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- Materials and Methods
- Supporting Information
Anthropogenic nitrogen (N) deposition poses a global threat to ecosystems and their floristic diversity (Sala et al., 2000; Phoenix et al., 2006). While much research to date has focused on the responses of higher plants, bryophytes have received far less attention despite these being major components of many ecosystems and known to be particularly sensitive to acid deposition (Bates, 2002). Furthermore, while major declines in bryophyte abundance resulting from increased N deposition have been reported (Lee & Caporn, 1998; Gunnarsson & Rydin, 2000; Bergamini & Pauli, 2001; Gordon et al., 2001; Pearce & van der Wal, 2002; Carroll et al., 2003; Pearce et al., 2003), elucidation of physiological responses driving such bryophyte losses have tended to focus on one or a few variables, often tissue N and the resulting N saturation measured through nitrate reductase activity (Gordon et al., 2001; Pearce & van der Wal, 2002; Pearce et al., 2003). While such studies have been invaluable in providing the first insights into likely causes of bryophyte decline, we currently lack a much broader analysis of physiological responses (such as responses to increased P limitation, photosystem damage, stress and shading) which would allow us to determine which variables are the most important drivers of bryophyte loss and understand their relative importance compared with those measured previously.
The importance of this fuller elucidation of bryophyte responses has further value since the sensitivity of bryophytes to N deposition means they are likely to be particularly useful sensitive bioindicators of the impacts of N deposition in ecosystem surveys and may provide earlier warning of the onset of N deposition impacts in comparison with higher plants. Physiological responses may also be particularly useful here since they may be more responsive and readily detectable, especially compared with measurements of growth or abundance, which can be harder to quantify with high precision in bryophytes. Furthermore, given that some previously heavily polluted areas (such as parts of the UK: NEGTAP, 2001; Fowler et al., 2004) are now experiencing declines in N deposition rates, as legislation and emission abatement strategies begin to take effect, bryophytes may also be valuable as sensitive and early indicators of ecosystem recovery from N deposition.
Surprisingly, few studies have assessed the effect of N deposition on photosynthesis or photosynthetic machinery, despite the likelihood that this is a sensitive target given that most leaf N is invested here (Lambers et al., 1998). Nitrogen pollution may also indirectly affect bryophytes through increased shading where higher plant cover increases (Bergamini & Peintinger, 2002; van der Wal et al., 2005) and may also exacerbate phosphorus (P) limitation of plant growth, the alleviation of which (though P additions) can counteract N deposition effects in higher plants (Phoenix et al., 2003) and Sphagnum species (Limpens et al., 2004).
Despite this knowledge, no broad analysis of multiple physiological responses on more than one species within the same ecosystem exists to assess the relative importance of different physiological responses in driving bryophyte loss and which may represent the best indicators of N deposition impacts and recovery.
With these concerns in mind, here we report a wide-ranging analysis of multiple physiological responses of two common terrestrial European bryophytes of contrasting growth form to long-term (11 yr) N deposition and recovery from N deposition in field plots (22 months ambient deposition following cessation of treatments). We also assess how these responses are effected by P fertilization since N deposition can enhance P limitation – a limitation that bryophytes may be particular susceptible to given their lack of roots and reliance on atmospheric deposition for nutrients.
Since any broad analysis cannot include measures of all possible parameters, our measurements were chosen to include those most commonly made in past studies and new measures that we predicted to be both responsive to N deposition and be likely drivers of bryophyte loss. Specifically, we assessed the nutritional status and requirements of the bryophytes through tissue chemistry (NPK) and activities of inducible nitrate reductase and phosphomonoesterase. We included tissue K since membrane damage can lead to K leakage. Furthermore, we undertook in vivo chlorophyll fluorescence measurements to evaluate the physiological health of the plants and impacts on photosynthetic machinery; we also assessed changes in the composition of photosystem II (through photosynthetic pigment analysis) and responses to the light environment where the mosses were growing (using de-epoxidation of the xanthophyll cycle pool (DES) and nonphotochemical quenching (NPQ)). We measured the ‘sclerophylly index’ to determine whether N deposition altered the proportion of nonphotosynthetic tissue. These physiological studies were coupled with measurements of the abundance of the bryophyte species under a range of different N deposition, P addition and recovery treatments in long-term field plots.
The study was undertaken in acidic Festuca–Agrostis–Galium grassland classified as U4e in the National Vegetation Classification (Rodwell, 1992), and located in the Derbyshire Dales National Nature Reserve, UK. This is the most widespread type of acid grassland in the UK and is known to be sensitive to N deposition; this type of grassland is in decline throughout Europe as a result of agricultural improvement and N deposition impacts (Preston et al., 2002; Carroll et al., 2003; Stevens et al., 2004). The Derbyshire Dales area has historically received among the highest rates of N deposition in the UK, with a cumulative total of up to 3000 kg N ha−1 over the last century (Fowler et al., 2004). The field site still experiences a high ambient deposition of c. 2.5 g N m−2 yr−1.
By analysing multiple physiological responses in the same two species at the same time we are able to (1) determine the relative sensitivity of different physiological variables to N deposition and recovery, (2) relate these to changes in abundance and hence begin to propose physiological mechanisms behind bryophyte species change, and (3) determine which physiological responses may provide the most sensitive indicators of both impacts of N deposition and the onset of recovery.
- Top of page
- Materials and Methods
- Supporting Information
This study has undertaken a wide-ranging analysis of multiple physiological variables in two widely distributed bryophytes to N deposition impacts, P additions and recovery.
We found that both species were highly sensitive to simulated enhanced N deposition, showing major and significant reductions in % cover at all levels of N addition. A number of key physiological variables were also shown to be highly sensitive to enhanced N deposition, providing insight into possible mechanisms causing the decline in abundance and which may have value as sensitive indicators of bryophyte responses to N loading. Importantly, while 22 months cessation of N treatments resulted in no clear recovery of cover, some physiological variables – particularly PME, shoot N, N : P and sclerophylly index (SI) – show clear recovery in P. purum, revealing their potential to provide sensitive early indicators of recovery from historic N loading.
Percentage cover and sclerophylly
The decline in abundance of the two dominant bryophytes within this acidic grassland supports earlier work undertaken on a neighbouring experiment in the same ecosystem (Lee & Caporn, 1998; Carroll et al., 2003). Our study further found important interspecific differences in sensitivity since R. squarrosus lost c. twofold more cover than P. purum. As discussed later, our physiological measurements suggest the declines in cover may be partially driven by increased P stress, increased fragility (reduced SI) and a decline in maximum potential photosynthetic rates despite investment of N in tissue leading to increased chlorophyll concentrations.
Interspecific differences in response to P addition (i.e. an alleviation of P limitation) were also seen with +P treatments, further reducing P. purum cover while having little impact on R. squarrosus. While P amendment has been shown to benefit bryophytes in some N addition studies (Limpens et al., 2004; Pilkington et al., 2007), our study suggests that such benefits may not occur in slow-growing species such as P. purum, possibly because they may become outcompeted by species that are not adversely affected by, or perhaps benefit from, P amendment.
The SI in P. purum showed a similar pattern of response to N deposition as percentage cover but also showed recovery that could not be detected in percentage cover. Reduction of cell wall thickness has been observed in some bryophytes when grown in a nutrient-rich medium (Voth, 1943) and may partly explain the reduction of SI in response to N deposition seen in our study. Alternatively, because surface area is measured on prostrate shoots and leaves overlap, the decreased SI may reflect a lowering of leaf density along the shoot (pers. obs.) resulting in less leaf overlap and a lower density of tissue per unit surface area. Both mechanisms of reduced SI, however, would suggest an increase in fragility (i.e. less sclerophylly), which could contribute to the decline in cover of P. purum.
Overall though, since SI of R. squarrosus was, in contrast, found to be unresponsive to N deposition, we cannot suggest SI as a good indicator of N deposition impacts in bryophytes in general.
Tissue N, K and P
In P. purum, increased N deposition resulted in increased tissue N and tissue N : P ratios whereas R. squarrosus showed no such changes. This latter absence of response has also been observed in R. squarrosus along an N deposition gradient across the UK (Stevens et al., 2006). These findings suggest tissue N may not be a robust indicator of N deposition impacts in bryophytes generally. Higher accumulation of N in P. purum compared with R. squarrosus was also observed by Solga & Frahm (2006) in a short-term fertilization experiment. Possibly a higher desiccation tolerance and opportunistic strategy for nutrient uptake in P. purum might confer a more direct relationship between N supply and N uptake (Bates, 1997). Furthermore, since R. squarrosus was already N saturated under +3.5N (as suggested by the significant decrease in NR), this would limit its N assimilation capacity and increase in tissue N. In this case, while the presence of rhizoids in R. squarrosus does not seem to be exacerbating increases in tissue N (by increasing uptake from the soil) they may be contributing to this moss becoming readily N saturated even under the lower rates of N deposition.
The reduction in shoot K concentration in R. squarrosus resulting from increased N deposition may result from exchange competition between and K+ ions or, more probably, by a direct toxic effect of N that leads to leakage of base cations through damaged cell membranes (Pearce et al., 2003). Direct toxic effects of N could also be exacerbated where decreased NR activity limits the bryophyte's capacity to assimilate N (Pearce et al., 2003). Since, in contrast, significant reduction in K was not observed in P. purum, this might indicate higher tolerance of this species to N deposition, which is consistent with the lesser reduction in % cover of this species compared with R. squarrosus.
Phosphomonoesterase and nitrate reductase
Our work reports the first evidence that PME activity is significantly stimulated in bryophytes under increased N loads – a finding consistent with similar responses seen in higher plant roots (Phoenix et al., 2004). Since this enzyme may increase the availability of phosphate (Turner et al., 2001), greater stimulation of PME by N loads in P. purum compared with R. squarrosus may also confer greater ability of the former species to enhance P uptake (and so reduce P limitation) under N enrichment. This response might also facilitate the higher assimilation of N in P. purum compared with R. squarrosus since the potential for bryophytes to use N can depend on the availability of other nutrients, especially P and K (Carfrae et al., 2007).
Reductions in induced NR under increased N loads have been well documented and are known to indicate reduced demand for N and the onset of N saturation since its activity is end-product inhibited (Glime, 2007). Interestingly, the magnitude of change in NR was much smaller than for PME. This difference may result if the assay for PME is more sensitive than that for NR, but more likely it suggests that exacerbation of P limitation is a more important driver of bryophyte responses to N deposition than N saturation per se.
In contrast to N loads, additions of P considerably reduced PME and increased NR activity in both species (changes that are indicative of a shift from P to N limitation under P amendment). Since decreases in shoot K caused by N loading in R. squarrosus disappeared with the addition of P, this supports the beneficial effect of P in this species: a possible explanation being the reduction of N toxicity through P amendments facilitating N assimilation.
The sensitivity and magnitude of response of PME and NR suggest that these have value as indicators of N deposition impacts. They may also provide good indicators of recovery since PME activity showed a marked return towards control levels in P. purum in N recovery plots (and some return was also seen in R. squarrosus– though not significantly so) and NR showed apparent recovery in R. squarrosus (although this should be viewed with caution since ‘recovery’ of NR was also seen in control plots suggesting the shift may be an artefact of spatial or temporal variation). Overall, while NR activity has previously been used as good indicator of N deposition impacts (Woodin & Lee, 1987; Pearce et al., 2003; Pitcairn et al., 2006), our work suggests that PME may actually provide a clearer (and possibly more sensitive) indicator of both N deposition impacts and recovery than NR.
Pigments and chlorophyll fluorescence
Increases in total chlorophyll (both chl a and b) in P. purum under enhanced N deposition suggests an investment of accumulated N in these pigments. The accompanying reductions in the Chl a/b ratio and increases in the (neoxanthin + lutein)/β-carotene ratio show that increased N loads alter the composition of pigment protein complexes in the thylakoid membranes and increase the relative proportion of light harvesting complexes compared with reaction centres. Interestingly, the increased pigment concentrations were associated with a significant decrease in the efficiency of PSII as both Fv/Fm and ΦPSII decreased under increased N deposition. An indirect shade effect of higher plants could explain the reduction of ΦPSII and the observed changes in pigment composition (Lambers et al., 1998). However, the N dose-dependent response and fast recovery detected in these variables (Fv/Fm, ΦPSII, total Chl, and Chl a/b and (neoxanthin + lutein)/β-carotene ratios) suggest that direct N loading is the main driver and not higher plant shading: in these P-limited grasslands, increased productivity of higher plants either does not occur or occurs little under increased N loads (and therefore also cannot show rapid recovery) (Carroll et al., 2003; Phoenix et al., 2003).
Overall, these changes suggest impairment of maximum potential photosynthetic rates, particularly as indicated by evidence of photo-inhibition (decline in Fv/Fm), and decline in energy utilization in photosynthesis (declines in ΦPSII). Since these responses are seen in both species, impairment of photosynthesis and hence growth, may be an important driver of decline in bryophyte cover.
In contrast to that direct effect, increased higher plant productivity that occurs with +P amendment in this grassland (Phoenix et al., 2003) would cause a shading effect of higher plants explaining the lower DES and NPQmax of P. purum in +P amended plots and possibly contributing to the reduction of cover of this moss under +P treatments.
In the case of R. squarrosus it was more difficult to assess the impacts of N loading on pigment composition because of limited plant material in the +14N plots. However, significant +N effects were still detected in the Chl a/b and (neoxanthin + lutein)/β-carotene ratios, following a similar pattern to that seen in P. purum. In R. squarrosus it was interesting to observe that total chlorophyll concentration seems to increase under combined +N +P treatments suggesting that +P addition might facilitate N assimilation as mentioned previously for this species: this is also consistent with increases in NR under +N +P treatments compared with +N only treatment.
Again, in contrast to the lack of recovery from N loading seen with percentage cover, a rapid recovery was observed in Fv/Fm, ΦPSII and total chlorophyll concentration in P. purum, NPQmax in R. squarrosus and Chl a/b and (neoxanthin + lutein)/β-carotene ratios in both species. Photosynthetic pigments and nondisruptive techniques such as chlorophyll fluorescence could be good complementary methods for early and sensitive indication of recovery from N pollution impacts and could be used to support other approaches such as PME measurements.
This study has undertaken an extensive assessment of bryophyte physiological responses to simulated enhanced N deposition, P amendment and recovery. In doing so, we have elucidated some of the drivers of sensitivity and changes in abundance of two common grassland bryophytes, as well as suggesting physiological parameters that may prove useful as sensitive indicators of N deposition impacts and recovery in national air-pollution monitoring schemes.
This work found P. purum to be more tolerant of simulated enhanced N deposition than R. squarrosus, a difference that may be attributable to a higher tolerance to P stress in P. purum, enabling this bryophyte to accumulate and invest the extra N supply in its photosynthetic apparatus (see the Supplementary Material, Table S1 for summary of interspecific differences in sensitivity). Intriguingly, since this greater tolerance of P. purum is conferred by a greater capacity to respond to N loading than R. squarrosus (in showing the greater physiological responses), it means P. purum is the better bioindicator species of N pollution impacts and recovery (and certainly over the range of N deposition rates we have tested). We also highlight that while 22 months cessation of N treatments resulted in no clear recovery of cover, many physiological variables did show recovery, in particular, changes in PME, N : P ratio and SI in P. purum and NR in R. squarrosus. Overall, the physiological variable of greatest value as a sensitive indicator of N deposition impacts and recovery in P. purum is PME, with tissue N : P ratios, Fv/Fm, and pigment analysis being useful as additional corroborating pollution indicators.
Validation of these variables as indicators of N deposition impacts and recovery in landscape and national scale surveys warrants further attention.