Tropical reef-building (scleractinian) corals are holobionts consisting of the coral animal host, dinoflagellate algae of the genus Symbiodinium, and a diverse assemblage of other microbes (Rohwer et al., 2002). These microbes form host-specific associations and provide key functional traits within the coral holobiont. Among these traits, biological nitrogen fixation is considered to be of high ecological significance, as nitrogen fixing Bacteria and Archaea, the diazotrophs, are widely associated with corals (Lema et al., 2012, 2014). Indeed, coral-associated nitrogen fixation provides an important source of nitrogen for Symbiodinium (Lesser et al., 2007; Benavides et al., 2016; Pogoreutz et al., 2017), thereby helping to sustain holobiont productivity when nutrients are scarce (Cardini et al., 2015).

While nitrogen fixation rates in scleractinian corals exhibit species-specific variation (Shashar et al., 1994; Cardini et al., 2015), the molecular and ecological drivers of these patterns remain unexplored. Scleractinian coral holobionts are mixotrophic, i.e., can draw energy and nutrients from both autotrophic and heterotrophic sources. Importantly, coral holobionts range from being mostly heterotrophic to mostly autotrophic (Rahav et al., 1989; Muscatine, 1990). As nitrogen fixation is a highly energy-consuming functional trait (Mortenson, 1964), its contribution (and thereby relevance) to holobiont metabolism may differ among trophic functional groups of corals. In this study, we therefore set out to compare rates of nitrogen fixation with coral-associated diazotroph abundance and activity in a comparative coral taxonomic framework assaying highly heterotrophic (Pleuractis granulosa, Ctenactis echinata) and mostly autotrophic (Pocillopora verrucosa, Stylophora pistillata) corals (Muscatine et al., 1984; Muscatine and Kaplan, 1994; Houlbrèque and Ferrier-Pagès, 2009; Ziegler et al., 2014). To do this, we indirectly quantified coral holobiont-associated microbial nitrogen fixation activity from acetylene reduction assays (ARA) (Rädecker et al., 2014). In addition, we assessed coral tissue-associated relative gene copy numbers and expression of the nifH gene, a common biomarker for diazotrophs (Gaby and Buckley, 2012) using quantitative PCR (qPCR) to investigate relative diazotroph community sizes along with their activity.

Both species of the mostly autotrophic Pocilloporidae (P. verrucosa, S. pistillata) exhibited detectable gross (i.e., holobiont) nitrogen fixation (0.03 ± 0.04 and 0.01 ± 0.00 nmol C₂H₄ day^-1 cm^-2, respectively) (Figures 2A,B). In contrast, nitrogen fixation remained below the detection limit for both species (P. granulosa, C. echinata) of the highly heterotrophic Fungiidae (Figures 2A,B). This pattern of differential nitrogen fixation activity between the two coral families aligned with nifH abundance and expression. Corals in the Pocilloporidae family showed a significantly higher abundance of nitrogen-fixing bacteria (assessed via relative nifH gene copy number) compared to both members of the Fungiidae family (ANOVA, F = 40.61, p < 0.001; Figure 3A and Supplementary Table S1). A similar pattern was confirmed for diazotroph activity, with the Pocilloporidae having higher relative nifH gene expression than the two fungiid species (ANOVA, F = 12.30, p < 0.001; Figure 3B and Supplementary Table S1). Specifically, P. verrucosa and S. pistillata showed 23- and 431-fold higher relative diazotroph abundance and 95- to 480-fold higher relative gene expression compared to P. granulosa, respectively (Supplementary Table S1). Non-linear regression revealed a strong significant relationship between nifH copy numbers and expression in the two species of Pocilloporidae (R² = 71.49, p = 0.0339), but only a weak although significant relationship in the two Fungiidae (R² = 0.07, p = 0.0288).

The present study investigated physiological and molecular patterns of nitrogen fixation along with diazotroph abundance in corals representing two different trophic functional groups (autotrophy vs. heterotrophy). This was achieved by assessing nitrogen fixation activity (as assessed via ARA) as well as tissue-associated relative gene copy numbers and expression of the nifH gene (as assessed via qPCR) in a comparative species framework. The investigated corals exhibited similar patterns within functional groups, but marked differences between functional groups for all measurements.

Our data are within the range of nitrogen fixation rates published for pocilloporid corals (Shashar et al., 1994; Rädecker et al., 2014; Cardini et al., 2015; Pogoreutz et al., 2017). Similarly, an absence of detectable nitrogen fixation was previously reported for the fungiid coral Fungia fungites (Shashar et al., 1994). Our present study shows that physiological differences in holobiont nitrogen fixation rates align with relative nifH gene abundance and activity, and hence, likely reflect differences of nitrogen-fixing bacteria in the tissues of these corals, highlighting molecular differences on the coral family level.

To understand the distinct physiological and molecular patterns in diazotrophy, the ecological context of the investigated coral families, particularly the trophic functional groups, must be considered. The observed patterns in diazotrophy align with differential heterotrophic feeding capacities of the investigated coral holobionts. While Fungiidae are highly heterotrophic and can exploit a large variety of food sources (Houlbrèque and Ferrier-Pagès, 2009), shallow-water Pocilloporidae have a very limited heterotrophic capacity and strongly rely on photoautotrophy (Muscatine et al., 1984; Muscatine and Kaplan, 1994; Ziegler et al., 2014). Given that the investigated functional groups align with family association, it remains to be determined whether the observed differences are reflecting ecological differences in autotrophic and heterotrophic corals at large, e.g., by the incorporation of further species. In any case, photosynthetically fixed carbon (photosynthate) is characterized by high C:N ratios compared to heterotrophic food sources (Falkowski et al., 1984; Muscatine et al., 1984). Therefore, high carbon availability coupled with strong nitrogen limitation in mostly autotrophic coral holobionts may select for relatively larger diazotroph populations compared to mostly heterotrophic coral holobionts. These results suggest that the importance of microbial nitrogen fixation depends on the heterotrophic capacity of the coral holobiont. This view is supported by previous studies showing differential partitioning of δ¹⁵N in corals along a water depth gradient, indicating a higher importance of nitrogen fixation products in low-light adapted phototrophs compared to heterotrophs (Muscatine and Kaplan, 1994; Bednarz et al., 2017).

The observed physiological and molecular patterns in nitrogen fixation have important implications for our understanding of holobiont nutrient cycling and functioning. For instance, changes in nutrient cycling and stoichiometry can be involved in coral symbiotic breakdown as recently shown (Wiedenmann et al., 2012; Ezzat et al., 2016; Pogoreutz et al., 2017). Indeed, while the input of ‘new’ nitrogen from diazotrophy may be beneficial for holobiont functioning in oligotrophic reef waters (Cardini et al., 2015; Benavides et al., 2016), stimulated nitrogen fixation activity may be linked to detrimental effects on Symbiodinium photophysiology and coral bleaching (Santos et al., 2014; Rädecker et al., 2015; Pogoreutz et al., 2017). Hence, diazotrophs may be considered beneficial microorganisms for corals (BMCs) under stable, oligotrophic conditions (Peixoto et al., 2017). Yet, their presence may affect the corals’ susceptibility to stress during environmental change highlighting the importance of understanding the role of microbes in holobiont functioning and breakdown (Sweet and Bulling, 2017). Indeed, Pocilloporidae are highly sensitive to thermal stress (McClanahan, 2004; Pinzón et al., 2013; Swain et al., 2016), whereas Fungiidae are resistant to a range of environmental stressors (Chadwick and Loya, 1990; Hoeksema, 1991; Philipp and Fabricius, 2003; Baria et al., 2015; Röthig et al., 2016; Swain et al., 2016). We thus argue that a strong coral host dependency on microbial nitrogen fixation may render corals more vulnerable to the effects of environmental change. Hence, future research efforts should be directed to disentangle a potential link between coral-associated nitrogen fixation and holobiont stress susceptibility. Understanding this link will assist to improve current reef conservation measures.

Taken together, our study provides insight into the ecological underpinnings that may underlie differences in diazotrophy of the coral holobiont with implications for coral host environmental resilience. As such, our work contributes to the growing body of literature that suggests the importance of microbes to metaorganism function. Future work assessing the diversity and composition of coral-associated diazotroph communities in a comparative coral taxonomic framework, e.g., via nifH gene sequencing (Lema et al., 2012; Santos et al., 2014) will provide additional insight into host-microbe interactions.

CP, NR, CW, and CV conceived and designed the experiment. CP and NR conducted the experiments, analyzed, and interpreted the data. AC, CW, and CV contributed reagents and tools. CP, NR, CW, and CV drafted and all authors revised the article. All authors have agreed to and approved the final article.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.