*5.1. Cultivation-Dependent E*ff*ort*

*S. adareanum* samples collected by SCUBA in 2004 and 2007 were used for cultivation (Table S5). The 2004 specimen were archived in 20% glycerol at −80 ◦C until processing by manual homogenization using sterilized mortar and pestle prior to plating a suspension onto marine agar 2216 (BD Difco ™, Franklin Lakes, NJ, USA) plates in 2006. The 2007 samples were homogenized immediately following collection using sterilized mortar and pestle, and suspensions were prepared in 1X marine broth 2216

or filter-sterilized Antarctic seawater then transferred at 4 ◦C to DRI. Shortly after (within 1 month of collection), the 2007 isolates were cultivated on three types of media in which suspensions initially stored in marine broth 2216 were plated onto marine agar (2216), while homogenate preparations stored in seawater were plated onto VNSS agar media [56] and Antarctic seawater agar plates amended with 3 g yeas<sup>t</sup> extract (BD Difco ™), 5 g peptone (BD Difco ™), and 0.2 g casein hydroslyate (BD Difco ™) per liter. Colonies were selected from initial plates, and purified through three rounds of growth on the same media they were isolated on.

### *5.2. Field Sample Collections for Cultivation-Independent E*ff*orts*

Next, a spatial survey of *Synoicum adareanum* was executed in which samples were collected by SCUBA in austral fall between 23 March and 3 April 2011. Seven sampling sites (depths 24.7–31 m; Table S6) around the region accessible by Zodiac boat from Palmer Station were selected in which we sampled in a nested design where three multi-lobed colonies were selected from each site, and three lobes per colony were sampled (Figure 1). At each dive site, multi-lobed colonies were collected by hand into separate mesh collecting bags. Underwater video [57] was taken at each site, then video footage was observed to note general ecosystem characteristics (% cover of major benthic species and algae). In total, 63 *S. adareanum* lobes were sampled (9 from each site). Samples were transported to Palmer Station on ice and multi-lobed colony samples were placed in sterile Whirl-Pak ®bags (Nasco, Fort Atkinson, WI, USA) and frozen at −80 ◦C, until processing at DRI and USF. At DRI, frozen pedunculated *S. adareanum* lobes were separated, then cut longitudinally in half for parallel processing through DNA and palmerolide detection pipelines.

Then, to address whether the composition of the SaM was distinct from the free-living bacterioplankton (< 2.5 μm fraction), we considered the overlap in membership between the SaM and bacterioplankton in the water column. To accomplish this, we used a reference seawater data set represented by samples that had been collected in the period February–March 2008 (five samples) and in the period August–September 2008 (nine samples) from LTER Station B near Anvers Island (east of the Bonaparte Point dive site), and at a few other locations in the region (Table S7). Seawater samples were collected by a submersible pump and acid washed silicone tubing at 10 m at Station B, and using a rosette equipped with 12 L Niskin bottles for the o ffshore samples at 10 and 500 m (2 samples each depth). The February–March seawater samples were processed using in-line filtration with a 2.5 μm filter (Polygard, MilliporeSigma, Burlington MA, USA) to screen larger organisms and bacterioplankton were concentrated using a tangential flow filtration system and the cells were harvested on 25 mm and 0.2 μm Supor filters (MilliporeSigma). The August–September seawater samples were processed using inline filtration with a 3.0 μm filter (Versapor, MilliporeSigma), and then bacterioplankton was collected onto 0.2 μm Sterivex (MilliporeSigma) filters using a multichannel peristaltic pump (Masterflex ®, Cole-Parmer, Vernon Hills, IL, USA). All filters were immersed in sucrose:Tris:EDTA bu ffer [58] and stored frozen at −80 ◦C until extraction.

### *5.3. Palmerolide A Screening*

Sixty-three frozen *S. adareanum* lobes were cut in half. Half lobes were lyophilized and then exhaustively extracted using dichloromethane for three days, followed by methanol for three days. The extracts were combined and dried on a rotary evaporator. The extracts were filtered, dried down, and reconstituted at 1.0 mg/mL to ensure the injected concentration was consistent. The residue was subjected to liquid chromatography-mass spectrometry (LC–MS) analysis using a H2O:ACN gradient with constant 0.05% formic acid. The high-resolution mass spectra were recorded on an Agilent Technologies 6230 electrospray ionization Time-of-Flight (ESI-ToF) spectrometer. LC–MS was performed using a C-18 Kinetex analytical column (50 × 2.1 mm; Phenomenex, Torrance, CA, USA). The presence of PalA was verified using MS/MS on an Agilent Technologies 6540 UHD Accurate-Mass QTOF LC–MS.

The *S. adareanum*-associated microbial culture collection was screened for the presence of PalA. Isolates were cultivated in 30 mL volumes, and the resulting biomass was lyophilized and screened by HPLC. Each sample was analyzed in ESI-SIM mode targeting masses of 585 amu and 607 amu. The reversed-phase chromatographic analysis consisted of a 0.7 mL × min−<sup>1</sup> solvent gradient from 80% H2O:CH3CN to 100% CH3CN with constant 0.05% formic acid using the same C-18 column as above. The analysis was conducted for over 18 min. PalA had a retention time of approximately 14 min. The twenty-four sample sequence was followed by a PalA standard to confirm its analytical characteristics.

### *5.4. S. Adareanum-Associated Microbial Cell Preparation*

The outer 1–2 mm of the ascidian tissue, which could contain surface-associated microorganisms, was removed using a sterilized scalpel before sectioning tissue subsamples (~0.2 g) of frozen (−80 ◦C) *S. adareanum* ( 12 lobe sections). Tissue samples were diced with a sterile scalpel before homogenization in sterile autoclaved and filtered seawater (1 mL) in 2 mL tubes. Each sample was homogenized (MiniLys, Bertin Instruments, Montigny-le-Bretonneux, France) using sterile CK28 beads (Precellys, Bertin Instruments) 3 times at 5000 rpm for 20 s, samples were placed on ice in between each homogenization. Homogenates were centrifuged at 500× *g* at 4 ◦C for 5 min to pellet tissue debris. The supernatant was removed to a new tube for a second spin at the same conditions. This supernatant was decanted and the cell suspension centrifuged at 12,000× *g* at 4 ◦C for 5 min to collect the microbial cells. Suspensions were stored on ice, then entered an extraction pipeline, in which 12 samples were processed in parallel on the QIAvac 24 Plus Manifold (Qiagen Inc., Germantown, MD, USA).
