Chemical prospection on the Antarctic moss Sanionia uncinata (Hedw.) Loeske, Amblystegiaceae and its relationship with biological activity and the altitude. A pilot studyAbstract
Twenty-four ethanolic extracts obtained from the Antarctic sickle-leaved-hook-moss <italic>Sanionia</italic> <italic>uncinata </italic>were tested for their chemical profile by gas chromatography-mass spectrometry, revealing the presence of fatty acids and their metabolites, diterpene, triterpene, and four steroids. Canonical correspondence analysis (CCA) indicated a significant correlation between the metabolite content and biological activities as antibacterial, β-carotene and diphenyl-picryl-hydrazyl DPPH antioxidant, cytotoxicity to breast and prostate cancer cell lines, and acetylcholinesterase inhibitory activity, and the principal component analysis (PCA) showed a variation in the metabolite content according to the altitude from which the mosses’ samples were collected, indicating that the altitude of 30 m was determinant for the metabolite variation in <italic>S. uncinata.</italic> The present findings constitute a pilot study supporting further plant prospection in the maritime Antarctica.
Key words
chemical profile; fingerprint; gas chromatography; mass spectrometry; multivariate analyses
INTRODUCTION
Bryophytes are avascular, terrestrial, and gametophyte-dominant plants comprising three lineages: mosses, liverworts, and hornworts (Clarke et al. 2011). These tiny plants have thin leaves or leaves-like organs that conduct water and nutrients and capture carbon dioxide, which allows photosynthesis and plant adaptation to many environmental conditions (Mischler & Oliver 2009), including those observed in the Antarctic Peninsula, despite diminished sun exposure and limitations related to the bryophyte’s cell wall thickness (Roig-Oliver et al. 2021). Bryophyte environmental adaptation is facilitated by morphological strategies and synthesizing various secondary metabolites.
The chemical complexity of the bryophytes is one of the main factors involved in the capacity of the small plants to survive under extreme environmental conditions, such as those found in Antarctica, where bryophytes - expressed in the local flora only by mosses and liverworts - survive after an extended freezing period (Lu et al. 2023).
A study concerning the metabolic adaptations of the moss Pholia nutans to cold suggested that several major biochemical pathways responded differently after 24h or 60h of cold exposition through the analyses of metabolome and transcriptome, indicating that fatty acids and very-long-chain fatty acids biosynthesis were stimulated by cold (Liu et al. 2022a). They also studied the variations in the Jasmonate signaling. They observed changes in the lipid and fatty acids biochemistry, Ca++ signaling, abscinic acid, antioxidant enzymes, and detoxification proteins. They related Jasmonate signaling variation to the adaptation mechanisms of the moss to the frozen environment (Liu et al. 2022b). The same group cloned the flavonoid 3’-hydroxylase gene from P. nutans into Arabidopsis to access oxidative and salt stress tolerance, and they discovered that the gene participated in the regulation of oxidative tolerance and that provoked a variation in the abscinic acid, a hormone interfering with plant stress, as a strategy of the moss to adapt to the Antarctic environment (Liu et al. 2022c). Also, they cloned the gene CPD photolyase and verified that its overexpression in Arabidopsis elevated the resistance to UV-B radiation and to salinity stress (Wang et al. 2021).
It is well described that the fatty acid variation is deeply involved with the adaptation to the Antarctica environment. So, lipids such as glycerolipids, phospholipids, neutral lipids, sterols, sphingolipids, phosphoinositides, arachidonic acid (AA), and eicosapentaenoic acid (EPA) play a pivotal role in bryophytes’ environmental adaptation considering that AA and EPA are closer related to marine organisms other than to vascular plants (Liu et al. 2022a; Resemann et al. 2019). Terpenes (Karunanithi & Zerbe 2019) and flavonoids (Panche et al. 2016) are widely distributed in the plant kingdom, including bryophytes, and the presence of such metabolites is also crucial to plant environmental adaptation. In bryophytes, terpenes and flavonoids are important to abiotic and senescence pathways (Kulshrestha et al. 2022). Terpenoids such as mono-, di-, sesquiterpenes, and carotenoids are commonly found in bryophytes (including hornworts, liverworts, and mosses), while flavonols, flavones, bi- and triflavonoids are commonly found in mosses only. Although not an issue to be approched in the present work, these metabolites show potential to be used as medicines due to their wide structural variety.
The chemical variations observed for Pholica nutans, an Antarctic moss, are also observed in mosses from other locations, as demonstrated in a previous study that reported the seasonal lipid variation in the moss Hypnum cupressiforme Hedw. (Hypnaceae Schimp.) extracts. The authors concluded that the highest content of fatty acids occurred during summer in Serbia (Lunić et al. 2022).
Considering the efforts made in the last years, there is a gap in evaluating how the variation of secondary metabolites is related to altitude. Some research has been done with bryophytes from Poland (Chmura et al. 2022), and in this study, the authors described the importance of altitude for the epixylic bryophytes developing in deadwood substrate at different sea levels, and they found that the altitude interfered with moisture and nutrients for liverworts and with light and soil acidity for mosses. No such studies were conducted with some moss from the Antarctic continent.
Recently, mosses have gained attention due to the identification of biological activities such as antibacterial and anti-inflammatory activities (Ivanova et al. 2007), antioxidant activity (Bhattarai et al. 2009), and mutagenicity (Fernandes et al. 2015), and inhibitory activity against acetylcholinesterase, the enzyme involved in the Alzheimer’s Disease (Teodoro et al. 2024), but yet, further information regarding the potential of Antarctic bryophytes as source of new active compounds is lacking and urges to be accessed.
To fill that gap, we assessed the ethanolic extracts obtained from Sanionia uncinata (Hedw.) Loeske (Amblystegiaceae), the sickle-leaved hook-moss, a common species in Antarctica being profoundly studied as a reference species largely inhabiting the Frozen Continent, were assessed and tested for their chemical profiles. Despite the focus on secondary metabolites, S. uncinata was previously used to access information regarding the genetic and molecular mechanisms related to the plant to adapt to a year of seasonal variation in King George Island, Antarctica. The authors reported that genes related to cell stress and abscinic acid signaling were highly expressed during the winter and otherwise in the summer (Yu et al. 2024). An interesting study reported that mosses in the Antarctic Continent have their photosynthesis apparatus optimized to warm temperatures as high as 15 to 20o C despite the extreme cold conditions (Perera-Castro et al. 2020).
The present study aimed to assess the chemical profiles of 24 extracts obtained from the plant collected in different spots in the Keller Peninsula considering the altitude and to evaluate the possible relationships with the biological activities previously assessed by our team as cytotoxic, antimicrobial, acetylcholinesterase inhibition, and antioxidant. The present findings were obtained from a convenience sample. The results will be interpreted within the proposed experimental design and be used as a pilot study exploring novel information regarding the bryophyte chemical profile variations concering altitude.
MATERIALS AND METHODS
Sanionia uncinata collection
Twenty-four samples of Sanionia uncinata (Hedw.) Loeske were collected on King George Island at the Keller Peninsula, South Shetlands (Fig. 1a), and the herborized vouchers were deposited at the UB Herbarium. The plant material was first collected to be submitted to a wide range of biological assays, so, it was cleaned from soil residues and any other alien material. Then, these samples were submitted to a 96-hour static maceration with consecutive solvent changes at each 24 h using analytical grade ethanol. The 24 moss extracts listed in Table I were kept frozen at -20 °C until use.
Results obtained from the biological assays performed with the 24 ethanolic extracts obtained from Sanionia uncinata (ADS#). (*) extracts ADS04 and ADS14 showed the best combination of significant biological activity and low cytotoxicity to the normal cell line. DeB=diffusion in bioautography; DPPH=diphenyl-picryl-hydrazyl; FCRu and FCTr=Folin Ciocalteu total phenolic content based on rutin or Trolox; Ach=acetylcholinesterase; MCF-7=non-metastatic breast cancer cell line; MDA-MB-231=metastatic breast cancer cell line; MCF-10A=normal breast cell line; PC-3=prostate cancer cell line. Altitute information from plant collection is also given.
a. King George Island at the Keller Peninsula, South Shetlands. b. Canonical correspondence analysis for the ethanolic extracts and biological assays, based on the chemical profiles. c. Principal component analyses to evaluate the relationship between chemical profile and ethanolic extracts from Sanionia uncinata in relation to the altitude from where the plants that originated the extracts were collected.
To limit and expose any bias in the analysis, the plant samples used to obtain the 24 extracts were obtained by a convenience-non-probability sampling method. This means that plants were collected based on their accessibility to the collector. Consequently, the results can be used to support further analyses reporting the altitude influence on mosses’ chemical constituents.
Biological and biochemical assays
Table I provides biological, biochemical, and data related to the altitude of the plant collection sites. The number of replicates and standard substances are also given (Teodoro et al. 2024).
Antimicrobial analysis
Diffusion in bioautography (DeB) method was used to evaluate the antimicrobial activity of the extracts (100 mg/mL) against Candida albicans, Escherichia coli, Streptococcus mutans, and Staphylococcus aureus (Suffredini et al. 2023). Those microorganisms are important pathogens to both men and companion animals. A 10 µL drop of each extract, diluted at 100 mg/mL, was applied on a thin-layer chromatography (TLC) plate. Afterward, a proper inoculated agar medium was poured on the TLC under sterile conditions and incubated accordingly. Afterward, MTT viability dye, 3-4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide, a tetrazolium salt, was used to highlight the antimicrobial activity of each extract. Culture conditions for each microorganisms: Candida albicans (ATCC10231; concentration of 1.5x10⁵ CFU/mL; Sabouraud Dextrose agar medium; incubation at 36 °C for 24 h; Escherichia coli (ATCC 29212; concentration of 1.5 x 10⁷ CFU/mL; Mueller-Hinton agar medium; incubation at 36 °C for 24 h); Staphylococcus aureus (ATCC29213; concentration of 1.5 x 10⁸ CFU/mL; Mueller-Hinton agar medium; incubation at 36 °C for 24 h); Streptococcus mutans (ATCC25175 concentration of 1.5 x 10⁷ CFU/mL; Brain Heart Infusion agar medium; incubation at 36 °C for 48 h). A solution of 1% chlorhexidine was used as the reference drug.
Cytotoxicity test against breast and prostate tumor cells
Cell culture and samples –Plant extracts (40 mg/mL) were tested against one human prostate cancer cell line, PC-3, the breast cell lines MCF-7 (non-metastatic cancer cell) and MDA-MB-231 (metastatic cancer cell), and MCF-10A, a normal cell line. The sulforhodamine B (SRB) cell viability test was adopted (Monks et al. 1991). The results were expressed as the percentage of cell growth [(T-T0)/(C-T0)*100], where T=optical density of treatment C=optical density of cell growth control; T0=optical density of 24-h cell growth, which is the time the cells set up before receiving treatments. Doxorubicin 25 mM was used as the reference substance.
Antioxidant activity of Sanionia uncinata extracts
To assess the antioxidant activity, β-carotene and diphenyl-picryl-hydrazyl (DPPH) antioxidant activity tests were performed according to classic techniques described in the literature (Duarte-Almeida et al. 2006, Sherma 2018). Drop autography was used in the qualitative analyses, and the quantification was assessed by obtaining the absorbance of β-carotene and DPPH after the treatment with the 24 moss extracts (Duarte-Almeida et al. 2006, Sherma 2018) diluted at 40 mg/mL. Trolox and rutin were used as the reference substances.
Total phenolic content analysis
The Folin-Ciocalteu (FC) test quantified the total phenolic content of each of the 24 moss extracts (40 mg/mL). Trolox and rutin were the reference substances (Zhang et al. 2006).
Acetylcholinesterase inhibitory activity
An autography assay was performed to assess the enzyme inhibitory activity of the 24 moss extracts (Rhee et al. 2001) diluted at 4 mg/mL. A 10 µL drop of each extract, diluted to 4 mg/mL, was placed in a silica gel GF254 chromatographic plate (TLC). After that, the enzyme and the Ellman’s reagent were applied to the TLC to evaluate for enzyme inhibition. Physostigmine was diluted in methanol at a 1.5 µg/mL concentration and was used as the reference substance. The quantification of the enzyme inhibitory activity 50% related to each of the 24 extracts was also assessed by a spectrophotometric assay using the same material (Ellman et al. 1961).
Sanionia uncinata extract fingerprint by gas-chromatography-mass spectrometry (GC-MS)
Samples were dissolved in methanol at a concentration of 4 mg/mL and analyzed by gas chromatography (6850 Network GC System, Agilent) coupled with mass spectrometry (MS; Agilent 5975C VL MSD), HP5-MS capillary column (Agilent, length 30 m, ID 250 μm, 0.25 μm film thickness). The initial column temperature was adjusted to 100° C for 5 min, ramped at 5° C/min to a final temperature of 320° C, total run time of 57 min. The injection volume was 1 μL, carrier gas Helium at one mL/min. The injector, ion source, and quadrupole temperatures were 300° C, 280° C, and 180° C, respectively. MS detection was performed with electron ionization (EI) at 70 eV, working in the full-scan acquisition mode ranging between 50-800 m/z at 2.66 scan/s. Compounds were identified using commercial standards and fragmentation patterns, and mass fragmentation was compared using NIST digital library spectra 2.0 (2008).
The samples (0.1 mg) were derivatized using N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (50 μL) in pyridine (50 μL) for 1h at 70°C (Carvalho et al. 2021). GC-MS analyzed metabolites using the same equipment and conditions described previously. Compound identification was made by comparing the mass fragmentation using NIST digital library spectra using the same parameters described above.
Sanionia uncinata extract EDS analyses
Energy-dispersive spectroscopy (EDS) in scanning electron microscopy (SEM) apparatus was used to determine the chemical element occurring in the plant extracts. The extracts were diluted in ethanol at one mg/mL and 10 μL were added to a round-shape coverslip that was placed on an acrylic stub in preparation for analysis in an EDS (Noran Instruments, Middleton, WI, USA) coupled to a SEM (JSM 5600 LV, JEOL, Tokyo, Japan). The analyses were conducted by using the EDS (20 kVp, cursor at 4.500 kV; 44 counts), and the results are expressed as Atom percentages.
Statistics and Experimental Design
Considering that the extracts were obtained from plants collected by a convenience-non-probability sampling method, statistical analyses were performed with information regarding the chemical composition of the samples. The authors state that the sampling is hard to represent the overall population, which leads to speculative results. Based on those conditions, we speculated about the approximate altitude at which there was a significant change in the extracts’ chemical composition. To prospect the relationship between chemical results, biological results, and altitude, a canonical correspondence analysis (CCA) was performed based on the relative percentage of the compounds or elements occurring in at least two of the extracts to analyze how the extracts were distributed concerning the biological data. Also, a principal component analysis (PCA) was performed to evaluate the distribution of the extracts according to the altitude. To perform analyses, data were standardized to a range of continuous initial variables; the covariance matrix and the eigenvalues were calculated. Statistical material is added to the Supporting Information. The chemical compounds or elements were considered the 49 variables for both analyses, and the 24 extracts were the cases.
RESULTS AND DISCUSSION
Biological and chemical data were investigated by the canonical correspondence analysis, CCA, which resulted in the general distribution observed in Fig. 1b (cumulative constraint percentage 49.755 in the 2nd axis; 65.829 in the 3rd axis). The vectors represent the biological assay results, the cases represent the extracts (ADS#), and the numbers represent the chemical compounds and variables, as seen in Table I. Table I shows that ADS04 and ADS14 have antibacterial activity, while antioxidant activity is widespread among the 24 extracts. Also, acetylcholinesterase inhibition was observed for all extracts, but ADS06 and ADS20 showed diminished inhibitory activity. The extracts were more cytotoxic to MDA-MB-231, the breast metastatic cell line, than the non-metastatic MCF-7 or the normal breast cell line MCF-10A. Finally, the extracts showed a mild cytotoxic activity against the prostate cancer cell line PC-3.
All the extracts obtained from plants collected above 30 m (ADS02, ADS04, ADS06, ADS08, ADS09, ADS10, ADS11, and ADS13) showed antiproliferative activity against MCF-7, MDA-MB-231, and PC-3 cell lines and a better capacity of inhibiting acetylcholinesterase. The other extracts showed better antiproliferative activity against MCF-10A, and better antioxidant activity, considering the β-carotene test.
Extracts ADS04 and ADS14 showed antibacterial activity against Staphylococcus aureus despite being obtained from plants collected at different altitudes. Previous works reported the presence of the antioxidant phenolic compounds sanionins A and B in S. georgico-uncinata (Müll. Hal.) Ochyra & Hedenäs, a species closely related to S. uncinata, also occurs in Antarctica (Ivanova et al. 2007).
Table II shows the suggested compounds identified from the fingerprint GC-MS analyses of the 24 ethanolic extracts obtained from the moss.
Gas chromatography-mass spectrometry chemical profiles from the 24 ethanolic extracts from Sanionia uncinata collected in the Antarctic Peninsulae. (*) = compounds indicated to occur in all 24 ethanolic extracts from Sanionia uncinata.
The chemical profiles of the twenty-four samples were obtained from injection of the diluted extracts in GC-MS, and the suggested identification of 20 compounds were reported: (E)-dec-2-enal (1, RT 8.36 min); (Z)-dec-2-enal (2, RT 8.42 min); neophytadiene (3, RT 22.15 min); 6,10,14-trimethylpentadecan-2-one or hexahydrofarnesilacetone (4, RT 22.30 min); methyl palmitate or palmitic acid methyl ester (5, RT 23.91 min); palmitic acid (6, RT 24.65 min); methyl linolelaidate or linolelaidic acid methyl ester (7, RT 27.10 min); methyl oleate (8, RT 27.21 min); oleic acid (9, RT 27.33 min); methyl stearate (10, RT 27.69 min); 13-octadecenal (12, RT 27.99 min); stearic acid (13, RT 28.33 min); glycidyl palmitate (14, RT 30.64 min); methyl eicosanoate or eicosanoic acid methyl ester (15, RT 31.15 min glycidyl palmitoleate (17, RT 33.56 min); stigmast-5,22-dien-3-yl acetate (18, RT 41.95 min); ergost-5-en-3b-ol or campesterol (19, RT 44.18 min); squalene (20, RT 44.88 min); β-ergostenone or campestenone (21, RT 45.56 min); stigmast-3,5-dien-7-one (22, RT 46.18 min).
The extracts were derivatized and re-analyzed in GC-MS, and the following compounds are suggested to be present in the samples: glycerol (23, RT 10.088 min), 6-acetyl-2,5-dihydroxynaphtoquinone (24, RT 17.1773 min), frutofuranose isomer 1 (25, RT 23.445 min), frutofuranose isomer 2 (26, RT 23.612 min), frutopiranose (27, RT 23.684 min), galactopyranose (28, RT 25.213 min), D-manitol (29, RT 26.025 min), Glycopyranose (30, RT 27.015 min), palmitic acid (31, RT 27.519 min), stearic acid (32, RT 31.044 min), trealose (33, RT 36.525 min), turanose (34, RT 36.770 min), saccharose (35, RT 38.309 min), glyceryl stearate (36, RT 39.562 min), campesterol (38, RT 45.730 min), and β-sitosterol (39, RT 46.807 min). Compounds 11, a fat acid, 16a, a carboxylic acid diester, and 37 were not fully identified.
The EDS analyses of the chemical elements occurring in the extracts indicated that C, O, Na, and Fe occurred in all 24 extracts, while Al, K, Ti, Co, Ni, Cu, Zr, and Nb occurred in 23 or fewer extracts.
The PCA distribution can be seen in Fig. 1c, where the extracts are considered the cases and the substances, the variables. The cumulative percentage of the variance expressed in the second axis accounted for 63.840. Two distinct groups were identified, and they are composed of extracts obtained from plants collected in sites allocated in an altitude range from sea level up to 30 m (in red) and from 31 m up to 130 m (in green). The group formed by the extracts obtained from plants collected at altitudes lower than 30 m is discriminated in the first component and is formed by extracts ADS12, ADS16, ADS20, ADS21, ADS22, ADS28, ADS30, ADS32, ADS34, ADS36, ADS38, and ADS40. Compounds Stigmast-5,22-dien-3-yl acetate (18) and saccharose (35) influenced the formation of that group. A study described the importance of stigmasterol to plants (Valitova et al. 2024) in an extensive review. It indicated that stigmasterol and its derivatives are produced by plants under prolonged saline exposure, disturb ion homeostasis, and disrupt physiological and biochemical processes in those plants, also leading to altered lipid metabolism, diminish of the membrane fluidity modulation as a consequence of temperature adaptation, also interfering with membrane permeation. Such conditions follow the present findings, as the plants from altitudes lower than 30 m above sea level presented higher amounts of stigmasterol derivatives.
A second group is formed by extracts ADS 2, ADS4, ADS6, ADS09, ADS11, and ADS13. Methyl palmitate (5), galactopyranose (28, sugar), and glycopyranose (30) discriminated the group formed by the extracts that were obtained from plants located above 30 m from the sea level. Fatty acids are a class of secondary metabolites that remarkably occur in mosses (Klavina et al. 2015), together with sterols and triterpenes, which is of major importance for their survival and adaptation to different environmental conditions to protect from outside impacts (Gunstone 1996). Some authors studied the variation of fatty acids and correlated compounds as sterols and triterpenes in 12 Sphagnum species. They observed that there was variation but not in an extensive range (Baas et al. 2000), and such results are in accordance with our findings regarding the fact that we compared 24 samples of the same species while they compared different species. We suggest that variation must be considered within the species other than among species. Previous works with the Antarctic moss Pohlia nutans described the metabolism adaptation to temperature changes (Liu et al. 2022c). The authors report that the fatty acid biosynthesis is increased, and the correlated genes are overexpressed, including those related to the very-long-chain fatty acids (VLCFAs), indicated by the high amounts of fatty acids and their derivatives.
It was observed that there was a difference in the chemical compound distribution in the extracts obtained from plants collected in altitudes higher than 30 m from the sea level, which may be attributed to some variations in the biochemical processes of the plants to adapt to different environmental conditions, as seen in a recent study (Sytiuk et al. 2022), which describes the combination of information on the morphological and physiological traits, including the primary and secondary metabolites, that are related to the plasticity of the plant to adapt and survive in their environment. A previous study (Costa et al. 2018) was performed with 12 liverwort Syzygiella rubricaulis (Nees) Steph. (Jamesoniellacceae) populations collected from different altitudes across four countries in South America and prospected for differences in the secondary metabolite amounts. Considering nothing but the number of identified compounds, the authors concluded that the altitude did not influence the differences in the secondary metabolite expression among the 12 populations, despite the fact that they made the conclusion without taking the percentage of each compound among the populations into account. The present findings support an indicative altitude limit where the plants suffer environmental adaptation and a consequent alteration in their biochemistry. Previous works have shown that S. uncinata presents an elevated cellular plasticity medicated by proteins to tolerate extreme conditions such as lack of water (Pizarro et al. 2019). The authors observed diminishing respiration and photosynthesis during moss desiccation conditions due to reduced metabolism, including its response to the oxidative processes.
Twelve compounds identified in the present work are indicated to occur in the 24 ethanolic extracts: neophtadiene (3; a diterpene), methyl palmitate (5; a fatty acid ester), methyl oleate (8; a fatty acid ester, FAE), methyl stearate (10; a fatty acid ester), glycerol (23, polyol), frutofuranose isomer 2 (26, sugar), frutopyranose (27, sugar), galactopyranose (28, sugar), glycopyranose (30, sugar), palmitic acid (31, fatty acid), turanose (34, sugar ), saccharose (35, sugar), and the chemical elements C, O, Na, and Fe.
CONCLUSIONS
Sterols were observed to occur frequently in Sanionia uncinata collected under 30 m above sea level, while fatty acids occurred in the plants collected from altitudes above 31 m above sea level, which indicated that a possible adaptation to environmental conditions is occurring. Further omics analyses are needed to confirm our present findings.
Acknowledgements
The authors are indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP), Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Marinha do Brasil (MB), Força Aérea Brasileira (FAB), Universidade Paulista (UNIP), Universidade Católica de Brasília (UCB), Universidade de Brasília (UnB), for their financial and logistics support, which made this work a reality. Grants# CNPq44272020181; CNPq30469920187; CNPq30962820210; FAPDF 00193.00001055/2021-13; FAPDF 00193.00001117/2021-97; CAPES/PROSUP#001 for ALS; UNIP Full scholarships (as tuition fee exemption) for ALS, JSS, and SAF. This paper adds knowledge to other Antarctic research initiatives, and the government supports it through the Brazilian Antarctic Program (PROANTAR; Brazil, 2016), whose main objective is to support the establishment of Brazil as one of the Scientific Committee on Antarctic Research (SCAR) leaders in the current Century.
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