Field collections
Samples of Scleractinia from various families were sampled by SCUBA with a hammer and chisel throughout various sites across the Indo-Pacific including the reefs of Palau, the Andaman Sea of western Thailand, Zanzibar of Tanzania, the Phoenix Islands, and the southern Great Barrier Reef (GBR) of Australia, New Caledonia, and Brazil. The samples used were collected from 2002–2018, including some from previously published diversity surveys (Butler et al., 2023). Coral fragments of approximately 1-3 cm2 were preserved in 20% DMSO buffer or 100% ethanol and stored at -20°C.

Genetic analyses
A modified Promega Wizard protocol was used to extract genomic DNA from 0.5 cm2 of each fragment (LaJeunesse et al., 2003). All samples were identified using the PCR-DGGE profiling of the internal transcribed space region 2 (ITS2) rDNA of the ribosomal array (LaJeunesse, 2002; T. C. LaJeunesse et al., 2004). Amplified products were examined with gel electrophoresis with a CB Scientific system with 45–80% denaturing gradient gels. Fingerprints of each sample were visualized using SYBR Green stain and compared to known standards and/or sequenced to confirm the identity of the ITS2 ‘type’ designation. Cladocopium samples typed as C1, C3, C3u, and C40 were investigated by using additional genetic markers, including the large ribosomal subunit (LSU) (Zardoya, Costas, Lopez-Rodas, Garrido-Pertierra, & Bautista, 1995), mitochondrial cytochrome b (cob), mitochondrial cytochrome oxidase 1 (cox 1) (H. Zhang, Bhattacharya, Maranda, & Lin, 2008), partial chloroplast cp23S (Z. Zhang, Green, & Cavalier-Smith, 2000), and the non-coding region of the psbA minicircle (psbAncr) (Moore, Ferguson, Loh, Hoegh-Guldberg, & Carter, 2003). These conventional phylogenetic markers (i.e., LSU, cob, cox 1, cp23S) were used to resolve lineages within Cladocopium, and then concatenated together to further differentiate putative species lineages (LaJeunesse et al. 2015, Lee et al. 2020). For comparison, the psbAncr region is a more rapidly evolving locus that provides finer-scale inter-species and intra-species resolution (LaJeunesse & Thornhill, 2011; Moore et al., 2003).

Successful PCR amplicons of each genetic marker were Sanger sequenced using the BigDye Terminator 3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA), with sequences analyzed at the Penn State University Genomics Core Facility on an Applied Biosciences Sequencer. The psbAncr region was sequenced in both directions. Sequences were quality checked, assembled, and edited in Geneious v 11.0.5 (Biomatters Ltd, Auckland, NZ).

Microsatellite characterization
To test for reproductive isolation in the C3-radiation Cladocopium samples, a total of seven microsatellite loci were used to generate multilocus genotypes using the following primers: SgrSpl_22, SgrSpl_24, SgrSpl_25, SgrSpl_78, Spl_1a, and Spl_16 were developed in Drew C. Wham, Carmichael, and LaJeunesse (2014), while the remaining locus (C1.05) was described by Bay, Howells, and van Oppen (2009). These loci are unlinked and have sufficient allelic diversity for population genetic analyses (Drew C. Wham et al., 2014). Loci were amplified in reaction volumes of 10 μL according to their respective development studies. Fragment analysis was performed on an ABI 3730 Genetic Analyzer (Applied Biosystems) using a 500-bp standard (LIZ-labeled) at the Penn State University Nucleic Acid Facility. Fragment sizes were scored visually using Geneious (v.2020.2.4). While Cladocopium spp. are haploid organisms, two alleles are often observed in each sample (D. C. Wham & LaJeunesse, 2016). This observation is most likely the result of Cladocopium having a partial or extensively duplicated genome (Chen et al., 2022; Liu et al., 2018). Previous work has shown that psbAncr sequences are consistent with microsatellite data when differentiating lineages in the C1-radiation (Turnham et al. 2021). Microsatellite analyses were only conducted on symbionts from the C3-radiation, which, relative to the C1-radiation, were less differentiated by psbAncr sequence divergence (Table S2 in Butler et al., 2023).