**2. Results**

#### *2.1. Cultivable Fungi's Phylogeny and Diversity*

A total of 102 fungal isolates were obtained from *Acanthus ilicifolius* using the PDA medium with four salt gradients of 3%, 5%, 7% and 10%. Duplicated strains were removed using a detailed morphological approach. Consequently, eighty-four independent strains were selected for sequencing and identification based on ITS sequences. According to the sequences deposited into NCBI, the 84 strains belonged to the phylum Ascomycota including seven taxonomic orders: *Hypocreales*, *Xylariales*, *Diaporthales*, *Eurotiales*, *Pleosporales*, *Capnodiales*, *Botryosphaeriaceae* and 22 genera: *Trichoderma*, *Hypocrea, Acremonium*, *Verticillium*, *Fusarium*, *Neocosmospora*, *Pestalotiopsis*, *Diaporthe*, *Phomopsis*, *Valsa, Colletotrichum, Penicillium, Eupenicillium*, *Aspergillus, Talaromyces*, *Pyrenochaeta*, *Pleosporales*, *Curvularia*, *Alternaria*, *Cladosporium*, *Phyllosticta*, and *Lasiodiplodia* (Table 1). These identified fungi and their best matches in the NCBI database are summarized in Table S1. Most of the isolates matched their closest relatives with 98 to 100% similarity, except for HS-G-02 (97%) and HS-G-06 (95%), which indicated that they were new species. Both of the fungi HS-G-06 and HS-G-02 further enriched the diversity of mangrove fungi. Further analysis of the isolated fungi showed that *Eurotiales* was the dominant group with identified fungi, followed by *Hypocreales*. The fungal community was dominated by *Penicillium*, comprising 21 isolates, followed by *Fusarium*, *Aspergillus*, and *Eupenicillium* with 15, 14, and 10 isolates, respectively. Some of the genera, such as *Trichoderma, Phomopsis* and *Cladosporium* obtained six, five and five, respectively. Most of the remaining genera occurred as singletons or doubletons.


**Table 1.** The classification of cultivable fungi associated with *Acanthus ilicifolius*.

In addition, the species of fungi isolated from different parts of *A. ilicifolius* were quite different (Figure 2). The results showed that some genera of fungi were isolated only from one part. For example, *Phomopsis* and *Acremonium* were isolated only from the stem. *Colletotrichum*, *Curvularia*, and *Alternaria* were isolated only from the leaf. *Valsa*, *Hypocrea*, and *Neocosmospora* were isolated only from the soil. *Diaporthe*, *Talaromyces*, and *Pyrenochaeta* were isolated only from the leaf.

**Figure 2.** The distribution of each species in different parts.

Further phylogenetic analysis was carried out on 37 strains. These 37 independent individuals were selected as the representative strains because they belong to different fungal species after we aligned the sequences with the BioEdit software (Figure S1). The phylogenetic tree of fungi in the order *Hypocreales* based on ITS gene sequence is presented in Figure S2. Furthermore, the fingerprints of secondary metabolites of fungi from different species and genera were analyzed (Figure S4).

#### *2.2. The Cytotoxicity of Cultivable Fungal Extracts*

The organic extracts of 55 identified fungi were evaluated for their cytotoxic activities against human lung cancer cell line (A-549), human cervical carcinoma cell (HeLa), human hepatoma cells (HepG2) and Jurkat tumour cell lines at the concentration of 50 μg/mL (Figure 3a). To identify active strains for further research as potential cytotoxic strains, the relative inhibition rate of A-549, HeLa and HepG2 cell lines should greater than 70%, and the relative inhibition rate of Jurkat cell line should greater than 60%. The results showed that these fungi showed different inhibition rates to different cell lines. The number of the fungi showing activity against A-549, HeLa, HepG2, and Jurkat tumour cell lines were 17, 17, 19 and 24, respectively (Figure S3). The crude extracts of 31 fungi displayed cytotoxicity against the test cell lines, of which 21 fungi showed selective inhibitory activity on different tested cell lines,; for example, *Fusarium* sp. showed selective inhibitory activity on HeLa cell lines. Most fungi showed strong selective inhibitory activity on Jurkat cell lines. Interestingly, the remaining 10 fungi belonging to the two orders *Eurotiales* and *Hypocreales*, displayed a broad-spectrum strong cytotoxic activity, such as *Penicillium* sp. (HS-N-23, HS-N-27, HS-N-29, and HS-G-01), *Eupenicillium* sp. (HS-N-25), *Trichoderma* sp. (HS-01 and HS-N-04), *Aspergillus* sp. (HS-G-04 and HS-Y-27), and *Verticillium* sp. (HS-N-28).

The crude extracts were further reduced in concentration for the activity test. The results showed that only the two active strains of *Penicillium* sp. (HS-N-27 and HS-N-29) still showed strong inhibitory activity against all the texted cell lines at the concentration of 25 μg/mL. Cytotoxic metabolites were isolated from the endophytic fungus *Penicillium chermesinum*, leading to the discovery of a cysteine-targeted Michael acceptor as a pharmacophore for fragment-based drug discovery, bioconjugation and click reactions [25]. The heteroatom-containing new compounds 2-hydroxyl-3-pyrenocine-thio propanoic acid and 5,5-dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one, which were isolated from a deep-sea *Penicillum citreonigrum* XT20-134, showed potent cytotoxicity to the human hepatoma tumor cell Bel7402 [26]. Additionally, the active strains HS-N-28, HS-G-01, and HS-N-25 showed strong selective inhibitory activity against A-549, HeLa and HepG2 cell lines. The active strains HS-Y-27, HS-N-23, HS-G-04, and HS-N-28 showed strong selective inhibitory activity against HeLa cell lines. The active strains HS-G-01, and HS-N-25 showed strong selective inhibitory activity against HepG2 and A549 cell lines. Obviously, these

active strains are important microbial resources and have the potential for interesting cytotoxic compounds (Figure 3b).

**Figure 3.** Cytotoxicity of organic extracts of 55 identified fungi. (**a**) The cytotoxic activities of 55 identified fungi against A-549, HeLa, HepG2 and Jurkat tumour cell lines at the concentration of 50 μg/mL. (**b**) The cytotoxic activities of the active strains at the concentration of 25 μg/mL.

#### *2.3. Isolation and Identifcation of Compounds 1–7*

As the two active strains *Penicillium* sp. (HS-N-27 and HS-N-29) showed strong cytotoxic activity against all the tested cell lines at the concentration of 25 μg/mL, both of the *Penicillium* sp. fungi were selected as the target strains. Combining cytotoxic activityguided strategy with fingerprint analysis, compound **1** was obtained from the fermentation broth of the two active strains HS-N-27 and HS-N-29. By comparison of NMR data with the reported literature, the structure was identified as brefeldin A (Figure 5), which was a 13- membered macrolactone with a cyclopentane substituent [7]. BFA is a well-known natural Golgi-disruptor and Arf-GEFs inhibitor [8]. Combining morphological characteristics and fingerprint analysis of metabolites (Figure S5), the two fungi HS-N-27 and HS-N-29 were identified as different individuals of the same *Penicillium* sp. species. The neighbor-joining of the phylogenetic tree of the target active strain *Penicillium* sp. (HS-N-27) in *Hypocreales* order fungi from *A. ilicifolius* based on ITS sequences is shown in Figure 4.

The genus *Aspergillus* is one of the dominant producers of new natural products [13]. The fingerprint analysis showed that the metabolites of *Aspergillus flavus* (HS-N-06) were relatively single and that *A. candidus* (HS-Y-23) was rich in metabolites with strong special UV absorption peak (Figure S4). The secondary metabolites of the two fungal strains were further studied. Under the guidance of chemical technology, 5-hydroxymethylfuran-3- carboxylic acid (**2**) was obtained from the fermentation broth of *A. flavus* (HS-N-06) [27]. Terphenyllin (**3**) was obtained from the fermentation broth of *A. candidus* (HS-Y-23), which showed weak cytotoxic activity against HeLa cell lines with the IC50 value of 19.0 μM [28]. In addition, 5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (**4**), indolyl-3- carboxylic acid (**5**), and trichodermamides A (**6**) and D (**7**) were obtained from the fermentation broth of *Trichoderma harzianum* (HS-N-04) [29–31]. The structures of isolated and identified compounds were in Figure 5.

**Figure 4.** The neighbor-joining of phylogenetic tree of HS-N-27 fungi in *Hypocreales* order fungi. The values at each node represent the bootstrap values from 1000 replicates, and the scale bar = 0.01 substitutions per nucleotide.

**Figure 5.** Structures of isolated and identified compounds.
