Microbiome variation in corals with distinct depth distribution ranges across a shallow–mesophotic gradient (15–85 m)
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Mesophotic coral ecosystems (MCEs) are generally poorly studied, and our knowledge of lower MCEs (below 60 m depth) is largely limited to visual surveys. Here, we provide a first detailed assessment of the prokaryotic community associated with scleractinian corals over a depth gradient to the lower mesophotic realm (15–85 m). Specimens of three Caribbean coral species exhibiting differences in their depth distribution ranges (Agaricia grahamae, Madracis pharensis and Stephanocoenia intersepta) were collected with a manned submersible on the island of Curaçao, and their prokaryotic communities assessed using 16S rRNA gene sequencing analysis. Corals with narrower depth distribution ranges (depth-specialists) were associated with a stable prokaryotic community, whereas corals with a broader niche range (depth-generalists) revealed a higher variability in their prokaryotic community. The observed depth effects match previously described patterns in Symbiodinium depth zonation. This highlights the contribution of structured microbial communities over depth to the coral’s ability to colonize a broader depth range.
KeywordsMesophotic coral ecosystems Prokaryotic community 16S rRNA tag sequencing Indicator species
Mesophotic coral ecosystems (MCEs) represent an extension of shallow-water coral reefs and provide an extensive habitat for light-dependent corals in subtropical/tropical regions starting at 30–40 m and reaching down to depths of about 150 m (Lesser et al. 2009, Hinderstein et al. 2010). MCEs can be further divided into the “upper mesophotic” (30–60 m) and “lower mesophotic” (>60 m), with the first representing a transition zone between shallow-water and lower mesophotic communities (sharing species of opposing depth zones) and the latter representing a more specialized coral community (Bongaerts et al. 2010, 2015b; Kahng et al. 2010). Given the increased difficulty in accessing lower mesophotic depths, most studies have thus far been limited to visual surveys of the benthos, and consequently, little is known about the coral-associated microbial communities.
A recent molecular study demonstrated that lower mesophotic depths in the southern Caribbean harbor a genetically distinct coral and associated Symbiodinium community, likely reflecting specialization of both symbiotic partners to the mesophotic environment (Bongaerts et al. 2015b). The coral holobiont, however, harbors not only symbiotic phototrophic zooxanthellae but also numerous other microorganisms such as other protists, fungi, bacteria, archaea and viruses (Rohwer et al. 2002; Carlos et al. 2013). Prokaryotes, including bacteria and archaea, are of particular interest due to their diverse metabolic and functional capabilities within the coral holobiont and their potential to complement the metabolic needs of the coral host (Ainsworth et al. 2015; Thompson et al. 2015). Studies on shallow-water communities have shown that prokaryotic communities associated with corals are species specific (Rohwer et al. 2002) and have a significant influence on host resilience (Glasl et al. 2016). Another study has found coral microbiomes in Seriatopora hystrix to correlate to reef habitat (depth) and geographical location, but not to intrinsic factors such as host genetic lineage and Symbiodinium genotype (Pantos et al. 2015). Although the interest in coral–prokaryote interactions has increased over the last decade (reviewed by Thompson et al. 2015), only sparse information on the prokaryotic community associated with corals from the upper mesophotic is available (reviewed by Olson and Kellogg 2010) and data from lower MCEs are virtually non-existent (but see Ainsworth et al. 2015), despite the potential metabolic contribution of these communities (compared to Symbiodinium) to the energetic balance of corals given the extremely low light conditions at lower mesophotic depths (reviewed by Thompson et al. 2015).
Here, we provide a first assessment of the variation in the structure and composition of coral-tissue-dwelling prokaryotic communities from shallow reef habitats down to lower mesophotic depths in three common Caribbean coral species with broad, but distinct, depth distributions on the island of Curaçao. In this study, we aimed at (1) determining whether the lower mesophotic corals host a distinct prokaryotic community or indicator assemblages and (2) addressing the respective roles of depth and host in prokaryotic community structure.
Materials and methods
Overview of the number of collected samples, the number of retrieved sequences, alpha diversity (Shannon index), richness, evenness and richness estimations (Chao index) of the prokaryotic community associated with each of three studied coral species (Agaricia grahamae, Madracis pharensis and Stephanocoenia intersepta) over their natural depth distribution on the island of Curaçao
Number of samples
Number of sequences
OTUs in total
2824 ± 2037
2.81 ± 0.77
49 ± 15
0.72 ± 0.15
3286 ± 3502
2.77 ± 0.35
45 ± 5
0.73 ± 0.08
3570 ± 4106
3.11 ± 0.22
60 ± 4
0.76 ± 0.04
6730 ± 6682
2.99 ± 0.56
55 ± 16
0.75 ± 0.10
6686 ± 5891
3.09 ± 0.70
58 ± 14
0.76 ± 0.14
1842 ± 1823
2.35 ± 0.60
41 ± 9
0.63 ± 0.14
4223 ± 5345
2.96 ± 0.71
56 ± 20
0.74 ± 0.12
Sequencing and data analysis
Thawed samples were rinsed with Milli-Q water to remove loosely attached bacteria from the coral’s surface. After scraping the tissue off the skeleton using sterile scalpels, genomic DNA of 51 coral tissue samples was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals) and a 728-bp fragment of the 16S rRNA gene (amplified by primers U341F and U1053R) sequenced by IMGM using 454 GL FLX + technology (Roche). Barcoded sequence reads were de-noised in Acacia (version 1.52.b0; Bragg et al. 2012) and analyzed using QIIME (version 1.9.0; Caporaso et al. 2010b) following the protocol described in detail by Glasl et al. (2016). In brief, sequences were picked and clustered into operational taxonomic units (OTUs) based on >98% sequence similarity using USEARCH (version v5.2.236; Edgar 2010), checked for chimera, and singletons excluded. Representative sequences were picked and aligned with PyNAST (version 1.2.2; Caporaso et al. 2010a) using the Greengenes database (version 13.5). The taxonomy was assigned with the Ribosomal Database Project Classifier (version 2.2; Wang et al. 2007). Furthermore, chloroplast reads were removed and 409 sequences were randomly picked for each sample to compensate for different sequencing efforts. The rarefied OTUs were grouped at the family level, and their relative abundances per sample were used for statistical analyses in R (R Development Core Team 2008). Data were not normalized prior to multivariate analyses as no linear methods were applied. Demultiplexed 16S rRNA gene raw reads are available in the NCBI SRA database under accession number SRP092218.
The influence of depth and host species on the alpha diversity (Shannon–Weaver index), richness and evenness of the coral-tissue-associated prokaryotic community was assessed with analysis of variance (ANOVA). Canonical correspondence analysis (CCA) was used to determine whether location, depth and host species drive the prokaryotic community assemblage. The significance of those factors was verified using an ANOVA-like permutation test based on 9999 permutations.
Non-metric multidimensional scaling ordination (nMDS) based on quantitative and binary Bray–Curtis dissimilarities of relative abundance and presence/absence data (Anderson et al. 2006), respectively, was used to visualize the variation of the prokaryotic community among different host species for a single depth (at 55 m) and within each host species over their depth distribution. Differences in the community structure were tested by applying a permutational multivariate ANOVA (PERMANOVA) using Bray–Curtis dissimilarity matrices (Anderson et al. 2006). The homogeneity of multivariate dispersions was tested using a resemblance-based permutation test (PERMDISP).
Indicator values (IndVal) analysis (De Cáceres and Legendre 2009) was applied to identify prokaryotic families significantly associated (p < 0.05, when both specificity and fidelity have probabilities >0.5) with coral host species and depth zones.
Results and discussion
Prokaryotic communities associated with the tissue of the corals were structured according to coral species and depth (permutation test CCA; p < 0.001 and p < 0.01, respectively; Electronic Supplementary Material, ESM, Table S1). As sampling location did not influence prokaryotic community composition, samples from both locations (ESM Table S2) were merged for further analysis. There were no significant differences in alpha diversity, richness or evenness of the prokaryotic communities (based on prokaryotic families) among the three studied coral species or in response to depth (within each species; ESM Tables S3, S4, S5). Consequently, the community composition, rather than alpha diversity, is responsible for the observed variation in prokaryotic communities across sampling depths and among host species.
There were no significant differences in the prokaryotic community assemblage of A. grahamae, a deep-water specialist (Fig. 1b), between upper and lower depth populations (55 vs. 85 m; ESM Tables S8, S9). In contrast, S. intersepta, a depth-generalist (Fig. 1c), showed significant difference between depth zones, but only with presence/absence data (PERMANOVA, p < 0.01; ESM Tables S10, S11). This suggests that the rare or low-abundance prokaryotic families are driving the differences between depths within this species. Finally, M. pharensis, an “extreme” depth-generalist (Fig. 1d), showed significant depth variation (PERMANOVA, p < 0.01; ESM Tables S12, S13). The shallow (15 m) and the deep (85 m) populations of M. pharensis significantly varied in their prokaryotic community composition (PERMANOVA, p < ,0.01; ESM Table S14). Madracis pharensis samples from 55 m, however, seem to overlap with communities originating from the depth extremes and may therefore represent a transition from shallow water to the lower mesophotic reef. Similar results were obtained with presence/absence data (except for S. intersepta). Overall these results suggest that “depth-specialist” hosts, characterized by their restricted depth distribution (Bak 1977; Bongaerts et al. 2010), maintain a specific holobiont community. “Depth-generalist” hosts with a wide depth distribution (Bak 1977; Bongaerts et al. 2010), however, might host the most favorable prokaryotic composition for the surrounding environment (as also shown by Pantos et al. 2015). We hypothesize that this association of corals with a range of different prokaryotes over depth greatly contributes to host distribution and survival across the different depth habitats.
The observed spatial dynamics in prokaryotic community composition over depth for these three studied coral species closely resemble those reported earlier for their dominant Symbiodinium communities (as determined within the detection limits of ITS2-DGGE by Bongaerts et al. 2015a). Agaricia grahamae, for example, neither exhibits zonation in the genetic lineages of its associated Symbiodinium (type C3/C11 is ubiquitous, Bongaerts et al. 2015a) nor in the prokaryotic community over depth. In contrast, M. pharensis shows both a significant shift in the Symbiodinium community (type B7 in the shallow layers is completely replaced by type B15 in the mesophotic habitats; Bongaerts et al. 2015a) and in its prokaryotic community composition. Finally, S. intersepta exhibits a shift in the Symbiodinium community over its depth range (mixed communities changing from C16 to C3 and C1 as dominant types with increasing depth; Bongaerts et al. 2015a), as well as a shift in its prokaryotic community (based on presence/absence data). Although we cannot decisively differentiate between the effect of depth and the effects of the holobiont itself, there is evidence that the holobiont (Symbiodinium and/or coral host) modulates the prokaryotic community associated with the coral tissue (Ainsworth et al. 2015). This conclusion is in contrast with that of Pantos et al. (2015) for Seriatopora hystrix; they suggested that the variation in microbial communities associated with coral hosts is primarily driven by external environmental conditions. However, our study focused on the detection of prokaryotes associated with relatively stable intratissue microenvironment, whereas Pantos et al. (2015) likely included a large portion of coral surface mucus, whose associated prokaryotic communities are more exposed to the ambient reef environment and, therefore, more likely to vary spatially.
This study provides the first detailed assessment of the prokaryotic community associated with multiple scleractinian corals toward the lower mesophotic reef. The depth distribution range of coral species seemed to affect the overall variability of the prokaryotic community associated with coral tissue. Coral species with narrower depth distribution ranges retained a stable prokaryotic community, whereas corals with a broader depth distribution revealed higher taxonomic flexibility in their associated prokaryotic community. The observed depth effects are consistent with earlier published Symbiodinium variation (Bongaerts et al. 2015a). This highlights the contribution of structured microbial communities over depth to the coral’s ability to colonize a broader depth range.
Open access funding provided by Austrian Science Fund (FWF). We thank Kyra Hay, Norbert Englebert and the submersible team at Substation Curaçao for their help with sample collections and Mark Vermeij and the CARMABI Research Station for logistical support. The study was part of the Catlin Seaview Survey funded by Catlin Group Limited, with support from the Global Change Institute. Submersible dives were funded through an Eddie Bauer Grant for Expeditions by The Explorers Club (NY), and molecular work was funded via the Marie Curie Fellowship FP7-299320 and the Lise Meitner Program of the Austrian Science Fund (FWF) Project: M1363-B20 to PRF. The study is Ocean Heritage Foundation/Curaçao Sea Aquarium/Substation Curaçao (OHF/CSA/SC) contribution #21. We are grateful to the editor and three anonymous reviewers for their comments.
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