Characterization of the Microbiome of Corals with Stony Coral Tissue Loss Disease along Florida’s Coral Reef

Stony coral tissue loss disease (SCTLD) is an emergent and often lethal coral disease that was first reported near Miami, FL (USA) in 2014. Our objective was to determine if coral colonies showing signs of SCTLD possess a specific microbial signature across five susceptible species sampled in Florida’s Coral Reef. Three sample types were collected: lesion tissue and apparently unaffected tissue of diseased colonies, and tissue of apparently healthy colonies. Using 16S rRNA high-throughput gene sequencing, our results show that, for every species, the microbial community composition of lesion tissue was significantly different from healthy colony tissue and from the unaffected tissue of diseased colonies. The lesion tissue of all but one species (Siderastrea siderea) had higher relative abundances of the order Rhodobacterales compared with other types of tissue samples, which may partly explain why S. siderea lesions often differed in appearance compared to other species. The order Clostridiales was also present at relatively high abundances in the lesion tissue of three species compared to healthy and unaffected tissues. Stress often leads to the dysbiosis of coral microbiomes and increases the abundance of opportunistic pathogens. The present study suggests that Rhodobacterales and Clostridiales likely play an important role in SCTLD.

Climate change, overfishing, and pollution are among the many stressors contributing to the decline of coral reef health worldwide [16,17]. As a result of these stressors and others (e.g., sedimentation), there has been an increase in widespread bleaching events, disease incidence, and subsequent mortality among coral communities in recent decades [18][19][20][21][22]. Techniques and methods developed previously [40] were used to collect coral tissue/mucus and water samples at each of the sites by divers on SCUBA. Divers used a sterile, 10 mL syringe to scrape and remove tissue from most colonies, except S. siderea. Because syringe tips would frequently break when attempting to sample S. siderea colonies, divers used a sterile corer to scrape off the tissue while simultaneously using a syringe to capture the scrapings and mucus. In the epidemic zone, three types of coral samples were collected per species: lesion tissue of diseased colonies (DL; along the disease margin of active lesions; Figure 2), unaffected tissue of diseased colonies (DU; the areas not showing obvious external signs of SCTLD on visibly diseased colonies), and tissue of apparently healthy colonies (AH; colonies with no external signs of SCTLD). For each diseased colony, the DU tissue sample was collected before the DL tissue sample to minimize the risk of contamination from sampling the lesion tissue. At every epidemic site, divers collected DL and DU tissue samples from three to five colonies per species and AH tissue samples from three colonies per species (Table 1). At the three sites in the vulnerable zone, AH tissue samples were collected from two to three colonies per species. Additionally, at each site in each zone, three water samples were collected by inverting sterile, 1 L bottles approximately 0.5 m above the benthos, in proximity of the coral colony sampling area. Table 1. Summary of coral tissue/mucus and water samples collected from sites (n = 8; Figure 1) along Florida's Coral Reef. Shown are the dates of sampling, stony coral tissue loss disease (SCTLD) zone, site letter codes, and coral species sampled: Colpophyllia natans (CNAT), Pseudodiploria strigosa (PSTR), Montastraea cavernosa (MCAV), Orbicella faveolata (OFAV), and Siderastrea siderea (SSID). For each coral species, the number of lesion and unaffected tissue samples from colonies with SCTLD, and the number of samples from apparently healthy colonies, collected per site are shown. The number of water samples collected per site is also shown.

Sample Processing
DNA was isolated from all samples using DNeasy PowerSoil Kits (QIAGEN, Ger- On the boat, coral tissue samples were transferred from syringes into plastic tubes before being transported, along with the water sample bottles, on ice directly from the sampling sites to the South Florida Regional Laboratory of the Florida Fish & Wildlife Conservation Commission's Fish and Wildlife Research Institute (FWC-FWRI, Marathon, FL, USA). At FWRI, water samples were filtered through 0.2 µm filters. All water filters and coral tissue samples were flash-frozen using liquid nitrogen before being immediately transferred to −80 • C for storage.

Sample Processing
DNA was isolated from all samples using DNeasy PowerSoil Kits (QIAGEN, Germantown, MD, USA) and modifications to the manufacturer's protocol. The protocol optimized and used previously [40] was employed to isolate DNA from all samples in the present study. However, to standardize concentrations of DNA isolated from the five coral species, the amount of starting material processed for DNA varied depending on the coral species. For S. siderea, DNA was isolated from 6 mL of the coral tissue/mucus slurries, but 2 mL of samples were used for the other four species. Each slurry was then centrifuged and the supernatant was discarded so that only a pellet remained in each tube. For the water samples, half of every 0.2 µm filter was cut into small pieces, which were then transferred to their respective tubes. Next, phenol: chloroform: isoamyl alcohol (pH 7-8; Fisher Scientific Company LLC, Hanover Park, IL, USA) and Solution C1 of the DNeasy PowerSoil Kit were added to every tube, vortexed for 10 min, and centrifuged. The supernatants were then transferred to new tubes to which Solutions C2 and C3 of the DNeasy PowerSoil Kit were added. The process of adding reagents and centrifuging samples continued, as described previously [40], until left with 60 µL of isolated DNA. DNA concentrations and quality were measured with a NanoDrop One TM Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Data Analysis
Sequences were demultiplexed using the MR DNA free software application, FASTqProcessor (version 20.03.02; www.mrdnalab.com (accessed on 12 October 2021), Shallowater, TX, USA). All other data analyses were completed in the program R (version 4.0.5; R Foundation for Statistical Computing, Vienna, Austria) [44]. The DADA2 pipeline [45] was used to determine amplicon sequence variants (ASVs) by checking read quality, filtering and trimming sequences, dereplicating, merging sequences, and removing chimeras. Since samples were sequenced on two lanes of an Illumina MiSeq, each lane was processed through the DADA2 pipeline individually. Taxonomy was assigned using the Silva (version 132; European Organization for Nuclear Research, Geneva, Switzerland) [46] reference database. Taxonomy tables, ASV tables, and sample data from the two sequencing lanes were then merged and further analysis and processing was completed using the phyloseq package [47]. Following taxonomic assignment, sequences from the coral host, algal symbionts, and eukaryotes were removed. The resulting ASV count data were filtered so that only ASVs present in a minimum of four samples were included in the analysis (similar to Rosales et al. [40]). ASV count data were then normalized using a centered log-ratio (CLR) transformation.
Differences in microbial communities were assessed between zone (epidemic vs. vulnerable), sites within each zone, and tissue sample type (DL, DU, and AH) for each coral species. All tissue sample types (DL, DU, and AH) collected in the epidemic zone were combined for each coral species when comparing sites within the epidemic zone. To measure beta diversity differences, the vegdist function of the R package 'vegan' [48] and the CLR-transformed data were used to calculate dissimilarity indices. These indices were then tested for the homogeneity of groups dispersion using the betadisper function and were, subsequently, tested for significance using a permutation test (Permutest) and a Tukey's honest significant difference (HSD) test. Next, permutational multivariate analysis of variance (PERMANOVA) and pairwise PERMANOVA tests were used to measure differences in bacterial communities among zones, sites, and sample types for each coral species. All PERMANOVA tests were performed using the adonis function in the R package 'vegan' with a Euclidean distance. Next, a pairwise comparison was conducted and the p-values generated from this comparison were adjusted using a Bonferroni correction [49]. Nonmetric multidimensional scaling (NMDS) with a Euclidean distance was used to generate ordination plots and further evaluate the bacterial community differences among samples.
Alpha diversity metrics (species richness and Shannon diversity) were analyzed using the 'vegan' package. Shapiro-Wilks tests were used to test for normality conditions. If the data met normality assumptions, a one-way analysis of variance (ANOVA) and Tukey's post-hoc test were used. If the data did not meet normality assumptions, then a Kruskal-Wallis rank sum test was used. When significant differences were detected, a post-hoc Wilcoxon signed rank test was then used to determine which groups were significantly different.
An analysis of composition of microbiomes (ANCOM) was used to identify significant ASVs that were differentially abundant among the tissue sample types for each coral species. To perform the ANCOM, a phyloseq object containing the ASV count data and taxonomy was used [50]. The ANCOM results were considered significant if the calculated W-statistic exceeded a detection threshold of 0.7. For each coral species, ANCOM was run with tissue sample type as the independent variable tested and, for water samples, disease outbreak zone was the independent variable. Bar plots displaying the mean relative abundance of bacterial classes present in each species were generated.

Microbial Communities among Sites within Zones
Among sites in the epidemic zone, there were significant differences in the microbial communities of all three tissue sample types (DL, DU, and AH) pooled (Table S1). However, there were no significant differences in the microbial communities of AH tissues among sites in the vulnerable zone. There were significant differences in the bacterial communities of water collected in the vulnerable zone compared to the epidemic zone ( Figure 4F; F 1,16 = 3.3523, R 2 = 0.11, p = 0.001).

Beta Diversity among Sites within Zones
There were no significant differences in dispersion among sites within the epidemic zone or within the vulnerable zone for each species: C. natans (epidemic: There were no significant differences in the beta diversity of water samples among sites in the epidemic zone (F 4,10 = 3.2959, p = 0.066) nor the vulnerable zone (F 2,6 = 2.8082, p = 0.14).

Relative Abundance Analysis of Bacterial Class
The relative abundance of bacterial classes revealed interesting patterns with coral species. Of the highly susceptible species C. natans and P. strigosa, Clostridia was the most dominant bacterial class in the DL tissue (45.11% and 56.22%, respectively; Figure 7A). Following Clostridia, Alphaproteobacteria (16.57%) and Bacteroidia (16.36%) were most abundant for C. natans DL tissue. Gammaproteobacteria constituted the highest relative abundance in DU (34.18%) and AH (33.26%) tissues of C. natans. In the DU and AH tissues of P. strigosa, however, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were similarly abundant.
Among the moderately susceptible species, Alphaproteobacteria was generally more dominant in DL tissues. Alphaproteobacteria was the dominant member (31.08%) of DL tissue of M. cavernosa even though they were also present, albeit at lower abun- As in the vulnerable zone, pairwise comparisons of AH tissue in the epidemic zone showed that C. natans and M. cavernosa had a lower species richness than O. faveolata, P. strigosa, and S. siderea (p_adj < 0.05 for all comparisons, range: < 0.00001-0.011; Figure 6B). Dissimilar to the vulnerable zone, S. siderea in the epidemic zone had a higher species richness than P. strigosa (p_adj = 0.0082) as well as O. faveolata (p_adj = 0.0029). Siderastrea siderea also had a higher Shannon diversity than C. natans (p_adj = 0.00010), M. cavernosa (p_adj = 0.0038), and O. faveolata (p_adj = 0.022). All other comparisons among AH tissue collected in the epidemic zone were not significant.

Relative Abundance Analysis of Bacterial Class
The relative abundance of bacterial classes revealed interesting patterns with coral species. Of the highly susceptible species C. natans and P. strigosa, Clostridia was the most dominant bacterial class in the DL tissue (45.11% and 56.22%, respectively; Figure 7A). Following Clostridia, Alphaproteobacteria (16.57%) and Bacteroidia (16.36%) were most abundant for C. natans DL tissue. Gammaproteobacteria constituted the highest relative abundance in DU (34.18%) and AH (33.26%) tissues of C. natans. In the DU and AH tissues of P. strigosa, however, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were similarly abundant.
this class was more abundant in DL tissue (42.56%) compared to AH and DU tissues (27.17% and 15.32%, respectively). Conversely in O. faveolata, AH and DU tissues had higher levels of Bacteroidia (30.54% and 29.09%, respectively) than DL tissue (13.21%). Relatively high abundances of Chlamydia were also present in AH and DU tissues (17.63% and 11.1%, respectively) but not in DL tissue (<1%) of O. faveolata. Though not at the same relative abundance levels as in C. natans and P. strigosa, Clostridia was more abundant (8.6%) in DL tissue than AH and DU tissues (<1%) of O. faveolata. Finally, for S. siderea, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were the most abundant groups of bacteria across all tissue sample types.
At every site in the epidemic and vulnerable zones, the bacterial community of the water column was dominated by Alphaproteobacteria, followed by Gammaproteobacteria, Bacteroidia, and Oxyphotobacteria ( Figure 7B).  Among the moderately susceptible species, Alphaproteobacteria was generally more dominant in DL tissues. Alphaproteobacteria was the dominant member (31.08%) of DL tissue of M. cavernosa even though they were also present, albeit at lower abundances (<13.86%), in AH and DU tissues. These two tissue sample types of M. cavernosa had relatively high levels of Bacteroidia (19.02% and 23.28%, respectively) compared to DL tissue (10.49%). In all three O. faveolata tissue sample types, Alphaproteobacteria, Bac-teroidia, and Oxyphotobacteria were among the highest relatively abundant taxa. While Alphaproteobacteria was ubiquitous across all tissue sample types of O. faveolata, this class was more abundant in DL tissue (42.56%) compared to AH and DU tissues (27.17% and 15.32%, respectively). Conversely in O. faveolata, AH and DU tissues had higher levels of Bacteroidia (30.54% and 29.09%, respectively) than DL tissue (13.21%). Relatively high abundances of Chlamydia were also present in AH and DU tissues (17.63% and 11.1%, respectively) but not in DL tissue (<1%) of O. faveolata. Though not at the same relative abundance levels as in C. natans and P. strigosa, Clostridia was more abundant (8.6%) in DL tissue than AH and DU tissues (<1%) of O. faveolata. Finally, for S. siderea, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were the most abundant groups of bacteria across all tissue sample types.
At every site in the epidemic and vulnerable zones, the bacterial community of the water column was dominated by Alphaproteobacteria, followed by Gammaproteobacteria, Bacteroidia, and Oxyphotobacteria ( Figure 7B).
The order Rhodobacterales was consistently present at high relative abundances in DL tissue compared to AH and DU tissues for all coral species except S. siderea ( Figure 8A-E). Eight significantly differentiated Rhodobacterales ASVs were detected in the DL tissue of at least two of the five species of coral: ASV6, ASV21, ASV22, ASV57, ASV116, ASV124, ASV148, and ASV208 (Table 2); all eight of these sequences have been reported in previous studies of SCTLD [39,40,51,52]. ASV148, an unclassified Rhodobacteraceae, was the only ASV detected in the lesions of all five coral species examined here. Rhodobacteraceae, Nioella [39,51] The order Clostridiales was also consistently found in the bacterial community of disease tissues. The DL tissues of C. natans, P. strigosa, and M. cavernosa had higher abundances of significantly differentiated Clostridiales compared to AH and DU tissues. ASV50, a Tepidibacter belonging to the order Clostridiales, was enriched in the DL tissues of these three coral species. In addition, the abundance of ASV11, a Halodesulfovibrio belonging to order Desulfovibrionales, was more abundant in the DL tissue of M. cavernosa and P. strigosa compared to AH and DU tissues. Water samples collected in the epidemic zone were enriched in Flavobacteriales compared to the vulnerable zone ( Figure 8F).

Discussion
The stony coral tissue loss disease (SCTLD) outbreak has caused the widespread

Discussion
The stony coral tissue loss disease (SCTLD) outbreak has caused the widespread mortality of important reef-building species in Florida and in the Caribbean Region. To date, the identity of the presumed pathogen(s) responsible for SCTLD remains unknown, despite many studies on microbial communities associated with SCTLD [39,40,51,52]. In the present study, the microbiomes of five coral species (Colpophyllia natans, Pseudodiploria strigosa, Montastraea cavernosa, Orbicella faveolata, and Siderastrea siderea) were analyzed to determine if a specific microbial signature exists across different species of corals with active SCTLD lesions. The lesions of four of these five coral species (except S. siderea) had significantly higher abundances of Rhodobacterales, a finding that is consistent with studies of other susceptible coral species (Table 2). In addition to Rhodobacterales, Clostridiales was also a significant and ubiquitous member of the lesion microbial community, especially for C. natans, M. cavernosa, and P. strigosa.

Diversity Indices
In general, the microbial beta diversity dispersion between zones (i.e., vulnerable and epidemic) and within zones was not significant in either water or apparently healthy (AH) coral samples, except for those from P. strigosa, which were more dispersed in the vulnerable zone than the epidemic zone. These results, with the exception of P. strigosa, are similar to Rosales et al. [40], who found no difference in the AH coral microbial beta diversity dispersion between the vulnerable and epidemic zones. Coral microbiomes are sensitive to environmental perturbations (e.g., thermal stress, nutrient pollution), which can cause an increase in the microbial beta diversity dispersion [53]. It is possible that AH P. strigosa colonies in the vulnerable zone had recently been exposed to an environmental stressor, which caused an increase in the beta diversity dispersion. However, longitudinal studies are needed to parse out the influence of environmental conditions on beta diversity dispersion through time.
The present study did not find a difference in the dispersion of microbial communities in the water column between zones. In Rosales et al. [40], the microbial beta diversity dispersion of water samples was significantly different between zones, with greater dispersion observed within the epidemic zone compared to the vulnerable zone. Even though the beta diversity dispersion of environmental samples (i.e., water samples) did not differ between zones in the present study, differences in environmental conditions between zones should not be ruled out as a possible factor in driving these differences. Additionally, the water sample sizes in the present study were lower than those used in Rosales et al. [40], potentially limiting the ability to observe meaningful differences between the zones in this study. This discrepancy between findings may also be attributed to differences in how the water samples were gathered: in the present study, water sample bottles were held approximately 0.5 m above the benthos and were not specifically gathered above coral colonies. In Rosales et al. [40], water sample bottles were held directly over the benthos, gathering water approximately 20 cm or less from the bottom, and in the epidemic zone, water samples were collected directly over colonies with SCTLD. The distance at which water samples are collected above the bottom influences the microbial signature of the water samples, as illustrated by Weber et al. [54]. Water samples collected within the coral ecosphere, or the environment immediately surrounding an individual coral colony (e.g., water < 30 cm above the colony), can have a different microbial signature compared to water collected >1 m above the reef. Therefore, bacterial beta diversity dispersion may be related to the sampling location (i.e., distance above the benthos or individual coral colonies) in the water column.
Among the tissue sample types of most coral species, the epidemic zone had a dispersed microbial community. While dispersion was not significantly different among the three tissue sample types in P. strigosa, as previously identified in Diploria labyrinthiformis, Dichocoenia stokesii, and Meandrina meandrites [40], the lesion (DL) tissue of the other coral species in this study were more dispersed than AH (in four coral species) and unaffected (DU) tissue (in three coral species, except C. natans) tissues. In Meyer et al. [39], microbial dispersion was similar between the DL tissue and DU tissue of diseased colonies from three susceptible species, Montastraea cavernosa, Diploria labyrinthiformis, and Dichocoenia stokesii. However, in M. cavernosa, the DL and DU tissues did have a higher dispersion compared to AH tissue. Similarly, in the present study, there were no significant differences between the DL tissue and the DU tissue of C. natans. Therefore, colonies showing signs of SCTLD may have a disrupted microbiome even far away from the lesion, suggesting a systemic effect [55]. It has been reported in other studies that stress, such as heat stress, often has a stochastic effect on the microbial community composition that can result in an increase in the beta diversity [56]. In Rosales et al. [57], for example, Acropora cervicornis exposed to diseased ramets had a higher beta diversity compared to control corals not exposed to disease. Eaton et al. [55] also showed that visibly unaffected areas (DU tissue) on diseased corals later showed signs of tissue loss after coral fragments were separated and isolated from the active disease border on the parent colony, and Landsberg et al. [30] found lytic necrosis characteristic of SCTLD lesions within some samples of DU tissue, again suggesting that SCTLD may be systemic within coral colonies.
In the epidemic zone, there were also significant differences in the species richness among tissue sample types, with DL tissues having a higher species richness compared to both DU and AH tissues in three species (C. natans, M. cavernosa, and S. siderea) and compared to the AH tissue of two species (O. faveolata and P. strigosa). This difference in alpha diversity may be a result of microbial dysbiosis, or an imbalance in the natural microbiome that can disrupt coral-microbe interactions and lead to disease [8,58]. The microbiomes of corals exposed to stressful environmental conditions (e.g., acidification, and increased temperature) often experience a shift in microbial community composition and, consequently, an increase in the species richness [8,59]. Microbial shifts may be attributed to a loss in beneficial bacteria; thus, freeing up niche space for putative pathogens to inhabit [13,60]. In a study conducted by MacKnight et al. [58], disease-resistant corals exposed to white plague disease (WPD) had a higher dysbiosis threshold compared to corals that developed WPD lesions. The authors hypothesized that certain bacteria may be helping to prevent pathogens from colonizing disease-resistant corals; thus, also preventing dysbiosis and the onset of WPD. In the present study, the higher microbial alpha diversity of coral DL tissues was potentially due to the decreased stability of the coral microbiomes and the hosts' inability to prevent pathogenic infection [59], or due to an increased propensity for diseased tissues and surface mucus to become colonized by diverse opportunistic bacteria, including Rhodobacteraceae [61,62].

Differences Were Detected among Sites and Tissue Sample Types
Bacterial communities of all but one species (O. faveolata) were different among at least three sites of the epidemic zone (Table S1). Even though sites within the epidemic zone were similarly dispersed, there were significant groupings among sites, suggesting a sitelevel effect on the bacterial signature of corals within the epidemic zone. There are likely several factors driving these site-wide differences. In a previous study, Williams et al. [34] showed that the coral species diversity, coral cover, and size of coral colonies affect SCTLD prevalence and severity. Sites with higher abundances of M. cavernosa and O. faveolata compared to four other susceptible species (C. natans, P. strigosa, Diploria labyrinthiformis, and Dichocoenia stokesii) had a greater disease prevalence. In addition, colonies that ultimately became diseased were significantly larger than colonies that did not display signs of SCTLD over the course of the study. This finding was also observed by Sharp et al. [63]. Both studies [34,63] observed that the coral density did not likely play a role in the spatiotemporal dynamics of SCTLD. While coral density may not be a factor, differences in the coral species diversity and size of colonies among sites in the present study may explain the site-level differences observed in microbial communities. Even though the microbial communities of these corals are site-specific (Table S1), there were consistent signatures within the DL tissues of corals among sites.

Rhodobacterales and Clostridiales in SCTLD Lesions
The DL tissues of four species in this study (C. natans, P. strigosa, O. faveolata, and M. cavernosa) had significantly higher relative abundances of Rhodobacterales compared to both DU and AH tissues. In previous studies, Rhodobacterales was also differentiated in the lesions of Stephanocoenia intersepta, Diploria labyrinthiformis, Dichocoenia stokesii, Meandrina meandrites, and Montastraea cavernosa in Florida [39,40], and, although not significant, Rhodobacteraceae was enriched in the lesion tissues of Meandrina meandrites and O. franksi in the U.S. Virgin Islands [52]. Other studies have reported high abundances of Rhodobacterales, specifically Rhodobacter, in disease tissue of black band disease, white plague, and white band disease [64][65][66][67][68][69]. This group of bacteria play an important role in colonizing submerged marine surfaces and is often considered the primary and most common colonizer [70,71]. The fast-growing nature of Rhodobacterales allows members of this group to thrive in areas that are rich in amino acids and other nutrients [72]. These bacteria also have the ability to produce antibiotic compounds [73]. Under stressful conditions, Rhodobacterales appear to be typical and abundant opportunistic bacteria associated with corals [61,62,74].
In this study, an exception to the pattern of higher relative abundance of Rhodobacterales in DL compared to both DU and AH tissues was the coral S. siderea. The DU tissues of S. siderea had a lower abundance of Rhodobacterales compared to both DL and AH tissues, but the latter tissue sample types had statistically similar abundances. Siderastrea siderea often shows signs of SCTLD that are distinct from other susceptible species, including areas of pinkish tissue discoloration and mucus strands [30], leading to some speculation about whether this species has the same disease or a generalized stress response [25]. However, the characteristic hallmark lytic necrosis of SCTLD as described in the lesion tissues of other susceptible species has been reported in S. siderea tissue [30], so differences in how the tissue loss progresses through the colony in S. siderea (generally originating in polyp mouths instead of moving across the colony [30]) or its species-specific holobiont response may affect the gross presentation of the disease and the relative abundances of bacteria across tissue sample types compared with other species. However, it should be noted that the method of sampling for S. siderea (predominantly coring and scraping versus scraping only) may have influenced the composition of the microbial community in the samples examined, especially if less tissue and mucus were potentially sampled. Surface mucus on corals has a diverse microbial community, the composition of which changes during the course of disease [61]. Scraping with a coring device may have obtained slightly deeper tissue samples which may have a different microbial composition. Studies to evaluate the vertical and horizontal distribution and abundances of the microbial flora in SCTLD-affected colonies over time in relation to lesion progression, coral species, sampling method, and mucus quantity may resolve this question.
In the present study, Clostridiales was another significantly differentiated group of bacteria in lesion tissue. ASV50 specifically was enriched in the lesion tissue of C. natans, M. cavernosa, and P. strigosa. Of these corals, C. natans and P. strigosa are highly susceptible to SCTLD, and exhibit acute to subacute tissue loss [25,30]; M. cavernosa is considered moderately susceptible, but the rate of lesion progression varies widely by colony, and most of the diseased colonies sampled in this study were experiencing subacute tissue loss (authors' pers. obs.). It is possible that Clostridiales is a signature of faster lesion progression, given that this order has been documented in other studies in M. cavernosa and C. natans [39,52] in addition to two other highly susceptible species: Dichocoenia stokesii and Diploria labyrinthiformis [39]. However, Rosales et al. [40] did not observe significantly differentiated Clostridiales in DL tissue of Dichocoenia stokesii or Diploria labyrinthiformis, but only within DL tissues of Stephanocoenia intersepta, another moderately susceptible species [25]. Similar to Rhodobacterales, Clostridiales is commonly found in the lesion tissue of other coral diseases, such as black band disease, white plague disease, and white syndrome [67,68,75]. When colonizing, these opportunistic anaerobic bacteria can necrotize host tissue. For example, humans and animals exposed to clostridial spores (e.g., through contaminated drinking water) can contract clostridial myonecrosis (gas gangrene) which is a lethal infection that causes severe necrosis of muscle and soft tissue [76]. The initial appearance of SCTLD lesions in deeper basal body wall tissues may indicate a role for anaerobic bacterial pathogenesis, although thus far, no histological evidence has been found for co-occurring bacteria in lesion initiation [30]. However, the exact role, if any, of Clostridiales in lesion progression and tissue necrosis of coral colonies with SCTLD, should be examined among highly susceptible species.

Conclusions
The goal of this study was to determine if the lesions of coral colonies showing signs of stony coral tissue loss disease (SCTLD) had a consistent microbial signature across five different coral species: Colpophyllia natans, Pseudodiploria strigosa, Montastraea cavernosa, Orbicella faveolata, and Siderastrea siderea. Diversity indices revealed that the lesion (DL) tissue of all but one species (P. strigosa) had a higher microbial beta diversity dispersion than apparently healthy (AH) tissue. In three species (M. cavernosa, O. faveolata, and S. siderea), DL tissue had a higher beta diversity dispersion than both AH tissue and unaffected (DU) tissue. Furthermore, DL tissue of every species had a higher species richness compared to AH tissue. In three species (C. natans, M. cavernosa, and S. siderea), the species richness of DL tissue was also higher than DU tissue. DL tissue consistently had higher relative abundances of the order Rhodobacterales compared to AH and DU tissues, except for S. siderea, a finding that has been observed in other coral species [39,40,51,52]. In addition, order Clostridiales was enriched in the DL tissue of three of the five species investigated in the present study, suggesting that Clostridiales may also play an important role in SCTLD. The beta diversity dispersion and species richness of water samples did not differ between the vulnerable and epidemic zones, suggesting that a bacterial signature of SCTLD was not detected in the water column, potentially due to sampling location within the sites. The results presented herein expand our understanding of SCTLD as it relates to the coral microbiome of critical reef-building species along Florida's Coral Reef. Understanding how SCTLD, coupled with changing environmental conditions, can affect host-microbe interactions is an important step towards developing practical and effective disease mitigation strategies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9112181/s1, Table S1: results of permutational multivariate analysis of variance (PERMANOVA) and pairwise PERMANOVA tests comparing microbial communities among sites in the epidemic zone and vulnerable zone, Figure S1: microbial alpha diversity metrics (species richness and Shannon diversity) comparing five corals species between the epidemic zone and vulnerable zone, Figure S2: microbial alpha diversity metrics (species richness and Shannon diversity) comparing five corals species among sites (Sites A-E) in the epidemic zone, Figure S3: microbial alpha diversity metrics (species richness and Shannon diversity) comparing five corals species among sites (Sites F-H) in the vulnerable zone.