Building consensus around the assessment and interpretation of Symbiodiniaceae diversity

Why is species-level resolution important for Symbiodiniaceae?

Species are evolutionarily independent lineages and therefore represent a fundamental level of biological organization. Species-level resolution provides insight into the ecological and evolutionary mechanisms that create diversity, and forms the basis of comparative physiological investigations (Kareiva & Levin, 2015). The delineation of species can affect nearly all scales of inquiry, from biochemical pathways to ecosystem processes. Species-level diversity in Symbiodiniaceae has been discussed in the literature since the description of Symbiodinium microadriaticum in 1962 by Freudenthal (1962). As more diversity was uncovered and more species were recognized (LaJeunesse, 2001; LaJeunesse & Trench, 2000; Rowan & Powers, 1992, 1991; Schoenberg & Trench, 1976; Trench & Blank, 1987), controversy arose as to where to draw species boundaries (Apprill & Gates, 2007; Correa & Baker, 2009; Cunning, Glynn & Baker, 2013; LaJeunesse et al., 2014; LaJeunesse, Parkinson & Reimer, 2012; Stat et al., 2012; Thornhill, LaJeunesse & Santos, 2007; Wham & LaJeunesse, 2016). At present, there is general consensus among Symbiodiniaceae specialists about the need for species-level resolution, as well as support for current taxonomic methodologies that are underpinned by genetic, ecological, and morphological data (LaJeunesse et al., 2018; Voolstra et al., 2021b). Such taxonomic descriptions facilitate scientific communication and are necessary for establishing legal frameworks for conservation (IUCN, 2021). The recent elevation of most Symbiodiniaceae “Clades” to genera has provided some clarity (LaJeunesse et al., 2021, 2018; Nitschke et al., 2020; Pochon & LaJeunesse, 2021), but the small number of formal species-level descriptions for the large genetic diversity found within most Symbiodiniaceae genera constitutes a formidable barrier to progress.

Without robust species delineation, functional differences can inadvertently be ascribed to incorrect taxonomic levels or non-existent biological entities. For example, the genus level may be too coarse and lead to over-generalizations regarding the physiology or function of Symbiodiniaceae variants (see “Beyond Genotype: Phenotyping Symbiodiniaceae”). A statement such as “the genus Cladocopium consists of heat-sensitive species” overlooks the superior stress tolerance of some Cladocopium species, including the dominance of Cladocopium thermophilum in corals on some of the world’s hottest reefs in the Persian/Arabian Gulf (Abrego et al., 2008; Hume et al., 2015; Varasteh et al., 2018). However, diversity assessments based on gene sequence variants may recover both interspecific variation (resolving distinct species) and intraspecific variation (sequence diversity within a single genome). This is a major issue for the commonly used multi-copy ITS2 gene. Consequently, a statement such as “Symbiodiniaceae harboring the ITS2 D13 sequence variant are adapted to temperate environments” overlooks the fact that ITS2 sequence variants D8, D8–12, D12–13, and D13 are all characteristic of the same species, Durusdinium eurythalpos (LaJeunesse et al., 2014). The statement could give a false impression that entities harboring the D8 variant are phylogenetically and ecologically distinct from those harboring D13. In this scenario, because we know the ITS2 profile of D. eurythalpos, we can clarify that the four sequence variants belong to the same species. However, for many undescribed species, the profiles are not yet resolved. Such issues are problematic because they may confuse ecological interpretations of sequence data, particularly in datasets composed of communities of different symbiont species where some consist of overlapping ITS2 intragenomic variants.

What types of data can identify Symbiodiniaceae species?

Although taxonomic descriptions are fundamental, describing a new species is not the same as recognizing a new species or identifying a known species. Describing should be based on multiple lines of evidence, whereas recognizing or identifying may require generating and interpreting data from only one or two diagnostic methods. At minimum, there are six major components of a valid Symbiodiniaceae species description: (1) information on at least two congruent genes (see our recommendations below in “How can we Resolve Symbiodiniaceae Species with Genetic Markers?”), (2) comparison of genetic data against that from other Symbiodiniaceae, (3) morphological description (e.g., comparison of cell size measurements against that from other Symbiodiniaceae), (4) a holotype or name-bearing type specimen (at minimum, an image of cells under light microscopy, but preserved cells are preferable), (5) deposition of the type specimen in a permanent archive (e.g., a museum or herbarium for preserved cells, but if only images are available, their publication in a peer-reviewed journal is sufficient), and (6) proposition of a valid name (according to the International Code of Nomenclature for Algae, Fungi, and Plants; Turland et al., 2018). Where possible, ecological descriptions such as host associations and biogeographic ranges are also encouraged, although sometimes such information is not available.

The Biological Species Concept dictates that if two organisms cannot reproduce and create viable offspring, they should be considered different species (Mayr, 1942). Unfortunately, it has been impossible to apply this criterion to Symbiodiniaceae, as no direct observation of sexual reproduction has been made to date (but see Figueroa, Howe-Kerr & Correa, 2021; Shah et al., 2020). Fortunately, many other species concepts exist, each placing emphasis on different criteria (De Queiroz, 2007; Leliaert et al., 2014). Robust species descriptions satisfy multiple species concepts using independent lines of evidence. For Symbiodiniaceae, the field has largely applied three key types of data: morphological (cell size and cell wall features), ecological (host specificity and biogeographic distribution), and phylogenetic (divergence across multiple DNA markers), along with the assignment of type material (Fig. 2). The taxonomic framework for describing species has matured since the earliest effort by Freudenthal (1962). For example, in line with the Morphological Species Concept, Trench & Blank (1987) proposed three new species based on Symbiodiniaceae cell ultrastructure. They used transmission electron microscopy (TEM) to reveal features such as the nucleus, chromosomes, pyrenoid, chloroplast thylakoid membranes, and cell size; additionally, they used scanning electron microscopy (SEM) to observe thecal plates and the arrangement of the two flagella. Technological advancements in SEM resolution now enable complete morphological characterization of amphiesmal vesicles in the cell wall (Jeong et al., 2014; LaJeunesse, Lee & Gil-Agudelo, 2015; Lee, Jeong & LaJeunesse, 2020; Lee et al., 2015; Nitschke et al., 2020), though such plate tabulations tend to be variable within species (LaJeunesse et al., 2018).

As an increasing number of host species are sampled, it has become clear that the Ecological Species Concept can also be used to support Symbiodiniaceae species descriptions. Although not diagnostic in all cases (Cunning, Glynn & Baker, 2013), symbiosis ecology can be particularly useful for Symbiodiniaceae species that exhibit host-specificity or coadaptation with their hosts (Davies et al., 2020; Finney et al., 2010; Howells et al., 2020; Santos et al., 2004; Smith, Ketchum & Burt, 2017; Thornhill et al., 2014). For example, Cladocopium pacificum and Cladocopium latusorum are found exclusively within corals of the genus Pocillopora (Turnham et al., 2021). Ultimately, because Symbiodiniaceae do not always have distinct morphological characteristics, nor do they always exhibit host specificity, the collection of genetic data to satisfy the Phylogenetic Species Concept has also become a necessity in the description of species (see “How can we Resolve Symbiodiniaceae Species with Genetic Markers?”).

Researchers who do not endeavor to describe Symbiodiniaceae species can encourage and incentivize those who do by accurately treating taxa names as hypotheses and citing the work of taxonomists at the first mention of previously described taxa within manuscripts. We encourage incorporating existing taxonomy (i.e., species names) into current research whenever possible. Due to a general lack of funding for taxonomic descriptions, formal species names are not always available for a given entity, and therefore accommodating sequence variant terminology in the literature will continue to be important. Providing synonyms (e.g., the ITS2 sequence variant and its species name) when a species is first mentioned will improve clarity. Ensuring that community resources consolidate current and past taxonomic assignments will be challenging, but it is critical for connecting historical and future research. Guidance for vouchering Symbiodiniaceae genomic data sets from cultures or holobiont tissues has recently been put forth (Voolstra et al., 2021b). Minimum recommendations include (but are not limited to) high quality DNA voucher material, comprehensive metadata, and common phylogenetic marker sequences. Work is underway to develop a robust ‘Rosetta Stone’ that can translate between different marker designations and species names. Such efforts should be expanded in the future (e.g., through incorporation into analysis pipelines for molecular data) to better facilitate efficient Symbiodiniaceae identification in complex samples.

How can we resolve Symbiodiniaceae species with genetic markers?

No single marker is likely able to distinguish species across all Symbiodiniaceae genera reliably (Table 1). Instead, ecological and physiological studies will benefit from adopting a multi-gene approach where possible, given funding and resource limitations (see “Guidance for Community-Level Assessment of Symbiodiniaceae”). Congruence among sequence data from different cellular compartments (nuclear, chloroplast, and mitochondrial; Table 1) indicates that classifying Symbiodiniaceae using a lineage-based species concept is achievable (De Queiroz, 2007; LaJeunesse & Thornhill, 2011; Sampayo, Dove & LaJeunesse, 2009). This multi-gene approach, supported with ecological, morphological, and sometimes physiological data, has led to the formal description (or re-validation) of 39 Symbiodiniaceae species in 11 genera thus far (Table 1; Hume et al., 2015; Jeong et al., 2014; LaJeunesse, 2017; LaJeunesse et al., 2021, 2018; LaJeunesse, Lee & Gil-Agudelo, 2015; LaJeunesse et al., 2014; LaJeunesse, Parkinson & Reimer, 2012; Lee, Jeong & LaJeunesse, 2020; Lewis, Chan & LaJeunesse, 2019; Nitschke et al., 2020; Parkinson, Coffroth & LaJeunesse, 2015; Pochon & LaJeunesse, 2021; Ramsby et al., 2017; Turnham et al., 2021; Wham, Ning & LaJeunesse, 2017; Xiang et al., 2013). This taxonomic list will continue to grow, and recent whole-genome data already point toward the potential need for further revision of some genera and species (Dougan et al., 2022; González-Pech et al., 2021).

The rate of evolution of gene markers dictates their respective power to resolve distinct genetic entities and whether these entities are likely to represent distinct species (Table 1). In addition, genetic differentiation may vary among genera for the same marker region (Pochon, Putnam & Gates, 2014). Efforts are underway to develop a taxonomic key for Symbiodiniaceae species based on genetic and ecological data. We envision a dynamic dichotomous key that would guide users to the appropriate markers and characteristics for a particular host organism of interest, or alternatively, suggest combinations of markers and characteristics most likely to provide species-level resolution within specific sets of closely related Symbiodiniaceae. Such a key would also reduce project costs by identifying the most informative minimal set of markers.

How many Symbiodiniaceae species exist?

The current best estimate for the total number of symbiotic Symbiodiniaceae species is in the range of hundreds based on phylogenetic (e.g., ITS2) sequence variants (Thornhill et al., 2014). However, these species numbers are likely a significant underestimate because sampling efforts have mainly focused on scleractinian coral hosts living at shallow depths in tropical and subtropical waters. It will be important to continue describing Symbiodiniaceae species in non-scleractinian hosts, including other cnidarians; e.g., octocorals (Goulet et al., 2017; Ramsby et al., 2014), zoantharians (Fujiwara et al., 2021; Mizuyama et al., 2020), actiniarians (Grajales, Rodríguez & Thornhill, 2016), corallimorpharians (Kuguru et al., 2008; Jacobs et al., 2022), hydrocorals (Rodríguez et al., 2019), jellyfish (Vega de Luna et al., 2019); as well as sponges (Hill et al., 2011; Ramsby et al., 2017), acoelomorph flatworms (Kunihiro & Reimer, 2018), molluscs (Baillie, Belda-Baillie & Maruyama, 2000; Banaszak, García Ramos & Goulet, 2013; Lim et al., 2019), ciliates (Mordret et al., 2016), and foraminifera (Pochon et al., 2007). Further collections from undersampled habitats and sources such as benthic sediment and rubble (Fujise et al., 2021; Nitschke et al., 2020; Sweet, 2014; Takabayashi et al., 2012), seagrasses and macroalgae (Porto et al., 2008; Yamashita & Koike, 2013), mesophotic depths (Frade et al., 2008; Goulet, Lucas & Schizas, 2019), the water column (Manning & Gates, 2008; Pochon et al., 2010; Sweet, 2014), and predator feces (Castro-Sanguino & Sánchez, 2012; Grupstra et al., 2021; Parker, 1984) will likely yield many undiscovered species and possibly even novel genera (Yorifuji et al., 2021). These efforts should not be limited to subtropical and tropical waters, as Symbiodiniaceae have been reported in more temperate locations (LaJeunesse et al., 2021; Lien, Fukami & Yamashita, 2012). Systematic and wide-ranging effort to better describe the genetic diversity of Symbiodiniaceae (such as the Tara Oceans expedition; Sunagawa et al., 2020) will lead to a better understanding of the drivers of taxonomic and functional diversity of Symbiodiniaceae.

What steps can be taken to enhance our understanding of Symbiodiniaceae species?

Expanding publicly accessible Symbiodiniaceae culture collections can drive not only taxonomic but also ecological, physiological, and genomic research (LaJeunesse et al., 2018; Voolstra et al., 2021b; Xiang et al., 2013). Most of the diversity in culture constitutes just a handful of species, predominantly from the Symbiodinium and Breviolum genera. More targeted and consistent funding to support further development, maintenance, and sharing of culture collections is critical to the field. Progress toward protocols for Symbiodiniaceae cryopreservation can help conserve biodiversity through the generation of cryogenic archives (Di Genio et al., 2021) and support research in laboratories that cannot maintain continuous cultures. Depositing live specimens in national and organizational archives can alleviate the burden on individual research groups. Examples of national archives include the Provasoli-Guillard National Center for Marine Algae and Microbiota at Bigelow Laboratory in the USA, (https://ncma.bigelow.org/), the Symbiont Culture Facility at the Australian Institute of Marine Science in Australia (https://www.aims.gov.au/), the National Institute for Environmental Studies, (https://mcc.nies.go.jp/) and Biological Resource Center at National Institute of Technology and Evaluation (https://www.nite.go.jp/nbrc/catalogue/) in Japan, the Central Collection of Algal Cultures in Germany (https://www.uni-due.de/biology/ccac/), the Roscoff Culture Collection in France (https://roscoff-culture-collection.org/), and the Culture Collection of Algae and Protozoa in the United Kingdom (https://www.ccap.ac.uk/).

Live cultures established from single cells can benefit taxonomic studies by providing relatively homogeneous strains to establish baselines of diversity and morphology. Monocultures can be confirmed molecularly through fragment analysis of microsatellites. As they are haploid, Symbiodiniaceae monocultures should only show single microsatellite peaks, except in taxa with evidence for broad duplications, such as Durusdinium trenchii (LaJeunesse et al., 2014). Molecular data from cultured isoclonal strains are less noisy than data from host tissues, since such tissues may contain multiple Symbiodiniaceae genera, species, or strains (Fig. 1; Voolstra et al., 2021b). Cultures are also superior for holotype depositions, and they facilitate morphometric analysis, for example, on swimming behavior (motility). However, live culture is not a prerequisite for formal species description, especially because many Symbiodiniaceae are currently difficult to culture (Krueger & Gates, 2012). Furthermore, many strains cultured from host tissue do not represent the dominant Symbiodiniaceae in a host species (Santos, Taylor & Coffroth, 2001). We encourage efforts toward testing new media and bringing new species into culture (Nitschke et al., 2020), as well as documenting and sharing successful and failed attempts. “Culturability” itself may be a useful phenotype to track, as it may reflect the degree of host-specificity, and influence media or antibiotic choice (Ishikura et al., 2004; Nitschke et al., 2020; Reimer et al., 2010; Yorifuji et al., 2021). Motility, cell division rates (growth), bacterial communities (microbiomes) and viral consortia (viromes) are also informative characteristics that can vary within and among symbiont species (Grupstra et al., 2022a; Lawson et al., 2018; Levin et al., 2017; Parkinson & Baums, 2014; Yamashita & Koike, 2016). Constructing a global phenotypic database for cultures, much like the Coral Trait Database (Madin et al., 2016) is another priority for Symbiodiniaceae research, as is exploring the culturable fraction of coral-associated bacteria that may interact directly with Symbiodiniaceae and impact their performance (Frommlet et al., 2015; Lawson et al., 2018; Matthews et al., 2020; Sweet et al., 2021; Li et al., 2022).

Finally, it would be advantageous to identify and culture model Symbiodiniaceae lineages to test species boundaries. For example, measuring DNA sequence differences between sibling species separated by a geological barrier (e.g., the Isthmus of Panama; LaJeunesse et al., 2018; Pochon et al., 2006) would provide molecular-divergence cutoffs that could then be applied to better resolve sympatric lineages. Additionally, cultures of closely related, putative sibling species could be used to explore cytological evidence for sexual recombination (Figueroa, Howe-Kerr & Correa, 2021), evaluate potential hybridization (Brian, Davy & Wilkinson, 2019), and characterize the role symbiotic interactions play in genome evolution (González-Pech et al., 2019).

How can we design population-level studies?

Studies evaluating the distribution of genetic variation within species, often across spatiotemporal gradients or among host taxa, seek to understand how populations are influenced by evolutionary processes such as gene flow, genetic drift, and selection (Aichelman & Barshis, 2020; Davies et al., 2020; Forsman et al., 2020; Prada et al., 2014; Reich et al., 2021; Thornhill et al., 2017; Turnham et al., 2021). Here, we define a population as a group of individuals belonging to the same species that live and interbreed with each other in a given space and time. The study of Symbiodiniaceae populations is fundamental to improving the resolution at which phenotypes of interest are differentiated. Thus, here we focus on allele-based identification and quantification of genetic variation.

Because a single host can contain a mixture of multiple species and/or genera, a first step in experimental design should include assessing sample sets for the presence of multiple distinct Symbiodiniaceae that may confound the interpretation of population-level genetic variation (see “Guidance for Community-Level Assessment of Symbiodiniaceae”). Such assessment can be done pre- and post-population-level analysis with established genetic markers (e.g., ITS2, cp23S) and may be guided by published literature for some regions or host species. Pre-screening is especially advantageous where information on the community composition of Symbiodiniaceae is also sought and especially for hosts which tend to associate with multiple genera or species. Quantitative PCR (qPCR) is one potential technique to pre-screen Symbiodiniaceae samples for the presence of particular lineages (Correa, McDonald & Baker, 2009; Mieog et al., 2007; Saad et al., 2020). After pre-screening, population-level studies typically target genetic variation from the numerically dominant symbiont associating with a particular host or set of hosts (Baums, Devlin-Durante & LaJeunesse, 2014), while excluding any confounding genetic variation from additional species that may be present within host samples (Baums et al., 2010; Thornhill et al., 2006). Post-screening of samples is also possible using tests of assignment to genetic clusters (Davies et al., 2020) or identifying and excluding samples with outlier allelic profiles. Post-screening may be more time- and cost-effective as verification can be performed on a subset of the total sample set.

The ideal number of samples to collect and analyze will depend on the particular aim(s) of the study (e.g., delineating populations vs. characterizing the degree of admixture among them), the scale of comparison (e.g., reef, habitat, colony, intra-colony, etc.), and the markers being employed. However, studies leveraging more traditional markers, such as microsatellites, tend to benefit from robust sample sizes with minimum ranges of 20–30 individual hosts per level of interest (e.g., habitat and location) (Hale, Burg & Steeves, 2012). Although this is a good target, studies limited by permit authorizations, budgets, and other constraints are still informative in some contexts.

How can we best use microsatellite loci?

Microsatellite loci (or simple sequence repeats; SSRs) are segments of DNA where 1–6 base pairs are repeated in a tandem array; these loci are distributed abundantly across genomes of nearly all eukaryotic organisms (Tautz, 1989). Variations in the length of repeats are generated by polymerase slippage during DNA replication, resulting in homologous regions (i.e., loci) of differing lengths (i.e., alleles) among individuals. Microsatellites are generally thought to represent neutral loci with high mutation rates. Their single-locus, multiallelic, and codominant properties can yield valuable information regarding ploidy and reveal genetic structure among populations within and between species. Furthermore, microsatellite analyses are generally a PCR-based technique, making them cost-effective relative to other methods (Sweet et al., 2012). With the advent of high-throughput sequencing and transcriptomics, the generation of hundreds of potential microsatellite loci is now comparatively straightforward (e.g., Abdelkrim et al., 2009). For species or lineages where numerous loci are available, costs and effort can remain low by multiplexing primer sets (Davies et al., 2013). Taken together, these features make microsatellites attractive for studying Symbiodiniaceae populations. These markers have been used to address questions related to overall diversity, population structure within and between reefs, gene flow, dispersal, and relatedness between symbionts (see Table 1 in Thornhill et al., 2017).

Once the target Symbiodiniaceae species or lineage has been identified within a dataset, these samples can be tested for variability using previously developed microsatellite loci via PCR amplification (Fig. 3). Primers for such loci have been developed for Symbiodiniaceae species across at least five genera: Symbiodinium (Pinzón et al., 2011), Breviolum (Andras, Kirk & Drew Harvell, 2011; Grupstra et al., 2017; Pettay & LaJeunesse, 2007; Santos, Taylor & Coffroth, 2001; Santos, Gutierrez-Rodriguez & Coffroth, 2003; Wirshing, Feldheim & Baker, 2013), Cladocopium (Bay, Howells & van Oppen, 2009; Davies et al., 2020; Howells, van Oppen & Willis, 2009; Magalon et al., 2006; Wham & LaJeunesse, 2016), Durusdinium (Pettay & LaJeunesse, 2009; Wham, Pettay & LaJeunesse, 2011), and Philozoon (Molecular Ecology Resources Primer Development Consortium et al., 2010). Importantly, these loci tend to have narrow phylogenetic ranges, with primers developed for a given species typically working only on other closely-related species within the same genus. Therefore, it is necessary to screen existing primers for utility with a given target species, to ensure that allelic variability among the chosen suite of microsatellite loci is sufficient, and to develop novel primer sets if existing primers fail or prove insufficiently specific. Ideally, new Symbiodiniaceae primers should be tested against monoclonal cultures of species within the same genus (positive controls) as well as against symbiont-free sperm or apo-symbiotic larvae (negative controls) to rule out off-target PCR amplification of host DNA. Although more loci will generally increase discriminatory power in population-level studies, as few as 2–3 loci have provided sufficient discriminatory power for some questions (Santos, Gutierrez-Rodriguez & Coffroth, 2003; Thornhill et al., 2009).

What other markers can resolve Symbiodiniaceae populations?

The ITS2 region of rDNA is repeated in tandem arrays within all known Symbiodiniaceae genomes. For population-level assessments, this universality presents an advantage over microsatellites, but the multi-copy nature of this marker poses unique challenges. As long as appropriate analytical frameworks are applied (see “Guidance for Community-Level Assessment of Symbiodiniaceae”), ITS2 data can be used to resolve strains within species. Such assessments require consideration of similarities in the assemblages of ITS2 sequences and their relative abundances within each genome. For example, genetic structure among Cladocopium thermophilum strains in the Persian/Arabian Gulf has been characterized (Hume et al., 2019; Smith, Ketchum & Burt, 2017) and patterns of IGV obtained from amplicon sequencing data show fine-scale spatial structure among C. thermophilum populations separated by tens to hundreds of kilometers (Howells et al., 2020). However, recombination (i.e., whether two populations are interbreeding) is often considered sufficient for operational recognition that those entities are members of the same species (Andras et al., 2009; Grupstra et al., 2017; Santos, Gutierrez-Rodriguez & Coffroth, 2003; Santos & Coffroth, 2003; Thornhill et al., 2017, 2009). Therefore, it is difficult to determine whether ITS2-based genotypes correspond to distinct populations of the same species or different species. Other markers are also able to resolve at the population level, but their application to Symbiodiniaceae population biology is limited. Examples include the chloroplast psbA minicircle noncoding region (psbA^ncr; Moore et al., 2003) and the chloroplast 23S ribosomal region (cp23S; Santos, Gutierrez-Rodriguez & Coffroth, 2003).

What are the next steps for understanding Symbiodiniaceae population biology?

Advancing our understanding of Symbiodiniaceae population biology will be greatly informed by leveraging samples that have single Symbiodiniaceae MLGs (Prada et al., 2014). For example, available monoclonal cultures of Symbiodiniaceae from several species could be used to develop and test new technologies and markers (including validation of copy number, see “Accounting for Copy Number Variation”) and these technologies could then be extended to more complex associations in hospite (within a host organism). To overcome the challenges of widespread gene duplication in Symbiodiniaceae genomes (González-Pech et al., 2021; Pochon et al., 2012; Prada et al., 2014), efforts should be directed toward identifying new low copy markers (or preferably single copy markers). Discovery of single copy loci may be informed by screening for universal single copy markers collated in the Benchmarking Universal Single-Copy Orthologs (BUSCO) database (Seppey, Manni & Zdobnov, 2019; Simão et al., 2015), although many BUSCOs are undetected in Symbiodiniaceae genomes (González-Pech et al., 2021). Restriction-associated DNA sequencing may serve as a low-cost method for generating single copy markers for population-level assessments in Symbiodiniaceae (Kitchen et al., 2020; Suyama & Matsuki, 2015); however, these methods require further development.

Whole-genome sequencing (WGS) is also becoming more affordable, especially at low coverage (<5X), opening the possibility of evaluating genome-wide variation in Symbiodiniaceae (González-Pech et al., 2021; Reich et al., 2021), although Symbiodiniaceae genomes are large (>Gbp) and few chromosome-scale assemblies exist (Marinov et al., 2021; Nand et al., 2021). We suggest that WGS first be applied to isoclonal cultures, where possible, to ensure reads derive from one genetic entity (Voolstra et al., 2021a; McKenna et al., 2021). Subsequently, this approach can be applied to multispecies assemblages where different Symbiodiniaceae lineages within the same genus could be mapped to these reference genomes. These types of analyses would allow for simultaneous quantification of gene flow and divergence among Symbiodiniaceae populations of co-occurring species and improve estimates of effective population sizes and clonality within and among species, hosts, and reefs. Another major advantage of genome-wide data is the potential to evaluate adaptive (non-neutral) genetic variation and signatures of selection across the genome (Ladner, Barshis & Palumbi, 2012; Liu et al., 2018; Voolstra et al., 2009). For example, identifying associations between traditional markers, genomic regions, Symbiodiniaceae functional traits, and/or environmental variables–including those that are important for the survivorship of corals under warmer, more acidic, and more eutrophic oceans–remains a research priority (see “Beyond Genotype: Phenotyping Symbiodiniaceae” and “Integrating Multiomic Technologies to Study Symbiodiniaceae”).

What is a Symbiodiniaceae community?

Generally defined, ecological communities are composed of more than one species that live together and interact. However, what is meant by terms such as “together” and “interact” can vary (Konopka, 2009), particularly when considering free-living vs. symbiotic Symbiodiniaceae. Typically one to two (but up to 10) Symbiodiniaceae cells reside in a coral gastrodermal cell (Davy Simon, Allemand & Weis Virginia, 2012; Muscatine et al., 1998), potentially restricting direct interactions between the endosymbiont cells within a coral host. Here, we use the term “local Symbiodiniaceae community” to refer to two or more Symbiodiniaceae species within a single host, whereas “macroscale Symbiodiniaceae community” (see “phenomenological community” in Konopka (2009)) describes the diversity of Symbiodiniaceae across some larger scale (e.g., conspecific hosts or multiple host species). Environments that include multiple free-living Symbiodiniaceae species also constitute macroscale communities; e.g., benthic sediments (Nitschke, Davy & Ward, 2016; Quigley, Bay & Willis, 2017; Sweet, 2014), the water column (Fujise et al., 2021; Porto et al., 2008), and macro-algal surfaces (Fujise et al., 2021; Porto et al., 2008).

Macroscale Symbiodiniaceae communities contain more species and encompass higher genetic diversity than local Symbiodiniaceae communities because symbiotic diversity accumulates with increased host colony and habitat sampling (Swain et al., 2020). Environmental samples include cells of symbiotic Symbiodiniaceae expelled from hosts as well as non-symbiotic, free-living species. In contrast, a given adult host typically harbors only one or two dominant Symbiodiniaceae species (Goulet, 2006), often from distinct genera, as well as other species at low relative abundances (Hume et al., 2020; Silverstein, Correa & Baker, 2012). In hospite Symbiodiniaceae communities can be transmitted vertically (promoting higher fidelity), reassembled horizontally (allowing for greater flexibility), or some combination of both (mixed-mode transmission) with each host generation (Quigley, Willis & Bay, 2017). The diversity of the free-living component of macroscale Symbiodiniaceae communities and the symbiotic component of local Symbiodiniaceae communities are each likely to be underestimated (e.g., Baker & Romanski, 2007), but for different reasons. Free-living communities are relatively diffuse and are therefore more difficult to exhaustively sample. In contrast, local Symbiodiniaceae community assessments are prone to sampling bias (but see, e.g., Goulet & Coffroth, 2003). Characterizations of local communities are often based on a single sample from a well-lit, “top” surface of a colony. Sampling across a host’s surface has revealed heterogeneous distributions of dominant Symbiodiniaceae within colonies of Caribbean stony corals such as Colpophyllia, Montastraea, Orbicella, Porites, and Siderastrea (e.g., Correa et al., 2009; Kemp, Fitt & Schmidt, 2008; Rowan et al., 1997; Ulstrup & van Oppen, 2003), as well as some Pacific stony corals (e.g., Fifer et al., 2022; Innis et al., 2018; Kemp, Fitt & Schmidt, 2008; Rowan et al., 1997; Ulstrup & van Oppen, 2003) and zoantharians such as Zoanthus (Fujiwara et al., 2021) and Palythoa (Wee, Kobayashi & Reimer, 2021). Whether local Symbiodiniaceae communities exhibit structure over smaller spatial scales in hospite (e.g., oral vs. aboral host surfaces) is unknown, but could be resolved with single-cell techniques (see “Integrating Multiomic Technologies to Study Symbiodiniaceae”).

Why study Symbiodiniaceae community diversity?

Studying macroscale communities can provide insights into cnidarian-Symbiodiniaceae dynamics along environmental gradients (Cunning et al., 2015; Rossbach et al., 2021; Silverstein et al., 2011; Terraneo et al., 2019). Regional macroscale Symbiodiniaceae community structure (i.e., beta diversity) may also reflect chronic disturbance from anthropogenic activity (Claar et al., 2020a) and help identify more resilient or resistant reefs (Ziegler et al., 2015). Additionally, macroscale communities in reef seawater, sediments, feces, and on macro-algal surfaces may be important sources of symbiotic Symbiodiniaceae that can be acquired horizontally by prospective hosts (Adams, Cumbo & Takabayashi, 2009; Ali et al., 2019; Castro-Sanguino & Sánchez, 2012; Coffroth et al., 2006; Cumbo, Baird & van Oppen, 2013; Fujise et al., 2021; Granados-Cifuentes et al., 2015; Grupstra et al., 2022b, 2021; Nitschke, Davy & Ward, 2016; Porto et al., 2008; Quigley et al., 2018; Quigley, Bay & Willis, 2017; Sweet, 2014; Umeki et al., 2020; Venera-Ponton et al., 2010). Symbiodiniaceae in a free-living mode may influence important processes, such as sexual reproduction, hybridization, and gene flow within Symbiodiniaceae (Figueroa, Howe-Kerr & Correa, 2021).

Positive and negative species interactions can occur within local Symbiodiniaceae communities resulting in resource and niche partitioning (Davy Simon, Allemand & Weis Virginia, 2012; Howe-Kerr et al., 2020; Matthews et al., 2020). Quantifying these interactions may help disentangle the factors and processes governing Symbiodiniaceae community assembly in early host life history stages (McIlroy et al., 2019; Quigley, Willis & Bay, 2016), as well as successional dynamics (or stability) in adult hosts. Studying local Symbiodiniaceae communities can also identify conditions that trigger symbiotic breakdown (i.e., dysbiosis). Dysbiosis has frequently been documented in the bacterial communities of stressed hosts (e.g., Zaneveld, McMinds & Vega Thurber, 2017; Ziegler et al., 2017; Boilard et al., 2020), and may also be evident in local Symbiodiniaceae communities. Generally speaking, dysbiosis can manifest itself in the host as: (1) an increase in symbiont richness (invasion or proliferation of low abundance symbionts), (2) a decrease in symbiont richness (loss of symbionts), or (3) more complex changes in community structure or beta diversity (Egan & Gardiner, 2016). For example, Symbiodinium necroappetens (LaJeunesse, Lee & Gil-Agudelo, 2015; Stat, Morris & Gates, 2008) and some symbionts in the genera Durusdinium (Bay et al., 2016; Manzello et al., 2018), Breviolum (LaJeunesse et al., 2010b), and Cladocopium (Wee, Kobayashi & Reimer, 2021) can opportunistically increase or decrease their abundance in bleached or stressed hosts. Stony coral juveniles in the field (Quigley, Willis & Bay, 2016) and adults in tank-based experiments (Howe-Kerr et al., 2020) have exhibited decreased survival in conjunction with more diverse local Symbiodiniaceae communities. Additional experiments to assess how frequently different types of dysbiosis occur in local Symbiodiniaceae communities are needed, including in non-scleractinian hosts, some of which can harbor up to 60 symbionts per host cell (Fitt, 2000). Testing the extent to which different types of dysbiosis are associated with specific cnidarian hosts, as well as specific environmental contexts, should also be prioritized.

Current challenges in understanding local Symbiodiniaceae community diversity and dynamics include: (1) determining actual and relative abundances of Symbiodiniaceae species given IGV and copy number issues (see “Accounting for Copy Number Variation”); and (2) understanding the roles (if any) that low abundance Symbiodiniaceae play in holobiont survival and fitness (see Arif et al., 2014; Bay et al., 2016; Lee et al., 2016). This knowledge is key to connecting Symbiodiniaceae genotypes to phenotypes (see “Beyond Genotype: Phenotyping Symbiodiniaceae”). Low abundance Symbiodiniaceae may serve as a reservoir of in hospite algal genotypes that may increase to dominance (at least ephemerally) during or following a change in environmental conditions (Bay et al., 2016; Berkelmans & Van Oppen, 2006; Boulotte et al., 2016; Buddemeier & Fautin, 1993; Claar et al., 2020a; Jones et al., 2008; Lewis, Neely & Rodriguez-Lanetty, 2019; Thornhill et al., 2006; Ziegler et al., 2018). The mechanisms controlling this turnover in hospite remain poorly understood, but involve host rewards and sanctions (Kiers et al., 2011, 2003) and competitive interactions among symbionts (Palmer, Stanton & Young, 2003). Competition among Symbiodiniaceae affects the initial uptake of symbionts in early coral ontogeny (McIlroy et al., 2019) and influences longer-term persistence in experimentally-generated symbioses (Gabay et al., 2019), but the relative importance of competition in shaping in hospite communities once they are established remains poorly understood. Beyond their potential to shift in hospite following bleaching events (Jones et al., 2008; Thornhill et al., 2006), low abundance Symbiodiniaceae could also contribute to emergent holobiont properties (Howe-Kerr et al., 2020; Ziegler et al., 2018). Quantification of holobiont traits with and without the addition of low abundance homologous Symbiodiniaceae (i.e., lineages that typically enter into a symbiotic relationship with a given host taxon) from a range of inoculation sources constitutes a critical next step to understanding the functional role these symbionts play in the host.

How can we optimize the study of Symbiodiniaceae community diversity?

Improving our understanding of the processes shaping Symbiodiniaceae communities is critical to predicting their distributions and potentially mitigating coral reef decline driven by global change. The methods below constitute suggested approaches for analyzing the diversity of macroscale and local Symbiodiniaceae communities. In some circumstances, identifying numerically dominant Symbiodiniaceae lineages (as opposed to the total diversity of a Symbiodiniaceae community) may be sufficient for the question at hand because hosts are generally selective in the symbionts they harbor, and some are highly specific to particular symbiont lineages (e.g., Hume et al., 2020; Thornhill et al., 2014). Whether quantifying numerically dominant lineages or total Symbiodiniaceae community, the selection of molecular marker(s) and the approach(es) to data generation and analysis have implications for the interpretation of diversity. Molecular markers available for assessing Symbiodiniaceae community diversity are multicopy, and thus, present the challenge of distinguishing intragenomic from intergenomic variation. Inclusion of symbiont taxa above or below the species level in the calculation of alpha and beta community diversity is problematic as these metrics are designed for species-level input. Including anything but species-level data in the calculation of these metrics can obscure patterns and lead to under- or over-estimation of diversity.

Accounting for copy number variation

Copy number variation (CNV) is any genetic trait involving the number of copies of a gene in the genome of an individual. Efforts to quantify the absolute and relative abundances of different species in a local Symbiodiniaceae community are complicated by the presence of high CNV across taxa for key markers such as ITS2 (Correa, McDonald & Baker, 2009; Mieog et al., 2007; Saad et al., 2020; Stat, Carter & Hoegh-Guldberg, 2006; Thornhill, LaJeunesse & Santos, 2007). Thus, relative rDNA-PCR amplicon abundance does not necessarily equate to actual abundance of Symbiodiniaceae cells in a sample (especially when inter-genus comparisons are being made; Arif et al., 2014; Correa, McDonald & Baker, 2009; Quigley et al., 2014; LaJeunesse et al., 2022). For instance, some symbiont taxa that have been reported to possess a considerably higher rDNA copy number than others (e.g., Cladocopium spp.; Saad et al., 2020); these high copy number taxa can appear to be abundant in mixed communities even though they might represent a low fraction of cells in hospite, leading to inaccurate estimation of actual symbiont abundances (Fig. 4). The incorrect classification of low abundance vs. dominant taxa can impact interpretations related to biogeography and ecology. Such errors could be avoided if a correction factor is applied (e.g., dividing the abundance value by the number of copies present in the genome of the relevant species, Fig. 4; Correa, McDonald & Baker, 2009; Mieog et al., 2007; Rubin et al., 2021; Saad et al., 2020), but such corrections rely on accurate copy number reference values, which are not currently available for the majority of Symbiodiniaceae taxa. Most studies that have quantified CNV have been limited to comparisons between genera, and there is considerable variation in the values reported across studies (e.g., Gong & Marchetti, 2019; Loram et al., 2007; Mieog et al., 2007; Quigley et al., 2014; Saad et al., 2020; Thornhill, LaJeunesse & Santos, 2007). Inconsistencies in reported CNV values may be attributed to variation among strains or species (within-genus differences can be as large as between-genus differences) or to methodological differences between studies. Therefore, the extent of CNV within Symbiodiniaceae genera or across populations largely remains to be established.

Lineage-specific qPCR assays have helped to quantitatively characterize mixed communities at the genus level (Correa, McDonald & Baker, 2009; Cunning, Silverstein & Baker, 2018; Cunning & Baker, 2013) and species level (Fujiwara et al., 2021). These targeted qPCR assays are more quantitative than sequencing approaches, but still require correction for CNV. When applied to systems with known symbiont diversity, qPCR can accurately and cost-effectively quantify local symbiont community structure and dynamics; however, these primer sets must be developed on a per-taxon basis. Another approach is the use of flow cytometry to quantify and/or physically separate cells of interest. While the natural variability in cell characteristics (e.g., size, shape, fluorescence) cannot distinguish taxa (Apprill, Bidigare & Gates, 2007), the use of fluorescent probes to tag taxa of interest has successfully quantified the absolute and relative abundance of co-occurring taxa (McIlroy, Wong & Baker, 2020; McIlroy, Smith & Geller, 2014). Importantly, these methods are also conducive to subsequent genetic and physiological analyses of sorted cells. The development of further resources to account for CNV is an important priority within the field (see “Integrating Multiomic Technologies to Study Symbiodiniaceae”).

When should researchers use multiple Symbiodiniaceae genetic markers for community-level analyses?

As each marker has its own evolutionary history and methodological bias (e.g., primer bias, CNV, etc.), congruence among multiple independent markers should enable more robust characterization of Symbiodiniaceae community ecology (Fujiwara et al., 2021; Kavousi et al., 2020; Noda et al., 2017; Pochon et al., 2019; Reimer et al., 2017; Smith et al., 2020; Smith, Ketchum & Burt, 2017; LaJeunesse et al., 2022). Where possible, when multiple markers are used, the markers should complement each other’s taxonomic scope and power to resolve. For example, it may be productive to pair the cp23S (larger taxonomic scope, lower resolving power) with the psbA^ncr (smaller taxonomic scope, higher resolving power). Instances where multiple markers produce conflicting results may help identify a Symbiodiniaceae lineage that cannot be accurately characterized with a single broad taxonomic marker. Additionally, combining multiple markers can provide greater resolution and improved interpretability compared to single-marker approaches. For example, the use of psbA^ncr can help overcome issues associated with interpretation of IGV and high copy number in ITS2 and provide support for ITS2-type profiles (Smith et al., 2020) by confirming which ITS2 IGVs are most likely part of a single lineage (LaJeunesse & Thornhill, 2011; Smith, Ketchum & Burt, 2017). Budget and logistics permitting, it is recommended to use multiple markers in some situations. For some ITS2 lineages (e.g., Breviolum B1 or Cladocopium C15; Hoadley et al., 2021; Parkinson, Coffroth & LaJeunesse, 2015), available markers that behave as if single copy do not distinguish ecologically relevant variants, but universal primers have yet to be designed for gene regions that capture this variation (e.g., psbA^ncr). Therefore, a marker that behaves as if single copy can first be applied to determine the dominant Symbiodiniaceae genera present to assist in the selection of the correct primer set for a higher resolution marker. We recognize that each additional marker can greatly increase high-throughput sequencing project costs, so such designs are only recommended when resources are available. Single marker studies can still provide great insight into symbiont community diversity as long as they are interpreted carefully.

How can we interpret Symbiodiniaceae diversity while acknowledging the pitfalls of common markers?

Given the complexities associated with common methodological approaches and how they influence ecological interpretations of Symbiodiniaceae genetic information in community-level studies, it is critical to provide sufficient methodological details when reporting and interpreting results. We encourage the field to follow reproducible research standards, which include making analysis pipelines and raw data available after publication (see Lowndes et al., 2017 for a comprehensive guide to open science tools). At minimum, commented code (including filtering thresholds, analysis decision points, and processing steps) should be deposited in each article’s Supplemental Materials or in a publicly accessible repository (e.g., GitHub) with a DOI (e.g., procured through GitHub and Zenodo). Raw sequencing data must be deposited in a dedicated archive such as NCBI SRA for amplicon sequencing data, or NCBI Genbank for single sequence data. Alongside the code and sequences, additional metadata (e.g., environmental and physiological parameters, as well as trackable information regarding the hosts’ ID, if applicable; Voolstra et al., 2021b), should be deposited either with the publishing journal or with a data repository (e.g., Dryad, Zenodo, the National Science Foundation’s BCO-DMO). Finally, all of these deposition options can be integrated. For example, a Zenodo deposition can be linked to a GitHub repository so that new code releases are automatically updated in the Zenodo repository. By making published data widely available, we can accelerate our understanding of cnidarian-dinoflagellate symbioses and their responses to a changing environment.

There continues to be dialogue amongst members in the Symbiodiniaceae field regarding the interpretation of gene amplicon data produced by metabarcoding (e.g., Illumina MiSeq). Specifically, there is an ongoing debate about if and how to incorporate Symbiodiniaceae taxa present at low abundances, and how certain parameters are built into existing analytical pipelines. For example, SymPortal will not attempt to predict profiles for a Symbiodiniaceae genus in a given sample if there are less than 200 reads for that genus/sample combination (Hume et al., 2019); this can potentially contribute to systematic underestimation of total Symbiodiniaceae community diversity. As such, we encourage authors to consider carefully what their data can and cannot discern (e.g., Table 1), report assumptions associated with their data interpretation, acknowledge that other interpretations exist, and discuss whether or not these other interpretations change the biological or ecological conclusions of their study. Diversity metrics merit careful attention and gene sequence diversity should not be conflated with species diversity. Authors (and reviewers and editors) can weigh what results, tables, or figures might be included in the Supplemental Material to acknowledge and address these additional interpretations in order to facilitate the inclusion of diverse perspectives (see “Ensuring an Inclusive Symbiodiniaceae Research Community”).

Why do we need to characterize Symbiodiniaceae phenotypic diversity?

Not all genetically distinct Symbiodiniaceae taxa exhibit physiological differences (e.g., through functional convergence; Goyen et al., 2017; Suggett et al., 2015), whereas unique isolates of the same taxon may be functionally divergent (e.g., Beltrán et al., 2021; Díaz-Almeyda et al., 2017; Hawkins, Hagemeyer & Warner, 2016; Howells et al., 2011; Mansour et al., 2018; Parkinson et al., 2016; Parkinson & Baums, 2014; Russnak, Rodriguez-Lanetty & Karsten, 2021). This is not surprising given the effects of strong local selection within reef habitats (Howells et al., 2011; Kriefall et al., 2022; Marhoefer et al., 2021; Suggett, Warner & Leggat, 2017; van Oppen et al., 2018) and the role of acclimatization (Torda et al., 2017). Stringent functional interrogation is therefore critical to determining how healthy cnidarian-symbiont associations will survive the climate crisis. This goal rests on advancing physiological descriptions, increasing the number of cultured isolates from diverse hosts, and extending the methodological toolbox to characterize Symbiodiniaceae differences. Thus, a more comprehensive functional characterization can accompany taxonomic assignment, helping to build greater community consensus on methods and standards for describing phenotypes of interest (Fig. 5).

What do we need to consider when assessing Symbiodiniaceae phenotypes in hospite?

An overwhelming interest among Symbiodiniaceae researchers to date has been identifying thermal threshold phenotypes based on bio-optics (e.g., Goyen et al., 2017; Hennige et al., 2009; Voolstra et al., 2021d), targeted biochemistry (e.g., Tchernov et al., 2004) or “-omics” metrics (e.g., Olander et al., 2021; Roach et al., 2021). These varied foci illustrate that phenotypes are operationally defined. Consequently, the detected functional diversity (the extent and range of phenotypes resolved) may appear different depending on the metrics used. For example, descriptions of phenotype diversity for heat stress sensitivity based on photobiological properties may not align with those based on metabolic indicators (Goyen et al., 2017) or light adaptation (Suggett et al., 2015, 2022). Thus, reconciling genetic diversity with functional diversity must be carefully contextualized based on the measurement criteria and scientific questions at hand.

While it is valuable to confirm symbiont traits when in symbiosis, the presence of local Symbiodiniaceae communities and “secondary” symbionts in cnidarian holobionts complicate this effort. In local Symbiodiniaceae communities, it can be difficult to determine the relative abundance of each lineage present. Although accounting for copy number can help determine symbiont cell number and density in such cases (see “Accounting for Copy Number Variation”), other algal-centric physiological metrics, which reflect the combined average of all symbionts present within the host (Cunning, Silverstein & Baker, 2018), will be difficult to interpret. Single-cell sorting techniques may help assess unique phenotypic distinctions across different symbiont species from the same host (Snyder et al., 2020), but these techniques constitute additional effort and cost. Moreover, cnidarians host a variety of other microeukaryotic, prokaryotic, and viral symbionts (Ainsworth, Fordyce & Camp, 2017; Hernandez-Agreda, Gates & Ainsworth, 2017; Thurber et al., 2017), some of which are associated with colony health and resilience to environmental stress (Bourne, Morrow & Webster, 2016; Voolstra et al., 2021c; Ziegler et al., 2017). Some viruses even infect Symbiodiniaceae cells themselves (Grupstra et al., 2022a; Levin et al., 2017), with diverse potential impacts on Symbiodiniaceae phenotypes (Correa et al., 2021; van Oppen, Leong & Gates, 2009). The degree to which these “secondary” symbionts impact the observed phenotype of Symbiodiniaceae cells in hospite is an active area of research (Maire et al., 2021; Matthews et al., 2020). Finally, coral tissue thickness, pigmentation, skeletal reflectance, or other coral-associated microorganisms can affect irradiance levels reaching Symbiodiniaceae in hospite (Dimond et al., 2013; Dimond, Holzman & Bingham, 2012; Enríquez, Méndez & Iglesias-Prieto, 2005; Marcelino et al., 2013; Smith et al., 2013; Titlyanov et al., 2009; Wangpraseurt et al., 2014, 2012). Variation in these physiological metrics can therefore affect symbiont phenotype, and lead to variable responses to climate stress (Hoadley et al., 2019).

Traits where variability across species exceeds that within populations are ideally suited for phenotypic analysis, but are presently unknown to the field or are challenging to measure in consistent and ecologically meaningful ways. Consequently, high-throughput approaches for assessing Symbiodiniaceae phenotypes need to consider tradeoffs that are constrained by end goals. For example, recent high-throughput approaches for assessing thermal tolerance at the whole coral level—such as coral bleaching automated stress systems (CBASS; Voolstra et al., 2020)—and the single cell level (Behrendt et al., 2020) have incorporated short thermal challenges followed by stress characterization through the measurement of 1–2 physiological variables such as maximum PSII photochemical efficiency (F_v/F_m) and cell density. While single-phenotype assays can be informative within the context of ecosystem service values (e.g., identifying thermally tolerant corals for nursery propagation; Cunning et al., 2021), identification of functionally distinct Symbiodiniaceae phenotypes will benefit from measuring a broader spectrum of physiological metrics (Hoadley et al., 2021). Phenotypic characterization using multiple photosynthetic metrics can provide some species-specific resolution (Suggett et al., 2015), and the non-invasive nature of chlorophyll a fluorometry lends itself to high-throughput approaches. However, poor contextualization of photosynthetic parameters with respect to cnidarian resilience currently limits the use of these techniques alone for large-scale phenomic studies, and may ultimately require integration of fitness metrics influenced by resource availability such as elemental composition via nutrient acquisition. While specific consensus on measurement protocols is beyond the scope of this perspective, taking a multidisciplinary approach and transparently documenting important methodological choices will help move the field forward.

What do we need to consider when assessing Symbiodiniaceae phenotypes in culture?

Axenic or bacteria-depleted cultures are promising tools for connecting Symbiodiniaceae genotypes to phenotypes because their genetic identity is readily determined (see “Guidance for Species-Level Assessment of Symbiodiniaceae Diversity”) and morphological, physiological, and behavioral diversity are readily discernible among such algal isolates (Costa et al., 2019; Xiang, 2018; Xiang et al., 2013). In terms of photo-physiology, fluorometry has become a convenient and accessible tool to gauge “culture health” (Hennige et al., 2009; Robison & Warner, 2006; Suggett et al., 2009). Fluorometry is also used in studies examining phenotypic variation focused on photosynthetic traits and how they are affected by resource availability (light, nutrients) and temperature (Díaz-Almeyda et al., 2017; Suggett et al., 2015), and has been inferred to reflect holobiont health (Voolstra et al., 2020). While photosynthetic traits are informative of cellular functioning, they are insufficient in isolation of other measurements to explain phenotypic variation in growth (e.g., Brading et al., 2011; Hennige et al., 2009; Suggett et al., 2015). Recent data point to variable photo-physiological tolerance and thermal plasticity of genetically divergent Symbiodiniaceae grown in monoculture, which has contributed to a deeper understanding of the algal symbiont response to increasing sea surface temperatures (Grégoire et al., 2017; Klueter et al., 2017; Russnak, Rodriguez-Lanetty & Karsten, 2021; Suggett et al., 2015). However, a large number of genetically distinct algal symbionts identified in hospite have resisted sustained growth in culture (e.g. Krueger & Gates, 2012; Santos, Taylor & Coffroth, 2001). Furthermore, physiological and functional ’omics data indicate that when in culture or freshly isolated, Symbiodiniaceae exhibit responses to thermal stress that differ from those of the same population in hospite (Bellantuono et al., 2019; Gabay et al., 2019; Goulet, Cook & Goulet, 2005). These data suggest that some physiological traits measured from culture-based studies may not be easily extrapolated to the symbiotic state. Such issues are particularly pronounced when measuring nutrient-associated phenotypes, as most culture media are nutrient-replete while Symbiodiniaceae in hospite appear to be nutrient-limited (Maruyama & Weis, 2021).

Emergent properties are novel characteristics that smaller units of organization gain when they become part of a larger complex system. Research focusing on core emergent properties expressed in culture, that can also be easily assessed in nature (in hospite), is logical given that phenotypes will consistently be the result of specific environmental conditions operating on the underlying molecular machinery. However, in decades of studies on Symbiodiniaceae cultures, the environmental conditions imposed have not consistently been reported at the time of, or prior to, sampling. Examples of such metadata include the growth phase (steady state vs. non-steady state; Tivey, Parkinson & Weis, 2020) or cell cycle phase (Fujise et al., 2018; Tivey, Parkinson & Weis, 2020), as well as the actual environments in the cultures (light quality/quantity, nutrients) as opposed to those measured in the incubators or assumed from the recipe of the medium used (e.g., Camp et al., 2020; Reich et al., 2020), and the extent of bacterial loading. Consequently, developing guidelines for rigorous reporting of environmental (experimental) conditions when phenotypes are quantified is a key priority. Ensuring inter-comparability among studies in the future will similarly depend on operating under a more consistent set of measurement protocols for phenotypic traits.

How can genomics and high-throughput sequencing be leveraged?

With continued cost reductions and increases in computational power and accessibility, advanced “-omic” technologies, including genomics, transcriptomics, proteomics, and metabolomics, are rapidly enhancing our ability to understand biological mechanisms (Krassowski et al., 2020). Coupled with powerful, multivariate statistical techniques and machine learning, omics technologies have greatly refined our understanding of Symbiodiniaceae biology.

Whole-genome sequencing (WGS) remains the gold standard for capturing genetic diversity (Aranda et al., 2016; González-Pech et al., 2021; Lin et al., 2015; Liu et al., 2018; Reich et al., 2021; Shoguchi et al., 2018, 2013). While individual and concatenated genes can help resolve phylogenetic relationships and define taxonomic lineages (LaJeunesse et al., 2018; Parkinson, Coffroth & LaJeunesse, 2015), WGS data provide more comprehensive phylogenomic signals, which can be used to investigate divergent selection. For example, while a Symbiodiniaceae phylogeny reconstructed using k-mers (short, sub-sequences of defined length k) derived from whole-genome sequences is largely consistent with the phylogeny reconstructed with LSU rDNA data (González-Pech et al., 2021), different genomic regions exhibit distinct phylogenetic signals (Lo et al., 2022). Further comparison of WGS data indicate that the similarity shared between different species within the genus Symbiodinium is comparable to that between different Symbiodiniaceae genera, revealing more extensive divergence than anticipated and suggesting a need for future revision (Dougan et al., 2022).

WGS efforts employ short- or long-read technologies, or a combination of both strategies. Short-read sequencing technologies (e.g., Illumina) have typically offered a cost-effective approach for deep sequencing with low error rates (<1%) and have provided valuable insights into Symbiodiniaceae diversity, including gene family expansions across different Symbiodiniaceae lineages (Aranda et al., 2016; Lin et al., 2015; Liu et al., 2018). Though short-read sequencing can identify lineage-specific divergence, short reads are difficult to assemble, especially with highly repetitive genomic regions. Long-read sequencing technologies such as Pacific Biosciences (PacBio) or Oxford Nanopore Technologies offer a viable alternative to improve the contiguity of highly fragmented short-read genomes. However, long-read sequencing technologies are more expensive and error-prone relative to short-read platforms, though these technologies are rapidly advancing (Karst et al., 2021). Long-read data allow us to observe chromosome structure, a greater number of genomic elements, such as DNA transposons, long terminal repeats, or chromosomal enrichment for genes with similar biological functions (González-Pech et al., 2021; Li et al., 2020; Nand et al., 2021). Chromosome-level assemblies represent a major milestone in dinoflagellate genomics as they confirm that many genes are encoded in unidirectional clusters which correspond to large topological domains (Marinov et al., 2021; Nand et al., 2021). Not only does this discovery provide insights into the structure of genes in Symbiodiniaceae, but the structure can be correlated with the encoded genes and their expression patterns to observe their interactions, elucidating novel insights into the evolution of diverse Symbiodiniaceae lineages at the chromosome level (Lin, Song & Morse, 2021).

Efforts are underway to expand the number of high-quality short- and long-read assemblies for cnidarian-associated and free-living Symbiodiniaceae and incorporate these data into taxonomic descriptions (Dougan et al., 2022; McKenna et al., 2021; Voolstra et al., 2021b). Additionally, the two read types can be coupled (e.g., Illumina with PacBio HiFi) to incorporate both the contiguous sequences (>20 kb) of long reads with the low error rate of short reads, allowing robust comparisons of sequence divergence within and across genomes (Ebert et al., 2021). Once more data become available, it may be feasible to incorporate whole-genome information into future taxonomic and systematic revisions to the family (Dougan et al., 2022). However, it will be crucial to achieve consensus on how to use these data to study Symbiodiniaceae diversity and taxonomy. A lack of consistency in methodology and quality standards persists, making cross-study analyses difficult (e.g., Chen et al., 2020). First steps in such standardization have been taken (Voolstra et al., 2021b; McKenna et al., 2021), but will need to be expanded as more genome data become available and the community using these data grows. Additionally, the current costs of completely sequencing the genomes of hundreds of potential Symbiodiniaceae species remains prohibitive. For the near future, feasible alternatives for WGS include using reduced representation phylogenomic approaches (such as those targeting ultraconserved elements; Cowman et al., 2020; Quattrini et al., 2020), full-length rDNA gene amplicons (Tedersoo, Tooming-Klunderud & Anslan, 2018), or entire organellar genome sequences (Liu et al., 2020). These alternatives may represent a compromise between WGS and phylogenetic marker studies by providing an intermediate amount of sequence information for taxonomic accuracy.

How can transcriptomics, proteomics, and single-cell techniques advance our knowledge?

Researchers are keen to make functional inferences about Symbiodiniaceae, which requires focusing on coding regions. To do so requires sequencing the collection of RNAs within the cells (i.e., transcriptomics). Transcriptome sequencing characterizes molecular phenotypes, such as transient responses to the environment, and can reveal differential gene expression among taxa that could reflect selective pressures driving Symbiodiniaceae diversification at the functional level (Avila-Magaña et al., 2021; Bayer et al., 2012; Parkinson et al., 2016). However, the extent of gene expression changes among Symbiodiniaceae is often surprisingly subtle (e.g., Barshis et al., 2014; Davies et al., 2018; Parkinson et al., 2016), although this is a matter of current debate (e.g., Bellantuono et al., 2019; Voolstra et al., 2021d). Furthermore, transcription can be influenced through alternatively spliced transcripts (Lin, 2011; Méndez, Ahlenstiel & Kelleher, 2015), RNA editing (Liew et al., 2017; Mungpakdee et al., 2014; Shoguchi et al., 2020), microRNA interactions (Baumgarten et al., 2018), and methylation of mRNAs (de Mendoza et al., 2018; Lohuis & Miller, 1998; Yang, Li & Lin, 2020). These post-transcriptional modifications create variation in the transcriptome, which can complicate transcriptomic interpretation, but tracking the conservation and divergence of these variations across the Symbiodiniaceae phylogeny may elucidate novel insights into the evolution of diverse lineages.

The primary concern with bulk transcriptomic analysis is that methods often pool transcriptomes from all Symbiodiniaceae cells within a host sample, so only “average” expression profiles can be generated (Traylor-Knowles, 2021). This approach may obscure nuances in the interactions between specific symbiont and host cells, especially for less abundant symbionts. Single-cell transcriptomics (i.e., isolating individual cells and sequencing their transcriptomes) could solve this issue as gene expression could be explored within and among each symbiont cell in hospite. The generation of a cell atlas for the coral Stylophora pistillata has enabled the characterization of fine-scale metabolic interactions between symbionts and host gastrodermal cells (Levy et al., 2021). Single-cell sequencing can also enable high-resolution interrogations of how Symbiodiniaceae and host cells interact during symbiosis establishment, maintenance, and breakdown, particularly when Symbiodiniaceae cells can be isolated from different parts of the host coral that exhibit contrasting physiologies. By comparing expression from symbiont cells derived from different positions in the coral colony, the location and ecological role of Symbiodiniaceae can be characterized, which is a major priority for improving our understanding of symbiont communities within cnidarians.

Proteomic analyses provide an alternative mechanism to explore Symbiodiniaceae physiology and the functional impacts of different symbionts on cnidarian-algal associations, such as metabolic mismatches that occur when hosts associate with atypical (heterologous) symbionts (Sproles et al., 2019). Proteomic analyses use liquid chromatography-mass spectrometry to identify and quantify proteins, which are more directly linked to phenotype than transcript abundance (Feder & Walser, 2005). “Bottom-up” or “shotgun” approaches are commonly employed to identify and quantify as many proteins as possible in a sample in an untargeted manner, and with modern instrumentation, 3,000–4,000 proteins are commonly quantified in established model systems as well as Symbiodiniaceae (Camp et al., 2022; Richards, Merrill & Coon, 2015). Two features of proteomic analyses are particularly powerful. One is the characterization of post-translational modifications, such as protein phosphorylation, oxidation, acetylation, or ubiquitination (Witze et al., 2007). Post-translational modifications help regulate protein activity and are crucial in many biological processes, yet they cannot be detected by pre-translational analyses. The second is the ability to localize proteins to particular cellular compartments by either selectively enriching such compartments (Tortorelli et al., 2022) or fractionating the global cell content. Spatial proteomics aims to resolve the compositional architecture of cells by mapping the subcellular location of thousands of proteins simultaneously (Lundberg & Borner, 2019). Spatial resolution is achieved by separation and differential enrichment of cell content via density gradients or step-wise centrifugation and the subsequent quantitation of relative protein abundance across these fractions (Dunkley et al., 2004; Geladaki et al., 2019). Protein populations from differentially enriched organelles, membranes, and molecular complexes will have consistent but distinct distribution profiles and can thus be classified and mapped accordingly. Global proteome maps are powerful blueprints of the cell and can provide functional context for many uncharacterized proteins. This may be of particular use with Symbiodiniaceae, where sequence homology-based gene annotation is less effective due to their phylogenetic distance from well-studied organisms, and due to the highly derived genomes of dinoflagellates. However, these methods still require high-quality protein model search databases derived from genomic or transcriptomic sequences as they cannot identify proteins from complex samples de novo. Protein expression studies in combination with spatial proteomes will be powerful tools that can, for example, provide insights into the known architectural and physiological changes that accompany the symbiotic engagement of Symbiodiniaceae with their hosts. In the broader context, proteomics as a means to study the functional phenotype of Symbiodiniaceae under various conditions will likely overcome many of the current limitations of gene expression studies in dinoflagellates.

What other omic technologies are promising?

Epigenomics, genome editing, metabolomics, and volatilomics are emerging areas within Symbiodiniaceae research. Epigenomic mechanisms, such as DNA methylation or chromatin modification, can modulate gene expression via gene suppression, gene enhancement, alternative mRNA splicing, or the regulation of spurious transcription without requiring any changes to genomic sequences (Bossdorf, Richards & Pigliucci, 2008; Feil & Fraga, 2012; Foret et al., 2012). DNA methylation is one epigenetic modification that occurs when methyl groups are added to DNA nucleotides, altering how transcriptional proteins bind to promoter regions thereby altering gene expression (Suzuki & Bird, 2008). Symbiodiniaceae have unusually high levels of genome methylation (Lohuis & Miller, 1998). Originally, the high level of methylation raised uncertainty about whether methylation actually played a role in gene regulation, but methylation has been linked to differential gene expression with varying irradiance (Yang, Li & Lin, 2020). Thus far, epigenomic analyses have largely been focused on the host animal, and questions are often centered around how methylation contributes to environmental tolerance (Dixon et al., 2018; Dixon, Bay & Matz, 2014; Dixon & Matz, 2021; Durante et al., 2019; Liew et al., 2018; Putnam, Davidson & Gates, 2016; Putnam & Gates, 2015; Rodriguez-Casariego et al., 2021; Rodríguez-Casariego et al., 2020; Rodriguez-Casariego et al., 2018). Therefore, determining how methylation contributes to Symbiodiniaceae functional diversity requires further exploration.

Overall, Symbiodiniaceae genomes are very difficult to annotate. At present, dinoflagellate genome and transcriptome projects rarely manage to annotate >50% of putative coding sequences via homology searches against genes that have been functionally characterized in other organisms (González-Pech et al., 2021; Stephens et al., 2018). In the future, genome editing could be better developed to knock out Symbiodiniaceae genes with unknown functions, making it easier to determine their biological roles. UV mutagenesis is a classic method for introducing mutations; it was used recently to create photosynthesis mutants via screening of colored mutants (Jinkerson et al., 2022), but its random nature is less than ideal for reverse genetics. RNA silencing is a more targeted approach that could potentially be exploited in Symbiodiniaceae studies (Zhang & Lin, 2019), but the rapidly advancing CRISPR/Cas9 technology is most desirable for its ability to knock out specific genes. Although genome editing efforts for protists have made encouraging progress (Faktorová et al., 2020), success with Symbiodiniaceae remains elusive (Chen et al., 2019; but see Gornick et al., 2022).

Biochemical analyses of Symbiodiniaceae are also in the early stages. Characterization of metabolic products (metabolomics) and volatile organic compounds (volatilomics) can provide insights into molecular cross-talk between partners. Among Symbiodiniaceae, both metabolomic and volatilomic profiles are species-specific, but they also fluctuate with environmental conditions (Klueter et al., 2015; Lawson et al., 2019; Roach et al., 2021). Distinct biochemical profiles reflect the interactions and coadaptation (or lack thereof) between the host and symbiont (Matthews et al., 2017), so biochemical assays can lead to a greater understanding of the drivers of Symbiodiniaceae evolution. As with all the omics methods mentioned so far, if metabolomics and volatilomics are to be used to understand Symbiodiniaceae divergence, more data spanning the phylogeny will be required.

How can we integrate omic technologies?

Integrative approaches that use more than one type of technology may be required to answer intricate research questions about Symbiodiniaceae biology. For example, transcriptomics informs us of gene expression patterns, but cannot reveal protein end-products and how they are used for symbiosis, particularly because transcript abundance does not correlate well with protein levels (Cziesielski et al., 2018; Liang et al., 2021). However, when transcriptomics is integrated with proteomics or metabolomics, phenotypes can be directly observed. Then, tools that make use of multivariate statistics to combine these different types of biological data across studies, such as mixOmics (Rohart et al., 2017) or weighted gene coexpression network analysis (WGCNA; Langfelder & Horvath, 2008), can improve our ability to elucidate molecular mechanisms associated with phenotypes of interest. Thus, integration across several omic technologies (“multiomics”) holds great promise for advancing our understanding of cnidarian-algal symbiosis (e.g., Camp et al., 2022), yet the financial costs associated with using multiple technologies in parallel remains a limiting factor. Additionally, the expertise of one laboratory may be restricted to one major type of analysis or instrument. Therefore, collaborations among different research groups are essential (see “Ensuring an Inclusive Symbiodiniaceae Research Community”). Logistical hurdles to collaborations include how to house and share samples and data, along with the major financial burden. Integrative approaches will remain constrained until these issues are resolved or facilitated through funding agencies.

How can we improve inclusivity in Symbiodiniaceae research?

Despite an increased recognition of the benefits of and need for more diverse representation in science, systemic biases continue to persist in science, limiting our creativity and innovation potential (Ahmadia et al., 2021). In coral reef science, where reefs are mostly found in non-industrial nations, capacity-building through “leveling the playing field” is required to facilitate a more inclusive research community and advance novel and important discoveries (O’Brien, Bart & Garcia, 2020). Marginalized groups within science have been and continue to be excluded from access to many opportunities, including funding, publishing, resources, collaborations, and networking. This exclusion is driven by limited resource availability and systemic racism, sexism, and ableism (Davies et al., 2021; Dzirasa, 2020; Ginther et al., 2011; Hoppe et al., 2019; Taffe & Gilpin, 2021; Brown & Leigh, 2018; Yerbury & Yerbury, 2021). While some progress has been made in the scientific community more broadly, there are still many deeply entrenched biases and critical gender, race, and ethnicity gaps that exist with respect to resource access; these need to be addressed by researchers, including those whose work focuses on Symbiodiniaceae, to ensure a more inclusive scientific community.

Research institutions, hiring committees, and organizers of panels, seminars, and conferences must actively work to change the demographics of scientists by increasing diversity at all career levels–from trainees to senior research scientists in positions of power. Gender, race, and ethnicity biases are rampant in the scientific-hiring process (e.g., Barber et al., 2020; Bennett et al., 2020; Huang et al., 2020); for example, in the United States these biases are particularly strong against Black and Latin scholars (Eaton et al., 2020) and in New Zealand biases are stronger against people of Māori and Pasifika descent (Naepi et al., 2020). In addition to recruitment difficulties, if scholars from these backgrounds are hired, they often face continued challenges that hinder their retention. Recognizing this, people in positions of power in the Symbiodiniaceae scientific community should (1) invest in retaining a diverse workforce by promoting the academic work of minority scientists; (2) provide spaces where researchers can safely report aggressions and other challenges (Valenzuela-Toro & Viglino, 2021); and (3) create programs that provide strong multidimensional mentorship, which serve to support and retain these scholars throughout each career stage (Davies et al., 2021; Montgomery, 2017a, 2017b; Montgomery, Dodson & Johnson, 2014). Moving forward, Symbiodiniaceae researchers need to understand and implement the strategies and proposals that already exist and continue to be put forward regarding increasing recruitment and retention of historically marginalized scholars (e.g., Barber et al., 2020; Chaudhary & Berhe, 2020; Greider et al., 2019). Increasing the diversity of perspectives at the decision-making table leads to more innovative discoveries (Hofstra et al., 2020; Nielsen, Bloch & Schiebinger, 2018), which are desperately needed to meet the formidable challenges of the coral reef crisis.

How can we ensure an equitable publication process for everyone?

Equity and diversity issues exist in the scholarly publication process at multiple levels and across different areas of research. Men are first authors more often than women (Casadevall et al., 2019), notably even when both authors are identified as having contributed equally to the work (Broderick & Casadevall, 2019). Such systematic and implicit gender biases are also evident in the peer-review processes (Calaza et al., 2021). Manuscript authors, irrespective of gender, are also less likely to suggest women reviewers (Fox et al., 2017). Unprofessional reviews disproportionately impact members of underrepresented groups, who report greater self-doubt after receiving such reviews, ultimately reducing scientific productivity overall (Silbiger & Stubler, 2019). Beyond peer evaluation, more men serve in editorial roles than women (Fox et al., 2019; Grinnell et al., 2020; Hafeez et al., 2019; Palser, Lazerwitz & Fotopoulou, 2022; Pinho-Gomes et al., 2021), and editors tend to invite men more often than women to write invited reviews or perspectives. For example, the journal Molecular Ecology, which often publishes research from the Symbiodiniaceae community, found significant gender bias in authorship of invited ‘perspective’ articles, with women only authoring between 17.2–28.6% of these pieces (Baucom, Geraldes & Rieseberg, 2019).

Language biases are also pervasive. English is currently the default language of science, which disadvantages scientists who do not consider English as their primary language (Gordin, 2015). Non-native English speakers spend on average 97 more writing hours than native English speakers on preparation for each manuscript (Ramírez-Castañeda, 2020). In addition, ideas may be lost in translation or are often challenging to explain in a secondary language (Flowerdew, 2001). There are also costs associated with publishing in a non-native language: for example, paying for translation and editorial services. Conversely, fluent English speakers publish more research articles at higher rates than non-English speakers (Taubert et al., 2021). To address English-centric journals, regional journals publish in their native languages (Bordons, 2004), but these publications are read by a smaller readership and are cited less (Di Bitetti & Ferreras, 2017), and thereby viewed as less impactful, and are less likely to be shared widely in the Symbiodiniaceae community.

General actions that can be taken within our community to ensure a more equitable publication process include: (1) increasing diversity on editorial boards, (2) increasing the diversity of invited reviewers, as well as the authors we review for; (3) promoting cost reduction strategies (as implemented in journals like Frontiers, PeerJ, and PLoS One) whereby publication fees are prorated by country or institution type, as well as other strategies that reduce editorial costs for non-native speakers (Taubert et al., 2021); (4) promoting double-blind review processes (Budden et al., 2008); (5) intentionally citing articles led by diverse colleagues (e.g., from diverse gender identities and geographical areas), thereby increasing the diversity of perspectives in the field that contribute to discussion; and (6) gathering data about where and how these inequities exist and working together as a community to take actionable steps for equity in science.

How can we avoid parachute science?

Parachute science, sometimes referred to as “helicopter” or “colonial” science, is a common practice whereby members of the scientific community from higher-income countries fail to involve local/indigenous/native people in an equitable fashion when performing research in lower-income countries (Haelewaters, Hofmann & Romero-Olivares, 2021; Stefanoudis et al., 2021). These practices tend to be more common in ecology and conservation research (de Vos, 2020), including coral reef studies. As a community, we need to understand the largely exploitative history of our discipline and avoid perpetuating it. We should stay informed regarding the history of the lands and peoples who live in the areas in which we conduct our studies. Our specific recommendations include: (1) developing laboratory manuals that include sections outlining values and best practices (including ethics approval and necessary permits); (2) adequately training students to conduct transparent research and develop equitable relationships with members of the host region; (3) supporting the establishment of long-term collaborations and exchange programs to involve local students in research; and (4) including the development of these relationships as important components of the tenure and promotion process in departments and institutions.

Importantly, researchers from institutions in high-income regions (e.g., North America, Australia, Western Europe) should: (1) be sensitive to the many challenges their colleagues in lower-income regions experience, such as a lack of funding, infrastructure, and institutional support; (2) be respectful of these collaborators by treating them as peers and not as assistants, involving them in all steps of the science, and acknowledging their intellectual contributions during discussions, and; (3) be fair to these researchers and their contributions through continued involvement in planning, manuscripts, projects, and grants generated through these collaborations. Together, these strategies can facilitate a more inclusive and collaborative Symbiodiniaceae community (Armenteras, 2021; Belhabib, 2021). Importantly, integration of members of the local community provides a long-term context to the collected scientific data, such as anecdotal observations (e.g., episodes of bleaching) that may be critically informative to the research.

How might we increase accessibility and collaboration?

There is an urgent need to increase the accessibility of Symbiodiniaceae science and foster collaboration, as innovation is necessary to address the coral reef crisis. Toward this goal, we have developed a living, global database of Symbiodiniaceae researchers and their key research expertise (http://symcollab.reefgenomics.org/; Supplemental Information). The database can be queried based on topic and methodology to aid scientists in diversifying their networks. Researchers joining the database are then also invited to participate in an open Slack channel (‘Sym Slack’, https://symslack.slack.com). These resources connect and promote diverse researchers and facilitate discussions of science from different perspectives. The COVID-19 pandemic also showcased the effectiveness of virtual conferences (case in point: this perspective is the product of a virtual workshop). Maintaining hybrid conferences with reduced costs for virtual attendance along with staggered schedules and access to presentation recordings to accommodate different time zones would ensure that the sharing of scientific information is more inclusive, alongside continued efforts to drop conference charges for lower-income countries (e.g., the 15th International Coral Reef Symposium in 2022). Therefore, we encourage conference organizers to facilitate virtual attendance and funding sources for those who have difficulties traveling to foster equitable networking opportunities across people from diverse backgrounds and academic stages. Additionally, incentivizing consortia of Symbiodiniaceae researchers across diverse career stages and locations and explicitly engaging researchers from marginalized backgrounds would lead to stronger capacity building and greater transfer of knowledge. Lastly, we encourage sponsors to continue expanding their funding schemes to support international collaborations. Several examples of these efforts exist, including a new grant solicitation from the United States National Science Foundation, which calls for collaborations with Brazilian scientists through the São Paulo Research Foundation. Similar schemes (e.g., Deutsche Forschungsgemeinschaft) also exist to foster collaboration between researchers in developing countries. The United States Fulbright Program and the European Union Marie Sklodowska-Curie Program are other examples that support collaborations across countries. The Japanese Society for the Promotion of Science provides funding for international exchange and research for graduate students, postdoctoral scholars, and early career scientists. These types of funding mechanisms are important because they promote wealth sharing across countries, encourage collaboration while thwarting parachute science, help with international challenges including research permitting, and ultimately lead to more open sharing of data and ideas within our Symbiodiniaceae research community and beyond.