Ecosystem consequences of a nitrogen-fixing proto-organelle

Abstract

Microscale symbioses can be critical to ecosystem functions, but the mechanisms of these interactions in nature are often cryptic. Here, we use a combination of stable isotope imaging and tracing to reveal carbon (C) and nitrogen (N) exchanges among three symbiotic primary producers that fuel a salmon-bearing river food web. Bulk isotope analysis, nanoSIMS (secondary ion mass spectrometry) isotope imaging, and density centrifugation for quantitative stable isotope probing enabled quantification of organism-specific C- and N-fixation rates from the subcellular scale to the ecosystem. After winters with riverbed-scouring floods, the macroalga Cladophora glomerata uses nutrients in spring runoff to grow streamers up to 10 m long. During summer flow recession, riverine N concentrations wane and Cladophora becomes densely epiphytized by three species of Epithemia, diatoms with N-fixing endosymbionts (proto-organelles) descended from a free-living Crocosphaera cyanobacterium. Over summertime epiphyte succession on Cladophora, N-fixation rates increased as Epithemia spp. became dominant, Cladophora C-fixation declined to near zero, and Epithemia C-fixation increased. Carbon transfer to caddisflies grazing on Cladophora with high densities of Epithemia was 10-fold higher than C transfer to caddisflies grazing Cladophora with low Epithemia loads. In response to demand for N, Epithemia allocates high levels of newly fixed C to its endosymbiont. Consequently, these endosymbionts have the highest rates of C and N accumulation of any taxon in this tripartite symbiosis during the biologically productive season and can produce one of the highest areal rates of N-fixation reported in any river ecosystem.

Keywords: Epithemia; N-fixation; diazoplast; endosymbiosis; epiphytic microbiome.

Fig. 1.

Photosynthesis and N-fixation rates of the three successional stages of Cladophora measured using ¹³CO₂ and ¹⁵N₂ in bottles (n = 6) incubated in the Eel River, CA (A). In a separate experiment, we fed labeled algae to grazing caddisflies (Gumaga nigricula) in flow-through stream-side chambers (n = 5) and measured their C and N assimilation after 3 d. C and N fluxes to grazing caddisflies increased through succession as Cladophora becomes covered in Epithemia (B). Trophic efficiency, measured as the percent of fixed ¹³C and ¹⁵N transferred to grazers, increased as Epithemia becomes the dominant primary producer (C). Error bars indicate 1 SE above and 1 SE below the mean.

Fig. 2.

Cross sections, secondary electron, and NanoSIMS images of early successional green (Top row) mid successional yellow (Middle row) and late successional red (Bottom row) Cladophora, labeled with ¹³CO₂ and ¹⁵N₂. Epithemia diatom cells are denoted with white arrows. In Green Cladophora (A), high levels of ¹³C around the periphery of the Cladophora cell indicate photosynthate is concentrated in the parietal chloroplast and cell wall (B), with no detectable ¹⁵N (C). In the yellow cross-section, two Epithemia diatoms are present (D), and photosynthate (¹³C) is distributed in both Epithemia and Cladophora (E). Epithemia cells are labeled with ¹⁵N, demonstrating N-fixation (F), with no detectable ¹⁵N in the Cladophora. In the red assemblage the Cladophora cell is surrounded by a halo of Epithemia cells (G). Isotopic labels of ¹³C (H) and ¹⁵N (I) are concentrated in Epithemia cells with small amounts also found in Cladophora and the interstitial spaces between diatoms.

Fig. 3.

Secondary electron and NanoSIMS images of ¹³C and ¹⁵N in representative cells show reciprocal transfer of C and N between Epithemia and its diazoplasts. White arrows point to diazoplasts. After 2 h (midday), C fixed by the diatom is already concentrated in the diazoplast, indicating the cell is prioritizing N-fixation. ¹⁵N is dispersed throughout the cell. The vertical white line shows where two adjacent NanoSims images were spliced and cropped. After 24 h (2nd and 3rd rows) both ¹³C and ¹⁵N concentrations are highest in diazoplasts and some cells show significant “sharing” of fixed N from diazoplast to diatom. Across multiple Epithemia cells, concentrations of ¹³C and ¹⁵N within the diatom’s cytoplasm and the diazoplasts are positively correlated. Concentrations of both isotopes are, on average, higher in diazoplasts compared with the diatom’s cytoplasm (Bottom row, Mann–Whitney, P < 0.0001). Data shown are after 24 h. See SI Appendix, Fig. S3 for 2 h incubations.

Fig. 4.

qSIP of Cladophora samples was used to measure growth rates and population sizes of individual prokaryote species capable of N-fixation, including diazoplasts (A). Samples of Cladophora and its microbiome were incubated in ¹⁸O-H₂O. Each taxon’s growth rate correlates with the amount of ¹⁸O incorporated into its DNA. Diazoplast growth can occur both when diazoplasts replicate within a host cell and when the host cell divides and diazoplasts duplicate and are vertically transferred to daughter cells with no change in diazoplast numbers per host cell (B). We estimated the relative contribution of diazoplasts, free-living cyanobacteria, and other bacteria to new growth (and hence N-fixation), based on each taxon’s biovolume and growth rate. The number of N-fixing cells (C), biovolume of N-fixers (D), and new growth of N-fixers (E) increased with epiphyte load. Diazoplasts dominated new growth of N-fixers accounting for 88% of new growth at the red stage (E).