The diversity of corals in the Aleutian archipelago is exceptional, and the region may harbor the highest abundance and diversity of cold-water corals in the world (Heifetz et al., 2005). Their bacterial communities, however, are almost entirely unknown and have not been sampled by either cultivation or cultivation-independent means. Compared with the cold-water scleractinian L. pertusa, the gorgonians of the Aleutian Islands have received very little attention.
The Gammaproteobacteria (the Vibrionaceae in particular) were dominant under all cultured conditions compared with the clone libraries. Cultured isolates from the samples grown at both 21 °C and at 4 °C indicated the importance of replicating the original growth conditions of the isolates, with half as many isolates growing at room temperature and slightly skewing selection even more toward Vibrionaceae (21% in the 4 °C isolates, 27% in the 22 °C isolates). The different bacteria recovered from cultured vs. clone library and the dominance of the Vibrionaceae within the cultured isolates indicate a notable selection bias arising from the GASWA medium. The presence of Vibrionaceae has been previously demonstrated from cold-water corals as disease-causing agents in temperature-stressed Eunicella verrucosa (Hall-Spencer et al., 2007), as has their association with disease in shallow-water corals (Kushmaro et al., 2001; Ben-Haim et al., 2003). However, Vibrios have also been shown to be a normal component of healthy tropical corals (Bourne & Munn, 2005).
There were few recovered isolates that were present in both of the samples from a given coral species. Among the cultured bacteria, all of the corals (with the exception of 6205, which failed to yield cultivable bacteria) had Vibrionaceae isolates. Both of the P. arborea samples had isolates that grouped closely with Colwellia psychrerythaea, a motile, psychrophilic heterotroph with a large repertoire of carbon-cycling abilities, as well as other nutrient-cycling functions (Methe et al., 2005) that may be important components of the biochemistry of the coral holobiont. All three species hosted close relatives to Photobacterium phosphoreum, the bacteria responsible for light production in many deep-sea fishes and other animals. Photobacterium species (also isolated on GASWA) have been determined to be common residents of the shallow-water, tropical coral Acropora palmata (Ritchie, 2006).
The clone libraries were dramatically different from each other, with samples 6203 and 6205 showing far more diversity than samples 6206 and 6215 (Fig. 2). Rarefaction curves (Fig. 1) generated from the clone libraries from samples 6206 and 6215 tapered off quickly, reaching a plateau at approximately 25 operational taxonomic units. This is a pattern very similar to that seen in the white L. pertusa sampled by Neulinger et al. (2008), whereas samples 6205 and 6203 showed more diversity, generating a curve more similar to that of red L. pertusa. The rarefaction curve generated by Penn et al. (2006) for their libraries from deep-water corals (both bamboo and black corals) reaches a higher plateau. However, it is difficult to make direct comparisons because the libraries in that study were combined. The reason for the differences between these samples is not immediately clear. Samples 6203 and 6206 are both from P. superba, whereas samples 6205 and 6215 are from C. koolsae. It is striking that the differences between the corals could be so drastic, regardless of the coral species sampled.
The sites where samples 6205 and 6206 were collected are very close to each other, separated by <300 m, whereas the 6203 collection site is 790 m to the northwest, and the 6215 collection site is approximately 122 km to the southwest and all were within 3.5 km of land. Additionally, there is no temporal explanation for the differences between the clone libraries because 6203, 6205, and 6206 were all collected the same day, and 6215 was collected 5 days later. The Delta does not have the equipment to measure current speed, and so we can only note that currents were relatively strong at all locations. A notable difference between the grouping of 6203–6205 and 6206–6215 is depth. Samples 6203 and 6205 were collected deeper (138 and 110 m, respectively) than 6206 and 6215 (96 and 86 m, respectively). This forces the question of whether there is a significant difference in oceanographic environments that are separated by only 14 m depth (6205 vs. 6206, 14 m depth and 300 m horizontal distance), that may account for the observed differences. Historically, the area has a strong seasonal halocline, found at a depth of approximately 100–140 m (e.g. Coyle, 1998; Miura et al., 2002), that may affect microbial communities at various depths in the Aleutian Islands. Oceanographic measurements made during the time of collection did reveal the presence of a moderately strong thermocline at 100–110 m depth, however, that might be responsible for the observed differences in the microbial communities.
Of the clones recovered, approximately 23% have a high-ranking blast match to another deep-water coral clone, and an additional 13% have a match to a shallow-water coral clone. Very few of the sequences recovered from the clone libraries are shared between samples of the same coral species. This makes it especially difficult to determine whether there are specific bacterial symbionts for these coral species. A few sequences were found in both of the samples from C. koolsae: one group nearest to Steroidobacter denitrificans (clones 6205-B10 and 6215-B83) and an Alphaproteobacterium (clones 6205-B78 and 6215-B73) that was most closely related by blast to Anaplasma phagocytophilum, a pathogenic Rickettsiales. Rickettsiales have been described in close relationship with both diseased and healthy A. palmata and Acropora cervicornis (Casas et al., 2004). The only closely grouped clones recovered from P. superba were the Spirochaete-like sequences. These isolates and clones may make up some of the microfauna that are unique to the coral species, or at least common enough in the species to be captured by our sampling strategies.
This study is the first to report spirochete-like sequences from a gorgonian coral in the deep sea, with samples from both of the P. superba corals containing one spirochete-like clone each. The two Spirochaete-associating sequences that were recovered (6203-B80 and 6206-B24) grouped with a clone from Montastraea faveolata (Sunagawa et al., 2009) and an isolate from the Peruvian continental margin oligochaete worm Olavius crassitunicatus (Blazejak et al., 2005). Spirochaete-like sequences have been reported from a deep-sea scleractinian L. pertusa (Kellogg et al., 2009), but those sequences only share approximately 80% similarity with the clones in this study.
The large variability of bacterial assemblages between samples of even the same species of coral has been observed in previous deep-sea studies. Penn et al. (2006) noted that there were significant differences between bamboo corals sampled from the same seamount, with one coral having primarily Alphaproteobacteria and Acidobacteria, whereas the other had mostly Firmicutes (Penn et al., 2006), including Mycoplasma that were reclassified as Tenericutes at a later date. In deep-water scleractinian corals, bacterial composition has also been shown to be quite variable within the same species of coral. In L. pertusa, samples from different areas grouped together based more on collection area, with most of the samples from the deeper area (VK826) dominated by Tenericutes, whereas the shallower site was dominated by Gammaproteobacteria (Kellogg et al., 2009).
All of the clone libraries had clones that grouped into a collection of two clades within the Tenericutes (Fig. 3). One of the clades grouped together with clones from both a deep-sea octocoral from the Gulf of Alaska (DQ395563, Penn et al., 2006) and a shallow-water octocoral, Muricea elongata (DQ917875 and DQ917898; L.K. Ranzer, P.F. Restrepo, & R.G. Kerr, unpublished data). A second clade consists only of clone sequences generated in this study with representative sequences from both the C. koolsae 6215 clone library and the P. superba 6206. These two octocoral-associated clades grouped together with the clone sequences from the deep-sea scleractinian L. pertusa in the Gulf of Mexico (Kellogg et al., 2009) and ‘Candidatus Mycoplasma corallicola’ (AM911412.1) from L. pertusa in Norwegian fjords (Neulinger et al., 2009). None of the sequences recovered in this study grouped closely with the scleractinian-associated Mycoplasma, indicating that each coral group may have a group of Mycoplasma with which they coexist. Additional Tenericutes sequences were recovered from sample 6205 that grouped with Spiroplasma, Lactobacillus, and Shimazuella, and not with any other marine or coral clones.
Through the use of FISH, Neulinger et al. (2009) demonstrated that the ‘Candidatus Mycoplasma corallicola’ was located near the nematocysts within the tissue of the L. pertusa from Norway, possibly to absorb nutrients from the sloppy feeding of the L. pertusa polyps. Kellogg et al. (2009) showed that these mycoplasmas are conserved within L. pertusa, based on identical and similar Mycoplasma-like sequences in the Gulf of Mexico. Mycoplasmas have also been found in clone libraries from a shallow-water gorgonian (Fig. 3; L.K. Ranzer, P.F. Restrepo, & R.G. Kerr, unpublished data) and in metagenomic analyses of Porites compressa, a shallow-water scleractinian (Thurber et al., 2009). The prevalence of mycoplasmas in all of the clone libraries from the current study is of some note, because it is one of the few groups shared in all of the clone libraries as well as being commonly found in coral clone libraries from both deep-sea octocorals and scleractinians (Penn et al., 2006; Neulinger et al., 2008; Kellogg et al., 2009). This would indicate a possibly significant role may be played by the mycoplasmas in these corals. Mycoplasmas seem to be a key, yet understudied, part of the coral microbiome.
Many of the Gammaproteobacteria-affiliated clones (Fig. 4) grouped very closely with several isolates from various other deep-sea investigations. There was also a cluster of clones grouping with the anaerobic denitrifier S. denitrificans (Fahrbach et al., 2008), indicating that there may be many microenvironments within the coral colonies that allow for anaerobic or microaerophilic metabolism, further increasing the available niches and possibly increasing microbial diversity.
Clones clustering with the Verrucomicrobia grouped with a ridge-flank crustal-fluid clone (Huber et al., 2006), a Bering Sea sediment clone, and with another deep-sea octocoral clone (Penn et al., 2006). Verrucomicrobia have been described from both the white and the red L. pertusa from Norwegian fjords (Neulinger et al., 2008), although they did not make up nearly as high a percentage in this study (6203, 8%; 6205 10%) as in the red L. pertusa (28%). Verrucomicrobia have been isolated previously from seawater near coral (e.g. Yoon et al., 2007) and from black-band-diseased Siderastrea siderea (Sekar et al., 2006).
Actinobacteria are frequently found in corals, in both shallow (Lampert et al., 2006) and deep water (Neulinger et al., 2008), and are a target for novel natural-products research (e.g. Bull & Stach, 2007). The Actinobacteria represented 19% and 10% of the clone library sequences from the white and red (respectively) L. pertusa in a Norwegian fjord, whereas in the present study, they comprise approximately 10% of both 6203 and 6205. This indicates that, although they are not necessary to the survival of the corals (6206 and 6215 both lacked Actinobacteria), they may be common coral associates.
Several chloroplast sequences were recovered and clustered with a clone from a white-plague-diseased M. faveolata (Sunagawa et al., 2009) and were most closely related to diatom plastids. The recovery of sequences that grouped with diatom plastids in samples 6203 and 6205 is interesting, considering that the sample sites are well below the photic zone. Phototrophs such as cyanobacteria have been observed in the deep sea before, in L. pertusa clone libraries from the Gulf of Mexico (Kellogg et al., 2009), as well as shallow-water (40-m depth) L. pertusa from a Norwegian fjord (Neulinger et al., 2008). Both diatoms and cyanobacteria have been shown to be able to live heterotrophically (Lewin, 1953; Zehr et al., 2008). Although the sequences grouped with a pair of Cyanobacteria sequences, both those sequences (AM501739 and AY711425) and the sequences recovered in this study cluster more tightly in blast searches with diatom plastids than with Cyanobacteria. However, the sequences detected in this study may represent components of the corals' last meals, the remains of diatoms in the marine snow from the surface. Clones that grouped with the Chloroflexi may also be facultatively heterotrophic because Chloroflexi species have been described from deep-sea sediments (Inagaki et al., 2006), indicating more phenotypic diversity within the group than is generally assumed.
The variability between the clone libraries from the same species is large enough that we must consider that there is no ‘typical’ bacterial community associated with these species, although Tenericutes will likely be found to play a major role. It is possible that, like the ‘low-bacterial abundance’ sponges (Taylor et al., 2007), their bacterial assemblages are largely a function of the water column and their diet. However, it can be suggested that the association of bacteria such as Colwellia sp., with their nutrient- and carbon-cycling abilities, indicates that the corals may be dependent upon bacterial processes for a portion of their nutrition and that we simply need to sequence more thoroughly and attempt culture with a greater variety of media to more accurately capture bacterial diversity.
The North Pacific Fisheries Management Council and National Marine Fisheries Service recently closed vast areas of seafloor in the Aleutian archipelago to bottom trawling in order to protect sensitive deep-sea coral habitat (71 FR 36694). By understanding the microbial consortia associated with these corals, we gain a better understanding of their relationship with their environment and the ecosystem services they provide. Additional research is needed on these corals to further characterize the microbial consortia and to understand the expanded biochemical repertoire afforded to the corals by their associated bacteria. Repeated sampling would help us understand the differences we observed within the coral species and their microbial consortia.