**1. Introduction**

Since the discovery of Easter Island, compelling explorations have characterized the flora [1] and fauna [2–4] of this geographically isolated location. However, little has been done to understand the chemistry harbored in this territory. The best-known finding is the discovery of rapamycin in a soil dwelling actinobacterial representative, which serves as an immunosuppressive drug [5,6]. Beyond that, little progress has been made in exploiting the chemical diversity harbored by marine invertebrates and microorganisms dwelling in this territory, despite the high degree of endemism found [7].

Marine invertebrates are immensely diverse, well distributed in the world oceans [8], and widely known to contain medicinally relevant molecules [9–11]. While these metabolites play different roles in nature, e.g., they act as chemical defense, chemical communication or reproductive signaling molecules, they also find application as human medicines [12]. During the last decades, much effort has been made to identify and characterize the chemicals contained in marine invertebrates. This has resulted in the discovery of astonishing chemicals with novel biological activities and chemical scaffolds [12–14].

Further, the immense progress in DNA sequencing technologies, the development of bioinformatics, and an improvement in analytical techniques enables the identification of the source of the chemicals. Thus, several molecules contained in marine invertebrates have now been shown to actually be of microbial origin [15,16]. It is expected that with the increasing availability of metagenomic information more identifications of the real producers of these metabolites will be made and will establish the metabolite relevance for the interaction of host, symbiont, and the environment.

Marine sponges represent a classic example of marine invertebrates that harbor microbes producing secondary metabolites. They have been studied in detail to determine the origin of the metabolites [17,18]. Another example is the producer of the approved anticancer drug Yondelis (Ecteinascidin-743). This compound was first assigned to the tunicate *Ecteinascidia turbinata*, but later identified as the product of a microbe, Candidatus *Endoecteinascidia frumentensis* [16].

Anthraquinones have been characterized in different marine invertebrates, for example crinoids [19] and sponges [20]. Anthraquinones have broad biological activity and are substances of pharmaceutical relevance for revealing antitumor [21], antibacterial [22], antifungal [23] and epigenetic modulator activities [24]. Two recently isolated anthraquinones, Lupinacidin A (**1**) and Galvaquinone B (**2**), have been reported to be produced by Actinobacteria belonging to the genera *Streptomyces* and *Micromonospora*. Lupinacidin A (**1**) was firstly reported as a specific inhibitor on murine colon 26-L5 carcinoma cells [25], and Galvaquinone B (**2**) showed moderate cytotoxicity against non-small-cell lung cancer cells Calu-3 and H2887, in addition of epigenetic modulatory activity [24].

Herein, we report the identification of two known anthraquinone molecules, Lupinacidin A (**1**) and Galvaquinone B (**2**), contained in an Easter Island sea anemone, *Gyractis sesere*. Interestingly, so far these molecules have been only characterized from microbial origin. Therefore, we undertook the isolation and culture of the Actinobacteria associated with this marine invertebrate. The culture, chemical and genomic evaluation of these bacteria showed that the real producer of the metabolites was an Actinobacterium belonging to the genus *Verrucosispora* and not the sea anemone.

### **2. Results and Discussion**

### *2.1. Sea Anemone Dereplication*

Samples of the sea anemone *Gyractis sesere* [26], also known as *Actiniogeton rapanuiensis* [27], were collected in the intertidal zone of Easter Island, South Pacific Ocean, and extracted with chloroform to give 8 mg of a yellowish residue. The HRLCMS analysis of this crude extract showed the presence of two previously identified antitumor anthraquinones (Figure 1) [24,25], Lupinacidin A (**1**) ([M + H]+ m/z 341.1378) and Galvaquinone B (**2**) ([M + H]+ m/z 369.3510), which were confirmed by complete NMR-spectroscopic characterization.

**Figure 1.** Identified molecules from the Easter Island sea anemone *Gyractis sesere*.

Interestingly, the crude extract of the sea anemone (Figure 2) shows five resonances above 10 ppm; a region which is characteristic for hydroxyl protons. The resonances at δ 14.18 and 12.96 ppm are assigned to the groups at C-1 and C-6 of Lupinacidin A (**1**), as their vicinity and consequential hydrogen bonding to the ketogroups slows down their exchange. Similarly, the resonances at δ 13.49, 12.50, and 12.14 ppm originate from the three hydroxylprotons in Galvaquinone B (**2**).

**Figure 2.** Chemical analysis of the crude extract of the sea anemone *Gyractis sesere*. (**A**) 1H NMR spectra of the crude extract of marine anemone *Gyractis sesere* acquired in CDCl3, 600 MHz. Highlighted in the zoomed area are the frequencies of characteristic resonances originating from hydroxyl exchangeable protons in vicinity to ketogroups. (**B**) UV chromatogram (254 nm) of the crude extract of the sea anemone *Gyractis sesere* highlighting the specific peaks for -RT: 24.3 min, and : RT: 25.2 min. (**C**) High resolution mass for - (m/z [M + H]+ 341.1378) and (**D**) high resolution mass for (2) (m/z [M + H]+ 369.3510). \*RT: Retention Time.

Other main peaks found in the sea anemone crude extract were peaks at RT 14.2 min with a HRMS [M + H]+ m/z 295.19009 and at RT 23 min with a HRMS [M + H]+ m/z 256.26312. Both exact masses were evaluated using the MarinLit database, however their HRMS did not match any known compound to-date.

### *2.2. Bacterial Metabolites and Harbored Bacteria*

Lupinacidin A (**1**) and Galvaquinone B (**2**) have so far only been characterized in actinobacterial representatives, specifically from the genera *Streptomyces* [24,28] and *Micromonospora* [25], raising the question of the origin of these compounds in the sea anemone extract. Thus, we cultivated the Actinobacteria harbored by this sea anemone to determine if the anthraquinone producer was a bacterium or the sea anemone. Isolation media and the respective obtained strains are specified in Supplementary Table S1. Ten strains were identified through analysis of the 16S rRNA gene sequences as members of the genera *Micromonospora*, *Streptomyces*, *Verrucosispora*, *Dietzia*, *Arthrobacter*, *Rhodococcus*, and *Cellulosimicrobium* (Figure 3). Remarkably, *Gyractis sesere* harbors a high number of Actinobacteria genera, in total seven; the most abundant genus being *Micromonospora* with three different species, followed by *Arthrobacter* with two different species. Other actinobacterial genera were present with only one species each. Outstandingly, the only isolate belonging to the *Verrucosispora* genus was the most abundant single Actinobacterium in the sea anemone.

**Figure 3.** Genera and number of Actinobacteria species strains isolated from the sea anemone *Gyractis sesere*.
