The mutualistic association between symbiotic dinoflagellates (with trivial name zooxanthellae) and corals, which form the basis of all shallow-water coral reefs on Earth, is one of the most widely studied examples of such a relationship (LaJeunesse, 2020). The biogeography of both corals and their symbionts, as well as the phylogeny of those symbionts, has been extensively studied in recent decades (Baker, 2003; Stat et al., 2006; LaJeunesse et al., 2018). In contrast, less attention has been paid to the association between scyphozoan jellyfishes and their symbionts. This association provide an important key traits as mixotrophic way of nutrition, because zoxanthellate medusae are holobionts and derive their nutrition from predation and photosynthesis. Possession of dinoflagellate symbionts in polyps is rarely necessary for surviving of polyps, but notable trait is a key role of symbionts in the life cycle of the jellyfish by allowing or facilitating strobilation of polyps to secure medusa with suitable symbionts (Djeghri et al., 2019). Of the 79 valid genera in Scyphozoa, only 11 harbour symbionts: Linuche, Nausithoe (Coronamedusae), Bazinga, Cephea, Cassiopea, Cotylorhiza, Netrostoma, Mastigias, Phyllorhiza, Thysanostoma, and Versugia, most belonging to the suborder Kolpophorae and all of which with a metagenic life cycle (Djeghri et al., 2019). The best-studied genus among the zooxanthellate Scyphozoa is Cassiopea, with its symbionts serving as a holobiont model to reveal various aspects of their mutualism (Lampert, 2016; Ohdera et al., 2018). The symbionts of the species Cotylorhiza tuberculata (Macri, 1779) and Phyllorhiza punctata (von Lendenfeld, 1884) have not yet been described in detail (LaJeunesse et al., 2021). The fried egg jellyfish Cotylorhiza tuberculata is one of the rare symbiont-bearing scyphozoans from temperate latitudes, distributed in the Mediterranean Sea and around the Canary Islands (Collins et al., 2021). It is predominantly distributed in oligotrophic environments; however, it can also occur in eutrophic areas, as in the Mar Menor Lagoon in the Balearic Sea (Pérez-Ruzafa et al., 2002). Phyllorhiza punctata is an invasive species introduced into the eastern Mediterranean Sea in the 1990s from oligotrophic tropical seas from the south-central coast of eastern Australia (Galil et al., 1990; Galil et al., 2009). Cassiopea xamachana (Bigelow, 1892) is sedentary in oligotrophic, shallow waters and is distributed in the Gulf of Mexico, the Caribbean Sea, and warmer areas of the western Atlantic Ocean (Verde & McCloskey, 1998). While the symbionts of Cassiopea xamachana have already been studied in terms of their mode of transmission (Thornhill et al., 2006; Mellas et al., 2014; Ohdera et al., 2018) and parasitic potential (Sachs & Wilcox, 2006), the symbionts of Cotylorhiza tuberculata and Phyllorhiza punctata have not yet been characterised in detail (LaJeunesse et al., 2021).

The dinoflagellate family Symbiodiniaceae is characterised by a rich genetic diversity within evolutionary divergent lineages (LaJeunesse et al., 2018; LaJeunesse et al., 2021). Members of this family are symbionts in foraminifera, ciliates, poriforans, cnidarians and molluscs (Venn et al., 2008; Hansen and Daugbjerg, 2009). The taxonomy of the flourishing algae of the family Symbiodiniaceae have recently been resolved by the 28S rDNA and ITS2 regions (LaJeunesse et al., 2018; LaJeunesse et al., 2021) which provide sufficient resolution to species delimitation (Moestrup and Daugbjerg, 2007). In details, the 28S rDNA ribosomal markers and the ITS2 regions of nuclear and chloroplast (cp23S region domain V) DNA have a phylogenetic signal that can resolve clades at the species level (Savage et al., 2002; Casado-Amezúa et al., 2014; LaJeunesse et al., 2018). Both markers (28S rDNA and cp23S region domain V) have similar levels of resolution (Sampayo et al., 2009), although the 28S rDNA region has been widely used to assign clades and phylotypes to zooxanthellae isolates and infer relationships between them (Coffroth and Santos, 2005; Barbrook et al., 2006). ITS markers are more commonly used to obtain phylogenetic resolution at the subclade level, especially at the species level (LaJeunesse, 2001; Rodriguez-Lanetty et al., 2001; Meron et al., 2012; Grajales et al., 2016). The relevance of ITS2 is based on the different secondary structure of ITS2 in the specific clades B, C, F and H (Meron et al., 2012). LaJeunesse et al. (2018) published a phylogenetic analysis of the Symbiodiniaceae (previously assigned Symbiodinium clades from A to H), interpreted the evolution of the group, and provided a redescription of clades A, B, and C into new genera. The former clade A is recognised now as the genus Symbiodinium (Hansen and Daugbjerg, 2009), with Symbiodinium natans as the type species (LaJeunesse et al., 2018). The most studied zooxanthellate jellyfish is Cassiopea xamachana and the majority of symbiont phylotypes associated with Cassiopea xamachana belong to the genera Symbiodinium (previously clade A, especially phylotype A1) (Thornhill et al., 2006; Lampert et al., 2012), Breviolum (previously clade B with common phylotype B1), or Cladocopium (previously clade C with phylotype C3) (LaJeunesse et al., 2003; LaJeunesse et al., 2018). The former phylotype “Temperate A”, which occurs in a variety of hosts from temperate zones, was recently redescribed as Philozoon (Geddes, 1882), containing several species, most closely related to the genus Symbiodinium (LaJeunesse et al., 2021).

Previous studies on mutualistic association between Symbiodiniaceae members pointed out that some of its members are host generalists (Baker, 2003; LaJeunesse et al., 2018), while others are thought to be specialists due to their rarity (LaJeunesse et al., 2004a; LaJeunesse et al., 2004b). Host specialists spread through preferential vertical transmission, while host generalists are transmitted horizontally from the pool of free-living cells into the host (Fabina et al., 2012). Horizontal transmission gives the host the opportunity to acquire locally adapted algal cells (Van Oppen, 2004), thus increasing its fitness to occupy available niches and respond to environmental changes (Bongaerts et al., 2015). This assumption is based on the high genetic diversity of species within the genera of Symbiodiniaceae, which supports their functional diversity as light harvesting and utilization under variable conditions (Stat et al., 2008; see review Suggett et al., 2008). In vertical transmission (Fabina et al., 2012), symbiont diversification is maintained by high genetic plasticity and twinning when the symbiont is isolated from the external population (Sachs & Wilcox, 2006). Meanwhile, horizontally transmitted symbionts are translocated into the host in each generation, limiting the possibility of coevolution.

In the present study, we used a phylogenetic approach using nuclear 28S rDNA and ITS2 markers to identify symbionts in Cotylorhiza tuberculata and Phyllorhiza punctata from the Mediterranean Sea during blooming period and Cassiopea xamachana collected at Cabo Frio (Rio de Janeiro, Brazil). In addition, symbionts were isolated from Cotylorhiza tuberculata and Cassiopea xamachana and cultivated symbiotic cells were used in cloning experiment to reveal the diversity of the ITS2 region of the dinoflagellate cells within individual host medusa. Photosynthetic pigments of two different genera of symbionts from Cotylorhiza tuberculata were characterised.

We identified symbionts in the scyphomedusae Cotylorhiza tuberculata, Phyllorhiza punctata and Cassiopea xamachana by phylogenetic analyses of 28S rDNA and ITS2 haplotypes as these markers provide sufficient resolution to species delimitation (Moestrup et al., 2007) and have recently been used to resolve the evolution of the flourishing algae of the family Symbiodiniaceae (LaJeunesse et al., 2018; LaJeunesse et al., 2021). In the present study, medusae of Cotylorhiza tuberculata collected in the Mediterranean Sea (Adriatic, Ionian, and Balearic Seas) were confirmed to harbour symbionts of the genus Philozoon or the genus Breviolum. In more details, medusae of Cotylorhiza tuberculata collected at Mar Menor (Balearic Sea) harbour only symbiotic dinoflagellate cells of Breviolum. We also confirmed presence of Breviolum in Cassiopea xamachana (type B1), while Breviolum (type B2) was found in Cotylorhiza tuberculata (Gulf of Trieste). Type B2 is common in temperate latitudes and tolerates a wide range of temperature, light, and other conditions (photosynthetic optimum at 25°C and minimum at 10°C), being able to recover quickly from low temperatures (Thornhill et al., 2006). In this regard, cultures offer the possibility to work on the free-living stage of the species, which are essential for a valid description of dinoflagellate species. Symbiotic dinoflagellates are difficult to isolate from a host in axenic culture (Liu et al., 2017). Therefore, antibiotics are used by default during the cultivation, as it is almost impossible not to transfer the organic matter of the host inhabited with other symbionts and commensals together with symbiotic cells (Agatha et al., 2004). Bacteria that grow on the host’s organic matter usually attack the symbionts as well. We have only managed to isolate one strain without antibiotics. In addition, there are many other conditions that influence the success or failure of isolating dinoflagellates, for example: salinity, light intensity and medium used (Agatha et al., 2004). This means that we could overlooked other types of symbionts. However, we would like to emphasise that the identity of isolated strains and symbionts taken from the host prior the cultivation were confirmed by phylogenetic analysis during study. But no distinctive features at light microscopic level could be found for the isolated strains. For additional morphological information a careful examination at ultrastructural level (SEM) of the different clades and species is indicated as an important completion of the genetic, physiological, and ecological data cited above.

Medusae of the non-native species Phyllorhiza punctata collected in the Mediterranean Sea (Ionian Sea) harbour symbionts of Symbiodinium microadriaticum (type A1), as confirmed in our study. Moreover, our analysis confirmed the presence of the same type of symbionts as in the native range of Phyllorhiza punctata. The sequences of the symbionts from the Mediterranean Sea, together with the sequences AY574347 and AY574348 (from Lake Illawarra, New South Wales, Australia), are combined in a 28S phylogenetic tree (see Figure 2 ), indicating the widespread occurrence of Symbiodinium microadriaticum. Closely related to the symbionts of Phyllorhiza punctata are those of Cotylorhiza tuberculata (see Figure 2 and Tables S2 and S3 in Supplementary Material ), now classified as the genus Philozoon (Geddes, 1882) (LaJeunesse et al., 2021). In our 28S rDNA phylogenetic tree, all sequences of Philozoon are grouped together, though the sequences of Philozoon in the ITS2 tree revealed finer structure into two subgroups (see Figure S1 in Supplementary Material ). The first subgroup contained sequences isolated from anemone and coral hosts (Paranemonia and Cladocora), a pattern that has pointed to more specialised group of symbionts (Meron et al., 2012). The other subgroup is formed by Philozoon medusarum from a variety of hosts such as Cotylorhiza tuberculata and hydrozoan species like Anemonia viridis, with the mean distance within this group measured as 0.007 ± 0.003, indicating very little difference between haplotypes of ITS2 (Raspor Dall’Olio, 2016). Philozoon includes earlier groups with the trivial names “A temperate” and “Mediterranean A” (Hunter et al., 2007), both groups are now reclassified into several species (LaJeunesse et al., 2021). They are identified in many hosts (Savage et al., 2002; Lee et al., 2015) with a known range in the Mediterranean Sea and circumglobally in temperate latitudes (Visram et al., 2006; Casado-Amezúa et al., 2014). Well known hosts are the marine snail Pteraeolidia ianthina from South Australia (Loh et al., 2006) and the anemone Anthopleura hermaphroditica along the coast of New Zealand (Howe, 2013).

We noted that the abundant Mediterranean blooming species Cotylorhiza tuberculata can establish a symbiosis with species of Breviolum or Philozoon (see Figures 2 , S1 ). However, the symbiosis in individual medusae only develops with one species at a time, as confirmed for Cotylorhiza tuberculata (Thornhill et al., 2006; Astorga et al., 2012; Newkirk et al., 2018). This is an important indication of the co-existence of free-living species and subsequent selection of symbionts in polyps, with the best-adapted symbionts subsequently transferred by strobilation in ephyra ( Astorga et al., 2012; Newkirk et al., 2018). Polyps play a crucial role in infection with symbionts and can be infected with several types of them at the same time, leading to the selection of the most suitable symbiont (Ohdera et al., 2018). To investigate the diversity of the ITS2 haplotypes of dinoflagellate symbionts within the single host medusa Cotylorhiza tuberculata we constructed a clone library from amplified ITS2 region to gain insight into the diversity of ITS2 haplotypes. Cloning of ITS2 haplotypes was performed in a subset of the samples to check for the possibility that more than one type of symbiont is present in the host medusa, which could compromise the analysis and be a source of chimaeras. In addition, cloning all samples would be very tedious and time-consuming (preparation of clone libraries, selection of positive colonies and sequence analysis). The results show that all cloned ITS2 haplotypes form the same phylogenetic group within Breviolum and the differences were negligible and do not reveal differences over the species level. Unique ITS2 haplotypes from the clone library were included into the phylogenetic analysis (see Table S3 in the Supplementary Material ).

Successful acquisition of symbionts at the polyp stage also affects the efficiency of strobilation, and in some species (e.g., Cassiopea xamachana), only symbiotic scyphystomae strobilate (Newkirk et al., 2018). Moreover, the efficiency of strobilation depends on the phylotype of the symbiont (cf. Mellas et al., 2014). All of the species investigated in the present study have a metagenic life that includes a scyphistoma, strobilation reproduction, and adult medusae, which reproduce sexually and produce planulae that develop into polyps. According to experimental models, Cassiopea polyps show extreme flexibility in the types of symbionts they acquire from free-living species in their external environment (Mellas et al., 2014; Lampert, 2016; Newkirk et al., 2018). Free-living species of Symbiodiniaceae provide the largest reservoir of symbiotic cells (Coffroth et al., 2006; Pochon & Gates, 2010; Newkirk et al., 2018). Cotylorhiza tuberculata reproduces sexually in the Gulf of Trieste in late August and early September, and polyps are grown from planulae within a few days (A. Ramšak, personal observation). Moreover, environmental conditions like sea temperature during this period are conducive to symbiont infection (hot summer, high light intensity vs colder summer with fewer sunny days). When the polyps acquire symbionts in a temperate climate in a shallow Mediterranean coastal environment is still unknown, though two possibilities emerge: i) immediately after the transformation of planulae into polyps that survive the winter period with colder temperatures, or ii) the polyps acquire the symbionts after the winter. Furthermore, symbiosis is not solely required for strobilation: other factors (e.g., increased temperatures) also trigger strobilation (Prieto et al., 2010; Astorga et al., 2012). Kikinger (1992) observed that the planulae were asymbiotic in a stationary population of Cotylorhiza tuberculata from the Ionian Sea and that the scyphystomae already had symbionts. Besides, the author found that the scyphystomae strobilated only if they had symbionts. In the laboratory, asymbiotic polyps did not strobilate for years and only reproduced by budding (maintaining the population of polyps that can acquire symbionts), and that strobilation occurred after they were fed on tissues of Anemonia sulcata with their symbionts (Kikinger, 1992). During ephyra development, symbionts are clustered in the mesoglea and are particularly numerous along the endodermal lining of the gastrovascular system, indicating their importance for feeding (Kikinger, 1992).

Breviolum psygmophilum has been recorded in Cotylorhiza tuberculata from the Adriatic Sea (Gulf of Trieste), an environment known for its marked temperature variability with minimum values around 8°C and maximum values around 26°C (Boicourt et al., 2021). Most evidence of seasonal and geographical distribution of cnidarian symbionts is available from anthozoan hosts such as Bunodeopsis strumosa (Visram et al., 2006) and Oculina patagonica (Casado-Amezúa et al., 2014). Breviolum psygmophilum is widespread in the temperate latitudes of the western Atlantic Ocean and the Mediterranean Sea (Thornhill et al., 2008; LaJeunesse et al., 2012). We have confirmed that Cassiopea xamachana from the coast of Cabo Frio (Rio de Janeiro, Brazil) harbours symbionts of the genera Breviolum and Cladocopium. Several symbionts harboured by Cassiopea xamachana in different parts of the world (Florida, Hawaii, Bermuda, Japan, and Australia) have been identified as Breviolum minutum (AF333511, JN602457, HQ317740, and AF184940), Breviolum antillogorgium (formerly Symbiodinium antillogorgium KT149341), Breviolum endomadracis (formerly Symbiodinium endomadracis KT149342), and Breviolum pseudominutum (formerly Symbiodinium pseudominutum KT149344). In addition, Symbiodinium type A1 was detected in Cassiopea xamachana and Cassiopea andromeda from the Gulf of Mexico and the Red Sea (see Figure 2 ). The reported diversity of symbionts of Cassiopea xamachana in Mexican coral reefs was much higher than the present study, and symbiosis with several species of the genus Symbiodinium, Breviolum and Cladocopium has been confirmed (Zardoya et al., 1995; LaJeunesse, 2001; Garcia-Cuetos et al., 2005). Previous studies have found that Symbiodinium type A1 is most likely to be harboured by Cassiopea xamachana in the adult phase, which has been sampled in Puerto Morelos (LaJeunesse, 2002), Florida (LaJeunesse, 2001; Thornhill et al., 2006), the Bahamas (LaJeunesse et al., 2009), Bermuda and the Caribbean (Rowan & Powers, 1991). Furthermore, even clade F symbionts have been detected in Cassiopea xamachana (Santos et al., 2002). Several conditions influence the acquisition of symbionts and their selection in the host. Benthic corals or anemones have different symbionts at the same time e.g., mixed infection with Philozoon sp. and Breviolum psygmophylum detected in Cladocora caespitosa from the island of Ischia (Meron et al., 2012), while scyphozoans have only one type of symbiont. Although exclusive host specificity is very rare (Fabina et al., 2012; Silverstein et al., 2012), some anthozoan hosts are capable of associating with a limited number of species, as in the case of Plesiastrea versipora, which hosts only Cladocopium (Rodriguez-Lanetty et al., 2001) and Echinophyllia aspera (LaJeunesse et al., 2004a). However, other studies have confirmed instances of multiple infections in corals (Coffroth and Santos, 2005; Bongaerts et al., 2011). The fitness of the symbiosis can be optimised in host animals if the symbiont is transferred vertically from the mother to the daughter of the host animal, such that the partners become specialist symbionts (Fabina et al., 2012).

We found only one symbiont species in the host medusae at a time, although many host species in coral reefs are capable of symbiosis with more than one species of Symbiodiniaceae. Mosaicism in coral colonies is a well-known process that depends on local light conditions (Thornhill et al., 2006). Their different environmental preferences have also been noted in cultures and during cultivation (Santos et al., 2001). Such ecological adaptation (mosaicism) is not necessary for individual medusae due to their motile lifestyle. However, adaptation to highly fluctuating light conditions and transition between heterotrophic and mixotrophic states is advantageous. A recent study investigated the adaptability and invasion potential of the holobiont Cassiopea sp. (Mammone et al., 2021). Cassiopea polyps were infected by Cladocopium sp., which showed efficient photosynthetic plasticity at both low and high irradiance as well as during sudden changes in light exposure. Both partners are under strong constraints that lead to the selection of certain morphological, biochemical, and physiological traits (Mammone et al., 2021). The optimisation of photosynthesis is a clear example of these constraints. The dilemma between optimal light exposure and low UV radiation requires balancing and adaptations by both partners.

The advantage of scyphozoan jellyfish with symbiotic medusae is the increase in fitness through physiological changes and their ability to switch between mixotrophic and heterotrophic states. Symbiotic scyphozoans are polytrophic (planktivorous, ingest particulate food, capable of autotrophy) because they grow very fast and have high nutrient requirements to ensure rapid medusa growth (Pitt et al., 2005). Algal endosymbionts accelerate the excretory process of their hosts by recycling ammonium-rich animal wastes (Cates & McLaughlin, 1976) and contributing to the host’s energy requirements through the direct transfer of algal photosynthates (Hofmann & Kremer, 1981). Symbiotic species such as Mastigias sp. probably have little predatory influence on zooplankton and co-occur with the zooplanktivorous Aurelia in the saltwater lakes of Palau (Hamner & Hauri, 1981), while blooms of Cotylorhiza tuberculata also co-exist with zooplanktivorous jellyfish such as Rhizostoma pulmo (Pestorić et al., 2021).

The stability of cnidarian-algal symbiosis depends on symbionts photosystem and on protective role of host mediated by colour pigments, which influence photosynthetic activity in endosymbionts by either providing the photosystem with irradiance of appropriate wavelength or protecting it from excessive and potentially harmful light (Lampert et al., 2012). The main aim of this study was the identification of the symbionts, while the analysis of pigments content and their ratio in the genera Breviolum and Philozoon, which are common symbionts of Cotylorhiza tuberculata from the Adriatic Sea and the Balearic Islands, especially the Mar Menor lagoon, provides valuable complementary data. Details on this part of symbiotic association between symbiotic dinoflagellate cells and Cotylorhiza tuberculata are scarce and limited on analysis of composition and ratio of pigments. Recent advance in taxonomy of Symbiodiniaceae enable to connect knowledge from photobiology with identified symbiont species, in our study case being Cotylorhiza tuberculata and its symbionts Breviolum and Philozoon. The pigment profile of dinoflagellate symbionts Breviolum and Philozoon had a high content of chlorophyll c₂ , peridinin, and diadinoxanthin, as well as chlorophyll a. The differences observed in the proportions of chlorophyll a, chlorophyll c₂ , and peridinin were notable between dinoflagellate cells of Breviolum and Philozoon hosted by Cotylorhiza tuberculata collected in two different environments. In details, a higher proportion of peridinin was detected in dinoflagellate cells of Breviolum from Cotylorhiza tuberculata collected at the Mar Menor Lagoon and a higher proportion of chlorophyll c₂ was detected in cells of Philozoon from Cotylorhiza tuberculata at the Gulf of Trieste (see Figure 3 ). Pigment analyses have revealed differences between both genera, and according to the study of Enrique-Navarro et al. (2022), the pigment ratio does not change between free-living symbionts and the cells living “in hospite”, and in this regard we think that the number of samples analysed is sufficient to confirm differences between both symbiotic genera. The recent study provide evidence that the light environment influence on differences in the amount of pigments as chl a, chl c₂ and peridinin (Enrique-Navarro et al., 2022) and dinoflagellate cells have adaptations enable them to survive in variable light conditions and intensity as PCPs are light-collecting complexes made of chl a and peridinin, on the other side host provide stable and more protected environment for symbionts. We collected Cotylorhiza tuberculata specimens in two different environments: Mar Menor, a shallow coastal lagoon (4 m average depth), and Gulf of Trieste, the northernmost part of the Mediterranean Sea (15 m average depth). However, the optimal depth for holobiont Cotylorhiza tuberculata can be affected by light penetration, water turbidity, seasonal light intensity, and cloud cover. Therefore, zooxanthellate jellyfish will respond to changes in the vertical water column by selecting a depth that matches their optimal physiological tolerance and maximises photosynthesis (Enrique-Navarro et al., 2022). We pointed out the higher peridinin content in symbionts from Mar Menor. Symbionts therefore occur in both pigmented and non-pigmented host tissues. As reported by Enrique-Navarro et al. (2022) the dinoflagellate symbionts are heterogeneously distributed within the host body being more concentrated in the medusa oral arms as a consequence of the tissue role, their high metabolic activity and their sinuous folding. This study also reveal that the endosymbionts revealed no significant differences in size neither chlorophyll a concentration within the oral arms and the umbrella.

Symbionts in medusae of Cotylorhiza tuberculata, Phyllorhiza punctata, and Cassiopea xamachana collected in the Mediterranean Sea and from the coast of Cabo Frio (Rio de Janeiro, Brazil) were identified by haplotypes of 28S rDNA and ITS2. We confirmed that the predominant symbionts in the investigated scyphozoan jellyfishes Cotylorhiza tuberculata belong to genera Breviolum and Philozoon, while non-indigenous species Phyllorhiza punctata harbour Symbiodinium. Cassiopea xamachana hosted Breviolum and Cladocopium symbiotic dinoflagellates. The individual medusae studied harbour only one phylotype of symbionts at a time as confirmed with detailed analysis of the ITS2 region within the ribosomal operon of the symbiont, although symbionts in zooxanthellate jellyfish can be different between years as we confirmed in Cotylorhiza tuberculata from the eastern Mediterranean Sea. The population of medusae from Mar Menor hosted only symbionts of Breviolum, while Breviolum and Philozoon were present in medusae from the Adriatic Sea. Furthermore, the possession of zooxanthellae is a unique feature of scyphozoans jellyfish that allows them to occupy several niches compared to non-zooxanthellate species due to mixotrophic way of nutrition.

Haplotypes from this study are deposited in GeneBank under accession numbers listed in Tables S2 , S3 in the Supplementary Material .

Conceptualisation, AR and AM. Data analysis, AR, LD’O, and VF-P. Writing, AR, AB, VF-P, LD’O and AM. All authors contributed to the article and approved the submitted version.

The work of A. Ramšak, A. Malej, and V. Flander-Putrle was funded by the ARRS Research Program P1-0237 (Marine Coastal Research) and by the bilateral cooperation between Slovenia and Brazil (agreements BI-BR /10-12-005) and project RI-SI-2 LifeWatch financed by Ministry of Education, Science and Sport of Slovenia and the European Regional Development Fund. The work of L. Raspor Dall’Olio was funded by the ARRS grant for young researchers (MR -33223).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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