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AOBPreview originally published online on October 19, 2007
Annals of Botany 2007 100(7):1537-1545; doi:10.1093/aob/mcm249
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© The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Growth and Nitrogen Relations in the Mat-forming Lichens Stereocaulon paschale and Cladonia stellaris

M.-M. Kytöviita* and P. D. Crittenden

School of Biology, University of Nottingham, Nottingham NG7 2RD, UK

* For correspondence. Present address: Department of Biological and Environmental Science, Jyväskylä University, PO Box 35, FIN-40014 Jyväskylä University, Finland. E-mail minna-maarit.kytoviita{at}oulu.fi

Received: 10 March 2007    Returned for revision: 20 April 2007    Accepted: 14 August 2007    Published electronically: 19 October 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

Background and Aims: Mat-forming lichens in the genera Stereocaulon and Cladonia have ecosystem-level effects in northern boreal forests. Yet the factors affecting the productivity of mat-forming lichens are not known. The aim of the presented work was to investigate whether mat-forming lichens adapted to low N availability employ N-conserving mechanisms similar to those of vascular plants in nutrient-poor ecosystems. Specifically, the following questions were asked: (a) Do lichens translocate N from basal areas to apical growth areas? (b) Are the quantities of N translocated of ecological significance. (c) Is lichen growth dependent on tissue N concentration [N].

Methods: Two different, but complementary, field experiments were conducted using the mat-forming N2-fixing Stereocaulon paschale and non-fixing Cladonia stellaris as model species. First, N translocation was investigated by feeding lichens with Na15NO3 either directly to the apex (theoretical sink) or to the basal part (theoretical source) and observing the redistribution of 15N after a growth period. Secondly, growth and variation in [N] in thalli of different lengths was measured after a growth period.

Key Results: 15N fed to lower parts of lichen was translocated towards the growing top, but not vice versa, indicating physiologically dependent translocation that follows a sink–source relationship. In the growth experiment where thalli were cut to different lengths, the significant decrease in [N] in apices of short vs. longer thalli after a growth period is consistent with internal relocation as an ecologically important source of N.

Conclusions: The presented results demonstrate that internal recycling of N occurs in both species investigated and may be ecologically important in these mat-forming lichens under field conditions. The higher nitrogen use efficiency and relative growth rate in C. stellaris in comparison with S. paschale probably enable C. stellaris to dominate the ground cover vegetation in dry boreal coniferous forests under undisturbed conditions.

Key words: Cladonia, Stereocaulon, nitrogen translocation, relative growth rate, nitrogen use efficiency, mat-forming lichens


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Lichens are one of the most important vegetation components in northern boreal and sub-arctic ecosystems. In sub-arctic conifer forests, such as in open woodlands in northern Canada, lichens can contribute up to 97 % ground cover and contain about 20 % of the total ecosystem biomass (Auclair and Rencz, 1982). Similar values are reported for Arctic ecosystems (Shaver and Chapin, 1991). Under undisturbed conditions, Cladonia stellaris and Stereocaulon paschale, the dominant lichen species in many northern lichen-dominated ecosystems, form deep and persistent mats. Mat-forming lichens in the genus Cladonia are important as winter fodder for reindeer and caribou, whereas Stereocaulon spp. are less favoured by herbivores (Svihus and Holand, 2000; den Herder et al., 2003). Lichen mats affect ecosystem productivity by influencing water, nutrient, thermal and microbial characteristics in the underlying soil (Kershaw and Field, 1975; Stark et al., 2000; Olofsson et al., 2001). Therefore, conservation and management of lichen-dominated boreal coniferous forests requires an understanding of factors that affect the growth of the lichen component.

There is debate about factors that might limit lichen productivity. Lichens are composite organisms comprising a fungal partner and one or more algal and/or cyanobacterial partners. A widely promulgated argument is that opportunities for photosynthesis might be growth limiting. This limitation could be alleviated by increasing N investment into the photobiont (Palmqvist and Dahlman, 2006). An alternative suggestion is that lichen growth might be limited by the availability of key nutrients such as N or P. In this case lichens should respond positively to nutrient enrichment and might be expected to have a high capacity to take up, retain and recycle nutrients within the thallus. Terricolous lichen mats do indeed have high nutrient capture efficiencies for N and P, a 25-mm layer of Cladonia stellaris or Stereocaulon paschale being able to capture ≥85 % inorganic N and P in intercepted rainfall (Crittenden, 1989; Hyvärinen and Crittenden, 1998). However, attempts to increase lichen growth with fertilizers have resulted in variable responses (Crittenden et al., 1994). Fertilizing C. stellaris under ecologically relevant conditions did not increase its growth (Kytöviita, 1993; Hyvärinen et al., 2003), whereas the cyanobacterial species S. paschale (Kytöviita, 1993) and Peltigera aphthosa (Hallingbäck and Kellner, 1992) responded positively (but see Dahlman et al., 2002). The marked vertical gradient in N concentration [N] within thalli of mat-forming lichens is suggested to be partly caused by internal N recycling (Crittenden 1989; 1991; Ellis et al., 2005) in which N is remobilized from older lower strata and translocated to the apices following a source–sink relationship. This process would increase the mean residence time of N in the living lichen thallus and elevate nitrogen use efficiency (NUE, rate of dry matter production per unit N) (Berendse and Aerts, 1987; Aerts, 1990). In vascular plants, the relative growth rate (RGR) is positively related to tissue [N] (Poorter and Bergkotte, 1992; Reich et al., 1992), but whether this is the case in lichens is not known.

Accordingly, the aim of the present work was to investigate whether mat-forming lichens adapted to environments with low N availability employ mechanisms similar to those of vascular plants in nutrient-poor ecosystems. Specifically, we asked whether (a) mat-forming lichens translocate N from old senescing basal tissues to apical growth areas; (b) the quantities of N translocated are of ecological significance; and (c) lichen growth rate is related to tissue [N]. These questions were investigated in two different, but complementary, experiments. First, N translocation was investigated by feeding Na15NO3 either to the apex (putative sink) or to the basal parts (putative source) of mat-forming lichens and observing the redistribution of 15N after a period of growth. According to our hypothesis, if the growing apices act as a sink, 15N fed to basal parts should be translocated to the apices during growth. Secondly, growth and change in [N] were measured in thalli of different lengths. Assuming that new growth occurs primarily in the apices, two possible results of the experiment were predicted: (a) if growth in the apical regions was partially dependent on N supplies from basal regions (i.e. N recycling occurs), then the [N] in apices of short thalli in which basal strata had been removed would decline during growth compared with the [N] in the apices of longer thalli; and (b) if growth in the apices was independent of N supplies from basal regions (i.e. N recycling does not occur), then growth should not induce differences in [N] amongst the apices of thalli cut to different lengths. Cladonia stellaris and S. paschale were chosen as model species for this study because of their ecological importance and because they differ in their main source of N. Stereocaulon paschale contains the N2-fixing cyanobacterium Stigonema as a secondary photobiont in cephalodia and the green alga Trebouxia as the primary photobiont in the phylocladia, while C. stellaris contains only Trebouxia. It has also been shown that lichens with green algae have N allocation patterns that differ from those in lichens with cyanobacterial symbionts (Palmqvist et al., 1998). Such differences in N allocation patterns might also be reflected in the N translocation within the lichen thallus. Therefore, the two model lichens were expected to have different N translocation patterns.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Field sites and lichen material
Field work was conducted in a mixed birch and pine woodland near the Kevo Subarctic Research Station (69 °45'N, 27 °E) in Finnish Lapland. Cladonia stellaris and S. paschale were collected from a lichen-rich location beside the Teno River approx. 20 km NW of Kevo and transferred to the experimental site. Care was taken to maintain the lichen material in as natural state as possible. Because of its extreme fragility when dry, C. stellaris was handled only in the wet state. Lichens were manipulated under field conditions and only transferred to the laboratory to be weighed.

Vertical movement of 15N in lichen thalli
Field procedures
Pseudopodetia of S. paschale and podetia of C. stellaris >50 mm long were collected on 17 June 1990. Each thallus was cut to 50 mm length (i.e. at 50 mm below the apex) and the dry mass measured on 22 June as described under ‘Growth experiments’ below.

Labelling of S. paschale was undertaken on 5 July in the field during a period of natural rainfall as follows. Hydrated thalli were blotted with tissue to remove excess surface water and then stood vertically in a 5 mL beaker containing 3·24 mM 15N-NaNO3 (99 % enrichment) to a depth of 25 mm (3·5 mL), i.e. immersing one half of the thallus length. The quantity of N in solution was selected to equal approx. 10 % of the total N in the 50 mm long thalli. Nitrate was chosen as the N source of label because (a) precipitation is believed to be the principal source of N for mat-forming lichens (Ellis et al., 2003, 2004); (b) it is one of the principal forms of N in precipitation [the molar ratio of NO3–: NH4+ in precipitation is frequently close to unity (Prado-Fiedler, 1990; Tuovinen et al., 1990; Garban et al., 2004)]; (c) it is readily taken up by lichens (Lang et al., 1976; Crittenden, 1996; Dahlman et al., 2004); and (d) unlike NH4+ (cf. Brown et al., 1995), NO3– is unlikely to bind passively to cell wall exchange sites.

Thalli were incubated in the 15N solution for 1·5 h under field conditions during which time the solution was agitated periodically to disrupt diffusion gradients. Either the lowermost 25 mm of thalli [top up (TU) treatment] or the uppermost 25 mm [top down (TD) treatment] were exposed to the isotope. Ground level temperature during the incubation was approx. 17 °C. At the end of the incubation, lichens were removed from the labelled solutions and excess surface solution removed by placing the exposed end of the thallus in contact with absorbent tissue. A sub-sample (12 TU and 12 TD) of the replicates was air dried in the laboratory and stored at this point to determine the quantity and distribution of 15N in thalli immediately after labelling. The remaining 20 thalli in each treatment were placed within a reconstructed lichen mat to grow in the field as described under ‘Growth experiments’ below. TU thalli were grown in their natural orientation while TD thalli were grown inverted (apex down). A set of control thalli were exposed to 3·23 mM NaCl solution (TU) to test whether the N in NaNO3 solution had a fertilizing effect. These control thalli were otherwise treated in a manner identical to those exposed to 15N.

Thalli of C. stellaris were exposed to 15N on 7 July in a manner similar to that described above for S. paschale except that the ground level temperature was approx. 12 °C. Owing to the larger dimensions of thalli of C. stellaris and their lower tissue [N], a 25 mL beaker containing 15 mL of 0·67 mM 15N-NaNO3 was used to produce the same depth of solution as above, the total quantity of N in solution again being approx. 10 % of the total N in a typical thallus cut to this length. 15N-labelled and NaCl-exposed thalli were inserted within a reconstructed mat of C. stellaris for 2 months as described under ‘Growth experiments’ below.

On 17 September, after the lichens had been rehydrated by an episode of natural rainfall, the experimental thalli of both species were removed from the lichen mats, dried for 2 d on a laboratory bench and stored dry in airtight containers until chemical analysis 6–9 months later.

15N determination
Twelve thalli of both species in the TU and TD treatments were analysed for 15N contents. The dry lichen thalli were rehydrated overnight in a water-saturated atmosphere (over water in a desiccator) and then cut horizontally into strata at 10 and 25 mm from the apex of TU thalli or at 10 and 25 mm from the base of TD thalli. In order to obtain sufficient mass of lichen for chemical analysis, it was necessary to combine corresponding strata in pairs of thalli from the same treatment, which resulted in six pairs per treatment to be analysed. The pairs of strata thus obtained were oven dried (overnight at 80 °C), weighed and analysed for total N content following the Kjeldahl digestion and distillation method of Bremner and Breitenbeck (1983). Between each distillation, an additional 100 mL of water was allowed to distil through the glass apparatus in order to eliminate any carry-over of 15N between samples (Bergersen, 1980). After determination of total N by titration with 0·1 N H2SO4, a sub-sample of the distillate solution containing about 0·1 mg of N was transferred into a 10 mL capacity glass vial and evaporated to dryness at 80 °C. Nitrogen gas was generated from these vials by addition of sodium hypobromite using the glass assembly described by Ross and Martin (1970). The N2 gas was introduced into an AEI MS2 mass spectrometer and the relative abundance of masses 28 and 29 were recorded. The percentage abundance of 15N in lichen samples was calculated as 100/2R + 1, where R is the mass ratio of 28/29 in N2 generated from the digests. Atom % excess 15N was then calculated by subtraction of the 15N abundance in equivalent strata of S. paschale and C. stellaris thalli which had not been exposed to 15N-NaNO3.

Growth experiments
Field procedures
Dry pseudopodetia of S. paschale ≥50 mm in length were collected on 25 May 1989. Each thallus was cut to one of six lengths (10, 15, 20, 25, 35 or 50 mm from the apex downwards), and the remaining lower portions discarded. The dry mass of each pruned pseudopodetium was measured both before and after the growth period as follows. Thalli were allowed to air-dry in the field under natural conditions, then transferred to the laboratory for 12 h to equilibrate with laboratory conditions prior to being weighed. In each thallus length class, ‘dummy’ thalli were included which were treated in a similar manner to the experimental thalli but which were also oven dried; their oven dry: air dry mass ratio was then used to estimate the oven dry mass of experimental material. Each weighed thallus was labelled with a small plastic tag attached by nylon thread, and then all such thalli were returned to the field.

At the experimental site, intact mats of S. paschale were reconstructed on 2·5 m2 plots of ground cleared of aboveground vegetation. The plot was in an inter-tree location without a tree overstorey. On 7 June 1989 the weighed thalli were each inserted into a 25 mm diameter gap in the Stereocaulon mat. The gaps were maintained by cylindrical cages of stainless steel mesh (0·25 mm diameter wire, eight holes per 25 mm). The bottoms of the mesh cages were lined with glass balls and then a quantity of opaque plastic beads added sufficient to raise the apices of the experimental thallus to the same level as those in the surrounding lichen mat. The glass balls and plastic beads also served to provide a uniform inert substratum for the experimental lichens. There were two sets of lichens. Set 1 was kept in the field during the summer of 1989 only and included replicates in each of the six length groups. Set 2 was kept in the field for 2 years (1989–1990) and included replicates in three length groups: 10, 25 and 50 mm. The number of replicates in each treatment (length group) was initially 20 and the thalli were placed within the lichen mats in a randomized block design. On five occasions between 1989 and 1990, thalli from Set 2 were removed, re-weighed as above and then re-inserted into the lichen mats (Kytöviita and Crittenden, 2002). Thallus [N] was measured at the start of the experiment, but only Set 2 was analysed for the N at the end of the experiment. Therefore, herein a comparison is made between (a) the RGR in Set 1 between 5 May 1989 and 25 September 1989 and thallus [N] at the start of the experiment; and (b) the RGR in Set 2 between 21 June 1990 and 13 September 1990 and thallus [N] at the end of the experiment.

A similar experiment was set up for C. stellaris collected on 17 June 1990, but in which there was only one set of lichens. Tagged podetia of 10, 15, 20, 25, 35 and 50 mm length were inserted into 32 mm diameter gaps in a reconstructed C. stellaris mat on 26 June and removed after 3 months (14 September).

At the end of each experiment, a further collection of both species was made at the Teno River site. These samples served as unmanipulated controls and are referred to as the ‘intact control’. All material was stored at –20 °C until chemical analysis.

Total N determination
Lichen samples were rehydrated as above and cut horizontally into strata in a contiguous manner from top to bottom: 5, 5, 5, 10, 10 and 15 mm. Total nitrogen in each stratum was determined using the indophenol blue method following acid digestion in a 1:1·2 (v/v) mixture of H2O2 and H2SO4 (Allen 1989).

Growth functions
RGR was calculated for each length group as (ln massend – ln massstart)/time (days) (Hunt, 1990). NUE was calculated as dry mass produced during the growth period per g N at the start of the growth period.

Statistical analyses
The effect of thallus size (mg) on the total quantity of 15N (µmol) taken up during the initial exposure was examined by linear regression analysis. Variation in 15N uptake rate was analysed by two-way analysis of variance (ANOVA) with lichen species and thallus orientation (TD or TU) as factors, followed by one-way ANOVA due to significant interactions. Differences between the four factor levels were further assessed with Tukeys's test. 15N enrichment in the end and start thalli was analysed separately for top, middle and bottom parts with one-way ANOVA followed by Tukey's test.

It could not be assumed that [N] values in different lichen strata are independent, and therefore differences in [N] in the 0–5 and 5–10 mm apical strata were tested with repeated measures ANOVA followed by Tukey's test. Homogeneity of variances and normality were studied with Levene's test and Kolmogorov–Smirnov test. All analyses were carried out using the SPSS 8·0 statistical package.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Vertical movement of 15N in lichen thalli
The quantity of 15N-NO3– taken up by S. paschale during the 1·5 h incubation period was between 10 (basal parts exposed) and 22 % (apices exposed) of the total label supplied in the solution, and uptake in C. stellaris was between 13 and 19 %, respectively. In both species, total 15N uptake (u, µg) was proportional to the mass (m, mg) of the exposed stratum of the thallus (S. paschale, u = 0·236m + 1·066, r2 = 0·87, P < 0·01; C. stellaris, u = 0·121m – 11·5, r2 = 0·42, P < 0·02, n = 12). The uptake rates were higher in S. paschale (TU 4·82 ± 0·38 and TD 9·44 ± 0·61 µmol N g–1 h–1) than in C. stellaris (TU 2·80 ± 0·90 and TD 3·77 ± 0·22 µmol N g–1 h–1) (P < 0·05). Uptake rates were also significantly higher in apical tissue (TD) than in basal parts (TU) in both species (P < 0·05). Attempts to estimate loss of 15N from thalli during the growth period were confounded by both species having a lower mean mass at time zero in samples harvested after 2 months than those that were stored immediately after labelling. However, values of 15N enrichment were broadly similar at both harvests, suggesting that loss of label had been relatively small.

Movement of 15N within the lichen thallus was indicated as changes during growth in 15N enrichment in different horizontal strata (Fig. 1). Immediately after exposure to 15N, the label was located principally in the exposed half of the thallus in both species. In TD S. paschale, however, 15N enrichment was also notable in the uppermost stratum and was significantly higher than in the top stratum of TU thalli (P < 0·01). After 2 months growth in S. paschale, the top stratum of TU, but not TD thalli had a significantly increased 15N content (P < 0·05, Fig. 1A, B). In C. stellaris, increased 15N enrichment following growth is evident in the TU middle stratum (P < 0·01), but not in the apices (P = 0·201). As in S. paschale, there was no indication of 15N translocation in TD C. stellaris during the growth period (Fig. 1C, D).


Figure 1
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  		FIG. 1. Relative 15N enrichment in top, middle and bottom strata in Stereocaulon paschale and Cladonia stellaris before and after a 2-month growth period in top up (TU) or top down (TD) position in the field. Mean values ± s.e.m. are shown, n = 6. Different letters indicate statistically significantly different values ({alpha} < 0·05).

 
The mean RGR in TU thalli during the 2 month growth period was 0·444 ± 0·096 mg g–1 d–1 in S. paschale and 0·607 ± 0·040 mg g–1 d–1 in C. stellaris. Growth was significantly depressed in TD thalli in both species: S. paschale ceased growing and RGR in C. stellaris was reduced to 0·290 ± 0·076 mg g–1 d–1. The quantity of N added in the 15N solution had no fertilizing effect, and TU thalli exposed to ionically equivalent solutions of NaCl had growth rates similar to those of TU thalli exposed to 15N-NaNO3 (data not shown).

Effect of thallus length on [N], RGR and NUE
In both S. paschale and C. stellaris, thallus [N] values decreased with increasing distance from the apices (Fig. 2). After 15 months' growth in S. paschale and 4 months in C. stellaris, [N] values in all strata in 50 mm long experimental thalli were similar to those of reference material (Fig. 2). However, as thallus length was reduced, there was a trend towards progressively lower [N] values such that [N] in the apical stratum of 10 mm thalli of both species was significantly lower than in reference thalli (Fig. 2).


Figure 2
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  		FIG. 2. Vertical distribution in [N] in lichens cut to 10, 25 or 50 mm length, or intact field material. (A) Stereocaulon paschale after a 15-month growth period and (B) Cladonia stellaris after a 4-month growth period. Mean values ± s.e.m. are shown, n = 20. Different letters indicate statistically significantly different values ({alpha} < 0·05).

 
All experimental thalli increased in mass during the measurement periods. Cutting the thalli into different lengths affected both relative and absolute growth. The largest absolute mass increment occurred in the longest thalli (50 mm) in both species (data not shown). However, the largest relative mass gain occurred in the shortest thalli (10 mm) (Fig. 3). The relationship between RGR and mean whole thallus average [N] was very much stronger in C. stellaris than in S. paschale, which showed a relatively flat response in RGR to whole thallus [N]. The relationship between NUE and RGR is overall similar in both species: NUE increases nearly linearly with decreasing thallus length (Fig. 4). In C. stellaris but not in S. paschale, the longest thalli (50 mm) deviate from this overall pattern and both RGR and NUE are higher in the 50 mm thalli in comparison with the 35 mm long thalli (Fig. 4).


Figure 3
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  		FIG. 3. Relationship between RGR (June to September 1990) and average thallus N concentration at the end of the experiments in Stereocaulon paschale and Cladonia stellaris cut to 10, 25 or 50 mm length. Mean values ± s.e.m. are shown, n = 13–20.

 


Figure 4
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  		FIG. 4. Relationship between NUE (mg dry mass produced per g thallus N at start) and RGR in (A) Stereocaulon paschale (June–September 1989) and (B) Cladonia stellaris (June–September 1990). Mean values ± s.e.m. are shown for lichens cut to various lengths as shown, n = 18–20 for each group.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
15N capture and translocation
There is abundant evidence that lichens effectively assimilate NO3– (see Crittenden, 1998; Dahlman et al., 2004; Ellis et al., 2004). Uptake rates in C. stellaris exposed to 0·67 mM NaNO3– (2·8–3·8 µmol N g–1 h–1) in the present study compare favourably with rates reported by Dahlman et al. (2004) in the same species exposed to 1 mM KNO3– solution (approx. 5 µmol N g–1 h–1). However, uptake values reported here might be affected by part of the labelled bathing solution remaining on the lichen surface, and so the true uptake rates might be a little lower. Thalli of S. paschale were exposed to NO3– concentrations approx. 4 times higher than those used in labelling C. stellaris and, consequently, calculated uptake rates in S. paschale were higher (4·8–9·48 µmol N g–1 h–1). In both species, uptake rates were significantly lower in basal than in apical strata, indicating that the apices are better adapted for NO3– uptake than basal parts. This might be explained, in part, by a higher surface area to mass ratio in the apices. However, NO3– acquisition in lichens is an active process (Crittenden, 1996; Dahlman et al., 2004) and, hence, higher uptake rates in the apices might reflect the higher metabolic rates in these younger tissues.

The total quantity of label taken up was broadly similar in both lichen species because the lower uptake rates in C. stellaris were compensated by the larger thallus mass in this species. However, the rate of label migration differed markedly between the two species. During the 1·5 h incubation in labelled solution and the subsequent period of drying in the laboratory, label readily moved into unexposed upper strata of both TU and TD S. paschale, and label was readily measurable in the apical 10 mm. In comparison, migration of label into unexposed strata in C. stellaris was small, and only a trace was detected in the apices. These distribution patterns did not change markedly after 2 months although in both species there was significant further upward migration in TU thalli. The greater extent of label migration in S. paschale might partly be due to the greater linearity in thallus morphology in this species, typically comprising one or several vertical stems. In contrast, the repeatedly dichotomously branched thallus of C. stellaris creates a tortuous pathway for vertical solute migration. It is also possible that the free space compartment is enlarged in the lower and older strata in S. paschale, favouring transport of ions by mass flow over and above assimilation. At the same time, upward migration of 15N in S. paschale roughly equates with a linear rate of 10 mm h–1, which agrees favourably with cytoplasmic streaming rates observed in other fungi such as Glomus sp. (Bago et al., 2002).

The general vertical patterns of 15N distribution illustrated in Fig. 1 were established during the 1·5 h incubation and subsequent period of drying in the laboratory. It is not known whether any subsequent migration occurred equally as rapidly (e.g. during the following 12 h) or whether it occurred gradually during the 2 month growth period. However, Hyvärinen and Crittenden (1998) conducted a comparable experiment on the uptake and vertical migration of 33P-PO43– in C. portentosa using a molecular imager to map the vertical location of radiolabel in thalli, and they observed that migration occurred between both 0 and 3 months and 3 and 6 months after exposure. Similarly, Ellis et al. (2005) found that 15N introduced into the bases of 50 mm long thalli of C. portentosa continued to migrate vertically between 1 and 2 years following exposure. These results suggest that in the present study gradual 15N migration probably occurred throughout the 2 month growth period.

Since the direction of this subsequent, and probably gradual, migration was dependent on orientation, it cannot be readily explained by diffusion in the free space. Rather, it is compelling evidence of intracellular translocation following a source–sink relationship. The short distances over which 15N migration occurred in C. stellaris in the present study (5–50 mm) could probably be accounted for by intracellular diffusion (Jennings, 1995; Boswell et al., 2002). At the same time, there is no evidence to suggest that the fungal mycelium within the lichen differs anatomically from other Ascomycetes in terms of the structure of septa and the operation of cytoplasmic streaming between cells; cytoplasmic streaming within hyphae facilitates bidirectional movement of metabolites (e.g. Uetake et al., 2002). Although cytoplasmic streaming in lichen-forming fungi has not been studied, there seems to be no a priori reason why lichens should differ in this respect from other filamentous fungi. The situation in mat-forming lichens appears similar to that in mycorrhizal fungi. In mycorrhizas, carbon is assimilated by the photobiont host and translocated to the fungus, while nutrients are taken up by the fungus and translocated to the photobiont (Smith and Read, 1997). Similarly, N and possibly other nutrients from basal regions could be transferred to the growing apical regions while carbon would move downwards from the apical regions with the highest algal densities to lower strata comprising mainly fungal tissue.

Nitrogen and lichen growth
The present results suggest that growth is predominantly apical in C. stellaris and is at least maximal in the apices of S. paschale. They are therefore consistent with Kärenlampi's (1971) predicted partitioning of RGR in different horizontal strata of C. stellaris based on growth measurements on thalli of different lengths conducted in a manner similar to that employed in the present work. However, these interpretations assume that growth in these lichens is unmodified by dissection and that RGR in 10 mm long thalli was the same as in the 0–10 mm stratum of intact specimens. There is no evidence that this is the case. In C. stellaris, the top 10 mm apical stratum has been shown to account for 50 %, and the top 35 mm for 90 %, of the total thallus carbon gain (Moser et al., 1983). It is possible that a proportion of this photosynthate is translocated to lower strata in intact thalli and that removal of lower strata by dissection removes a carbon sink and modifies the zonal growth rate.

RGR was negatively related to thallus length despite a potential counter effect of more rapid desiccation of smaller thalli (cf. Larson and Kershaw, 1976; but see also Gaio-Oliveira et al., 2006). If thallus water relations are more growth-limiting than tissue [N], then growth in the apical stratum might decline when disconnected from the main body of the lichen. Since N uptake during rainfall in the apices will be independent of connection to the rest of the lichen mat, reduced RGR in disconnected apical strata induced by more rapid desiccation should therefore result in an increase in apical [N]. In the current work, [N] was actually reduced in the apical fragments when compared with intact apices, supporting the notion of N translocation from basal areas to support growth in the apices and the importance of N limitation in the growth in both species. Also, in C. stellaris, the 50 mm tall experimental thalli had unexpectedly high RGR and NUE when compared with shorter thalli. High NUE in the 50 mm thalli might suggest that the lower strata act as water reservoirs during desiccation and thus facilitate extended carbon fixation periods in the upper lichen strata. On the other hand, high RGR in 50 mm tall thalli points to the possibility that the lower strata serve as N sources to upper strata and in that way facilitate high production in the upper strata. Since both NUE and RGR were enhanced in the 50 mm tall lichens, it seems that the mat-forming growth habit is an adaptation to both N and water limitation in their natural habitat.

As a consequence of the higher tissue [N] and lower RGR, biomass production per unit N was overall lower in S. paschale than in C. stellaris. There are several ecophysiological mechanisms that might potentially underlie the lower nitrogen productivity in S. paschale. First, respiratory costs are likely to increase with increasing [N] and, although photosynthesis also increases with increasing [N], photosynthetic gain per unit N probably declines (Palmqvist et al., 2002). Secondly, N2 fixation consumes more energy than uptake of inorganic N in ionic form (Pate, 1984). This is particularly true for lichens since they do not construct specific structures for nutrient absorption. The high tissue [N] acquired by symbiotic N2 fixation is associated with a carbon cost not covered by the carbon-fixing activity of the N2-fixing secondary photobiont. Thirdly, it has been shown that cyanobacterial lichens invest more chitin in their cell walls (Boissiere, 1987; Schlarmann et al., 1990; Crittenden et al., 1994; Palmqvist et al., 2002). Chitin is energetically expensive to construct, and N allocation to chitin will compete with N allocation to the photobiont photosynthetic N pool. The higher chitin concentration in cyanolichen mycobiont cell walls could be a co-evolutionary adaptation to improve the performance of the photobiont. Increased chitin concentration may improve thallus water-holding capacity (Palmqvist et al., 1998) and, since cyanobacteria require higher moisture levels than green algae for active photosynthesis (Lange et al., 1986, Green et al., 1993), higher chitin levels in the mycobiont might increase time for photosynthesis in the photobiont, thus offsetting some of the costs of higher thallus [N]. The higher N demand in cyanolichen mycobionts lowers the lichen RGR and consequently NUE, but presumably is associated with other beneficial effects. Considering NUE as a critical component of lichen productivity, C. stellaris is more tolerant of fragmentation by trampling than S. paschale. Small fragments of C. stellaris had high RGRs but the highest lichen biomass productivity was produced by the 50-mm tall lichens.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Nitrogen can migrate vertically in thalli of mat-forming lichens. Both 15N translocation and growth data support the hypothesis that apical growth in mat-forming lichens is supported by internal recycling in which N is remobilized in older parts of the thallus and translocated to the apices in a source–sink relationship. Therefore, both lichen species studied showed an N conservation strategy similar to that of vascular plants. The quantities translocated are ecologically relevant as indicated by a decline in [N] in thalli disconnected from basal supplies. The nitrogen-fixing species S. paschale translocated N further to the apical regions in comparison with the non-fixing species C. stellaris. The difference in RGR–thallus length relationships indicated, however, that the N translocation might be more important within the apical regions in S. paschale whereas in C. stellaris the lower regions are also important in supplying both water and N to apical regions. This indicates that relatively deep C. stellaris cover is necessary for maximal lichen production in the reindeer lichen grounds.

The higher NUE and RGR in C. stellaris in comparison with S. paschale probably enable C. stellaris to dominate the ground cover vegetation in dry boreal coniferous forests under undisturbed conditions. Tolerance to fragmentation caused by reindeer trampling does not seem to contribute to the relatively higher abundance of S. paschale than C. stellaris in areas disturbed by intensive reindeer grazing. Instead, this pattern seems to be caused solely by reindeer feeding selectively on C. stellaris.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Dr John W. Millbank for his invaluable contribution in 15N determination, and Kevo Subarctic Research Station for support during fieldwork. This work was financed by Nottingham University and Academy of Finland.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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