Dataset: Sequences for symbiont psbA and ORF host sequences

Genetic analyses

A small chip (2‒3mm) of each fragment (preserved in 100% ethanol and stored at -80°C) was used to extract gDNA, cf. methods described in (LaJeunesse et al., 2003) and the DNA eluted in ddH2O. The coral species field-based identification using morphological characteristics (sensu Pocillopora damicornis) was confirmed with a genetic assay (Schmidt-Roach et al., 2013) using the mitochondrial ORF region. The DNA was diluted 1:20 and PCR was done using the FATP/RORF primers cf. (Flot and Tillier, 2007) and NEB TAQ Polymerase (M0320) following the manufacturers routine PCR protocol under cycling conditions: 95°C 2 min initial denaturation, 35 cycles of 94°C 30s, 53°C 30s, 68°C 1 min, and a final extension of 68°C for 10 min. All but two specimens (one from each habitat) were successfully amplified; these were cleaned using ExoSap-IT and Sanger sequenced in a single direction using the forward FATP primer at the Australian Genome Research Facility (Brisbane, Australia). In addition, the species of resident coral endosymbionts (Symbiodiniaceae) were identified using PCR amplification of the ITS2 rDNA region followed by denaturing gradient electrophoresis alongside local ‘marker’ specimens from Pocillopora species with previously confirmed symbiont identifications (cf. Sampayo et al., 2009). To cross-confirm, dominant bands of characteristic profiles were excised, re-amplified and sequenced using the forward ITS2intfor and reverse ITS2reverse primers (cf. LaJeunesse et al., 2003; Sampayo et al., 2009). In addition, the chloroplast minicircle psbA non-coding and partial coding region were amplified using two different primer sets and conditions. Initially, the 7.4-Forw/7.8-Rev primers were used cf. (Moore et al. 2003) but some samples failed to amplify. Some of these yielded successful PCR amplicons with the psbAFor-1/psbARev-1 primers (LaJeunesse and Thornhill, 2011). Successful reactions were cleaned using ExoSap-IT and bi-directionally Sanger sequenced with their respective primers at the Australian Genome Research Facility. Sequence chromatograms for all regions (host mtORF; symbiont ITS2 rDNA and chloroplast psbA) were visually inspected and compared to holotype sequences from described Pocillopora species (Schmidt-Roach et al., 2013) and recently described symbiont species derived from these coral species (Turnham et al., 2021). The mtORF sequences from the study specimens all had 100% match with the P. damicornis (GenBank Accession numbers OP296503 – OP296521; 100% match to Pocillopora type alpha cf. (Schmidt-Roach et al., 2013), GenBank accession numbers JX985598 and JX985606). The ITS2 rDNA profiles as well as sequences matched previously described Cladocopium type ‘C1b-c, 42a’ from Heron Reef (Sampayo et al. 2007). For the psbA, 4 out of 24 samples returned poor sequence reads (2 from each environment). The remaining 20 psbA sequences were aligned with inclusion of reference sequences for recently described Cladocopium latusorum and C. pacificum (GenBank Accession numbers MW819755 - MW819779 and MW861711 - MW861727, respectively) as well as the sequence for C. goreaii (Turnham et al., 2021). Sequences were aligned using Geneious (MAFFT), checked and adjusted by eye, which was particularly important given the large number of indels present in the psbA sequence dataset. All sequences were truncated to the 7.4-Forw and 7.8-Rev primer binding sites, given that no differences were seen in the sequence regions extending outward to the psbA coding region psbAFor1/psbARev1 binding sites. The truncates sequences were submitted to GenBank under accession numbers OP279755 – OP279774 (https://www.ncbi.nlm.nih.gov/popset/2310683771).   All indels were adjusted to represent a single base change, using gaps as a 5th character state, in subsequent phylogenetic analyses. A psbA (partial coding, and entire non-coding region) maximum parsimony majority-rule phylogeny was constructed using a heuristic search with bootstrap values calculated (TBR branch swapping, random addition, 1000 replicates) in PAUP vs. 4.0a (build 169; Turnham et al., 2021).

For more detailed information, please see: Brown et al. (2022).