**1. Introduction**

Coral reefs are both among the most biodiverse of marine habitats and among the most productive ecosystems on Earth [1]. However, most of our information 'about them is based on our knowledge of shallow reefs (<30 m depth). Light-dependent reefs can thrive in deeper waters though. Mesophotic coral ecosystems (MCEs) are light-dependent coral reefs found typically from 30 to 150 m in tropical and subtropical regions [2]. Hereinafter, MCEs are also referred to as 'mesophotic reefs' for the ease of the reader, a phrase considered analogous with the term 'shallow reefs'. Mesophotic reefs have been estimated to represent about 80% of potential coral reef habitat by area worldwide; however, we know little about them compared to shallow reefs [3]. Scleractinians (hard corals) are present in MCEs, but to a much lesser extent than in shallow reefs [3–5]. Instead, antipatharians, octocorals, sponges and macroalgae provide most of the available habitat structure at these depths [3–5].

**Citation:** Gress, E.; Eeckhaut, I.; Godefroid, M.; Dubois, P.; Richir, J.; Terrana, L. Investigation into the Presence of *Symbiodiniaceae* in Antipatharians (*Black Corals*). *Oceans* **2021**, *2*, 772–784. https://doi.org/ 10.3390/oceans2040044

Academic Editors: Rupert Ormond, Peter Schupp, Ronald Osinga and Michael W. Lomas

Received: 29 October 2020 Accepted: 18 November 2021 Published: 25 November 2021

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The order Antipatharia (subclass Hexacorallia) consists of seven families and around 270 species [6,7]. These corals occur in most oceans at depths ranging from 2 to 8900 m, generally favouring strong currents and low-light environments [8,9]. Antipatharians do not produce a calcium carbonate skeleton; instead, the thorny axial skeleton is composed of a proteinaceous complex called antipathin [10,11]. It has been suggested that antipatharians tend to increase in diversity and abundance with depth, reaching a peak at mesophotic depths (30–150 m) [12]. Nonetheless, dense aggregations of antipatharians have also been observed in shallow (<30 m depth) clear water environments [13,14], Gress pers. obs. Further, they are important habitat-providing corals with which an array of marine fauna associates [9,13,15–18]. For instance, in the Philippines, the average density of invertebrate macrofauna associated with antipatharians ranged from 82 to 8313 individuals m−<sup>2</sup> [14]. Thus, antipatharians can be considered ecosystem engineers supporting and enhancing marine biodiversity at shallow, mesophotic and deep-sea depths.

However, limited studies have evaluated their current condition under increasing threats, such as those associated with global warming. In the closely related Hexacorallian order of scleractinia, global warming has led to extensive mortality due to temperaturerelated coral bleaching. Bleaching is the consequence of a disturbance of the symbiotic relationship between dinoflagellates and scleractinians on both shallow and mesophotic reefs as a result of climate change—a causal relationship that is well established [19–22]. Nevertheless, our understanding of the physiological mechanisms underlying the endosymbiotic association between dinoflagellates and their cnidarian host has been frequently revised, and this relationship is still not fully understood. It now appears that four of the seven genera of Symbiodiniaceae are found in symbioses with scleractinian corals (*Symbiodinium*, *Breviolum*, *Cladocopium* and *Durusdinium*) [23,24]. The translocation of photosynthetically fixed carbon from the symbiont to the host is considered the best-known aspect of the coral–algae symbiosis. However, the amount of photosynthetic carbon translocated to the host and the identity of the compounds are not fully known [25].

In the coral–algae symbiotic association, Symbiodiniaceae harvest sunlight for photosynthesis and dissipate excess energy so as to prevent light-induced oxidative stress [26–28]. Under ambient conditions (i.e., not heat and light-stressed), Symbiodiniaceae absorbs light that can be (1) used to drive photochemistry, (2) re-emitted as fluorescence, (3) dissipated as heat or (4) decayed via the chlorophyll triplet state [26–29]. Experiments have shown that Symbiodiniaceae in scleractinians, under typical irradiances at shallow coral reefs (640 μmol photons m−<sup>2</sup> s<sup>−</sup>1), dissipate 96% of the energy and use only 4% of the absorbed light energy for photosynthesis [26]. However, over prolonged periods of water temperature alterations, the invertebrate host needs to lower the number of its symbionts because the oxidative stress, which results from the production and accumulation of reactive oxygen species (ROS), can damage lipids, proteins and DNA [27,30]. When corals reduce the number of their symbionts, the main source of ROS production is removed, although the coral host itself may also produce ROS as a result of light and temperature [27]. Oxidative stress on corals results in a lack or low number of dinoflagellates and their photosynthetic pigments, an effect known as 'coral bleaching'. Three potential mechanisms have been suggested for the regulation of symbiont numbers under such stressful conditions: (i) expulsion of excess symbionts; (ii) degradation of symbionts by host cells; and (iii) inhibition of symbiont cell growth and division controlled by the pH of the host cell [25,31].

The earliest suggestion of dinoflagellates being present in antipatharian tissues comes from the *Report on the Antipatharia collected by H.M.S. Challenger* by Brook [32]. A few years later, in a report on *The Antipatharia of the Siboga Expedition*, van Pesch [33] documented six species containing what he referred to as 'symbiotic Algae' ranging from 7–10 μm in diameter present in the gastrodermis. Significantly, he reported observing these cells in only six out of the thirty species he examined [33]). With no more empirical studies or reports for many decades and given antipatharians ability to thrive at abyssal depths and in low-light environments, they were assumed to lack Symbiodiniaceae, which is commonly referred to as 'being azooxanthellate' [8,34]. Moreover, a few later reports using molecular techniques did not find dinoflagellates in the antipatharian species examined. For instance, after intense morphological studies, dinoflagellates were reported absent in *Antipathes grandis* VERRILL, 1928, from Hawaii [35]. Likewise, dinoflagellate-specific primers and spectrophotometric methods that detect dinoflagellate chlorophyll absorbance patterns did not reveal any found microalgae in the species *Stichopathes luetkeni* (BROOK, 1889) (formerly called *Cirrhipathes lutkeni*) [36].

In contrast, in accordance with the early historical suggestions, two more recent studies have confirmed the presence of Symbiodiniaceae in various antipatharian species. A histological analysis of 14 antipatharian species collected from a depth between 10 and 396 m from Hawaii and Johnston Atoll revealed low densities (0–92 cells mm−3) of Symbiodiniaceae cells inside antipatharian gastrodermal tissues, suggesting that the dinoflagellates are endosymbiotic [34]. Additionally, dinoflagellates sequences retrieved from the antipatharians confirmed the presence of Symbiodiniaceae in the genera *Cladocopium*, *Gerakladium* and *Durusdinium*. However, it was concluded that the endosymbiotic dinoflagellates had no significant role in the 'nutrition' of the species examined and suggested more research to determine whether the association might be parasitic [34]. The conclusion was based on the low density of microalgae cells within the antipatharians and their presence in colonies at depths where light penetration does not enable photosynthesis. They did not find any pattern in the types of Symbiodiniaceae present in the different antipatharian species; therefore, they suggested that endosymbiont acquisition might occur opportunistically and not be host-specific. In another recent study conducted on a single species of the genus *Cirrhipathes* from Indonesia, two colonies were sampled at 38 m and one at 15 m and evidence of abundant (~107 cells cm–2) Symbiodiniaceae cells in the gastrodermis of the corals was found [37]. Among these, the authors identified two genera—*Cladocopium* and *Gerakladium*—the latter commonly found in association with clinoid sponges. They concluded that a mutualistic endosymbiosis existed based on the presence of the dinoflagellates inside the antipatharian gastrodermis and a symbiosome surrounding the microalgal cell, combined with evidence of its division inside the host [37]. These findings led us to reconsider our view of the vulnerability of antipatharians to global change and prompted us to further investigate the occurrence of dinoflagellate symbionts in antipatharian species.

Most lineages in the subclass Hexacorallia are believed to have evolved photosymbioses independently [38], with the order Antipatharia being one of the exceptions until evidence of antipatharian species hosting dinoflagellates inside the coral gastrodermis was found [34,37]. However, these two studies presented two contrasting conclusions. In one case, it was concluded that the presence of the Symbiodiniaceae was opportunistic, and in the other, it was concluded that a mutualistic endosymbiosis existed. Those conclusions were based on the presence and abundance of the Symbiodiniaceae cells and their location in the host tissue, although from a very limited number of specimens. The present study was therefore undertaken with the objective of gaining further insight into the possibility of antipatharians hosting high abundances of dinoflagellates, expanding the geographic range of the species studied and the number of colonies examined. We used an integrated methodological approach, combining both microscopy and molecular techniques to investigate the presence, abundance, location and identity of Symbiodiniaceae in two antipatharian species—*Cupressopathes abies* (LINNAEUS, 1758) and *Stichopathes maldivensis* COOPER, 1903. Our samples represent two different morphologies and were collected from both shallow and mesophotic reefs in SW Madagascar.
