skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Pitfalls and pointers: An accessible guide to marker gene amplicon sequencing in ecological applications
Abstract Next‐Generation Sequencing (NGS) is a powerful tool that has been rapidly adopted by many ecologists studying microbial communities. Despite the exciting demonstration of NGS technology as a tool for ecological research, cryptic pitfalls inherent to its use can obscure correct interpretation of NGS data. Here, we provide an accessible overview of a NGS process that uses marker gene amplicon sequences (MGAS) that will allow scientists, particularly community ecologists, to make appropriate methodological choices and understand limits on inference about community composition and diversity that can be drawn from MGAS data.We describe the MGAS pipeline, focusing specifically on cryptic sources of variation that have received less emphasis in the ecological literature, but which may substantially impact inference about microbial community diversity and composition. By simulating communities from published microbiome data, we demonstrate how these sources of variation can generate inaccurate or misleading patterns.We specifically highlight sample dilution without researcher awareness and lane‐to‐lane variability, two cryptic sources of variation arising during the MGAS pipeline. These sources of variation affect estimates of species presence and relative abundance, particularly for species with moderate to low abundances. Each of these sources of bias can lead to errors in the estimation of both absolute and relative abundance within, and turnover among, microbial communities.Awareness and understanding of what happens and, specifically, why it happens during MGAS generation is key to generating a strong dataset and building a robust community matrix. Requesting sample dilution information from the sequencing centre, including technical replicates across sequencing lanes, and understanding how sampling intensity and community taxa distribution patterns shape the measurement of community richness, evenness and diversity are critical for drawing correct ecological inferences using MGAS data.  more » « less
Award ID(s):
2129332
PAR ID:
10446139
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Methods in Ecology and Evolution
Volume:
13
Issue:
2
ISSN:
2041-210X
Page Range / eLocation ID:
p. 266-277
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract I asked whether Grime's triangle of competitive, stress tolerance and ruderal ecological strategies—which was originally developed for plants—applies to microbes.I conducted a synthesis of empirical studies that tested relationships among microbial traits presumed to define the competitive, stress tolerance and ruderal, and other ecological strategies.There was broad support for Grime's triangle. However, the ecological strategies were inconsistently linked to shifts in microbial communities under environmental changes like nitrogen and phosphorus addition, warming, drought, etc. We may be missing important ecological strategies that more closely influence microbial community composition under shifting environmental conditions.We may need to start by documenting changes in microbial communities in response to environmental conditions at fine spatiotemporal scales relevant for microbes. We can then develop empirically based ecological strategies, rather than modifying those based on plant ecology.Synthesis. Microbes appear to sort into similar ecological strategies as plants. However, these microbial ecological strategies do not consistently predict how community composition will shift under environmental change. By starting ‘from the ground up’, we may be able to delineate ecological strategies more relevant for microbes. 
    more » « less
  2. Summary Microbial communities can rapidly respond to stress, meaning plants may encounter altered soil microbial communities in stressful environments. These altered microbial communities may then affect natural selection on plants. Because stress can cause lasting changes to microbial communities, microbes may also cause legacy effects on plant selection that persist even after the stress ceases.To explore how microbial responses to stress and persistent microbial legacy effects of stress affect natural selection, we grewChamaecrista fasciculataplants in stressful (salt, herbicide, or herbivory) or nonstressful conditions with microbes that had experienced each of these environments in the previous generation.Microbial community responses to stress generally counteracted the effects of stress itself on plant selection, thereby weakening the strength of stress as a selective agent. Microbial legacy effects of stress altered plant selection in nonstressful environments, suggesting that stress‐induced changes to microbes may continue to affect selection after stress is lifted.These results suggest that soil microbes may play a cryptic role in plant adaptation to stress, potentially reducing the strength of stress as a selective agent and altering the evolutionary trajectory of plant populations. 
    more » « less
  3. Abstract Anthropogenic habitat fragmentation—the breaking up of natural landscapes—is a pervasive threat to biodiversity and ecosystem function world‐wide. Fragmentation results in a mosaic of remnant native habitat patches embedded in human‐modified habitat known as the ‘matrix’. By introducing novel environmental conditions in matrix habitats and reducing connectivity of native habitats, fragmentation can dramatically change how organisms experience their environment. The effects of fragmentation can be especially important in urban landscapes, which are expanding across the globe. Despite this surging threat and the importance of microbiomes for ecosystem services, we know very little about how fragmentation affects microbiomes and even less about their consequences for plant–microbe interactions in urban landscapes.By combining field surveys, microbiome sequencing and experimental mesocosms, we (1) investigated how microbial community diversity, composition and functional profiles differed between 15 native pine rockland fragments and the adjacent urban matrix habitat, (2) identified habitat attributes that explained significant variation in microbial diversity of native core habitat compared to urban matrix and (3) tested how changes in urbanized and low connectivity microbiomes affected plant community productivity.We found urban and native microbiomes differed substantively in diversity, composition and functional profiles, including symbiotic fungi decreasing 81% and pathogens increasing 327% in the urban matrix compared to native habitat. Furthermore, fungal diversity rapidly declined as native habitats became increasingly isolated, with ~50% of variation across the landscape explained by habitat connectivity alone. Interestingly, microbiomes from native habitats increased plant productivity by ~300% while urban matrix microbiomes had no effect, suggesting that urbanization may decouple beneficial plant–microbe interactions. In addition, microbial diversity within native habitats explained significant variation in plant community productivity, with higher productivity linked to more diverse microbiomes from more connected, larger fragments.Synthesis. Taken together, our study not only documents significant changes in microbial diversity, composition and functions in the urban matrix, but also supports that two aspects of habitat fragmentation—the introduction of a novel urban matrix and reduced habitat connectivity—disrupt microbial effects on plant community productivity, highlighting preservation of native microbiomes as critical for productivity in remnant fragments. 
    more » « less
  4. Summary Interactions between plants and soil fungi and bacteria are ubiquitous and have large effects on individual plant fitness. However, the degree to which spatial variation in soil microbial communities modulates plant species’ distributions remains largely untested.Using the California native plantClarkia xantianassp.xantianawe paired glasshouse and field reciprocal transplants of plant populations and soils to test whether plant–microbe interactions affect the plant’s geographic range limit and whether there is local adaptation between plants and soil microbe communities.In the field and glasshouse, one of the two range interior inocula had a positive effect on plant fitness. In the field, this benefit was especially pronounced at the range edge and beyond, suggesting possible mutualist limitation. In the glasshouse, soil inocula from beyond‐range tended to increase plant growth, suggesting microbial enemy release beyond the range margin. Amplicon sequencing revealed stark variation in microbial communities across the range boundary.Plants dispersing beyond their range limit are likely to encounter novel microbial communities. InC. x. xantiana, our results suggest that range expansion may be facilitated by fewer pathogens, but could also be hindered by a lack of mutualists. Both negative and positive plant–microbe interactions will likely affect contemporary range shifts. 
    more » « less
  5. Summary Stress often induces plant trait plasticity, and microbial communities also alter plant traits. Therefore, it is unclear how much plasticity results from direct plant responses to stress vs indirect responses due to stress‐induced changes in soil microbial communities.To test how microbes and microbial community responses to stress affect the ecology and potentially the evolution of plant plasticity, I grew plants in four stress environments (salt, herbicide, herbivory, and no stress) with microbes that had responded to these same environments or with sterile inoculant.Plants delayed flowering under stress only when inoculated with live microbial communities, and this plasticity was maladaptive. However, microbial communities responded to stress in ways that accelerated flowering across all environments. Microbes also affected the expression of genetic variation for plant flowering time and specific leaf area, as well as genetic variation for plasticity of both traits, and disrupted a positive genetic correlation for plasticity in response to herbicide and herbivory stress, suggesting that microbes may affect the pace of plant evolution.Together, these results highlight an important role for soil microbes in plant plastic responses to stress and suggest that microbes may alter the evolution of plant plasticity. 
    more » « less