Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO₂ per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO₂ levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.

Diurnal measurements of RuBPCase (expressed in micromoles CO₂ per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO₃⁻ and Mg²⁺, but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO₃⁻ and Mg²⁺-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO₃⁻ and Mg²⁺-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.

These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO₂ and Mg²⁺.

     

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase(RuBPCase) was estimated after rapidly extracting it from intactwheat leaves pretreated under different light and CO₂ levels.No HCO₃^– was added to the extraction buffer since it isshown to inhibit RuBPCase. The activity increased as light intensityor CO₂ concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased.Light activation did not affect the affinity of RuBPCase forCO₂. A K_m of 30 µM CO₂ under air level O₂ was determined.CO₂, light and temperature are three main limiting factors ofphotosynthesis. It seems that the activity of RuBPCase is regulatedby these factors according to the requirements for CO₂ fixation.     

Effect of Carbonic Anhydrase on the Activity of Ribulose Bisphosphate Carboxylase

At concentrations of CO₂ less than saturating, carbonic anhydrase(EC 4.2.1.1 [EC] ) stimulates the carboxylation of ribulose bisphosphatecatalysed by ribulose bisphosphale carboxylase (EC 4.1.1.3 [EC] .9)in vitro. This is not through any beneficial association ofthe two enzymes but is a consequence of the increased rate ofconversion of HCO₃^– ion to CO₂, the substrate for thecarboxylation. Carbonic anhydrase should always be includedin reaction mixtures used to determine the Michaelis constantof ribulose bisphosphate carboxylase for CO₂ where fixationof radioactive CO₂ into phosphoglycerate is the basis of rateestimation. The effect is to decrease the value obtained forthe Michaelis constant.     

Ribulose Bisphosphate Carboxylase Activity and Photosynthesis during Leaf Development in the Tomato

Besford, R. T., Withers, A. C. and Ludwig, L. J. 1985. Ribulosebisphosphate carboxylase activity and photosynthesis duringleaf development in the tomato.—J. exp Bot. 36: 1530–1541. The carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenaseand of phosphoenolpyruvate carboxylase, and the light saturatedrate of net photosynthesis were measured in the developing 5thleaf of tomato plants. Values for light saturated net photosynthesiswere also calculated from the measured carboxylase activitiesand estimates of internal CO₂ and oxygen concentrations. Thecalculated rate using the activity of ribulose bisphosphatecarboxylase alone for net CO₂ assimilation in 300 mm³ dm^–3CO₂ was greater than the measured rate at 80% and full expansionbut less than the measured rate in younger leaves. When theactivities of both the carboxylases were taken into accountbetter agreement was evident for young leaves but the rate wasfurther overestimated for older leaves The calculated rate forphotosynthesis in 1200 mm³ dm^–3 CO², assuming saturationof ribulose bisphosphate carboxylase with RuBP, was an overestimatefor young leaves but was close to the observed values for leavesnear full expansion. The results are discussed in terms of measuredconductances for CO₂ and the availability of RuBP in the leaf Key words: Tomato, leaf development, photosynthesis, RuBP carboxylase, oxygenase     

External and Internal CO2 Transport in Lemanea: Interactions with the Kinetics of Ribulose Bisphosphate Carboxylase

The rate of photosynthesis by the freshwater alga Lemanea mamillosais proportional to CO₂ concentration, virtually to the pointof saturation, and inversely proportional to the radius of thethallus. By contrast, the CO₂ response curve of very thin slicesof the thallus is a rectangular hyperbola with a (lower) halfsaturation concentration of 10 mmol m^–3. For the intactplant, the kinetics of CO₂ fixation are strongly masked by internalCO₂ transport limitations, although the maximum rate of photosynthesisis probably determined by the rate of supply of ribulose bisphosphate(RuBP). The flow of water over the alga becomes turbulent atwater velocities greater than about 90 mm s^–1 and thethallus stretches significantly at higher water velocities.In its natural habitat, therefore, the external unstirred layerwill be thin (< 10 µm) and the thallus will be stretched,leading to rapid external and increased internal rates of CO₂transport from the bulk solution. The estimated maximum rateof CO₂ transport is commensurate with the maximum rate of photosynthesis(i.e. the rate of supply of RuBP). Key words: Transport limitations, Kinetics of CO₂ fixation     

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO₂ (1200 microliters per liter) than in ambient CO₂ (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO₂. Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO₂. The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO₂ compared with 50% in the ambient atmosphere. While CO₂ enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO₂.     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.     

Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates

Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO₂ per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO₂ exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO₂-enrichment and drought stress reduced the total (HCO₃⁻/Mg²⁺-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K_m(CO₂) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO(2) Exchange

The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO₂ assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light-mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed.     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Photosynthesis by Flag Leaves of Wheat in Relation to Protein, Ribulose Bisphosphate Carboxylase Activity and Nitrogen Supply

The effects of nitrate supply on the composition (cell numbers,protein and chlorophyll contents) of flag leaves of winter wheatgrown with two amounts of N fertilizer and of spring wheat grownin the glasshouse under controlled nitrate supply are describedand related to photosynthesis. Nitrogen deficiency decreasedthe size of leaves, mainly by reducing cell number and, to asmaller extent, by decreasing cell volume. Protein content perunit leaf area, per cell and per unit cell volume was largerwith abundant N. Total soluble protein, ribulose bisphosphatecarboxylase-oxygenase (RuBPc-o) protein and chlorophyll changedin proportion irrespective of nitrogen supply and leaf age.Photosynthesis per unit area of flag leaf and carboxylationefficiency in both winter and spring wheat were proportionalto the amount of total soluble protein up to 7.0 g m^–2and to the amount of RuBPc-o protein up to 4.0 g m^–2.However, photosynthesis did not increase in proportion to theamount of total soluble or RuBPc-o protein above these amounts.In young leaves with a high protein content the measured ratesof photosynthesis were lower than expected from the amount andactivity of RuBPc-o. Carboxylation per unit of RuBPc-o protein,measured in vitro, was slightly greater in N-deficient leavesof winter wheat but not of spring wheat. RuBPc-o activity perunit of RuBPc-o protein was similar in winter and spring wheatleaves and remained approximately constant with age, but increasedin leaves showing advanced senescence. RuBPc-o protein fromN-deficient leaves migrated faster on polyacrylamide gels thanprotein from leaves with high N content. Regulation of the rateof photosynthesis in leaves and chloroplasts with a high proteincontent is discussed. The conductance of the cell to the fluxof CO₂ from intercellular spaces to RuBPc-o active sites iscalculated, from cell surface areas and CO₂ fluxes, to decreasethe CO₂ partial pressure at the active site by less than 0.8Pa at an internal CO₂ partial pressure of 34 Pa. Thus the decreasein partial pressure of CO₂ is insufficient to account for theinefficiency of RuBPc-o in vivo at high protein contents. Otherlimitations to the rate of photosynthesis are considered. Key words: Wheat, photosynthesis, nitrogen, ribulose, bisphosphate carboxylase     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

A Non-radioisotopic Anion-exchange Chromatographic Method to Measure the CO2/O2 Specificity Factor for Ribulose Bisphosphate Carboxylase/Oxygenase

A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/ oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: ν_c = ([3-PGA] – [PG])/2 and ν_o = [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.     

Relation between Ribulose Bisphosphate Carboxylase Content and Chloroplast Number in Naturally Senescing Primary Leaves of Wheat

The amounts of ribulose bisphosphate carboxylase protein decreasedrapidly with leaf age to a low content by the middle stage ofsenescence. In contrast, the decrease in chloroplast numberwas slight during the same period. This indicates that the enzymecan be degraded within the chloroplast before the chloroplastsdisintegrate during senescence. (Received September 3, 1983; Accepted November 24, 1983)     

Changes of Ribulose Bisphosphate Carboxylase/Oxygenase Content, Ribulose Bisphosphate Concentration, and Photosynthetic Activity during Adaptation of High-CO(2) Grown Cells to Low-CO(2) Conditions in Chlorella pyrenoidosa

Changes of some photosynthetic properties of high-CO₂ grown cells of Chlorella pyrenoidosa during adaptation to low-CO₂ conditions have been investigated. The K_m value of photosynthesis of the high-CO₂ grown cells for dissolved inorganic carbon was 3.3 millimolar and decreased to 25 to 30 micromolar within 4 hours after transferring to air. In the presence of saturating CO₂ concentrations the photosynthetic activity of the high-CO₂ grown cells was 1.5 times as high as that of the low-CO₂ grown cells. There was a significant rise of the photosynthetic activity during adaptation of the high-CO₂ grown cells to air, followed by a steady decrease. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in both the high- and low-CO₂ grown cells was close to the photosynthetic activity of the cells. The concentration of ribulose 1,5-bisphosphate (RuBP) was higher in the low-CO₂ adapting and low-CO₂ grown cells than in the high-CO₂ grown cells regardless of the photosynthetic rate. This seems to be due to an increased RuBP regeneration activity during adaptation followed by maintenance of the new higher concentration. The RuBP level always exceeded the concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP binding sites in both the high- and low-CO₂ grown cells at any dissolved inorganic carbon concentration.     

Changes in Contents of Adenine Nucleotides and Development of Ribulose Bisphosphate Carboxylase Activity During Shooting of Mulberry Saplings

At the onset of budding in mulberry saplings (Morus alba L.,cv. Shin-ichinose), the ATP, ADP and carbohydrate contents beganto decline rapidly. This decline continued until RuBPCase activitybegan during the development of the leaves. The concentrationsof these constituents and the value for the adenylate energycharge, though partially restored, were lower than the initialvalues even eight weeks after planting. (Received March 7, 1983; Accepted May 25, 1983)     

Molecular Evolution of the Small Subunit of Ribulose Bisphosphate Carboxylase: Nucleotide Substitution and Gene Conversion

The nucleotide sequences encoding the mature portion of 31 ribulose 1.5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is approximately 6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species.