**2. Results and Discussion**

## *2.1. Sample Evaluation*

An environmental sample of marine filamentous cyanobacteria, HAINAN-19SEP17- 3, was collected near Wenchang, Hainan, China. Based on colonial morphology and light microscopy, the sample was initially classified as cf. *Neolyngbya* sp. (Figure 2). To validate this and determine the microbiome composition, a portion of the sample was analyzed by 16S rRNA gene sequencing using universal PCR primers, and this further supported the genetic identity of the predominant biomass as cyanobacteria categorized under *Phormidiaceae* (57%; certainly includes basionyms in *Oscillatoriaceae*) along with additional associated microbes from *Bacteroidetes* (22%), *Proteobacteria* (14%), and others at a lower abundance (Figure 2). The higher taxonomic order Oscillatoriales is presented for the majority of the cyanobacterial 16S gene sequence data in Figure 2 to avoid confounding basionyms that occur within its members, i.e., parts of the families *Oscillatoriaceae* and *Phormidiaceae*. The 16S gene sequence V3-V4 amplicon of the organism that dominates this consortium was found to clade with *Neolyngbya*. *Neolyngbya* is a recently described genus of the family *Oscillatoriaceae*, and was established from the *Lyngbya*-like morphotype that has historically also been a misclassification for some *Phormidium* organisms [16,17,21]. There is great difficulty in growing axenic cultures of cyanobacteria; therefore, it is important

to refer to the collected consortia as a whole. While several studies demonstrated that the microbiome of cyanobacteria is relatively stable between environmental samples and non-axenic cultures (mainly *Proteobacteria* and *Bacteroidetes*) [37], little is known about the microbiome associated with *Lyngbya*-like and *Phormidium*-like organisms [38].

**Figure 2.** Sample information. (**A**) Collection site and morphology of HAINAN-19SEP17-3. (**B**) Microbiome analysis; the order Oscillatoriales contains the families *Oscillatoriaceae* and *Phormidiaceae*, and this higher taxonomy is presented here to avoid confounding basionyms within these two. (**C**) Phylogeny of the environmental assemblage dominated by cf. *Neolyngbya* sp. from the South China Sea that was evaluated in this study. Map generated with Google Earth. Taxonomy and phylogeny were evaluated using Silva and EMBL-EBI databases. *Gleobacter* was used as the outgroup.

An LC-MS/MS untargeted metabolomic approach [28] was utilized to overview the chemical potential of the prioritized South China Sea cf. *Neolyngbya* sp. sample. Feature detection and annotation analyses were done using the Global Natural Products Social (GNPS) Molecular Networking platform. This method aligns the fragmentation patterns obtained by MS/MS against various spectrometric databases and allows for the putative annotation of structural characteristics and chemical classifications [33,36]. Nearly 750 molecular features were present in the initial evaluation of this sample; however, reported cyanobacterial specialized metabolites were not able to be detected. Some common pigments (mainly chlorophylls and breakdown products thereof) were annotated in the dataset. Together, these data highlighted the potential for discovery of novel compounds and, at the same time, allowed ubiquitous pigment molecules to be avoided in the isolation procedure. Furthermore, most of the chemistry had no match to any known structure in the spectrometric libraries (84%), yet some had putative annotations to general chemical classes (5 super-classes; Figure 3A), based on the associated fragmentation patterns. The subset of classified molecules were further delineated into 19 putative chemical subclasses (Figure 3B) that highlight the chemical diversity and discovery potential of this complex extract. The main prevalent classes that were detected and annotated include peptides

(42%) and terpenoids (17%). Though databases on such molecules are largely incomplete, or hard to access, these molecular families are known to contain many types of bioactive natural products [39–41]. Nonribosomal peptides are a diverse group of natural products that have complex chemical structures and a vast array of bioactivity potentials as anticancer, anti-parasitic, anti-fungal, and cytotoxic agents, protease inhibitors and more [39]. The structures of natural products resulting from non-ribosomal peptide synthetase (NRPS) biosynthesis can be linear or cyclic, possess typical and/or unusual amino acids, and may even be hybridized with modules from polyketide synthase (PKS) genes. NRPS and PKS biosynthetic gene clusters are mostly common in bacteria, and many such hybridized biosynthetic mechanisms have been uniquely found in cyanobacteria or are rarely described from other organisms [39,42]. The metabolomic annotation of unknown peptides, depsipeptides and derivatives from the cyanobacteria sample here studied was accordingly encouraging for the potential to discover new bioactive molecules.

**Figure 3.** Chemical space of the organic extract of cf. *Neolyngbya* sp. HAINAN-19SEP17-3 as evaluated by data-dependent LC-MS/MS. Samples were analyzed via the GNPS platform using NAP, Dereplicator and MolNetEnhancer workflows to yield putative annotations of (**A**) SuperClasses and (**B**) SubClasses of annotated molecular features based on observed fragmentation patterns.
