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1. Isolation, growth, and nitrogen fixation rates of the Hemiaulus-Richelia (diatom-cyanobacterium) symbiosis in culture

   https://doi.org/10.7717/peerj.10115  

   Pyle, Amy E.; Johnson, Allison M.; Villareal, Tracy A. (January 2020, PeerJ)

   Nitrogen fixers (diazotrophs) are often an important nitrogen source to phytoplankton nutrient budgets in N-limited marine environments. Diazotrophic symbioses between cyanobacteria and diatoms can dominate nitrogen-fixation regionally, particularly in major river plumes and in open ocean mesoscale blooms. This study reports the successful isolation and growth in monocultures of multiple strains of a diatom-cyanobacteria symbiosis from the Gulf of Mexico using a modified artificial seawater medium. We document the influence of light and nutrients on nitrogen fixation and growth rates of the host diatom Hemiaulus hauckii Grunow together with its diazotrophic endosymbiont Richelia intracellularis Schmidt, as well as less complete results on the Hemiaulus membranaceus - R. intracellularis symbiosis. The symbioses rates reported here are for the joint diatom-cyanobacteria unit. Symbiont diazotrophy was sufficient to support both the host diatom and cyanobacteria symbionts, and the entire symbiosis replicated and grew without added nitrogen. Maximum growth rates of multiple strains of H. hauckii symbioses in N-free medium with N 2 as the sole N source were 0.74–0.93 div d −1 . Growth rates followed light saturation kinetics in H. hauckii symbioses with a growth compensation light intensity (E C ) of 7–16 µmol m −2 s −1 and saturation light level (E K ) of 84–110 µmol m −2 s −1 . Nitrogen fixation rates by the symbiont while within the host followed a diel pattern where rates increased from near-zero in the scotophase to a maximum 4–6 h into the photophase. At the onset of the scotophase, nitrogen-fixation rates declined over several hours to near-zero values. Nitrogen fixation also exhibited light saturation kinetics. Maximum N 2 fixation rates (84 fmol N 2 heterocyst −1 h −1 ) in low light adapted cultures (50 µmol m −2 s − 1) were approximately 40–50% of rates (144–154 fmol N 2 heterocyst −1 h −1 ) in high light (150 and 200 µmol m −2 s −1 ) adapted cultures. Maximum laboratory N 2 fixation rates were ~6 to 8-fold higher than literature-derived field rates of the H. hauckii symbiosis. In contrast to published results on the Rhizosolenia-Richelia symbiosis, the H. hauckii symbiosis did not use nitrate when added, although ammonium was consumed by the H. hauckii symbiosis. Symbiont-free host cell cultures could not be established; however, a symbiont-free H. hauckii strain was isolated directly from the field and grown on a nitrate-based medium that would not support DDA growth. Our observations together with literature reports raise the possibility that the asymbiotic H. hauckii are lines distinct from an obligately symbiotic H. hauckii line. While brief descriptions of successful culture isolation have been published, this report provides the first detailed description of the approaches, handling, and methodologies used for successful culture of this marine symbiosis. These techniques should permit a more widespread laboratory availability of these important marine symbioses. 

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2. Seasonal and spatial patterns in diazotroph community composition at Station ALOHA

   https://doi.org/10.3389/fmars.2023.1130158  

   Turk-Kubo, Kendra A.; Henke, Britt A.; Gradoville, Mary R.; Magasin, Jonathan D.; Church, Matthew J.; Zehr, Jonathan P. (May 2023, Frontiers in Marine Science)

   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. 

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3. UCYN-A/haptophyte symbioses dominate N2 fixation in the Southern California Current System

   https://doi.org/10.1038/s43705-021-00039-7  

   Turk-Kubo, Kendra A.; Mills, Matthew M.; Arrigo, Kevin R.; van Dijken, Gert; Henke, Britt A.; Stewart, Brittany; Wilson, Samuel T.; Zehr, Jonathan P. (December 2021, ISME Communications)

   Abstract The availability of fixed nitrogen (N) is an important factor limiting biological productivity in the oceans. In coastal waters, high dissolved inorganic N concentrations were historically thought to inhibit dinitrogen (N 2 ) fixation, however, recent N 2 fixation measurements and the presence of the N 2 -fixing UCYN-A/haptophyte symbiosis in nearshore waters challenge this paradigm. We characterized the contribution of UCYN-A symbioses to nearshore N 2 fixation in the Southern California Current System (SCCS) by measuring bulk community and single-cell N 2 fixation rates, as well as diazotroph community composition and abundance. UCYN-A1 and UCYN-A2 symbioses dominated diazotroph communities throughout the region during upwelling and oceanic seasons. Bulk N 2 fixation was detected in most surface samples, with rates up to 23.0 ± 3.8 nmol N l −1  d −1 , and was often detected at the deep chlorophyll maximum in the presence of nitrate (>1 µM). UCYN-A2 symbiosis N 2 fixation rates were higher (151.1 ± 112.7 fmol N cell −1  d −1 ) than the UCYN-A1 symbiosis (6.6 ± 8.8 fmol N cell −1  d −1 ). N 2 fixation by the UCYN-A1 symbiosis accounted for a majority of the measured bulk rates at two offshore stations, while the UCYN-A2 symbiosis was an important contributor in three nearshore stations. This report of active UCYN-A symbioses and broad mesoscale distribution patterns establishes UCYN-A symbioses as the dominant diazotrophs in the SCCS, where heterocyst-forming and unicellular cyanobacteria are less prevalent, and provides evidence that the two dominant UCYN-A sublineages are separate ecotypes. 

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4. Cell-specific measurements show nitrogen fixation by particle-attached putative non-cyanobacterial diazotrophs in the North Pacific Subtropical Gyre

   https://doi.org/10.1038/s41467-022-34585-y  

   Harding, Katie J.; Turk-Kubo, Kendra A.; Mak, Esther Wing; Weber, Peter K.; Mayali, Xavier; Zehr, Jonathan P. (December 2022, Nature Communications)

   Abstract Biological nitrogen fixation is a major important source of nitrogen for low-nutrient surface oceanic waters. Nitrogen-fixing (diazotrophic) cyanobacteria are believed to be the primary contributors to this process, but the contribution of non-cyanobacterial diazotrophic organisms in oxygenated surface water, while hypothesized to be important, has yet to be demonstrated. In this study, we used simultaneous 15 N-dinitrogen and 13 C-bicarbonate incubations combined with nanoscale secondary ion mass spectrometry analysis to screen tens of thousands of mostly particle-associated, cell-like regions of interest collected from the North Pacific Subtropical Gyre. These dual isotope incubations allow us to distinguish between non-cyanobacterial and cyanobacterial nitrogen-fixing microorganisms and to measure putative cell-specific nitrogen fixation rates. With this approach, we detect nitrogen fixation by putative non-cyanobacterial diazotrophs in the oxygenated surface ocean, which are associated with organic-rich particles (<210 µm size fraction) at two out of seven locations sampled. When present, up to 4.1% of the analyzed particles contain at least one active putative non-cyanobacterial diazotroph. The putative non-cyanobacterial diazotroph nitrogen fixation rates (0.76 ± 1.60 fmol N cell −1 d −1 ) suggest that these organisms are capable of fixing dinitrogen in oxygenated surface water, at least when attached to particles, and may contribute to oceanic nitrogen fixation. 

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5. The Sponges of the Carmel Pinnacles Marine Protected Area

   https://doi.org/10.11646/zootaxa.5318.2.1  

   TURNER, THOMAS L.; LONHART, STEVE I. (July 2023, Zootaxa)

   California's network of marine protected areas was created to protect the diversity and abundance of native marine life, but the status of some taxa is very poorly known. Here we describe the sponges (phylum Porifera) from the Carmel Pinnacles State Marine Reserve, as assessed by a SCUBA-based survey in shallow waters. Of the 29 sponge species documented, 12 (41%) of them were previously unknown. Using a combination of underwater photography, DNA sequencing, and morphological taxonomy, we greatly improve our understanding of the status and distribution of previously described species and formally describe the new species as Hymedesmia promina sp. nov., Phorbas nebulosus sp. nov., Clathria unoriginalis sp. nov., Clathria rumsena sp. nov., Megaciella sanctuarium sp. nov., Mycale lobos sp. nov., Xestospongia ursa sp. nov., Haliclona melissae sp. nov., Halichondria loma sp. nov., Hymeniacidon fusiformis sp. nov., Scopalina carmela sp. nov., and Obruta collector gen. nov., sp. nov. An additional species, Lissodendoryx topsenti (de Laubenfels 1930), is moved to Hemimycale, and H. polyboletus comb. nov., nom. nov. is created due to preoccupation by H. topsenti (Burton, 1929). Several of the new species appear to be rare and/or have very restricted distributions, as they were not found at comparative survey sites outside of Carmel Bay. These results illustrate the potential of qualitative presence/absence systematic surveys of understudied taxa to discover and document substantial novel diversity.  

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