GRAZER-INDUCED COLONY FORMATION IN SCENEDESMUS ACUTUS (CHLOROPHYCEAE): ECOMORPH EXPRESSION AT DIFFERENT TEMPERATURES

Scenedesmus acutus Meyen was cultured at four temperatures (9.5°, 16.5°, 24°, and 29° C) in standard medium or in medium with filtered water from a Daphnia culture. Growth was significantly reduced at low temperatures. At 9.5° C it took more than a week before formation of eight-celled coenobia occurred in both the absence and presence of water from a Daphnia culture. At higher temperatures, formation of four- and eight-celled coenobia occurred more rapidly and was already observed in the presence of Daphnia water within 2 days. As cultures aged, also in the absence of Daphnia water, four-celled coenobia became dominant. At cold temperature, cell volume initially was significantly larger but declined after 3–4 weeks. Grazer-induced colony formation had occurred independent of the incubation temperature, but the number of cells per colony was increased with declining temperature. The morphological expression may be interpreted as a cyclomorphosis driven by nutrients, temperature, and chemical cues from grazers.     

GRAZER-INDUCED COENOBIAL FORMATION IN CLONAL CULTURES OF SCENENDESMUS OBLIQUUS (CHLOROCOCCALES, CHLOROPHYCEAE)

Five strains of the phenotypically plastic alga Scenedesmus obliquus (Turpin) Kützing were cultured in standard medium or in medium containing filtered water from a Daphnia culture to examine grazer-induced coenobia formation. Strains varied considerably in their response to Daphnia chemicals. A rapid formation of four- and eight-celled coenobia was observed in the presence of Daphnia water in three strains. One strain showed numerous irregular aggregates with more than eight cells per colony, and no colony formation occurred in a fifth strain. In standard medium, all cultures remained unicellular. Growth rates were affected by Daphnia water in four strains and were different among strains. The chl a concentration was significantly different among strains but was not influenced by Daphnia water. The specific absorption coefficient was reduced at larger colony sizes, suggesting a possible cost associated with colony formation. The phenomenon of grazer-induced coenobia formation seems widespread but not universal within the species S. obliquus.     

CYCLOMORPHOSIS IN SCENEDESMUS ARMATUS (CHLOROPHYTA): AN ORDERED SEQUENCE OF ECOMORPH DEVELOPMENT1

Scenedesmus armatus (Chod.) Chod, growth and morphology were monitored in medium 7 (oligotrophic) and Bristol's medium (eutrophic); cultures in both media were incubated at 10 and 22° C. Growth rate at 10° C was reduced, i.e. only one doubling in 7 days in medium 7 and 2.3 doublings in Bristol's, compared to 4.3 and 6 doublings at 22° C over the same period. Unicells as well as cells of colonies were larger at the cold temperature. The lengths of cells were not significantly different regardless of temperature or medium, but cell width was markedly increased at the lower temperature. Additionally, an arcuate, eight-celled, multispined ecomorph, which resembled several previously described taxa, was produced at 10° C. It becomes a component of a previously published ordered sequence of ecomorph development for this species. Based on data now accumulated in both the laboratory and the field, these temporal changes are interpreted to be a cyclomorphosis, driven by a coupling of nutrient availability and temperature. Within the addition of new cold temperature (spring) ecomorphs, the ordered sequence of ecomorphs for S. armatus is a succession from unicells to multispined eight-celled colonies to quadricaudate colonies, ending with acaudate four-celled ecomorphs.     

PHENOTYPIC PLASTICITY IN SCENEDESMUS COMMUNIS (CHLOROPHYCEAE). I. RELATIONSHIP OF S. COMMUNIS TO S. KOMAREKII1

When scenedesmus communis Hegew. (UTEX 76) was transferred daily in dilute media and a low cell density was maintained (ca. 1000 cells · mL^?1), up to 30% unicells were produced in that population. Unlike previously described uncell-coenobium-unicell transformation with other species, these unicells never produced S. communis coenobia (large coenobium type, LCT) but rather small coenobium type (SCT) resembling S. komarekii Hegew. Growth and morphological development of the paratype strain of S. komarekii (UTEX 1236) was compared with an isolated SCT strain (SCT 76–8). SCT 76–8 never produced LCTs and grew significantly faster than UTEX 1236. Both SCT 76–8 and UTEX 1236 produced uncells at low cell densities. Coenobia formed when cell densities increased over time in batch cultures. SCT 76–8 and UTEX 1236 did not differ morphologically when viewed with the light microscope. Under scanning electron microscopy, an outer opaque layer covered an inner warty layer on unicells. The outer layer was reduced or absent in coenobia from batch cultures in stationary growth. In addition, long spikelets, not present on the walls of unicells, were prominent on coenobial walls. The spikelets of UTEX 1236 appeared smaller and more uniformly distributed than in strain 76–8. In contrast, the surface wall morphology of LCT S. communis was composed of an outer reticulate layer supported by spikelets and appeared as a pentagonal meshwork covering the cell walls. This phenotypic plasticity, as demonstrated by SEM and light microscopy, provides further evidence needed for an understanding of Scenedesmus evolution.     

INFLUENCE OF PHOSPHORUS NUTRITION ON COPPER TOXICITY TO THREE STRAINS OF SCENEDESMUS ACUTUS (CHLOROPHYCEAE)1

Three strains of Scenedesmus acutus f. alternans Hortobagyi with markedly different sensitivities to copper were examined to determine the relative importance of cellular polyphosphate content on acute copper toxicity to photosynthesis. By manipulating the phosphate concentration in semicontinuous cultures, the response of each strain was assessed at three cellular phosphorus states: P-loaded, P-sufficient, and P-deficient. The results demonstrated the importance of cellular polyphosphate content in reducing the toxic effect of copper on photosynthesis; the greater the cellular P content, the less inhibition of photosynthesis occurred during copper exposure. This relationship was evident for both Cu-tolerant strains (XCu and B-4) and a Cu-sensitive strain (X72). The ranges of response to 9.9 μM Cu (measured as the percentage of control rate of photosynthesis within strains) were, from P-loaded to P-deficient cells, X72, 78–54%; XCu, 95–77%; and B-4, 99–94%. The data suggest that polyphosphate plays a passive role in protecting cells from copper; however, with respect to the mechanism of Cu tolerance, polyphosphate appeared to be relatively unimportant because the sensitivity of the Cu-tolerant strains showed less dependence on cellular polyphosphate than did the Cu-sensitive strain.     

THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). II. CELL DIVISION AND COLONY FORMATION1

Cell division in Scenedesmus is fairly typical of other chlorococcalean genera. The closed spindle has centrioles at polar fenestrae and apparently a series of nuclear divisions precedes cytokinesis. The phycoplast system of cytokinetic microtubules predicts the path of cleavage furrows whose mode of formation is obscure. Before and during cell division, the endoplasmic reticulum invariably accumulates granular material which later, during cytokinesis, appears to he secreted via the golgi bodies. Similar dense granular material then at accumulates outside the forming daughter cells but inside the parental wall, as the latter begins eroding away. By the end of colony formation, the cellulosic parental wall has disappeared, leaving its outer sheath and attached ornamentative features (spines, combs, reticulate or warty layer, etc.) intact as a “ghost.” The spines and combs of new colonies appear to condense out of the extracellular aggregate; their precise mode of formation is obscure. As they form, the daughter cells, having become rearranged within the parental wall, stick to one another apparently at specific sites on their outer surface. A trilaminar (sporopollenin-containing) layer arises first in each cell at these adhesive sites and immediately afterwards, dense material aggregates between the adjacent layers to give rise to the coenobial adhesive. Plaques of the trilaminar layer later appear over the rest of the cell's surface; they grow and fuse so that eventually each cell is enclosed by one continuous Trilaminar Sheath (TLS). While the plaques are forming, another dense layer materializes around the whole coenobium. Depending on the species, this layer turns into either the warty layer, in which instance it is applied directly on to the surface of the TLS except near the coenobial adhesive, or else it becomes the reticulate layer, in which instance it remains entirely separate from the TLS, soon acquiring the complex system of propping spikelets which suspend it from the coenobial surface. When fully farmed, the daughter coenobium is tightly compressed within the parental TLS, with its spines folded lengthwise along the daughter cells. Release of the colony follows a quite explosive rupturing of the parental TLS, and immediately upon release, the daughter colony flattens out and erects its spines.     

COENOBIAL CELL NUMBER IN SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AS A FUNCTION OF GROWTH RATE IN NITRATE-LIMITED CHEMOSTATS1

The relative numbers of cells growing as coenobia of different cell number are functions of growth rate when Scenedesmus quadricauda (Turp.) Bréb. is grown axenically in nitrate-limited, steady-state chemostats in continuous light. Unicells decreased from a maximum fraction of 0.04–0.05 of the total number of cells at 0.1 day^?1 to 0.01 or less as growth rate increased. The fraction of cells that grew as two-celled coenobia decreased from about 0.2 to 0.01–0.02. The fraction that grew as 4-celled coenobia increased from about 0.7–0.8 at 0.1 day^?1 to near unity at 0.5–0.6 day^?1, and then decreased sharply. The fraction of 8-celled coenobial cells increased from very small values below 0.6 day^?1, to ca. 0.9 at 1.0 day^?1.     

THE ROLE OF UNICELLS IN THE POLYMORPHIC SCENEDESMUS ARMATUS (CHLOROPHYCEAE)1

The UTEX 2193 strain of Scenedesmus armatus (Chod.) Chod, when cultured in any of several media (whether natural or artificial, concentrated or dilute) produced a variety of colonial morphologies as well as a unicell population. Morphological expression was related to culture ape. When the initial cell density was just a feu1 hundred cells per mL. the culture first produced a unicell population, then spiny colonies, and as stationary phase was approached, spine-less colonies. Two classes of spiny colonies were detected. Type I colonies had elongate cells with the terminal cells shorter than median cells. Spines were longer than cell length. The wider, oval, grainy cells of Type II colonies were uniform m length. Spines were shorter and thicker than those on Type I colonies. Only Type I colonies produced unicells: the latter appeared as two morphs. The smaller unicell was obovoid with four delicate spines: the larger had ovate cells bearing four thicker spines. Control of unicell development in all media was achieved by carefully monitoring colony type and cell number used for the inoculum. A unicellular population developed in batch culture in defined media, both concentrated and dilute, when the initial cell density (either Type I or Type II colonies) was low (below 1000 cells-mL^?1), as well as in synchronous cultures. With higher initial cell densities, e.g. 2 × 10⁴ cells·mL^?1, the inoculum had to contain Type I colonies to produce unicells. Unicells were also produced in water from Agronomy Pond, where the strain originated. We discuss the role of unicell populations in the distribution of Scenedesmus.     

REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

LAGERHEIMIA HINDAKII IS THE UNICELLULAR STAGE OF A SCENEDESMUS1

Extensive variability in spine number and position was emphasized in the protologue for the spiny unicell Lagerheimia hindakii Hegew. et A. Schmidt. Using an axenic clone established from culture Hindk 1975/95, which provided the type material of that organism, we initiated a morphogenetic study. While in the unicellular stage, three short spines were usually formed at each cell pole. A colonial morph was first observed during the initial isolation procedure after unicells were dispersed on firm agar and allowed to grow. We propose that L. hindakii is a Scenedesmus, close to S. subspicatus R. Chod. This organism is unlike most representatives of the genus Scenedesmus because colonies do not readily appear in dilute media; they also fail to form in Bristol's liquid medium unless there is an organic supplement, e.g. glucose. Hindk 1975/95 may be more closely related to truly unicellular taxa than to other Scenedesmus.     

STIMULATION OF GROWTH IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) BY HUMIC ACIDS UNDER IRON LIMITED CONDITIONS1,2

Stimulatory affects of humic acids of molecular weight 30,000 or greater on iron-starved Scenedesmus obliquus (Turp.) Kütz. in association with bacteria were studied by growth and Fe uptake experiments. Humic acids stimulated growth of Fe-starved cells by decreasing the lag phase and extending the growth phase. Humic acids stimulated increased algal growth in medium containing EDTA as well as in medium containing no EDTA, indicating humic acids are not stimulating algal growth under Fe limiting conditions by creating a soluble Fe pool. Humic acids decreased Fe availability to Fe-starved S. obliquus. Iron bound to humic acids is unavailable for uptake by Fe-starved cells indicating growth stimulation is not due to chelation effects alone. Stimulation of growth is not a membrane phenomenon as humic acids show the same stimulatory effect when in contact with cells or separated by dialysis membrane. Humic acids also stimulate growth in the dark, with and without aeration, indicating use as a heterotrophic substrate. A photoheterotrophic mechanism is indicated by increased algal growth caused by illuminating cultures, containing humic acids but excluding CO₂.     

SURVIVAL OF SCENEDESMUS ACUMINATUS (CHLOROPHYCEAE) IN DARKNESS1

Survival of the green alga Scenedesmus acuminatus Lagerh. in complete darkness was studied in axenic batch cultures at 7°C and 22°C for three months. The decrease in cell numbers was insensitive to temperature and slower than the loss of dry weight. However, the lag phase before cells began to lyse was more than twice as long at 7° C than at 22°C. The decline in cellular carbohydrates and proteins occurred in two phases. During the first 3-4 days, the decrease in cellular carbohydrate levels was significantly accelerated and temperature-sensitive. Pyrenoids disappeared within 5 days of darkness. Proteins showed 20-fold higher degradation rates at 22°C than at 7°C during the first 4 days. Thereafter, the rates of carbohydrate and protein decomposition were slow and temperature-independent. By contrast, lipids degraded only little at virtually constant and temperature-insensitive rates over the entire experimental period. After three months of dark incubation, about 40% of the remaining cells had retained their growth potential. However, the lag phase, after which cell division was resumed when exposed to light, increased with the duration of the previous dark period. The decrease in photo synthetic potential, which was more pronounced at 22° C than at 7° C, was apparent both in declining maximum assimilation numbers and maximum quantum yields. Cellular chlorophyll a concentrations in surviving cells decreased only slightly. We conclude that the primary means by which S. acuminatus survives extended dark periods is by reduction of catabolic reactions. This was suggested by the slow loss of cell weight. No evidence of significant heterotrophic acetate uptake was found. The initial temperature-dependence of most observed processes indicates that in natural environments chances for survival of algae are augmented by the prevailing low water temperatures.     

ETHEPHON (2-CHLOROETHYLPHOSPHONIC ACID) EFFECTS ON SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE)1,2

Growth of Scenedesmus quadricauda (Turp.) Bréb. was significantly increased when the cultures were grown in Ethephon 68–241 (2-chloroethylphosphonic acid) at concentrations of 0.001–1.0 μg · ml^?1. At 0.1 μg · ml^?1, Ethephon caused a significant increase in RNA levels on a dry weight basis. Total protein and DNA levels increased but not significantly. These data indicate that ethylene, the decomposition product of Ethephon at physiological pH, has some effect on the metabolism of algae.     

EFFECT OF IRRADIANCE ON GROWTH AND REPRODUCTIVE PROCESSES DURING THE CELL CYCLE IN SCENEDESMUS ARMATUS (CHLOROPHYTA)1

Synchronized populations of the chlorococcal alga Scenedesmus armatus (Chod.) Chod. were grown under five irradiance levels. During the cell cycles of these populations, reproductive processes such as DNA replication, nuclear division, protoplast fission, and daughter cell release and growth processes such as RNA and protein accumulation were followed. The amount of RNA and proteins increased stepwise with a short time interval between individual steps during which the rate of RNA and protein accumulation decreased. At each of the steps, the amount of RNA and protein approximately doubled and the number of steps increased with irradiance. At the end of each of the growth steps, a commitment to trigger the sequence of reproductive events (DNA replication, nuclear division, protoplast fission) was attained. After attaining the commitment point, the cells were able to trigger and terminate the whole reproductive sequence without any further growth, that is, even in the dark when the external supply of energy was cut off. With increasing irradiance, the number of commitment points attained during one cell cycle increased from one to four. Consequently, one to four sequences of the reproductive steps were triggered, and each of them ended by doubling the reproductive structures, which resulted in the formation of 2, 4, 8, or 16 daughter cells. The length of the precommitment periods shortened with increasing irradiance as the result of an increasing rate in growth. The length of postcommitment periods showed light independence and remained constant at the range of irradiances at which the number of growth steps and, consequently, the number of sequences of reproductive events did not change. At higher irradiances, the number of sequences of reproductive events increased, which caused a prolongation of postcommitment periods. The length of the cell cycle varied as a result of this distinct effect of irradiance on pre- and postcommitment periods.     

SECONDARY CAROTENOID ACCUMULATION IN SCENEDESMUS KOMAREKII (CHLOROPHYCEAE, CHLOROPHYTA)

As Scenedesmus komarekii Hegewald was cultured under high light intensity and nitrogen limitation, the color of cells progressed from green to brown, and finally through orange to brick red. The secondary carotenoids astaxanthin and canthaxanthin, as well as apolar carotenoids, were detected in the brown and orange cells. These carotenoids were contained in lipoidal globules that were first formed at the periphery of the cell and progressively propagated toward its inside, eventually filling most of it. The chloroplast was single and parietal in the green cells. As the cells turned brown, the chloroplast divided into several small lobes and was pushed toward the interior by the accumulating lipoidal globules. Sometimes the outer layer of the wall of the brown cell developed one or two diametrically opposed swellings. Once the cells became orange or red, neither lipoidal globules nor any major organelles were distinguishable. The cell wall in the orange cells became thick because of the formation of electron-dense granules between its outer and inner layers. The mode of the secondary carotenoid accumulation in S. komarekii differs from that of Haematococcus, an alga well known for its ability to accumulate secondary carotenoids, but resembles that of " Chlorella " zofingiensis (= Mychonastes zofingiensis (Dönz) Kalina et Punčochářová).     

SEQUENCE ANALYSIS OF THE ITS-2 REGION: A TOOL TO IDENTIFY STRAINS OF SCENEDESMUS (CHLOROPHYCEAE)

The genetic distances between several strains of Scenedesmus obliquus (Turp.) Kütz., S. acutus Hortobagyi, and S. naegelii Chod. calculated from ITS-2 sequences were found to be smaller than the genetic distances within other strains of Scenedesmus —that is, in S. acuminatus (Lagerh.) Chod. and S. pectinatus Meyen. These results confirm that the studied strains were not properly identified and should be renamed S. obliquus , as already suggested in other studies.     

PHOSPHORUS STATUS AND CYTOPLASMIC STRUCTURES IN SCENEDESMUS (CHLOROPHYCEAE) UNDER DIFFERENT METABOLIC REGIMES1

Phosphorus deficiency affects the anatomy of the unicellular green alga Scenedesmus obtusiusculus Chad. and influences the distribution of other inorganic elements in the cell in addition to phosphorus. Scenedesmus was grown under standard conditions with or without phosphorus. Cells were then cultured with phosphorus under conditions favouring glycolysis, respiration, or photophosphorylation for 2 h or photosynthesis for up to 8 h. The dominating features of phosphorus starvation, were loss of phosphorus and coions from polyphosphate bodies, accumulation of starch, decrease in the volume density of ribosomes both in the chloroplast and cytoplasm, and an increase in wall thickness. Under conditions favoring photosynthesis the mass fraction for phosphorus is low after 1 h, exceptionally high by 2 h, and diminishes by 8 h. High amounts of phosphorus are also regained under conditions favoring glycolysis and photophosphorylation but not respiration. After 2 h under photosynthetic conditions the volume densities of the chloroplast, cytoplasmic ribosomes, the vacuole, and the mitochondrion increased over controls. By 8 h the relative volume of the single ramified mitochondrion had decreased slightly and recognizable segments of it were sequestered within the vacuome. The autophagic nature of the vacuole was further evidenced by the presence of ribosomes and whorls of lamellae within it. Serial sections showed that all polyphosphate granules and sequestered materials were located within a continuous vacuolar cisterna.     

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)1

Effects of CO₂ concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low-CO₂ cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO₂ conditions (high-CO₂ cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO₂ cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low-CO₂ cells of C. vulgaris 11h, while a grana-like structure was found in high-CO₂ cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low-CO₂ cells than in high-CO₂ cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO₂ conditions, whereas CO₂-effects on lamellar stacking are species dependent.     

EFFECT OF TEMPERATURE AND IRRADIANCE ON GROWTH OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)1

The growth characteristics of Haematococcus pluvialis Flotow were determined in batch culture. Optimal temperature for growth of the alga was between 25° and 28°C, at which the specific growth rate was 0.054 h^?1. At higher temperatures, no cell division was observed, and cell diameter increased from 5 to 25 μm. The saturated irradiance for growth of the alga was 90 μmol quanta · m^?2·s^?1; under higher irradiances (e.g. 400 μmol quanta·m^?2·s^?1) astaxanthin accumulation was induced. Growth rate, cell cycle, and astaxanthin accumulation were significantly affected by growth conditions. Careful attention should be given to the use of optimal growth conditions when studying these processes.     

COMPARISON OF METHYL JASMONATE AND CADMIUM EFFECT ON SELECTED PHYSIOLOGICAL PARAMETERS IN SCENEDESMUS QUADRICAUDA (CHLOROPHYTA,CHLOROPHYCEAE)1

Effect of methyl jasmonate (MeJA) in the concentrations of 10 or 100 μM on selected physiological parameters in Scenedesmus quadricauda (Turp.) Bréb. after 24 h of exposure was studied. Results were compared with the application of general toxic metal (cadmium, Cd) to identify MeJA‐specific responses. Accumulation of reactive oxygen species (ROS; hydrogen peroxide and superoxide) was the most elevated by 10 μM MeJA and 100 μM Cd, while total chls showed decrease (Cd) and increase (MeJA) in these variants. The amount of carotenoids and cell viability were affected neither by MeJA nor by Cd application. The sum of free amino acids was considerably elevated by 10 μM Cd (increase in histidine, threonine, arginine, leucine, and lysine mainly) but depleted by 100 μM MeJA (14 from 17 compounds decreased), while accumulation of soluble proteins was unaffected by Cd and enhanced by MeJA. Cadmium application reduced the amount of Ca and also Mg in the case of 100 μM Cd, while MeJA had no effect on the content of mineral nutrients. Total Cd content reached 557 and 1,334 μg · g^?1 dry weight (dwt) in 10 and 100 μM Cd variant, respectively. Intracellular Cd uptake was ca. 55% from total Cd content in both Cd variants. The present findings are discussed in the context of the available literature, and possible explanations are suggested.