Molecular studies of the microbial communities associated with Sargassum are relatively limited [25-28, 49] and to our knowledge absent from near shore Atlantic coasts. Here, we uncover both the prokaryotes and the eukaryotic communities associated with 100 samples corresponding to Sargassum seaweeds from tide sites, the seawater surrounding stranded Sargassum or near-shore rafts, and to Sargassum seaweeds from inland storage sites.
Specific prokaryotic diversity associated with Sargassum samples
The prokaryotic communities associated with the Sargassum tide-related samples were dominated by Proteobacteria (39.3%), Bacteroidetes (30.9%), Cyanobacteria (7.0%), Spirochaetes (5.5%) and Firmicutes (3.7%). Considering the three different compartments studied here, while the higher taxonomic ranking of diversity and abundance appeared quite similar (Figure 3A), some differences were visible in the order level (Additional file 2). The most abundant families associated with the marine Sargassum samples were the Vibrionaceae (Proteobacteria) (14.18%), a family of ubiquitous marine bacteria. Within the Flavobacteriaceae (11.23% of M.Sarg), we identified putative fish (Flavobacterium, 36 OTUs; Tenacibaculum, 3 OTUs) and human (Elizabethkingia, 4 OTUs) pathogens. The third one is the Saprospiraceae (Bacteroidetes) (8.14% of M.Sarg), a family that might contribute to the degradation of complex macromolecular release (i.e., polysaccharides) from the seaweeds and their associated algae. The fourth family is the Spirochaetaceae (4.83% of M.Sarg), which was shown to dominate the microbial community associated with corals [50] and contained anaerobic species living in coastal marine sediments [51]. The next most abundant families were the Prolixibacteraceae (4.35% of M.Sarg), and the Rhodobacteraceae (2.88% of M.Sarg). Overall, our results show that the bacterial communities, which could be a mix of epiphytes and probably endophytes, associated with tide-associated Sargassum, are distinct from those in the surrounding seawater, confirming findings previously obtained for various macroalgae (for Sargassum sp. see [25, 26, 28]).
Interestingly, even with only nine samples of Sargassum collected from inland storage sites, striking differences were found at the highest taxonomy levels. In T.Sarg, Acidobacteria was the sixth most abundant phylum (4.06%), whereas these bacteria were barely present in the two other compartments analyzed (0.14 and 0.31% for the SW and M.Sarg, respectively). This phylum has been recognized as one of the most abundant and diverse on Earth, predominantly colonizing soil habitats [52]. The other striking difference was for the FBP candidate division, with average abundance of 1.75% in T.Sarg and below 0.002% in the other samples. Members belonging to this taxon are known to have the capacity to degrade cellulose and hemicellulose [53] and are potential candidates for algae biomass degradation. At family level, we found the most abundant ones were the Flavobacteriaceae (9.31% of T.Sarg), the Prolixibacteraceae (5.36% of T.Sarg), and the Halomonadaceae (4.73% of T.Sarg). Interestingly, the two most abundant species correspond to the genera Cobetia and Halomonas. Some members of this genera were isolated in marine and coastal environments, but also in acid mine drainages which are environments with low pH, high salinity, toxic heavy metals and metalloids [54]. Interestingly, storage sites were shown to present high salinity levels and high concentrations of arsenic, which is bioaccumulated by S. fluitans and S. natans (Devault et al., unpublished data). The other most widely represented families were the Chitinophagaceae (4.21% of T.Sarg), the Pirellulaceae, which contain ammonia-oxidizing bacteria and bacteria associated with biofilms colonizing microplastics [55] and surface sediments, the Spirochaetaceae (3.57% of T.Sarg), and Acetobacteraceae (2.96% of T.Sarg). The most abundant OTU of the latter family was assigned to the genus Acidiphilium, containing aerobic bacteria producing acetic acid. Overall, our results highlight changes in prokaryotic diversity associated with the Sargassum holobiont upon terrestrial storage.
Metabolic predictions of Sargassum-associated microbiota
To gain insights into the roles of prokaryotic taxa, their putative functions were inferred for 2,831 OTUs. We found that 1,260 OTUs were deemed to be chemoheterotroph, representing 23.2% of the overall abundance (Figure 6). They belong to several bacterial genera, with the most abundant one being Vibrio, followed in M.Sarg by Reinekea, Desulfomicrobium, Spirochaeta, Polaribacter, Cobetia and Alteromonadaceae. Interestingly, the genus Cobetia harbors strains capable of crude oil degradation and producing biosurfactants [56], while Reinekea members were shown to use numerous molecular weight sugars and algal polysaccharides for growth, and are capable of oxidizing thiosulfate and fermenting under anoxic conditions [57]. The predominant bacteria predicted to perform nitrate reduction corresponds to Vibrio. Due to the complexity of the N cycle, with numerous possible substrates and product makes, it is difficult to assume the role of these bacteria in this instance, but they may make a significant contribution to N assimilation, either as free-living bacteria or associated with particles, zooplankton or other eukaryotes [58]. Phototrophy, corresponding mainly to abundant OTUs of the Oxyphotobacteria (Cyanobacteria) class, represented 241 OTUS and 6.9% of the total abundance. Cyanobacteria have long been described on the surface of Sargassum [59] and might also correspond to symbiotic association across the diverse prokaryotic and eukaryotic lineages present in Sargassum rafts.
When washed ashore, Sargassum will decompose and provoke significant environmental issues and health concerns due to the hydrogen sulfide (H2S) and ammonia (NH3) gas emissions. Functional prediction identified 404 OTUs involved in the respiration of sulfur compounds. These OTUs were assigned to several orders including Thermococcales (1 OTU), Clostridiales (51 OTUs), Desulfarculales (35 OTUs), Desulfobacterales (257 OTUs), Desulfovibrionales (87 OTUs), Desulfuromonadales (3 OTUs) and Syntrophobacterales (3 OTUs). We also found OTUs that were not predicted by FAPROTAX but are likely to contribute to the S cycle. These corresponded to the genus Sulfurovum or Sulfurimonas. Some of these prokaryotes, which might originate from sediments on the marine surface layers, may contribute to Sargassum decomposition and hydrogen sulfide emissions. While preliminary studies of the Desulfobacterales family reveal a large number of putative novel species, understanding the diversity of sulfate-reducing microorganisms and sulfur-oxidizing microorganisms associated with the Sargassum decomposition process will require further study. In particular, investigation of the presence of functional S cycling-related genes (see, for example, Pelikan, 2016 #9032) will be needed. The sulfur cycle is linked to other cycles including carbon, metals and metalloids such as arsenic (As) I [60, 61]. This is of particular interest and another important issue given the large amount of As found in S. natans and S. fluitans along the coastlines of these islands (Devault et al., unpublished data).
A total of 491 OTUs were predicted to be symbiotic, with parasites or human and animal pathogens, including OTUs assigned to the Coxiellales order (128 OTUs of the genus Coxiella), Rickettsiales and Legionellales (55 OTUs of the genus Legionella). Members of these genera can cause severe disease in humans, and their presence in Sargassum-associated samples needs to be investigated with special care since they could well present potential risks. Coxiella were also shown to correspond to stable symbionts associated with corals [62]. The presence of prokaryotic symbionts and eukaryotic parasites can partially explain the significant correlation observed between prokaryotic and eukaryotic community structures.
In the terrestrial samples (T.Sarg), we found OTUs potentially involved in methanogenesis (Additional file 5) among the archaeal Methanomicrobiaceae, Methanosarcinaceae and Methanococcaceae families. These families are often members of microbial communities involved in methane production from algal biomasses [63]. Due to the anaerobic conditions associated with inland storage sites, especially those sampled which were essentially piles of Sargassum, our findings also suggest putative methanogenesis from Sargassum decomposition. This process could be of great interest in the biomass valorization of Sargassum.
Commonality and differences in eukaryotic diversity
Several taxonomic investigations devoted to specific classes or to the global eukaryotic communities have revealed the micro- and meiofauna associated with Sargassum spp. [19, 49, 64-67]. The present study reports the largest molecular inventory of eukaryotic species associated with Sargassum.
The largest group found corresponded to the opisthokonts, representing 24.9% of the total diversity and about half (48.9%) of the abundance. With a total of ~3,600 OTUs, our data highlight the significant diversity of metazoans, with considerable differences depending on the Sargassum samples (Additional file 4). With ~2,200 OTUs, the surrounding water was dominated, in decreasing order, by Arthropoda, Bryozoa and Annelida (Additional file 3). A contrasting result was found for the M.Sarg samples that were dominated by Bryozoan, Arthropoda, Platyhelminthes, Cnidarian and Nematodes (Additional file 3). Bryozoa, a large group of often calcified aquatic invertebrates [68], which is known to contain abundant epibionts on Sargassum species [15, 66], were largely present on the surface of the collected Sargassum (Additional file 5). Phylogenetic analyses revealed that among the 490 OTUs assigned to Bryozoan, 438 of them were clustering with Membranipora membranacea (accession: JN680943.1) (not shown), including the most abundant OTU that largely dominates our dataset with 19.6% of the total abundance. Other previously described eukaryotes associated with Sargassum sp. were found such as Fungi (~410 OTUs; with putative endophythes of the genus Corollospora or Arthrinium) and Choanoflagellates (~190 OTUs) [64, 69].
Nematodes, which represented the most diverse group of Metazoans (21.4% of their total diversity), showed striking differences depending on the substrate type. They were largely dominant in terrestrial samples, with ~92% of the metazoan’s abundance. The association between nematodes and floating Sargassum from the Atlantic region was already noted a century ago [70]. While colonization of such usually benthic meiofauna could be the result of accidental encounter, benthic nematode species were found attached to the surface of pelagic or benthic Sargassum spp [71-74]. In our dataset, we found that the most abundant OTUs could be present in the three different compartments, although with potentially specific patterns (Figure 4). Marine Sargassum-associated nematodes belong to the Enoplia and Monhysterida clades that have previously been described in marine habitats and present bacteriovore and algivore-omnivore-predator trophic behaviors [39, 75]. Stranding and decaying stranded Sargassum along the shore could induce alterations in the structure of the intertidal soft-bottom and help nematode fauna to colonize the macroalgae. On the other hand, Halicephalobus (Tylenchina) and Rhabditidae, which are bacterivores [39], presented exclusive or specific patterns associated with terrestrial sites. Regarding the Halicephalobus OTUs, the best blast hit for Otu00067 and Otu00111 was Halicephalobus mephisto (accession number GQ918144), a bacterivore nematode found a the terrestrial deep subsurface that presented hypoxic conditions and a temperature of 37°C [76]. Similar conditions are expected to occur in T.Sarg samples.
After the Opisthokonts, the Alveolata were the second most diverse superphylum in our samples, including 1,932 dinophyceae and 1,557 ciliate OTUs, corresponding to 15.4% and 5.4% of the total abundance, respectively. Among the ciliates, the Oligohymenophorea, which corresponded to the third most abundant class, with 586 OTUs, were abundant in all sample types (Additional file 3). The Spirotrichea and Plagiopylea were especially present in the seawater samples and the Colpodea in the terrestrial ones (Additional file 3). This latter ciliophoran class is mainly present in terrestrial and semiterrestrial habitats, although there is some evidence of its presence in marine environment surveys [77, 78]. Among the Oligohymenophorea, some Scuticociliates are known to invade and colonize marine hosts’ internal organs and can induce severe systemic infections. For instance, Scuticociliatosis has been reported in a broad range of teleosteans [79-81]. Here, we found a number of OTUs which could be assigned to known causative agents of scuticociliatosis: Pseudocohnilembus persalinus or P. longisetus (37 OTUs were assigned to this gender), Uronema marinum (2 OTUs), and Miamiensis avidus (1 OTU), suggesting health concerns or at least surveillance.
Ciliates are versatile organisms and display considerable functional diversity, including in food webs, acting as predators of bacteria, algae, other protists and even some metazoans. Some ciliates are known to be sensitive to oxic/anoxic conditions [82-85] which can occur upon heavy accumulation of Sargassum on the shoreline. Ciliated protozoans were found to be abundant in mats dominated by sulfide-oxidizing bacteria [86], or in sulfur cycling in marine sediments [87]. Ciliates can also present chemosynthetic symbioses with bacteria and archaea [88], including endosymbionts that are involved in methane metabolism [89]. In particular, members of the Plagiopylea and Armophorea classes correspond to ciliates living in oxygen-depleted habitats, which have been shown to host endosymbiotic methanogens [40, 41, 90-93]. Here, we found a total of 134 OTUs belonging to these two classes of ciliates, with the most abundant OTUs present mainly in SW and M.Sarg samples (Supplementary Figs. 7-8). Our data suggest oxygen concentration depletion at least in marine tide sites, and that some of these organisms could be involved in methane and/or sulfate cycles along the shore.
The third most abundant supergroup, the Stramenopiles, was largely dominated by Bacillariophyceae, with ~2,000 OTUs or ~11% of the total diversity. After the diatoms came the Labyrinthulomycetes, which are protists found as parasites on unicellular phototrophs, green algae, and diatoms, and which present pathogenic behavior towards shell organisms (Additional file 3). More generally, heterotrophic protists are organisms commonly found on seaweed surfaces, and are known to play various roles, including in the consumption of organic materials such as dissolved organic carbon (DOC), released by seaweeds such as Sargassum [94]. Thus, the large diversity of protists found here are likely to make a significant contribution to primary and secondary production and to nutrient cycling in the surf and the swash zones [95].
Identification of Sargassum biomarkers
We used the ALDEx2 algorithm on the prokaryotic and eukaryotic community matrices to identify abundant OTUs with differential abundance between substrates (SW versus M.Sarg) and media (M.Sarg versus T.Sarg). Interestingly, the OTUs with differential abundance in the M.Sarg samples were Dinophyceae, three unclassified Maxillipoda, and probably copepods, which have often been described as associated with Sargassum sp. [66]; three OTUs corresponding to Bryozoa and probably to the genus Membranipora (see [15]), a hydrozoan of the genus Zanclea that was described as being common on a hydroid assemblage on holopelagic Sargassum from the Sargasso Sea [65], and one OTU of flatworm of the Rhabdocoela order, some of whose species are known to live in association with Sargassum [96]. Among the 24 OTUs differentially expressed in the M.Sarg sites (Figure 5), somewhat remarkably, we found one OTU assigned to a toxic dinoflagellate of the Amphidinium carterae genus, another to Ulvella that are endophytic microalgae, one related to the raphid diatom genus Aneumastus, one OTU with the best BLASTN results as Sargassococcus simulans, an epiphyte on floating Sargassum thallus, isolated in the Sargasso Sea, one to the copepod genus Zaus, which are organisms living in the phytal zone, and five to Hydrozoans (Opisthokonta, Cnidaria), which are also frequently encountered as Sargassum epibionts [66].
We also found 21 prokaryotic biomarkers in the M.Sarg samples, including cyanobacteria (4 OTUs) and Saprospiraceae (4 OTUs), which are important in the breakdown of complex organic compound (Figure 6). The M.Sarg samples only showed 4 OTUs that were differentially present in this compartment compared to T.Sarg which had two of the three most abundant OTUs in the whole dataset (a Cyanobiaceae and a Cryomorphaceae). In the terrestrial samples, 11 OTUs were considered as biomarkers, with Saprospiraceae (3 OTUs) and new environmental members of the Rivulariaceae family. Altogether, our data revealed a number of potential new molecular markers associated with Sargassum racks from the Caribbean, with a number of them corresponding to potentially new or poorly described species involved in the trophic food webs at tide sites.