Diazotroph diversity and nitrogen fixation in the coral Stylophora pistillata from the Great Barrier Reef

Abstract

Diazotrophs, both Bacteria and Archaea, capable of fixing nitrogen (N₂), are present in the tissues and mucous, of corals and can supplement the coral holobiont nitrogen budget with fixed nitrogen (N) in the form of ammonia (NH₃). Stylophora pistillata from Heron Island on the Great Barrier Reef collected at 5 and 15 m, and experimentally manipulated in the laboratory, showed that the rates of net photosynthesis, steady state quantum yields of photosystem II (PSII) fluorescence (∆F_v/F_m') and calcification varied based on irradiance as expected. Rates of N₂ fixation were, however, invariant across treatments while the amount of fixed N contributing to Symbiodinium spp. N demand is irradiance dependent. Additionally, both the Symbiodinium and diazotrophic communities are significantly different based on depth, and novel Cluster V nifH gene phylotypes, which are not known to fix nitrogen, were recovered. A functional analysis using PICRUSt also showed that shallow corals were enriched in genes involved in nitrogen metabolism, and N₂ fixation specifically. Corals have evolved a number of strategies to derive nitrogen from organic (e.g., heterotrophic feeding) and inorganic sources (e.g., N₂ fixation) to maintain critical pathways such as protein synthesis to succeed ecologically in nitrogen-limited habitats.

Fig. 1

Rates of photosynthesis and calcification normalized to surface area (mean ± SE) for Stylophora pistillata. LL = 150 µmol quanta m⁻² s⁻¹, HL = 500 µmol quanta m⁻² s⁻¹. Treatment groups (n = 7 samples per group) and for each independent parameter similar superscripts are not significantly different from each other

Fig. 2

Steady state quantum yield (∆F_v/F_m′) of PSII fluorescence (mean ± SE) for Stylophora pistillata. LL = 150 µmol quanta m⁻² s⁻¹, HL = 500 µmol quanta m⁻² s⁻¹. Treatment groups (n = 7 samples per group) with similar superscripts are not significantly different from each other

Fig. 3

Rates of N₂ fixation (n = 7 samples per treatment group) normalized to surface area (mean ± SE) for Stylophora pistillata. LL = 150 µmol quanta m⁻² s⁻¹, HL = 500 µmol quanta m⁻² s⁻¹

Fig. 4

Pie Charts representing average percent contribution of the most dominant prokaryotic phyla (large pies) to the total relative abundance in Stylophora pistillata at: a 5 m, 150 µmol quanta m⁻² s⁻¹, b 5 m, 500 µmol quanta m⁻² s⁻¹, c 15 m, 150 µmol quanta m⁻² s⁻¹, d 15 m, 500 µmol quanta m⁻² s⁻¹, e ambient seawater. Additionally, all Proteobacteria families (superimposed small pie charts) are presented across all treatment groups (n = 7 samples per group)

Fig. 5

nifH diversity recovered from Stylophora pistillata. a Maximum-likelihood topology of nifH sequences, rooted with non-nitrogen-fixing Cluster IV (see Fig. S1 for expanded nifH phylogeny). Novel nifH phylotypes from S. pistillata are denoted as “nifH novo” and boot strap >70% is indicated on applicable nodes. b Relative abundance of nifH phylotypes across S. pistillata samples by treatment. nifH phylotypes are hierarchically clustered by abundance similarity. Newly recovered nifH novo phylotypes are colored to match cluster of origin in b

Fig. 6

Symbiodinium spp. diversity recovered from Stylophora pistillata treatment groups (n = 7 samples per group). a. Relative abundances of Symbiodinium phylotypes across S. pistillata coral samples. b. Non-metric MDS plot of Symbiodinium diversity across S. pistillata samples. Vectors are shown for Symbiodinium spp. phylotypes that differed significantly across treatments

Fig. 7

PICRUSt nitrogen metabolism abundance differences. a Relative abundance of OTUs participating in nitrogen metabolism pathways based on PICRUSt prediction. b Relative abundances of PICRUST-predicted OTUs contributing nitrogen-fixing genes (i.e., anfG, nifDHK)