We investigated the bacterial communities associated with octocorals from the mesophotic zone of the Mediterranean and Red Seas, and assessed whether evolutionary patterns in host-microbe associations exist. Our study revealed that Mediterranean and Red Sea octocoral holobionts harbor specific bacterial communities, but phylosymbiosis was found only in octocorals from the Mediterranean Sea. However, cophylogenetic associations were detected in 13 of the 14 octocoral species investigated, suggesting that octocorals co-evolved with a few specific bacterial symbionts, primarily belonging to the Endozoicomonadaceae and Spirochaetaceae.
Octocorals are associated with specific bacterial communities.
The composition of the coral-associated bacterial communities differed significantly between corals from the Mediterranean Sea and those from the Red Sea. This difference may be related to the about 17 million-years separation of the Mediterranean Sea and the Red Sea before the opening of the Suez Canal in 1869 (Steininger & Rögl, 1984). But the different light, temperature, and salinity conditions in these two seas may also have contributed to the divergence in the microbiota of Mediterranean and Red Sea octocorals. Similarly, differences in the microbiota of coral species from different reefs or oceans have been observed (Lima et al. 2020; Roder et al. 2015; Pantos et al. 2015; Vohsen et al. 2023, van de Water et al. 2017).
Mediterranean octocorals from the mesophotic zone had distinct species-specific microbiota, but nevertheless two distinct ‘clusters’ could be observed – corals with a microbiota dominated by Endozoicomonas (P. clavata and Eunicella spp.) and corals with a Spirochaetaceae-dominated microbiota (A. coralloides, C. rubrum, C. verticillata). These patterns are consistent with reports on shallow populations (van de Water et al. 2016; 2017; 2018; Bayer et al. 2013; La Rivière, Garrabou, et Bally 2015; Ransome et al. 2014), suggesting that depth has little influence on the microbiota of these octocoral species. The latter ‘cluster’ was rather surprising as it was comprised of corals that belong to two different orders - the Malacalcyonaceae and Scleralcyonaceae, respectively. Each coral species did, however, harbor its own microbial phylotypes and taxa, explaining the overall species-specific hierarchical clustering observed here, but some species also shared phylotypes. For example, the microbiota of Eunicella spp. and P. clavata had high abundances of Endozoicomonas OTU1 and OTU3, and the core microbiome of A. coralloides contained both phylotypes of C. rubrum (e.g., Spirochaetaceae OTU4) as well as gorgonians (e.g., Endozoicomonas OTU1, OTU2, OTU5). This overlap in the core microbiota is rather striking as A. coralloides is called the ‘false red coral’ in common language because of the similarities in appearance with C. rubrum, while it is in fact a ‘parasite’ or ‘epibiont’ that overgrows gorgonians, like P. clavata and Eunicella spp. (McFadden 1999; Groot et al.1982; Quintanilla et al. 2013). This suggests that evolutionary patterns in host-microbiota associations may be masked by differences in species-specific life strategies and/or that associations with specific symbionts may have arisen multiple times during host evolution.
Other taxa found at relatively high abundances in the microbiota of Mediterranean octocorals here and previously belonged to the genus Rickettsia in P. clavata, the Spongiibacteraceae BD1-7 clade in C. rubrum (Prioux et al., 2023; Tignat-Perrier et al., 2022), and the Gammaproteobacteria BD72BR169 in E. cavolini and E. verrucosa (van de Water, Voolstra, et al., 2018). However, bacteria from these taxa are also present in the microbiota of various other Mediterranean gorgonians (Bayer et al., 2013; Tignat-Perrier et al., 2023; van de Water, Voolstra, et al., 2018).
The microbiota of octocorals from the Red Sea were distinct from those from the Mediterranean, but Endozoicomonas and Spirochaetaceae were also frequent and abundant symbionts of several of these tropical soft coral species. Overall, they could be divided into three main groups based on their microbiota composition: (1) Endozoicomonas-Spirochaetaceae-dominated (K. utinomi, S. glaucum, S. eilatensis, S. vrijmoethi); (2) Endozoicomonas-dominated (Ovabunda spp., P. thyrsoides); and (3) others with relatively high abundances of Rhodobacteraceae (S. polydactylum, S. loyai). While matching relatively well with the hierarchical clustering analysis, these groups were not as clear-cut, as Mollicutes were K. utinomi (Entoplasmatales) and Ovabunda spp. (Mycoplasmatales), whereas P. thyrsoides, S. eilatensis and S. vrijmoethi contained a relatively high abundance of Rhodospirillales (family Terasakiellaceae) in their microbiota. The microbial taxa identified here as the main symbionts of octocorals from the Red Sea are consistent with reports on octocorals from other tropical locations. For example, Endozoicomonas is a highly abundant symbiont of Sclerophytum and Sarcophyton species from the Indo-Pacific (Haydon et al. 2022; O’Brien et al. 2020; Park et al. 2022; Easson et al. 2024), but also, in many other octocorals from the Indo-Pacific (Wessels et al., 2017) and Caribbean (Morrow et al. 2012; Robertson et al. 2016; McCauley et al. 2016; Pike et al. 2013; Sunagawa et al. 2010). Spirochaetes have also been found relatively abundant in the microbiota of Indo-Pacific Sarcophyton spp. (Haydon et al., 2022; O’Brien et al., 2020), Lobophytum (Wessels et al., 2017) and in some Sclerophytum species (O’Brien et al., 2020; Park et al., 2022). But the holobionts of Sclerophytum species do not always harbor Spirochaetes in this region (Haydon et al., 2022). However, Sclerophytum spp. from both the Red Sea as the Indo-Pacific do associate with Rhodospirillales (Haydon et al., 2022) and Rhodobacteraceae (Lu et al. 2020; Chen et al. 2012; Alsharif et al. 2023).
Mollicutes, such as Entoplasmatales and Mycoplasmatales, were also highly abundant in the microbiota of C. verticillata and K. utinomii, and of Ovabunda spp. and C. rubrum, respectively. Members of these orders have previously been observed in association with deep-sea corals and gorgonians (Entoplasmatales: (Gray et al. 2011; Chapron et al. 2020; Weiler et al. 2018; van de Water et al. 2018)), and with other octocorals (Mycoplasmatales: (Gray et al., 2011; Holm & Heidelberg, 2016; Porporato et al., 2013; van de Water et al., 2017; van de Water, Voolstra, et al., 2018).
Overall, as for the hexacorals, octocorals are consistently found to have dominant associations with bacteria belonging to a few main taxa, particularly Endozoicomonas and Spirochaetaceae, but also harbor numerous other rare taxa. Of particular interest, are those phylotypes that belong to a coral species’ core microbiome and those that are unique to a coral species. Such consistent associations of various microbial taxa with octocorals underscore their importance as symbionts and suggest that there may be an evolutionary link between these holobiont partners.
Unclear signals of phylosymbiosis in octocoral holobionts
A phylosymbiotic signal, i.e. a link between the evolutionary history of the host and the diversity of the microbiome (Lim and Bordenstein 2020), has been observed in scleractinian corals (Pollock et al., 2018) and octocorals from Australia (O’Brien et al., 2020). However, we did not observe phylosymbiotic signals between octocorals and their microbiota, when we considered all octocoral species studied here. This may be explained by a clear separation in bacterial community composition according to the two geographic locations, the Mediterranean Sea and the Red Sea. Indeed, these seas are isolated for at least 17 million years (Steininger & Rögl, 1984), suggesting that the geographical separation of these coral populations (i.e., vicariance) influenced patterns in the structure of the microbiota more than host phylogeny.
However, when the coral populations from the two seas were assessed separately, signals of phylosymbiosis were observed in the temperate Mediterranean octocorals but not in corals from the tropical Red Sea. The Mediterranean octocorals C. rubrum, P. clavata and several Eunicella spp., have been extensively studied and possess a microbiota that is specific as well as temporally and spatially stable (van de Water et al., 2016, 2017; van de Water, Voolstra, et al., 2018). It was therefore previously hypothesized that the microbiota may have evolved closely with the host via co-diversification and/or via vertical transmission (maternal inheritance) of bacterial communities between coral generations in these larval-brooding species (van de Water, Voolstra, et al., 2018). Here, we confirm that phylosymbiosis exists in Mediterranean coral holobionts, but the mechanisms explaining this pattern remain to be investigated.
On the contrary, the tropical octocorals from the Red Sea and their microbiota did not demonstrate phylosymbiotic relationships. This contrasts with the findings in octocorals from the Great Barrier Reef (O’Brien et al., 2020) and to the patterns observed in the temperate Mediterranean in this study. Particularly the positioning of K. utinomi and S. loyai in the bacterial community dendrogram did not match with the host phylogeny. O’Brien et al. (2020) also observed three such ‘mismatches’ but phylosymbiosis signals were still detected. However, their dataset contained a greater diversity of octocoral species representing a wider range of octocoral taxonomy, which may have contributed to better detection of phylosymbiotic patterns (Lim and Bordenstein (2020)). The relatively thick layer of mucus surrounding their tissues is another factor that might have masked the phylosymbiosis signal in the Red Sea octocorals. In scleractinian corals, phylosymbiosis was in fact only found in bacterial communities associated with host animal tissues and skeleton, but not in the mucus (Pollock et al., 2018). To obtain a better understanding of the relationships between octocorals and their microbiome, it may be important to target different anatomical regions of the coral hosts to determine if this would result in different phylosymbiosis patterns. Besides, future studies into evolutionary links between tropical octocorals and their microbiota would likely benefit from expanding the dataset to include additional octocoral species representing higher diversity in octocoral taxonomy.
Overall, we detected signals of phylosymbiosis in octocoral holobionts in only one of the two seas studied. As populations in the Mediterranean Sea and Red Sea are geographically separated, this suggests that vicariance may explain the divergent evolutionary patterns between host phylogeny and microbiota structure. But also, that phylosymbiosis in octocoral holobionts is not evident and may be specific to oceanic regions. To obtain a broader picture, meta-analyses with more standardized methodologies are needed.
Cophylogenetic interactions are restricted to only a few host-symbionts associations.
While phylosymbiosis sheds light on how species that are closely related tend to have more similar microbiota, fine-scale tests of host-microbe cophylogeny help identify specific microbial lineages that may have coevolved with their hosts. These specific lineages are worth exploring as they may play an important role in shaping the dynamics of the microbiota.
Of the 244 most abundant OTUs (with a relative abundance of > 0.1%) that were examined, 101 OTUs were found to have significant associations contributing to the cophylogenetic signal with one or more of the 14 octocoral species investigated (0–24 phylotypes per coral species). This finding supports the idea that a limited group of bacterial phylotypes might play a significant role in host fitness. Similar findings were observed in two previous studies on octocorals, scleractinian corals and marine sponges, where only a small number of bacterial genera, such as Endozoicomonas, displayed cophylogenetic patterns and contributed to a phylosymbiotic signal (O’Brien et al., 2021; Pollock et al., 2018). The cophylogenetic signals observed for the host-bacteria associations listed below suggest that codiversification (i.e., parallel evolutionary changes and speciation events in two or more host and symbiont lineages, leading to a shared evolutionary history) is likely significant between corals and certain bacterial lineages. It can result from different mechanisms such as coevolution but also vicariance (specificity of certain bacterial strains according to geographical and ecological factors) and vertical transmission.
In this study, nearly half of the 101 OTUs that showed a cophylogenetic signal with octocorals belonged to the genus Endozoicomonas, and such associations were present in 9 of the 14 octocoral species studied here. Interestingly, cophylogeny was observed between the abundant Endozoicomonas OTU3 and both P. clavata and E. cavolini, and between OTU1 and both E. cavolini and E. verrucosa. And in octocorals from the Red Sea, cophylogenetic associations with Endozoicomonas were particularly found in Ovabunda spp. (24), S. vrijmoethi (6) and P. thyrsoides (7). This shows the importance of the Endozoicomonas-coral associations in the evolutionary history of octocorals from both seas and highlights the possibly important contributions of these symbionts to their host’s fitness, such as the digestion of complex molecules (Alex et al. 2019; Speck et Donachie 2012), the provision of amino acids (Neave et al., 2017), vitamins (Maire et al., 2023), and involvement in sulfur cycling (Tandon et al., 2022). However, others question the perception that Endozoicomonas is always a mutualist symbiont in coral holobionts (Pogoreutz and Ziegler 2024).
Spirochaetaceae were another main microbial taxon identified as contributing to the cophylogenetic congruence in our study. This was the case in three temperate octocorals (C. rubrum, C. verticillata and A. coralloides) and in two tropical octocorals (S. loyai and S. eilatense). The microbial associations showing a fit with cophylogeny were different for each coral species, and primarily involved the genus Spirochaeta as well as unclassified Spirochaetaceae. While some coral species may have co-evolved with their most abundant Spirochaetaceae symbionts, others may have an evolutionary relationship with lower abundant or rare phylotypes, as previously observed in other coral species and sponges from the Great Barrier Reef (O’Brien et al., 2021). For example, the most abundant Spirochaetaceae in C. rubrum did not contribute to cophylogenetic congruence, whereas a member of the genus Spirochaeta did. This cophylogenetic link is interesting as the Spirochaeta symbiont of C. rubrum has been hypothesized to be involved in the characteristic color of this red coral (Van De Water et al. 2024). Surprisingly, however, most of the tropical octocorals assigned to the ‘Endozoicomonas-Spirochaetaceae'-dominated microbiota group did not show cophylogenetic relationships with their Spirochaetes symbionts. The role of Spirochaetaceae within the coral holobiont remains to be investigated, but other symbiotic Spirochaetes are known to fix nitrogen (Lilburn et al., 2001), metabolize carbon sources (Lim et al. 2019), and produce vitamin B6 and the antimicrobial compound pyrroloiminoquinone (Waterworth et al. 2021).
Rhodobacteraceae also showed cophylogeny with octocorals in which they were a main symbiont (S. loyai, S. polydactylum) as well as S. vrijmoethi. O’Brien et al. (2021) also found cophylogenetic relationships between this taxon and corals from the Indo-Pacific. The function of these symbionts is yet unknown, but members of this taxon have been suspected to be opportunistic pathogens as they are often found in stress-impacted corals (Alsharif et al., 2023; Clark et al., 2021; Prioux et al., 2023; Rosales et al., 2020, 2023). This also shows that it is difficult to interpret the results on evolutionary links between corals and microbes, as these analyses cannot distinguish between the nature of these host-microbe relationships, and thus the importance of these microbes in host health.
Besides members of the three taxa that dominated the bacterial communities of the octocorals studied here, phylotypes belonging to other taxa were also found to contribute to cophylogenetic congruence. For example, cophylogeny was observed between A. coralloides and Thioglobaceae SUP05 cluster OTU73. This microbe is part of A. coralloides' core microbiome, and may be of importance for the health of this coral because of its amino acid and B vitamin biosynthesis capacities, as well as for its antiviral defense system and chemoautotrophic metabolism has been demonstrated (Keller-Costa et al. 2022). Cophylogenetic signals were also detected between Mollicutes and two corals from the Mediterranean Sea, particularly OTU101(Entomoplasmatales) and C. verticillata, and OTU27 (Mycoplasma) and C. rubrum. Mycoplasma has been proposed to feed commensally on remnants of prey captured by scleractinian cold-water corals (Kellogg et al. 2009; Neulinger et al. 2009), and may explain how this type of association evolved. Mycoplasma have also been identified to play a role in cophylogenetic in salmonids (Rasmussen et al., 2023). Moreover, associations between Spongiibacteraceae OTUs from clade BD 1–7 and the corals C. verticillata and E. verrucosa were detected to contribute to cophylogeny, which may not be surprising as these bacteria are commonly found at relatively high abundances in various gorgonian coral species (Quintanilla et al., 2022; van de Water et al., 2017; van de Water, Voolstra, et al., 2018). Characteristic for P. clavata were its cophylogenetic associations with various Alphaproteobacteria (Rhizobiaceae, Terasakiellaceae, Fokiniaceae, Sneathiella and Candidatus Megaira), which are not very abundant in its microbiome. These taxa have been observed, although in low abundance, within the microbiome of Mediterranean gorgonians (Prioux et al., 2023; Tignat-Perrier et al., 2022, 2023). Terasakiellaceae were also detected across multiple coral species (Parker et al. 2020; Quintanilla et al. 2022; Weiler et al. 2018). Terasakiellaceae as well as Sneathiellaceae may play a role in the cycling of nitrogenous compounds (Carareto Alves et al., 2014; Kurahashi et al., 2008) within the coral holobiont.
Interestingly, we also found cophylogenetic associations between the octocorals studied here from the Mediterranean Sea and Red Sea and several bacterial taxa that had previously been shown to have cophylogeny with octocorals from the tropical Indo-Pacific. Notably, O’Brien et al. (2021) found evolutionary links between Sclerophytum and Sarcophyton coral species and Cyanobacteria, Chloroflexi, Verrucomicrobiae and Thermoanaerobaculaceae. Compared with scleractinian corals, few similarities have been observed. While Pollock et al. (2018) found strong cophylogenetic signals between scleractinians and Endozoicomonas, they also revealed cophylogeny with Clostridiaceae, Kiloniellales and Myxococcales. Such relationships have, however, not been identified in octocorals so far.
The absence of cophylogenetic relationships between certain high abundant phylotypes and their coral hosts raises intriguing questions about the dynamics of host-symbiont interactions. For example, neither OTU1 in P. clavata nor OTU4 in C. rubrum exhibited significant cophylogenetic associations with their respective hosts, despite their high abundance within the holobionts. This suggests a lack of specific evolutionary relationships between them. This finding challenges the conventional assumption that dominance within the microbiome implies a strong and persistent relationship with the host organism. Other factors could thus play important roles in shaping the nature and efficacy of these associations.
The observations by us and others highlight the potential importance of relatively few but taxonomically diverse microbial taxa in the evolutionary history of coral-bacteria associations. Corals throughout the world appear to engage in symbioses with several specific microbial taxa, particularly Endozoicomonas. Altogether, these observations also suggest that it is not necessarily the most abundant symbiont or all members of the consistently associated core microbiome that may have co-evolved with their host.
Limitations in cophylogeny studies into microbe-coral associations
Our investigation of cophylogenetic patterns in host-bacteria relationships may present some limitations. First, as with most cophylogenetic studies, our assessment of bacterial phylogeny is based on variations within the variable regions of the 16S rRNA gene (O’Brien et al., 2021; Pollock et al., 2018; Youngblut et al., 2019). Although this approach is currently the most practical, it involves the use of amplicons, which are relatively short marker sequences with limited phylogenetic information and the results may have to be interpreted with some caution. For example, no overlap in Endozoicomonas symbionts was found between P. clavata and Eunicella spp. previously (van de Water et al., 2017), whereas these corals share a dominant Endozoicomonas phylotype (OTU1) in this study. The main difference between these studies is the use of a different primer set (targeting V5-V6 and V3-V4, respectively) to generate the 16S rRNA gene amplicons. Using full-length 16S rRNA gene sequencing approaches may provide better resolution and insights into evolutionary aspects in coral-microbe symbiosis.
Second, as emphasized by O’Brien et al. (2021), cophylogenetic models do not provide insights into the nature of the symbiotic interaction between host and microbe (i.e., whether it is mutualistic, parasitic, or commensal), nor into the underlying mechanisms responsible for these observed patterns. It is thus essential to recognize that associations showing cophylogenetic congruence may not always be beneficial. For instance, some Vibrionaceae and Rhodobacteraceae significantly contributed to the cophylogenetic signal in various coral species from both seas. Although their role in these coral holobionts is unclear, members of these families have been implicated in coral disease and mortality (Rosales et al. 2023; Clark et al. 2021; Gajigan et al. 2017; Martin et al. 2002; Sun et al. 2023), and their evolutionary relationship may thus also indicate a potentially harmful relationship. To understand the effect of symbiosis on host fitness, it is crucial to consider both parasitic and mutualistic symbiotic relationships as equally important factors.