*3.2. Collection and Taxonomy of Talaromyces sp. CMB-TU011*

The isolation and taxonomy of *Talaromyces* sp. CMB-TU011 from an unidentified tunicate collected from Tweed Heads, NSW, Australia, has been previously reported [7].

#### *3.3. Cultivation and Fractionation of Talaromyces sp. CMB-TU011*

A loop of spores from a 7-day old M1-salt culture of CMB-TU011 was streaked on ISP-4 agar plates (×200) and incubated for 20 days at 26.5 ◦C, after which the combined agar/mycelia was extracted with EtOAc (3 × 500 mL) and concentrated in vacuo to yield an extract (264 mg), which was partitioned between n-hexane and aqueous MeOH to give hexane (70 mg) and MeOH (194 mg) soluble fractions. The MeOH fraction was subjected to gel chromatography (Sephadex® LH-20 (Merck, Darmstadt, Germany) in MeOH) to obtain 15 fractions, which were combined based on HPLC-DAD-MS analysis to yield a talarolides-enriched fraction (30.5 mg). Further semi-preparative HPLC (Agilent Zorbax Eclipse C8 column, 5 μm, 9.4 × 250 mm, 32% MeCN/H2O isocratic elution at 3.0 mL/min inclusive of an isocratic 0.01% TFA/MeCN modifier) was used to yield talarolide A (**1**) (*t*<sup>R</sup> 25.6 min, 3.4 mg, 1.3%), talarolide B (**2**) (*t*<sup>R</sup> 21.3 min, 1.1 mg, 0.42%), talarolide C (**3**) (*t*<sup>R</sup> 16.1 min, 1.1 mg, 0.42%), and talarolide D (**4**) (*t*<sup>R</sup> 19.3 min, 0.9 mg, 0.34%). (Note: % yields are calculated as a weight to weight of the EtOAc extract) (Figure S2).

Talarolide A (**1**): white powder; [α]D<sup>25</sup> –17 (c 0.12, MeOH); 1D and 2D NMR (DMSO-*d*6) see Tables 1, 2 and S2, and Figures S3–S8; UPLC-QTOF (MS/MS) fragmentation see Figure S42; HRESI(+)MS *m*/*z* 740.3978 [M + Na]+ (calcd for C35H55N7O9Na 740.3953) (Figure S9).

Talarolide B (**2**): colorless amorphous solid; [α]D<sup>25</sup> –15 (c 0.059, MeOH); NMR (DMSO*d*6) see Tables 1, 2 and S3, and Figures S10–S16; UPLC-QTOF (MS/MS) fragmentation see Figure S43; HRESI(+)MS *m*/*z* 724.4008 [M + Na]<sup>+</sup> (calcd for C35H55N7O8Na 724.4004) (Figure S17).

Talarolide C (**3**): colorless amorphous solid; [α]D<sup>25</sup> –15 (c 0.072, MeOH); NMR (DMSO*d*6) see Tables 1, 2 and S4, and Figures S18–S25; UPLC-QTOF (MS/MS) fragmentation see Figure S44; HRESI(+)MS *m*/*z* 726.3827 [M + Na]<sup>+</sup> (calcd for C34H53N7O9Na 726.3797) (Figure S25).

Talarolide D (**4**): colorless amorphous solid; [α]D<sup>25</sup> –14 (c 0.054, MeOH); NMR (DMSO*d*6) see Tables 1, 2 and S5, and Figures S26–S32; UPLC-QTOF (MS/MS) fragmentation see Figure S45; HRESI(+)MS *m*/*z* 726.3827 [M + Na]<sup>+</sup> (calcd for C34H53N7O9Na 726.3797) (Figure S33).
