An official website of the United States government
Dinitrogen (N2) fixation is carried out by specialized microbes, called diazotrophs, and is a major source of nitrogen supporting primary production in oligotrophic oceans. One of the best-characterized diazotroph habitats is the North Pacific Subtropical Gyre (NPSG), where warm, chronically N-limited surface waters promote year-round N2fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N2fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (TrichodesmiumandRichelia), unicellular free-livingCrocosphaera, and the UCYN-A/haptophyte symbiosis, based on using microscopy and quantitative PCR (qPCR). However, the diazotroph community in this ecosystem is diverse and includes non-cyanobacterial diazotrophs (NCDs). We investigated the diversity, depth distributions, and seasonality of diazotroph communities at Stn. ALOHA using high throughput sequencing (HTS) ofnifHgene fragments from samples collected throughout the euphotic zone (0-175 m) at near-monthly intervals from June 2013 to July 2016. The UCYN-A symbioses andTrichodesmiumsp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a newCrocosphaera-like species, and several NCDs affiliated with γ- and δ-proteobacteria. Notably, some of the NCDs appear to be stable components of the community at Stn. ALOHA, having also been reported in prior studies. Depth and temporal patterns in microdiversity within two major diazotroph groups (Trichodesmiumand UCYN-A) suggested that sub-populations are adapted to time- and depth-dependent environmental variation. A network analysis of the upper euphotic (0-75 m) HTS data identified two modules that reflect a diazotroph community structure with seasonal turnover between UCYN-A/Gamma A, andTrichodesmium/Crocosphaera. It also reveals the seasonality of several important cyanobacteria and NCDs about which little is known, including a putative δ-proteobacterial phylotype originally discovered at Stn. ALOHA. Collectively, these results underscore the importance of couplingnifHgene HTS with other molecular techniques to obtain a comprehensive view of diazotroph community composition in the marine environment and reveal several understudied diazotroph groups that may contribute to N2fixation in the NPSG.
more »
« less
This content will become publicly available on July 29, 2026
Global phylogeography and microdiversity of the marine diazotrophic photoautotrophs Trichodesmium and UCYN-A
ABSTRACT Photoautotrophic diazotrophs, specifically the generaTrichodesmiumand UCYN-A, play a pivotal role in marine nitrogen cycling through their capacity for nitrogen fixation. Despite their global distribution, the microdiversity and environmental drivers of these diazotrophs remain underexplored. This study provides a comprehensive analysis of the global diversity and distribution ofTrichodesmiumand UCYN-A using the nitrogenase gene (nifH) as a genetic marker. We sequenced 954 samples from the Pacific, Atlantic, and Indian Oceans as part of the Bio-GO-SHIP project. Our results reveal significant phylogenetic and biogeographic differences between and within the two genera.Trichodesmiumexhibited greater microdiversity compared to UCYN-A, with clades showing region-specific distribution.Trichodesmiumclades were primarily influenced by temperature and nutrient availability. They were particularly frequent in regions of phosphorus stress. In contrast, UCYN-A was most frequently observed in regions experiencing iron stress. UCYN-A clades demonstrated more homogeneous distributions, with a single sequence variant within the UCYN-A1 clade dominating across varied environments. The biogeographic patterns and environmental correlations ofTrichodesmiumand UCYN-A highlight the role of microdiversity in their ecological adaptation and reflect their different ecological strategies. These findings underscore the importance of characterizing the global patterns of fine-scale genetic diversity to better understand the functional roles and distribution of marine nitrogen-fixing photoautotrophs.IMPORTANCEThis study provides insights into the global diversity and distribution of nitrogen-fixing photoautotrophs, specificallyTrichodesmiumand UCYN-A. We sequenced 954 oceanic samples of thenifHnitrogenase gene and uncovered significant differences in microdiversity and environmental associations between these genera.Trichodesmiumshowed high levels of sequence diversity and region-specific clades influenced by temperature and nutrient availability. In contrast, UCYN-A exhibited a more uniform distribution, thriving in iron-stressed regions. Quantifying these fine-scale genetic variations enhances our knowledge of their ecological roles and adaptations, emphasizing the need to characterize the genetic diversity of marine nitrogen-fixing prokaryotes.
more »
« less
- PAR ID:
- 10635989
- Editor(s):
- Campbell, Barbara J
- Publisher / Repository:
- ASM Journals
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 10
- Issue:
- 7
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Inputs of new nitrogen by cyanobacterial diazotrophs are critical to ocean ecosystem structure and function. Relative to other ocean regions, there is a lack of data on the distribution of these microbes in the western South Atlantic. Here, the abundance of six diazotroph phylotypes: Trichodesmium , Crocosphaera , UCYN-A, Richelia associated with Rhizosolenia (Het-1) or Hemiaulus (Het-2), and Calothrix associated with Chaetoceros (Het-3) was measured by quantitative PCR (qPCR) of the nifH gene along a transect extending from the shelf-break to the open ocean along the Vitória-Trindade seamount chain (1200 km). Using nifH gene copies as a proxy for phylotype abundance, Crocosphaera signals were the most abundant, with a broad distribution throughout the study region. Trichodesmium signals were the second most abundant, with the greatest numbers confined to the warmer waters closer to the coast, and a significant positive correlation with temperature. The average signals for the host-associated diazotrophs (UCYN-A, Het-1, and Het-2) were consistently lower than for the other phylotypes. These findings expand measurements of cyanobacterial diazotroph distribution in the western South Atlantic, and provide a new resource to enhance modeling studies focused on patterns of nitrogen fixation in the global ocean.more » « less
-
Abstract Exploring the diversity of diazotrophs is key to understanding their role in supplying fixed nitrogen that supports marine productivity. A nested PCR assay using the universal primer set nifH1-nifH4, which targets the nitrogenase (nifH) gene, is a widely used approach for studying marine diazotrophs by amplicon sequencing. Metagenomics, direct sequencing of DNA without PCR, has provided complementary views of the diversity of marine diazotrophs. A significant fraction of the metagenome-derived nifH sequences (e.g. Planctomycete- and Proteobacteria-affiliated) were reported to have nucleotide mismatches with the nifH1-nifH4 primers, leading to the suggestion that nifH amplicon sequencing does not detect specific diazotrophic taxa and underrepresents diazotroph diversity. Here, we report that these mismatches are mostly located in a single-base at the 5′-end of the nifH4 primer, which does not impact detection of the nifH genes. This is demonstrated by the presence of nifH genes that contain the nucleotide mismatches in a recent compilation of global ocean nifH amplicon datasets, with high relative abundances detected in a variety of samples. While the metagenome- and metatranscriptome-derived nifH genes accounted for 4.4% of the total amplicon sequence variants from the global ocean nifH amplicon database, the corresponding amplicon sequence variants can have high relative abundances (accounting for 47% of the reads in the database). These analyses underscore that nifH amplicon sequencing using the nifH1-nifH4 primers is an important tool for studying diversity of marine diazotrophs, particularly as a complement to metagenomics which can provide taxonomic and metabolic information for some dominant groups.more » « less
-
Abstract Diazotrophic microorganisms regulate marine productivity by alleviating nitrogen limitation. However, we know little about the identity and activity of diazotrophs in deep-sea sediments, a habitat covering nearly two-thirds of the planet. Here, we identify candidate diazotrophs from Pacific Ocean sediments collected at 2893 m water depth using 15N-DNA stable isotope probing and a novel pipeline for nifH sequence analysis. Together, these approaches detect an unexpectedly diverse assemblage of active diazotrophs, including members of the Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria, and Deltaproteobacteria. Deltaproteobacteria, predominately members of the Desulfobacterales and Desulfuromonadales, are the most abundant diazotrophs detected, and display the most microdiversity of associated nifH sequences. Some of the detected lineages, including those within the Acidobacteria, have not previously been shown to fix nitrogen. The diazotrophs appear catabolically diverse, with the potential for using oxygen, nitrogen, iron, sulfur, and carbon as terminal electron acceptors. Therefore, benthic diazotrophy may persist throughout a range of geochemical conditions and provide a stable source of fixed nitrogen over geologic timescales. Our results suggest that nitrogen-fixing communities in deep-sea sediments are phylogenetically and catabolically diverse, and open a new line of inquiry into the ecology and biogeochemical impacts of deep-sea microorganisms.more » « less
-
Abstract. Marine dinitrogen (N2) fixation is a globally significant biogeochemical process carried out by a specialized group of prokaryotes (diazotrophs), yet our understanding of their ecology is constantly evolving. Although marine N2 fixation is often ascribed to cyanobacterial diazotrophs, indirect evidence suggests that non-cyanobacterial diazotrophs (NCDs) might also be important. One widely used approach for understanding diazotroph diversity and biogeography is polymerase chain reaction (PCR) amplification of a portion of the nifH gene, which encodes a structural component of the N2-fixing enzyme complex, nitrogenase. An array of bioinformatic tools exists to process nifH amplicon data; however, the lack of standardized practices has hindered cross-study comparisons. This has led to a missed opportunity to more thoroughly assess diazotroph diversity and biogeography, as well as their potential contributions to the marine N cycle. To address these knowledge gaps, a bioinformatic workflow was designed that standardizes the processing of nifH amplicon datasets originating from high-throughput sequencing (HTS). Multiple datasets are efficiently and consistently processed with a specialized DADA2 pipeline to identify amplicon sequence variants (ASVs). A series of customizable post-pipeline stages then detect and discard spurious nifH sequences and annotate the subsequent quality-filtered nifH ASVs using multiple reference databases and classification approaches. This newly developed workflow was used to reprocess nearly all publicly available nifH amplicon HTS datasets from marine studies and to generate a comprehensive nifH ASV database containing 9383 ASVs aggregated from 21 studies that represent the diazotrophic populations in the global ocean. For each sample, the database includes physical and chemical metadata obtained from the Simons Collaborative Marine Atlas Project (CMAP). Here we demonstrate the utility of this database for revealing global biogeographical patterns of prominent diazotroph groups and highlight the influence of sea surface temperature. The workflow and nifH ASV database provide a robust framework for studying marine N2 fixation and diazotrophic diversity captured by nifH amplicon HTS. Future datasets that target understudied ocean regions can be added easily, and users can tune parameters and studies included for their specific focus. The workflow and database are available, respectively, on GitHub (https://github.com/jdmagasin/nifH-ASV-workflow, last access: 21 January 2025; Morando et al., 2024c) and Figshare (https://doi.org/10.6084/m9.figshare.23795943.v2; Morando et al., 2024b).more » « less
