Phytochemical Analysis and Molecular Identification of Green Macroalgae Caulerpa spp. from Bali, Indonesia

The studies of the Bulung Boni and Bulung Anggur (Caulerpa spp.) species and secondary metabolites are still very limited. Proper identification will support various aspects, such as cultivation, utilization, and economic interests. Moreover, understanding the secondary metabolites will assist in developing algae-based products. This study aimed to identify these indigenous Caulerpa algae and analyze their bioactive components. The tufA sequence was employed as a molecular marker in DNA barcoding, and its bioactive components were identified using the GC-MS method. The phylogenetic tree was generated in MEGA 11 using the maximum likelihood method, and the robustness of the tree was evaluated using bootstrapping with 1000 replicates. This study revealed that Bulung Boni is strongly connected to Caulerpa cylindracea. However, Bulung Anggur shows no close relationship to other Caulerpa species. GC-MS analysis of ethanolic extracts of Bulung Boni and Bulung Anggur showed the presence of 11 and 13 compounds, respectively. The majority of the compounds found in these algae have been shown to possess biological properties, such as antioxidant, antibacterial, anticancer, anti-inflammation, and antidiabetic. Further study is necessary to compare the data obtained using different molecular markers in DNA barcoding, and to elucidate other undisclosed compounds in these Caulerpa algae.


Introduction
Seaweeds, also known as macroalgae, are eukaryotic and non-flowering plants with no true stem, leaves, or root surrounding their reproductive systems [1,2]. As with terrestrial plants, seaweeds also subsist by photosynthesis. Seaweeds include a variety of pigments and are taxonomically classified into Chlorophyta (green algae that have chlorophyll pigment), Rhodophyta (red algae that have phycocyanin, and phycoerythrin pigments), and Phaeophyta (brown algae that have fucoxanthin pigment) [3].
Seaweed has been consumed for centuries throughout the world, most notably in Asian countries such as Japan, Korea, China, and Indonesia, but on a smaller scale. Seaweeds have also been used for numerous food, fertilizer, and pharmaceutical products [2]. Seaweed species, such as Caulerpa spp., Gracilaria spp., and Euchema spinosum, are utilized and widely consumed in Bali [4][5][6]. Caulerpa seaweeds, locally known as Bulung Boni and Bulung Anggur, have a wide range of biological properties. Julyasih et al. [4] discovered that Caulerpa sp. possesses the highest antioxidant activity compared to Gracilaria spp. and Euchema spinosum. Moreover, Caulerpa extract could help prevent photoaging through its inhibitory effect on MMP-1 and its protective mechanism against oxidative DNA damage, according to Wiraguna et al. [7].
Although it has been widely taken and used, to date, no research has identified the taxonomic profile of Bulung Boni and Bulung Anggur. According to Farnsworth et al., [11] species identification is essential to documenting, managing, and sustaining organism diversity. The Caulerpa algae are well-known for their great phenotypic plasticity, which refers to the ability of the same species to exhibit a variety of morphological structures in response to changing environmental conditions [12,13]. Accurate identification and characterization of seaweed species are critical for resolving their taxonomic uncertainty, especially for those with commercial interests. Thus, molecular identification is essential to correctly identifying the Caulerpa species. DNA barcoding is a precise and rapid molecular technique for identifying species through the use of short genetic markers [14]. The barcode regions rbcL, matK, and tuf A are frequently employed in green macroalgae. tuf A is a chloroplast gene that encodes the elongation factor TU. Numerous studies have utilized tuf A as a genetic marker for identifying green algae due to its high rate of amplification and sequencing success [9,[15][16][17][18][19][20][21][22][23].
Additionally, the chemical compositions of these Caulerpa seaweeds have not been investigated. Identifying the secondary metabolites and their properties in Caulerpa algae will assist in their utilization and the production of algae-based cosmetics and pharmaceutical medications. Therefore, it is necessary to study the bioactive components of these two Caulerpa algae. The objective of this study was to identify two indigenous Caulerpa seaweed species in Bali, Bulung Boni and Bulung Anggur, using tuf A sequence as a molecular marker in DNA barcoding, and to analyze their bioactive components using GC-MS analysis.

Morphological Characterization
Bulung Boni and Bulung Anggur, like other Caulerpa algae, have green-colored thalli composed of stolons, rhizoids, and erect fronds or assimilators ( Figure 1). Bulung Boni has a distichous assimilator with a maximum height of 10 cm. It also features uncrowded cylindrical or clavate ramuli radially and distichously arranged. On the other hand, Bulung Anggur has an 8 cm irregular assimilator with vesiculated ramuli that have no distinct arrangement.
Caulerpa spp. are known as macroalgae with a high degree of phenotypic plasticity, making precise identification difficult based on morphological observations alone. Morphologically, Bulung Boni resembles Caulerpa cylindracea. C. cylindracea showed a straightforward morphology, with creeping stolons and spherical branchlets with upright shoots and grape-like ramuli, distributed radially or distichously [10,[24][25][26]. In contrast, the morphological characteristics of Bulung Anggur are similar to those of Caulerpa macrophysa. Both Bulung Anggur and Caulerpa macrophysa have thallus that can grow up to 3-5 m in length and are found in a lower intertidal and upper subtidal area with strong water movement. Colorless rhizoidal holdfasts hold the prostrate terete, bare branching stolon and erect, terete branches of this seaweed in thick clusters on the sandy-muddy substrate [24]. and erect, terete branches of this seaweed in thick clusters on the sandy-mudd [24].   [27]), and temperature °C) of the saltwater in the Serangan island region are within the acceptable ran rine biota; however, salinity (29.9-32.7 ppt) is inadequate [28]. Stress generated can trigger plants and algae to produce higher secondary metabolites [29]. H condition might affect the growth of Bulung Boni and Bulung Anggur, resul production of several secondary metabolites.  The pH (7.74-7.92), BOD5/Five-Day Biological Oxygen Demand (2.8-5.4 mg/L calculated using the Delzer and McKenzie standard method [27]), and temperature (28.9-30.5 • C) of the saltwater in the Serangan island region are within the acceptable range for marine biota; however, salinity (29.9-32.7 ppt) is inadequate [28]. Stress generated by salinity can trigger plants and algae to produce higher secondary metabolites [29]. Hence, this condition might affect the growth of Bulung Boni and Bulung Anggur, resulting in the production of several secondary metabolites.  Several compounds with the highest peak in the chromatogram could not be matched to the database library (they have low quality or probability percentage). These compounds could be quite novel and require additional analysis to clarify their nature. However, various compounds in these algae have been proven to have biological activities. Cyclohexanamine is considered the dominant chemical of Bulung Boni. This chemical can be found in several plants, such as Duddingtonia flagrans and Rhazya stricta [30,31]. D. flagrans showed the dominant presence of cyclohexanamine. This plant possesses a potent nematicidal activity [30]. Cyclohexanamine is also present in Rhazya stricta, which showed antidiabetic activity by inhibiting various hyperglycemic key enzymes [32]. Eicosane, in Bulung Boni, has been identified in several studies. Eicosane, an alkane compound ordinarily present in wax, acts as the plant protector against physical damage and prevents the plant from dehydration [33,34]. Eicosane possesses anti-inflammatory, antibacterial, and antitumor properties [35][36][37]. The compound 1-Dodecanol is also found in the ethanolic extract of Bulung Boni, which has been proven to have insecticide and antibacterial properties [38,39]. Neophytadiene, present in both Bulung Boni and Bulung Anggur, is a terpene compound with potent antibacterial, antifungal, anti-inflammatory, antioxidant, antipyretic, and analgesic properties [40].
This research establishes that Bulung Boni and Bulung Anggur are chemically distinct. The difference in the number of compounds could be attributable to the environment wherein they grow (temperature, light intensity, salinity, nutrition, and pH), the biotic factors (pathogen or predator), or their genetic structure. Bulung Boni and Bulung Anggur are presumably different variations or species of Caulerpa that are genetically distinct and produce different secondary compounds. In GC-MS analysis, ethanolic extracts of Caulerpa mexicana and Caulerpa racemosa exhibit different compounds; however, these two Caulerpa algae have simulant biological activities, such as potent anti-inflammatory, antidiabetic, and antioxidant properties [51,52].
Seaweed, or macroalgae, is an excellent source of protein, polysaccharides, lipids, and fiber. Multiple studies have demonstrated the high lipid content of various green macroalgae, especially Caulerpa. In addition, seaweed is also a source of minerals [2]. Further research to investigate protein, amino acids, total sugar, lipid content, and mineral profiles will support the utilization of this Caulerpa seaweed.

Molecular Identification
The correct and successful identification of seaweed strains is aided by identification based on comprehensive taxonomy. DNA barcoding is a quick and accurate method. This method is considered the best approach for species identification. DNA barcoding does not require the whole genome sequence but only a short strand of DNA sequences created from the standard marker region of the entire genome [14]. The Bulung Boni and Bulung Anggur were successfully amplified using the tuf A primer (Figure 4). The products of PCR amplification of Bulung Boni and Bulung Anggur with tuf A primers were 903 and 930 bp, respectively. There were 18 species with an identity percentage of more than 90% (against Bulung Boni and Bulung Anggur) selected from the NCBI-BLAST results and used in the phylogenetic analysis (Table 3).
Molecules 2022, 27, x FOR PEER REVIEW (against Bulung Boni and Bulung Anggur) selected from the NCBI-BLAST resu used in the phylogenetic analysis (Table 3).    (2) with tuf A as a primer.
The amplification of the tuf A sequence performed well in our investigation. The tuf A gene is an excellent candidate for a molecular marker due to its conserved nature across various taxa [9]. tuf A encodes protein synthesis elongation factor Tu. Tu was moved from the chloroplast to the nucleus within the green algal lineage that gave rise to terrestrial plants [53]. tuf A, ITS (internal transcribed spacer), and rbcL (rubisco large subunit) molecular markers are frequently used in phylogeny identification. Nevertheless, tuf A produced the best results in identifying Chlorophyceae compared to ITS and rbcL [54]. Numerous other studies have also demonstrated that tuf A has excellent amplification and sequencing results for identifying green algae compared to other markers [16][17][18].
According to the phylogenetic tree reconstruction results ( Figure 5), Bulung Boni is closely related to Caulerpa cylindracea, with a 100 percent bootstrap value. Thus, morphological and molecular identification of Bulung Boni produced similar findings. Additionally, Bulung Boni and Caulerpa cylindracea have a genetic distance of 0.000 (Table 3). Caulerpa cylindracea has been raised from Caulerpa racemosa var. cylindracea because of its genetic independence [12]. C. cylindracea is one of the most invasive algae species that has been reported by several researchers [55][56][57][58]. As a result of its invasive character and high productivity, Bulung Boni has the potential to be utilized and commercialized. various taxa [9]. tufA encodes protein synthesis elongation factor Tu. Tu was moved from the chloroplast to the nucleus within the green algal lineage that gave rise to terrestrial plants [53]. tufA, ITS (internal transcribed spacer), and rbcL (rubisco large subunit) molecular markers are frequently used in phylogeny identification. Nevertheless, tufA produced the best results in identifying Chlorophyceae compared to ITS and rbcL [54]. Numerous other studies have also demonstrated that tufA has excellent amplification and sequencing results for identifying green algae compared to other markers [16][17][18].
According to the phylogenetic tree reconstruction results ( Figure 5), Bulung Boni is closely related to Caulerpa cylindracea, with a 100 percent bootstrap value. Thus, morphological and molecular identification of Bulung Boni produced similar findings. Additionally, Bulung Boni and Caulerpa cylindracea have a genetic distance of 0.000 (Table 3). Caulerpa cylindracea has been raised from Caulerpa racemosa var. cylindracea because of its genetic independence [12]. C. cylindracea is one of the most invasive algae species that has been reported by several researchers [55][56][57][58]. As a result of its invasive character and high productivity, Bulung Boni has the potential to be utilized and commercialized.
Bulung Anggur, on the other hand, seems to be an outgroup on the phylogenetic tree, indicating that it is genetically distinct from other Caulerpa homologs determined by NCBI-BLAST. Bulung Anggur shares morphological traits with Caulerpa racemosa, particularly with Caulerpa racemosa var. macrophysa (Sonder ex Kützing) [58]. Moreover, Bulung Anggur has the closest genetic relationship distance to Caulerpa racemosa, at 1.200 (Table  3). Thus, it is assumed that Bulung Anggur is a species that is still understudied, given that there are few taxa in the GenBank database that have similar sequences to Bulung Anggur. The majority of seaweed species growing in Bali's waters are poorly described taxonomically. These limitations can hinder various aspects, especially in cultivation and nature conservation. In this study, the application of DNA barcodes with the tufA marker has successfully identified one species of Caulerpa seaweed in Bali. This study has been able to locate the proximity of the Bulung Boni species to other Caulerpa species. However, Bulung Anggur, on the other hand, seems to be an outgroup on the phylogenetic tree, indicating that it is genetically distinct from other Caulerpa homologs determined by NCBI-BLAST. Bulung Anggur shares morphological traits with Caulerpa racemosa, particularly with Caulerpa racemosa var. macrophysa (Sonder ex Kützing) [58]. Moreover, Bulung Anggur has the closest genetic relationship distance to Caulerpa racemosa, at 1.200 (Table 3). Thus, it is assumed that Bulung Anggur is a species that is still understudied, given that there are few taxa in the GenBank database that have similar sequences to Bulung Anggur.
The majority of seaweed species growing in Bali's waters are poorly described taxonomically. These limitations can hinder various aspects, especially in cultivation and nature conservation. In this study, the application of DNA barcodes with the tuf A marker has successfully identified one species of Caulerpa seaweed in Bali. This study has been able to locate the proximity of the Bulung Boni species to other Caulerpa species. However, the tuf A marker was insufficient to distinguish Bulung Anggur accurately. Before concluding that Bulung Anggur is a novel indigenous strain, it is necessary to conduct additional research to examine its phylogeny using various markers utilized to identify Caulerpa algae, including ITS, rbcl, and rDNA [17].

Sample Collection
The Caulerpa algae, Bulung Boni and Bulung Anggur, were collected from the coastal area around Serangan Island, Bali ( Figure 6). The samples were subsequently washed with clean water to remove dirt and epiphytes. Bulung Boni and Bulung Anggur were morphologically identified by referring to the morphological descriptions of several previous studies [10,24,25,59]. Fresh samples were stored in a refrigerator to be used afterward in further analysis.
Molecules 2022, 27, x FOR PEER REVIEW the tufA marker was insufficient to distinguish Bulung Anggur accurately. Befo cluding that Bulung Anggur is a novel indigenous strain, it is necessary to condu tional research to examine its phylogeny using various markers utilized to i Caulerpa algae, including ITS, rbcl, and rDNA [17].

Sample Collection
The Caulerpa algae, Bulung Boni and Bulung Anggur, were collected from the area around Serangan Island, Bali ( Figure 6). The samples were subsequently wash clean water to remove dirt and epiphytes. Bulung Boni and Bulung Anggur we phologically identified by referring to the morphological descriptions of several p studies [10,24,25,59]. Fresh samples were stored in a refrigerator to be used afterw further analysis.

Maceration and GC-MS Analysis
Clean Bulung Boni and Bulung Anggur were air-dried in the shade for 2 da then dried in the oven for 7 days at 45 °C. Dried samples were then powdered u electric blender. The samples of each alga (15 g) were macerated in ethanol solve mL). The mixtures were kept for 96 h, then filtered and concentrated using a rota uum evaporator to produce the crude extract.
The crude extract was then subjected to phytochemical analysis using GC-M chromatography analysis was carried out on the Agilent 7890B GC (Santa Clara, CA coupled with a mass detector Agilent 5977B GC/MSD (Santa Clara, CA, USA). A m of 1.0 µL of the extract was injected into the chromatograph at an injector temper 250 °C. The column (Wakosil-II5C18 4.6*200 mm, Richmond, VA, USA) oven temp was programmed to increase from 70 °C to 290 °C at a rate of 10 °C/min. The run t GC was 17 min. The identification of chemical compounds and the interpretation spectra of GC-MS were carried out using the Wiley Spectral library.

Maceration and GC-MS Analysis
Clean Bulung Boni and Bulung Anggur were air-dried in the shade for 2 days and then dried in the oven for 7 days at 45 • C. Dried samples were then powdered using an electric blender. The samples of each alga (15 g) were macerated in ethanol solvent (150 mL). The mixtures were kept for 96 h, then filtered and concentrated using a rotary vacuum evaporator to produce the crude extract.
The crude extract was then subjected to phytochemical analysis using GC-MS. Gas chromatography analysis was carried out on the Agilent 7890B GC (Santa Clara, CA, USA) coupled with a mass detector Agilent 5977B GC/MSD (Santa Clara, CA, USA). A measure of 1.0 µL of the extract was injected into the chromatograph at an injector temperature of 250 • C. The column (Wakosil-II5C18 4.6*200 mm, Richmond, VA, USA) oven temperature was programmed to increase from 70 • C to 290 • C at a rate of 10 • C/min. The run time for GC was 17 min. The identification of chemical compounds and the interpretation of mass spectra of GC-MS were carried out using the Wiley Spectral library.

DNA Extraction and PCR Amplification
Total genomic DNA of fresh Bulung Boni and Bulung Anggur samples were extracted using the Plant Genomic DNA Mini Kit (GP100, Geneaid, New Taipei City, Taiwan), following the manufacturer's procedure.
The tufA region was amplified using published primers by Fama et al. [9], i.e., tufA F 5 TGAAACAGAAMAWCGTCATTATGC-3 and tuf A R 5 -CCTTCNCGAATMGCRAAWCGC-3 . PCR amplification was conducted using MyTaq HS Red Mix (Bioline, Bio-25048, London, UK). The PCR reactions were performed with the following profiles: pre-denaturation (94 • C for 2 min), 35 cycles of denaturation (98 • C for 15 s), annealing (50 • C for 30 s), extension (72 • C for 45 s), and followed by termination at 4 • C. Exactly 2 µL of PCR products were visualized using an agarose gel and selected for the sequencing process. The Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) was used to determine the sequence bi-directionally.

Computational Analysis
Sequences obtained in this study were compared to sequences in NCBI (National Center for Biotechnology Information) by BLASTN analysis. Accessions with the highest identity percentage were selected, and multiple sequence alignment was carried out with ClustalW (MEGA 11th version, Philadelphia, PA, USA). The phylogenetic tree was constructed using the maximum likelihood method and the Tamura 3-parameter model in MEGA 11 [60]. The robustness of the tree was evaluated using bootstrapping with 1000 replicates.

Conclusions
In conclusion, the ethanolic extracts of Bulung Boni and Bulung Anggur may be a source of valuable pharmaceuticals with antioxidant, antibacterial, anticancer, antiinflammatory, antitumor, and antiaging properties that could be manufactured as cosmetics or healthy processed foods. However, further study is necessary to elucidate other undisclosed compounds in these Caulerpa algae. Furthermore, this study revealed that the Balinese Caulerpa algae, Bulung Boni and Bulung Anggur, are genetically distinct species, with Bulung Boni being strongly connected to Caulerpa cylindracea and Bulung Anggur showing no close relationship to other Caulerpa species. Although the tuf A sequence was amplified effectively and generated a robust phylogenetic tree, additional research is required to compare the results obtained using different molecular markers.  Data Availability Statement: This study uses data published in GenBank, which can be accessed on https://www.ncbi.nlm.nih.gov/ (accessed on 9 December 2021). These data are used in the BLAST application to compare the sequences in the GenBank database with the sequences obtained in this study.