Beyond the Symbiodiniaceae: diversity and role of microeukaryotic coral symbionts

Many corals form intimate symbioses with photosynthetic dinoflagellates in the family Symbiodiniaceae. These symbioses have been deeply studied, particularly in reef-forming corals. The complex microbial community that is associated with corals contains other members that have also been well characterized such as bacteria. However, our understanding of the coral holobiont and subsequently coral reef ecosystems is not complete if we do not take into consideration the microeukaryotes like protists and fungi. Microeukaryotes are currently the greatest enigma within the coral microbiome. Only a handful of them have been characterized, very few have been cultured and even less have genomes available. This is a reflection of a smaller community of scientists working on this particular group of organisms when compared with bacteria or Symbiodiniaceae, but also of the many technical challenges that we face when trying to study microeukaryotes. Recent advances in the use of metabarcoding are revealing the importance of microeukaryotes in corals in terms of abundance and presence, with notable examples being the green algae Ostreobium and the apicomplexans Corallicolidae. We believe that it is timely and necessary to present what we know so far about coral microeukaryotes before the expected flow of high-throughput metabarcoding studies exploring the microeukaryotic fraction of the coral microbiome.


Introduction
Corals reefs support hyper-diverse ecosystems and build natural offshore structures that protect our coastlines from storm surge and coastal inundation (Reaka-Kudla et al. 1997;Harris et al. 2018). The corals that build these structures are able to do so by forming intimate symbioses with dinoflagellates in the family Symbiodiniaceae, which provide photosynthetically fixed carbon to the coral host as an energy source (Schlichter et al. 1995). Apart from their photosynthetic symbiotic algae, corals are known to host a complex microbial community. The coral and all its microbial partners are known as the coral holobiont (Rohwer et al. 2002). Some elements of the holobiont have been studied in detail such as the aforementioned Symbiodiniaceae or bacteria. However, the microeukaryotes (microscopic fungi and protists) associated with corals have been less studied, limiting our capacity to fully understand the coral holobiont and subsequently the coral reef ecosystem (Ainsworth et al. 2017). The most prevalent approaches to study the holobiont are 'molecular techniques', principally those based on DNA and RNA sequencing. Among those methods, metabarcoding is the most common one used to explore the biodiversity within the holobiont (Rohwer et al. 2002). Even today 1 3 microeukaryotes tend to be an overlooked, if not forgotten, part of most microbiome studies. This largely reflects the challenges that culturing microeukaryotes presents as compared to bacteria and also the technical limitations in the use of metabarcoding approaches that preclude an extensive exploration of the coral microeukaryote community del Campo et al. 2020).
Identifying and assigning roles or functions to all microbial components of coral microbiomes, from viruses to microeukaryotes, is key for the understanding of coral biology (van Oppen and Blackall 2019). Here, we will summarize the diversity of microeukaryotes (beyond Symbiodiniaceae) that have been identified in association with corals so far, mostly through isolation and microscopical characterization (Fig. 1). As well, we propose solutions to the limitations we currently have to exploring this particular component of the coral holobiont.

Phototrophic microeukaryotes
The Symbiodiniaceae dinoflagellates are the best studied microbial members of the coral holobiont. That said, they have only recently been characterized properly and described to the species level (LaJeunesse et al. 2018). However, Symbiodiniaceae are not the only algae known to be associated with corals. Algae are key players in coral reef ecosystems. For example in temperate reefs, calcifying algae are dominant instead of reef building corals (Ballesteros 2006). As well, algae compete for space and resources with the corals in tropical reefs, and since both are sessile they are in permanent competition and biochemical "warfare" (Roach et al. 2020). As a result, most algae other than Symbiodiniaceae are commonly considered coral foes, competitors, or bioeroders of coral skeletons. In recent years, however, some of these relationships have been re-evaluated, in particular green and red algae, which are most commonly reported in direct association with corals.

Chlorophyta
The chlorophytes, or green algae, are photosynthetic eukaryotes with double membrane-bound plastids containing chlorophyll a and b. They are ubiquitous in aquatic habitats and have a variety of morphologies and lifestyles ranging from free-swimming unicellular forms (e.g., Chlamydomonas or Micromonas) to macroscopic benthic forms (e.g., Ulva or Caulerpa) (Lewis and McCourt 2004). The chlorophyte lineage is typically split into four classes: the Chlorophyceae and Trebouxiophyceae are both well-supported clades by molecular phylogenies, Fig. 1 Eukaryotic tree showing the groups that contain microeukaryotes that have been isolated from corals (purple dots). Some examples of these organisms are presented above the tree. Tree based on EukProt (Richter et al. 2022) while the Prasinophyceae and Ulvophyceae are traditional classes which are not supported as monophyletic clades in molecular phylogenies. The Ulvophyceae are very diverse (Leliaert et al. 2012) and stand out because they include many microbial members but are more typically found as large, macroscopic forms. Ostreobium sp. (Bryopsidales, Ulvophyceae) have been shown to be a pervasive member of the endolithic community of coral (Tandon et al. 2022). This makes them unique among the chlorophytes in that they thrive in the low-light and variable pH and O 2 environment of the coral skeleton where they are often seen as a visible green ring just below the coral tissue. They have also been shown to interact with prokaryotes like the cyanobacteria Halomicronema (Williams et al. 2015). Ostreobium are capable of exchanging nitrogen and carbon with the coral and are most likely horizontally transmitted from adult to recruit (Massé et al. 2018). Genomic evidence now suggests that Ostreobium supplements their carbon needs by feeding on the organic skeletal matrix of the coral host while relying upon other (likely bacterial) holobiont members for vitamin B12 (Iha et al. 2021). Considering it shares similar biogeographical patterns and distinct subclasses with host-species preference, like Symbiodiniaceae, Ostreobium has been hypothesized to have co-evolved with corals and Symbiodiniaceae ). However, whether Ostreobium are indeed beneficial to the coral or a parasite of the coral remains to be confirmed (Verbruggen and Tribollet 2011). The former seems most likely given it absorbs light at a wavelength not used by Symbiodiniaceae and provides photosynthates to the coral (Schlichter et al. 1995). It has even been suggested that Ostreobium can replace the Symbiodiniaceae function during bleaching events and temporarily sustain the coral prior to a possible return of the Symbiodiniaceae (Fine and Loya 2002). Notably, recent metatranscriptomics work has shown Ostreobium blooms to become the dominant oxygenic phototroph of the coral holobiont during bleaching (Iha et al. 2021). As well, according to the most recent research on Ostreobium, the endolithic algae can contribute to coral recovery after bleaching when it blooms near the skeletal surface, alleviating the high light stress of the bleached corals by reducing skeleton reflectance (Galindo-Martínez et al. 2022). Besides Ostreobium, a few other chlorophytes have been recovered from coral holobionts, including Phaeophila sp. (Phaeophilales, Ulvophyceae), Ochlochaete sp. (Ulvales, Ulvophyceae), Ulvella sp. (Ulvales, Ulvophyceae), and several unidentified Cladophorales (Marcelino and Verbruggen 2016). Phaeophila is a known microborer, while Ulvella has been associated with pathogenesis in some corals (Goldberg et al. 1984). Despite these advances in our knowledge, the full diversity of chlorophytes and their potential role in the coral holobiont is still unclear.

Rhodophytes
The Rhodophyta, or red algae, are a highly diverse group of eukaryotic algae which cells lack flagella and centrioles. Their chloroplasts contain phycobilins which give them their characteristic red color; they also contain other pigments such as chlorophyll a (Yoon et al. 2006). Although they primarily live in marine environments, ranging from deep oceanic depths to shallow intertidal zones, they are known to flourish in freshwater and on land (Yoon et al. 2016). Remarkably the plastids of apicomplexans and dinoflagellates, both of which have tight associations with cnidarians, originated from a common red algal endosymbiont (Janouškovec et al. 2010)-a fact alone that seals the importance of the red algae to the health of coral reefs. Rhodophytes are ecologically important in their own right as primary producers and habitat structurers. Today, the best known rhodophytes on the reef are the crustose coralline algae (CCA). Certain CCA have been identified as primary inducers of metamorphosis for coral larvae (Morse et al. 1988). Some species of CCA that have been shown to induce metamorphosis include Lithophyllum insispidum, Hydrolithon onkodes, Neogoniolithon brassica, Mesophyllum sp., and Penyssonnelia sp. (Heyward and Negri 1999). However, the relationship is not always so straight forward and coral larvae face a battle with the CCA and try not to be overgrown during the first few weeks of development (Craggs et al. 2019). Like corals, the CCA appear to have their own distinct microbiome with specific bacteria that reside within the algae and appear to be paramount to the facilitation of metamorphosis in the coral larvae (Webster et al. 2004). Beyond their role in coral settlement, rhodophytes have been found in abundance within the coral's microbiome. Similar to Ostreobium they often reside in the coral skeleton. This association was first identified in 1995, when Le Campion-Alsumard and colleagues highlighted conchocelis life stages of rhodophytes within corals. They noted how the presence of the conchocelis filaments appeared to exclude other endolithic algae in the skeleton, such as Ostreobium (Le Campion-Alsumard et al. 1995). Red algae within coral microbiomes were then mostly overlooked until 2016, when Pica et al. identified the conchocelis stage of the rhodophyte Porphyra to be the most abundant endolith within stylasterid hydrozoans (Pica et al. 2016). Stylasterids are tropical azooxanthellate lace corals with unusual coloration ranging from red to purple on the photophilic portions of the colony. When researchers dug a little deeper, they found that the calcareous skeletons of stylasterids are most likely invaded and eroded by the conchocelis stages of bangialean rhodophytes, such as Porphyra marcosii and Pyropia vietnamensis . Whether these rhodophytes confer any advantage to the coral host remains to be uncovered.

Heterotrophic microeukaryotes
Heterotrophic protists and fungi have historically been associated with coral disease. The most notorious of these would be the fungi Aspergillus sydowii which for a long time was thought to be responsible for the massive Caribbean sea fan mortalities at the end of the nineties . Other well-known microeukaryotic pathogens of corals are the ciliate Halofolliculina corallasia which is responsible for eroding band syndrome (AKA Caribbean ciliate infection) and Philaster guamensis and Philaster lucinda which are involved in the pathogenesis of white syndrome, white band disease and brown band disease (Sweet and Séré 2016). As the focus has primarily been on disease, the general impression is that most heterotrophic microeukaryotes associated with corals are pathogenic, but less targeted approaches have led to a more complicated picture. Certain protists that could be easily suspected of being parasites, such as apicomplexans, are now known to be widespread across both coral geographic distribution and taxonomic diversity, and are present in apparently healthy coral colonies while not being associated with any disease (Kwong et al. 2019). In addition, molecular sampling of the diversity of eukaryotes in coral communities shows a broad range of heterotrophic eukaryotes associated with corals, interestingly, many of which are alveolates (Brener-Raffalli et al. 2018;del Campo et al. 2019).

Apicomplexans and related lineages
Apicomplexans are a large group of parasitic alveolates that cause devastating human diseases such as malaria. These parasites evolved from photosynthetic ancestors and retained a relict non-photosynthetic plastid. Some close relatives, called chromerids, are still photosynthetic, and the only two chromerids that have been formally described, Chromera velia and Vitrella brassicaformis, were both isolated from coral reefs (Moore et al. 2008;Oborník et al. 2012). That the photosynthetic relatives of apicomplexans are somehow linked to coral reefs has prompted a major re-evaluation of the ecological conditions and symbiotic associations that drove the evolution of parasitism Woo et al. 2015). However, the chromerids relationship with corals has been disputed. Chromerids appear to be residents of the biogenous sediment of coral reefs rather than part of the coral holobiont and perhaps only live with or in the actual coral for limited periods of time (Mathur et al. 2018). Historically, corals have also not been considered a common host for apicomplexans: sporadic reports in the last 30 yrs include a morphological description of a single coccidian, Gemmocystis cylindrus (Upton and Peters 1986), the detection of apicomplexan 18S rRNA gene sequences (called "Type-N") (Toller et al. 2002;Šlapeta and Linares 2013), and plastid 16S rRNA gene of uncharacterized Apicomplexan-Related Lineages (ARLs) (Janouškovec et al. 2012(Janouškovec et al. , 2013. In a recent study, Type-N and ARL-V were proven to be the same group of organisms, and structurally similar to G. cylindricus (Kwong et al. 2019). These symbionts, informally known as corallicolids, are widespread across corals (in some cases as abundant as Symbiodiniaceae), and although the corallicolid plastid is non-photosynthetic, it nevertheless retains genes for chlorophyll biosynthesis (Kwong et al. 2019). Electron microscopy further confirmed that these symbionts live intracellularly within the gastrodermis of coral hosts, just like Symbiodiniaceae. In 2021, the Corallicolida order was officially designated with Corallicola aquarius as the type species. All members of the clade, which now includes the reassigned G. cylindrus along with Anthozoaphila gnarlus, are exclusively found in association with Anthozoans (Kwong et al. 2021). The ubiquity of these general anthozoan symbionts is further emphasized by the finding that corallicolids can be associated with coral hosts at depths of 1400 m (Vohsen et al. 2020). Corallicolids and chromerids are promising candidates in which to study the evolutionary transition to parasitism in apicomplexans and how the loss of photosynthesis affected this process.

Fungi
The kingdom Fungi encompasses diverse forms of saprotrophs, including both unicellular and multicellular taxa that can reproduce both sexually (spores) and asexually. Fungi present a cell wall made of β-glucan and usually chitin. The majority of aquatic fungi belong to two phyla, Ascomycota and Chytridiomycota, which are known to play critical functional roles within their ecosystems, for example infecting and consuming normally non-digestible phytoplankton (e.g., colonial diatoms), contributing organic matter to the microbial loop that would otherwise sink (Grossart and Rojas-Jimenez 2016). Fungi have been observed in association with corals, and are suggested to have both beneficial and saprophytic symbioses (Paulino et al. 2020). Beneficial ecological roles of the mycobiome (collective term of the fungal associates and their host) are thought to surround the heterotrophic conversion of reef biomass to nutrients (Morrison-Gardiner 2002). Saprophytic relationships are best known with the yeast Aspergillus sydowii, the causative agent of the Caribbean Sea fan disease (Smith et al. 1996;Alker et al. 2001;Troeger et al. 2014). Aspergillus has also been shown to be present in apparently healthy coral, suggesting that the 'pathogenic' nature of this fungi might not be fully understood. Similarly, fungi including Cladosporium and Fusarium for example have been linked via both histological evidence and ITS sequences to a newly described disease in Micronesia -named gray patch disease (Sweet et al. 2013. Another fungal genus, Rhytisma, has been associated with dark spot syndrome in Caribbean corals (Sweet et al. 2013). That said, similar to Aspergillus, confirmation of pathogenesis is lacking for all these examples. Regardless of the exact role these fungi are playing in the coral holobiont, we do know that many boring endolithic fungi are associated with the coral skeleton (Golubic et al. 2005). However, it should be noted that although sequencing has highlighted the presence of these fungi only relatively few studies have attempted to culture the fungi and described those obtained to the species level (Kendrick et al. 1982;Bak and Laane 1987;Ravindran et al. 2001). Even though ITS-based analysis is a straightforward option for the characterization of fungi in corals (Schoch et al. 2012), there are issues with the sequencing specifically with regard to cross-amplification of the host and fungal cells-which likely underestimates the fungal diversity in any given sample (Amend et al. 2012;Bonthond et al. 2018;Góes-Neto et al. 2020;Paulino et al. 2020).

Ciliates
Ciliates are unicellular heterotrophic alveolates that inhabit nearly all environments on Earth, most as free-living organisms, but also including many that specialize as ecto/endosymbionts of an animal host (Foissner et al. 2008). Freeliving ciliates have even been suggested to play critical roles in the likes of marine trophic dynamics for example, linking primary producers and consumers (Gifford 1991). Characterized by their relatively complex body plan and the presence of hair-like organelles called cilia, these organisms have a wide range of morphologies that vary in sizes from 10 µm to a few millimeters (Foissner et al. 2008). Ciliates have been found to form associations with a wide variety of hosts, in many cases apparently pathogenic or opportunistic feeders on host tissues during pathogenesis (Sniezek et al. 1995;Poynton et al. 2001;Song et al. 2009). While there are a variety of ciliates observed in association with corals (Sweet and Séré 2016), they are rarely seen in healthy individuals. Instead, most ciliates are either associated with disease or found at the front of advancing lesions in diseased specimen (Katz et al. 2014;Sweet and Séré 2016), such as Caribbean ciliate infection (Cróquer et al. 2006a(Cróquer et al. , 2006bRodríguez et al. 2009;Sweet et al. 2014;Sweet and Séré 2016;Montano et al. 2020), white band disease (Sweet et al. 2014), white plague disease (Randall et al. 2015), white syndrome Bythell 2012, 2015), skeletal eroding band disease (Antonius 1999;Antonius and Lipscomb 2001;Anthony et al. 2008;Montano et al. 2020), or brown band disease Katz et al. 2014;Randall et al. 2015), and brown jelly syndrome Randall et al. 2015). Unfortunately, many early studies lack molecular surveys, a critical method to recover the diversity of the ciliate community. That said, the more recent work has described a number of ciliates present in corals and on a global scale (Sweet and Séré 2016). Ciliates of particular interest that have been observed in association with coral disease are the folliculinids (Cróquer et al. 2006b) and the scuticociliates . Halofolliculina corallasia was the first protistan pathogen identified in corals and is responsible for skeletal eroding band syndrome (Antonius and Lipscomb 2001). The story with the scuticociliates is also complicated and members of the Philaster genus have a global reach like Halofolliculina Bythell 2012, 2015;Sweet et al. 2014;Sweet and Séré 2016). However, confusion arises when these ciliates are discussed as they were originally morphologically described as members of the genera Porpostoma or Helicostoma . The combination of metabarcoding and the construction of phylogenetic trees will help us to avoid this kind of confusion and to improve our knowledge on the ciliate diversity associated with corals.

Labyrinthulomycetes
Members of this group of ubiquitous and diverse unicellular stramenopiles are characterized by their production of an ectoplasmic network: a branched membrane network secreted through a unique organelle called the bothrosome (sagenogenetosome) that is involved in saprotrophic nutrient uptake (Bennett et al. 2017). Labyrinthulomycetes are found in diverse habitats, including marine and freshwater, and from the euphotic zone to the deep-sea. They have also been isolated from various substrates, including but not limited to algae, mangrove leaves, seagrass, mollusks and of course coral (Pan et al. 2017). The production of high levels of omega-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has made Labyrinthulomycetes commercially valuable (Carmona et al. 2003;Lee Chang et al. 2012). In some cases, labyrinthulids have been described as parasites, specifically in clams, nudibranchs and seagrass (McLean and Porter 1982;Ragone Calvo et al. 1998;Ragan et al. 2000;Muehlstein et al. 2018). Five Labyrinthulomycetes species have been isolated from corals (Raghukumar and Raghukumar 1991;Ben-Dov et al. 2009;Siboni et al. 2010), and diverse members have also been reported to be widely associated with the coral Fungia sp., but the nature of their interactions has not been elucidated (Kramarsky-Winter et al. 2006;Ben-Dov et al. 2009;Siboni et al. 2010). Additionally, there has been a report proposing that labyrinthulids play a pathogenic role in the multifocal purple spot disease (MFPS) that affects Caribbean sea fans. However, their causal role is doubtful due to their presence in healthy tissues as well (Burge et al. 2012;Dennis et al. 2020). There is still a debate as to the differences between MFPS disease and the other known sea fan disease, aspergillosis, albeit recent MFPS disease histology shows numerous opportunistic organisms in association with MFPS disease (Dennis et al. 2020), suggesting a microbial differentiation throughout pathology compared to aspergillosis.

Syndiniales
The Syndiniales, also called Marine Alveolate (MALV) Groups I-V, are a diverse collection of parasitic dinoflagellates that are known to infect a vast diversity of marine organisms, including metazoans, radiolarians, ciliates, and even other dinoflagellates. The Syndiniales kill their host resulting in the release of free-living parasitic dinospores that can last up to three days in the environment (Guillou et al. 2008). The diversity of Syndiniales within coral reef environments has not been fully explored, but a few studies have noted their presence in reef plankton, sediments, and the coral themselves Qiu et al. 2021). Clerissi and colleagues discovered that a subgroup of Syndiniales (Group I) were one of the four main microeukaryotic taxa found within the cauliflower coral, Pocillopora damicornis. Other Group I species include Ichtyodinium, an endoparasite of fish embryos, and Duboscquella, a parasite of ciliates (Harada et al. 2007). Given their huge diversity in marine environments and their cosmopolitan range of known host organisms, Syndiniales represent a major lineage in need of additional exploration in reef environments, and in particular more data linking Syndiniales known only from environmental sequencing to their hosts to see which members of the reef environment are infected. The widespread parasitism of this clade, along with their accompanying ability to exert top-down control on host populations, suggests an important ecological role in coral holobionts.

Other dinoflagellates
Outside of the Syndiniales and Symbiodiniaceae, there are other dinoflagellates with potential roles in the coral holobiont. Alexandrium spp., Gymnodinium catenatum, and Prorocentrum lima have all been found in coral samples with signs of black band disease (BBD). Furthermore, Alexandrium and Gymnodinium are known to produce paralytic shellfish toxins (Hold et al. 2001;Green et al. 2004;Sala et al. 2005). While not described as etiological agents in BBD, the disease is characterized as being caused by a microbial mat consortium and it may well be influenced by these dinoflagellates presence (Sekar et al. 2008). There is also indirect evidence that dinoflagellates may play a role in Stony Coral Tissue Loss Disease-a coral disease rampaging through the Caribbean reefs. Indeed one of the bacteria named as a possible causal agent is known to be isolated from the ichtyopathogenic dinoflagellate Cochlodinium polykrikoides (Kim et al. 1999). Further evidence of the role of algae in coral disease stems from early reports back in 1985 where blooms of Margalefidinium polykrikoides and Gonyaulax monilata in Panama caused 100% mortality of surveyed corals (López-Cortés et al. 2019). However, some dinoflagellates do certainly appear to offer benefits to the host like their famous Symbiodiniaceae cousins. Taxa from the genus Gyrodinium for example have been recovered from corals and these species are known to produce mycosporine-like amino acids (MAAs). MAAs function as a UV-B absorbing compound (Klisch et al. 2001) and may therefore assist corals by reducing damage from UV (Gleason 1993). The genus Blastodinium has also been reported in corals (Sebens et al. 1996). Members of this genus are well known for parasitizing copepods for example (Skovgaard et al. 2012). We do not know if they play a similar role in the coral host or if they are only observed because the coral feeds on parasitized copepods. Similarly, Amphidinium sp. is another member from the family Gymnodiniaceae that has been recovered from corals; however, this taxa is also commonly observed as part of the coral diet (Sorokin 1973).

Molecular approaches to unveil the coral eukaryome
The majority of studies focused on coral-associated microeukaryote diversity have centered around disease dynamics with researchers isolating and identify associated microbes via microscopy. This low-throughput approach of microscopy and culturing is by necessity targeted and, as a result, relatively narrow in scope. It was not until the use of non-targeted, culture-independent approaches such as high-throughput metabarcoding (Taberlet et al. 2012) that we could retrieve from an environment not only what we might be looking for, but also everything else we did not even know was there. However, the vast majority of metabarcoding studies remain exclusively focused on prokaryotes. Some examine fungi (Cui et al. 2013), but few address the microeukaryotic community as a whole . Indeed, thousands of publications have revealed a great diversity of bacteria associated with an almost equally diverse range of hosts (McFall-Ngai et al. 2013). This includes corals, where prokaryotic diversity has been extensively sampled and analyzed (Pollock et al. 2018), but few such studies have focused on microeukaryotes (Ainsworth et al. 2017). The paucity of DNA-based studies of coral-associated microeukaryotes reflects not only research biases, but also a serious technical issue that arises because symbiotic microeukaryotes are evolutionarily related to their animal hosts, both being eukaryotes (del Campo et al. 2019). Among the barcoding genes used to infer the identity of microorganisms, the most widely used is the Small Subunit ribosomal RNA (SSU rRNA) gene, which is amplified by polymerase chain reaction (PCR) using primers that target the barcode gene. These approaches have been widely utilized to define phylogenetic diversity of free-living microeukaryotes for over 20 yrs (López-García et al. 2001;Moonvan der Staay et al. 2001), but their application to analyzing microeukaryotic components of communities associated with eukaryotic hosts (like animals) is challenging because host-derived sequences dominate the data (Parfrey et al. 2014;Wampach et al. 2017;Wilcox and Hollocher 2018). One solution to this is to develop PCR primers that amplify the SSU rRNA from wide range of eukaryotes, but exclude all or most metazoans. One such primer set developed to investigate parasites was recently shown to work on diverse microeukaryotes associated with diverse animal hosts ). More recently, improved versions of these primers have been released that will reduce the primer bias within certain eukaryotic groups and will increase the reported microeukaryotic diversity in microbiome samples Minardi et al. 2021). Other recent studies have used alternative methods to explore the eukaryome, such as blocking primers ). Both strategies have revealed novel microeukaryotes in the holobiont. Furthermore, metagenome assembled genomes can also be used and cases have already provided insights into the functional characteristics of this newly characterized diversity (Kwong et al. 2019).
The microeukaryotic component of the coral microbiome is the last major element that needs to be characterized in order to have a full picture of the holobiont. Metabarcoding and metagenomics, as well as promising methodologies to study the coral holobiont such as single-cell transcriptomics (Hu et al. 2020;Levy et al. 2021), will all play a crucial role in revealing the coral holobiont microeukaryotic diversity and function. We will likely see how the image we have of protist and fungi will move from being the usual suspects in disease to common members of the healthy holobiont. As part of the holobiont, microeukaryotes may also have an impact on the dynamics of other members of the coral microbiome. So, in time, they could potentially be utilized as a tool to assist corals through a changing climate in years to come (Reshef et al. 2006;Peixoto et al. 2021).
Funding This study has been suppor ted by project PID2020-118836GA-I00 financed by MCIN/ AEI /10.13039/501100011033 and startup funds from the University of Miami, Rosenstiel School of Marine, Atmospheric and Earth Sciences. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

Conflict of interest
The authors declare no competing interests to report.
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