*3.1. Structure Elucidation of Compounds* **1**–**7**

In total, seven novel compounds (**1**−**7**) and xanthoquinodin B4 (**8**) [22], were isolated from the ex-type strain of *Jugulospora vestita* (Figure 1). Their structures were elucidated by 1D- and 2D-NMR spectroscopy (Supplementary Figures S1–S6, S8–S13, S15–S20, S22–S27, S29–S34, S36–S41, and S43–S48), HR-MS (Supplementary Figures S7, S14, S21, S28, S35, S42, and S49), and ECD spectra.

**Figure 1.** Chemical Structures of Compounds **1**–**8**.

Compound **1** was obtained as a yellow powder and its molecular formula was established as C35H32O12 (20 degrees of unsaturation) according to the mass ion peak at *m*/*z* 645.1967 [M + H]<sup>+</sup> in the HRESIMS spectrum. The 1H- and 13C-NMR spectra (Table 1), accompanied with heteronuclear single quantum coherence (HSQC) correlations, revealed signals of two methyl (δ<sup>C</sup> 22.0, 13.5), one methoxy (δ<sup>C</sup> 53.3), five sp3 methylenes (δ<sup>C</sup> 36.2, 35.1, 27.5, 23.7, and 18.4), three sp<sup>3</sup> methines

(δ<sup>C</sup> 72.8, 71.8, and 37.3), five aromatic methines (δ<sup>C</sup> 132.4, 131.9, 123.1, 119.3, and 110.9), seventeen sp<sup>2</sup> quaternary carbons, and two sp<sup>3</sup> quaternary carbons (δ<sup>C</sup> 41.5 and 84.3 (oxygenated)). In the 1H–1H correlation spectroscopy (1H–1H COSY) spectrum, there were three isolated spin systems (H-3–H-4–H-5, H-11 –H-12 –H-13 , and H-19 –H-20 –H-21 ). The heteronuclear multiple bond correlation (HMBC) spectrum showed correlations from H-21 (δ<sup>H</sup> 0.86) to C-20 and C-19 , from H-16 (δ<sup>H</sup> 2.38) to C-3 , C-4 , and C-5 , from H-3 (δ<sup>H</sup> 6.89) to C-1 , C-5 , C-7 , and C-16 , from H-5 (δ<sup>H</sup> 6.81) to C-3 , C-6 , C-7 , and C-16 , from H-1 (δ<sup>H</sup> 5.96) to C-2 , C-3 , C-7 , C-9 , C-13 , C-14 , C-15 , C-18 , and C-8 (weak correlation), and from H-13 (δ<sup>H</sup> 6.05) to C-1 , C-9 , C-11 , C-14 , and C-10 (weak correlation), above analysis, indicating an anthraquinone moiety (ABC-ring) with 1 -butyrate group. Moreover, the HMBC correlations from H-3 (δ<sup>H</sup> 4.25) to C-2, C-4, C-5, and C-15, from H-5 (δ<sup>H</sup> 2.65) to C-3, C-4, C-6, and C-7, from H-13 (δ<sup>H</sup> 6.07) to C-9, C-11, and C-15 , and from 15-OCH3 (δ<sup>H</sup> 3.67) to C-15 revealed the rest part as the xanthone moiety (DEF-ring). In addition, the key HMBC correlations from H-15 (δ<sup>H</sup> 2.74 and 2.68) to C-1 , C-9 , C-13 , C-14 , C-11, C-12, and C-13, and from OH-10 (δ<sup>H</sup> 11.76) to C-9, C-14, C-11, and C-11 (weak correlation) indicated that a methylene (C-15 ) linked these two moieties at C-12 and C-14 , as well as C-11 connected to C-11 .

Based on the combined above NMR analysis data and the molecular formula, the planar structure of **1** was elucidated as xanthone–anthraquinone heterodimer similar to xanthoquinodin A6 [21] and xanthoquinodin A9 [23]. The difference is at C-1 , where the hydroxyl is replaced by the butyl side chain of **1**. Noticeably, the β-keto-enol tautomeric system showed at C-8 (δ<sup>C</sup> 185.8) and C-10 (δ<sup>C</sup> 186.0), which both displayed keto carbonyl property in carbon chemical shift data. The Δ12 ,13 double-bond was revealed as *Z* for the small coupling constant (*J*H12 H13 = 8.5 Hz). There are five chiral centers (C-2, C-3, C-1 , C-11 , and C-14 ) in compound **1**, whose relative configuration was assigned by analysis of NOESY correlations and 1H,1H coupling constants. Since bridging carbons C-12 and C-13 must be on the same side, the relative configurations at C-11 and C-14 were deduced as *S* and *R*, respectively. In the NOESY spectrum, the strong intensity correlations of H-1 with Hα-15 and Hβ-15 indicated the *S\** configuration at C-1 . The methyl ester group was in axial bond orientation positioned between the F and G ring. A diaxial orientation was deduced for both H-3 and H-4a due to the large coupling constant (*J*H3H4a = 12.3 Hz) between these protons. For the assignment of absolute configuration of **1**, the ECD spectrum (Figure 2a) was measured, and showed a similar pattern as the spectrum of xanthoquinodin A6 [22], proving that both compounds possessed the same stereochemistry. On the basis of the above data, the absolute configuration of compound **1** was assigned as 2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*, and named xanthoquinodin A11.

Compound **2** was obtained as a yellow amorphous solid. The molecular ion cluster at *m*/*z* 645.1967 [M + H]<sup>+</sup> in the HRESIMS spectrum indicated that the molecular formula of **2** was C35H32O12 (20 degrees of unsaturation). The 1H- and 13C-NMR spectra, accompanied with HSQC correlations, revealed signals of two methyl (δ<sup>C</sup> 22.1, 13.5), one methoxy (δ<sup>C</sup> 53.3), five sp3 methylenes (δ<sup>C</sup> 36.2, 35.0, 27.7, 23.9, and 18.4), three sp<sup>3</sup> methines (δ<sup>C</sup> 72.7, 71.7, and 38.6), five aromatic methines (δ<sup>C</sup> 132.3, 131.8, 123.2, 119.3, and 114.2), seventeen sp<sup>2</sup> quaternary carbons, and two sp<sup>3</sup> quaternary carbons (δ<sup>C</sup> 41.6 and 85.3 (oxygenated)). The same molecular formulae and the resemblance of NMR spectroscopic data of **1** and **2** (Table 1) suggested that they were isomers. The main differences between 13C NMR spectrum of **1** and **2** were the upfield shifts at C-13 (Δδ –2.0) and C-14 (Δδ –3.6) in **2**, as well as the clearly downfield shifts at C-10 (Δδ +3.3) and C-11 (Δδ +3.3). Moreover, the strong HMBC correlations from OH-10 (δ<sup>H</sup> 11.05) to aromatic methine δ<sup>C</sup> 114.2 indicated that apart from the C-12–C-15 –C-14 bridge, the two moiety (ABC-ring and FEG-ring) linked by C-13 connected to C-11 , which is similar to xanthoquinodin B series of structures. Meanwhile, the configurations of C-11 , C-14 , C-2, C-3, and 1 -butyrate were assigned as the same as those of **1**. Furthermore, the experimental ECD curves (Figure 2b) of **2** displayed the same as those previously given for xanthoquinodin B4 [23].

Therefore, the absolute configuration of **2** was assigned as 2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*. The trivial name of xanthoquinodin B10 was given for compound **2**.

Compound **3** was obtained as a yellow amorphous solid with a molecular formula of C35H32O12 (20 degrees of unsaturation) based on the mass ion peak at *m*/*z* 645.1966 [M + H]<sup>+</sup> in its HRESIMS spectrum. The 1H- and 13C-NMR spectra, accompanied with heteronuclear single quantum coherence (HSQC) correlations, revealed signals of two methyl (δ<sup>C</sup> 22.1, 13.5), one methoxy (δ<sup>C</sup> 53.6), five sp<sup>3</sup> methylenes (δ<sup>C</sup> 36.2, 35.0, 24.4, 22.9, and 18.4), three sp3 methines (δ<sup>C</sup> 72.7, 66.9, and 37.9), five aromatic methines (δ<sup>C</sup> 132.7, 131.6, 123.2, 119.3, and 114.7), seventeen sp2 quaternary carbons, and two sp3 quaternary carbons (δ<sup>C</sup> 41.4 and 84.5 (oxygenated)).

**Figure 2.** Electronic Circular Dichroism (ECD) spectra of compounds **1**–**7** measured in MeOH, (**a**) ECD spectrum of **1** (2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*), (**b**) ECD spectra of **2** (2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*), **4** (2*S*, 3*S*, 5*S*, 1 *S*, 11 *S*, and 14 *R*), and **5** (2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*), (**c**) ECD spectrum of **3** (2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R*), (**d**) ECD spectra of **6** (2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R*) and **7** (2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R*).

The NMR spectroscopic data of **3** (Table 1) resembled those of **2**, with the main chemical shift difference (δ<sup>C</sup> and δH) located at the positions 2–7 in the F-ring, which indicated epimerization at C-2 or C-3. The small coupling constant (4.0 and 2.0 Hz) of the C-3 methine proton demonstrated an equatorial bond of H-3. Therefore, configurations of chiral carbons C-2 and C-3 were opposite, indicating that compound **2** and **3** were epimers at C-2. In addition, the experimental ECD spectra of compound **2** (Figure 2b) and **3** (Figure 2c) were different at 200–250 nm, which displayed similar patterns to those differentiated for xanthoquinodin B4 and xanthoquinodin B5 [22]. Therefore, the absolute configuration of **3** was assigned as 2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R*. Xanthoquinodin B11 was the name chosen for compound **3**.

Compound **4** was obtained as a yellow crystalline solid. The mass ion peak at *m*/*z* 661.1915 [M + H]<sup>+</sup> in its HRESIMS spectrum indicated that the molecular formula of **4** was C35H33O13 (20 degrees of unsaturation). The 1H- and 13C-NMR spectra, accompanied with HSQC correlations, revealed signals of two methyl (δ<sup>C</sup> 22.1, 13.5), onrfde methoxy (δ<sup>C</sup> 53.4), four sp<sup>3</sup> methylenes (δ<sup>C</sup> 36.2, 35.1, 32.7, and 18.4), four sp<sup>3</sup> methines (δ<sup>C</sup> 72.7, 68.6, 65.9, and 38.7), five aromatic methines (δ<sup>C</sup> 132.4, 131.7, 123.3, 119.4, and 114.3), seventeen sp2 quaternary carbons, and two sp3 quaternary carbons (δ<sup>C</sup> 41.6 and 85.5 (oxygenated)). The NMR spectroscopic data of **4** (Table 1) resembled those of **2**, except for the main chemical shift difference (δ<sup>C</sup> and δH) located at the positions 3–6 in the F-ring, and C-5 (δ<sup>C</sup> 65.9), which was oxygenated methine. Compared to **2**, there were one more oxygen and hydrogen atom in **4** according to the molecular formulae. On the basis of the above analysis, **4** was proposed as a new xanthoquinodin compound that possesses one additional hydroxyl substituent at the C-5. The configurations at C-2, C-3, C-1 , C-11 , and C-14 were assigned as the same as those of **1** and **2** because of the similar corresponding NMR data (Table 1). The configuration at C-5 was confirmed as *S* based on the strong intensity 1H–1H COSY correlations of H-5 (δ<sup>H</sup> 4.55, d (4.7)) with Ha-4 (δ<sup>H</sup> 2.42, m). However, it barely showed correlations with Hb-4 (δ<sup>H</sup> 2.23, m), indicating the dihedral right-angle of H-5–C-5–C-4–Hb-4 and the bond of H-5 at the axial orientation consistent with the axial bond of Ha-4 (δ<sup>H</sup> 2.42, m), which showed large coupling constant (12.3 Hz) with H-3. Furthermore, compound **4** shared the similar experimental ECD curve with compound **2** (Figure 2b). Based on the above data, the absolute configuration of **4** was identified as 2*S*, 3*S*, 5*S*, 1 *S*, 11 *S*, and 14 *R*, and named xanthoquinodin B12.

Compound **5** was obtained as a yellow amorphous solid with a molecular formula of C33H28O12 (20 degrees of unsaturation) according to the mass ion peak at *m*/*z* 617.1650 [M + H]<sup>+</sup> in its HRESIMS spectrum. The 1H- and 13C-NMR spectra, accompanied with HSQC correlations, revealed signals of two methyl (δ<sup>C</sup> 35.0, 21.1), one methoxy (δ<sup>C</sup> 53.3), three sp<sup>3</sup> methylenes (δ<sup>C</sup> 35.0, 27.7, and 23.9), three sp3 methines (δ<sup>C</sup> 73.0, 71.8, and 38.6), five aromatic methines (δ<sup>C</sup> 132.2, 131.8, 123.3, 119.4, and 114.3), seventeen sp<sup>2</sup> quaternary carbons, and two sp<sup>3</sup> quaternary carbons (δ<sup>C</sup> 41.5 and 85.3 (oxygenated)). The NMR spectroscopic data of **5** (Table 2) were similar to those of **2** (Table 1), except for the absence of the butyrate group, replaced by acetate group at C-1 . In addition, the ECD spectrum of compound **5** showed the same pattern as those of compounds **2** and **4** (Figure 2b), corroborating that these compounds possessed the same stereochemistry. Besides, based on the same methods of analysis, the absolute configuration of **5** was proposed to be the same as that of **2**, being assigned as 2*S*, 3*S*, 1 *S*, 11 *S*, and 14 *R*. Compound **5** was named xanthoquinodin B13.

Compound **6** was obtained as a yellow amorphous solid. The mass ion peak at *m*/*z* 645.1967 [M + H]<sup>+</sup> in its HRESIMS spectrum indicated that the molecular formula of **6** was C35H32O12 (20 degrees of unsaturation). The 1H- and 13C-NMR spectra, accompanied with HSQC correlations, revealed signals of two methyl (δ<sup>C</sup> 22.1, 13.5), one methoxy (δ<sup>C</sup> 53.8), six sp<sup>3</sup> methylenes (δ<sup>C</sup> 38.6, 36.2, 35.1, 27.7, 22.2, and 18.4), three sp<sup>3</sup> methines (δ<sup>C</sup> 80.8, 72.6, and 37.9), five aromatic methines (δ<sup>C</sup> 132.9, 131.4, 123.2, 119.3, and 114.9), seventeen sp2 quaternary carbons, and two sp3 quaternary carbons (δ<sup>C</sup> 41.5 and 84.9 (oxygenated)). The NMR spectroscopic data of **6** (Table 2) were similar to those of xanthoquinodin B6 [23], except for the substituent of C-1 , where the hydroxyl was replaced by the butyrate-like compound **1**. Furthermore, the NOESY correlations from H-1 to H-15 confirmed that 1 -hydroxyl was at the same orientation with the double bond of C-12 and C-13 , consistent with all found xanthoquinodins. Likewise, the experimental ECD spectrum of compound **6** (Figure 2d) displayed a similar pattern at 200–250 nm to that of compound **3**, suggesting the same absolute configurations at C-2 and C-3, i.e., *R* and *S*, respectively. Thus, the absolute configuration of compound **6** was assigned as 2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R,* and the name given to it was xanthoquinodin B14.

Compound **7** was obtained as a yellow amorphous solid with a molecular formula of C35H34O13 (19 degrees of unsaturation) based on the mass ion peak at *m*/*z* 663.2072 [M + H]<sup>+</sup> in its HRESIMS spectrum. The 1H- and 13C-NMR spectra, accompanied with HSQC correlations, revealed signals of two methyl (δ<sup>C</sup> 22.1, 13.5), one methoxy (δ<sup>C</sup> 53.4), six sp<sup>3</sup> methylenes (δ<sup>C</sup> 38.2, 36.2, 35.1, 30.0, 25.6, and 18.4), three sp3 methines (δ<sup>C</sup> 73.8, 72.6, and 38.0), five aromatic methines (δ<sup>C</sup> 132.9, 131.4, 123.2, 119.3, and 114.1), seventeen sp<sup>2</sup> quaternary carbons, and two sp3 quaternary carbons (δ<sup>C</sup> 41.4 and 87.3 (oxygenated)). The degrees of unsaturation and the observably different chemical shifts of C-2 to C-6 compared to compound **6** suggested the opening of the γ-lactone ring. The analysis of their NMR

spectroscopic data (Table 2) and same experimental ECD curves (Figure 2d) revealed that **6** and **7** share the same absolute configuration as 2*R*, 3*S*, 1 *S*, 11 *S*, and 14 *R*, and **7** was named xanthoquinodin B15.
