The genes and spacers of the rDNA operon have provided useful genetic markers disclosing phylogenetic relationships in the Symbiodinium genus. Small subunit (SSU) rDNA markers first revealed different lineages or ‘clades’ within the genus Symbiodinium (Rowan and Powers 1991), which is now divided in nine broad genetic clades, A-I (Pochon and Gates 2010). The capital letters A, B, C, D, E, F, G H or I indicate clade designation. The use of the more variable internal transcribed spacer unit 1 and 2 (ITS1/ITS2) revealed significant diversity within each of the described broad cladal groupings, which are described using a alphanumerical system, A1, 2, 3, etc. and similarly for each of the other clades. The use of such molecular markers with a higher taxonomic resolution has facilitated further description of the genus Symbiodinium and its huge diversity of genetically and ecologically distinct sub-cladal types (van Oppen et al. 2001; Iglesias-Prieto et al. 2004; LaJeunesse et al. 2004a; LaJeunesse et al. 2004b; Warner et al. 2006; Pochon et al. 2007; Sampayo et al. 2007; Frade et al. 2008).
Considering the multi-copy nature of the ribosomal gene regions, and incomplete or ongoing homogenization of faster evolving areas such as the ITS regions, some types contain more than one co-dominant repeat within their genome. These co-dominant repeats are typically closely related to the ancestral sequence, containing a single derived change such as a base substitution or indel. Co-dominant intragenomic ITS variants are further identified by designation of a lowercase letter (C1a, C1b, C1c, …, C1z, and continuing onto double letter (C1aa, C1bb etc. signifying a single sequence after the number of letters in the alphabet had been exceeded by the number of types, cf. (LaJeunesse 2005). If the ancestral sequence (for instance C1 or C3) has more than one codominant sequence variant within the genome of a single symbiont these are separated by a hyphen (for instance C1m-aa contains three co-dominant sequences within the genome of a single Symbiodinium type, namely C1, C1m and C1aa). The use of lowercase letters to designate co-dominant sequence variants is also applied in other clades (for example A3a, B1n or G2a) but clade C types appear to contain by far the highest levels of heterogeneity across ribosomal repeat regions.
While the same system was applied initially, the nomenclature was altered for clade D Symbiodinium because similar sequences occurred as co-dominant repeats across various types (LaJeunesse et al. 2010a). The use of an extended numerical system, such as that used for clade D might have, in hindsight been more practical considering the high amount of co-dominant repeats in association with ancestral C types (such as C1 and C3). This is why it is possible to find D1a in the literature that is now referred to as D1-4 or Symbiodinium trenchi (LaJeunesse et al. 2010b). A D1-4 profile includes a D1 sequence with an additional D4 sequence. Combinations of more intragenomic variants exist as well, such as D4-5-9 (LaJeunesse et al. 2010b).
Mixed Symbiodinium populations either from the same clade or different clades are found, and mixture of different clades are far less common on the GBR compared to Caribbean symbioses. While mixes of different clades are easily recognized, this is more complex when trying to distinguish whether closely related types within a clade are present within a single host individual since this could also be the result of co-dominant copies occurring within the same genome. As a rule of thumb, mixtures are assigned following a set of rules (LaJeunesse et al. 2003; Sampayo et al. 2009): 1) Most symbionts types are distinguished using fingerprinting techniques that take into account the entire fingerprint but symbiont names are assigned according to the (co-dominant) sequence because of its evolutionary relevance. When two symbionts are present both fingerprints are visible independently of each other. 2) Generally, co-dominant variants differ only by several base pair changes and form characteristic heteroduplexes. 3) Stability in band intensity across various samples from similar or different hosts within and amongst locations from large biogeographic scales indicates the presence of co-dominant rDNA repeats within the genome of a single symbiont rather than a mix of symbionts. Generally band intensities are not variable, and if a mixture of two or more symbionts were to be present in this manner, this would require concentrations of each symbiont to be equal across widely distant regions and spanning highly diverse environments. This does however, at times, require multiple samples to be analyzed of the same organism.
As a final note, the multi-copy nature and intragenomic variation associated with the rDNA region have the potential to overestimate sequence diversity, when methodologies cannot distinguish between intragenomic variations versus distinct organisms, and thereby impede ecological interpretations. For example, this can occur when the different Symbiodinium sequences that are obtained using bacterially cloning of the rDNA are interpreted as if representing distinct Symbiodinium types (Thornhill et al. 2007). As such, a distinction is made between the occurrence of rare intragenomic variants and dominant sequence variants (Thornhill et al. 2007; LaJeunesse and Thornhill 2011), which have evolutionary relevance.
The database contains taxonomic information on Symbiodinium identity obtained using different molecular marker regions (18S rDNA, D1/D2 LSU rDNA, psbA minicircle (psbAncr), ITS1 and ITS2) analyzed with a variety of molecular techniques (restriction fragment length polymorphism (RFLP), single stranded conformation polymorphism (SSCP) and denaturant gradient gel electrophoresis (DGGE). As a result the data differs in its level of taxonomic resolution (Sampayo et al. 2009) and different nomenclatures are used for genetically identical Symbiodinium (namely ITS1 and ITS2). For example ITS1 type C2 refers to ITS2 type C3. It should be noted that the earlier mentioned methods generally pick up the most dominant sequences and background types may not be detected if they constitute less than 5-10% of the total population (Mieog et al. 2007).
Recently sequences of the non-coding region of the psbA minicircle (psbAncr) were used to provide detailed genetic resolution within the ITS lineage (LaJeunesse and Thornhill 2011). The psbAnc displays low intragenomic variability in Symbiodinium and is more comparable to single-copy markers. Although this rapidly evolving marker is limited to within clade comparison it has already shown to be valuable by allowing validation of widely used ITS1/ITS2 markers and their accuracy to identify the dominant sequence in a coral host. Moreover the added resolution in combination with more conserved genetic markers has the potential to provide the resolution necessary to resolve Symbiodinium species classifications (LaJeunesse and Thornhill 2011).