Dataset: Cultures isolated from A. bipinnata 2008-2016
Deployment: Coffroth_2008-16

Symbionts were isolated from adult colonies of Antillogorgia bipinnata following the protocol outline in Santos et al 2001. Briefly, a small piece (1-2 cm) of the branch was ground in a glass tissue homogenizer with 2 ml of filtered seawater (FSW) and poured through a series of meshes (250 µm on top, then 120 µm, then 70 µm mesh) into a 15 ml tube.  The mesh was washed with 1 ml FSW several times for a final volume between 3 and 10 ml. This slurry was spun for 5 min at 500-800 rpm on a Beckman J6-HC centrifuge to pellet symbiont cells, the supernatant removed and resuspended in 10 ml of FSW.  This step was repeated again and then the pellet was resuspended in 1.0 ml of F/2 (Gulliard and Ryther 1962).  Cultures were started by using 20-50 µl of the resuspended pellets to inoculate 30ml of F/2 and incubated at the appropriate temperature.     

Clade identity and Cp-type were determined following the protocols outline in Santos et al (2003). Briefly, DNA was amplified using the primers HYPERUP and HYPERDN on and MJ96 or BioRad thermocyclers. PCR products were visualized and scored using size standards on a LI-COR 4200 NEN® Global IR2 DNA sequencing system as specified in Santos et al (2003). Putative species identity was based on sequence analysis of B7SYM15 flanking region (LaJeunesse et al. 2012, Parkinson et al. 2015).  Briefly, the B7SYM15 flanking region was amplified and directly sequenced in 5’ and 3’ directions on a 3730XL DNA Analyzer (High Throughput Genomics Center, University of Washington). Sequences were compared to known species within the GenBank database using BLAST- Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).