The Coral Holobiont Madracis Decactis Associates With Multiple Types of Symbiodiniaceae


 The scleractinian reef building coral Madracis decactis is a cosmopolitan species. Understanding host–symbiont associations is critical for assessing the coral’s habitat occupancy and it’s response to environmental changes. In this study, we performed fine grained phylogenetic analyzes of Symbiodiniaceae algae associated with Madracis across a broad latitudinal gradient in the SW Atlantic (Abrolhos Bank, and St. Peter and St. Paul Archipelago). Previous studies have argued that Madracis is a specialist coral, with colonies harboring a single symbiont type B. However, these previous studies have not precisely addressed if Madracis is colonized by several types of Symbiodiniaceae simultaneously, or if whether this coral is a specialist. The hypothesis that Madracis is a generalist coral host was evaluated in the present study. A total of 1.9 million reads of ITS2 nuclear ribosomal DNA were obtained by Illumina MiSeq sequencing. ITS2 sequences were classified into Clades A, B, and C in Abrolhos, and only clade B symbionts recorded in St. Peter and St. Paul. This study also demonstrate that a single Madracis colony can host different symbiont types with >30 Symbiodiniaceae species. Abrolhos corals presented a higher photosynthetic potential as a possible result of co-occurrence of multiple Symbiodiniaceae in a single coral colony. Multiple clades of Symbiodiniaceae possibly confer coral hosts with broader environmental tolerance and ability to occupy diverse or changing habitats.


Introduction
Symbiodiniaceae dino agellates belonging to the genus Symbiodinium (former Symbiodinium Clade A) and other related phylogenetic lineages are pivotal cnidarian endosymbionts [1]. These are important primary producers and also form a crucial symbiosis with reef-building corals [2]. Global and local environmental disturbances may cause losses of symbionts from the hosts (coral bleaching), with potential mortality for the coral [3,4]. Currently, nine evolutionary lineages of Symbiodiniaceae (clades A to I), each with many subgroups (subclades), are recognized based on relatively conserved markers [5,6]. The ability to harbor different Symbiodiniaceae clades and subclades may provide an effective way to withstand severe and extended stressful conditions [7,8]. The scleractinian reef building coral genus Madracis inhabits shallow and mesophotic regions across the West Atlantic. Species-speci c studies documented only Symbiodiniaceae type B (with different subgenus, B7 and B13) in species of Madracis in Caribbean, Mexico and the Bahamas [9-10-11-12, 13] and they suggest that the brooding coral is reproductively isolated and specialist. However, the ne grained diversity of Symbiodiniaceae associated with the reef-building coral Madracis is remained unknown.
The present study analyzed the diversity of Symbiodiniaceae associated with Madracis decactis (only species of Madracis in Brazil) across a broad latitudinal gradient (Abrolhos, and St. Peter and St. Paul). Abrolhos is a large continental reef complex that consists of a topological barrier for the southward warm waters of the Brazil Current.
While reefs in the Northeastern Region are composed mostly by patch or elongated banks on a narrow continental shelf (~ 50 km), those in the central Brazilian coast are predominantly formed by large platforms and isolated pinnacles with expanded tops [14,15]. St. Peter and St. Paul comprises the eastern most Brazilian oceanic islands and is one of the smallest and most isolated tropical oceanic islands in the world. The islets are devoid of shore and consist entirely of steep drop-offs extending to 60-150 m depth [16], with the most limited area of shallow habitat among oceanic islands (∼200m 2 ) [17]. Madracis decactis is an important contributor of reef structures in Abrolhos, while it is the dominant scleractinian in the rocky reefs of the St. Peter and St. Paul [18]. Previous studies on M. decactis did not assess species-speci c diversity and indicated the exclusive presence of clade C in shaded habitats (pinnacles) in the Abrolhos, a pattern similar to that recorded in other regions [19,20], and the presence of the clade A from shallow well-lit waters of the Abrolhos [21], again corroborating previous studies [22,10].
Coral species are considered generalists when associated with more than one symbiont clade [1]. The knowledge about the diversity of symbionts of shallow water scleractinian corals in the south Atlantic has been largely neglected, and it has been mainly restricted to the report of clades A (Symbiodinium), B (Breviolum), C (Cladocopium) [23-26-19, 20]. Cladocopium Clade C is one that confers the most resistance to bleaching in scleractinians and has high resistance and resilience to environmental disturbances [8,23]. Symbiodinium A was found to be associated with high temperatures [20]. South Atlantic corals occupy greater depth zones, are tolerant to higher turbidity and nutri cation, are morphologically resistant, engaged in exible symbiotic associations, and most importantly, are less affected by bleaching [25]. The south Atlantic has proportionally more generalist coral species. For species that have been evaluated for symbiont diversity, approximately 60% of Symbiodiniaceae phylotypes in the South Atlantic are generalists, such as Favia, Millepora, Mussismilia and Siderastrea [25]. Meanwhile, Montastraea and Madracis were considered as specialists [25]. However, a previous study suggested Madracis decactis metagenomes from St. Peters and St. Paul had Clades A1 and F [27].
The aim of this study was to evaluate the diversity of Symbiodiniaceae in Madracis decactis in Abrolhos and in St. Peter and St. Paul. The presence of possible new endosymbionts lineages was also analyzed. The results of this study showcase that M. decactis exhibits some degree of exibility in its symbiotic association with zooxanthellae as a generalist. In addition, high Symbiodiniaceae diversity may lead to greater resistance to environmental changes and plasticity in habitat occupancy, as well as less bleaching susceptibility.

Amplicon Sequencing Analysis with SymPortal
Demultiplexed forward and reverse fastq les were analyzed locally with SymPortal [30] and were subjected to standard sequence quality control protocols implemented with MOTHUR 1.39.5 [31], the BLAST suite of executables [32], and minimum entropy decomposition [33] to lter non-Symbiodiniaceae and sequencing artifacts from the dataset [30]. Sequences were clustered by clade. Sequences occurring in a su cient number of samples within both, the dataset being analyzed and the entire database of samples run through SymPortal, were identi ed as DIVs which were then used to characterize ITS2 type pro les [30]. All sequences were queried against the SymPortal database using the nucleotide BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Best hits were used to classify ITS2 sequences into subclades. Sequence alignments were performed with Muscle [34]. Phylogenetic reconstruction with a maximum likelihood model was inferred with Fast Tree 2 [35]. Nodal support tested by bootstrap analysis (1000 times). initial uorescence) between regions, while controlling for the effect of depth which was included as covariate in the model [36].

Results And Discussion
A total of 1.9 million ITS2 nuclear ribosomal DNA reads were obtained (Table 1). Three Clades and eight subclades of Symbiodiniaceae were detected (Table 1 and Supplementary Table S1). Symbiodiniaceae of clades A, B and C occurred in Abrolhos, while Symbiodiniaceae B was found in St. Peter and St. Paul (Fig. 1a and 1b). Moreover, the ITS2 results identi ed variants within the clades A, B and C (Figs. 1c and 2 Abrolhos. These differences may be related to co-evolutionary processes to enhance holobiont survival. The Analysis of Covariance (ANCOVA) showed a signi cant difference in photosynthetic yield between Abrolhos and St. Peter and St. Paul corals (Fig. 3), with higher values recorded at Abrolhos (DF, 1; MS, 0.054; F, 6.20; p, 0.013). The effect sampling depth was not signi cant (DF, 1; MS, 0.002; F, 0.24; p, 0.619). The higher photosynthetic potential in Abrolhos than in St, Peter and St. Paul is a possible result of multiple Symbiodiniaceae co-occurrence in a single coral colony in Abrolhos (Fig. 1a and 1b). Many species of coral harbor multiple clades of Symbiodiniaceae, and the symbiont composition may vary both within and/or between colonies depending on environmental conditions and geographic location [19-13-37, 38]. The enhanced symbiont diversity observed in Abrolhos, represented by multiple clades of Symbiodiniaceae (A, B, and C), possibly leads to greater resistance to environmental changes and plasticity in habitat occupancy.

Conclusion
South Atlantic Madracis decactis is a generalist coral and contains an ample diversity of Symbiodiniaceae types that may contribute to coral holobiont health. Collectively, south Atlantic corals are not only typical generalists, but also tend to associate with generalist symbionts. This symbiotic exibility may allow for adaptive shifts in the holobiont community, which is critical in increasing resistance and resilience to bleaching. It has adapted for thriving in turbid environments that may prove critical for surviving the increasing impacts of climate change and mass bleaching episodes. concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Maximum likelihood tree of sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. This analysis involved 127 nucleotide sequences. There were a total of 839 positions in the nal dataset. Evolutionary analyses were conducted in MEGA X [48]. Samples in the present study are shown in red circle.