In four shallow and mesophotic tropical reef sponges from Guam the microbial community largely depends on host identity

Sample processing and sequencing

Samples of four different sponge species R. globostellata (n = 6 specimens), Callyspongia sp. (n = 7), A. cavernosa (n = 3), Rhaphoxya sp. (n = 5) and seawater (n = 5) were collected from closely connected Guam reef sites and depths (shallow exposed reef sites and a deep drop-off) via snorkelling and technical diving (Table 1). The tropical island Guam is known for the presence of a marine karstic limestone sinkhole (i.e., Guam Blue Hole); Taborosi, Jenson & Mylroie (2003). The vertical Blue Hole sinkhole has a vertical shaft of more than 90 m depth at which it merges with a deep drop-off running along the southern part of the Orote peninsula. This site harbours a rich and diverse associated coral reef fauna (Paulay, 2003) with very different environmental conditions from the shallow coral slopes of the surrounding areas. All shallow water samples for R. globostellata and Callyspongia sp. were collected from either Western Shoals or Gab Gab, which are both inside Apra Harbor and separated less than 1 nautical mile. The samples were collected in the same habitat, a shallow fore reef slope, which is dominated by Porites rus corals. Sample collection took place between June 25th 2010 and July 6th 2010, except for the samples C4 & C5 (March 2010) and A2 & A3 (November 2010 and March 2008, respectively) (see Table 1 for the sample accession codes). Sampling was carried out by technical diving, using trimix of helium, nitrogen and oxygen with mixes varying between target depths (oxygen 10–15%, helium 25–45%, nitrogen making up the balance). Decompression stops were carried out with nitrox mixes of 32–40%, which was switched to 75–82% oxygen once a decompression depth of 9 m was reached. Due to the various gas mixes, dive equipment included 5 independent tanks. Given that bottom times were limited to ca. 10–15 min at 90 m including descent time, searching for replicate sponges was very limited, explaining the somewhat limited number of replicates for the deep drop-off sponges. Sea surface temperatures (SST) were obtained from the Coastal Data Information Program (CDIP, http://cdip.ucsd.edu/themes/). SST averaged 29.8 °C in June and 29.7 °C in July 2010. Temperatures at depth (90 m) averaged 25 °C (taken with Dive rite-Intel HE and VR3-trimix dive computers). All samples were frozen, freeze-dried, and then stored at −20 °C prior to further processing. Sponges were identified by Dr Nicole J. de Voogd and vouchers of each species were preserved in 70% ethanol at the Naturalis Biodiversity Center, Leiden, Netherlands. Genomic DNA was extracted from sponge tissue and water filters (1 L each, 0.22 µm filter) by bead-beating in an ammonium acetate buffer, as previously described (Taylor et al., 2004). 16S rRNA gene amplification with primers targeting the V4-V5 region (454MID_533F: GTGCCAGCAGCYGCGGTMA and 454_907RC: CCGTCAATTMMYTTGAGTTT) and purification for pyrosequencing was performed as previously described by Simister et al. (2012b). Pyrosequencing was performed by Macrogen Inc. (Seoul, South Korea) using the Roche GS FLX Titanium system. The obtained raw sequence data can be accessed via the NCBI Sequence Read Archive under accession number SRX838037.

Raw sequence processing

Sequences were processed using mothur v.1.33.0 (Schloss, Gevers & Westcott, 2011; Schloss et al., 2009). Pyrosequencing flowgrams were filtered and denoised using the mothur implementation of AmpliconNoise (Quince et al., 2011). Adaptor, MID, and primer sequences were removed from raw sequences. Sequences were removed from the analysis if they were ≤200 bp or contained ambiguous characters, homopolymers longer than 8 bp, more than one MID mismatch, or more than two mismatches to the reverse primer sequence. Unique sequences were aligned against a SILVA alignment (available at http://www.mothur.org/wiki/Silva_reference_alignment). After chimera-checking with UCHIME (Edgar et al., 2011), unique sequences were identified using the Greengenes “gg_13_8_99” reference taxonomy (available at http://www.mothur.org/wiki/Greengenes-formatted_databases). Non-target sequences (e.g. chloroplasts, mitochondria, eukaryotic 18S rRNA) were removed.

Sequence data analyses

After raw data processing, mothur was used to group the obtained high quality sequences into 97% average neighbour sequence-similarity threshold operational taxonomic units (i.e., 97%-OTUs), for calculation of Shannon & inverse Simpson diversity, and rarefaction curves. Reads were evenly subsampled to 2,387 sequences per sample for all alpha diversity calculations (mothur command summary.single & subsample=T; 1,000 iterations). For visualization and interpretation of the microbial community data, we used standardized 97%-OTU abundance information (vegan command decostand & method= hellinger or pa; Bray-Curtis dissimilarities for relative abundance & Jaccard dissimilarities for presence/absence analyses). To estimate the variance of beta diversity, two hypothetical treatments were applied to the dataset: (a) ‘habitat’ (shallow reef slope, deep drop-off) and (b) ‘group’ (Rhabdastrella, Rhaphoxya, Callyspongia, Acanthella, seawater) (Table S1). These treatments were used for analysis of multivariate homogeneity of group dispersions (variances) (Anderson, 2006) with the betadisper (followed by pairwise Tukey’s Honestly Significant Difference tests) and permutest function from the vegan package in R (v. 3.0.2) (Oksanen et al., 2012; R Development Core Team, 2013). We used the adonis function (1000 permutations) from the vegan package to estimate the variances in beta diversity for both treatment groups (Anderson, 2001). Visualization of variations in sponge composition among habitats (i.e., shallow reef slope, deep drop-off) and sponge hosts (R. globostellata, Callyspongia sp., Rhaphoxya sp. and A. cavernosa) were assessed with multivariate non-metric multidimensional scaling (nMDS) using the metaMDS function from the vegan package. Hypothesis-based treatments were added as dispersion ellipses to the ordination plots with the vegan function ordieellipse (0.95 confidence interval). All multivariate analyses were performed with relative abundance and presence/absence data. The contribution of OTUs to average overall pairwise sample dissimilarity in R. globostellata, Callyspongia sp. and seawater specific datasets (‘habitat’ treatment) was assessed using the vegan function simper for similarity percentages (SIMPER). Relative abundance of the 30 most abundant OTUs (each ≥0.5% relative abundance) and relative phylum abundance were visualized with JColorGrid (Joachimiak, Weisman & May, 2006). Representative sequences of the 30 most abundant OTUs were assembled via the get.oturep command in mothur and BLAST searched against the NCBI Nucleotide collection (discontiguous megablast & Models excluded). The best hits and representative sequences for the most abundant OTUs can be accessed via the Figshare online repository (https://dx.doi.org/10.6084/m9.figshare.2366827). Hierarchical clustering based on all OTUs was performed using the vegan package in R via the function vegdist (Bray-Curtis dissimilarity) and hclust (method = average) and subsequently added onto the phylum fingerprint. As described above, all samples were included in the group-based (i.e., sponge and seawater samples) multivariate community (adonis and betadisper) and ordination (nMDS) analysis. In the subsequent habitat-based (shallow reef slope vs. deep drop-off) comparison, A. cavernosaand Rhaphoxya sp. samples were omitted from all analyses, and seawater samples from multivariate community analysis and ordination analyses, due to insufficient numbers of replicates in the dataset for the particular habitats. The 97% OTU abundance table combined with the Greengenes classification for each individual OTU can be accessed via the Figshare online repository (https://dx.doi.org/10.6084/m9.figshare.2063280).

Host specificity of the microbiota of four MCE sponges

In the present study divergent patterns between two different habitats were visible in the tropical sponge and seawater microbiota. Callyspongia sp. and seawater samples appear to have an intrinsic microbial community composition, which is variable enough to significantly separate the intra-species communities by their shallow reef slope or deep drop-off habitats. On the other hand R. globostellata exhibits an observable difference in community variation that is not significant. In addition, we could also observe significant microbial specificity across all analyzed sponge taxa independent of habitat. Since the shallow collection sites for Callyspongia sp. and R. globostellata inside Apra Harbor represent the same shallow water fore reef slope habitat dominated by Porites rus corals, it is unlikely that the observed differences in beta diversity between shallow reef slope or deep drop-off habitats are due to collection of Callyspongia sp. from the two shallow Apra Harbor sites.

Little is known about the R. globostellata and Rhaphoxya sp. microbial communities. Culture-dependent approaches reported only Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria (Alpha & Gamma) from R. globostellata (Lafi, Garson & Fuerst, 2005; Steinert et al., 2014). In addition, Lafi et al. (2009) found the candidate phylum “Poribacteria” in this demosponge. While the first high-throughput-sequencing amplicon screening detected 16 bacterial phyla associate with R. globostellata (Schmitt et al., 2012), we can now increase the number to a total of 23 microbial phyla. For Rhaphoxya sp., virtually nothing has been known about the associated microbiota. However, this sponge taxon is already the focus of natural products research, which hints at a chemically active symbiotic microbiota (Wright et al., 2012). Here we present phylum and OTU composition patterns in R. globostellata and Rhaphoxya sp. that are surprisingly similar. While designation of the two sponge taxa as either high microbial abundance (HMA) or low microbial abundance (LMA) sponges is lacking in the literature, the associated microbial phylotypes are congruent with the microbial phyla commonly found in association with HMA sponges (i.e., Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, and the candidate phylum “Poribacteria”) (Schmitt et al., 2011; Gloeckner et al., 2014).

Recent transmission electron microscopy, DAPI cell-counting and 16S rRNA gene amplicon sequencing results assigned different members of the demosponges A. cavernosa and Callyspongia sp. to the LMA group, with abundant Proteobacteria (Alpha, Beta, & Gamma) and Cyanobacteria (Synechococcus) microbial community members (Gloeckner et al., 2014; Giles et al., 2013; Jeong, Kim & Park, 2013). We also found that our Callyspongia sp. specimens exhibited an abundant occurrence of Synechococcus (Cyanobacteria), Actinobacteria, Bacteroidetes and Proteobacteria (Beta & Gamma). Within the Betaproteobacteria, the order EC94 exhibited the highest overall abundance in our Callyspongia sp. samples and among all OTUs. Reports on this order are rare, but recent community analyses on different demosponges found this microbial taxon to be dominant in the deep sea sponge Inflatella pellicula and several shallow water sponges from Korea (Jeong, Kim & Park, 2015; Jackson et al., 2013; Jeong, Kim & Park, 2013).

Overall, sponge and seawater community structures correlated significantly with host identity. This underlines the common view that host identity is an important factor for the composition of sponge-associated microbial communities (e.g., Cárdenas et al., 2014; Cleary et al., 2013; Easson & Thacker, 2014; Pita et al., 2013). Recent studies on several sponge species showed that the microbiota of LMA sponges, in particular, exhibits a low degree of similarity among the investigated sponge species (Giles et al., 2013; Blanquer, Uriz & Galand, 2013). The sponge genera Acanthella and Callyspongia were previously found to include LMA sponges (Gloeckner et al., 2014), and the bacterial community ordination, phylum composition and OTU structures of the present study separates these two sponge taxa very distinctly from the other two investigated sponges (Rhabdastrella, Rhaphoxya). In addition, the observed microbial community patterns in Callyspongia sp. appear to be more closely related to the seawater samples (see OTU and phylum composition). This high similarity with seawater communities is a well-known feature of LMA sponges (e.g., Taylor et al., 2007; Schmitt et al., 2011; Blanquer, Uriz & Galand, 2013). In contrast, HMA members are commonly more closely related to each other, especially after the removal of potential environmental sources of variation (Blanquer, Uriz & Galand, 2013). Finally, four sponge samples (two A. cavernosa and two Callyspongia sp.) have been sampled at different points in time. However, a temporal effect on the community structure is not evident, which is in accordance with recent research showing the low seasonal variability of the sponge-microbiota (Erwin et al., 2012; Erwin et al., 2015), although other similar studies have indicated the temporal variability of sponge-associated bacteria (White et al., 2012; Anderson, Northcote & Page, 2010).

Depth-dependent microbial community patterns in MCE sponges

Knowledge about variability of the sponge microbiota along environmental gradients on local spatial scales is still scarce (Olson & Kellogg, 2010). A T-RFLP and clone library study on three MCE sponges identified a trend in community composition along a depth gradient, but could not identify the bacteria which caused these variations (Olson & Gao, 2013). Correspondingly, a recent 16S rRNA gene amplicon analysis found significant shifts in the X. muta-associated microbial community along a depth gradient from 10 to 90 m and demonstrated that environmental factors may influence the sponge microbiota (Morrow, Fiore & Lesser, 2016). A sponge transplantation experiment showed little overall effect between different habitats (Cárdenas et al., 2014), and comparisons between sponges obtained from different habitats (marine lake vs. coastal system & intertidal vs. subtidal) showed the importance of host relatedness and habitat as determinants of microbial community structure (Weigel & Erwin, 2015; Cleary et al., 2013).

In the present study, Callyspongia sp. and seawater microbial communities were significantly different when comparing shallow reef slope and deep drop-off habitats. In contrast, based on the multivariate statistical tests, R. globostellata microbial communities were not significantly affected by the different habitats (shallow versus deep), which leads to the conclusion that in this particular case the observed habitat-specific nMDS and hierarchical clustering patterns are random variations of the microbial community composition. In order to sample in two environmentally very distinct but closely related habitats, we focused sampling on “shallow” and “very deep” depths as categorized by Brazeau, Lesser & Slattery (2013). Irradiance, but also nutrient availability and water temperature, are dependent on both depth and changing environmental factors along spatial gradients from shallow to mesophotic coral reefs (see Lesser, Slattery & Leichter, 2009; Olson & Gao, 2013). The observed temperature difference between shallow reef slope and deep drop-off sites averaged 4 °C, similar to that reported by Lesser et al. (2010); difference of 4 °C between 3 m and 91 m depth). While temperature is one indication for environmental differences between the two habitats, it seems unlikely that the observed difference of 4 °C will affect sponge-microbe communities between the two habitats to the extent reported in this study. Studies examining the effect of elevated temperatures found no change (at sub-lethal temperatures) in sponge bacterial communities during short-term experiments (Webster, Cobb & Negri, 2008; Simister et al., 2012b). It is likely that other local environmental factors such as light have a considerable effect on the small-scale patterns observed here, especially as similar studies on mesophotic reefs by Lesser, Slattery & Leichter (2009) and Lesser et al. (2010) did observe pronounced differences in light along similar depth gradients.

Within Callyspongia sp. and seawater samples, either Cyanobacteria or Proteobacteria OTUs were mainly responsible for the differences observed by SIMPER. In particular, cyanobacterial Synechococcus OTUs were among the main contributors to the shallow microbiota, whereas only one Prochlorococcus OTU was a dominant cyanobacterium contributing to the deep group from mesophotic depths. The predominance of photoautotrophs in tropical filter-feeding sponges is intuitive given the widespread prevalence of cyanobacteria in the ocean. LMA sponges in particular are known to harbour cyanobacteria in high abundance (Bayer, Kamke & Hentschel, 2014), and cyanobacteria comprise one of the most abundant sponge-associated phyla, with well-established sponge-specific symbionts (e.g., Synechococcus spongarium) (Hentschel, Usher & Taylor, 2006; Taylor et al., 2007; Simister et al., 2012a). Moreover, LMA sponges exhibit higher water filtering capabilities, presumably due to a less dense mesohyl and less complex aquiferous system compared to HMA sponges (Weisz, Lindquist & Martens, 2008). Therefore, the congruent microbial community patterns between Callyspongia sp. and seawater samples observed here, in contrast to those of R. globostellata, could be correlated with physiological differences between HMA and LMA sponges (see Weisz, Lindquist & Martens, 2008; Thacker & Freeman, 2012). Besides the potential phototrophic activities in the sponge pinacoderm (outer tissue) and mesohyl (inner sponge matrix), nitrogen-fixing cyanobacteria may inhabit a niche within the complex nitrogen cycle in sponges, with a mutual benefit due to nutrient supply by the sponge and secondary metabolite production by the cyanobacteria (Taylor et al., 2007; Wilkinson & Fay, 1979; Arillo et al., 1993). Given the dominance of Cyanobacteria in our Callyspongia sp. specimens and seawater samples, with Synechococcus dominant in the shallow and Prochlorococcus in the deep sponges, the spatial pattern could be shaped predominantly by cyanobacterial lineages. This distribution pattern of microbes in Callyspongia sp. could also indicate a seasonal vertical distribution pattern, in which one genus dominates the shallow high-light water column, while the other genus is temporarily mainly present in the low-light area below. While temporal shifts of Cyanobacteria in sponges have been observed to varying degrees previously, the combined effect of time and depth on these and other chlorophototrophs in sponges remains uncertain (White et al., 2012; Erwin et al., 2012; Hardoim & Costa, 2014; Taylor et al., 2004). A recent analysis on stable isotopes indicated that, with increasing depth, the inorganic nutrients dependency in sponges shifts from photoautotrophy to heterotrophy, in accordance with a significant shift in the associated microbiota (Morrow, Fiore & Lesser, 2016). Conversely, in the same sponge species at a different location an observed stable isotopic enrichment correlated with a larger microbial community similarity across different sampling depths. In addition, other factors should be considered to explain the observed differences. Since the same sponge species show differences in growth rates and species richness at different depths (Lesser, Slattery & Leichter, 2009), different biotic and abiotic niches may be available for symbionts depending on their habitat. Such ecologically-based niche differentiation for symbionts and hosts is known for the tropical corals Seriatopora hystrix (Bongaerts et al., 2010) and Montastraea cavernosa (Lesser et al., 2010; Brazeau, Lesser & Slattery, 2013). Recent oligotyping of Nitrospira symbionts associated with sponges collected along large horizontal and vertical gradients provided further evidence for such patterns of differential enrichment of closely related microbial variants (Reveillaud et al., 2014).

In R. globostellata, one cyanobacterial OTU (OTU0016) is predominant within the deep sheltered communities, but also present at high abundance in the shallow specimens of this species (Table 4). Moreover, it was also largely absent from all other sponge and seawater samples. The BLAST search against the NCBI nucleotide collection revealed that this particular OTU is highly similar to Candidatus Synechococcus spongiarum, a symbiotic cyanobacterium found in many sponges (Hentschel, Usher & Taylor, 2006). Compared to this, the main cyanobacterial OTUs found in Callyspongia sp. and seawater (OTU0002 & OTU0003) yielded different BLAST results (uncultured Synechococcus sp. clone & Prochlorococcus sp., respectively). This corresponds to the theory that LMA sponges generally acquire their microbial symbionts via horizontal transmission from the surrounding environment, while HMA sponges possess a more individual microbial community that does not mirror the surrounding seawater microbiome as closely as their low abundance counterparts (Gloeckner et al., 2014; Hentschel, Usher & Taylor, 2006). The detection of microbes within different reproductive stages of seven sponge species led to the hypothesis that HMA sponges can maintain parts of their symbiotic microbiota via vertical transmission (Schmitt et al., 2008). Interestingly, the major microbial drivers contributing to the observed differences in beta diversity are all located in the deep sponges. Given that the abundant microbial phyla in R. globostellata (i.e., Acidobacteria, Chloroflexi, Cyanobacteria, Gemmatimonadetes, and Alphaproteobacteria) are known to contain (bacterio)chlorophyll-based phototrophic lineages (Zeng et al., 2014), it is possible that photoheterotrophic bacteria also play a considerable role in this host-specific microbiota. However, while the function of Chloroflexi in sponges is yet unclear, the distribution of members of this phylum within their hosts from different depths suggests that they may not be phototrophically active within the sponges (Olson & Gao, 2013).