JXB Advance Access originally published online on November 1, 2005
Journal of Experimental Botany 2005 56(422):3121-3127; doi:10.1093/jxb/eri309
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Physiological and molecular diversity of feather moss associative N2-fixing cyanobacteria
1UPSC, Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden
2Department of Forest Vegetation Ecology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden
3Department of Ecosystem and Conservation Sciences, The University of Montana, Missoula, MT 59812, USA
* To whom correspondence should be addressed. Fax: +46 90 7866676. E-mail: Francesco.Gentili{at}plantphys.umu.se
Received 3 June 2005; Accepted 9 September 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Cyanobacteria colonizing the feather moss Pleurozium schreberi were isolated from moss samples collected in northern Sweden and subjected to physiological and molecular characterization. Morphological studies of isolated and moss-associated cyanobacteria were carried out by light microscopy. Molecular tools were used for cyanobacteria identification, and a reconstitution experiment of the association between non-associative mosses and cyanobacteria was conducted. The influence of temperature on N2 fixation in the different cyanobacterial isolates and the influence of light and temperature on N2-fixation rates in the moss were studied using the acetylene reduction assay. Two different cyanobacteria were effectively isolated from P. schreberi: Nostoc sp. and Calothrix sp. A third genus, Stigonema sp. was identified by microscopy, but could not be isolated. The Nostoc sp. was found to fix N2 at lower temperatures than Calothrix sp. Nostoc sp. and Stigonema sp. were the predominant cyanobacteria colonizing the moss. The attempt to reconstitute the association between the moss and cyanobacteria was successful. The two isolated genera of cyanobacteria in feather moss samples collected in northern Sweden differ in their temperature optima, which may have important ecological implications.
Key words: Acetylene reduction assay (ARA), Calothrix, cyanobacteria, moss, N2 fixation Nostoc, Stigonema
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The feather moss, Pleurozium schreberi, has recently been shown to form an association with the N2-fixing cyanobacteria, Nostoc sp. (DeLuca et al., 2002
). However, there is currently little that is understood regarding the physiology of this association. P. schreberi is ubiquitous across the boreal forest region of the world (Carleton and Read, 1991) where carpets of this moss can account for up to 90% of total ground cover; furthermore P. schreberi is one of the most common mosses on earth (DeLuca et al., 2002). This broad global distribution highlights the ecological importance of the P. schreberi–cyanobacteria association. The presence of this association at low latitudes has received no attention. However, the occurrence of feather mosses is far less in temperate regions and N deposition (a phenomenon more prevalent at lower, urban-dominated latitudes) clearly reduces N2 fixation by this association (Zackrisson et al., 2004). This N2-fixing association has a great impact on the N cycle of mature boreal forests (Zackrisson et al., 2004). In addition, it is generally believed that the majority of reduced organic and inorganic N in boreal forest ecosystems originates from cyanobacteria, either free-living or symbiotic in lichens, or in association with bryophytes (Weber and Van Cleve, 1981; Alexander and Billington, 1986; Cleveland et al., 1999; DeLuca et al., 2002). Therefore, it is of great importance to discover if it is possible to reconstitute the moss and the cyanobacteria after the isolation of the latter (Enderlin and Meeks, 1983).
The effects of abiotic factors on N2 fixation in mosses have been studied at the vegetative community level at high latitudes (Alexander et al., 1974; Dickson, 2000; Rastetter et al., 2001; Zielke et al., 2002). These studies are of great ecological value for the entire N2-fixing community; however, they do not reveal which organisms are most affected by the abiotic factors actually considered. An early study has shown a clear influence of abiotic factors, such as light and temperature, on N2-fixation in some mosses such as Sphagnum sp. and Drepanocladus exannulatus in subarctic mire (Basilier et al., 1978). Basilier et al. (1978) showed that, in the mosses Sphagnum riparium and Drepanocladus exannulatus, N2 fixation was light-dependent and had peaks around 16 °C and 11 °C, respectively. However, in the cited work only a part, and not the entire, moss shoot was tested for N2 fixation; furthermore only one abiotic factor was changed at the time and no cyanobacteria were isolated.
Although, N2 fixation in the P. schreberi–cyanobacteria association has been clearly demonstrated (DeLuca et al., 2002; Zackrisson et al., 2004), there is no information available regarding the taxonomy and physiology of the associated cyanobacteria. Pandey et al. (1992) have shown that the moss communities of an Antarctic oasis can be colonized by different species of N2-fixing and non-N2-fixing cyanobacteria. However, in this study, the activity of the N2-fixing cyanobacteria community was detected only at one temperature and the cyanobacteria were not isolated. In Sphagnum sp. moss from Swedish mires N2 fixation was found to be related mainly to the presence of intracellular Nostoc sp. filaments; even in this work no cyanobacteria were isolated (Granhall and Hofsten, 1976). Although, some cyanobacteria have been characterized with DNA-fingerprinting techniques (Paulsrud and Lindblad, 1998; Rasmussen and Svenning, 1998), most were in symbioses with lichen, Gunnera, and Cycas and a few in association with hornwort (West and Adams, 1997).
The purpose of the work reported was to investigate the physiological and morphological characteristics of cyanobacteria that live in association with feather mosses in northern Europe. The specific objectives of this work were (i) to isolate and characterize the N2-fixing cyanobacteria that colonize the leaves of P. schreberi; (ii) to investigate the combined influence of light and temperature on N2 fixation in the feather moss and the influence of temperature in the cyanobacterial isolates in pure culture; and (iii) to demonstrate reconstitution of the association between non-N2-fixing mosses and cyanobacterial isolates.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Isolation of cyanobacteria and reconstitution of the association
Samples of P. schreberi were collected from a late post-fire succession of boreal forest at the Reivo natural forest preserve in northern Sweden (65°46'65'' N, 19°05'71'' E; for site characteristics see the Ruttjeheden site; Zackrisson et al., 2004
). Ten leaves from each shoot in a total of five shoots were placed on sterile agar plates with BG11 (N free) medium (Rippka et al., 1979). The plates were incubated at 22 °C with a light regime of 50 µE m–2 s–1. The plates were stored in a constant temperature and light chamber for 1 month after which cyanobacteria colonies were visible and could be transplanted subsequently three times prior to microscopy and isolation of DNA. The isolated cyanobacteria were collected and five loops (10 µl each) separately transferred from the BG11 media to five individual shoots of the moss collected from a site at Reivo (Reivo site 1), which had no visible cyanobacteria present and had consistently not shown any nitrogenase activity over a 3 year period. The inoculated moss shoots were then grown at 22 °C in sterile tubes with the addition of sterile deionized water. Reconstitution was evaluated by the use of ARA (acetylene reducing activity), and measurements were performed on the reconstituted moss 2 weeks after inoculation, as described below.
Moss and cyanobacteria used for N2 fixation
Mosses were collected from the same site as described above at the beginning of May and then kept in 50 ml sterile tubes containing sterile deionized water in a growth chamber at 22 °C with 17 h of light at 30–40 µE m–2 s–1 and 7 h of darkness for a few weeks before the experiments started. The cyanobacteria were grown on sterile agar plates with BG11 medium without N for 1 month at 20 °C with 17 h of light at 50–60 µE m–2 s–1 and 7 h of darkness.
Morphology of free-living and associated cyanobacteria, colonization study
Light microscopy was used to aid in the identification of the isolated cyanobacterial strains in accordance with Rippka et al. (1979). The strains isolated as above described were individually observed under the microscope (Olympus BX 60 light microscope, Olympus, Japan). Cyanobacteria were also observed in association with the moss P. schreberi used to measure N2 fixation. The colonization, expressed as the percentage (%) of moss stems having cyanobacteria, was determined by randomly selecting two stems per shoot in three replicates (every replicate had five shoots) for a total of 30 stems for each treatment. The stems were mounted in water and observed under the microscope. Photographs of the cyanobacteria were taken with an Axiocam camera (Zeiss, Jena, Germany) mounted on an Axioplan 2 microscope (Zeiss, Jena, Germany) and were adjusted using Adobe Photoshop 7.0 software (Adobe, San Jose, Ca, USA, 2002).
Isolation of DNA, PCR amplification and sequence analysis
Cyanobacteria DNA was isolated according to Lind et al. (1985) except that no lysozyme was added, rather the cells were disrupted by freezing and thawing in TE-buffer with the addition of 200 µl 10% SDS and 20 µl 3 M NaAc. Thereafter the cells were placed in liquid N2 and ground with a mortar and pestle. The cells were also treated with RNA:se A (10 µg ml–1) in 37 °C for 30 min. Approximately 60 ng of DNA was isolated from each sample. To study differences between cyanobacterial genera and species, it has been demonstrated that the intron in the tRNALeu (UAA) gene is suitable (Xu et al., 1990).
A nested PCR was performed on a PC 960G Thermal cycler. Reaction mixtures of 100 µl contained DNA, 1x Taq buffer, and 1 U Taq DNA polymerase. All PCR reactions were performed as described by Paulsrud and Lindblad (1998) using 30 cycles (with annealing temperature of 55 °C for the first four cycles and 60 °C for the 26 next cycles) as described in Sellstedt et al. (1992). The primers used were designed by Jeff Elhai (Department of Biology, University of Richmond, Richmond, VA USA) and were outer primers 5'-GGAATTCGGGGRTRTGGYGRAAT-3' and 5'-TCCCGGGRYRGRGGGACTT-3', while the inner primers were 5'-AGAATTCGGTAGACGCWRCGGACTT-3' and 5'-ACCCGGGTWTACARTCRACGGATTTT-3', respectively. The PCR-product was diluted 1000-fold in sterile water between the first reaction with the outer primers and the second reaction with the inner primers. The reactions were run three times for each sample.
The achieved fragment was approximately 300 kb and was purified with the QIAquick PCR purification kit (Qiagen, GmbH, D-40724 Hilden) according to the manufacturer's description. The purified fragment was then run in a sequencing reaction with 200 fmol of template and 10 pmol of primer 5'-AGAATTCGGTAGACGCWRCGGACTT-3' in a reaction (95 °C, 20 s; 50 °C, 15 s; 60 °C, 1 min in 28 cycles) in an ABI 377 sequencing apparatus (Applied Biosystems, USA).
The obtained sequences in this study were blasted in the NCBI, GenBank database (http://www.ncbi.nlm.nih.gov).
Influence of abiotic factors on N2 fixation
Nitrogen fixation was estimated using the ARA in both moss–cyanobacteria associations and in cyanobacterial isolates. A total of six replicate samples of the moss and eight replicate samples of the isolates were used in these studies. For each replicate, five moss shoots were placed in 22 ml sterile tubes and wet with 2 ml of sterile deionized water. Moss treatments included two controls, one where no acetylene was added to the moss and another where 2 ml of water was added without moss, but acetylene was added. These controls were included to assess the endogenous moss ethylene production and the concentration of ethylene in the acetylene placed in the headspace of the tubes. The sum of the two sources was about 0.8 nmol ethylene d–1 sample.
Cyanobacteria isolates were picked from agar plates with a 1.0 µl sterile plastic loop and placed in a sterile Eppendorf containing 0.8 ml of N-free BG11 medium. The cell suspension was homogenized by repeated passage through 21 and 23 gauge needles. Then the cells were resuspended in N-free BG11 medium, and, whilst being stirred continuously, 0.4 ml of the cell suspension was placed in a 1.9 ml sterile glass vial.
Tubes and vials were closed and placed in different light (between 15 and 160 µE m–2 s–1) and temperature regimes (between 2 °C and 31.5 °C) for moss and at different temperature regimes (between 5 °C and 30 °C) for free-living cyanobacteria with the light at a constant 130 µE m–2 s–1. The light and temperature regimes used are similar to conditions often encountered in the Swedish boreal forest. Moss and cyanobacteria were acclimated for 64 h in growth chambers for mosses and in a water bath for cyanobacteria. During this time tubes and vials were opened once a day for few minutes in a sterile laminar flow hood. After 64 h in each tube and vial 10% of the air was removed and replaced by 10% of acetylene. Based on previous observations, where no acetylene-inducing decline was observed after a series of measurements during a full 24 h (data not shown) ARA was measured 24 h after acetylene injection. A Shimadzu GC-8A (Shimadzu, Kyoto, Japan) gas-chromatograph with a Porapak T column coupled to a flame ionization detector (FID) was used. Nitrogen was used as carrier gas using a flow rate of 300 ml min–1, column oven temperature was 90 °C and detector temperature was 220 °C. The values obtained were compared with a standard gas containing 570 ppm ethylene in N2 gas. In free-living cyanobacteria, N2 fixation was related to protein content, while in the cyanobacteria–moss association N2 fixation was related to the dry weight obtained after drying the shoots for 24 h at 70 °C.
Protein extraction and determination in cyanobacteria
The cyanobacteria were precipitated by centrifugation at 10 600 g for 5 min, the medium was poured out and the precipitated cyanobacteria were resuspended in 80% acetone solution and sonicated in an Eppendorf tube using a sonifier cell disruptor (Branson, Danbury, Connecticut, USA). The sonicated samples were left overnight at 4 °C. The samples were then centrifuged at 10 600 g for 5 min and eight pellet replicates of each treatment were suspended in the solubilization buffer containing 7.5 ml of ultra-pure water, 2.5 ml of 1 M TRIS–HCl pH 6.8, 16 ml of 10% SDS, and 1 ml of 80% glycerol (v/v). Extract aliquots were used for protein determination using the BCA (bicinchoninic acid) method as described by Mattsson and Sellstedt (2000).
Statistical analysis
A central composite design in two factors (temperature and light) (Box et al., 1978; Eriksson et al., 2000) was used to reduce the number of treatments drastically while investigating a broad light and temperature range. Statistical analyses of the central composite design were performed by using analysis of variance with Modde 6 software (Umetrics, Umeå, Sweden). All other data were analysed by t-test using Minitab statistical software (Minitab Inc., State College Pennsylvania, PA, USA, 2000).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Characterization of isolated cyanobacterial strains
Two genera of cyanobacteria were successfully isolated from the feather moss, P. schreberi. After successive re-isolation, the cyanobacteria were viewed under a light microscope revealing the two genera: Calothrix and Nostoc (Fig. 1A, B). This, to our knowledge, is the first isolation of Calothrix sp. from P. schreberi. Two species of Calothrix, C. gracilis Fritsch and C. parietina Thuret, were recognized in moss communities in Schirmacher oasis in Antarctica (Pandey et al., 1992
). In addition, morphological studies of the moss–cyanobacteria association performed in this study revealed that the predominant cyanobacteria colonizing the P. schreberi are Nostoc sp. and Stigonema sp. (Fig. 1C). The diffuse presence of Nostoc-type cyanobacteria was also found in mosses in an Arctic environment (Solheim et al., 2004). The high frequency of moss colonization by Stigonema sp. is confirmed in a previous work, where Stigonema minutum Hassal was one of the most frequent colonizers of moss in the Antarctic (Pandey et al., 1992). However, Stigonema has not been successfully isolated from the moss samples.
|
The molecular analysis revealed the presence of two sequences (Table 1). The sequence from the first isolate showed 94% similarity to Calothrix desertica (NCBI, GenBank accession number U83252 version U83252 [GenBank] .1 GI:1857481), but only 83% identity to Nostoc sp. (GenBank accession number AF204065 version AF204065 [GenBank] .1 GI:16269524).
|
In addition, the sequence from the second isolate (Table 1) demonstrated 94% identity to Nostoc sp. ‘Placopsis parellina cyanobiont’ (NCBI, GenBank, accession number AY304283 version AY304283 [GenBank] .1 GI:32130652) and only 82% identity to Anabaena cylindrica (NCBI, GenBank, accession number AF105129 version AF105129 [GenBank] .1 GI:7688479).
Further sequence analyses revealed that the two cyanobacteria genera were completely different from each other, which was further confirmed by the differences in morphology (see above) as well as in nitrogenase activity.
Colonization and reconstitution of the association
Cyanobacteria were found in all ten treatments. Eight out of the ten treatments had cyanobacteria colonization of moss ranging from 77% to 83%, one had a lower value (57%), and one a higher value (100%). The uniform cyanobacterial colonization of moss samples allowed the N2 fixation to be presented on a moss dry weight basis.
To demonstrate the reconstitution of cyanobacteria isolates with P. schreberi, samples of P. schreberi were collected that have regularly demonstrated no measurable N2-fixation activity over the three years from a plot (designated Reivo 1) within the same site (Ruttledjan) from which the cyanobacteria were isolated (DeLuca et al., 2002; Zackrisson et al., 2004). Moss shoots from this plot seldom showed the presence of cyanobacteria when observed under a light microscope. The reconstitution experiment revealed that 2 weeks after the addition of cyanobacteria isolates, the samples produced measurable N2 fixation (Table 2), indicating that it is possible to reconstitute the association of cyanobiont with the host plant. Nitrogen fixation in the reconstituted association continued and was measurable until the end of the experiment, indicating that the reconstitution created an active N2-fixing association. By contrast, moss shoots, where no cyanobacteria were added, demonstrated no measurable N2 fixation (Table 2).
|
Interestingly, the reconstituted association of P. schreberi from Reivo 1 with Calothrix sp. fixed much less N2 when compared with the Reivo 1 samples reconstituted with Nostoc sp. (Table 2). This could be a result of a longer lag time for the Reivo 1–Calothrix sp. association to become physiologically active or perhaps the result of a higher sensitivity of Calothrix sp. to environmental factors (light, temperature, moisture).
Influence of abiotic factors on N2 fixation
The effect of abiotic factors on cyanobacterial N2 fixation in Arctic and subarctic regions has been the subject of many studies (Alexander et al., 1974; Basilier et al., 1978; Jordan, 1978; Davey, 1983; Christie, 1987; Nakatsubo and Ino, 1987; Chapin et al., 1991; Lennihan et al., 1994; Liengen and Olsen, 1997; Dickson, 2000; Solheim et al., 2002; Zielke et al., 2002) with the unifying concept that moisture, temperature, light, and nutrient conditions are the most important environmental factors influencing N2 fixation in the Arctic and subarctic.
In this study, moisture was held constant and the effect of temperature and light in cyanobacteria–moss associations and the effect of temperature in isolated cyanobacteria in pure culture were studied. Nitrogen fixation was detected in moss samples at temperatures as low as 2 °C (Fig. 2A). These results are in agreement with the seasonal field observations previously made by DeLuca et al. (2002) wherein N2 fixation was measured under snow pack in northern Sweden in May. Furthermore, the low temperature N2 fixation underscores the significance of this association in the boreal forest, where temperatures are low during extended periods of the year (Van Cleve and Alexander, 1981). Nitrogen fixation in feather moss was found to increase with temperature, reaching peaks at 13 °C and 22 °C and found to decline at 31.5 °C (Fig. 2A). Temperature effects on N2 fixation have been studied in Drepanocladus exannulatus and Sphagnum riparium (Basilier et al., 1978) and it was found that N2 fixation had a maximum at approximately 11 °C and 16 °C, respectively. The cyanobacteria inhabiting D. exannulatus were, however, not identified, while for S. riparium the presence of Nostoc sp. was reported (Granhall and Hofsten, 1976). Nitrogen fixation was also measured in D. trichophyllus and was estimated as 170 nmol C2H4 g–1 dry wt h–1 at 12.7 °C (Granhall and Selander, 1973), which seems comparable with what was found in these studies (Fig. 2A). It must be noted, however, that these studies were performed under laboratory conditions while that of Granhall and Selander (1973) was performed in situ.
|
Interestingly, the moss–cyanobacteria association was found to be active at low light intensities (Fig. 2A). By contrast, work involving S. riparium and D exannulatus from a Swedish mire suggested that N2 fixation was light-dependent and it had a light optimum much higher than in the present work (Basilier et al., 1978
). Even in arctic vegetation including moss, N2 fixation activity greatly decreased at a light intensity lower than 80 µmol m–2 s–1 (Zielke et al., 2002). This discrepancy could be explained by adaptation to different environmental conditions, such as low light intensity under the forest canopy in the present work, and a higher light intensity in mire as well as arctic tundra where the tree canopy is absent or limited.
To obtain a profile of temperature effects on N2 fixation in the moss-cyanobacteria association, two separate cyanobacteria genera were studied (Fig. 2B, C), recognizing that there are clearly more than one cyanobacteria genus that colonizes feather mosses. The focus was on temperature, because preliminary experiments (data not shown) and the study presented below indicated temperature as a particularly important factor influencing N2 fixation. Furthermore, previous studies indicate that temperature is a more important driver of N2 fixation than light (Alexander et al., 1974; Jordan, 1978; Davey, 1983; Chapin et al., 1991; Lennihan et al., 1994; Liengen and Olsen, 1997; Solheim et al., 2002). There was a great difference in the amount of N2 fixed between temperature treatments in Calothrix sp. (P <0.001; Fig. 2B) and Nostoc sp. (P <0.001; Fig. 2C). Moreover there were clear differences between the two cyanobacteria genera (Fig. 2B, C). Indeed, at the lowest temperature of 5 °C, Nostoc sp. were observed to fix low levels of N2, while Calothrix sp. showed no N2-fixation activity. By contrast, at the highest temperature of 30 °C, Calothrix sp. had the highest N2-fixation activity, a level approximately three times greater than the Nostoc sp. at this temperature (Fig. 2B, C). Clearly the isolated cyanobacteria had different temperature optima, as indicated by the fact that Nostoc sp. had a peak at 13 °C (Fig. 2C) while Calothrix sp. had a peak at 30 °C (Fig. 2B).
The fact that N2 fixation in the moss–Nostoc association was much higher at 13 °C than at 31.5 °C is potentially explained by the fact that Nostoc sp. were more predominant than Calothrix sp. in the moss colonization.
This is, to our knowledge, the first detailed report on the biology of a feather moss–cyanobacteria association. These differences in temperature optima are of great physiological and ecological relevance. Indeed, it has been shown that the moss–cyanobacteria association can fix N2 at a variety of temperature conditions and, consequently, can adapt to a variety of environmental situations. Furthermore, it is possible that these different temperature optima help explain some of the seasonal and spatial variability in N2 fixation in feather mosses (DeLuca et al., 2002; Zackrisson et al., 2004). The ecological importance at a global scale of the moss–cyanobacteria associations should not be overlooked because they are ubiquitous in the boreal forests and are common in Arctic and Antarctic environments (Pandey et al., 1992; Solheim et al., 1996; Zackrisson et al., 2004).
In conclusion, taxonomic differences were confirmed by morphological and molecular studies. The isolates can be reconstituted with non-N2-fixing moss and effectively induce N2 fixation. The two cyanobacteria genera differ in their temperature optima for fixing N2, which may have important ecological implications, functioning as a N source under a wide range of temperatures. Indeed, DeLuca et al. (2002) found N2 fixation under snow-pack in subnivion spaces where Nostoc sp. may be a more active genus, whereas in midsummer to early autumn Calothrix sp. may be one of the more active N2-fixing genera. These differences in temperature optima may also partially explain the seasonal and spatial heterogeneity of N2 fixation in the P. schreberi–cyanobacteria association. Future work should be devoted to the isolation of other cyanobacteria from P. schreberi and cyanobacteria from other feather mosses (e.g. Hylocomium splendens) in which active N2 fixation (data not shown) has been observed. In particular, an effort should be made to isolate the Stigonema sp. that is frequently observed to colonize moss shoots. In these studies Nostoc and Stigonema were found to be the predominant cyanobacteria genera observed on the feather moss P. schreberi.
|
Acknowledgements |
|---|
We thank Paavo Lundberg, and Kjell Olofsson for technical help and Dr Anasuya Mohapatra and Melakeselam Leul for valuable discussion. We greatly appreciated the help of Dr. Michael Sjöström on the statistical design.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Alexander V, Billington M. 1986. Nitrogen fixation in the Alaskan taiga. In: Van Cleve K, ed. Forest ecosystems in the Alaskan taiga. New York: Springer-Verlag, 112–121.
Alexander V, Billington M, Schell D. 1974. The influence of abiotic factors on nitrogen fixation rates in the Barrow, Alaska, arctic tundra. Reports from the Kevo Subarctic Research Station 11, 3–11.
Basilier K, Granhall U, Stenström T-A. 1978. Nitrogen fixation in wet minerotrophic moss communities of a subarctic mire. Oikos 31, 236–246.
Box GEP, Hunter WG, Hunter JS. 1978. Statistics for experimenters. New York, USA: John Wiley and Sons Inc., 510–539.
Carleton TJ, Read DJ. 1991. Ectomycorrhizas and nutrient transfer in conifer-feather moss ecosystems. Canadian Journal of Botany 69, 778–785.
Chapin DM, Bliss LC, Bledsoe LJ. 1991. Environmental regulation of nitrogen fixation in high arctic lowland ecosystems. Canadian Journal of Botany 69, 2744–2755.
Christie P. 1987. Nitrogen in two contrasting Antarctic bryophyte communities. Journal of Ecology 75, 73–93.
Cleveland CC, Townsend AR, Schimel DS, et al. 1999. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochemical Cycle 13, 623–645.[CrossRef]
Davey A. 1983. Effects of abiotic factors on nitrogen fixation by blue-green algae in Antarctic. Polar Biology 2, 95–100.
DeLuca TH, Zackrisson O, Nilsson MC, Sellstedt A. 2002. Quantifying nitrogen-fixation in feather moss carpets of boreal forests. Nature 419, 917–920.[CrossRef][Medline]
Dickson LG. 2000. Constraints to nitrogen fixation by cryptogamic crusts in a polar desert ecosystem, Devon island, NWT, Canada. Arctic, Antarctic, and Alpine Research 32, 40–45.
Enderlin CS, Meeks JC. 1983. Pure culture and reconstitution of the Anthoceros–Nostoc symbiotic association. Planta 158, 157–165.[CrossRef]
Eriksson L, Johansson E, Kettaneh-Wold N, Wikström C, Wold S. 2000. Design of experiments: principles and applications. Umeå, Sweden: Umetrics Academy, 165–190.
Granhall U, Selander H. 1973. Nitrogen fixation in a subarctic mire. Oikos 24, 8–15.
Granhall UV, Hofsten A. 1976. Nitrogenase activity in relation to itracellular organisms in Sphagnum mosses. Physiologia Plantarum 36, 88–94.[CrossRef]
Jordan DC, McNicol PJ, Marshall MR. 1978. Biological nitrogen fixation in the terrestrial environment of a high Arctic ecosystem (TrueLove Island, Devon Island, N. W. T.). Canadian Journal of Microbiology 24, 643–649.[Web of Science][Medline]
Lennihan R, Chapin DM, Dickson LG. 1994. Nitrogen fixation and photosynthesis in high arctic forms of Nostoc commune. Canadian Journal of Botany 72, 940–945.
Liengen T, Olsen RA. 1997. Seasonal and site-specific variations in nitrogen fixation in a high arctic area, Ny-Ålesund, Spitsbergen. Canadian Journal of Microbiology 43, 759–769.
Lind LK, Kalla R, Lönneborg A, Öquist G, Gustafsson P. 1985. Cloning of the ß-phycocyanin gene from Anacystis nidulans. FEBS Letters 188, 27–32.[CrossRef]
Mattsson U, Sellstedt A. 2000. Hydrogenase in Frankia KB5: expression of and relation to nitrogenase. Canadian Journal of Microbiology 46, 1091–1095.[CrossRef][Web of Science][Medline]
Nakatsubo T, Ino Y. 1987. Nitrogen cycling in an Antarctic ecosystem. I. Biological nitrogen fixation in the vicinity of Syowa station. Memoirs of the National Institute for Polar Research, Series E 37, 1–10.
Pandey KD, Kashyap AK, Gupta RK. 1992. Nitrogen fixation by cyanobacteria associated with moss communities in Schirmacher oasis, Antarctica. Israel Journal of Botany 41, 187–198.
Paulsrud P, Lindblad P. 1998. Sequence variation of the tRNAleu intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens. Applied and Environmental Microbiology 64, 310–315.
Rastetter EB, Vituosek PM, Field C, Shaver GR, Herbert D, Agren GI. 2001. Resource optimation and symbiotic nitrogen fixation. Ecosystems 4, 369–388.[CrossRef]
Rasmussen U, Svenning M. 1998. Fingerprinting of cyanobacteria based on PCR with primers derived from short and long tandemly repeated repetitive sequences. Applied and Environmental Microbiology 64, 265–272.
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111, 1–61.
Sellstedt A, Wullings B, Nyström U, Gustafsson P. 1992. Identification of Casuarina–Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers. FEMS Microbiology Letters 93, 1–6.[CrossRef]
Solheim B, Endal A, Vigstad H. 1996. Nitrogen fixation in Arctic vegetation and soils from Svalbard, Norway. Polar Biology 16, 35–40.[CrossRef]
Solheim B, Johanson U, Callaghan TV, Lee JA, Gwyn-Jones D. 2002. The nitrogen fixation potential of arctic cryptogam species are influenced by enhanced UV-B radiation. Oecologia 133, 90–93.[CrossRef]
Solheim B, Wiggen H, Röberg S, Spaink HP. 2004. Associations between Arctic cyanobacteria and mosses. Symbiosis 37, 169–187.
Van Cleve K, Alexander V. 1981. Nitrogen cycling in tundra and boreal ecocsystems. In: Clark FE, Rosswall T, eds. Terrestrial nitrogen cycle. Ecology Bulletin 33. Stockholm, 375–404.
Weber MG, Van Cleve K. 1981. Nitrogen dynamics in the forest floor of interior Alaskan black spruce ecosystems. Canadian Journal of Forest Research 11, 743–751.
West NJ, Adams DG. 1997. Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site. Applied and Environmental Microbiology 63, 4479–4484.
Xu MQ, Kathe SD, Goodrich Blair H, Nierzwicki Bauer SA, Shub DA. 1990. Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria. Science 250, 1566–1570.
Zackrisson O, DeLuca TH, Nilsson M-C, Sellstedt A, Berglund L. 2004. Nitrogen fixation increases with successional age in boreal forests. Ecology 85, 3327–3334.[CrossRef][Web of Science]
Zielke M, Ekker AS, Olsen RA, Spjelkavik S, Solheim B. 2002. The influence of abiotic factors on nitrogen fixation in different types of vegetation in the High arctic, Svalbard. Arctic, Antarctic and Alpine Research 34, 293–299.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. G. Adams and P. S. Duggan Cyanobacteria-bryophyte symbioses J. Exp. Bot., March 1, 2008; 59(5): 1047 - 1058. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||