The hologenome theory of evolution (HTE) considers the host and its associated microbiota genomes combined as one unit of natural selection in evolution (Rosenberg et al., 2007; Zilber-Rosenberg and Rosenberg, 2008). However, different aspects regarding the evolution of the genomes of hosts and their symbionts, as well as complex aspects of the evolutionary processes, remain under discussion (Leggat et al., 2007; Rosenberg et al., 2007; Moran and Sloan, 2015). In fact, assumptions underpinning this theory that the microbiome, or parts thereof, can be transmitted from one generation to the next and may influence the evolution of the coral holobiont have not been completely elucidated and remain under debate.

Corals depend on their associated microbiome (Rosenberg et al., 2007). Previous studies have indicated that the majority of broadcast spawning coral species do not vertically transfer their microalgal symbionts (Symbiodinium spp. Freudenthal, 196) (Freudenthal, 1962; Knowlton and Rohwer, 2003; Fabina et al., 2012) and other microbial symbionts (Sharp et al., 2010) via their gametes. They generally acquire symbionts as larvae or during post-settlement and metamorphosis as a juvenile polyp via horizontal transmission (from seawater) (Weis et al., 2001; Knowlton and Rohwer, 2003; Apprill et al., 2009). However, new data have reported the vertical transmission (VT) (from a parental colony) in broadcast spawning coral Montipora capitata, which transfers its Symbiodinium through its eggs (Padilla-Gamiño et al., 2012). Many studies have also confirmed that brooded coral transmit their Symbiodinium vertically from parent to offspring through their larvae (Fadlallah, 1983; Harrison and Wallace, 1990; Hirose et al., 2001; Thornhill et al., 2006). Less information is available about the transmission of other important coral microbial symbionts, such as bacteria. Sharp et al. (2011) demonstrated that Roseobacter spp. are vertically translocated from the parental colony to newly released larvae in the brooded coral Porites astreoides. On the other hand, some larvae seem to remain free of bacteria after fertilization, being capable of acquiring bacteria directly from the seawater during larval or polypoid stages (Apprill et al., 2009). Moreover, horizontal transmission of coral-associated bacteria in seven broadcast spawning coral species was reported by Sharp et al. (2010). Littman et al. (2009) and Lema et al. (2014) have also reported changes and uptake of bacteria in early stages of corals but not inside the oocytes.

Considering the controversial data regarding Symbiodinium transmission and the absolute lack of information concerning bacterial core diversity, dynamics and transmission in broadcast spawning corals, specifically the lack of data on this transmission through coral gametes, we here investigate and identify the (core) bacteria and Symbiodinium associated with different life stages of the Brazilian endemic coral Mussismilia hispida (Verrill, 1902). M. hispida is a hermaphrodite species that releases gametes in the water column in a seasonal spawning event that occurs over three consecutive months (Pires et al., 1999; Pires et al., 2016). Approximately 1/3 of the stony coral reef fauna of Brazil are endemic, and most are major builders of Brazilian reefs, including the Mussismilia genus.

To the best of our knowledge, this is the first report that shows the bacterial core composition and dynamics at early stages of coral life, including gametes, as discussed below. We showed that the spawner coral M. hispida can vertically transfer its bacteria and Symbiodinium cells.

Considering the transmission of bacterial symbionts, there is no consensus or pattern on the transmission of these microorganisms. A study on the gametes and planulae larvae of other seven broadcast spawner coral species did not find any bacterial association in these life stages (Sharp et al., 2010). Thus, these corals may acquire their associated microbiota from the seawater (horizontal transmission). On the other hand, Ceh et al. (2012) investigated the microbiome of three different broadcast spawner and brooder corals (Acropora tenuis, Pocillopora damicornis, and Tubastrea faulkneri) before and after spawning and indicated that the bacterial diversity increased after spawning in all coral species. The authors highlighted that Roseobacter spp. was an important genus transmitted during coral reproduction. They also observed a decrease in some key groups that could have evolved through bacterial VT. However, the authors could not prove the occurrence of VT because only adult corals were sampled.

Here, not only adult colonies but also gametes and coral planula larvae at 3 and 5 days a.f. were sampled. The sequencing data showed that the M. hispida microbiome was composed by bacteria and Symbiodinium cells, even in gametes, although we could not verify the presence of bacterial and Symbiodinium cells in association with the (female and male) gametes of M. hispida through microscopy. Sequencing data also showed a stable core bacterial microbiome from the gametes to planula larvae stages that was also present in the parental coral colony. In addition, we verified that the stable microbiome represents a greater proportion of the associated bacteria in gametes and planula larvae stages. In contrast, the core microbiome represents a low proportion in the adult colonies, indicating microbial transmission once these microorganisms have left the parental colony for the bundles. The core microbiome presence could indicate a potential key function of this group in early life stages of coral development. Therefore, the obtained data suggest that this coral could use parallel and different strategies for microbiome acquisition. Considering our sequencing data, the bacterial core is transmitted vertically, potentially via the mucus that visually binds the gametes together in a bundle (observed in vivo). Most likely, the offspring can be partly colonized post-spawning through the uptake of microbial associates released by the parental colony into the surrounding seawater, as previously described (Apprill et al., 2009; Sharp et al., 2011; Ceh et al., 2012, 2013), and partly colonized by parental colonies, which transfer their bacterial core by seeding the mucus with parental-derived bacterial symbionts, as indicated by our data.

Thus, while Symbiodinium cells could be transmitted inside gametes, as shown in literature and suggested by our molecular data, the core bacterial community – being present in abundance within the mucus, which will form the bundle – could be vertically transmitted ‘from outside’ of the gametes, inside the coral. This does not exclude that some bacteria are eventually transmitted inside the gamete or even inside the Symbiodinium cells being transmitted in the eggs; we could not detect these bacteria due to poor resolving power of our methods. The presence of bacterial symbionts living in the Symbiodinium was previously reported by Ainsworth et al. (2015) in tissues of adult corals. On the other hand, Symbiodinium cells, which can be transmitted inside oocytes, can also be acquired from seawater in the planula larvae stage or even by coral adults (Boulotte et al., 2016).

Herein, we propose the model presented in Figure 6 for the transmission of microorganisms in this coral species. M. hispida is a broadcaster spawner and a hermaphroditic species; therefore, its eggs and spermatic cysts develop inside the same mesenteries, but oogenesis and spermatogenesis start at different periods, with oogenesis beginning several months before spermatogenesis (Pires et al., 1999). During gamete development and spawning, the gametes are initially in the gastrodermis, migrating to the mesoglea during the vitellogenesis period. When the eggs and spermatic cysts are mature, the rupture of the mesentery wall occurs, and the bundles that formed (eggs, cysts, and mucus) reach the gastrovascular cavity, passing through the mouth to reach the surface (Pires et al., 1999). This mural contains knowledge already available in the literature (Apprill et al., 2009; Sharp et al., 2011; Ceh et al., 2012, 2013) and includes our new data, providing this mural a very comprehensive view of coral microbiome transmission in M. hispida, potentially applicable to other coral species.

Regarding the preliminary survey of the dominant Symbiodinium clades diversity, according to Picciani-de-Souza et al. (2016), M. hispida colonies from Bahia, Brazil, shelter only Symbiodinium belonging to clade C, while possessing clades A and B at other locations along the Brazilian coast. In contrast, only clades B, C, and G were found in our parental colonies, which suggests that this coral can acquire new clades or have them in lower abundance under normal field conditions, including the rare association with clade G (commonly found in Foraminifera hosts (Pochon et al., 2001; Coffroth and Santos, 2005). However, when conditions change (i.e., aquarium confinement or pre-spawning event), the abundance of less abundant clades can be enhanced or simply detected by PCR, a technique that detects mostly dominant sequences. Additionally, although Symbiodinium clades are transferred vertically, clade selection seems to occurs early, since only clade B was found in the embryonic and planula larvae stages. Abrego et al. (2009) demonstrated the presence of only Symbiodinium of clade C in parental colonies of acroporid corals, whereas only Symbiodinium of clades A or D were found in the larvae, though the mechanisms behind these associations remain unknown. Our data corroborate the findings of Padilla-Gamiño et al. (2012), demonstrating that Symbiodinium can be transmitted vertically, even in coral species where fertilization of gametes and larval development occur in seawater. Boulotte et al. (2016) demonstrated that Symbiodinium cells can also be acquired from the environment by adult corals, and Hester et al. (2016) indicated the presence of a stable core microbiome comparing different coral species and locations but also demonstrated that coral–microbiome associations are not exclusive and therefore could not be considered heritable as a unit. Altogether, these results reinforce the idea that the mechanisms for coral–microbial symbiosis could be more flexible and dynamic than first thought (Boulotte et al., 2016).

Not only are the symbiotic mechanisms not yet fully understood, but there is also a great paucity of knowledge on the potential beneficial traits of microbial holobiont members that are vertically or horizontally transferred during spawning and this remains an important issue for ongoing studies. Antimicrobial activity of symbiont microbes may serve as a protective function for coral spawned gametes or larvae. As noted by Marquis et al. (2005), Montipora digitata seeds its eggs inside bundles with Symbiodinium, which might suggest a potentially protective role for Symbiodinium-derived metabolites. A recent study of Lin et al. (2015) found biochemical complementarity between the genomes of the symbiont and the host, which is indicative of coevolution. These data seem to underpin the need to guarantee the transfer of the microbiota, even if complementary strategies of transmission are needed. Most studies have indicated Roseobacter and Alteromonas as genera that occur in the early stages of coral life (Sharp et al., 2011; Ceh et al., 2012, 2013). However, we found here that at least six genera are associated with these stages, with Burkholderia sp. being the most abundant genus (approximately 75–85%). Recent studies have suggested that members of this genus are also vertically transmitted in plants (Sphagnum spp.) (Bragina et al., 2013). Several Burkholderia species are capable of fixing nitrogen and degrading aromatic compounds (Suárez-Moreno et al., 2012), whereas others strongly interact with fungi and/or cold-adapted plants. The presence of N-fixing bacteria was previously observed in the initial stages of coral life (Lema et al., 2014). Thus, bacteria can potentially benefit the coral host, since these features seem to be important to coral symbionts (Santos et al., 2014) and to the protection of coral from environmental impact (Santos et al., 2015).

Some studies have described a Pseudomonas sp. isolated from corals with antimicrobial and anti-pathogenic potentials, which could be important to regulate the presence of some microorganism or prevent the establishment of pathogens during early stages of coral life (Radjasa et al., 2007; Bakkiyaraj et al., 2013). Members of the genus Pseudomonas are also genetically very plastic, including different abilities that are important to corals, such us nitrogen fixation, pollutant degradation, use of different carbon sources to provide nutrients and production of antibiotics (Garcia-Valdes et al., 1988; Das and Mukherjee, 2007; Rojo, 2010). These features seem to provide clues about the importance of this genus being transmitted to coral offspring.

Corals from the Southwestern Atlantic are located in waters with higher sedimentation rates compared to those of the Pacific Ocean (Ricardo et al., 2015). Suspended sediment can represent a risk to the reproductive processes of corals because high sediment concentrations in suspension are capable of removing sperm from the seawater surface during reproduction events (Ricardo et al., 2015). This would decrease the contact time between the male and female gametes and might compromise recruitment rates and, in turn, have a negative population effect (Ricardo et al., 2015). We believe that corals from the Brazilian coast have developed specific strategies to live and survive in turbid waters. In its embryonic and planula larvae stages, the coral M. hispida, besides being able to transfer Symbiodinium cells to its offspring, also hosts bacterial genera that have not yet been described as part of the microbiome of other coral species.

The specific microorganisms transmitted by coral from generation to generation are assumed to be key to the sustenance of the coral life. It is likely that such transmission reflects some level of selection and influences host fitness. The HTE hypothesizes that the holobiont is the unit of selection in evolution and that any genetic variation in the holobiont, occurring in the host and/or in the microbial symbiont genomes, in cases of selective advantage is successfully transmitted to the offspring. This theory places as much emphasis on cooperation as it does on competition according to the principle of ‘survival of the fittest’ or as a result of stochastic events (Rosenberg et al., 2009). Even though our data indicate VT in M. hispida, we agree that this is not enough to assume co-evolution, as stated by other authors, including Hester et al. (2016), although it can be considered as additional evidence. More data are required to indicate whether the stable transmitted populations and the hosts are indeed units of natural selection or symbiotic assemblages of independently evolving organisms. The organisms that constitute the holobiont need to fit together in a tight relationship (including the vertical microbiome transmission demonstrated here in M. hispida) so that the holobiont adapts to the environment.

The spawner coral M. hispida can transfer its bacterial and Symbiodinium cells to its offspring through VT. The core bacterial populations found in the bundles, planula larvae and parental colonies were identified as members of the genera Burkholderia, Pseudomonas, Acinetobacter, Ralstonia, Inquilinus, and Bacillus. The abundance of these populations was shown to be dynamic and related to coral life stages. While the stable microbiome represents a greater proportion of the associated bacteria in embryonic and planula larvae stages, the core microbiome represents a low proportion in the adult colonies. This result suggests that M. hispida has parallel and different strategies for its microbiome acquisition. We hypothesized that coral offspring can be partly post-spawning colonized through the uptake of microbial associates released by the parental colony into the surrounding seawater and partly colonized by parental colonies, which transfer their bacterial core and Symbiodinium cells by seeding the mucus and the gametes, respectively. Being dynamic, the coral holobiont may incorporate part of its associated microbiota from the water column every time an adaptation to new environmental conditions is required, and this plasticity and capacity to modulate the microbiome uptake could be an important strategy of corals that could explain their billions of years ancient existence and persistence.

Study conception and design: RP, AR, DL, HS, CC, DP. Acquisition and identification of coral samples: DL, CC, DP. Acquisition of data (experimental development): DL, PL, UL, AG, CZ. Analyses and interpretation of data: RP, AR, DL, PL, UL, AG, JvE, CZ, CC, DP. Drafting of the manuscript: RP, DL. Critical revision: all authors. Financial support: RP, AR, UL, CC, CZ.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.