3.1. The Environmental Context and Seasonal Variation of MAAs
The site of sample collection (Bahía Mansa, Magellan Strait in the Subantarctic region) exhibited wide seasonal difference in PAR and UV levels, with maximum values in summer and lowest values in winter. Additionally, this latitude in the Southern Hemisphere is exposed to an increase of biologically harmful UVR because of ozone depletion [
2], which takes place in early spring. On the other hand, the mean surface water temperature recorded in Bahía Mansa varied between 6.7 °C in winter-spring to 8.7 °C in summer. However, at local level, in the intertidal of Magellan Strait, other factors can vary according to tide and season; this environmental variation could impose constraints on the physiology and growth of seaweeds. In the intertidal zone of Magellan Strait, the species
Nothogenia fastigiata and
Iridaea tuberculosa grow well in the mid-intertidal, even when exposed to high solar radiation and desiccation. However,
I. tuberculosa occupies more shaded and wet places (e.g., cracks), and
Corallina officinalis occurs in the lower intertidal where variations in environmental conditions could be less severe.
MAA compounds were presented throughout the year in the three species; however, the concentration and composition of MAAs followed strong seasonal changes. In the case of
N. fastigiata and
C. officinalis, MAA content increased during spring and summer, respectively. This seasonal pattern in MAA content with maximum concentrations under high incident levels of solar radiation is consistent with the idea that these compounds are synthesized and accumulated in response to solar irradiance stress (mainly UV). In fact, MAA content in these two species correlated positively with solar radiation. Additionally, the fact that ozone depletion takes place particularly in the Southern Hemisphere, which results in an increase of biologically harmful UVR reaching the Earth’s surface, magnifies the importance of MAAs in algae from these regions. We know that mycosporine-glycine (UV-B-absorbing MAA) increased to the detriment of the UV-A-absorbing MAAs in the red alga
Mazzaella laminarioides during a period of ozone depletion in southern Chile, indicating that this seaweed can acclimate to increased UV-B radiation [
45].
This is one of a few studies reporting concentrations and composition of MAAs relative to seasonal changes in seaweed species from high latitudes. Seasonal variation with high MAA content correlating with high solar irradiance was reported in the red alga
Gracilaria vermiculophylla [
46] cultured outdoors and planktonic organisms from lakes [
47,
48]. Furthermore, it was reported that UV-absorbing MAAs and antioxidant enzyme activities experience strong seasonal changes in
Palmaria palmata and
Devaleraea ramentacea from the Kongsfjorden on Spitsbergen. Both species exhibited a significant increase in the concentration of MAAs coinciding with the increase in underwater radiation during sea ice breakup in summer [
49]. However, although a seasonal variation in the MAA concentration was reported in the seaweed
Pyropia plicata from New Zealand, the increased total MAA content was not correlated with solar radiation [
18]. In fact, the authors observed that fronds collected from April to August (autumn-winter period) exhibited the highest MAA content, while fronds collected from August to November (spring) showed a strong decrease in MAA content. This last report is consistent with our study with respect to
I. tuberculosa data, which shows high total MAA concentration in winter.
According to the classification of Hoyer et al. [
16,
24],
N. fastigiata,
I. tuberculosa, and
C. officinalis can be characterized as seaweeds with a basic MAA concentration that is adjusted according to environmental conditions. However, each species exhibited high MAA content in different periods of the year:
I. tuberculosa in winter,
N. fastigiata in spring, and
C. officinalis in summer. This difference could be attributed to local environmental factors associated with the microhabitat where each species grows in the intertidal, which could, in turn, reflect MAA composition and accumulation. In a given seasonal period, it has been suggested that some UV-absorbing secondary metabolites (e.g., phenolic compounds) have a stronger correlation with local environmental factors than large-scale factors (e.g., months, seasons, latitude) [
50,
51]. The content of MAAs in red seaweeds from the Chilean coast (mid latitude) was higher in organisms collected from the eulittoral area compared to the sublittoral area in relation to higher daily integrated irradiance and other stress conditions, such as desiccation events [
17]. Other variables, such as nitrogen supply in upwelling coastal areas of Brazil, produce an increase of MAAs, whereas in areas with high irradiance and nitrate-poor waters, the level of MAAs decreased [
44]. In the same context, in the upper intertidal,
N. fastigiata is exposed to higher solar irradiance and thermal stress during spring and summer compared to the other two studied species, and for this reason, an increase of MAA content would take place during these seasons. Additionally, porphyra-334 is the quantitatively most important MAA in
N. fastigiata at over 60% in all seasons, except in winter, with palythinol between 15% and 40%. The presence of porphyra-334 as the main component is reported in species that preferentially are often exposed to high solar radiation and grow in the upper littoral to supralittoral zones [
16,
18,
19]. On the other hand,
N. fastigiata collected during summer in Valdivia (Chile) exhibited higher MAA content (3.8 mg g
-1 of dried mass weight) and different composition as follows: porphyra-334 (80%), shinorine (10%), and palythinol, asterina-330, palythine and mycosporine-glycine with less than 4% each [
17]. This suggests that concentration and composition could vary with latitude and that this variation could be related to levels of solar irradiance. Interestingly, in our study, even though total MAA concentration of
N. fastigiata decreased from spring to summer, asterina-330 was still synthetized (5%) in summer. This type of MAA has an important antioxidant activity against lipid peroxidation, similar to porphyra-334 [
11], which could be important to quenching oxygen reactive species promoted by light excess.
Iridaea tuberculosa showed the highest MAA content among the studied species. This species accumulated more MAAs during winter. On the other hand, unlike
N. fastigiata,
I. tuberculosa has palythine (more than 50%) as the most representative MA but also palythinol (ranging from 18% to 50%). This is consistent with the report that palythine (and shinorine) are usually the dominant MAAs in red seaweed species inhabiting more shaded places (e.g., subtidal) [
16]. The high MAA content in
Pyropia plicata during winter was related to nitrogen availability in seawater [
18] on account of the positive correlation between N content and MAAs observed in several species [
9,
25,
40,
45,
52]. However, seawater of the Magellan Strait can be considered mesotrophic waters since NO
3- concentration is rather uniform at about 0.005 mM, and no annual significant variation has ever been reported [
53]. Thus, the induction and synthesis of MAAs in winter in
I. tuberculosa could be influenced not only by specific wavelengths during short light periods but also by any possible endogenous biological controlling factor. It should be noted that
I. tuberculosa is more abundant during autumn and winter seasons.
N. fastigiata and
I. tuberculosa decreased their concentration of MAAs in summer when compared to previous seasons. Both species exhibited a high concentration of palythinol, which varied differently in both species during the year. Specifically, the former exhibited a high palythinol concentration in winter, while the latter showed high concentration in the summer-autumn seasons. In addition, short-term variation of MAAs was reported. For example,
Porphyra columbina increased its MAA content 3 h before high solar radiation [
52]. In studies on action spectra for the induction of MAAs in
Chondrus crispus from Helgoland (Germany), short wavelength UV-B exhibited the highest quantum efficiency in its synthesis as well as in asterina-330 and palythine [
54].
The calcareous
C. officinalis exhibited the lowest total MAA concentration among the studied species, ranging from 0.1 to 0.4 mg g
-1 of dried mass weight. The low total MAA content could be a reflection of its acclimation to low radiation levels. On the other hand, it should be noted that the cell wall of
C. officinalis contains calcium carbonate, which effectively absorbs UV radiation, thus minimizing the exposure of important biomolecules in that alga [
13,
55]. MAA concentration varied seasonally, with high levels being observed during spring-summer and the lowest recorded during autumn-winter. The most abundant MAAs detected in this species were shinorine (25%–50%) and palythine (approx. 35%–40%); other MAAs, such as asterina-330, were also present (4%–6%). Shinorine was also the main and only MAA reported in
Corallina officinalis from Argentinian Patagonia [
55]. Similar results were reported in
Ellisolandia elongata (before
Corallina elongata), with shinorine (50% to 60%) and palythine (approx. 40%) being the most abundant MAAs, while other MAAs, such as asterina-330, were present in trace amounts [
14]. On the other hand, the inventory and percentage of MAAs in
C. officinalis are consistent with those of
C. officinalis from New Zealand [
19]. Interestingly, asterina-330 increased in spring (6%) when compared to the previous season (3.8%). Similarly, porphyra-334 was synthetized from zero (in winter) to 8% in spring to the detriment of shinorine. Under light stress, all of these MAAs could prevent photodamage by their UV screening and antioxidant capacity [
11].
3.2. MAA Composition by HPLC-ESI-MS
Seven different MAAs were identified using HPLC-ESI-MS, with palythine-serine and mycosporine-glutamic being recorded for the first time in seaweed of Subantarctic ambient (
Iridaea tuberculosa). Another “unidentified” UV-absorbing compound was also found. The identification of MAAs by HPLC using secondary standards agreed well with HPLC-ESI-MS, mainly in
Nothogenia fastigiata (palythinol, porphyra-334, shinorine, and asterina-330). For
Corallina officinalis, the techniques differed by the identification of only one MAA, i.e., palythine (HPLC) and palythine-serine (HPLC-ESI-MS). However, for
I. tuberculosa, HPLC-ESI-MS turned out to be a good tool because three more MAAs were recorded (palythine-serine, mycosporine glutamic acid, and mycosporine-glycine), including an unidentified potential MAA, as noted above. Shinorine, porphyra-334, palythine, and asterina-330 are the most common MAAs described in seaweeds [
8], while mycosporine-glycine, mycosporine-glutamic acid, and palythine-serine are less frequent. In fact, palythine-serine had not been previously reported in seaweeds.
Palythine-serine was previously described in corals [
22,
56,
57,
58,
59], cyanobacteria [
60,
61,
62], and in dinoflagellates [
6]. Although the wavelength of the absorption maximum of palythine-serine and palythine are similar (λ
max = 320 nm), palythine-serine would be a secondary MAA synthesized from shinorine after decarboxylation and demethylation of the glycine subunit, as suggested in the literature [
57,
58], whereas palythine would be synthetized from a more primary MAA: mycosporine-glycine [
6]. In the cyanobacterium
A. variabilis PCC 7937, the synthesis of palythine-serine could be regulated by sulfur deficiency [
60,
61].
To the best of our knowledge, mycosporine-glutamic acid was reported a few times in the literature, mainly in fungal organisms, e.g.,
Glomerella cingulate [
63] and
Heluella leucomelaneue [
64] but more recently in the red seaweed
Bostrychia scorpioides from the coast of France [
31]. Mycosporine-glutamic acid is a quantitatively less important MAA in the species studied, and its contribution to UV protection and as an antioxidant agent is still unknown. According to Wada et al. [
65], the reaction of mycosporine-glutamic acid with singlet oxygen can be expected, though not much experimental detail has been reported [
63].
According to its characteristics (λ
max: 330 nm;
m/
z: 261.14189), the unidentified UV-absorbing compound could correspond to an aplysiapalythine C. However, further studies should be conducted for better identification. Aplysiapalythine A, B, and C were reported for the first time in the sea hare
Aplysia californica [
66], and the authors suggested that these marine animals acquire MAAs from their algal diet. More recently, Orfanoudaki et al. [
19] reported aplysiapalythine A and B, but not aplysiapalythine C, in several seaweed species from New Zealand and Australia. The same authors explain that aplysiapalythine A and B were identified from MAA-enriched extracts and in trace concentrations.
3.3. The Subantarctic Red Seaweeds as Source for Cosmetic Application
Currently, a few red seaweeds, such as
Porphyra spp.,
Bostrychia spp., and
Bangia spp. with high MAA concentration, supply the biotechnological industry with compounds to produce sunscreens [
67,
68,
69]. Still, such compounds have yet to be fully exploited because of the inherent difficulty of ensuring sufficient algal biomass for MAAs extraction [
70]. In the case of
Porphyra spp., the alternation between macroscopic and microscopic phases makes it difficult to get sufficient algal biomass throughout the year, particularly in cold waters. In this sense, the diversification of sources for MAAs extraction among various seaweed species (e.g.,
N. fastigiata and
I. tuberculosa) could result in a plentiful supply of algal biomass for extraction of these compounds, mainly in seasons when
Porphyra spp. are in microscopic stages. In the case of
C. officinalis, its low MAA concentration does not allow it to be used as source of MAAs; however, further investigation is warranted because many extracts of this species are now being used in the cosmetic industry based on its diverse properties, such as anti-aging, anti-inflammation, smoothing agent, and UV filter [
71,
72].
Even though the three species studied in the present work have a low concentration of MAAs when compared to
Porphyra spp., they have the ability to modify not only their concentration but also their composition, depending on the season. Thus, modifying the factors in controlled culture systems could make it possible to change the MAA composition and content. Our research group reported the increase of MAA content in seaweeds cultured in tanks under controlled conditions [
39,
45,
46,
52].
Such diversification not only implies finding species with high MAA concentration but also species with different MAA composition or species with novel MAAs. In this respect, although low in concentration, I. tuberculosa possesses two MAAs uncommon in seaweeds, namely mycosporine-glutamic acid and palythine-serine. The ecophysiological and biotechnological relevance of these MAAs is still uncertain and further research is necessary. A detailed screening of MAAs and their biological activity should be carried out in seaweeds of the extreme environments. Because these organisms inhabit extreme environmental conditions, they could be an excellent source for ecologically and pharmacologically relevant natural compounds.