Stratification of endolithic communities in the coral skeleton
The ɑ-diversity (observed diversity and Pielou’s evenness) of the bacterial community increased from shallower to deeper skeletal layers (Fig. 2a-d). In the vertical cross-section of the coral skeleton, the observed bacterial diversity in Po. lutea and Pa. australensis increased gradually (Fig. 2a and 2b), while Pielou’s evenness of the bacterial communities of both coral species first increased steeply between 0 and 8 mm (more pronounced in Po. lutea than in Pa. australensis) and then slightly between 8 and 20 mm (Fig. 2c and 2d). Principal component analysis (PCA) biplots of the β-diversity of the bacterial communities of Po. lutea and Pa. australensis show a progressive increase in dispersion of samples moving from shallower to deeper skeletal layers (Fig. 2e and 2f). In biological terms, these ɑ- and β diversity metrics show that the endolithic bacterial communities of both coral species were more diverse and evenly distributed in deeper skeletal layers. The data suggest that the deep skeleton may offer a suitable environment to a wider range of bacteria, a similar pattern to that observed in stromatolites, where deeper and more physicochemically stable layers have been shown to harbour a broader bacterial diversity (Toneatti et al. 2017).
Hyperspectral reflectance imaging on vertical cross-sections of Po. lutea and Pa. australensis coral colonies revealed a diversity of chlorophylls (Chl) and bacteriochlorophylls (Bchl) sustaining both oxygenic and anoxygenic photosynthesis in the coral skeleton (Fig. 3; Supp. Figure 3). Our 16S and 18S rRNA gene datasets contained 375 and 203 unique ASVs attributable to bacterial and eukaryotic phototrophs (Supp. Table 1), respectively, supporting the hyperspectral imaging results. However, the distribution of photosynthetic pigments was different in the two coral species, where pigments showed a clear stratification in Po. lutea and a more patchy distribution in Pa. australensis (Fig. 3; Supp. Figure 3). This suggests that the skeletal architecture dimmed the light environment differently (Marcelino et al. 2013) and consequently affected the distribution of the photosynthetic pigments. These results support previous findings (Fordyce et al. 2021) where chlorophyll concentrations correlated with the skeletal morphology characteristics of each coral species. Chl a was more abundant in shallower layers in the proximity of the tissue than in deep skeletal layers (> 2 cm from the coral tissue; Fig. 3; Supp. Figure 3). Although signals of Chl a in the deep skeleton indicate the presence of oxygenic phototrophs such as Cyanobacteria and endolithic algae, our data cannot confirm whether these chlorophyll pigments were photosynthetically active or rather remaining pigments from older microbial populations.
In both coral species, the phyla Proteobacteria (Orders: Rhizobiales, Cellvibrionales, Rhodobacterales and Rhodospirillales) and Chlorobi (Order: Chlorobiales) dominated the phototrophic community (Supp. Table 1). Signals of Bchl a and c were present across the vertical cross-sections of both Po. lutea and Pa. australensis skeletons (Fig. 3; Supp. Figure 3), albeit with stronger Bchl a signals in deeper skeletal layers of Po. lutea. These results suggest that members of the Chlorobi that have both Bchl a and c may colonize the whole Po. lutea skeleton, while members of the Proteobacteria that only have Bchl a were mainly confined to deeper skeletal layers. In line with the hyperspectral imaging data, the number of phototrophic bacterial ASVs increased with increasing depth in the coral skeleton (Supp. Figure 4), an environment generally regarded as more stable in comparison to shallower skeletal layers (Magnusson et al. 2007; Kühl et al. 2008). The lower biodiversity of shallower phototrophic communities in the coral skeleton could also be explained by coral host-bacterial interactions. For instance, it has been proposed that members of the Chlorobi could interact with the coral host and remove H2S from shallower skeletal layers (Cai et al. 2017). H2S is toxic for corals but necessary for chemolithotrophic sulphur oxidising bacteria (Pokorna and Zabranska 2015).
Physicochemical gradients in the skeleton
Chemical imaging shows that the coral skeleton microenvironment was characterized by O2 and pH gradients (Fig. 4; Supp. Figure 5; Supp. Table 2). Chemical imaging provides fine-scale measurements in structurally complex systems (Santner et al. 2015) but our experimental setup had limitations. For instance, in our experiments the coral cross-sections were illuminated through the transparent planar optodes and the skeleton cross-section borders were sealed using black plasticine. These procedures prevented us to take into account the natural vertical light gradient that penetrates the skeleton and measuring the influence of the tissue layer on the O2 and pH gradients.
Hyperspectral imaging data showed apparent absorption in the ranges 600–640 nm (phycobiliproteins), 660–680 nm (Chl a) and 700–740 nm (far-red shifted Chl a, Chl d and/or Chl f) in the skeletons of both coral species (Fig. 3; Supp. Figure 3; Robertson et al. 2001; Antonaru et al. 2020) that suggest the presence of oxygenic phototrophs and align with the detection of Chlorophyta, Stramenopiles, and Rhodophyta in our 18S rRNA data (Supp. Table 1). Our results also support previous studies showing that skeletal oxygenic phototrophs absorb far-red wavelengths (Magnusson et al. 2007; Kühl et al. 2008; Fordyce et al. 2021; Ricci et al. 2021) and through their metabolism influence the physicochemical environment of the skeleton (Fig. 4; Shashar and Stambler 1992; Kühl et al. 2008).
The cross-section of all coral skeleton samples exhibited some O2 production by endoliths when illuminated, but the bulk skeletal environment remained anoxic (Fig. 4). Peak concentrations of dissolved O2 occurred in shallower skeletal layers of Po. lutea (0–8 mm) but was found deeper in the skeleton of Pa. australiensis (4-16mm; Fig. 4; Supp. Table 2). Our results confirm that O2 production can be observed inside the coral skeleton (Bellamy and RiSK 1982; Shashar and Stambler 1992; Kühl et al. 2008) and show that different coral species are characterized by different O2 gradients. Oxygenic phototrophs induce oxygenation and alkalinization of the skeleton through photosynthesis, but this is not the only functional group influencing the O2 and pH gradients through their metabolism. For instance, denitrifiers and sulphate reducers induce alkalinization (Rust et al. 2000; Tran et al. 2021) and microbial respiration limits build-up of O2 and an increase in pH (Berggren et al. 2012). In Po. lutea, O2 and pH gradient peaks were in the same skeletal layers under irradiance levels of ~ 8 and ~ 21 µmol photons m− 2 s− 1, while the two gradients seemed disconnected in Pa. australensis (Fig. 4). In Po. lutea the pH peaks were between 4 and 16 mm from the colony surface (Fig. 4; Supp. Table 2), while the pH in Pa. australensis increased consistently from shallower to deeper skeletal layers (Fig. 4; Supp. Table 2). In the natural environment, the physicochemical properties of each coral species and colony thus result from more complex interactions between coral tissue thickness, skeletal architecture, autotrophic and heterotrophic metabolisms.
The inferred functional profile of the endolithic community
Using computational predictions of function from the 16S rRNA gene, we predicted that the bacterial community was involved in carbon, nitrogen and sulphur metabolic pathways (Supp. Fig. 6) and potentially could provide pyruvate, acetyl-CoA, fixed nitrogen and sulphur to the coral holobiont. These metabolic pathways are tightly interwoven and some of their reactions are O2 and pH-sensitive (Šimek et al. 2002; Pratscher et al. 2011), therefore their spatial-temporal rates are likely to be affected by the physicochemical gradients of the skeletal environment.
Skeletons of Po. lutea and Pa. australensis showed high numbers of ASVs potentially involved in the transformation of inorganic and organic carbon compounds via pathways like 3-hydroxypropionate bicycle, acetate kinase-PO43- acetyltransferase, Wood-Ljungdahl and reverse Krebs cycle throughout the coral skeleton cross-sections (Supp. Fig. 6). These pathways are thought to encompass various physicochemical requirements (e.g. presence/absence of O2, low pH, high temperature) and trophic strategies (e.g., chemoautotrophs, chemoheterotrophs) (Ingram-Smith et al. 2006; Tabita 2009; Bar-Even et al. 2012; Weiss et al. 2016). Microbes use these pathways to convert inorganic and organic carbon compounds to energy and molecules such as acetyl-CoA and acetate (Quayle 1972; Ingram-Smith et al. 2006; Tabita 2009; Pratscher et al. 2011; Bar-Even et al. 2012; Weiss et al. 2016). For example, the 3-hydroxypropionate bicycle is used by bacteria in aerobic environments to autotrophically fix CO2 and produce pyruvate through a series of reactions (Hügler and Fuchs 2005). This bicycle was mainly predicted by our data in the deep skeleton (Supp. Fig. 6), an environment where we measured low O2 but a high abundance of Chloroflexi (Supp. Table 1), which are the only bacteria known to use this bicycle (Bar-Even et al. 2012). By way of another example, bacteria use the reverse Krebs cycle to fix carbon and, through a series of reactions, synthesise molecules such as acetyl-CoA and pyruvate (Tang and Blankenship 2010; Bar-Even et al. 2012). This cycle is thought to be restricted to anaerobic environments but studies suggest that bacteria can also operate it in presence of O2 (Bar-Even et al. 2012) and accordingly our data predicted the presence of this cycle both in aerobic and anaerobic skeletal areas (Supp. Fig. 6).
We predicted the involvement of endolithic bacteria in six pathways associated with nitrogen metabolism (Supp. Fig. 6). Our data show that Po. lutea had more ASVs associated with diazotrophic bacteria than Pa. australensis and their abundance was higher in the deeper and less oxygenated skeleton (Supp. Fig. 6). Assimilatory and dissimilatory NO3- reduction were among the two most abundant predicted nitrogen pathways in both coral species (Supp. Fig. 6). Through the assimilatory pathway, NO3- is reduced to NH4+ and incorporated as organic nitrogen (Sias et al. 1980). In the dissimilatory pathway, NO3- is used as an electron acceptor and reduced through a series of steps to N2 (Sias et al. 1980), thus allowing bacterial growth in environments that lack O2 like the coral skeleton. Many bacterial ASVs could be responsible for reductive pathways (e.g. dissimilatory and assimilatory NO3- reduction, anammox and denitrification), while there were few ASVs that could be responsible for nitrification, whose end product is NO3- (Supp. Fig. 6). Previous studies that measured NO3- in the skeleton found contrasting results on the concentration of this form of inorganic nitrogen (Risk and Muller 1983; Ferrer and Szmant 1988). One study (Risk and Muller 1983) suggested that skeletons characterized by nearly anoxic conditions, like those measured in our study (Fig. 4), promote reductive pathways, while skeletons that show higher O2 concentrations facilitate oxidative pathways. Our results are in line with this concept. However, since we found few ASVs that could be responsible for nitrification (Supp. Fig. 6) our data leave an open question regarding the origin of NO3- necessary to feed the reductive pathways occurring within the skeleton. Although nitrogen is a growth-limiting nutrient (Kuypers et al. 2018), our data show that endolithic bacteria had the potential to contribute to the nitrogen budget of the coral holobiont with essential compounds like NH4+ (Supp. Fig. 6).
Metabolic pathways associated with sulphur metabolism were among the most predicted in deep skeletal layers of both coral species (Supp. Fig. 6). Sulphate reduction is a predominant pathway largely restricted to anaerobic environments like the deep skeleton (Wasmund et al. 2017; Fig. 4; Supp. Fig. 6). However, we also predicted the presence of this pathway in shallower skeletal layers (Supp. Fig. 6) that, when illuminated, showed higher O2 build-up (Fig. 4). These results suggest that part of the skeleton could be characterized by temporal compartmentalization of metabolic functions (e.g. photosynthesis in daylight and sulphate reduction in darkness). The coral skeleton is an environment enriched in sulphur (Clode and Marshall 2003; Cuif et al. 2003) and accordingly, metabolic pathways involving the processing of sulphur compounds of intermediate oxidation states were predicted in abundance across the whole coral skeleton cross-section (Supp. Fig. 6). The carbon, nitrogen and sulphur metabolic pathways predicted through our data analysis support the hypothesis that endolithic bacteria can be considered major nutrient recyclers (Fine and Loya 2002; Sangsawang et al. 2017; Moynihan et al. 2021) and, by showing how the abundance of these pathways changes across the skeleton depth gradient, we provide new insight into the spatial distribution of the coral skeleton biogeochemical cycle.
The ecological microniches of the coral skeleton
The skeletal microniches were characterized by the presence of dynamic O2 and pH gradients (Fig. 4; Supp. Fig. 5; Supp. Table 2) and harboured microbial communities that varied in their composition with depth in the skeleton (Fig. 5). PERMANOVA showed that dissolved O2 concentrations and pH distributions measured in Po. lutea and Pa. australensis skeletons correlated with the β-diversity of their endolithic bacterial communities (Supp. Table 3). Considering these results, we investigated whether the abundance of specific bacterial taxa showed associations with the physicochemical properties of the skeleton, and we found that bacterial ASVs correlated with dissolved O2 and pH gradients in the CCA biplots in both coral species (Fig. 6; Supp. Fig. 7 and 8; Supp. Table 4 and 5). These results suggest that the overall bacterial community composition and the presence and abundance of certain endoliths are influenced by the physicochemical environment of the skeleton.
The spatial heterogeneity of the physicochemical environment differed between the two coral species and was possibly influenced by their skeletal architecture (Fig. 5). In Po. lutea, O2 and pH peaks were constrained to skeletal layers with abundant endolithic algae (Fig. 4a and 4b; Fig. 5a; Supp. Table 2), suggesting that the more homogeneous and denser skeleton of this species limited gas and solute diffusion. In Pa. australensis, peaks in dissolved O2 concentration corresponded to skeletal areas with abundant endolithic algae and the shape of the O2 gradients was possibly determined by the more perforated skeletal architecture of this species (Fig. 4c and 4d; Fig. 5b; Supp. Table 2). Interestingly, the pH of Pa. australensis increased deeper in the skeleton (Fig. 4c and 4d; Fig. 5b), where we found ASVs belonging to Bacillus and Spirochaetaceae (Fig. 5d) that may increase the skeletal matrix pH through denitrification (Wei et al. 2015).
Increased O2 and pH values in Po. lutea skeletons correlated with ASVs belonging to the nitrogen fixing genera Spirochaeta and Tistlia (Fig. 6a) and in Pa. australensis with Alteromonas and Pseudoalteromonas ASVs that are thought to take part in nitrogen cycling and antibacterial activity (Fig. 6b; Shnit-Orland et al. 2012; Ceh et al. 2013a; Ceh et al. 2013b). In Po. lutea, ASVs of the obligate anaerobes Chlorobi correlated with higher O2 values, suggesting that these bacteria were likely to be found in skeletal layers characterized by higher but still hypoxic O2 concentrations (Fig. 6a). The occurrence of these presumed obligate anaerobes in presence of O2 is unexpected, but Chlorobi have been previously reported in coral tissue (Cai et al. 2017) and skeletons with abundant endolithic algae (Marcelino and Verbruggen 2016). It is possible that in our study the presence of Chlorobi in hypoxic skeletal layers resulted from their interactions with other holobiont members rather than being a direct response to the O2 gradients. For instance, it has been proposed that these bacteria could remove toxic H2S generated by sulphate-reducing bacteria (Cai et al. 2017). Accordingly, we found that predicted sox system pathways (Fig. 5a; Supp. Fig. 6), through which bacteria like Chlorobi oxidize H2S (Friedrich et al. 2001), were present in shallower layers of Po. lutea skeletons. In Pa. australensis, ASVs of another presumed strictly anaerobic bacterium, Paramaledivibacter, correlated with elevated O2 concentrations and higher pH values (Fig. 6b). These bacteria have also been found in the tissue of other coral species (Santoro et al. 2021; Ricci et al. 2022), but identification of their physiological requirements in the coral holobiont awaits further investigation.