VEGETATIVE AND REPRODUCTIVE MORPHOLOGY OF EUCHEUMA ISIFORME (SOLIERIACEAE,GIGARTINALES, RHODOPHYTA)1

Eucheuma isiforme (C. Agardh) J. Agardh exhibits a combination of vegetative and reproductive features that distinguish it from other critically studied genera in the Solieriaceae. The development of the multiaxial thallus, emphasizing the arrangement of periaxial cells around each axial file; presence of reproductive nemathecia that contain carpogonial branches and auxiliary cells; and post-diploidization stages, including gonimoblast and pericarp initiation, stages of fusion cell formation, and carposporophyte development are described and illustrated for the first time in this species. The vegetative and reproductive features observed in E. isiforme are not diagnostic of any of the recently erected tribes in the Solieriaceae. Eucheuma appears most closely related to the Indian Ocean genus, Sarconema.     

REPRODUCTIVE DEVELOPMENT OF THE PARASITIC RED ALGA GARDNERIELLA TUBERIFERA (SOLIERIACEAE,GIGARTINALES)1

Examination of the reproductive morphology of the adelphoparasitic red alga Gardneriella tuberifera Kylin reveals that this monotypic genus is correctly placed in the family Solieriaceae (Gigartinales), to which its host Agardhiella gaudichaudii (Montagne) Silva et Papenfuss also belongs. Gardneriella is multiaxial, nonprocarpic and has an inwardly directed, three-celled carpogonial branch. The large, reniform uninucleate auxiliary cell is distinct prior to and after fertilization. It is diploidized by an unbranched, multicellular connecting filament which lacks pit connections. One or two connecting filaments arise from each fertilized carpogonium. From the diploidized auxiliary cell, the gonimoblast initial is cut off obliquely toward the interior of the thallus. The cells of the gonimoblast fuse with adjacent unpigmented vegetative cells of Gardneriella and pigmented cells of the host. These cells become incorporated into the developing cystocarp and, from those of Gardneriella, additional short chains of gonimoblast cells arise. The mature cystocarp is placentate, radiately lobed, and lacks a surrounding involucre. Carposporangia are borne in short chains and the unpigmented carpospores are released upon the dissolution of outer vegetative cells. No ostiole is present. Gardneriella appears to be most closely related to the placentate solieriacean genera Agardhiella, Sarcodiotheca, and Meristiella and therefore this genus should be placed in the tribe recently erected for these taxa, the Agardhielleae.     

SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES,RHODOPHYTA)1

This study evaluates the hypothesis that spore coalescence may cause intraclonal variation. Spore coalescence might allow the occurrence of unitary thalli that in fact correspond to genetically different, coalesced individuals. Plant portions simultaneously derived from these chimeric individuals may exhibit dissimilar growth responses even when incubated under similar abiotic conditions. Testing of the hypothesis included various approaches. Transmission electron microscopy observations of early stages of sporeling coalescence indicated that polysporic plantlets were formed by groups of spores and their derivatives. Even though adjacent cells in two different groups may fuse, these groups maintained an independent capacity to grow and form uprights. Laboratory-grown plantlets showed a significant correlation between the initial number of spores and the total number of erect axes differentiated from the sporeling. Construction and growth of bicolor individuals indicated the chimeric nature of the coalesced individuals. Coalesced, bicolor holdfasts had green and red cells, which subsequently produced green and red uprights, respectively. Individuals fronds were also chimeric, as indicated by the production of green and red branchlets from single, red uprights. The existence of mixed tissues was further substantiated by random amplified polymorphic DNA analysis. The banding pattern produced by branchlets of a unisporic thallus was consistently monomorphic, whereas the patterns produced by the polysporic thallus were polymorphic. Growth rates of polysporic thalli had larger data dispersal and variation coefficients than oligosporic or monosporic thalli. Therefore, all results support the original hypothesis and suggest that coalescence might be ecologically more important than previously thought.     

DIFFUSION BOUNDARY LAYER TRANSPORT IN GRACILARIA CONFERTA (RHODOPHYTA)1

A theoretical framework on the combined effect of water velocity and solute concentration on the photosynthetic performance of the red alga Gracilaria conferta (Rhodophyta) is developed. This is based on the balance between the rate of transport through boundary layers and Michaelis-Menten-type equations for carbon consumption and for production of oxygen and hydroxyl ions. By comparing the theoretical models with experimental data, we found that the mechanism of enhancing photosynthetic rates by increasing water velocity cannot be attributed to enhanced bicarbonate and CO₂ transport, nor to CO₂ as a sole source of carbon. Velocity-facilitated photosynthesis may be due to the enhanced removal of OH^? ions, which inhibit photosynthesis when accumulated on the algal surface. Oxygen had no inhibitory effect on Gracilaria conferta.     

QUANTIFICATION OF THE EFFECTS OF SPORELING COALESCENCE ON THE EARLY DEVELOPMENT OF GRACILARIA CHILENSIS (RHODOPHYTA)1

Sporeling coalescence in species of Gracilariales and Gigartinales is predicted to result in larger basal areas of growing disks as well as earlier initiation, increased abundance, and faster growth rates of erect shoots as compared to noncoalescent sporelings. These responses have been interpreted as providing mutual benefits for organisms living in aggregation, counterbalancing disadvantages associated with crowding. Quantitative evaluations of sporelings of Gracilaria chilensis failed to support several of these predictions. Sporelings were grown in the laboratory from a range of single sporelings to coalescent masses of 20 sporelings. Coalescent sporeling masses of G. chilensis exhibited larger basal areas than noncoalescent ones, but because the specific growth rates were inversely related to the original number of carpospores, no significant differences in actual area increments, during most of the experiment, were found among sporelings derived from one, two, or three to five coalescing sporelings. Initiation of erect shoots occurred at a similar time, regardless of their origin, i.e. coalescent or noncoalescent. Abundance of erect shoots was only loosely related to the number of coalescing sporelings. Even though by the end of the experiment (week 18), the total length of the longer erect shoots arising from coalescent sporeling masses was significantly greater than that of shoots arising from noncoalescent sporelings, total length was independent of the original number of coalescing sporelings. Furthermore, specific elongation rates between week 12 and week 18 were significantly greater for noncoalescent sporelings than for coalescent sporeling masses. Quantitative screening of other species seems necessary before generalizations on the ecological advantages of sporeling coalescence in seaweeds can be made.     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.     

THE GENUS PLATOMA (GIGARTINALES,RHODOPHYTA) WITH A DESCRIPTION OF P. ABBOTTIANA SP. NOV.1

Platoma abbottiana (Gymnophlaeaceae; Gigartinales), a new species of benthic red algae, is described from Isla Mejía and is the second member of this genus reported from the Gulf of California, Mexico. Vegetative anatomy and reproductive details of the carpogonial and auxiliary cell branches of the new species are described and illustrated. Geographical distributions of the seven previously described Platoma species are given. Their distinguishing morphological characteristics are compared to those of the new species. The correct time and place of valid publication of the generic name, Platoma, and some of its binomials are discussed.     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

DESICCATION TOLERANCE OF GRACILARIA TIKVAHIAE (RHODOPHYTA)1,2

The photosynthesis of Gracilaria tikvahiae McLachlan, normally a subtidal plant, returned to initial levels within a day following single desiccations to 50% and repeated desiccations to at least 15%. In contrast, growth rates were decreased by even very mild desiccation.     

THE EFFECTS OF LIGHT AND NITROGEN ON GROWTH,PIGMENT CONTENT,AND BIOCHEMICAL COMPOSITION OF GRACILARIA FOLIIFERA V. ANGUSTISSIMA (GIGARTINALES,RHODOPHYTA)1

The combined effects of light intensity and nitrogen (NO₃^?) on growth rate, pigment content, and biochemical composition of Gracilaria foliifera v. angustissima (Harvey) Taylor was investigated using outdoor continuous cultures. Growth of Gracilaria increased linearly with increasing light to 0.43 doublings d^?1 at high light levels (383 ly d^?1 of in situ light), suggesting that light may often limit growth of this plant in nature. Chlorophyll a and phycoerythrin contents were inversely proportional to light level and growth rate. However, pigment content did not affect the growth capacity of Gracilaria. There was no increase in growth or pigment content with increasing additions of nitrogen. The low nitrogen treatment was unenriched seawater that had higher NO₃^? levels than most coastal waters (influent = 8.61 μM; residual = 0.94 μM). When growing near its maximum rate under high light intensities, Gracilaria had a significantly (P < 0.001) lower phycoerythrin: chlorophyll a ratio (phyco: Chl a) than did Gracilaria growing more slowly under lower light (Phyco:Chl a of 2.8 ± 0.2 vs. 3.8 ± 0.3). Faster growing plants also had C:N ratios above 10, indicating N- limitation. In addition to harvesting light the phycobiliproteins of Gracilaria may store nitrogen. Growth rates of Gracilaria correlated negatively with ash (r =–0.85) and positively with the carbon: phycoerythrin ratio (r = 0.85), suggesting that these two indices can be used to estimate growth in the field.     

BETA/KAPPA-CARRAGEENANS AS EVIDENCE FOR CONTINUED SEPARATION OF THE FAMILIES DICRANEMATACEAE AND SARCODIACEAE (GIGARTINALES,RHODOPHYTA)1

The Dicranemataceae consists of five species in four genera of macroscopic red algae endemic to the southern half of Australia plus a single species from southern Japan. Investigations of the nonfibrillar wall components of five of the six species show that all are composed mainly of hybrid (or mixed) beta (β)/kappa(κ)-type carrageenans. Detailed studies of Tylotus obtusatus (Sonder) J. Agardh show that it produces the largest dry-weight percentage of β-carrageenan yet recorded. Monosaccharide composition, total sulfate content, sulfation pattern revealed by infrared and ¹³C-nuclear magnetic resonance spectroscopy and a positive specific optical rotation ([α]_D+ 54°) are indicative of a low-sulfate-containing carrageenan with gelling properties similar to those of agar and furcellaran. β-carrageenan is recorded in only five other red algal species belonging to relatively unrelated families, and we conclude that its uniform occurrence in the highly specialized family Dicranemataceae has phylogenetic significance. Chemical and anatomical examination of the genus Sarcodia, which produce lambda-type carrageenan in both its gametophytic and tetrasporophytic phases, suggests that, despite the recent proposal to incorporate the Dicranemataceae into the Sarcodiaceae, the two families should continue to be separated.     

A REVISION OF THE SYSTEMATICS OF THE NIZYMENIACEAE (GIGARTINALES,RHODOPHYTA) BASED ON POLYSACCHARIDES,ANATOMY, AND NUCLEOTIDE SEQUENCES1

The Australian endemic family Nizymeniaceae, based on Nizymenia australis Sonder, consists of three species in the two genera Nizymenia (1 sp.) and Stenocladia (2 spp.). We have reassessed the generic composition of the family based on evidence from nonfibrillar wall polysaccharides, vegetative anatomy, and the nucleotide sequences of an internal transcribed spacer, ITS 2, of the nuclear ribosomal cistron. Investigation of the polysaccharides by constituent sugar analysis, sulfate content determination, and methylation analysis, combined with gas chromatography-mass spectrometry and infrared analysis, showed that the polysaccharides elaborated by the three species were branched, highly sulfated xylogalactans. These polysaccharides also contained significant amounts of mono-O-methyl galactose (5–8 mol% of total sugars), mainly 4-O-methyl galactose. Although no discrete chemical structures could be assigned to the polysaccharides, the analyses showed that those from Nizymenia australis and Stenocladia australis (Sonder) Silva were more alike than either was to that from S. furcata (Harvey) J. Agardh. This polysaccharide affinity was echoed by a suite of vegetative anatomical features. However, the only likely synapomorphy was the presence of refractive, thick-walled medullary rhizines in both N. australis and S. australis. The ITS 2 sequences were inferred from direct sequencing of the products of polymerase chain reaction amplification. Comparison of the ITS 2 sequences of its three species with those of two outgroups indicated that the family Nizymeniaceae is monophyletic but that interspecific relationships within the family could not be resolved. We conclude that there is insufficient evidence to separate any of the species from the rest at the genus level. Therefore, all three species are consolidated into the genus Nizymenia. This necessitates nomenclatural changes of Stenocladia australis to Nizymenia conferta (Harvey) Chiovitti, Saunders, et Kraft comb. nov.     

DIURNAL FLUCTUATION OF NITRATE REDUCTASE ACTIVITY IN THE MARINE RED ALGA GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The biochemical characteristics and diurnal changes in activity of the enzyme nitrate reductase (NR; EC 1.6.6.1) from the marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia are described. Different assay conditions were tested to determine the stability of NR. The crude extract of G. tenuistipitata has a NR specific activity of 10.2 U.mg⁻¹, which is higher than the NR activities found for other algae, plants, and fungi. This NR is highly active at a slightly alkaline pH and is stable over a wide range of temperature, with an optimal activity at 20° C. The apical portions of the thallus contain 64.9 ± 6.6% of the total NR specific activity. The apparent Michaelis-Menten (K_m) constant found for KNO₃ was 197 μM, and it was 95 μM for NADH. The NR from G. tenuistipitata can be included in the NADH-specific group, because no activity was found when NADPH was used as an electron donor. In extracts of algae grown under either continuously dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at the middle of the light phase, when activity is 30-fold higher than during the night phase.     

SCHMITZIA EVANESCENS SP. NOV. (RHODOPHYTA,GIGARTINALES) A NEW SPECIES OF CALOSIPHONIACEAE FROM THE NORTHEASTERN NEW ZEALAND COAST1

A new red alga, Schmitzia evanescens sp. nov. (Gigartinales), is described from the northeastern coast of North I., New Zealand. Gametophytes are diminutive, delicate, mucilaginous spring annuals which grow subtidally on cobble substrata. The uniaxial construction and carposporophyte development place this new taxon in the Calosiphoniaceae, a family of only two genera, Calosiphonia H. M. Crouan et P. L. Crouan and Schmitzia P. C. Silva. This is the first report of this family in New Zealand. The new species has vegetative features of both these genera, but is assigned to Schmitzia on the basis of its unusual carposporophyte development in which the gonimoblast initial arises directly from the connecting filament at some distance from the undifferentiated intercalary auxiliary cell. Because recent investigators of other red algal genera with a similar carposporophyte development have questioned the validity of site of gonimoblast initiation as a generic criterion it is suggested that the separation of Schmitzia and Calosiphonia should be re-evaluated. These and several other genera in the Gymnophlaeaceae, Pseudoanemoniaceae, Dumontiaceae and Gloiosiphoniaceae, can be considered, on morphological and reproductive grounds, to be relatively simple and therefore perhaps primitive members of the Rhodophyta.     

AN INCREASE OF ORNITHINE δ-AMINOTRANSFERASE-MEDIATED PROLINE SYNTHESIS IN RELATION TO HIGH-TEMPERATURE INJURY IN GRACILARIA TENUISTIPITATA (GIGARTINALES, RHODOPHYTA)

Changes of proline biosynthesis in relation to high-temperature (35° C) injury were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia. On exposure to 35° C, the specific growth rate decreased after 5 days while free proline levels increased gradually after 2 days and reached the maximal level on days 4–6 but decreased at day 7. The repair ability of thalli treated at 35° C by measuring the growth rate after transfer to 25° C for another 5 days decreased in thalli that had been grown at 35° C for more than 2 days, and the extent increased as treatment at 35° C was prolonged. After 4 days of treatment at 35° C, the activities of both ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) and Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) increased, but that of γ-glutamyl kinase (γ-GK; EC 2.7.2.11) remained unchanged, and that of glutamate-5-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) decreased. The application of 10 μM gabaculine, an irreversible inhibitor of δ-OAT, at 35° C recovered the growth ability but inhibited the increase of both δ-OAT activity and free proline level; its effects were reversed by 1 mM proline. G. saliconia, which is relatively tolerant to high temperature, showed a decrease of both δ-OAT activity and free proline level at 35° C. It seems that a stimulation of proline synthesis from the ornithine pathway via an increase in both δ-OAT and P5CR activities might be associated with high-temperature injury in G. tenuistipitata.     

DISSECTION OF TWO DISTINCT DEFENSE‐RELATED RESPONSES TO AGAR OLIGOSACCHARIDES IN GRACILARIA CHILENSIS (RHODOPHYTA) AND GRACILARIA CONFERTA (RHODOPHYTA)1

The two agar‐producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H₂O₂) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H₂O₂‐dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membrane‐located NADPH oxidase, appears to be responsible for the release of H₂O₂ in G. conferta. Subcellular examination of G. chilensis showed that the H₂O₂ release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 17°C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 12°C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.     

THE ATLANTIC SPECIES OF SOLIERIA (GIGARTINALES,RHODOPHYTA): THEIR MORPHOLOGY,DISTRIBUTION AND AFFINITIES1

Solieria chordalis (C. Agardh) J. Agardh and S. tenera (J. Agardh) Wynne et Taylor exhibit multiaxial growth from a cluster of four to eight obconical apical cells. A single periaxial cell is cut off from each axial cell and successive periaxial cells are rotated 120° in a zig-zag pattern along each axial filament. Periaxial cells produce branched, laterally diverging filaments which form the cortex. The medulla is composed of axial cells, elongate cells of lateral filaments, stretched interconnecting cells, and secondary rhizoids. The two species are nonprocarpic. Carpogonial branches are 3-celled, inwardly directed, with a reflexed trichogyne. The auxiliary cell together with associated darkly-staining inner cortical cells form an association, the auxiliary cell complex, that is recognizable prior to diploidization. A single, unbranched, non-septate connecting filament issues from the fertilized carpogonium and fuses with the inner, lateral side of an auxiliary cell. Production of an involucre from surrounding vegetative cells is stimulated and a gonimoblast initial is cut off toward the interior of the thallus which divides to form a compact cluster of gonimoblast cells. A fusion cell is produced through fusion of inner gonimoblast cells with the auxiliary cell that, in turn, fuses progressively with cells of the lateral file bearing the auxiliary cell. Mature cystocarps have terminal carposporangia cut off from gonimoblast cells at the periphery of the fusion cell and are surrounded by an involucre with a distinct ostiole. Tetrasporangia are cut off laterally from surface cortical cells which then cut off one or two additional derivatives toward the outside. A lectotype is designated for Solieria chordalis, but the lectotypification of S. tenera is questioned. We conclude that Solieria is closely related to Rhabdonia and place the Rhabdoniaceae in synonomy with the Solieriaceae.     

MORPHOLOGY OF PLACENTOPHORA (SOLIERIACEAE,GIGARTINALES: RHODOPHYTA), A NEW GENUS BASED ON SARCODIOTHECA COLENSOI FROM NEW ZEALAND1

The only member of the red algal family Solieriaceae known from New Zealand is the endemic Sarcodiotheca colensoi (Hook. & Harv.) Kylin. This study shows that it differs in several respects from the type S. furcata (Setch. & Gard.) Kylin; thus a new genus Placentophora is created for the New Zealand alga. Although P. colensoi nov. comb. is retained in the Solieriaceae on the basis of vegetative, spermatangial, tetrasporangial, carpogonial-branch and early gonimoblast features, it differs from typical members of that family in its pattern of later carposporophyte development. After a single gonimoblast initial is cut off from the auxiliary cell towards the center of the thallus, further gonimoblasts develop from the initial as ramifying, radiating filaments. These filaments enter an extensive “nutritive-cell” region surrounding the auxiliary cell, form, numerous connections to the “nutritive” cells, and incorporate most of them into a central placenta of interconnected, and variously-fused vegetative and gonimoblast cells. Carpo-sporangia then form in short chains around the periphery of the placenta. The cystocarp lacks both a central fusion cell and a sterile-celled investment, or “Faserhülle.” The distinctive carposporophyte of Placentophora is compared to patterns of gonimoblast development, known in other members of the Solieriaceae.     

ECOLOGY AND NATURAL HISTORY OF IRIDAEA CORDATA (GIGARTINALES,RHODOPHYTA) GROWTH1

The in situ growth, maturation and senescence, of central California Iridaea cordata (Turner) Bory was studied. This ontogenetic progression was quantified by measuring development and growth of: i) individually tagged blades, and ii) populations of blades within cumulative (1-yr duration) and seasonal experimental plots. Greatest growth and longest life span were exhibited by winter-spring initiated blades, and were correlated with a rapid increase, in irradiance, but not with either seawater temperature or nutrients. Tagging studies showed that reproductive maturation and senescence of blades occurred throughout the year, irrespective of date tagged, growth rate or size. Moreover, the majority of blades continued to elongate following maturation, and some matured within 3 mo of initiation at all seasons but winter. At the population level maturation took place primarily during summer-autumn when 90 ±2% of the population was mature. The majority of the population senesces or “dies back” during autumn-winter. It is concluded that in situ, the blades are derived almost totally via vegetative means involving perennation. This indirectly suggests that sexual reproduction or the success of sporeling development are marginal. Additionally, the species is perennial with annually deciduous blades, characterized by both rapid growth and maturation.