Factors affecting akinete differentiation in Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria)

1. Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium which can produce akinetes (reproductive spores) that on germinating can contribute to future populations. To further understand factors controlling the formation of these specialised cells, the effects of diurnal temperature fluctuations (magnitude and frequency), in combination with different light intensities and phosphorus concentrations were investigated under laboratory conditions. 2. Akinete differentiation was affected by the frequency of temperature fluctuations. Maximum akinete concentrations were observed in cultures that experienced multiple diurnal temperature fluctuations. 3. Akinete concentrations increased with increasing magnitude of temperature fluctuation. A maximum akinete concentration was achieved under multiple diurnal temperature fluctuations with a magnitude of 10 °C (25 °C to 15 °C). 4. A fourfold increase in light intensity (25–100 μmol m^?2 s^?1) resulted in an approximate 14‐fold increase in akinete concentration. 5. High filterable reactive phosphorus (FRP) concentrations (>70 μg L^?1) in the medium, combined with a multiple diurnal temperature fluctuation of 10 °C, supported the development of the highest akinete concentration.     

Potassium deficiency triggers the development of dormant cells (akinetes) in Aphanizomenon ovalisporum (Nostocales,Cyanoprokaryota)1

Akinetes are spore‐like nonmotile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Various environmental factors were reported to trigger the differentiation of akinetes including light intensity and quality, temperature, and nutrient deficiency. Here, we report that deprivation of potassium ion (K⁺) triggers akinete development in the cyanobacterium Aphanizomenon ovalisporum. Akinetes formation is initiated 3 d–7 d after an induction by K⁺ depletion, followed by 2–3 weeks of a maturation process. Akinete formation occurs within a restricted matrix of environmental conditions such as temperature, light intensity or photon flux. Phosphate is essential for akinete maturation and P‐limitation restricts the number of mature akinetes. DNA replication is essential for akinete maturation and akinete development is limited in the presence of Nalidixic acid. While our results unequivocally demonstrated the effect of K⁺ deficiency on akinete formation in laboratory cultures of A. ovalisporum, this trigger did not cause Cylindrospermopsis raciborskii to produce akinetes. Anabaena crassa however, produced akinetes upon potassium deficiency, but the highest akinete concentration was achieved at conditions that supported vegetative growth. It is speculated that an unknown internal signal is associated with the cellular response to K⁺ deficiency to induce the differentiation of a certain vegetative cell in a trichome into an akinete. A universal stress protein that functions as mediator in K⁺ deficiency signal transduction cascade, may communicate between the lack of K⁺ and akinete induction.     

Phosphorus deficiency,nitrogen assimilation and akinete differentiation in the cyanobacteriumAnabaena torulosa

Anabaena torulosa is unable to fix N₂ and to differentiate akinetes in a P-deficient nitrate-free medium. In a P-deficient medium with nitrate, the NO₃ ^? assimilation and period of akinete differentiation are of the same order of magnitude as in a P-containing nitrate medium. It is suggested that regulation of akinete differentiation in P-deficient organism proceeds through the regulation of the N-assimilating system. At the time of akinete differentiation, cellular P is excreted into the medium which leads to a decrease of cellular P.     

The influence of light quality on akinete formation and germination in the toxic cyanobacterium Anabaena circinalis

Physiological control of akinete formation and subsequent germination is likely to be important in understanding and predicting how natural populations of cyanobacteria respond to their environment. While previous research has indicated nutrient limitation may be important in akinete formation new results presented here indicate that in the toxic and bloom-forming species Anabaena circinalis there was a profound effect of spectral quality. Under 40 μmol photons m^?2 s^?1 photosynthetically active irradiance (PAR) of predominately red irradiance akinete production was maximal at 2.1 × 10^?4 akinetes vegetative cell^?1 d^?1, some 3000 times greater than the 6.5 × 10^?8 akinetes vegetative cell^?1 d^?1 observed under equivalent PAR but predominately blue light. For cells grown under a range of predominantly red, white and green irradiance even short exposures to blue light reduced akinete formation rates by a factor of ten relative to controls, indicating that exposure to blue light inhibits akinete formation. Germination of akinetes was not influenced by the irradiance under which akinetes were formed: 88 ± 4.1% (mean ± 1 S.D.) of akinetes germinated with no evidence of an effect on germination success due to their production under predominately red, white or green irradiance (germination of akinetes produced under blue light was not tested). Spectral quality had a significant impact on both vegetative cell and germling growth rates. The results indicate a significant reduction in the cellular differentiation of A. circinalis vegetative cells into akinetes that is mediated by blue light. In an ecological context the production of akinetes will be greater in environments with less blue light; potentially including those with slower flow, more stratification, less vertical mixing and more turbidity. The resulting spatial pattern of akinete production is likely to influence the location of akinetes in sediments and the development of subsequent blooms from excysting germlings.     

Potential triggers of akinete differentiation in Nodularia spumigena (Cyanobacteriaceae) isolated from Australia

Nodularia spumigena, like many cyanobacteria, produces specialised reproductive structures, known as akinetes, which are believed to allow survival under unfavourable conditions. This study investigated the effects of salinity, nitrogen and phosphorus concentration at two irradiances on akinete differentiation in a N. spumigena isolate from the Gippsland Lakes, Victoria, Australia. A computer image analysis program was used to photograph filaments and assess production of akinetes over time in separate experiments for each environmental parameter. Heterocyst production and cell morphology were also examined. The results suggest that akinete production increases over time. Production of akinetes is further increased at low and high salinities and with the addition of nitrate. Higher irradiance increases akinete differentiation, although in combination with different phosphorus concentrations causes varied effects. The development and sedimentation of akinetes may provide an inoculum for reoccurring blooms. Heterocysts were only observed during experiments with varying salinity and nitrogen exposures. Light quantity appeared to play a large role in heterocyst production. The ability of N. spumigena to produce akinetes and heterocysts is likely to be part of the reason for its success and continual occurrence in estuarine environments low in nitrogen, such as the Gippsland Lakes, Victoria, Australia. Factors known to reduce heterocyst and akinete production will provide new insight to possible management controls for this species.     

AKINETE PRODUCTION OF ANABAENA LEMMERMANNII AND A. CYLINDRICA (CYANOPHYCEAE) IN NATURAL POPULATIONS OF N‐ AND P‐LIMITED COASTAL MESOCOSMS1

A strong biomass increase of two Anabaena species was observed in natural plankton community enclosed into nine large mesocosms (51 m³) and manipulated with mineral nutrients and an organic carbon source during a 3‐week period in the coastal Baltic Sea. The water column and settled material from the bottom of the mesocosms were sampled at 2‐day intervals. Planktonic populations of Anabaena lemmermannii Richter and A. cylindrica Lemmermann and sedimentation rates of akinetes to the bottom were quantified. Comparing mesocosms with artificially induced nitrogen and phosphorus limitation, we found that during the third week of the experiment, the population size of A. lemmermannii was clearly higher in nitrogen‐limited units (by a factor of 2.4), whereas the production rate of akinetes was higher in the phosphorus‐limited units (by a factor of 2.5). Input of freshly produced A. lemmermannii akinetes to the benthos was on average 15 × 10⁶ and 6 × 10⁶ cells· m^?2·d^?1 in the P^? and N^? limited mesocosms, respectively. Our estimates of specific akinete production rate of A. lemmermannii in P^? and N^? limited mesocosms revealed an even larger divergence (a factor of 5.5), being on average 2.4 and 0.4 akinetes·10^?3 vegetative cells^?1·d^?1, respectively. The phosphorus addition effectively reduced akinete production of A. lemmermannii. Differences in the nutrient manipulation had no apparent effect on the biomass and akinete production of A. cylindrica. The akinete production pattern of A. cylindrica revealed a 1‐week delay compared with the vegetative population peak, whereas such a delay was not obvious in A. lemmermannii.     

Characterization of akinete differentiation in the cyanobacterium Anabaena azollae

Differentiation of akinetes was investigated in the filamentous cyanobacterium Anabaena azollae Stras. In this organism all pre-existing vegetative cells are capable of developing into akinetes. Standard sporulation medium (SSM) was used to synchronously induce the formation of akinetes, while cultures in Allen and Arnon (AA/8) medium were used as controls.This paper describes the changes in photosynthetic pigments and total soluble proteins in these cultures over a 25-day period encompassing akinete differentiation. Heterocyst frequencies and nitrogenase activity were also monitored during the same period in both media. SDS-PAGE results indicated that specific proteins were synthesized in a manner correlated with akinete differentiation. The results demonstrate that in cultures undergoing akinete development, some of the photosynthetic pigments are maintained, nitrogen-fixation and heterocyst differentiation are suppressed, and the cells synthesize a variety of specific proteins.     

Cyanobacterial akinete induction and its application as biofertilizer for rice cultivation

Nostoc sp. VICCR1-1 was induced in order to form akinetes on the basis of nutrient modification. Phosphorus and iron were found to be the critical for akinete differentiation, especially when both elements were omitted. The number of akinete cells increased up to 20% when compared with culturing in BG11₀ medium (without N source). In addition, CaCl₂ played a role in heterocyst differentiation, and was able to induce heterocyst ranging between 30% and 46%. In order to prepare akinetes as inoculum, the dried form of akinetes was prepared by mixing it with montmorillonite clay. The inoculum with the amount of 2.8 × 10⁶ cells m⁻² was applied to rice (Oryza sativa) fields. After harvesting, the grain yields from chemical N fertilizer, vegetative cells, and akinete inoculum treatments were not significantly different. To monitor the persistence of Nostoc sp. VICCR1-1 after harvesting, the most probable number-denaturing gradient gel electrophoresis technique using 16S rRNA gene was employed. The results indicated that the remaining population is at 2.5 × 10⁵ and 1.62 × 10⁶ cells m⁻² in treatments supplied with vegetative cells and akinete inocula, respectively. Akinete induction might be one of the appropriate approaches for producing cyanobacterial inoculum.     

Environmental influences on akinete germination of Anabaena circinalis and implications for management of cyanobacterial blooms

Certain cyanobacteria, including the noxious bloom-forming species Anabaena circinalis Rabenhorst, produce thick-walled reproductive structures (akinetes) which may serve as a resting stage and ensure survival during adverse growth conditions. The effect of certain environmental variables (temperature, salinity and desiccation) on akinete germination of A. circinalis was investigated under laboratory conditions, to determine the conditions under which germination was inhibited. The overall aims were to provide a broader understanding of the life history and ecology of this species and to assess suppression of akinete germination as a potential management strategy for control of cyanobacterial blooms in the lower Murray River, Australia. The results indicated a marked threshold of temperature and salinity tolerance for germination of A. circinalis, but the latter was not within a range that could be successfully manipulated in a natural ecosystem. However, it was found that desiccation of akinetes for moderately short periods can significantly impair their capacity to germinate. It is, therefore, speculated that allowing periodic drying of shallow wetlands adjacent to the Murray River and in other areas may reduce the size of the inoculum for population growth by reducing viability of akinetes in surface sediments.     

PHOTOSYNTHETIC CHARACTERIZATION OF DEVELOPING AND MATURE AKINETES OF APHANIZOMENON OVALISPORUM (CYANOPROKARYOTA)1

Akinetes, differentiated resting cells produced by many species of filamentous, heterocystous cyanobacteria, enable the organism to survive adverse conditions, such as cold winters and dry seasons, and to maintain germination capabilities until the onset of suitable conditions for vegetative growth. Mature akinetes maintain a limited level of metabolic activities, including photosynthesis. In the present study, we have characterized changes in the photosynthetic apparatus of vegetative cells and akinetes of the cyanobacterium Aphanizomenon ovalisporum Forti (Nostocales) during their development and maturation. Photosynthetic variable fluorescence was measured by microscope‐PAM (pulse‐amplitude‐modulated) fluorometry, and the fundamental composition of the photosynthetic apparatus was evaluated by fluorescence and immunological techniques. Vegetative cells and akinetes from samples of Aphanizomenon trichomes from akinete‐induced cultures at various ages demonstrated a gradual reduction, with age, in the maximal photosynthetic quantum yield in both cell types. However, the maximal quantum yield of akinetes declined slightly faster than that of their adjacent vegetative cells. Mature akinetes isolated from 6‐ to 8‐week‐old akinete‐induced cultures maintained only residual photosynthetic activity, as indicated by very low values of maximal photosynthetic quantum yields. Based on 77 K fluorescence emission data and immunodetection of PSI and PSII polypeptides, we concluded that the ratio of PSI to PSII reaction centers in mature akinetes is slightly higher than the ratio estimated for exponentially grown vegetative cells. Furthermore, the cellular abundance of these protein complexes substantially increased in akinetes relative to exponentially grown vegetative cells, presumably due to considerable increase in the biovolume of akinetes.     

AKINETE GERMINATION IN ANABAENA CIRCINALIS (CYANOPHTA)

Akinetes of a clonal culture of Anabaena circinalis Rabenhorst from Mt. Bold reservoir (eutrophic), South Australia, were isolated and the effects of light, phosphorus, and nitrogen availability on their germination were investigated. Light was required but there was no significant difference in percentage of germination after 72 h if akinetes were incubated in ASM-1 medium at irradiances of 15, 30, or 50 μmol.m^-2.s^-1. Maximum akinete germination occurred by 48 h. Nitrogen was not required, as 88% of akinetes germinated in the flasks without combined nitrogen added to the medium and without N₂ in the air. NH₄⁺-N at 28 mg N.L^-1 completely suppressed germination, whereas 28 mg NO₃ N.L^-1 had no effect relative to the controls without nitrogen. Phosphorus was required, and at 48 h percentage of germination in the flasks with 0.6 mg P.L^-1 added (78%) was significantly greater than in the flasks with 0.06 P.L^-1 (58%) and 0 mgP.L^-1 (24%) added. Germlings in the 0 mg P.L^-1 flasks were only 2–4 cells long and stunted in appearance, whereas germlings at all other P concentrations were 8–16 cells long. It is likely that the isolation process exposed some akinetes to intracellular phosphorus released from lysing vegetative cells, but this was insufficient to allow normal development in the 0 mg P.L^-1 flasks. The plot of percentage of germination vs. initial phosphorus concentration, in the medium showed a relationship analogous to Michaelis-Menten nutrient uptake kinetics, suggesting that a specific membrane-bound enzyme system(s) is involved, with phosphorus as the substrate. The half saturation value (K_S) for germination was 50 μg P.L^-1.     

Long-term observations on Anabaena circinalis Rabenhorst (Cyanophyta)

Field observations on Anabaena circinalis Rabenhorst over six summer seasonal appearances in two dams have shown that the frequency of occurence of heterocysts became fairly constant soon after the appearance of the species and decreased just before the end of the growing season. By contrast, akinete frequency reached a maximum, very early in the season, then decreased rapidly. Drought led to a decrease in occurrence of both Anabaena and of akinetes in several dams; this was possibly associated with an increasing concentration of NOx in the water. At Carcoar dam this akinete reduction was shown first in end-of-season figures. Drought also led to an end-of-season decrease there in the occurrence of heterocysts. Variations in morphology were noted. The coiling of the trichome, shape of akinete and relative position of heterocyst were all variable, although these characters are often assumed to be of taxonomic importance.     

Seasonal dynamics of akinetes of Anabaena flos-aquae in bottom sediments and water column of small Siberian reservoir

Seasonal dynamics of Anabaena flos-aquae (Lyngb.) Breb., including vegetative cells, akinetes and akinete envelopes, in bottom sediments and water column at both littoral and deeper central stations of a small Siberian reservoir was studied. Two types of akinetes were observed: in the first half of summer Anabaena formed akinetes, which served for vegetative reproduction and germinated in water column soon after differentiation, while in the second half of summer the akinetes produced served as a resting stages, which were deposited to bottom sediments. Canonical correlation analyses revealed that decrease of water temperature was the main environmental factor that stimulated the akinete formation. In contrast to the general opinion, concentration of inorganic phosphorus slightly, but positively influenced the akinete formation. Thus, akinetes formed in response to the temperature decrease, needs a certain level of this nutrient. At littoral and open-water stations abundance and seasonal dynamics of akinetes in water column and their sinking pattern were very similar. However, seasonal dynamics of abundance of akinetes in sediments in these two reservoir locations differed: whereas the abundance of akinetes in open water increased permanently during the summer, that in the littoral decreased soon after their sedimentation. The cause for decrease in abundance of akinetes in bottom sediments in winter is unknown.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

AKINETE FORMATION IN PLANKTONIC ANABAENA SPP. (CYANOBACTEFUA) BY TREATMENT WITH LOW TEMPERATURE1

The effects of temperature, light intensity and nutrient depletion on akinete formation in seven strains of planktonic Anabaena spp.: A. mucosa TAC426; A. crassa TAC436; A. spiroides TAC443 and TAC444; A. flosaquae TAC446; and A. ucrainica TAC448 and TAC449 were examined. A Marked Pfft of temperature on akinete formation was observed at 40 μmol photons·m^?2·sec^?1 and nutrient-sufficient conditions. At 20° C, akinetes did not develop in A. mucosa TAC426, A. crassa TAC436, A. spiroides TAC443, A. flos-aquae TAC446, or A. ucrainica TAC449 but were formed at frequencies of a little over 11% (ratio of filaments with akinetes to total filaments) in A. spiroides TAC444 and A. ucrainica TAC448. None of the strains fmd akinetes or heterocysts at 30° C and 35° C. At lower temperature (10° C and 15° C), akinetes developed in all the strains at maximum frequencies of 13.4–77.4% during the late exponential phase or late exponential to stationary phases of growth. With only one exception, low light or nutrient deletion did not lead to the induction of akinete diferentiation at 20° C. Only akinete formation in A. flosaquae TAC446 was induced by nitrogen deletion with a frequency of 12.1%, similar to that induced by low temperature, but the initiation of akinete formation in the strain was delayed compared to treatment with low temperature. These results show that temperature was the most important environmental factor triggering akinete formation in these species. In A. crassa TAC436 and A. spiroides TAC443 and TAC444, akinetes developed during the late exponential growth phase even though heterocysts were formed at a 100% frequency (ratio of filaments with heterocysts to total filaments) throughout the entire growth phase. In A. mucosa TAC426, A. flos-aquae TAC446, and A. ucrainica TAC448 and TAC449, there was a positive correlation between heterocyst and akinete formation, suggesting that the presence of a heterocyst may play a role in akinete formation.     

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.     

Phosphorus sorption characteristics of sediment in the Simiyu and Kagera River basins: implications for phosphorus loading into Lake Victoria

As part of a larger study to assess the influence of land use on riverine and atmospheric phosphorus (P) loading to Lake Victoria, P sorption characteristics of eight composite bottom sediment samples from the Simiyu and Kagera rivers were determined using the Langmuir equation. The samples had low to medium Langmuir adsorption maxima (Γ_m), ranging from 107 to 201μg g^?1. Langmuir binding energy co-efficient (K) ranged from 60 to 181μg l^?1 and the equilibrium P concentration at zero sorption (EPC₀) from 0.1 to 2.75μg g^?1. By using Langmuir co-efficients derived from P sorption experiments and soluble reactive phosphorus (SRP) concentrations measured in rivers as well as the in-shore waters of Lake Victoria, it was possible to determine the potential release of SRP into the lake by sediment from the two catchments. For the 2000 water-year, it was estimated that about 28.65 ± 0.89 (mean ± SD) and 66 ± 6.76 tons of SRP were released into Lake Victoria by sediment deposited by the Simiyu and Kagera rivers, respectively. The implications of these results to future management of cultural eutrophication in Lake Victoria are also discussed.     

Effect of nutrients and aeration on O2 evolution and photosynthetic pigments ofAnabœna torulosa during akinete differentiation

The addition of a nitrogen (nitrate) and carbon sources (acetate, citrate and fructose) and phosphate deficiency (nitrate medium deficient in phosphate) under unaerated conditions induced akinete differentiation inAnabœna torulosa. Aerated cultures of this organism in these nutrients did not differentiate akinetes. Oxygen evolution by aerated cultures was higher when compared to unaerated cultures, which concurred with high chlorophyll content of aerated cultures. Nitrate nitrogen supported high phycocyanin content in unaerated cultures, phycocyanin and allophycocyanin contents were low under aerated conditions. The contents of phycocyanin, allophycocyanin, phycoerythrin and carotenoids gradually decreased at the mature akinete phase. Under aerated conditions, chlorophyll content rose and the content of all the pigments increased with the growth rate of the organism.     

Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.     

Phosphorus and light colimit periphyton growth at subsaturating irradiances

1. This study investigated the combined effects of light and phosphorus on the growth and phosphorus content of periphyton. To investigate the potential for colimitation of algal growth by these two resources, diatom‐dominated periphyton communities in large flow‐through laboratory streams were exposed under controlled conditions to simultaneous gradients of light and phosphorus. 2. Periphyton growth rate was predictably light‐limited by the subsaturating irradiances (12–88 μmol photons m^?2 s^?1) used in this experiment. However, phosphorus concentration also limited growth rate: growth increased hyperbolically with increasing soluble reactive phosphorus (SRP), reaching a threshold of growth saturation between 22 and 82 μg L^?1. 3. Periphyton phosphorus content was strongly and nonlinearly related with SRP, reaching a maximum at 82 μg L^?1 SRP. Contrary to the Light : Nutrient Hypothesis, periphyton phosphorus content did not decrease with increasing light, even at the lowest concentrations of SRP. Periphyton phosphorus was highly correlated with periphyton growth rate (Spearman's ρ = 0.63, P < 0.005). 4. Multiple regression analysis reinforced evidence of simultaneous light and phosphorus limitation. Both light and periphyton phosphorus content were significant variables in multiple regressions with growth parameters as dependent variables. Light alone accounted for 67% of the variance in periphyton biomass, and the addition of periphyton phosphorus as an additional independent variable increased the total amount of variance explained to 81%. 5. Our results did not support the hypothesis that extra phosphorus is required for photoacclimation to low light levels. Rather, the effect of additional phosphorus may have been to accommodate increased requirements for P‐rich ribosomal RNA when growth was stimulated by increased light. The potential colimitation of periphyton growth by phosphorus and light at subsaturating irradiances has important implications in both theoretical and applied aquatic ecology.