Diversity and distribution of Actinobacteria associated with reef coral Porites lutea

Actinobacteria is a ubiquitous major group in coral holobiont. The diversity and spatial and temporal distribution of actinobacteria have been rarely documented. In this study, diversity of actinobacteria associated with mucus, tissue and skeleton of Porites lutea and in the surrounding seawater were examined every 3 months for 1 year on Luhuitou fringing reef. The population structures of the P. lutea-associated actinobacteria were analyzed using phylogenetic analysis of 16S rRNA gene clone libraries, which demonstrated highly diverse actinobacteria profiles in P. lutea. A total of 25 described families and 10 unnamed families were determined in the populations, and 12 genera were firstly detected in corals. The Actinobacteria diversity was significantly different between the P. lutea and the surrounding seawater. Only 10 OTUs were shared by the seawater and coral samples. Redundancy and hierarchical cluster analyses were performed to analyze the correlation between the variations of actinobacteria population within the divergent compartments of P. lutea, seasonal changes, and environmental factors. The actinobacteria communities in the same coral compartment tended to cluster together. Even so, an extremely small fraction of OTUs was common in all three P. lutea compartments. Analysis of the relationship between actinobacteria assemblages and the environmental parameters showed that several genera were closely related to specific environmental factors. This study highlights that coral-associated actinobacteria populations are highly diverse, and spatially structured within P. lutea, and they are distinct from which in the ambient seawater.


Introduction
Coral reef ecosystem is one of the most important tropical marine ecosystems, mainly distributed in the Indo-West Pacific, Eastern Pacific, Western Atlantic, and the Eastern Atlantic (Moberg and Folke, 1999). Corals provide habitats for numerous bacteria in their mucus layer, tissue, and calcium carbonate skeleton, as well as the dinoflagellates, fungi, archaea, and viruses (Rosenberg et al., 2007). Coral-associated bacteria not only take part in carbon, nitrogen, and sulfur biogeochemical cycles and provide necessary nutrient for coral, but also keep corals from being infected by pathogens (Rosenberg et al., 2007;Raina et al., 2009;Bourne and Webster, 2013).
Environmental conditions, coral species, colony physiology, and seasonal variation are considerable influencing factors on the coral-associated bacterial community (Hong et al., 2009). Moreover, due to various microhabitats provided by corals' biological structures, the spatial heterogeneity has been proved in bacterial communities associated with a single coral colony (Rohwer et al., 2002;Sweet et al., 2011;Li et al., 2014a). As a major coral-associated bacterial group, how actinobacteria is spatially and temporally organized in corals, and what is the connection between the actinobacteria communities in corals and in seawater remains poorly understood. Comprehensive investigation of the distribution of this ubiquitous group at spatial and temporal scales will help understanding the variation of coral associated bacteria and the potential function of actinobacteria, and will contribute a lot to bioprospect the actinobacteria resources for utilization as novel sources for bioactive natural products.
Coral reefs are widely distributed in the South China Sea (Liu et al., 2009;Wang et al., 2014). Porites lutea is the dominant, typical coral species in the Luhuitou fringing reef, which is located in the south end of the Hainan province (Zhao et al., 2008). In this study, the diversity and distribution of actinobacteria were investigated in coral P. lutea and in the surrounding seawater every 3 months for 1 year using cultureindependent method for the first time. We aimed to reveal the coral-associated actinobacteria community structures in three divergent coral compartments in different months, compare the actinobacterial communities in the coral and in the surrounding seawater, and research the actinobacteria community variation responds to the environmental factors.

Sample Collection
The coral and surrounding sea water samples were collected in four different months (February, May, August, and November) in 2012 from the Luhuitou fringing reef (109 • 28 ′ E, 18 • 13 ′ N). Coral fragments (approximately 10 × 10 cm) were collected from the side of three healthy P. lutea colonies at the depth of 3-5 m each time using punch and hammer. Coral mucus, tissues and skeleton were separated and stored according to the method described previously (Li et al., 2014a). One liter of seawater adjacent to the coral colonies was collected, and filtered through 0.22 µmpore-size filter membrane (Millipore). The filter membranes were stored at −80 • C until DNA extraction. As the samples were collected at the same time, environmental parameters including water temperature, salinity, dissolved oxygen, pH value, ultraviolet radiation intensity, and rainfall were cited from the published data (Li et al., 2014a).

DNA Extraction and PCR Amplification
The coral tissue and skeleton samples were homogenized thoroughly in liquid nitrogen with sterile mortar and pestle before added to the PowerBead Tubes. The filter membranes with adsorbed microbial cells were cut into pieces, and then added to the PowerBead Tubes. Total DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio, Solana Beach, CA, USA) according to the manufacturer's instruction.

Gene Library Construction and Sequencing
Sixteen clone libraries of actinobacterial 16S rRNA genes were constructed using the pMD18-T Vector Cloning Kit and E. coli DH5α competent cells (Takara, Dalian) following the manufacturer ′ s instructions. The positive clones from each library inoculated on MacConkey agar with ampicillin (100 µg/ml) were randomly picked and sequenced using M13F (−47) primer on ABI 3730xl capillary sequencers (Applied Biosystems, USA).

Libraries Analysis
The vector sequences were screened by the VecScreen tool provided in NCBI (http://www.ncbi.nlm.nih.gov/tools/ vecscreen/). Chimeras were checked by running chimera.uchime packaged in Mothur (Schloss et al., 2009), and potential chimeras were removed. All valid sequences were deposited in GenBank (accession numbers were shown in Data S1). All qualified sequences were identified by using the classify.seqs command in Mothur with Silva reference alignment database (http:// www.mothur.org/wiki/Silva_reference_files, Release 119) at a confidence level of 80%. The sequences, which do not belong to Actinobacteria, were removed from further analysis. Sequences were clustered into operational taxonomic units (OTUs) with a 97% threshold using the cluster command in Mothur. The relationships among actinobacterial communities associated with different coral compartments and in the ambient seawater in different months were analyzed by hierarchical cluster analysis. Based on Bray-Curtis similarity estimated from the OTU matrix, clustering was generated by using the complete linkage method with the PRIMER 5 software (Clarke, 1993). The shared OTUs were determined by using the online tool venny (Oliveros, 2007(Oliveros, -2015://bioinfogp.cnb.csic.es/tools/venny/ index.html).
The correlations between Actinobacteria assemblages of coral samples and the environmental factors were analyzed by using the software package CANOCO 4.5.1 (ter Braak and Šmilauer, 2002). Redundancy analysis (RDA) was carried out to determine the relationship between the actinobacteria community and the environmental factors including temperature, salinity, dissolved oxygen, pH value, rainfall, and UV radiation and in combination with two nominal variables including the coral divergent compartments and the different sampling months. The significance of the relation between the explanatory variables and the actinobacterial community compositions was tested using Monte Carlo permutation tests (9999 unrestricted permutations, P < 0.05).

Coral-associated Actinobacteria Diversity
A total of 2403 sequences were obtained from sixteen 16S rRNA gene clone libraries, resulting in 395 OTUs (stringency at 97%). The rarefaction analysis result showed that most of the curves did not flatten to asymptote, but climbed less steeply (Figure 1). The coverages ranged from 0.69 to 0.97 in 16 libraries, and the average coverage was 0.83 ( Table 1). The highest number of OTUs was found in the tissue collected in May, while the lowest OTUs was found in the skeleton collected in November ( Table 1). The Shannon indices in mucus collected in different months ranged from 2.32 to 3.44, from 2.45 to 3.55 in tissues, from 1.82 to 3.35 in skeleton, and from 1.53 to 2.82 in sea water (Table 1), and the diversity in the actinobacterial community associated with P. lutea was higher than which in the surrounding sea water (P = 0.045).

Coral-associated Actinobacterial Community Composition
At a confidence threshold of 80%, 2403 qualified reads were assigned to four classes, i.e., Acidimicrobiia, Actinobacteria, Thermoleophilia, and KIST-JJY010. Among them, Acidimicrobiia and Actinobacteria were ubiquitous and dominant in P. lutea and in the seawater samples. Thermoleophilia was not detected in corals collected in February, in the mucus and seawater in May, and in the mucus in August, while accounted for 0.5-48.8% in all other samples. KIST-JJY010 was detected only in the mucus in November (0.6%), and in the skeleton in August (2.6%).
Twenty-five described families and 10 unnamed families were detected in the 16 libraries (Figure 2). OM1_clade and Propionibacteriaceae (genera Friedmanniella and Propionibacterium) were ubiquitous, major groups in P. lutea. Meanwhile, OM1_clade was not detected in the seawater in  February and May, and rare in the other two seawater libraries, and Propionibacteriaceae was absent in all the seawater libraries.
Micromonosporaceae was the most abundant group in the tissue in February (47.4%) and in the mucus in August (46.2%), in which most of the reads were affiliated with an unclassified group. Nonetheless, Micromonosporaceae was absent in all other coral and seawater samples. Sva0996_marine_group was detected in all coral samples (5.2-50%) except in the skeleton collected in November, and which also was abundant in the ambient sea water (21.9-80%). Micrococcaceae was absent in the coral skeleton collected in August and in November, and in the sea water samples. Group 480-2 was abundant in the coral tissue in August (24.7%), as well as in the skeleton in May (26.9%) and in November (48.8%), but it was nearly absent in the surrounding seawater. In reverse, Microbacteriaceae and Ilumatobacter were major groups in sea water, while they were less abundant in P. lutea.

Spatial and Temporal Distribution of P. lutea-associated Actinobacteria
Results of hierarchical cluster analysis showed that the actinobacteria communities were significantly different between in the coral and in the surrounding seawater samples (p = 0.01, R = 0.993). The actinobacterial communities associated with the same coral compartments tended to cluster together (Figure 3). The season factor did not significantly influence the variation in the actinobacteria communities. The RDA results indicated that 38.9% of the total variance in the coral-associated actinobacterial Frontiers in Microbiology | www.frontiersin.org composition was explained by the environmental, spatial and temporal factors (Figure 4). The first and second axes differentiated the actinobacteria assemblages in the distinct coral compartments (Figure 4, Table S1). This result was consistent with the hierarchical cluster analysis. None of the environment parameters analyzed in this study was determined as the significant influencing factor in the variation of the P. lutea associated actinobacteria communities. A triplot map illustrated the relationship between major actinobacterial groups, with abundance more than 1%, and the environmental parameters (Figure 4). Friedmanniella and Micrococcus were positively related with the salinity. Microbacterium, Propionibacterium, and group 480-2 were positively correlated with seawater temperature, but negatively correlated with dissolved oxygen.
To investigate the distribution of OTUs in the three divergent coral compartments (mucus, tissue, and skeleton) and in the surrounding seawater, a venn diagram was constructed. The results showed that only 5 OTUs were present in all of P. lutea mucus, tissue and skeleton, and in sea water, which were identified as Sva0996_marine_group, Ilumatobacter, Corynebacterium, OM1_clade and Microbacterium (Table 2, Figure S1A). Another 17 OTUs, which were identified as Candidatus_Microthrix, Corynebacteriales, Friedmanniella, Micrococcus, Mycobacterium, OM1_clade, Propionibacterium, Sva0996_marine_group, Yonghaparkia and 480-2 were common in mucus, tissue, and skeleton (Table 2, Figure S1A). Twelve OTUs distributed in Propionibacterium, Friedmanniella, OM1_clade, Sva0996_marine_group, Kocuria, Mycobacterium, Corynebacteriales, Brevibacterium, and Brachybacterium were present in coral libraries in all four different months (Table 3, Figure S1B). The most abundant OTU0003, which was classified as Propionibacterium, was present in all coral samples with a high abundance (128 out of total 1687 reads in the coral libraries, 7.6%). The secondary abundance OTU0004 affiliated with Friedmanniella was present in all libraries except in skeleton collected in November.

Comparison of Actinobacterial Communities in the Corals and in the Ambient Seawater
Comparing the actinobacteria communities between in P. lutea and in the surrounding seawater will help us to understand the source of coral associated actinobacteria, and the interaction between the bacteria in sea water and in corals. Consisted with previous study on bacteria communities (Li et al., 2014a), the P. lutea associated actinobacteria communities were significantly different from which in the ambient seawater (Figure 3). Groups such as Propionibacteriaceae, Micromonosporaceae, and Micrococcaceae, were specifically associated with the corals rather than in the ambient seawater, where they originated from should be in doubt. Whether the wide distributed groups such as Sva0996_marine_group, OM1_clade, Microbacteriaceae and Ilumatobacter travel between the ambient seawater and the corals need to be investigated. When researchers make a general observation of the whole bacterial communities, which were observed significantly different in coral mucus, tissue, and skeleton (Rohwer et al., 2002;Bourne and Munn, 2005;Sweet et al., 2011;Lee et al., 2012). However, it is unclear whether actinobacteria has a similar distribution pattern. In this study, both the hierarchical cluster analysis (Figure 3) and the RDA analysis (Figure 4) showed that the actinobacteria communities from the same compartment tended to cluster together. The distinct physiochemical microenvironments provided by corals probably is one of the causes (Le Tissier, 1990;Brown and Bythell, 2005;Sweet et al., 2011;Tremblay et al., 2011). Only a small fraction of OTUs (22 out of 299 OTUs in the coral libraries) was common in the coral mucus, tissue, and skeleton libraries in this study ( Table 2). This result suggested that these members might have capabilities to adapt to different microenvironments in divergent compartments of P. lutea. A large amount of the OTUs was specifically associated with a certain coral compartment. Whether and how the properties of distinct actinobacteria assemblages in different coral compartments actually contribute to the close relationship constructed between these associates and corals should be addressed from a functional perspective.

Relationship of environmental factors and the P. lutea-associated Actinobacteria
It is different from previous conclusion of the distribution of coral-associated bacteria (Chen et al., 2011;Li et al., 2014a), actinobacteria associated with P. lutea did not show the apparent seasonal dynamic variations. We suggest that the actinobacteria compositions are relatively stable in distinct compartments in P. lutea. In addition, none of the environmental factors analyzed in this study was determined as the most significant influence on the actinobacteria communities. Even so, some genera were found closely correlated with specific environmental factors. For instance, Propionibacterium showed negatively correlation with dissolved oxygen, probably due to its capability of living in the anaerobic conditions (Patrick and McDowell, 2012). Moreover, the OTUs0003 and 0004 affiliated with Propionibacteriaceae was present in almost all 12 clone libraries with a very high abundance. Whether they are true symbionts, and what functions they play are worth further research.

Conclusion
The diversity and distribution of coral-associated actinobacteria were first comprehensively investigated in this study. Highly diverse actinobacteria was revealed in the 16S rRNA gene clone libraries of scleractinian coral P. lutea in the South China Sea. Twelve Actinobacteria genera were detected in corals for the first time as well as a large number of unclassified groups. The actinobacterial community compositions were distinct in P. lutea and in the surrounding seawater. Furthermore, the higher similarity of actinobacteria composition was observed in the same compartment (i.e., mucus, tissue, or skeleton) of P. lutea. This study will help attracting the attentions on the ecological role of actinobacteria in corals besides the natural products bioprospecting.