for Aminicenantia in subseafloor oceanic crust

Abstract After decades studying the microbial “deep biosphere” in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use “primordial” siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.

Environmental Sciences & Ecology↗
DOE OSTI2024

Dynamic nitrogen fixation in an aerobic endophyte of Populus

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte–poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15 N 2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant–endophyte interactions.

15 N-tracking metabolomics↗
DOE OSTI2024

Exon disruptive variants in Populus trichocarpa associated with wood properties exhibit distinct gene expression patterns

Abstract Forest trees may harbor naturally occurring exon disruptive variants (DVs) in their gene sequences, which potentially impact important ecological and economic phenotypic traits. However, the abundance and molecular regulation of these variants remain largely unexplored. Here, 24,420 DVs were identified by screening 1014Populus trichocarpafull genomes. The identified DVs were predominantly heterozygous with allelic frequencies below 5% (only 26% of DVs had frequencies greater than 5%). Using common garden‐grown trees, DVs were assessed for gene expression variation in the developing xylem, revealing that their gene expression can be significantly altered, particularly for homozygous DVs (in the range of 27%–38% of cases depending on the studied common garden). DVs were further investigated for their correlations with 13 wood quality traits, revealing that, among the 148 discovered DV associations, 15 correlated with more than one wood property and six genes had more than one DV in their coding sequences associated with wood traits. Approximately one‐third of DVs correlated with wood property variation also showed significant gene expression variation, confirming their non‐spurious impact. These findings offer potential avenues for targeted introduction of homozygous mutations using tree biotechnology, and while the exact mechanisms by which DVs may directly influence wood formation remain to be unraveled, this study lays the groundwork for further investigation.

Genetics & Heredity↗
DOE OSTI2022

Data from: "Lab and Field Warming Similarly Advance Germination Date and Limit Germination Rate for High and Low Elevation Provenances of Two Widespread Subalpine Conifers"

This archive contains data used to draw conclusions in “Lab and Field Warming Similarly Advance Germination Date and Limit Germination Rate for High and Low Elevation Provenances of Two Widespread Subalpine Conifers”, by Kueppers et al. 2017. Cone collection and field experiments were conducted on Niwot Ridge, Colorado, with the latter completed within the Alpine Treeline Warming Experiment (ATWE). Laboratory germination experiments were conducted at the University of California Merced. Geospatial files are also provided for additional geographical context.There are four data file formats in this package: comma-separated values (.csv), Microsoft Excel (.xlsx), keyhole markup language (.kml), and ESRI shapefile (.shp). The latter two are geospatial file formats. .csv files can be opened using any simple text editor program such as TextEdit (Mac) and Notepad (Windows), and .xlsx files can be opened using Microsoft Excel or Google Sheets. The .kml file contains coordinate data, and is compatible with Google Earth and Google Maps. ESRI shapefiles (.shp) are compatible with any geospatial software able to read the file type, such as QGIS and ESRI’s ArcGIS Suite. This data user’s guide is available in as a .pdf, and can be opened using software such as Adobe Acrobat Reader.-----------------------------------------------------------------------------------------------------------------------------------Accurately predicting upslope shifts in subalpine tree ranges with warming requires understanding how future forest populations will be affected by climate change, as these are the seed sources for new tree line and alpine populations. Early life history stages are particularly sensitive to climate and are also influenced by genetic variation among populations. We tested the climate sensitivity of germination and initial development for two widely distributed subalpine conifers, using controlled-environment growth chambers with one temperature regime from subalpine forest in the Colorado Rocky Mountains and one 5 °C warmer, and two soil moisture levels. We tracked germination rate and timing, rate of seedling development, and seedling morphology for two seed provenances separated by ~300 m elevation. Warming advanced germination timing and initial seedling development by a total of ~2 weeks, advances comparable to mean differences between provenances. Advances were similar for both provenances and species; however, warming reduced the overall germination rate, as did low soil moisture, only for Picea engelmannii. A three-year field warming and watering experiment planted with the same species and provenances yielded responses qualitatively consistent with the lab trials. Together these experiments indicate that in a warmer, drier climate, P. engelmannii germination, and thus regeneration, could decline, which could lead to declining subalpine forest populations, while Pinus flexilis forest populations could remain robust as a seed source for upslope range shifts.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Signatures of local nitrogen adaptation in the Brachypodium distachyon root microbiome

Plants associate with diverse microbiomes that impact their fitness, yet the contribution of the microbiome to plant adaptation is uncertain. As plant recruitment of its microbiome can be both highly variable and genetically determined, we hypothesized this recruitment process may be the result of adaptive evolution, and contributing to plant local adaptation. We investigated the evolution and adaptive benefit of plant–microbiome recruitment by characterizing the rhizosphere communities across a genotypic panel of Brachypodium distachyon in a common garden experiment. By linking microbial communities to their host genotype's historic environment, we identified signatures of selection on plant–microbiome recruitment. Plant–microbiome composition was significantly correlated with the host genotype's historic environment, with enrichment of microbial traits aligned to local resource conditions. For example, genotypes from low-nitrogen environments recruited communities enriched in nitrogen acquisition traits. In a complementary experiment evaluating plant nitrogen response, these same genotypes were well-adapted to low-nitrogen environments, contingent on the presence of key nitrogen-cycling microbes. These results suggest that local adaptation in plants may partially be mediated by recruitment of beneficial microbiomes. This perspective suggests that plant adaptation may be an emergent property of host–microbe interactions, where evolutionary responses favor traits that promote recruitment of locally beneficial microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Merging Fungal and Bacterial Community Profiles via an Internal Control

Integrated measurements of fungi and bacteria are critical to understand how interactions between these taxa drive key processes in ecosystems ranging from soils to animal guts. High-throughput amplicon sequencing is commonly used to census microbiomes, but the genetic markers targeted for fungi and bacteria (typically ribosomal regions) are domain-specific so profiling must be performed separately, obscuring relationships between these groups. To solve this problem, we developed a spike-in method with an internal control (IC) construct containing primer sites commonly used for bacterial and fungal taxonomic profiling. The internal control offers several advantages: estimation of absolute abundances, estimation of fungal to bacterial ratios (F:B), integration of bacterial and fungal profiles for holistic community analysis, and lower costs compared to other quantitation methods. To validate the IC as a scaling method, we compared IC-derived measures of F:B to measures from quantitative PCR (qPCR) using a commercial mock community (the ZymoBiomic Microbial Community DNA Standard II, containing two fungi and eight bacteria) and complex environmental samples. For both the mock community and the environmental samples, the IC produced F:B values that were statistically consistent with qPCR. Merging the environmental fungal and bacterial profiles based on the IC-derived F:B values revealed new relationships among samples in terms of community similarity. As a result, this IC method is the first spike-in method to employ a single construct for cross-domain amplicon sequencing, offering more reliable measurements.

59 BASIC BIOLOGICAL SCIENCES↗