Gertia stigmatica is a recently described member of the Kareniaceae with a peridinin-containing plastid rather than the aberrant, haptophyte-derived, tertiary plastid found in canonical Kareniaceae ...genera such as Karenia, Karlodinium, and Takayama. G. stigmatica provides a unique opportunity to compare biochemical traits, such as sterol composition, between these two fundamentally different types of Kareniaceae. To this point, canonical members of the Kareniaceae have been observed to typically produce a set of 4α-methyl-substituted, Δ8(14)-nuclear-unsaturated major sterols, such as (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol) and 27-nor-(24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (brevesterol), which are very uncommon throughout other members of the class Dinophyceae. Our objective was to compare the sterols of G. stigmatica to canonical Kareniaceae to elucidate whether these same distinctive sterols are found, with our hypothesis being that they would because G. stigmatica is indeed a member of the Kareniaceae. Contrary to our hypothesis, G. stigmatica lacks gymnodinosterol and brevesterol, with its sterols instead dominated by 4-desmethyl sterols, such as cholesterol, 24-methylcholesta-5,22E-dien-3β-ol, and the unusual tri-unsaturated sterols ergosta-5,8(14),22E-trien-3β-ol and cholesta-5,8(14),22E-trien-3β-ol. No sterols were found to possess a 4α-methyl substituent or a single Δ8(14) nuclear unsaturation. Thus, G. stigmatica's sterol composition as a member of the Kareniaceae is atypical.
The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ8(14)‐nuclear unsaturated 4α‐methyl‐5α‐cholest‐8(14)‐en‐3β‐ol (C28:1) and ...4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol; C29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest‐5‐en‐3β‐ol; C27:1), which is found in many other dinoflagellate genera, rather than Δ8(14) sterols. While the Δ8(14) sterols 4α‐methyl‐5α‐cholest‐8(14)‐en‐3β‐ol and 4α,23,24‐trimethyl‐5α‐cholest‐8(14),22E‐dien‐3β‐ol (C30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24‐trimethyl‐5α‐cholest‐22E‐en‐3β‐ol (dinosterol; C30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ8(14) sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ8(14) sterols, especially amphisterol, as Amphidinium‐specific biomarkers within these species where cholesterol is the predominant sterol.
The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ
-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C
) and ...4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C
) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C
), which is found in many other dinoflagellate genera, rather than Δ
sterols. While the Δ
sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C
) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C
), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ
sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ
sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.
Prorocentrum texanum var. texanum and its morphologically distinct yet genetically identical (as based on the sequences of five genes) variety P. texanum var. cuspidatum represent a species of ...Prorocentrum recently isolated from the Gulf of Mexico. Together, these two varieties represent a sister species to Prorocentrum micans. P. micans has had its sterols, which are ringed lipids common to eukaryotic cell membranes, shown in some studies to be comprised of cholesterol (cholest‐5‐en‐3β‐ol), 23,24‐dimethyl‐cholesta‐5,22‐dien‐3β‐ol, 23,24‐dimethyl‐5α‐cholest‐22E‐en‐3β‐ol, dinosterol, and 4α,23,24‐trimethyl‐5α‐cholestan‐3β‐ol (dinostanol) as major sterols, thus placing it within a previously identified cluster of dinoflagellates characterized by the predominance of cholesterol and dinosterol. In this study we have determined the sterol compositions of these two varieties of P. texanum to be abundant in cholesterol, 23,24‐dimethyl‐cholesta‐5,22‐dien‐3β‐ol, 23,24‐dimethyl‐5α‐cholest‐22E‐en‐3β‐ol, dinosterol, and dinostanol such that the varieties are virtually indistinguishable from each other, making them both in general agreement with the sterols of P. micans, its closest species relative. This expands our knowledge of the sterols of this environmentally important dinoflagellate genus.
SUMMARY
Zooxanthella nutricula
is a photosynthetic dinoflagellate symbiont of polycystine radiolarians. As such, it is hypothesized to provide fixed organic carbon, including in the form of ...acylglycerolipids and sterols, to its non‐photosynthetic host. We have previously characterized the sterols of
Z. nutricula
that may be transferred to its host and, in the present study, have turned our attention to three classes of fatty acid‐containing lipids, chloroplast‐associated galactolipids, betaine lipids, which are non‐phosphorylated phospholipid analogs present in many eukaryotes, and triglycerides.
Zooxanthella nutricula
was observed using positive‐ion electrospray/mass spectrometry (ESI/MS) and ESI/MS/MS to produce the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched in octadecapentaenoic (18:5(n‐3)) and octadecatetraenoic (18:4(n‐3)) acid to place it within a group of peridinin‐containing dinoflagellates in a C
18
/C
18
(
sn
‐1/
sn
‐2 fatty acid regiochemistry) cluster, as opposed to another cluster with C
20
/C
18
MGDG and DGDG, where the C
20
fatty acid is eicosapentaenoic acid (20:5(n‐3)) and the C
18
fatty acid is either 18:5(n‐3) or 18:4(n‐3).
Zooxanthella nutricula
was also observed to produce 38:10 (total number of fatty acid carbons:total number of double bonds), 38:6, and 44:7 diacylglycerylcarboxyhydroxymethylcholine (DGCC) as the sole type of betaine lipid. Although it is more difficult to determine which fatty acids are present in the
sn
‐1 and
sn
‐2 positions on the glycerol backbone of DGCC using ESI/MS/MS, gas chromatography/mass spectrometry (GC/MS)‐based examination indicated the putatively DGCC‐associated polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:6(n‐3)). Coupled with the C
18
PUFAs of MGDG and DGDG, and fatty acids associated with triglycerides (also examined via GC/MS),
Z. nutricula
could serve as a rich source of PUFAs for its radiolarian host. These data demonstrate that
Z. nutricula
produces a similar set of PUFA‐containing lipids as
Symbiodinium microadriaticum
, a photosynthetic dinoflagellate symbiont of cnidarians, indicating a metabolic commonality in these phylogenetically discrete dinoflagellate symbionts with unrelated host organisms.
Testudodinium testudo is a peridinin‐containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA ...genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ8(14) sterols, such as 4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ8(14) sterol, (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α‐methyl‐5α‐ergosta‐8,14,22‐trien‐3β‐ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.
Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA ...genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ
sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ
sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.
Some dinoflagellates, such as Symbiodinium, are able to form symbiotic relationships with larger marine organisms. An important aspect of dinoflagellate symbiosis involves the exchange of lipids, ...namely sterols, from the symbiont to the host. Much research has explored the lipid biochemistry of the symbiotic relationship between cnidarians and Symbiodinium dinoflagellates. However, no research has addressed the sterol biochemistry of the symbiosis between radiolarians and dinoflagellates such as Zooxanthella nutricula. To this end, we have provided the first sterol characterization of Z. nutricula isolated from a spumellarian polycystine radiolarian. Fifteen sterols and one steroidal ketone were observed where the major sterol identified was C27 22-dehydrocholesterol, which does not tend to be a dominant sterol among dinoflagellates, including closely related peridinioid species in the genus Heterocapsa. However, C30 dinosterol and dinostanol were major sterols in both Z. nutricula and Heterocapsa spp., thus indicating common sterols between closely related taxa. Major sterols of the distantly related genus Symbiodinium, a symbiont of foraminifera and cnidarians, have included C27 cholesterol and C30 gorgosterol, whereas in Z. nutricula these sterols were minor and absent, respectively. Our results indicate potentially different sterol pools available to cnidarian and radiolarian symbiont hosts during their respective relationships with symbiotic dinoflagellates.
SUMMARY
Gertia stigmatica is an atypical member of the Kareniaceae that has a peridinin‐containing plastid rather than a tertiary, (acyloxy)fucoxanthin‐containing, haptophyte‐derived plastid, as is ...found in the canonical genera Karenia, Karlodinium, and Takayama. While the origin of G. stigmatica's plastid is uncertain per its original published description, with alternative hypotheses stating that it could have originated before or after the acquisition of a haptophyte‐derived plastid, the description of the species indicates that it does possess ultrastructural and genetic features that firmly place it within the Kareniaceae. As a photosynthetic, plastid‐containing organism, G. stigmatica, like other algae, possesses two galactolipids, mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively), that form the basis of its plastid‐associated photosynthetic membranes. MGDG and DGDG have been extensively characterized in peridinin‐containing dinoflagellates, where it has been observed that these dinoflagellates can be segregated into two clusters. One cluster is characterized by MGDG and DGDG possessing the polyunsaturated C18/C18 fatty acids (sn‐1/sn‐2 regiochemistry) octadecapentaenoic acid (18:5(n‐3)) and octadecatetraenoic acid (18:4(n‐3)), while the second cluster possesses eicosapentaenoic acid (20:5(n‐3)) in the sn‐1 position while retaining a polyunsaturated C18 fatty acid in the sn‐2 position. By contrast, Karenia brevis and Karenia mikimotoi have been observed to be enriched in species of MGDG and DGDG, such as 18:5(n‐3)/14:0 MGDG and DGDG, uncommon to peridinin‐containing dinoflagellates. Our objective was to characterize the galactolipids of G. stigmatica to compare it to both peridinin‐containing dinoflagellates and other members of the Kareniaceae to search for insight, such as (a) remnant 14:0 fatty acid‐containing galactolipid(s), into the evolution of its plastid. Our results show that G. stigmatica possesses 20:5(n‐3)/18:5(n‐3) MGDG and DGDG as the primary galactolipids, with little evidence of those galactolipid species enriched in K. brevis and K. mikimotoi. The implications of this for the evolution of the G. stigmatica plastid are discussed.
The genus Amphidinium is shown in many phylogenies to be basal to other peridinin-containing, photosynthetic dinoflagellates as one of the first photosynthetic genera to arise after the evolution of ...heterotrophic genera. As part of our continuing examination of the plastid-associated galactolipids, namely mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), in dinoflagellates, we here examine the galactolipid composition of members of the genus Amphidinium. We show that this genus is characterized by an abundance of 20:5(n-3)/18:5(n-3) and 20:5(n-3)/18:4(n-3) forms of MGDG and DGDG (with sn-1/sn-2 regiochemical specificity of fatty acids), but also sometimes with generally lesser amounts of some polyunsaturated C
18
/C
18
forms, thus placing the examined species within a previously identified cluster of C
20
/C
18
MGDG- and DGDG-containing, peridinin-containing dinoflagellates. We also show that Testudodinium testudo, previously known as Amphidinium testudo, conversely falls within a previously identified C
18
/C
18
cluster, indicating a distinct difference in galactolipid biosynthesis capability. While it is likely that further revision of the genus may occur in the future and/or more basal peridinin-containing, photosynthetic genera may be discovered, at the current time Amphidinium is the currently agreed-upon most basal dinoflagellate genus for which isolates are available for biochemical characterization such as what we describe in this paper. Thus, because of the presumed basal position of the genus Amphidinium, we present a hypothesis that its galactolipids currently represent those that are ancestral to other genera of peridinin-containing dinoflagellates, including those within the C
18
/C
18
cluster.
Amphidinium species' galactolipids reside within the C
20
/C
18
peridinin dinoflagellate cluster.
Conversely, Testudodinium testudo (formerly Amphidinium testudo) falls within the C
18
/C
18
cluster.
We hypothesize Amphidinium's galactolipids as basal to other peridinin dinoflagellates.
