OCCURRENCE OF OCTADECAPENTAENOIC ACID IN LIPIDS OF A COLD STENOTHERMIC ALGA,PRYMNESIOPHYTE STRAIN B1

The fatty acid composition of the prymnesiophyte strain B, a cold stenothermic microalga, was examined. The major fatty acids derived from the total lipids in this strain were myristic (14:0), palmitic (16:0), oleic (18:1ω9), linoleic (18:2ω6), octadecatetraenoic (18:4ω3), octadecapentaenoic (18:5ω3), and docosahexaenoic (22:6ω3) acids. Octadecapentaenoic acid (18:5ω3) was an unusual component and was characterized by mass spectrometry, infrared absorption spectrometry, and proton nuclear magnetic resonance spectrometry. Saturated fatty acids (14:0 and 16:0) and 18:5ω3 were distributed at significant levels in the major classes of galactolipids (monogalacto-syldiacylglycerol, digalactosyldiacylglycerol, and sulfoqui-novosyldiacylglycerol), phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine), and neutral lipids with the exception that phosphatidylethanolamine contained only trace amounts of 14:0. By contrast, 22:6ω3 was distributed in phospholipids and neutral lipids. A decrease in growth temperature from 5°C to 2°C was accompanied by a significant increase in levels of 18: 5ω3 and 18:4ω3 with a concomitant decrease in the level of saturated fatty acids, whereas the level of 22:6ω3 was scarcely changed. These results suggest that, in prymnesiophyte strain B, eighteen-carbon polyunsaturated fatty acids with more than three double bonds, 18:5ω3 in particular, serve as modulators of membrane fluidity. The potential role of 18:5ω3 as a specific marker for prym-nesiophytes is also discussed.     

Changes in the fatty acid composition of Drosophila melanogaster during development and ageing.

In Drosophila melanogaster the saturated fatty acids increase in amount early in pupal development relative to the concentrations in late third instar larvae, then decline to the levels characteristic of one-day-old adults. Conversely, the monounsaturated fatty acids decline in content early in pupal development, then increase late in the pupal period. Lauric acid (12:0), myristic acid (14:0) and palmitoleic acid (16:1) become more prominent and oleic acid (18:1) and palmitic acid (16:0) less prominent as the adult ages. At about 40 days of adult age myristic acid (14:0) begins to decrease and oleic acid (18:1) to increase. Within 20 days of eclosion males and females contain different amounts of myristic acid (14:0), palmitic acid (16:0) and oleic acid (18:1).     

Fatty acid and lipid compositions of Conidiobolus

The fatty acid composition of the total lipids from two Conidiobolus species was studied by gas—liquid chromatography. The major fatty acids of C. lamprauges were palmitic acid (C_16:0), oleic acid (C_18:1), linolenic acid (C_18:3), and arachidonic acid (C_20:4). For C. eurymitus , myristic acid (C_14:0), C_16:0, and linoleic acid (C_18:2) were the most abundant acids. The fatty acid composition of C. eurymitus was quite different from that of the Conidiobolus species as mentioned in other reports. The lipid composition of the total lipids of C. lamprauges and C. eurymitus was also studied by thinchrography on quartz rods. Triglycerides and phospholipids were the major components in the two Conidiobolus species.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Changes of Lipid Composition with Growth Phase of Cryptococcus neoformans

Qualitative and quantitative profiles of phospholipids, neutral lipids, and fatty acid composition in Cr. neoformans during the growth phase were investigated in relation to pyrophosphatidic acid. A marked increase of the total lipid content, which depended on the accumulation of triglyceride in yeast cells with the growth, was observed. The total phospholipid contents in yeast cells remained almostly constant during the exponential phase and slightly decreased in the stationary phase. The major phospholipids of this yeast were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and cardiolipin, the next groups being pyrophosphatidic acid, phosphatidic acid, lysophos-phatidylcholine, and unidentified components. The amounts of phosphatidylcholine, phosphatidylinositol, and cardiolipin were fairly constant throughout the growth phase, but the amount of phosphatidylethanolamine increased and that of phosphatidylserine decreased with progressive growth. The pyrophosphatidic acid contents were 0.9~0.7% for total phospholipid during the growth phase. The major fatty acids of pyrophosphatidic acid were C_16:0, C_18:1, and C_18:2 acids. The changing patterns of fatty acid composition in pyrophosphatidic acid through the growth phase closely resembled that of phosphatidic acid, which contained larger amounts of C_18:1 acid (35~45%) than C_16:0 acid (30~25%) and C_18:2 acid (30~25%). Phosphatidylserine and phosphatidylinositol contained considerable amounts of saturated fatty acid (C_16:0 acid, more than 55%). On the other hand, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin contained extremely large amounts of unsaturated fatty acid (C_18:1 and C_18:2 acid, 85ç90%).     

Phospholipids and fatty acids of Neisseria gonorrhoeae.

The phospholipids and fatty acids of two strains of Neisseria gonorrhoeae of different penicillin susceptibilities were examined. The phospholipids, which comprise about 8% of the dry weight of the cells, consisted of phosphatidylethanolamine (70%) and phosphatidylglycerol (20%); small amounts of phosphatidylcholine and traces of cardiolipin were also present. Growing and stationary-phase cells were similar in content and composition of phospholipids except for phosphatidylcholine, which increased two- to fivefold in the stationary-phase cells. The fatty acids of the phospholipids were characterized by two major acids, palmitic and a C16:1, with myristic and a C18:1 acid present in smaller amounts. The fatty acids present in purified phospholipid fractions varied considerably in relative proportions from fraction to fraction. No significant difference in the composition of phospholipids from the two strains was evident. Large amounts of beta-hydroxy lauric acid were detected only after saponification of the organisms. Differences in the lipid composition between the gonococcus and other gram-negative bacteria are discussed.     

Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis

The identification and composition of the fatty acids of the major lipid classes (triacylglycerols and phospholipids) within Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae) nymphs were determined. Comparisons were made to fatty acids from the internal lipids of B. argentifolii adults. The fatty acids, as ester derivatives, were analyzed by capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). All lipid classes contained variable distributions of eight fatty acids: the saturated fatty acids, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0); the monounsaturated fatty acids, palmitoleic acid (16:1), oleic acid (18:1); the polyunsaturated fatty acids, linoleic acid (18:2), linolenic acid (18:3). Fourth instar nymphs had 5-10 times the quantities of fatty acids as compared to third instar nymphs and 1-3 times the quantities from adults. The fatty acid quantity differences between fourth and third instar nymphs were related to their size and weight differences. The percentage compositions for fatty acids from each lipid class were the same for the pooled groups of third and fourth instar nymphs. For nymphs and adults, triacylglycerols were the major source of fatty acids, with 18:1 and 16:0 acids as major components and the majority of the polyunsaturated fatty acids, 18:2 and 18:3 were present in the two phospholipid fractions, phosphatidylethanolamine and phosphatidylcholine. Evidence was obtained that whiteflies indeed synthesize linoleic acid and linolenic acid de novo: radiolabel from [2-(14)C] acetate was incorporated into 18:2 and 18:3 fatty acids of B. argentifolii adults and CGC-MS of pyrrolidide derivatives established double bonds in the Delta(9,12) and Delta(9,12,15) positions, respectively.     

Changes in the lipid and fatty acid composition of hemolymph and ovaries during the reproductive cycle of Labidura riparia

Lipid metabolism was investigated during the reproductive cycle of Labidura riparia (Pallas). The lipid classes and their constitutive fatty acids present in hemolymph and ovaries were measured using thin‐layer chromatography and gas‐liquid chromatography. In the hemolymph, total lipids increase steadily from the previtellogenic period to vitellogenic arrest. These lipids are predominantly diacylglycerols and phospholipids. In the ovaries, total lipids increase during vitellogenesis then decrease during the vitellogenesis arrest period. The major lipids are triacylglycerols, followed by phospholipids. In both hemolymph and ovaries, all lipid classes contained variable proportions of seven main fatty acids: the saturated fatty acids myristic acid (14:0), palmetic acid (16:0), and stearic acid (18:0); the monounsaturated fatty acids palmitoleic acid (16:1) and oleic acid (18:1); and the polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3). Unsaturated fatty acids predominate throughout the reproductive cycle. The percentage compositions of total and triacylglycerol fatty acids do not change markedly during the reproductive cycle in hemolymph nor in ovaries, with 18:2, 18:1 and 16:0 fatty acids being the major components. However, for diacylglycerols and phospholipids, the proportions of fatty acids vary systematically. For phospholipids during the vitellogenesis period, 18:2 increases considerably whereas other fatty acids decrease; for diacylglycerols, these fatty acids vary in the reverse way.     

The lipid composition of plasma membrane from goat mammary gland

Experiments were conducted to examine and characterize the lipid composition of the plasma membrane from the lactating goat mammary gland. The plasma membranes were purified by discontinuous sucrose density centrifugation. Lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The results of these studies demonstrate that (i) the principal phospholipids of mammary-gland plasma membranes are phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; (ii) the principal neutral lipids are triacylglyceride and cholesterol ester; (iii) the major glycolipids are globotetraosylceramide and globotriaosylceramide; and (iv) the major fatty acids are oleic (18:1), palmitic (16:0), stearic (18:0), and myristic (14:0) acids.     

Effect of growth temperature on lipid composition of Streptococcus faecium

The effect of growth temperature on the lipid and fatty acid composition of Streptococcus faecium has been studied. No differences in the qualitative composition of S. faecium lipids were observed. In all isolated fractions (neutral lipids, glycolipids, and phospholipids plus other polar lipids), the major fatty acids were palmitic (C-16:0), palmitoleic (C-16:1), octadecenoic (C-18:1), and cyclopropane (C-19:0). Changes in the fatty acid composition of the different fractions were observed which depended on growth temperature; the most significant one was the decrease of octadecenoic acid and the increase of palmitic acid in glycolipids and polar lipids as the temperature increased. The level of cyclopropane C-19:0 was approximately eightfold lower at 8 degrees C than at the other temperatures tested (20, 30, and 45 degrees C).     

Effect of sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of shape Candida albicans

The effect of a sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans was investigated. The total lipid content of this yeast grown in the presence of chlorhexidine was reduced whilst the total sterol content was increased compared with control-grown cells. Lipids and sterol analyses of this yeast grown in the presence and absence of chlorhexidine are presented. Chlorhexidine-grown yeast had a higher level of phosphatidylethanolamine, phosphatidylcholine and monogalactosyldiacylglycerol. Lower proportions of phosphatidylinositol plus phosphatidylserine, phosphatidic acid and cardiolipin were found in C. albicans grown in the presence of the drug when compared with control-grown yeast. The major fatty acids in control-grown cells were C16 and C18. Drug grown-cells had higher proportions of palmitic acid (16 : 0) and stearic acid (18 : 0), but lower proportions of palmitoleic acid (16 : 1) and oleic acid (18 : 1). Chlorhexidine also decreased the unsaturated-to-saturated fatty acid ratio, while the C16/C18 ratios increased compared to control-grown cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug and control-grown yeast were also detected. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (55.4% wt). A significant increase in ergosterol and obtusifoliol was observed in chlorhexidine-treated cells and a significant decrease in squalene and lanosterol. Our results suggested that chlorhexidine affected the lipid and sterol composition of C. albicans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of ethanol on the hopanoid content and the fatty acid pattern in batch and continuous cultures of Zymomonas mobilis

By thin layer chromatographic, gas-liquid chromatographic, and mass spectrometric methods 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) was shown to occur in Zymomonas mobilis. This compound contributed up to 20% to the total lipids.The fatty acid pattern and the content of hopanoids (hopene, hopanol, and THBH) were determined in batch and continuous cultures. In late exponential cells from batch cultures the relative amount of palmitic acid was increased partially at the expense of cis-vaccenic acid, when the initial glucose concentrations were increased. In a batch culture, THBH reached a maximum value in the early exponential growth phase.In an anaerobic continuous culture with a low glucose feed concentration, the THBH content and the relative amount of cis-vaccenic acid were low. The contribution of both compounds increased strongly with increasing glucose feed concentrations (i.e. at higher steady-state ethanol concentrations). The same result was found with aerobic continuous cultures which produced significant amounts of acetaldehyde and acetic acid, in addition to ethanol and carbon dioxide.It was concluded that stability and permeability of the cytoplasmic membrane of the ethanol producing bacterium Z. mobilis was regulated by variations in the distribution of hopanoids and fatty acids.Abbreviations 14:0 myristic acid - 16:0 palmitic acid - 18:1 cisvaccenic acid - THBH 1,2,3,4-tetrahydroxypentane-29-hopane     

Effect of Milk Components on IgA Production in Peyer’s Patch Cell Cultures from Mouse

The lipid composition of some commercial bakers’ yeasts having different freeze-sensitivity in frozen dough was investigated to clarify the correlation between their lipid composition and freeze-tolerance. The total lipid content including neutral lipid, free fatty acid, sterol, and phospholipid ranged between 23.0 to 32.2 mg/100 mg protein of the yeasts tested. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the main phospholipids found in all yeast strains, but no distinct difference in these components between freeze-tolerant and freeze-sensitive strains was observed. Palmitoleic (C16:l), oleic (C18:l), palmitic (16:0), and stearic (CI8:0) acids were the major fatty acids present in total lipid and phospholipid, and unsaturation indices of fatty acid in these lipid components were almost equal by the strains. The molar ratios of sterol to phospholipid of freeze-sensitive strains were higher than those of freeze-tolerant strains. The difference in the sterol-pho-spholipid ratio that influences the fluidity of plasma membranes in yeast cells was supposed to reflect the difference in freeze-sensitivity of bakers’ yeast.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Phospholipids of Clostridium perfringens: a reexamination

We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free β-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing β-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.     

The solid–liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids

For the first time, the solid–liquid phase diagrams of five binary mixtures of saturated fatty acids are here presented. These mixtures are formed of caprylic acid (C_8:0) + capric acid (C_10:0), capric acid (C_10:0) + lauric acid (C_12:0), lauric acid (C_12:0) + myristic acid (C_14:0), myristic acid (C_14:0) + palmitic acid (C_16:0) and palmitic acid (C_16:0) + stearic acid (C_18:0). The information used in these phase diagrams was obtained by differential scanning calorimetry (DSC), X-ray diffraction (XRD), FT–Raman spectrometry and polarized light microscopy, aiming at a complete understanding of the phase diagrams of the fatty acid mixtures. All of the phase diagrams reported here presented the same global behavior and it was shown that this was far more complex than previously imagined. They presented not only peritectic and eutectic reactions, but also metatectic reactions, due to solid–solid phase transitions common in fatty acids and regions of solid solution not previously reported. This work contributes to the elucidation of the phase behavior of these important biochemical molecules, with implications in various industrial applications.     

Differentiation of Three Varieties of Rhizoctonia circinata; var. circinata, var. oryzae and var. zeae on the Basis of Cellular Fatty Acid Composition

Cellular fatty acid analysis was employed to differentiate three varieties of Rhizoctonia circinata ; var. circinata , var. oryzae and var. zeae . Eight fatty acids including myristic (14 : 0), pentadecanoic (15 : 0), palmitic (16 : 0), palmitoleic (16 : 1  cis 9), stearic (18 : 0), oleic (18 : 1  cis 9), linoleic (18 : 2  cis 9,12) and linolenic (18 : 3  cis 9,12) acids were present in isolates of all three varieties of R. circinata . Heptadecanoic acid (17 : 0) was detected in isolates of R. circinata var. zeae but not in isolates of R. circinata var. circinata or R. circinata var. oryzae . Palmitic, oleic and linoleic acids were the major fatty acids found, comprising 94–98% of the whole-cell fatty acid content. The remaining fatty acids were present in small amounts. Based on the composition (%) of fatty acids, isolates of R. circinata var. circinata , R. circinata var. oryzae and R. circinata var. zeae were clearly differentiated into three groups as shown by principal component and cluster analyses. This finding agrees well with the grouping of R. circinata into three varieties based on differences in colony morphology of the vegetative state. In principal component and cluster analysis, isolates of R. circinata var. circinata from Japan and Alaska were indistinguishable.