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Chlorophyll d (Chl d) is only found in the cyanobacterium Acaryochloris marina, where it has replaced Chl a as the dominant pigment in the antennae and in the reaction centers extending the range of photosynthetic active radiation into the near infrared (NIR) light region (700–740 nm; Miyashita et al., 1996; Kühl et al., 2007). Only four A. marina strains have so far been isolated (Table 1), and they have been subject to detailed biochemical and genomic analysis (for example, Miller et al., 2005; Swingley et al., 2008). Although there is increasing evidence that Chl d is more widely distributed in both aquatic and terrestrial systems (Kashiyama et al., 2008; Table 1), the ecology of Chl d-containing cyanobacteria is still largely unknown and in only one case, has a detailed description of its habitat been considered with actual in situ measurements of photosynthetic activity (Kühl et al., 2005). Among cyanobacteria, A. marina has an unusually large genome with a significant proportion of its DNA localized to 9 plasmids (Swingley et al., 2008), and it was speculated that such high capacity for genetic mobility and genome expansion could facilitate its adaptation to a variety of ecological niches. In this study, we show that Chl d-containing cyanobacteria can colonize widespread endolithic habitats on coral reefs, where they occupy a distinct optically defined niche deprived of visible light.

Table 1 Habitats for Chlorophyll d-containing and/or Acaryochloris marina-like phototrophs
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We combined hyperspectral and variable chlorophyll fluorescence imaging (Kühl and Polerecky, 2008) with pigment extraction (Mohr et al., 2010), scanning electron microscopy and DNA pyrosequencing (see Materials and Methods in Supplementary Online Material) to investigate the occurrence of Chl d-containing phototrophs on patches of dead coral branches from the reef flat and shallow outer reef crest off Heron Island, Australia. Such branches were almost completely covered by a diversity of crustose coralline algae (predominantly Porolithon spp. and Lithophyllum spp.; Ringeltaube and Harvey, 2000) and other epiphytic macroalgae, sponges and didemnid ascidians. Careful mechanical removal of the coralline algal layer revealed a thin yellow–green, biofilm closely associated with the carbonate skeleton immediately below (Figures 1a, 2a and Supplementary Figure S1). Hyperspectral imaging in combination with variable chlorophyll fluorescence imaging and scanning electron microscopy confirmed that this biofilm contained photosynthetically active Chl d in patches of A. marina-like cells (1–2 μm) intermixed with other endolithic filamentous cyanobacteria and the green alga Ostreobium sp. (Figures 1b–d and Figures 2b–f).

Figure 1
figure 1

Endolithic habitat of Chl d -containing cyanobacteria. (a) Typical Chl d-containing yellow–greenish biofilm found directly under encrusting coralline algae. Labels indicate two different areas of interest (AOI) where pulse-amplitude modulated (PAM) variable chlorophyll fluorescence imaging was carried out: (1) Endolith AOI is dominated by filamentous cyanobacteria and green algae, and (2) Chl d AOI is dominated by Acaryochloris-like Chl d-containing cyanobacteria. Another circular AOI indicates a Chl d-containing area where no PAM analysis was carried out. Inserts show scanning electron microscopy pictures for the respective AOI; note the presence of round 1–2 μm large bacteria, similar in size and shape to Acaryochloris marina. (b) Hyperspectral reflectance image at 710 nm, that is, the peak absorbance of Chl d in situ. Dark areas indicate strong Chl d absorption. (c) Reflectance spectra of the two AOI showing distinct spectral signatures of Chl d around 710 nm. (d) Photosynthetic activity versus irradiance measured as the PSII related relative electron transport rate (rETR) in the two AOI.

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Figure 2
figure 2

Scanning electron microscopy and hyperspectral measurements performed on a dead coral sample with the crustose coralline algal layer removed. (a) Biofilm found under encrusting coralline algae. (b) Reflectance spectra extracted from hyperspectral imaging of three AOI in (a) showing Chl d absorption around 710 nm. (c–f) Scanning electron microscopy pictures portraying biofilms growing below encrusting corraline algae. Note frequent occurrence of 1–2 μm large A. marina like cells (c–f), along with filamentous cyanobacteria (c, d) and Ostreobium-like filamentous green algae (e).

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We did not attempt a quantification of light attenuation in the crustose coralline layer, but earlier studies of the light microenvironment in coral skeleton (Magnusson et al., 2007) showed a very strong light attenuation of visible light (down to 0.1–1% of the incident irradiance) in the uppermost millimetres, whereas NIR light penetrated significantly deeper reaching 10–100 times the level of visible wavelengths in the skeleton matrix. However, coral skeleton density varies with species affecting light penetration (Enriquez et al., 2005; Magnusson et al., 2007).

Vertical slices through the encrusted coralline layer further revealed that Chl d is found in a thin layer in between the coralline alga and deeper parts of the coral skeleton (Supplementary Figures S1A, B), which was harboring other phototrophic endoliths, such as the green algae Ostreobium sp. These siphonalean green algae can thrive on NIR (Halldal, 1968) due to antenna chlorophylls bound to the Lhca1-type protein (Koehne et al., 1999) that absorb light beyond the typical absorption maximum of Chl d (Kühl et al., 2007). We found shoulders between 710 and 725 nm in the reflectance spectra indicative of such far-red antenna pigments (Figures 1a, 2b, Supplementary Figure S1C). Pigment extraction analysis of crustose coralline samples with ∼1–2 mm of attached skeleton showed a distinct spectral signal of Chl d, albeit on a large background of coralline algal photopigments (Supplementary Figure S2). Pyrosequencing analysis of DNA extracted from similar samples revealed an endolithic microbial community with cyanobacteria as the dominant oxyphototrophs, of which ∼5% could be assigned to A. marina (Supplementary Figure S3); A. marina accounted for ∼1.8% of the total microbial community. In this first attempt of quantifying the presence of A. marina in a natural habitat, a total of five operational taxonomic units (OTUs) showed significant homology to known Acaryochloris species.

Crustose coralline algae are ubiquitous on coral reefs and are essential for stabilizing corals against mechanical erosion by sealing the porous carbonate skeleton matrix and consolidating the reef framework. Inside shallow water patches of coralline encrusted corals NIR light prevails (Kühl et al., 2007) and our data indicate that A. marina-like Chl d-containing cyanobacteria are much more common on coral reefs than previously thought. Interestingly, a new A. marina-like isolate was reported from similar habitats (Mohr et al., 2010). We have recently started a survey of Chl d underneath crustose coralline algae from different climatic zones, and first preliminary results obtained with pigment extraction and hyperspectral imaging (data not shown) confirmed the presence of Chl d underneath coralline specimens encrusting sandstone rock platforms around Sydney in more temperate coastal waters. Our results can also explain several sequence-based findings of A. marina and/or Chl d in other endolithic habitats (Table 1) and thus provide strong evidence for a more widespread occurrence of A. marina-like phototrophs, supporting the hypothesis that these unique oxyphototrophs exhibit a large capacity for niche adaptation (Swingley et al., 2008). Recently, a new chlorophyll, that is, Chl f, has been identified in cyanobacteria from stromatolites (Chen et al., 2010), and this pigment shows an even more red-shifted absorption than Chl d. It is not known whether this new pigment is indeed involved in active photosynthesis, but Chl f-containing phototrophs may colonize similar habitats where Chl d-containing cyanobacteria thrive. Thus Chl d-containing cyanobacteria and other phototrophs with far-red absorbing chlorophylls may have an important yet unexplored role as NIR-driven oxygenic primary producers in both terrestrial and aquatic ecosystems.