Macroalgae Significantly Alter the Structure of Coral-Associated Microbial Communities

Our findings suggest that direct competition with some macroalgal species increases the taxonomic variability of microbes on P. astreoides. Similar decreases in the specificity of the coral microbiome have previously been observed in corals that differed in their health state and/or proximity to sewage outflows [39]. These increases in microbial community variability may be a general response to disturbance as has been seen in communities of macro-organisms [43]. Although the overall microbial community significantly changed only on corals in competition with two macroalgal species (S. polyceratium and G. obtusata) (Figure 3B; Table S1), some microbial taxa were significantly altered on all corals challenged with macroalgal competitors (Figure 5). Four types of alterations in the microbial TRFs associated with corals in competition with macroalgae were identified. First, some taxa present on control corals increased in abundance on macroalgae-treated corals. This increase was the least frequently observed change to microbial TRFs, occurring only with TRF 63. This TRF was present on every coral and macroalgae tested, and drove much of the clustering among microbial communities (Tables S2 & S3). Yet, the abundance of TRF 63 was significantly altered only on S. polyceratium-exposed corals, where it increased 4-fold relative to control corals (Figure 5).

Conversely, a bacterial taxon previously documented [3] to be a member of the P. astreoides holobiont always declined on corals in competition with macroalgae. TRF 341 was the most abundant TRF comprising ∼50% of the community in control corals, but its presence was reduced in every macroalgal treatment to below the detection level. Further, this TRF was not observed on any of the macroalgal species in this study. Rohwer et al. [3] first discovered TRF 341 on P. astreoides in Panama. TRF 341 was the most commonly identified taxa [3] (comprising an average of 61% of clones detected), and it was therefore named P. astreoides 1 (PA1). Sequencing identified PA1 as a γ-proteobacteria (GenBank accession # AF365457) [3]; the TRF 341 observed in corals in this study also represents the PA1 phylotype. PA1 has also previously been identified in Diploria strigosa corals as TRF 342 [39], where it exhibited lower abundances in diseased colonies, relative to apparently healthy conspecifics. Together, these previous studies have indicated that TRF 341 (PA1) is a likely important, but context dependent, mutualistic symbiont of corals. With the observation that there is concomitant reduction in PA1 abundance and coral growth in this study, it is possible that the loss of coral-associated microbial symbionts can potentially lead to reduced coral health. Alternatively the loss of this symbiont may have arisen from altered coral physiology that resulted from exposure to the algae. Both hypotheses should be tested in the future.

A third group of TRFs were present in control corals and changed non-uniformly across the macroalgal treatments. For example, TRF 423 increased in two of the competition treatments (S. polyceratium and H. tuna) but decreased in another (L. variegata). These data suggest that competition with macroalgae can have idiosyncratic effects on coral-associated microbial communities depending on the species of macroalgal competitor and, potentially, the mechanisms that macroalgae use to compete with corals (e.g. allelopathy, abrasion, smothering).

The last and most commonly identified alteration to microbial communities was the detection of TRFs on macroalgal-treated corals that were not observed on controls. In our study, this included TRFs 75, 126, 227, 564, 802, 826, 828, 858, 879, and 899. We additionally searched for these TRFs within datasets generated from nine other P. astreoides colonies collected from this same reef in a long-term study (Burkepile & Vega Thurber, unpublished data), and TRFs 227, 564, 828, 858, 879, and 899 were never detected in these unmanipulated colonies (data not shown). This apparent relaxation of specificity in coral-microbial associations may represent colonization of corals by opportunist microbes during disturbance events.

The most significant taxon introduction to macroalgae-treated corals was TRF 879. This TRF was not detected from any control corals (n = 3) or local unmanipulated corals (n = 9) but represented up to ∼53% (mean 42.6% ±6.11 SE) of the microbial community on corals in competition with S. polyceratium. TRF 879 was not detected from thalli of S. polyceratium, and therefore does not appear to be vectored directly to the coral by the algae. In contrast, TRF 564 also was not observed on controls or local corals but was abundant on corals exposed to S. polyceratium (mean ∼26% of the coral-associated microbial community). Importantly, however, TRF 564 was detected from S. polyceratium thalli, albeit at lower relative abundances (∼1.5% of the algae-associated microbial community) than on treated corals, and TRF 564 was never detected on any other type of macroalgae. Taken together, these data indicate that TRF 564 was vectored from S. polyceratium to P. astreoides colonies and induced to proliferate. Although we currently do not know the identity of this TRF 564 microbial taxon, to our knowledge, this is first example of a macroalga vectoring a microbe to a coral. An alternative hypothesis is that TRF 564 also is present in the overlying water column and thus the surrounding seawater could have contributed to the increase in the relative abundance of this TRF on corals after exposure to the S. polyceratium algae. However, if that were the case, then TRF 564 should have been present in all of the samples (like TRF 63), yet, it was only ever detected on thalli of the S. polyceritium algae and corals exposed to that same algae. Therefore it is more parsimonious to suggest that TRF 564 is vectored from the algae to the corals.

While these data clearly demonstrate alterations in microbial diversity on corals, a caveat of TRFLP analysis is that it is not as sensitive at detecting rare members of the community, compared to other methods such as pyrosequencing [44]. It cannot be ruled out that rare members of the coral and macroalgal microbiomes were not detected using this technique. Therefore, it is possible that some microbial taxa, which appeared to be present on macroalgae-treated corals but not on controls, were in fact also present on control corals but below the detection threshold of our TRFLP analysis. For example, TRF 564 could normally be a rare member of the coral microbiome, whose growth is highly stimulated by the presence of S. polyceratium. Nevertheless, such dramatically large shifts in any one member of the microbiome in response to algal competition (e.g., from undetectable to 30% of the community) are likely to affect the metabolism of the coral holobiont [45], [46] with potentially adverse consequences for the coral.

Yet, our analysis did indicate that minor members of the coral microbiome were affected by interactions with macroalgae. While we found that the major (≥3% of any one community) microbial members of coral and algae were different among species and coral treatments, only 60% of the TRFs that were significantly altered on corals exposed to algae were from this majority. Rare taxa were also significantly altered, and contributed to 40% of all the significant individual TRF changes. These rare TRFs that were altered included: 51, 54, 199, 227, 802, 803, 839, 858, 882, 883, and 889. Together these data suggest that members of both the coral’s common and rare biosphere are impacted by algal interactions.