Carotenoids in five aeroterrestrial strains from Vischeria/Eustigmatos group: updating the pigment pattern of Eustigmatophyceae

Abstract Carotenoids have received particular attention both for their importance in algal systematics and hydrobiology and for their health benefits for humans, along with other applications in various industries. Here, we provide new data on the carotenoid content of five Vischeria/Eustigmatos strains isolated from aeroterrestrial habitats in Bulgaria and kept in the Algal Collection of Sofia University (ACUS). The obtained pigment pattern with nine carotenoids and generalization of literature data allowed us to update the knowledge on the chemotaxonomic characteristics of Eustigmatophyceae with a total of 47 pigments reported for the group, out of which 37 are carotenoids. The important photosynthetic pigment lutein, considered also a high-value product with extensive applications in feed, food, nutraceutical and pharmaceutical industries, was proved as a novel carotenoid for the group. The results also confirmed the presence of luteoxanthin, which is rarely reported in algae. All strains had a high content of the commercially valuable health-promoting xanthophyll astaxanthin. There were also different amounts of beta-carotene, violaxanthin, vaucheriaxanthin, canthaxanthin, zeaxanthin and antheraxanthin. Based on the differences in the pigment composition of the members of the ecological groups of aquatic and aeroterrestrial species, we propose the existence of two main pigment types in the class Eustigmatophyceae, with the aquatic type further divided into freshwater and marine pigment subtypes.


Introduction
Carotenoids are natural compounds, commonly known as plant pigments responsible for red, yellow and orange colours of fruits and vegetables. They are isoprenoids and contain a 40-carbon polyene chain backbone with cyclic groups [1,2]. Carotenoids are typically divided into two types: carotenes, or hydrocarbon carotenoids, which only have carbon and hydrogen atoms (e.g. a-carotene, b-carotene), and xanthophylls, which are oxidized carotenoid derivatives. In them, oxygen may be present as OH groups (e.g. lutein), as oxo-groups (e.g. canthaxanthin), or in a combination of both (e.g. astaxanthin) [3].
Besides in plants, carotenoids are widely distributed in photosynthetic bacteria, algae, archaea, fungi and animals [2,4,5]. Due to their photochemical properties [6] carotenoids play an important role in plant and algal photosynthesis and, being specifically distributed in algal classes, have been successfully used in chemosystematics [7][8][9][10]. As a result of these chemotaxonomic implications, at present, an unknown microalga may be assigned to a particular algal class or phylum on the basis of its detailed carotenoid composition. The extensive use of carotenoids in phylogenetic and taxonomic studies of photosynthetic organisms is easily explainable with their wide distribution, structure variability and relatively easy identification [11]. Since the end of the twentieth-century and the beginning of the twenty-first century, carotenoids, together with other photosynthetic pigments, have been widely applied in high-performance liquid chromatography (HPLC)-identification and quantification of aquatic algae [12][13][14][15]. In addition to their importance for algal systematics and hydrobiology, carotenoids are strong antioxidants, scavenging agents and compounds essential to vision, growth and development. Carotenoids have strong health benefits for humans in decreasing the risk of cancer, stimulating the immune system, cardiovascular disease prevention, antidiabetic and antiobesity effects, skin health, neuroprotective effects, positive impact on pregnancy and lactation, etc. [16][17][18][19][20][21][22]. Carotenoids have also received particular attention for their applications in food and drinks, aquaculture, cosmetic and pharmaceutical industries as colourants and feed additives [23].
Owing to the increasing demand, these healthpromoting compounds logically have become the focus of many studies related to human health and biotechnologies. Nowadays more than 750 structurally defined carotenoids are reported from nature [4,5]; out of these, about 200 from algal sources [24]. Nevertheless, in spite of general recognition of main pigment patterns in microalgal phyla (divisions) and classes, and the growing interest in the exploitation of novel sources of commercially valuable algal carotenoids [25], there are virtually no detailed analyses for the vast majority of freshwater and aeroterrestrial species [26]. The small monophyletic class Eustigmatophyceae of peculiar phototrophic eukaryotes which contain only chlorophyll a and carotenoids as photosynthetic pigments, is not an exception although some of its members have been recognized as important biotechnological sources, sparking a renewed interest in this group. For details, see [27]. However, due to lack of a recent monograph, all information is scattered in different sources and is difficult to track down, especially in cases of controversial pigment data.
The aim of the present study was to provide new data on the carotenoid content of five strains of the Vischeria/Eustigmatos group, isolated from aeroterrestrial habitats of Bulgaria and kept in the Algal Collection of Sofia University (ACUS) [28]. The obtained pigment pattern and generalization of literature data allowed us to update the knowledge on the chemotaxonomic characteristics of Eustigmatophyceae (proposing three pigment types as indicative of the group) and their commercially valuable and biotechnologically interesting carotenoid compounds.

Algal strains and culture conditions
The present study was based on five original Bulgarian strains of Vischeria/Eustigmatos group. They were isolated using standard collecting methods from soils in the protected areas of the UNESCO natural heritage Pirin Mt (PM) and in the nearby gorge Kresnensko defile (KD) [29,30], and using the direct collecting method [31] from the soils in the region of the Natural landmark Belogradchishki Skali (BS) [30,32]. They were initially identified by means of light microscopy [29,30,32]. Molecular-genetic methods confirmed their group and genetic affiliation [33] with Vischeria stellata (Chodat ex Poulton) Pascher included as Endangered species in the Red List of threatened microalgae in Bulgaria [34].
The strains were kept on agar plates with standard Bold-Basal Medium (BBM) in the Algal Collection of Sofia University (ACUS) [28]

Identification of carotenoids
Carotenoids were identified by liquid chromatography/ electrospray ionization tandem mass spectroscopy (LC-ESI-MS) analysis [35,36] with high-performance liquid chromatography (HPLC). The extracts were diluted 1:6 in 50% methanol, 0.1% formic acid. Two microliters of sample were introduced into the electrospray ionization (ESI) source of the mass-spectrometer (Thermo Scientific TM Orbitrap Elite TM Hybrid Ion Trap-Orbitrap) via direct infusion at a speed of the mobile phase (50% methanol, 0.1% formic acid) of 5 s min À1 . Full MS spectra were collected in the FT mode in a normal mass range from 500 to 650 m/z (mass-to-charge) range at a resolution of 30,000 and 50 ms ion injection time. The ion source was operated at 2.5 kV ion spray voltage and capillary temperature of 250 C. In the MS/MS analysis, precursor ions were selected with an isolation window of 1 and subjected to CID at either 20 V or 40 V. Fragment spectra (MS/MS) were collected at the same settings as described for MS except that the resolution was increased to 60,000. Data were processed using Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific Inc., Waltham, MA).
Identification and peak assignment of carotenoids were based on the comparison of their retention times, full scan mass spectra, and MS/MS fragmentation patterns with those of authentic standards analyzed under identical conditions [37][38][39][40][41][42][43][44][45][46][47][48]. Transitions to distinguish between compounds were monitored considering the m/z value of protonated molecules as precursor ions, and the m/z value of the product ions, which produce a fingerprint pattern specific to the compound under investigation. Mass spectral data were standardly collected in a positive ionization mode.

Carotenoid composition
The carotenoid composition in the pigment extracts from the algal cells of the five examined aeroterrestrial strains of Vischeria/Eustigmatos group are presented in Table 1. The compounds are listed in the order of the observed sequence of chromatographic peaks, which reflects the decreasing polarity of eluted components. . The ion at m/z 221 corresponds to the oxo-ring fused to the 3hydroxy-b-ring. This fragment is produced by cleavage between carbons 10 and 11. The fragments at m/z 352, 221 and 181 indicated epoxy substituents located in b-rings with hydroxyl groups, with cleavage between C12 and C13, C10 and C11, and C8 and C9, respectively. Compound (3) with a [M þ H] þ at m/z 601 was identified as luteoxanthin. This 5,8-epoxy xanthophyll was characterized by the major product ion at m/z 583 [M þ H-18] þ and at m/z 221, resulting from the cleavage of C10-C11 bond in the polyene chain from the epoxy end group. The diagnostic ion m/z 221 indicated the presence of an epoxy substituent in a b-ring with a hydroxyl group [39].
The mass spectra of 5,6-epoxyxanthophylls were very similar to those of 5,8-epoxyxanthophylls: the molecular ions and protonated molecules, and the fragmentation profile were the same.   þ , corresponding to the loss of one and two hydroxyl groups, respectively [40], and a fragment at m/z 505 [M þ H-92] þ , resulting from the loss of toluene from the polyene chain [41,42]. The mass spectrum of compound 5 had [M þ H] þ at m/z 585.4139. There was a fragment at m/z 567 [M þ H-H 2 O] þ , representing the loss of a water molecule, and also a fragment at m/z 493 [M þ H-92] þ , which corresponded to loss of toluene. This fragmentation indicated the presence of extensive conjugation within the molecule. The ions at m/z 221 and 181 showed that the epoxide group was in a ring with a hydroxyl group and compound 5 was identified as antheraxanthin [39]. The mass spectrum of compound 6, identified as lutein (with one b-ring and one e-ring as end-groups [43]), showed fragments at m/z 551 [M þ H-18] þ , corresponding to the loss of a water molecule. Owing to the presence of a double bond allylic to the hydroxyl group, the fragment at m/z 551 is more stable than the protonated molecule [44].  [38]. The peak at m/z 193 was consistent with a fragment having a hydroxyl group in a b-ring cleaved between C9 and C10 of the polyene chain [40,45]. The mass spectrum of compound 7, identified as zeaxanthin (with two b-ring end-groups [43]), in positive ion mode presented higher intensity of the protonated molecule peak at m/z 569 [M þ H] þ in comparison to the fragment at m/z 551 [M þ H-18] þ . This indicated that the hydroxyl group was not allylic to the double bond, whilst in the lutein spectrum the fragment at m/z 551 [M þ H-18] þ was more intense than the protonated molecule at m/z 569 [M þ H] þ [39]. In the case of compound 8, identified as canthaxanthin (a xanthophyll with one keto group in each ionone ring), the [M þ H] þ produced the neutral elimination of toluene and xylene at low intensities (m/z 473 and 459, respectively) [46]. The m/z 203 product ion is known as a characteristic signal of carotenoids containing a keto group (as the only substituent on the b-ring) conjugated to the polyene chain [47]. MS analysis of compound 9 showed molecular ion [M þ H] þ at m/z 537 and fragment ions MS2 (m/z 444, 333, 177), and was referred as b-carotene [48].

Chlorophylls in Eustigmatophyceae
Class Eustigmatophyceae has a unique combination of photosynthetic pigments, notable primarily for the absence of secondary chlorophylls b and c, and lack of the xanthophyll fucoxanthin, typical of most classes in the ochrophyte line of algal evolution [19]. The lack of c-type chlorophylls (or their precursors chlorophyllides c) was amply underlined as one of the main peculiarities of the class [72], by which it is similar to the relatively recently discovered heterokont classis Aurearenophyceae [73]. Recently, small amounts of chlorophyll c (ca. 0.18% of the dry weight) were found spectrophotometrically in a strain of the freshwater eustigmatophycean species Trachydiscus minutus (Bourrelly) Ettl. [74], which in the authors' opinion, needs to be confirmed chromatographycally. Detailed discussion on chlorophyll types and their content in Eustigmatophyceae is beyond the scope of the present study, although we would like to remind about the opinion that it is possible to find small amounts of chlorophyll c in the group [54]. This is noteworthy, due to the immense role of David Hibberd as one of the two outstanding experts who discovered the peculiarities of the group of eustigmatophycean algae and advocated their separation from other algal classes and phyla, and from yellowgreen algae (class Xanthophyceae ¼ Tribophyceae) in particular [75][76][77][78][79][80]. Similarly, possible presence of c-type chlorophyll was indicated for all eustigmatophycean algae, which were fondly called eustigs [51]. A summary of chlorophylls and their derivatives identified in research papers is provided in Table 2.

Carotenoids in Eustigmatophyceae
Following [81], the presence of chlorophyll a in combination with violaxanthin as major xanthophyll was highlighted as 'an important diagnostic character which is significant enough to be detected even in the total spectrum of extracted pigments' [54, p. 327]. The same two pigments were also outlined as indicative of the group also in [52,53]. Violaxanthin accounted for up to 60% of the carotenoid composition of eustigs in a study which discovered its light-harvesting (LH) function [82]. A subsequent study reported that violaxanthin apparently did not play the LH role in other phosynthethic organisms [55]. However, another report indicated that violaxanthin-Chl a/c 1 /c 2 -protein complex could act as a second LH complex which occurred in lesser amounts to the major fucoxanthin-Chl a/c 2 -protein LH in some brown algae [83]. Thus, violaxanthin, which was already known from higher plants and some other algal classes [84], became broadly accepted as the main accessory pigment of Eustigmatophyceae (which absorbs light to drive photosynthesis in addition to chlorophyll [85]). The predominance of violaxanthin in the members of the class was underlined also in [86,87]. Later, the eustigmatophycean violaxanthinchlorophyll a-protein complex was proved as the first example of an LH complex binding only non-carbonyl carotenoids with carotenoid-to-chlorophyll energy transfer efficiency of over 90% [88]. The earlier evidence accumulated in [89,90] indicated also the unique primary structure of the eustigmatophycean LH apoprotein, which differred from that of analogous proteins from non-green algae, green algae or higher plants. More recently, the high violaxanthin content and very low chl a-to-violaxanthin ratio were used as markers of Eustigmatophyceae in HPLC studies [91]. In addition, violaxanthin as a dominant xanthophyll could play an important role in the photoprotective xanthophyll cycle of eustigmatophyceans [27,[92][93][94]. This cycle appears to be an important protection mechanism of plant cells from photooxidative cell damage [95,96]. Only a few studies do not report violaxanthin in the unsaponified pigment extract of Vischeria sp. [97,98] and there is one report of small amounts of violaxanthin in the bizarre eustigmatophycean alga Pseudostaurastrum limneticum, which has no stigma in the zoospores in contrast to other members of the group [99].
Lubi an and Montero [92] highlighted the high content of beta-carotene, accounting for over 60% of total carotenoids, in Vischeria helvetica but did not even mention vaucheriaxanthin for the studied strain ( Table 2). It was supposed that similar to the green algae of genus Dunaliella, rich in beta-carotene, eustigs synthesized more beta-carotene to protect themselves from the damage caused by ageing or environmental stresses, such as nutrient depletion and high light irradiance [25]. The amount of betacarotene significantly varies in different studies, but its high relative concentration is commonly emphasized [97,124]. The predominance of beta-carotene over the xanthophylls firstly was outlined in [97,124] as a unique feature of the studied algae but then was confirmed for most eustigmatophycean species analyzed afterward. The highest published value showed that beta-carotene could account for more than 85% of the total carotenoid content and over 5% of the dry weight of Eustigmatos polyphem [25,122].
The main pigment composition (chlorophyll a, beta-carotene, violaxanthin and vaucheriaxanthin, free or in ester form) or at least the main accessory role of violaxanthin is repeatedly indicated for the whole class in phycological and protistan handbooks, as well as in different review papers [5,27,[57][58][59][60][61]87,125,126]. Some references [77,126] emphasize the replacement of anteraxanthin by violaxanthin as a significant difference with yellow-green algae (Xanthophyceae) from which Eustigmatophyceae were taxonomically derived [75]. However, further studies proved the presence of both pigments (Table 2) and this is a possible explanation for the opinion that 'minor details in pigmentation' distinguish eustigs from yellow-green algae [127]. This was supported by statements that 'in the pigmentation of their plastids, eustigs are indeed very much like the true yellow-green xanthophytes … Violaxanthin is commonly present; epoxanthin, diadinoxanthin, and diatoxanthins may also be present' [41, p. 198] and that in Eustigmatophyceae the 'accessory pigment (carotenoid) composition is similar to Xanthophyceae except for the absence of heteroxanthin' [128, p. 492]. For Xanthophyceae, the same authors mentioned that 'the principal accessory pigments are beta-carotene, vaucheriaxanthin, diatoxanthin, diadinoxanthin, and heteroxanthin' [128, p. 486]. Similarly, the absence of heteroxanthin (and of diadinoxanthin) in Pleurochloris commutata was underlined as a difference with Xanthophyceae, which supported the need for its transfer to the eustigmatophycean group [102]. Earlier, Thomas and Goodwin [98] pointed out the minor, only quantitative differences between the eustigmatophycean Vischeria sp. and five other species of Xanthophyceae in the main pigment pattern (namely beta-carotene, antheraxanthin, lutein 5,6-epoxide and an unidentified compound, which was possibly trollixanthin).
According to [81,101,120], the vaucheriaxanthin ester appeared to be the characteristic xanthophyll of all yellow-green coloured algae, both Xanthophyceae and Eustigmatophyceae. In these three, as well as in all other specified pigment studies cited above, vaucheriaxanthin in all his forms was quantitatively less important than violaxanthin. There are reports of exceptions with higher amounts of vaucheriaxanthin only in Chlorobotrys regularis [106], two peculiar eustigs strains (Tunis and GSB Sticho) [103] and Eustigmatos polyphem, E. vischeri and Vischeria punctata [25]. Later vaucheriaxanthin (commonly in the diesterified form) was underlined as the main characteristic carotenoid of Eustigmatophyceae [5,129]. It has a light-harvesting role, similar to that of violaxanthin, as supposed earlier [82] and confirmed in [116].
Isomers of neo-violaxanthins U and V, and neovaucheriaxanthin U and V esters are part of the pigment pattern of Pleurochloris [102] in addition to the major pigments listed above ( Table 2).
It was long believed that Eustigmatophyceae had no ketocarotenoids (carotenoids with ketonic groups [81,131]) or had them in minute concentrations at the biosynthetic oxidation level of canthaxanthin [103,132]. After the first report of 'ketocarotenoids' accounting for 5% and 4% of the total carotenoid content [104], a number of studies showed the presence of canthaxanthin [82,93,105,107,[109][110][111][112]118,119,121]. The content of canthaxanthin tends to increase during growth of the cultures even when violaxanthin and vaucheriaxanthin remain the major xanthophylls [107,110,111,125]. However, canthaxanthin and other ketocarotenoids never achieved ageing associated predominance in the total carotenoid mixture [107]. Parallel to the increase in ketocarotenoid amounts, the dynamics of relative pigment content in Eustigmatophyceae during growth show an increase in the total chlorophyll a/carotenoid ratio. This is visible as a change from the initially green colour of cultures to brown and finally bright orange, when cultures become older and nutrients are depleted [110,112,125]. The change in the amount of pigments reflected in culture colour is considered a good indicator of physiological stage and response of pigment concentrations (and thus of the photosynthetic efficiency) to factors like light intensity and nutrient concentrations [110]. Variations in cellular pigmentation in response to growth conditions were also shown in [93,133]. Orange colouration of cultures in the process of ageing was noted in [25,122] but was explained with the increase in intracellular beta-carotene content together with the decrease in the concentrations of other carotenoids and chlorophylls. Li et al. [25] suggested that further improving of culture conditions could raise the amount of this valuable pigment. Similar gradual increase in the b-carotene content by prolongation of the culture time resulting in its exclusive production at the end of cultivation was reported in [123].
In Eustigmatophyceae, the first documentation of in vivo 'production of astaxanthin (free plus monoesterified) and astacene in significant amounts, indicating the capacity of this algal type to synthesize the highest oxidation level of 4-keto carotenoids known from the algal kingdom and hitherto found only in the Chlorophyceae, Euglenophyceae and Dinophyceae … ' was in [107, p. 47]. Later other studies showed the presence of the ketocarotenoid astaxanthin in a few eustigmatophycean species [93,109,112]. Strain KGU-Y001 of Vischeria helvetica could utilize both the photoprotection activity of the violaxanthin cycle and the antioxidant activity of astaxanthin to resist highlight stress [93]. Aburai et al. [93] believed that its pathway for astaxanthin biosynthesis was different from those known for green algae and from the six eustigs (incl. V. helvetica) investigated in [25]. The 'unknown keto-carotenoids A, B, C and D' were detected and were suggested to be intermediates in the biosynthesis of astaxanthin and its ester [107]. In addition, Ishida and Hara [143] reported the 'unknown ketocarotenoid E' with a possible relationship with astacene ( Table 2).
Echinenone is an intermediate in the biosynthesis of canthaxanthin. It was found in Nannochloropsis oculata and Microchloropsis salina but was obtained in too small a quantity to be identified with certainty, and its spectrum suggested that it could be a cisisomer [107]. Despite missing from previous lists, the pigment was expected by the same authors to occur in the strains investigated earlier [103,126], which contained low amounts of canthaxanthin. Echinenone was reported much later in Nannochloropsis limnetica and in its three Baltic strains [119,121] - Table 2.
Another study [134] found a monoepoxide and a diepoxide, but the used chromatographic technique could not fully resolve the xanthophylls (Table 2). Afterward, the reporting of zeaxanthin and antheraxanthin could be traced back to [25,92,107,112,122] - Table 2. Some records indicate only zeaxanthin [67,81,82,99,[117][118][119][120][121], and in addition, a zeaxanthin-like pigment [119]. Zeaxanthin, inseparable from lutein, was listed in [93]. Antheraxanthin was reported in [98,102] and in [107], where its furanoid form was also found, and in [114, p. 437] with a note that the 'experimental procedure did not allow a distinction between vaucheriaxanthin and antheraxanthin'. Later, antheraxanthin was found in [25,92,112,116] - Table 2. The presence of zeaxanthin in algae which contain violaxanthin was underlined in [102]. Considering the LH role of violaxanthin [82], Stransky and Hager [102,135] suggested that there is a light-induced de-epoxidation cycle between violaxanthin, antheraxanthin and zeaxanthin in eustigmatophyceans. This cycle, named Viola cycle, is similar to that in the majority of land plants, brown and green algae and some species of red algae [14,[136][137][138]. The same interconversion between violaxanthin, antheraxanthin and zeaxanthin in the xanthophyll cycle was later confirmed for eustigs [101]. Experimental evidence demonstrated that it operates under high-irradiance-induced stress conditions, showing that under proper conditions of high irradiance (2000 mmol m À2 s À1 ) and temperature (40 C), up to 78% of violaxanthin was de-epoxidated in a short time-interval to render zeaxanthin [92]. In subsequent experiments [112], the violaxanthin content was restored (without any cell damage) after 24 hours exposure to dim light and lower temperatures (25 C). The same study also proved the major role of violaxanthin and vaucheriaxanthin with dynamics in their content in culture conditions: the decrease in violaxanthin correlated with the increase in zeaxanthin and antheraxanthin, while the decrease in vaucheriaxanthin correlated with the increase in the ketocarotenoids canthaxanthin and astaxanthin. An earlier report demonstrated the combined effect of light intensity and nitrate concentration on the cellular content of canthaxanthin in eustigs [111].
Whittle [106] reported that almost all eustigmatophycean species contained beta-carotene, whereas alpha-carotene was detected only in the authentic strain of Microchloropsis gaditana and served as a biochemical ground for distinguishing from Nannochloropsis species (Table 2). Afterward however, alpha-carotene was not discovered in other strains of the same species [120].
In addition to the pigments certainly identified in Eustigmatophyceae, different studies have reported some other minor carotenoids (Table 2). Such were mentioned as 'other carotenoids' (3-7% from the total content) in [82,104]. Five unknown carotenoids were shown with their absorption maxima and minima in [119]. They were in relatively high amounts when estimated as chl a/pigment ratio (up to 77) [119]. Some other unknown carotenoids were reported but were generally considered to be of minor importance [25,117,120].

General pigment pattern of Eustigmatophyceae
The qualitative pigment pattern of Eustigmatophyceae generalized on the basis of reviewed literature data and our results, is presented in Table 3.
The accumulated data on the pigment composition in Eustigmatophyceae, as summarized in Tables 1-3, show that out of 47 pigments reported in total (incl. the group of 'other/minor'), 37 are carotenoids. The highest number (34) comes from studies on aquatic (mostly marine and rarely freshwater) species. Fewer pigments (25) are known from the studies on the aeroterrestrial representatives of the group. The pigments common to both ecological groups, in alphabetical order, are antheraxanthin, astaxanthin, beta-carotene, canthaxanthin, vaucheriaxanthin, vaucheriaxanthinester, violaxanthin and zeaxanthin, in addition to the main chlorophyll a, as well as some unidentified minor pigments and possibly neoxanthin (which was proved in aeroterrestrial algae, but was only speculated to occur in the aquatic forms) - Table 3. However, even these common pigments, were not found in all studied species (Table 2) and have been recorded in different amounts. So far, auroxanthin, lutein, luteoxanthin, cryptoxanthin, cryptoxanthin monoepoxide, neo-violaxanthin isomers U and V, neoxanthin-like xanthophyll as well as three isomers of neo-vaucheriaxanthin esters and two unknown epoxides together with phaeophytin a and unidentified chlorophyll have been found only in aeroterrestrial forms (Table 3). Only aquatic species reportedly contain alpha-carotene, furanoid form of antheraxanthin, astacene, astaxanthinester, canthaxanthin-like xanthophyll, cryptoxanthin furanoepoxide and cryptoxanthin difuranoepoxide, echinenone, violaxanthin-like pigments 1 and 2, neochrome, isomer of vaucheriaxanthin ester, vaucheriaxanthin-like and zeaxanthin-like xanthophylls, as well as some other minor carotenoids together with various forms of chlorophyll a (derivatives, epimers, etc.) - Table 3. Similar to the pigments in aeroterrestrial species, those in different aquatic algae have various distribution (Table 2) in different amounts. Moreover, in aquatic algae, the number of pigments found in the marine species (30) is higher, in contrast to fewer pigments (10) reported in the freshwater eustigs ( Table 3). The only pigments common for these two ecological subgroups are betacarotene, canthaxanthin, zeaxanthin, vaucheriaxanthin, vaucherixanthin ester and violaxanthin ( Table 3).
The results obtained in this study generally revealed that: (1) vaucheriaxanthin, although present in all studied strains, was not their major xanthophyll; (2) beta-carotene and violaxanthin, which were commonly accepted as the major photosynthetic pigments in combination with chlorophyll a, were with different distribution in the members of Eustigmatophyceae ( Table 1). The minor role of beta-carotene was briefly noted also in [120] and the decrease in apha þ betacarotene content during the ageing of cultures was shown in [110].
By contrast, the ketocarotenoid astaxanthin, which has been quite rarely reported before for marine Eustigmatophyceae (Table 2) and was assigned mainly to green algae, as well as to a few dinoflagellate species and to some cyanoprokaryotes/cyanobacteria [5,14,130,140], was found in all five strains included in the present study. Astaxanthin accounted for 5-6% of their total carotenoid content, but in two of the strains it reached 9% and 13% (Table 1). Considering the broad usage of this lipid-soluble pigment as a safe dietary supplement [141], its detection in our aeroterrestrial strains could be of commercial interest. Our finding is in conformity with previous reports of its presence in a strain of the aerophytic Vischeria helvetica [93].
Another ketocarotenoid, canthaxanthin, was detected in four of our strains but always in lower amounts than astaxanthin (Table 1). Canthaxanthin is also recorded as a minor or trace pigment in some Pigments mentioned in generalized books and reviews, but not found by us in specialized papers are excluded from Table 3. A pigment was indicated as present (x) when it was found at least once in a species or strain from the relevant ecological group.
species of cyanoprokaryotes, dinoflagellates, diatoms, chlorophytes and prymnesiophytes [24]. Violaxanthin shows oscillating dynamics in its content and can convert reversibly into astaxanthin and canthaxanthin during the algal growth [92,102,111,112]. Hence, we believe that our results are in agreement with these findings, not necessarily indicating a minor role of violaxanthin. There is need for further more detailed studies to clarify the reason for these observations. In our study, we were not able to identify echinenone, the carotenoid intermediate of the biosynthesis of canthaxanthin. This pigment, discovered so far only in three aquatic eustigmatophycean species (Table 2), had been considered more typical of prokaryotic algae (phylum Cyanoprokaryota) and quite rare in eukaryotes, where, besides in eustigs, it was found in a few species of the phyla Euglenophyta, Haptophyta and Pyrrhophyta [14,24]. The results from our analysis did not show the presence of some other carotenoids reported rarely by earlier authors, like alpha-carotene, neoxanthin, cryptoxanthin and its esters, auroxanthin, neochrome, astacene or intermediates of astaxanthin and its ester (Tables 1 and 2).
Another rarely reported xanthophyll, the epoxycarotenoid luteoxanthin, was found in a relatively high content (13-15%) in three of the five studied strains (Tables 1 and 2). These findings confirm its previous records in marine and aeroterrestrial eustigmatophycean species [25,81,82,107,122]. This is noteworthy, since luteoxanthin has been extremely rarely reported from algae at all (e.g. chlorophyte Dunaliella tertiolecta) with a note in [24] that it could undergo rearrangement with violaxanthin in weak acid solutions.
Two other epoxycarotenoids, antheraxanthin and zeaxanthin, known as minor xanthophylls in some eustigmatophyceans (Table 2), were found in all the strains studied by us, with constant higher amounts of antheraxanthin (Table 1). The relative amount of antheraxanthin in the total carotenoid content in our strains was considerably higher in comparison with its content in other strains of the same genera obtained in [25]. Antheraxanthin is involved in the Viola xanthophyll cycle typical of land plants, brown and green algae [137]. It is known as one of the minor pigments in the small peculiar phylum Chlorarachniophyta with plastids derived from green algae [e.g. 61] and in some red and golden algae as well [14,24]. Zeaxanthin is commonly synthesized in higher plants. It was long believed to be a relatively rare xanthophyll in algae, generally referred to their prokaryotic counterparts (Cyanoprokaryota) as one of the dominant pigments, even among the prokaryotes containing chlorophyll b (prochlorophytes) [14,24,58] and in the few cyanoprokaryotes with chlorophyll f [130]. However, zeaxanthin is also found as important pigment in the eukaryotic phyla Glaucocystophyta, Rhodophyta, Pyrrhophyta (=Dinophyta), Chlorarachniophyta, in the green algal classes Chlorophyceae and Prasinophycea, and as being ubiquitously spread in the ochrophyte classes Dictyochophyceae, Pinguiophyceae, Pelagophyceae, Phaeophyceae and Raphidophyceae but disjunctively in some other ochrophytes from the classes Bacillariophyceae and Chrysophyceae [14,130]. It occurs in the novel ochrophyte class Aurearenophyceae [70,130]. Once zeaxanthin was indicated even as a dominant pigment in Eustigmatophyceae, as well as in 'cyanobacteria, chrysophytes, pelagophytes, rhodophytes and dinoflagellates Pigment Type 5; minor in pinguiophytes, raphidophytes, chlorarachniophytes, chlorophytes, prasinophytes and trebouxiophytes; and occasional in dictyochophytes and dinoflagellates Pigment Type 3' [24, p. 810]. Later however, the same author placed zeaxanthin among the accompanying pigments of eustigs [130].
Zeaxanthin has an isomer lutein, which differs just by the shift of a single double bond [2]. Similarly to zeaxanthin, lutein was normally assigned to higher plants and was thought to be less commonly distributed among algae [4,84,114], where it was usually considered the most important carotenoid of the eukaryotic green algae [14,21,51,55,61,83,87,102,103,130,142]. Lutein has been found also in the phyla Chlorarachniophyta [14,55,56,143,144] and Rhodophyta [14,21,51,55,61,130,145,146]. In Rhodophyta, most algae contain either zeaxanthin or lutein as the sole dihydroxy-xanthophyll [14]. Egeland [130] summarized the occurrence of lutein in dinoflagellates with endosymbiotic origin from green algae. The presence of lutein in Xanthophyceae (in 'Tribonema aequale, Heterococcus fuoreresis and Ophiocytium majus') was underlined in [97] as discovered by Jamikorn [147] but, to our knowledge, was not even discussed in the later works. Besides this finding, in the large ochrophyte/heterokont algal evolutionary line, lutein was detected in the marine silicoflagellates, or Dictyochophyceae s.str. [55] and was assigned to the class Raphidophyceae as lutein or lutein monoepoxide [148,149]. These two groups, despite possessing chlorophyll c, were thought to be relatively close phylogenetically to the Eustigmatophyceae [57,58,61,87,126,134,[150][151][152][153][154]. According to [98], yellow-green algae (to which eustigs belonged at that time before being assigned as a novel class) could synthesize lutein epoxide. This ability however, was mostly ignored in later eustigs studies. To date, lutein is not indicated in the pigment studies of Eustigmatophyceae except its first mentioning in [25], where it was included as a table footnote with four more pigments among the 'other carotenoids' without any explanation, and its later inseparable inclusion together with zeaxanthin in [93], also without discussion. The lack of lutein in the studied eustigs was underlined in [97,102,113,114] and was proposed as an explanation for the major role of violaxanthin [114]. In our study, lutein was detected in all investigated strains of the Vischeria/Eustigmatos group (Table 1). Moreover, in all of them it accounted for a considerable part (13-25%) to the total carotenoid content. This shows that it is necessary to update Eustigmatophyceae in a chemotaxonomical aspect. It also shows that the isolated strains could be further explored as potential commercial sources of lutein, which is a high-value product with extensive applications in feed, food, nutraceutical and pharmaceutical industries [21].
The updated general pigment pattern of Eustigmatophyceae (Table 3) shows the chemotaxonomic diversity of carotenoids found in the class. However, no pigment was commonly found in all studied strains (Table 2). Naturally, the knowledge on the photosynthetic pigments and their diversity have generally increased with the advancement of analytical techniques and the accumulation of more intensive studies on a given species (Tables 2 and 3). The results obtained by us allow to retool the general eustigmatophycean pigment pattern by proving the presence of the important pigment lutein and of luteoxanthin, which is rarely reported from algae. The data generalized in Tables 2 and 3 open a discussion of the differences in the carotenoid composition among the ecological groups of aeroterrestrial and aquatic algae, and even of the difference between freshwater and marine species. The pigment distribution strongly suggests the possibility to propose at least two main pigment types in Eustigmatophyceae (with further division of aquatic type into two subtypes) following the models accepted for other algal groups (e.g. cyanoprokaryotes with five pigment types (PT), haptophytes with eight PT, pyrrhophytes with five PT, chlorophytes with two PT and prasinophytes with five PT) [14,130,155,156]. The pigment pattern of each type/ subtype provided in Table 3 is logically explainable with differences in the light composition and intensity in the habitats. However, since the analytical methods applied up to now have different resolution and the cellular pigmentation varies in response to growth conditions [107,110,111,132], there is need of further more unified quantitative studies with more taxa investigated from all ecological groups to prove the two proposed PT in Eustigmatophyceae. At present, besides chlorophyll a, only beta-carotene, canthaxanthin, zeaxanthin, vaucheriaxanthin, vaucherixanthin ester and violaxanthin could be tentatively outlined as common for eustigs from all ecological groups, despite the strong variability in their quantities.
At the same time, all accumulated data in our study and previous ones point to the need of further analyses of these pigments and their eustigmatophycean bearers as potential valuable natural sources due to their high antioxidant capacity and high scavenging activity [157]. Our results also support the opinion of Lubi an et al. [112], who described Eustigmatophyceae as novel sources of valuable pigments related both to capacity for single pigment accumulation and to the availability of a broad range of carotenoids. Last but not least, we would like to stress the importance of further studies on eustigs regarding the trend towards increasing their number of members [27,59]. Another direction of future research is based on the fundamental particular roles that algal pigments play in the process of photosynthesis and photoprotection thereby determining algal biodiversity under a changing environmental and climatic scenario, and in the associated biogeochemical cycles [10,158].

Conclusions
The results from our study, with nine identified carotenoids, together with the generalization of literature data, allowed us to update the knowledge on the pigment pattern of Eustigmatophyceae with a total of 47 pigments, of which 37 are carotenoids. Lutein was proved as a novel pigment for this class. Based on the differences in the pigment composition of the members of the ecological groups of aquatic and aeroterrestrial species, we propose the existence of two main pigment types in the class Eustigmatophyceae, with the aquatic type further divided into freshwater and marine pigment subtypes. Besides this chemotaxonomic result, our data confirm the high relative amount and diverse combinations of commercially important pigments in the studied aeroterrestrial strains of the Vischeria/Eustigmatos group. Apart of the well-known and largely explored beta-carotene, they contain carotenoids (e.g. lutein, luteoxanthin, astaxanthin, canthaxanthin) considered as high-value functional products with extensive applications in human affairs. Their finding in the originally isolated strains from protected areas in Bulgaria proves the need of further phycoprospecting in the country and future studies on the eustigmatophycean microalgae as important commercial sources.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the America for Bulgaria Foundation under Grant number AGR.0050.20160121.