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Article

Diversity of Microbial Communities Associated with Epilithic Macroalgae in Different Coral Reef Conditions and Damselfish Territories of the Gulf of Thailand

by
Jatdilok Titioatchasai
1,2,
Komwit Surachat
3,
Jeong Ha Kim
4 and
Jaruwan Mayakun
1,2,*
1
Division of Biological Science, Faculty of Science, Prince of Songkla University, Songkhla 90110, Thailand
2
Molecular Evolution and Computational Biology Research Unit, Faculty of Science, Prince of Songkla University, Songkhla 90110, Thailand
3
Department of Biomedical Science & Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand
4
Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2023, 11(3), 514; https://doi.org/10.3390/jmse11030514
Submission received: 10 February 2023 / Revised: 21 February 2023 / Accepted: 24 February 2023 / Published: 27 February 2023
(This article belongs to the Topic Marine Microbiology: Resources, Ecology, and Biogeochemistry)
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Abstract

:
Reef degradation and algae-farming fish alter the structures and compositions of coral-algal-microbial communities. We collected epilithic macroalgae in different reef conditions and damselfish territories. The microbial communities were characterized by subjecting the V3-V4 hypervariable region of the 16S rRNA gene to amplicon sequencing. Metagenomic analysis revealed 2 domains, 51 phyla, 112 orders, and 238 families and the dominance of Proteobacteria and Bacteroidota in both fair and degraded reefs inside and outside territories. Chloroflexi on the degraded reef was dominant and its proportion was almost two and a half times compared to the fair reef, whereas Cyanobacteria was low on the degraded reef. Bacteroidota was dominant on the fair reef, whereas Actinobacteriota was scarce on this reef. For the damselfish territories, Chloroflexi was dominant inside the territory, whereas Bacteroidetes were found outside the territory. Differences in the microbial species diversity and richness were not apparent between all sites; however, species evenness was higher on the degraded reef condition and lower outside the territory. Important potential pathogens of reef organisms, such as Vibrio, Photobacterium, and Phormidium, were found on the degraded reef areas inside the damselfish territory. The farming behaviors of damselfish influenced microbial communities by changing the epilithic algal matrix that harbors many microbial communities. This study provides useful information on microbial biota in coral reef habitats which is further applicable to reef conservation and coastal management.

1. Introduction

Coral reefs play crucial ecological roles and provide many ecosystem services in coastal protection, nutrient cycling, and habitat provision [1,2]. They also harbor a huge diversity of eukaryotes and coral-associated prokaryotes [3]. Coral reefs have been degrading owing to human disturbances, such as coastal development and overfishing, together with global climate change [4,5,6,7]. These disturbances contribute to a decline in reef habitat complexity and a massive loss of coral cover that can shift the structure of reef benthic communities [8,9,10]. Where coral degradation and destructive overfishing coincide, coral reefs can shift from a coral-dominated to algal-dominated community, coupled with reef resilience reduction [11,12,13,14,15]. These changes are accompanied by a loss of marine biodiversity, declines in the composition and abundance of many reef organisms, and changes in coral-algal-associated microbes [8,16,17,18].
The absence of large-to-medium-sized coral reef fishes due to destructive overfishing can increase the number of herbivorous fishes such as the territorial damselfish (family Pomacentridae) [19,20]. The territorial damselfish can alter the reef benthic community with their gardening and territorial defense behavior [21,22]. Damselfish often cultivate filamentous turf algae (their preferred algae) and eliminate fleshy algae (non-preferred algae) [23]. Additionally, damselfish defend their foods from other reef fishes, then their territories are dominated by the turf algae and microbial assemblages associated with epilithic algae. Many studies have reported that the presence of damselfish can decrease coral cover and increase algal growth [24,25]. Further, it possibly facilitates the introduction of potential coral pathogens [26].
Shifts from coral-algal-dominated communities can alter coral-algal-microbial communities that might potentially affect coral and algal health, entraining coral disease, coral juvenile settlement, and coral metamorphosis [27,28]. Many studies have shown that different algal species or algal compositions support different microbial communities [26,29,30], indicating somewhat species-specific characteristics in the algal-microbial relationship. In addition, some studies also reported the possibility of dynamic changes in the characteristic relationship depending on coral reef habitat and the presence or absence of algae-farming fish and environmental stressors [18,26,31,32]. Nonetheless, few have focused on microbial composition and community dynamics between different reef conditions and algae-farming fish. It is also important to gain a better understanding of coral-algal-microbial relationships and microbial dynamics in response to reef degradation and overfishing for the conservation perspectives.
The aim of this study is to investigate the microbial communities associated with epilithic macroalgae in different coral reef conditions combined with the territory of algae-farming fish. Microbial species were identified by subjecting the V3-V4 hypervariable region of the 16S rRNA gene to amplicon sequencing for metagenomic analysis. After sequencing and analyzing, microbial composition and dynamics were characterized and compared as a function of reef conditions and the damselfish territory.

2. Materials and Methods

2.1. Study Site

The study was conducted on the shallow subtidal reef crest at Ko Taen, Mu Ko Thale Tai National Park, in the Gulf of Thailand in February 2021 (Figure 1). This area is under a typical climate condition of monsoonal influence, which represents two contrasting seasons: a rainy season from May to January dominated by the northeast monsoon, and a dry season from February to April dominated by the southwest monsoon. The study site supports a high abundance and diversity of coral reef fishes and macroalgae. The dominant macroalgae are Padina sp., Lobophora variegata, Sargassum, Turbinaria, Cladophora, and red turf algae. For reef fish diversity, these reefs are dominated by Neoglyphidodon nigroris, Abudeduf sexfasciatus, Abudeduf sexfasciatus, Abudefduf vaigiensis, Abudefduf bengalensis, Scarus rivulatus, and Chaetodon octofasciatus. Massive coral species of the genus Porites are the dominant corals in this area.
For our study, the study site was divided into two different coral reef conditions, i.e., a degraded reef (9°23′04″ N, 99°57′06″ E) and a fair reef (9°22′28″ N, 99°57′21″ E), each 1.2 km apart. Reef conditions were categorized according to the Department of Marine and Coastal Resources (DMCR) criteria, by the ratio of live coral and dead coral percentage cover: 1:1 for the fair reef and 1:3 for the degraded reef [33]. This study site is one of many spots for snorkeling, SCUBA diving, boating, and anchors. Stepping on and touching living corals are essential in these degradations, especially on the degraded reef area.
On the two reefs, damselfish territories were delimited after observations were made for 15 min to determine adjacent areas that were defended (inside the territory) or not defended (outside the territory) by damselfish species [34]. There were, therefore, four conditions in the factorial design for this study: (Ⅰ) degraded reef inside damselfish territory (DI); (Ⅱ) degraded reef outside the damselfish territory (DO); (Ⅲ) fair reef inside the damselfish territory (FI); and (Ⅳ) fair reef outside the damselfish territory (FO). There is a difference in algal compositions in the four different sites. The degraded reef at Ko Taen represented a high relative abundance of turf algae (Polysiphonia, Ceramium, and Cladophora), crustose coralline algae, Turbinaria and Lobophora, while the fair reef was dominated by turf algae (Polysiphonia and Ceramium), Padina, Lobophora, and Turbinaria. Inside the damselfish territory, red turf algae (Polysiphonia and Ceramium) were highly abundant macroalgae, while the damselfish outside the territory were dominated by frondose macroalgae (such as Turbinaria, Padina and Lobophora). Coral was found in a higher abundance on the fair reef. The coral-algal community composition is described in Figure 1 and Figure 2.
Using a hammer and chisel, three patches of epilithic macroalgae (20 × 20 cm) were randomly collected at 3 m depths from each condition. The distance between the patches collected was around five meters. All samples were kept in filtered seawater in 50 mL tubes and stored at −20 °C before DNA extraction.
For the environmental factor, light intensity and temperature were measured using the Onset-HOBO data logger (Model: UA-002-64, Onset Computer Corporation, Bourne, MA, USA).

2.2. DNA Extraction and 16S rRNA Sequencing

Microbial DNA was extracted from macroalgal samples using a DNeasy® PowerSoil® Pro Kit (QIAGEN Co., Ltd., Hilden, Germany) following the manufacturer’s instructions. The concentration of extracted DNA was then quantified using the DS-11 Series spectrophotometer (DeNovix Inc., Wilmington, NC, USA). Sequencing was performed at GENEWIZ Biological Technology Co., Ltd. (Suzhou, China). The V3–V4 variable regions of the 16S rRNA Bacteria and Archaea genes were amplified with pairing primers designed by GENEWIZ (South Plainfield, NJ, USA). The sequences of forward and reverse primers were 5′-CCTACGGRRBGCASCAGKVRVGAAT-3′, and 5′-GGACTACNVGGGTWTCTAATCC-3′, respectively. The total volume of each polymerase chain reaction was 25 µL, containing 2.5 µL of TransStart Buffer (TransGen, Beijing, China), 2 µL of dNTPs, 1 µL of each primer, and 20–30 ng of template DNA [35]. With the Index PCR product, the final libraries were purified using AMPure XP beads (Beckman Coulter, Indianapolis, UK) before quantification. The sequencing process was conducted on the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) in a 2 × 300 bp paired-end run.

2.3. Bioinformatic and Statistical Analysis

The results of specimen sequencing were processed with the QIIME2 pipeline v2021.4 [36]. Quality control and denoising were performed with DADA2 [37] to extract the feature table and a dataset of representative sequences was taken for subsequent analysis. Taxonomic classification was carried out using a Naive Bayes classifier trained on the SILVA database [38] at 95% similarity. Microbial data were reported as relative abundance data and the most abundance microbial taxa in each site was presented. Alpha diversity measures (Chao-1, Shannon, and Simpson indices) were calculated using alpha and alpha-phylogenetic methods in QIIME 2 software. Species richness and species evenness were calculated with observed features and Pielou’s evenness index [39], respectively. Species richness and evenness among conditions were verified with the pairwise Kruskal-Wallis test. Species diversity was calculated with the Shannon Diversity index (Shannon 1984). Principal coordinates analysis (PCoA) was based on an unweighted UniFrac distance matrix and the differences in microbial community between reef conditions and territories were quantified and tested with permutational multivariate analysis of variance (PERMANOVA) using beta and beta-phylogenetic methods of QIIME2 software [40]. To determine the significant difference for each group of specific prokaryotic microbial taxa, linear discriminant analysis for effect size (LEfSe) [41] was used. LEfSe was conducted on the website (http://huttenhower.sph.harvard.edu/galaxy/ (accessed on 7 November 2022)) with a LDA threshold score of 2.0. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) [42] was used for the prediction of microbial community function using 16S rRNA profiles.

3. Results

In this study, we only succeeded in sequencing 11 epilithic macroalgae samples, except one sample which was taken from the degraded reef inside the damselfish territory (DI). After demultiplexing, sequencing returned a total of 1,746,878 reads. The sequence depth ranged from a minimum of 107,302 to 225,492 reads per sample. The mean and median values were 158,897 and 155,372 reads per sample, respectively. Taxonomic classification by QIIME identified 2 domains, 51 phyla, 112 classes, 238 orders, 338 families, 488 genera, and 605 species. Only a small read (0.0115%) could not be classified to any division. In addition, there was a fraction (0.53%) that could not be classified to any phyla. According to the dataset, the most abundant phyla were distributed differently among the four sampling sites. Proteobacteria was the dominant phylum with a relative abundance of 34.94–56.40%, followed by Bacteroidota (6.44–27.92%), Chloroflexi (0.50–25.69%), Actinobacteria (2.57–9.09%), and Acidobacteria (1.63–6.99%). Nitrospirota (0.10–1.61%) and Cyanobacteria (0.31–3.38%) were at a lower abundance (Figure 3). At the class level, five dominant classes were found, including Gammaproteobacteria (22.55–39.63%), Alphaproteobacteria (8.02–33.28%), Bacteroidia (6.13–27.39%), Acidimicrobiia (2.43–8.50%), and Dehalococcoidia (0.02–20.08%).
Under different reef conditions, there was no significant difference in microbial species richness between reef conditions (p > 0.05, Figure 4A; Table 1), but there was a significant difference in microbial species evenness between the degraded reef and fair reef (p < 0.05, Figure 4B; Table 1). Species evenness was higher on the degraded reef (J’ = 0.884) than on the fair reef (J’ = 0.875). There was no difference in microbial species diversity between reef conditions; however, the species diversity of the fair reef was higher (H’ = 4.67) than the degraded reef (H’ = 4.55). Proteobacteria, Bacteroidota, and Actinobacteria were dominant phyla in both reef conditions; however, on the degraded reef, Chloroflexi, Acidobacteriota, and Gemmatimonadota were also dominant. In contrast, Verrucomicrobia, Cyanobacteria, and Desulfobacterota presented with high relative abundances on the fair reef. At the class level, the heat map (Figure 5) showed that Acidimicrobiia, Dehalococcoidia, BD2-11, AncK6, and Vicinamibacteria were dominant on the degraded reef while Alphaproteobacteria, Bacteroidia, Cyanobacteriia, Desulfobacteria, and NB1-j were dominant on the fair reef. At the species level, Vibrio sp., Candidatus tenderia, Bdellovibrio sp., and Nitrosopumilus sp. were found with high relative frequency on the degraded reef, while Ruegeria sp., Pseudoalteromonas sp., Perspicuibacter marinus, Magnetospiracea sp., Kordiimonas sp., Salinirepens sp., Robiginitalea sp., and Agaribacterium haliotis were dominant species on the fair reef.
For the absence and presence of damselfish, there was no significant difference in species richness outside and inside the damselfish territory on both the fair and degraded reefs (p > 0.05, Figure 4A; Table 1), but there was a significant difference in species evenness, showing that species evenness was higher inside (J’ = 0.882) than outside (J’ = 0.868) the damselfish territory, especially on the fair reef (p < 0.05, Figure 4B; Table 1). For the diversity, the inside damselfish territory exhibited greater Shannon-Weiner bacterial diversity (H’ = 4.723) compared to the outside damselfish territory (H’ = 4.634) on the fair reef. However, on the degraded reef, the outside damselfish territory (H’ = 4.684) had a greater diversity than the inside damselfish territory (H’ = 4.363). Proteobacteria was a dominant phylum both outside and inside the damselfish territory. Chloroflexi, Actinobacteriota, Gemmatimonadota, and Acidobacteriota coexisted with Proteobacteria in a high relative abundance inside the damselfish territory. At the same time, Bacteroidota, Myxococcota, NB1-j, and Verrucomicrobia were also dominant phyla outside damselfish territory. The heatmap (Figure 5) illustrates the high abundances of Acidimicrobiia, Anaerolineae, Dehalococcoidia, JG30-KF-CM66, Nitrososphaeria, and Nitrospiria inside the damselfish territory, and the high abundance of Alphaproteobacteria, Bacteroidia, Polyangia, and Gammaproteobacteria outside the territory. There were some differences in bacterial species found outside and inside the damselfish territory. Vibrio sp., Desulfatitale, Robiginitalea, SAR202 clade, TK17, Dadabacteriales, Ulvibacter, Taibaiella, Candidatus amoebophilus, and Marinoscillum were found outside the damselfish territory while Phormidium, PAUC43f, TK17, UBA10353, bacteriap25, Halieaceae, Coraliitalea coralii, Nitrospira, and Photobacterium rosenbergii were found inside the territory.
PCoA analysis using the Unweighted UniFrac distance matrix illustrates that there were significant differences in microbial species composition among four conditions, and species composition was clustered from each other (PERMANOVA, p = 0.020, Figure 6). Proteobacteria, Bacteroidota, and Actinobacteria were dominant phyla in all conditions. At the class level, Acidimicrobiia, Acidobacteriae, and Anaerolineae were found in high abundance inside the damselfish territory on the degraded reef. Rhodothermia were found with the highest relative abundance. Outside the damselfish territory on the fair reef, Fusobacteriia, Alphaproteobacteria, and Desulfuromonadia were dominant, but Gammaproteobacteria and Bacteroidia were dominant inside the damselfish territory on the fair reef. The dominant species of bacteria in each treatment is illustrated in Figure 7. According to LEfSe analysis, twelve prokaryotic clades were screened out with a 2.0 threshold score of LDA. The abundance levels of Woesearchaeales (member of Archea), the Steroidobacterales order (member of the Gammaproteobacteria class), the Phormidiaceae family, Balneola, and Bdellovibrio were higher outside the damselfish territory of the degraded reef. The Gemmatimonadales order (including the Gemmatimonadaceae family) was the taxa that is present with a high abundance inside the damselfish territory on the fair reef, while Cohaesibacter was the dominant genus outside the damselfish territory on the fair reef (Figure 8A,B).
For environmental factors and measurements, light intensity values were 877.65 ± 89.15, 820.24 ± 91.88, 850.37 ± 62.38, and 887.62 ± 20.43 µmole photon m−1·s−1 in the DI, DO, FI, and FO, respectively. The temperatures of each patch were 28.41 ± 0.04, 28.45 ± 0.05, 28.76 ± 0.07, and 28.61 ± 0.04 °C, respectively. However, there were significant differences in the light intensity and temperature among the sites (p > 0.05.)
For functional prediction, PICRUSt showed that 64 functional subgroups (level two) were identified in all treatments that affiliated with the main groups (level one) of Environmental information processing, cellular processes, genetic information processing, human diseases, metabolism, organismal systems, and cellular processes and signaling (Figure 9A). Moreover, 328 KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathways were detected across all samples. The top thirty-five most common abundances among treatments are presented in the heatmap (Figure 9B). The transporter, ABC transporter, general function, DNA repair and recombination proteins, and purine metabolism pathways were the five most dominant pathways in all treatments (Figure 9B). For differences in the relative abundance of gene functions, there was a higher relative abundance of xenobiotics biodegradation and metabolism gene classes in the degraded reef than in the fair reef. Meanwhile, the abundance levels of gene functions in relation to cellular processes and environmental information processes (such as translation, replication and repair, and membrane transport) were higher in the fair reef (Figure 9A).

4. Discussion

In this study, the composition and abundance of epilithic macroalgae-associated microbial communities seemed to be different among the different reef conditions. The algal-associated microbial communities on the degraded reef had a higher abundance and evenness than those on the fair reef. This might be because the more diverse assemblage of turf algae on the degraded reef can harbor distinct microbial-associated communities [43]. Additionally, it has been reported that benthic algal-dominated communities had higher abundances of microbes and potential pathogens than coral-dominated communities, leading to increases in coral disease and death [44,45,46].
Our results show that Proteobacteria, Bacteroidota, and Actinobacteria were the dominant phyla. This is consistent with previous reports, indicating that these three major bacterial phyla are common and widely distributed in marine habitats [31,44,45,46,47,48,49,50,51,52,53,54,55,56]. These phyla are associated with different types of algae and corals [31,47,49]. However, in this study, we found a low abundance of Cyanobacteria (3%) associated with epilithic algae or turf algae. Consistent with our findings, Barrott et al. [43] reported that the turf algae had an abundance of Cyanobacteria around 2% and the abundance of associated microbes varied by the algal functional group. It has been known that microbial communities are specific to their algal host that can lead to differences among reef conditions [43]. Many studies showed that turf algal hosts can positively and negatively influence microbial assemblages using a variety of mechanisms [43,46]. Algae can produce mucus and allelochemicals that can promote or inhibit the growth of microbes [43,46]. On the other hand, microbes likely provide some benefits to algal hosts, preventing fouling on algal surfaces, excluding algal pathogens, and providing nitrogen or nutrient sources for the algal host [43]. Some bacteria, e.g., Cyanobacteria, can protect the host from herbivory and provide nitrogen sources for algae [52,53,54]. Phyla in the Archaea domain represented less than 2% of the phyla found in this study. Many studies have been reported that Archaea are absent or in low abundances on coral reefs and other marine environments, but are normally found in contaminated or polluted areas [57,58]. In our study site, careless snorkeling and SCUBA divers can directly and indirectly damage coral, mainly by touching, kicking, stepping on, and smothering it. At the class level, the trend of Gammaproteobacteria and Alphaproteobacteria predomination in this study is consistent with the findings of coral-associated bacterial diversity in Thai waters [31,58]. Moreover, a high proportion of these bacterial classes would also be a common pattern at the global scale of coral reefs [49,59,60,61,62].
In this present study, Pseudoalteromonas was highly abundant on the fair reef. It has been reported that Pseudoalteromonas may play a protective role in the defense against Gram-positive coral pathogens by secreting antibacterial compounds, and can inhibit macroalgal settlement and spore germination [63,64]. Additionally, Pseudoalteromonas has an important impact on coral by inducing juvenile coral settlement and metamorphosis [29]. However, it has reported that Pseudoalteromonas piratica can play a role as a coral pathogen [65] and can supply macroalgae with various phytohormones (such as cytokinin and auxin hormones) [63]. On the degraded reef, Vibrio species were found in higher proportions and have been reported to be pathogenic and opportunistic in association with coral bleaching and coral disease [66]. The low abundance of Vibrio on the fair reef might be related with the high abundance of Ruegeria, which has the ability to inhibit Vibrio growth [67].
For the absence and presence of damselfish, our results show that microbes were more even inside the damselfish territory on the fair reef than outside. Meanwhile, on the degraded reef, the outside damselfish territory had a greater species evenness than the inside damselfish territory. The farming behaviors of herbivorous fish influence the reef benthic communities by excluding unpalatable turf algae and cultivating low-diversity and high-density communities of palatable turf algae [23,26], which can shift microbial communities associated with the turf algae to communities with a high abundance of core microbiomes. Moreover, territorial defense is also an important behavior that protects preferred macroalgae from other herbivorous fish. These feeding behaviors influence microbial communities because they change the benthic macroalgal community, especially the epilithic algal matrix, which harbors many microbial communities, especially coral disease-associated bacteria [26,43,68,69,70]. Both their farming behaviors and bite marks can damage corals, consequently expanding their territory areas [25]. Increases in the epilithic algal matrix, higher coral disease-associated bacteria prevalence, and bite marks inside the damselfish territory could enhance coral degradation [26].
In this study, as in other studies [71,72], Chloroflexi and Proteobacteria were common phyla on turf algae and macroalgal surfaces inside the damselfish territory. These core microbiomes have been reported from the algae-farming damselfish due to the richness of detritus and sediment that acts as a food source of these bacteria [73,74]. In previous studies, Phormidium, the main cause of black band disease [75], was highly abundant inside damselfish territories, while Vibrio sp., an important potential pathogen in coral reefs [76,77], was found in high abundance outside damselfish territories. Moreover, a higher abundance of Photobacterium rosenbergii, a potential coral pathogen, was found inside the damselfish territory as opposed to outside [78]. These high abundances could be related to the exposure of fish fecal matter [79]. Additionally, turf algae themselves can play role as a coral-associated pathogen harbor, and can trap detritus that is a food resource of bacteria [43,70,73].
For predicted functional profiles, there were differences in the relative abundance levels of some genes in different reef conditions. Xenobiotics biodegradation and metabolism genes were more abundant in the degraded reef. This enzyme was present in the area that was disturbed by human activities [80]. The presence of this detoxification gene may indicate that there was a higher anthropogenic pollution level in the degraded reef [80,81]. For the fair reef, there were higher abundance levels of genes used for translation, replication, and repair, as well as membrane transport in relation to bacterial growth. It might be shown that fair reefs had suitable surrounding environments for some groups of marine benthic bacteria.

5. Conclusions

This study indicates that microbial communities are different in their assemblage and dominance patterns among reef conditions and in relation to the presence of damselfish. Species evenness was higher on the degraded reef condition and lower outside the damselfish territory. The farming behaviors of damselfish can shift microbial communities associated with turf algae to communities with a high abundance of core microbiomes by cultivating low-diversity and high-density communities of palatable turf algae. Proteobacteria, Bacteroidota, and Actinobacteria were the dominant bacterial phyla in all areas. At the class level, Gammaproteobacteria, Alphaproteobacteria, Bacteroidia, Acidimicrobiia, and Dehalococcoidia were dominant in this study. Important potential pathogens of reef organisms, such as Vibrio and Phormidium sp., were found on the degraded reef and inside the damselfish territory areas. However, Pseudoalteromonas and Ruegeria were found in the fair reef area, which provides advantages for a coral reef ecosystem. Ruegeria and Pseudoalteromonas play a protective role in defense against coral pathogens and can induce juvenile coral settlement. However, some species of Pseudoalteromonas can be pathogens that induce diseases in coral. This study provides useful information on microbial biota in coral reef habitats with different reef conditions using metagenomics analysis, which is further applicable to reef conservation and coastal management.
Microbial communities can play different roles and functions in different habitats or reefs. This can make it more challenging to interpret the specific roles. Therefore, future studies should investigate the functional roles of coral-algal-microbial communities and the interactive effects of abiotic factors (e.g., light intensity, temperature, and nutrients) to evaluate their effects on coral-algal-microbial communities.

Author Contributions

Conceptualization, J.M., J.H.K. and K.S.; methodology, J.T., J.M., J.H.K. and K.S.; investigation, J.T., J.M. and K.S.; software, K.S.; formal analysis, K.S.; writing—original draft preparation, J.T.; writing—review and editing, J.M., J.H.K. and K.S.; supervision, J.M. and K.S.; project administration, J.T., J.M. and K.S.; funding acquisition, J.T. and J.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research and innovation activity is funded by the National Research Council of Thailand (NRCT) (grant no. N41D640040) and the Science Achievement Scholarship of Thailand (SAST). This research was supported by the National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (SCI6505009S) to J.M.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank the Seaweed and Seagrass Research Unit, and the Division of Biological Science, Prince of Songkla University, for their scuba diving equipment. We would also like to thank the Molecular Evolution and Computational Biology Research Unit for sample collection design and data analysis.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Maps of Thailand (A), Samui Island (B), and Taen Island (C): the degraded reef is located at 9°22′28″ N, 99°57′21″ E and the fair reef is located at 9°23′04″ N, 99°57′06″ E. The photographs show the benthic community structure on the fair reef (D), the benthic community structure on the degraded reef (E), and the benthic community structure in the damselfish territory area on the fair reef (F).
Figure 1. Maps of Thailand (A), Samui Island (B), and Taen Island (C): the degraded reef is located at 9°22′28″ N, 99°57′21″ E and the fair reef is located at 9°23′04″ N, 99°57′06″ E. The photographs show the benthic community structure on the fair reef (D), the benthic community structure on the degraded reef (E), and the benthic community structure in the damselfish territory area on the fair reef (F).
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Figure 2. Diagram of benthic biota in four different treatments. The size of the fonts illustrates the abundance of each benthic organism group: turf algae (including Polysiphonia, Ceramium, and Cladophora); frondose macroalgae (including Padina, Lobophora, and Turbinaria); and coral (including Porites).
Figure 2. Diagram of benthic biota in four different treatments. The size of the fonts illustrates the abundance of each benthic organism group: turf algae (including Polysiphonia, Ceramium, and Cladophora); frondose macroalgae (including Padina, Lobophora, and Turbinaria); and coral (including Porites).
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Figure 3. The chart shows relative abundances at the phylum level in four conditions. DI = inside the damselfish territory on the degraded reef; DO = outside the damselfish territory on the degraded reef; FI = inside the damselfish territory on the fair reef; FO = outside the damselfish territory on the fair reef.
Figure 3. The chart shows relative abundances at the phylum level in four conditions. DI = inside the damselfish territory on the degraded reef; DO = outside the damselfish territory on the degraded reef; FI = inside the damselfish territory on the fair reef; FO = outside the damselfish territory on the fair reef.
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Figure 4. Species richness (A) and species evenness (B) of microbial communities in all conditions. Inside degraded means inside damselfish territory on the degraded reef, while inside fair means inside the damselfish territory on the fair reef. Outside degraded means outside the damselfish territory on the degraded reef, while outside fair means outside the damselfish territory on the fair reef. Whiskers indicate the 10th and 90th percentiles. * p < 0.05.
Figure 4. Species richness (A) and species evenness (B) of microbial communities in all conditions. Inside degraded means inside damselfish territory on the degraded reef, while inside fair means inside the damselfish territory on the fair reef. Outside degraded means outside the damselfish territory on the degraded reef, while outside fair means outside the damselfish territory on the fair reef. Whiskers indicate the 10th and 90th percentiles. * p < 0.05.
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Figure 5. Heatmap analysis of 30 microbial dominant class distributions in each condition. The colors of the heatmap squares are varied from white to dark-red to represent the low to the high levels of relative abundance. DI = inside the damselfish territory on the degraded reef; DO = outside the damselfish territory on the degraded reef; FI = inside the damselfish territory on the fair reef; FO = outside the damselfish territory on the fair reef.
Figure 5. Heatmap analysis of 30 microbial dominant class distributions in each condition. The colors of the heatmap squares are varied from white to dark-red to represent the low to the high levels of relative abundance. DI = inside the damselfish territory on the degraded reef; DO = outside the damselfish territory on the degraded reef; FI = inside the damselfish territory on the fair reef; FO = outside the damselfish territory on the fair reef.
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Figure 6. The PCoA of the reef condition and territory microbiomes was based on an unweighted UniFrac distance matrix. Red = inside the damselfish territory on the degraded reef. Orange = outside the damselfish territory on the degraded reef. Blue = inside the damselfish territory on the fair reef. Green = outside the damselfish territory on the fair reef.
Figure 6. The PCoA of the reef condition and territory microbiomes was based on an unweighted UniFrac distance matrix. Red = inside the damselfish territory on the degraded reef. Orange = outside the damselfish territory on the degraded reef. Blue = inside the damselfish territory on the fair reef. Green = outside the damselfish territory on the fair reef.
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Figure 7. Diagram of dominant microbial species in four different treatments. The size of the fonts illustrates the abundance of each microbial species.
Figure 7. Diagram of dominant microbial species in four different treatments. The size of the fonts illustrates the abundance of each microbial species.
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Figure 8. The linear discriminant analysis effect size (LEfSe) analysis of microbial abundance among different treatments. (A) The cladogram of detected prokaryotic taxa for each treatment. (B) Taxa with significant differences in different treatments were detected by LEfSe analysis with an LDA threshold score of 2.0 and a significance rating of 0.05. LEfSe analysis was performed on the website http://huttenhower.sph.harvard.edu/galaxy/ (accessed on 9 February 2023). FI = inside the damselfish territory on the fair reef; DO = outside the damselfish territory on the degraded reef; FO = outside the damselfish territory on the fair reef.
Figure 8. The linear discriminant analysis effect size (LEfSe) analysis of microbial abundance among different treatments. (A) The cladogram of detected prokaryotic taxa for each treatment. (B) Taxa with significant differences in different treatments were detected by LEfSe analysis with an LDA threshold score of 2.0 and a significance rating of 0.05. LEfSe analysis was performed on the website http://huttenhower.sph.harvard.edu/galaxy/ (accessed on 9 February 2023). FI = inside the damselfish territory on the fair reef; DO = outside the damselfish territory on the degraded reef; FO = outside the damselfish territory on the fair reef.
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Figure 9. Predicted functional profiles for the microbial community by PICRUSt. (A) Histogram showing the predicted functional main group (level one) and subgroup (level two) in all treatments, and (B) heat map of the relative abundance of the predicted top thirty-five dominant functions among different treatments.
Figure 9. Predicted functional profiles for the microbial community by PICRUSt. (A) Histogram showing the predicted functional main group (level one) and subgroup (level two) in all treatments, and (B) heat map of the relative abundance of the predicted top thirty-five dominant functions among different treatments.
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Table
Table 1. Pairwise analysis of species richness (observed OTUs) and species evenness (Pielou’s evenness) among four treatments.
Table 1. Pairwise analysis of species richness (observed OTUs) and species evenness (Pielou’s evenness) among four treatments.
Kruskal-WallisGroup1Group2Hp-Value
Species richnessInside the territory of the
degraded reef		Inside the territory of the fair reef3.0000.083
Outside the territory of the degraded reef1.3330.248
Outside the territory of the fair reef3.0000.083
Inside the territory of the fair reefOutside the territory of the degraded reef0.4280.512
Outside the territory of the fair reef1.1900.275
Outside the territory of the
degraded reef		Outside the territory of the fair reef0.4280.512
Species evenness
Inside the territory of the
degraded reef		Inside the territory of the fair reef1.3330.248
Outside the territory of the degraded reef1.3330.248
Outside the territory of the fair reef3.0000.083
Inside the territory of the fair reefOutside the territory of the degraded reef0.0470.827
Outside the territory of the fair reef3.8570.049
Outside the territory of the
degraded reef		Outside the territory of the fair reef3.8570.049
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MDPI and ACS Style

Titioatchasai, J.; Surachat, K.; Kim, J.H.; Mayakun, J. Diversity of Microbial Communities Associated with Epilithic Macroalgae in Different Coral Reef Conditions and Damselfish Territories of the Gulf of Thailand. J. Mar. Sci. Eng. 2023, 11, 514. https://doi.org/10.3390/jmse11030514

AMA Style

Titioatchasai J, Surachat K, Kim JH, Mayakun J. Diversity of Microbial Communities Associated with Epilithic Macroalgae in Different Coral Reef Conditions and Damselfish Territories of the Gulf of Thailand. Journal of Marine Science and Engineering. 2023; 11(3):514. https://doi.org/10.3390/jmse11030514

Chicago/Turabian Style

Titioatchasai, Jatdilok, Komwit Surachat, Jeong Ha Kim, and Jaruwan Mayakun. 2023. "Diversity of Microbial Communities Associated with Epilithic Macroalgae in Different Coral Reef Conditions and Damselfish Territories of the Gulf of Thailand" Journal of Marine Science and Engineering 11, no. 3: 514. https://doi.org/10.3390/jmse11030514

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