Arbuscular mycorrhizal colonization increases with host density in a ...

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Arbuscular mycorrhizal colonization increases with host density in a heathland community Blackwell Science Ltd

David R. Genney1, Sue H. Hartley2 and Ian J. Alexander1 1

Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB24 3UU, UK; 2Centre for Ecology and Hydrology, Banchory Research Station,

Banchory, Aberdeenshire AB31 4BY, UK

Summary Author for correspondence: David R. Genney Tel: +44 (0) 1224 273 725 Fax: +44 (0) 1224 272 703 Email: [email protected] Received: 2 April 2001 Accepted: 4 July 2001

• The relationship is examined here between Nardus stricta root length colonized (RLC) by arbuscular mycorrhizal (AM) fungi (% RLC) and vegetation composition in a heathland community. • Two approaches were taken: a field survey of AM colonization of Nardus at two contrasting locations (Glen Clunie and Glen Shee, UK) in relation to vegetation composition; and an 11-month bioassay of AM inoculum potential in Calluna vulgaris and Nardus swards using uncolonized Nardus transplants. • In the field survey, % RLC of Nardus was positively related to Nardus density. This relationship was strongest in Glen Clunie, probably because overall colonization levels were lower at the more fertile Glen Shee site. In the bioassay, Nardus transplants within Calluna swards developed little or no AM colonization, whereas those within Nardus swards had a mean RLC of 23%. • This is the first report that degree of AM colonization is related to host density in the field. Isolated Nardus plants in Calluna swards might be disadvantaged compared with Nardus plants in Nardus swards because of lower of AM colonization. Key words: arbuscular mycorrhiza, Nardus stricta, Calluna vulgaris (heather), heathland. © New Phytologist (2001) 152: 355–363

Introduction Nardus stricta (L.) is a common component of upland heath and grassland communities in the UK. Colonization of Nardus roots in these communities by arbuscular mycorrhizal (AM) fungi has been reported in a number of studies (Ali, 1969; Read et al., 1976; Sparling & Tinker, 1978a). One of the main mechanisms by which AM colonization is thought to benefit plants is by increasing the volume of soil exploration and hence quantity of immobile ions available to host-plants (Bolan, 1991). In the acid heathland soils in which Nardus grows, inorganic nitrogen availability limits plant growth due to low rates of mineralization, high rates of leaching and retention of nitrogen in complex organic forms (Read, 1991). The low pH of these soils also results in particularly poor phosphorus availability ( Willard, 1979). In pot studies, Nardus exhibits a range of responses to AM colonization, from large increases in growth and nutrient uptake (Heijne et al., 1994) to no response (Ali, 1976; Sparling & Tinker, 1978b). AM colonization

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therefore has the potential to improve the growth and competitive ability of Nardus in heathland environments. AM colonization is known to influence plant growth in other natural ecosystems (Newsham et al., 1994; Merryweather & Fitter, 1995a). Pot studies have demonstrated that AM colonization can alter the outcome of competition between grass species (Fitter, 1977; West, 1996) and AM colonization can have profound impacts on plant community structure (Gange et al., 1993; van der Heijden et al., 1998; Wilson & Harnett, 1998). Positive correlations have been observed between the proportion of root length colonized by AM fungi (% RLC) and phosphorus inflow in both pot (Sanders et al., 1977) and field (Merryweather & Fitter, 1995b) studies. It is therefore useful to understand the extent, and spatial variation in, colonization of individual host species, such as Nardus, in natural plant communities. This allows a better assessment of the possible significance of AM colonization in plant growth and plant/plant interactions in the field. To date, few studies have investigated the basis of

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spatial variation of AM colonization in the field (but see Klironomos et al., 1993; Merryweather & Fitter, 1998b). In recent decades, Calluna vulgaris (L.) Hull. heathland has been replaced by graminoid dominated vegetation in which Nardus is a frequent and dominant component. In the Scottish uplands, over-grazing is thought to be the main cause of heathland loss, because Calluna is grazed in preference to unpalatable grasses such as Nardus, particularly at high nutrient levels (Hartley, 1997). The ability of Nardus to successfully invade Calluna swards may be influenced by AM colonization, however, colonization can be hampered by the limited dispersal capabilities of AM fungi (Brundrett, 1991; Smith & Read, 1997). This may result in a lack of AM inoculum where a host-plant establishes in a habitat where there are no plants that support a compatible endophyte (Reeves et al., 1979; Allen & Allen, 1980; Louis, 1990). Calluna is colonized only by ericoid mycorrhizal (ErM) fungi (Read & Stribley, 1973) and therefore does not contribute to the maintenance of an active AM hyphal network. The roots of Nardus plants that establish in vegetation dominated by Calluna may therefore encounter fewer AM propagules and develop less colonization per unit root length than plants that establish among AM host species. In addition to a lack of AM inoculum, mycorrhizal Calluna roots may also inhibit the formation of AM colonization. Such interference has not been demonstrated between ericoid roots and AM colonization, but the presence of ErM Calluna roots has been shown to inhibit the growth of a range of ecto-mycorrhizal fungi (Robinson, 1972). Reduced AM colonization of host-plants when grown with nonhost-plants has also been demonstrated (Fontenla et al., 1999). The aim of this work was to determine whether AM colonization of Nardus in the field depends on plant community composition, in as much as this was expected to determine AM inoculum potential. In particular, the influence of Nardus and Calluna density and proximity on the proportion of Nardus root length colonized by AM fungi was investigated. Two approaches were taken; the first, a field survey of AM colonization of Nardus in relation to vegetation composition; the second, a bioassay of colonization in defined field situations using uncolonized Nardus transplants.

Materials and Methods Field survey Site descriptions The survey was conducted at two locations of approx. 1.5 hectares in Aberdeenshire, UK: Glen Clunie (grid reference NO147865) and Glen Shee (grid reference NO123374). These locations were at an altitude of 400 m, had an easterly aspect of slope and consisted of a mosaic of Nardus and Calluna dominated communities (U5 and H12 classifications in the British National Vegetation Classification (Rodwell, 1991; Rodwell, 1992) ). In Glen Clunie the predominant soil type is a peaty podsol overlying a base-poor

schist (Welch & Scott, 1995). In Glen Shee the soil overlies dolomitic outcrops and is a brown-earth with a less well developed A0 horizon. Local abundance of calcicole species such as Briza media, Helianthemum numularium, and Saxifraga aizoides in Glen Shee are indicators of base enrichment. Mean nitrogen mineralization rates of −17 and 35 µg N mg−1 dry soil wk−1 and soil organic matter contents (LOI) of 85% and 15% have been recorded at Glen Clunie and Glen Shee, respectively (B. Emmett, pers. com.). The average annual rainfall (1980–1999) in both glens is approx. 890 mm (British Meteorological Office). Both are grazed by sheep and red deer, but levels of grazing are higher in Glen Shee (Hartley, 1997). Field sampling procedure In September 1997, 24 target Nardus plants were selected in each glen from Nardus/Calluna mosaics such that proximal vegetation cover (within a 0.25-m2 quadrat centred on the target plant) ranged from almost pure Calluna swards to Nardus swards containing no Calluna (Nardus /Calluna quadrats). Six isolated Nardus plants were also selected from Calluna-free swards dominated by other grass species in each glen (Nardus/grass quadrats). The minimum distance between target Nardus plants was 5 m. In addition to Nardus and Calluna, the cover of all other vascular plant species within the 0.25 m2 quadrats was recorded to the nearest 5%. Species with cover less than 5% were recorded as 1%. Distance from the target plant to the nearest Nardus and Calluna neighbour was also recorded. Each target Nardus plant was then removed in a 20-cm diameter × 20 cm deep soil core, sealed in a plastic bag and stored in a cold-room at 3°C before analysis. Nardus tissue analysis In the lab, green Nardus tillers were harvested, oven-dried for 48 h and milled before a c. 100 mg subsample was digested by the sulphuric acid/hydrogen peroxide method (Allen, 1989). Nitrogen content was measured as ammonium using the continuous flow, indophenol-blue method (Rowland, 1983), and phosphorus using the molybdenum blue method (Allen, 1989). Determination of field AM colonization The soil cores were carefully washed with a water jet, and Nardus roots extracted with tweezers after being traced back to the base of tillers. Roots were extracted from a depth of 1–10 cm. Lateral roots were removed from the coarse nodal roots, washed carefully, cleared in 10% KOH (20 min, 80°C), acidified in 1% HCl (1 h) and stained (20 min, 80°C) with 0.05% trypan blue in acid glycerol. After destaining in acid glycerol, roots were scored for % RLC by mycorrhizal endophyte using the line-intersect method (McGonigle et al., 1990). Two samples from Glen Clunie were rejected because insufficient root material could be extracted to allow an accurate assessment of colonization. Statistical analysis of field survey Minitab® (v. 11.21, Minitab Inc., PA, USA) was used to perform one-way ANOVA to determine differences in % RLC and shoot nutrient

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Sites Five 1 ha sites, at an altitude of c. 500 m were selected along the length of Glen Clunie. Each site contained a mosaic of Nardus grassland and Calluna heath. Preparation of Nardus bioassay plants Nardus seeds were collected in late August 1997 from Glen Derry, Aberdeenshire, UK (grid reference NO040958). In September 1998, after 5 min surface sterilization with 1.0% sodium hyperchlorite, the seeds were germinated on autoclaved sand in an unheated incubator. Plant pots (80 mm diameter, 60 mm depth) were lined with nylon curtain netting (mesh size approx. 1 mm) and filled with autoclaved, acid washed medium sand (mean particle size, 0.1 mm, Hepworth Minerals and Chemicals Ltd, Cheshire, UK; pH adjusted to 3.8 with HCl). Three evensized Nardus seedlings were carefully transplanted into each pot and watered with 20 ml half-strength, phosphorus-free Rorison’s solution (Hewitt, 1966). No further nutrients were applied and the substrate was kept moist with distilled water. The pots were covered with plastic bags to minimize the chance of contamination with AM propagules and grown for 3 wk in a glasshouse. After hardening off for a further week, the plants were transplanted to the field. Before field transplantation, five randomly selected plants were harvested to check for AM colonization; none was observed. Field transplantation of bioassay plants The sets of three plants were transplanted in October 1998 in their netting bags into holes dug into the field soil. This arrangement enabled roots to grow both into and out of the mesh bag. Each plant was protected from grazing by a PVC coated wire mesh cloche pegged down with plastic pegs. At each of the five bioassay sites, five transplants were planted into Calluna swards (> 80% Calluna) and five into Nardus swards (> 80% Nardus), with a minimum distance of 5 m between any two transplants. Transplants in Callunadominated vegetation were a minimum of 2 m from the

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Statistical analysis of bioassay A χ2 test was used to determine the effect of vegetation type on the frequency of AM colonized and uncolonized bioassay plants. % RLC (n + 1) data were Box-Cox transformed and GLM-ANOVA was then used to determine the effects of vegetation type and site. The design was unbalanced because plants were lost in the field due to vandalism and persistent grazing by sheep, resulting in 21 surviving plants from Nardus swards and 18 from Calluna swards.

Results Field survey In the following description of field survey results, ‘glen/s’ refers to the Glen Clunie and Glen Shee locations described above. AM colonization of field Nardus The overall mean proportion of root length colonized by AM fungi was 57.59% (S.E. = 2.25), with a range from 29 to 92%. % RLC in Glen Clunie was significantly higher than in Glen Shee (F1,56 = 12.09; P = 0.001) (Fig. 1a). All of the colonization was of the Paris-type and composed of broad hyphae (c. 3 µm diameter) that formed intracellular coils within cortical cells. Auxiliary arbuscules occurred on some coils, but the majority appeared to be arbuscule free. No intercellular hyphae, characteristic of Arum-type colonization, was observed. Tissue nitrogen and phosphorus concentrations of field Nardus There was no overall relationship between tissue nitrogen or phosphorus concentrations and cover of Nardus 80

0.20

(a)

(b)

0.16

60

Tissue P (%)

Bioassay

nearest individual Nardus plant and 5 m from the nearest Nardus sward (defined as > 1 m2 of Nardus cover). The reciprocal rules were applied to the positioning of transplants into Nardus dominated vegetation. Harvesting took place in September 1999, after 11 months of growth. Plants were removed in their nylon netting bags and stored at 3°C before analysis. Roots from the bioassay plants, growing in the sand within the nylon netting bags, were extracted, washed, stained with trypan blue (as above) and scored for % RLC.

RLC (%)

concentrations between glens. For each glen, Pearson’s correlation coefficient and best-subset regression (BSR) analyses (Minitab) were used to determine which variables, and combination of variables, correlated with, and described the greatest amount of variation in, % RLC of target Nardus plants in Calluna / Nardus quadrats. Nardus/grass quadrats were treated separately. Floristic data were subjected to detrended correspondence analysis (DCA) using PC-Ord® (MjM Software, Gleneden Beach, OR, USA) (McCune & Mefford, 1997) and the resultant axes were incorporated into the BSR analysis. Anderson-Darling normality tests (Minitab) were performed on residuals of all analyses to ensure their suitability for parametric statistics. Where residuals were not normal, Box-Cox analysis (Minitab) was used to determine the most suitable transformation function. Mean values given in the text are presented ±1 SE.

40 20

0.12 0.08 0.04 0.00

0

Glen Clunie

Glen Shee

Glen Clunie

Glen Shee

Fig. 1 Overall % root length colonized (RLC) (a) and shoot phosphorus concentration (b) of Nardus plants from the Glen Clunie and Glen Shee sites.

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(N: F1,58 = 1.22; P = 0.274 and P: F1,58 = 1.06; P = 0.308) or Calluna (N: F1,58 = 2.21; P = 0.143 and P: F1,58 = 0.120; P = 0.725). Tissue phosphorus concentrations were higher in Glen Shee than in Glen Clunie (F1,58 = 25.29; P < 0.001) (Fig. 1b) but there was no difference in tissue nitrogen (mean = 1.52 ± 0.05%) between glens (F1,58 = 2.09; P = 0.155). Vegetation analysis The pooled Glen Clunie and Glen Shee data on % cover of individual species (in the quadrats from which sample Nardus plants were taken) were subjected to DCA ordination to determine whether there were differences in vegetation composition between glens, and to examine in more detail the relationship between vegetation composition and % RLC at

each glen. Rare species (frequency < 20% that of the commonest species) were downweighted in proportion to their frequency. When Calluna and Nardus cover were incorporated into the data set, axis one (eigenvalue = 0.62) correlated with Calluna cover (F1,58 = 15.2; P < 0.001; R 2 = 20.7%) and axis two (eigenvalue = 0.23) represented a separation of samples by glen. Because Calluna and Nardus cover was determined by sampling strategy, further ordination was conducted with the exclusion of these species. This modified analysis demonstrates a clear division of samples by glen on axis one (eigenvalue = 0.80). Twice as many species were recorded in Glen Clunie than in Glen Shee, and while many of these were recorded only occasionally (Table 1), it is these species that appear to

Table 1 Site differences in vascular plant species composition associated with Calluna and Nardus (species names follow Stace (1991). Species in bold are site specific

Site

Glen Clunie

Glen Shee

No. of species

36

18

Dwarf shrubs

ErM ErM AM ErM ErM

Erica cinerea Erica tetralix* Myrica gale Vaccinium myrtillus Vaccinium vitis-idea*

ErM ErM ErM

Erica cinerea Vaccinium myrtillus Vaccinium vitis-idea*

Herbs

AM NM AM AM AM AM OrM AM AM AM AM AM EcM AM NM AM AM AM AM AM AM

Achillea millefolium* Achillea ptarmica* Alchemilla alpina* Antennaria dioica Campanula rotundifolia* Cerastium fontanum* Dactylorhiza maculata* Galium saxatile Gentianella campestris* Lathyrus linifolius* Lotus corniculatus Narthecium ossifragum* Persicaria vivipara Potentilla erecta Pedicularis sylvatica* Prunella vulgaris* Succisa pratensis Trichophorum caespitosum* Trientalis europaea* Veronica officinalis* Viola riviniana

AM AM AM AM AM AM AM AM

Achillea millefolium* Campanula rotundifolia* Cerastium fontanum* Galium saxatile Persicaria vivipara Potentilla erecta* Prunella vulgaris* Trientalis europaea*

Grasses, etc ....

AM AM NM AM AM AM AM NM AM AM

Agrostis canina Agrostis capillaris Carex spp. Danthonia decumbens Festuca ovina Festuca vivipara Holcus lanatus* Juncus squarrosus Luzula multiflora Molinia caerulea*

AM AM NM AM AM AM AM

Agrostis canina Agrostis capillaris Carex spp. Danthonia decumbens Deschampsia flexuosa Festuca ovina Luzula multiflora

*, occasional species with cover not exceeding 5% in any quadrat. Prefix denotes mycorrhizal status (following Harley & Harley (1987)): ErM, ericoid mycorrhizal; AM, arbuscular mycorrhizal; EcM, ectomycorrhizal; OrM, orchid mycorrhizal; NM, nonmycorrhizal.

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Research Table 2 Pearsons correlation matrix for variables analysed in model for Glen Clunie (a) and Glen Shee (b). DCA axes one-three use DCA I data set % Cover

Nearest neighbour

DCA I

% RLC

Nardus

Calluna

%N

%P

Nardus

Calluna

Ax. 1

Ax. 2

(a) Glen Clunie % Nardus % Calluna %N %P Nardus (nn)† Calluna (nn)† Ax.1 DCA I: Ax.2 Ax.3

0.46* – 0.55** – 0.02 – 0.01 0.13 – 0.53* 0.46* – 0.41 – 0.43*

– 0.74*** – 0.03 – 0.04 0.24 – 0.48 0.37 – 0.92*** – 0.42*

0.10 0.17 –0.07 0.37 –0.84*** 0.70*** 0.54**

0.37 –0.32 0.04 –0.15 –0.06 0.06

–0.29 0.02 –0.20 0.03 0.13

–0.21 –0.06 –0.20 –0.18

–0.37 0.47* 0.46*

– 0.37 – 0.50*

(b) Glen Shee % Nardus % Calluna %N %P Nardus (nn)† Calluna (nn)† Ax.1 DCA I: Ax.2 Ax.3

0.32 – 0.23 0.05 – 0.54** 0.12 – 0.09 0.18 0.36 – 0.13

– 0.93*** 0.33 0.15 0.30 – 0.78*** 0.82*** 0.96*** – 0.46*

–0.29 –0.24 –0.38 0.75*** –0.85*** –0.90*** 0.51*

0.15 0.40 –0.25 0.32 0.32 0.04

0.07 –0.15 0.10 0.17 0.07

–0.31 0.44* 0.20 –0.32

–0.76*** –0.71*** 0.38

0.72*** – 0.48*

0.28

– 0.26

†(nn), nearest neighbour. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Further analysis Following separate re-ordination of the vegetation data from each of the two glens, axis 1–3 scores, along with other vegetation measurements, were incorporated into best subset regression analysis and Pearson’s correlation matrices to ascertain which of the measured variables correlated with, and described the greatest degree of variation in, % RLC. Variables incorporated into the model were: • % RLC of the sample plant. • Local % Calluna and Nardus cover (i.e. % cover in 0.25 m2 quadrat). • Distance from sample plant to nearest Calluna and Nardus neighbour. • Tissue nitrogen and phosphorus concentrations of the sample plant. • Axis 1, axis 2 and axis 3 sample scores for DCA ordination of vegetation data from each site separately. DCA analysis was performed both with (DCA I) and without (DCA II) the inclusion of Calluna and Nardus cover values. A further analysis was conducted with the exclusion of

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100 80

RLC (%)

cause the separation of glens on axis one. The difference in species composition between the Glen Shee and Glen Clunie sites conform to the ‘species poor’ and ‘Carex panicea-Viola riviniana’ U5 subcommunities, respectively, as defined by Rodwell (1992). The moisture-loving species, Molinia caerulea, Myrica gale, Narthecium ossifragum, Juncus squarrosus and Erica tetralix, fell towards one end of axis two (eigenvalue = 0.37), suggesting that this axis describes a hydrological gradient.

60 40 20

% RLC = 0.27 × % Nardus + 55.0 R 2 = 0.21, F1,20 = 5.37, P < 0.05

0 0

20

40

60

80

100

Nardus cover (%) Fig. 2 Glen Clunie: relationship between % root length colonized (RLC) and % Nardus cover. Regression line and closed circles represent quadrats from Nardus/Calluna swards (n = 22). Open circles quadrats from Nardus/other grass swards (n = 6).

Calluna, Nardus and nonAM hosts species (see Table 2), but this did not describe any further variation in % RLC. Glen Clunie There was a strong negative correlation between the cover of Nardus and Calluna (P < 0.001, Table 2a) which was predetermined by the sampling strategy. % RLC was positively correlated with % Nardus cover (Fig. 2) (F1,20 = 5.37; P < 0.05; R 2 = 21.0%) and negatively correlated with % Calluna cover (% RLC = 75.9 – 0.316% Calluna; F1,20 = 8.60; P < 0.01; R 2 = 30.1%). That is to say that % RLC was greatest in Nardus dominated swards and lowest in isolated Nardus

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plants in Calluna swards. There were further significant correlations between % RLC and DCA axis 1, DCA axis 3 and distance to the nearest Calluna neighbour, however, these variables did not describe further variation in % RLC once variation due to Calluna cover was removed. There was no relationship between % RLC and tissue nitrogen or phosphorus concentrations. Glen Shee In Glen Shee, % RLC was negatively correlated with tissue phosphorus concentration (Fig. 3a, Table 2b) (F1,22 = 9.10;

AM colonization of bioassay transplants

100

(a)

80

RLC (%)

P < 0.01; R 2 = 21.3%). This was the only significant single correlation. There was no direct relationship between % RLC and Nardus cover (Fig. 3b) (F1,22 = 2.44; P > 0.10; R 2 = 10.0%), but % Nardus cover was significantly, and positively, related to the residual variation in % RLC once variation due to % P was removed (Fig. 3c) (F1,22 = 8.03; P = 0.01; R 2 = 27.2%). % Calluna cover accounted for less of the variation in these residuals (F1,22 = 4.93; P < 0.05; R 2 = 18.3%). The greatest amount of variation in % RLC was therefore explained by a combination of tissues phosphorus concentration and % Nardus cover (% RLC = 88.6 – 302 tissue P + 0.17% Nardus; F2,21 = 8.72; P < 0.01; R 2 = 45.4%).

60 40 20

% RLC = –272 × % P + 92.8 R 2 = 0.29, F1,22 = 9.10, P < 0.01

0 0.1

0.12

0.14

0.16

0.18

0.2

0.22

0.24

Nardus tissue P (%)

The roots of 80% of the Nardus transplants became colonized by a coarse endophyte which was morphologically indistinguishable from the endophyte encountered in the field survey. The % RLC of transplants was an order of magnitude greater in Nardus swards (mean, 23.1 ± 2.1%) than in Calluna swards (mean, 0.5 ± 0.3%) (F1,29 = 515; P < 0.001). All of the transplants in the Nardus swards were colonized, in contrast to only 39% of the transplants from Calluna swards (χ2 = 16.5, P < 0.001). There was no significant difference in % RLC between the five sites.

100

(b)

Discussion

RLC (%)

80 60 40 20

% RLC = 0.13 × % Nardus + 42.9 R 2 = 0.10, F1,22 = 2.44, P > 0.10

0 0

20

40

60

80

100

Nardus cover (%) 30

Residual RLC (%)

(c)

20 10 0 –10

Residual % RLC = 0.17 × % Nardus – 8.88 R 2 = 0.27, F1,22 = 8.03, P = 0.01

–20 –30 0

20

40

60

80

100

Nardus cover (%)

Fig. 3 Glen Shee: relationship between (a) % root length colonized (RLC) and % Nardus tissue phosphorus concentration (b) % RLC and % Nardus cover and (c) residual variation in % RLC (once variation due to tissue phosphorus was removed) and % Nardus cover; the significance of this regression was computed on Log (residual + 30) transformed data. Regression lines and closed circles represent quadrats from Nardus/Calluna swards (n = 24) and open circles quadrats from Nardus/grass swards (n = 6).

The field survey of AM colonization of Nardus roots demonstrated that % RLC is greatest where % Nardus cover is highest and decreased where Nardus is replaced by Calluna. If there is a positive relationship between % RLC and host performance, then the growth, and competitive ability, of isolated Nardus plants in Calluna swards may be lower than that of Nardus plants in Nardus swards. If this is true, Nardus may be better able to out compete and replace Calluna at discrete community boundaries than by dispersive seedling invasion. However, Nardus is shaded out by intact Calluna canopies at community boundaries and requires gaps (such as those caused by heavy grazing) in order to establish effectively (Alonso & Hartley, 1998). Colonization of such gaps requires that Nardus has the ability to grow in isolation from other Nardus plants. In addition, the rate of vegetative Nardus advancement at community boundaries is limited compared with dispersive advancement because typical tiller elongation is only in the order of 2 cm yr−1 (Chadwick, 1960). Reduced or delayed mycorrhizal colonization of isolated Nardus plants that disperse into Calluna swards may therefore be one mechanism that limits the rate at which Nardus can replace Calluna. The assumption that mycorrhizal benefit is linearly related to % RLC may not necessarily be true (Gange & Ayres, 1999), for example, it has been demonstrated that plant response to mycorrhizal colonization sometimes declines with increasing host root density (Koide, 1991; Allsopp & Stock, 1992).

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Calluna roots are able to exclude Nardus roots from surface organic layers (Genney et al., 2000) and may consequently restrict lateral spread of Nardus roots into Calluna swards. It is therefore probable that root systems of individual (< 5% cover) Nardus plants within Calluna dominated quadrats were indeed ‘isolated’ in that they had no contact with other Nardus root/mycorrhizal systems. In the context of inoculum availability to Nardus, it is clear that whereas seedlings/tillers that establish within the rooting zone of mycorrhizal Nardus plants have access to existing mycorrhizal networks, isolated plants must rely on rather limited AM fungal dispersal mechanisms as sources of inoculum. Spores were not found attached to external hyphae during endophyte quantification, or in soil surrounding colonized Nardus roots (although spore extraction was only attempted on five of the soil cores). The apparent absence of spores supports the notion that inoculum availability limits colonization of isolated Nardus plants. The endophyte/s observed in the Nardus roots in this study showed a high degree of morphological uniformity, and on the basis of the morphological characteristics used by Merryweather & Fitter (1998a) would be classified as a single morphotype. This suggests that Nardus was colonized either by a single endophyte or by a number of endophytes of identical morphology. The mean proportion of root length colonized was consistent with previous assessments of field colonization levels in this species (Read et al., 1976; Sparling & Tinker, 1978a). The abundance of intracellular coils and lack of arbuscules and intercellular hyphae agrees with the description of colonization given by Ali (1969) and conforms to the Paristype morphology described by Smith & Smith (1997). Previous studies have described low (< 10% RLC) colonization of Nardus roots by a fine, Arum-type endophyte that resembles Glomus tenuis (Ali, 1969; Sparling & Tinker, 1978a), but this type of colonization was not observed in this study. Although the colonization of isolated Nardus was lower than those in Nardus swards, these plants still attained c. 50% RLC. A criticism of the field survey may be that all plants were mature (of flowering age) and had therefore been exposed to the possibility of AM colonization for variable, but considerable, lengths of time. It could be argued that the probability of colonization of young seedlings is most important for successful competitive establishment. However, the bioassay confirmed that there was indeed a large difference in inoculum potential between Calluna and Nardus swards. Two further interesting points arose from the bioassay. First, because low % RLC was recorded in Calluna sward transplants by an endophyte/s morphologically similar to that found in the highly colonized plants from Nardus swards, small amounts of inoculum may have dispersed into Calluna from local Nardus populations. Second, by the end of the 11 month experiment, colonization of roots of the Nardus sward transplants was still much lower than that of roots from the indigenous Nardus population. This may have been due to the inhibition of colonization in the sandy substrate or to rapid root growth of the

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transplants, but is more likely to be due to naturally slow rates of growth of the dominant field endophyte/s. Slow growth rates of Paris-type colonization compared with Arum-type colonization have been noted elsewhere (Brundrett & Kendrick, 1990). At the Glen Clunie site, where Nardus shoot phosphorus concentrations were lowest, % RLC was positively related to % Nardus cover and negatively to % Calluna cover. Because of the unavoidable auto-correlation between % Calluna cover and % Nardus cover, the question arises as to whether the presence of Nardus or the absence of Calluna was most important in determining AM colonization. It could be argued that high % Calluna cover inhibited AM colonization, however, as the lowest % RLCs were observed in Nardus/grass swards, where there was no Calluna, it seems more likely that % Nardus cover is the controlling factor. To the best of our knowledge, this is the first time that levels of AM colonization have been related to host density in a natural plant community. Pot investigations into the effect of host density on % RLC have produced variable results, but there is a tendency for colonization to be greatest at low host densities (Abbott & Robson, 1984; Facelli et al., 1999). However, the conditions of such pots studied are very different from the field because inoculum is provided at a constant density, at a single point in time. In the field, inoculum availability is determined by propagule dispersal and the existing population of AM colonized roots. Such processes are likely to result in an increase in inoculum availability with time in natural plant communities, due to an increased chance of propagule import and subsequent increase in AM colonized root density. In a field survey of the AM colonization of bluebell, Merryweather & Fitter (1998b) found that the distribution of some AM endophyte types was related to the tree species under which the bluebell grew. It was suggested that these spatial correlations occurred because bluebells share common fungal partners with nearby tree species. % RLC did not correlate with the cover of other potential AM hosts in the present study and % RLC of isolated Nardus plants in swards of other grass species were as low as Nardus plants in Calluna swards. This suggests that the Nardus endophyte may not be shared by other species. Janos (1980) hypothesized that dispersal limitation of AM plant or fungal species is most likely to occur when the association is specific. Sanders (1993) found no evidence for AM specificity when pot plants were inoculated with a range of field inoculum, however, differential host colonization has been demonstrated in a grassland community (McGonigle & Fitter, 1990). If such specificity occurs between Nardus and its endophyte, host density dependent % RLC may not be confined solely to Nardus/ Calluna mixtures. By contrast to Glen Clunie, in Glen Shee, shoot phosphorus concentration was the best predictor of % RLC. There is a wealth of evidence that mycorrhizal colonization is inhibited by elevated tissue phosphorus concentration (Menge et al.,

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1978; Graham et al., 1981; Amijee et al., 1989; Bruce et al., 1994). We cannot be certain that elevated tissue phosphorus concentrations, rather than some other factors (such as different endophyte/s) in Glen Shee, were the cause of lower % RLC, but it must be a strong possibility. There was no relationship between shoot phosphorus concentration and % RLC in Glen Clunie, and lower shoot concentrations at that site may not have been high enough to inhibit colonization. If, as demonstrated in Glen Clunie, % RLC is related to host density, the response should be ubiquitous and demonstrable at both sites. Further analysis of the Glen Shee data demonstrated this to be the case, although the effect was much weaker and only apparent after analysis of the residual variation once variation due to phosphorus had been removed. The combination of survey and field bioassay has demonstrated that when Nardus disperses into Calluna dominated vegetation, mycorrhizal colonization may be reduced. Assuming that levels of colonization affect plant performance in the field, the competitive ability of Nardus would be reduced. This could be of particular importance for seedling establishment since Nardus planted among Calluna developed little or no mycorrhizal colonization over an 11-month period. From a mycorrhizal perspective, Nardus may have greater competitive success at a vegetatively advancing Nardus/Calluna boundary rather than by replacement of Calluna by seeddispersed individuals. Correlation of % RLC with host density was demonstrated in Glen Clunie. However this relationship was masked in Glen Shee by higher Nardus phosphorus concentrations, which probably suppressed mycorrhizal formation. This study has demonstrated that spatial variation in mycorrhizal colonization can be explained by ecologically meaningful parameters in a natural plant community.

Acknowledgements We would like to thank the Invercauld Estate for permission to work on their land. This work was funded by an NERC CASE studentship to DRG.

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Rodwell JS. 1991. British plant communities, vol. 2. Mires and heaths. Cambridge, UK: Cambridge University Press. Rodwell JS. 1992. British plant communities, vol. 3. Grass lands and montane communities. Cambridge, UK: Cambridge University Press. Rowland AP. 1983. An automated method for determining the ammonium-N in ecological materials. Communications in Soil Science and Plant Analysis 13: 49–63. Sanders IR. 1993. Temporal infectivity and specificity of vesicular– arbuscular mycorrhizas in co-existing grassland species. Oecologia 93: 349–355. Sanders FE, Tinker PB, Black RLB, Palmerley SM. 1977. The development of endomycorrhizal root systems: I. Spread of infection and growthpromoting effects with four species of vesicular–arbuscular endophyte. New Phytologist 78: 257–268. Smith SE, Read DJ. 1997. Mycorrhizal symbiosis. London, UK: Academic Press. Smith FA, Smith SE. 1997. Structural diversity in (vesicular)–arbuscular mycorrhizal symbiosis. New Phytologist 137: 373 –388. Sparling GP, Tinker PB. 1978a. Mycorrhizal infection in Pennine grassland I. Levels of infection in the field. Journal of Applied Ecology 15: 943–950. Sparling GP, Tinker PB. 1978b. Mycorrhizal infection in Pennine grassland II. Effects of mycorrhizal infection on the growth of some upland grasses on gamma-irradiated soils. Journal of Applied Ecology 15: 951–958. Stace C. 1991. New flora of the British Isles. Cambridge, UK: Cambridge University Press. Welch D, Scott D. 1995. Studies in the grazing of heather moorland in north-east Scotland VI. Twenty-year trends in botanical composition. Journal of Applied Ecology 32: 596–611. West HM. 1996. Influence of arbuscular mycorrhizal infection on competition between Holcus lanatus and Dactylis Glomerata. Journal of Ecology 84: 429–438. Willard LL. 1979. Chemical equilibria in soils. Chichester, UK: John Wiley & Sons. Wilson JM, Harnett DC. 1998. Interspecific variation in plant responses to mycorrhizal colonisation in tallgrass prairie. American Journal of Botany 85: 1732–1738.

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THE 13th CONGRESS OF THE FEDERATION OF EUROPEAN SOCIETIES OF PLANT PHYSIOLOGY WILL BE HELD IN CRETE, GREECE from 2 to 6 of September 2002 For more information please visit www.biology.uoc.gr/meetings/fespp www.maris.gr or contact [email protected] [email protected] [email protected]

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The New Phytologist Trust is dedicated to the promotion of plant science. New Phytologist Symposia – independent, international meetings – are one way in which the Trust fulfils this aim, supporting the research community in bringing together the top scientists in a given field. These meetings, established in 1995, have quickly gained a reputation for excellence through stimulating presentations and open discussion: • Plant–microbe symbiosis – molecular approaches York, UK, 1995 • Putting plant physiology on the map – genetic analysis of developmental and adaptive traits Bangor, UK, 1997 • Major biological issues resulting from anthropogenic disturbance of the nitrogen cycle Lancaster, UK, 1997 • At the crossroads of plant physiology and ecology – causes and consequences of variation in leaf structure Montpellier, France, 1998 • Root dynamics and global change – an ecosystem perspective Tennessee, USA, 1999 Held in collaboration with GCTE • Signalling in plants Wye, UK, 2000 Held in collaboration with The Biochemical Society • Stomata 2001 Birmingham, UK, 2001 2002 is the 100th Anniversary Year of New Phytologist and, celebrating this, the New Phytologist Trust will be funding three special symposia in 2002, located in three different countries and covering the three sections of the journal, Function (Section Editor Dale Sanders FRS), Environment (Section Editor Richard Norby) and Interaction (Section Editor Francis Martin). New Phytologist is committed to publishing top research across the breadth of plant science, and it is hoped that these symposia will provide focus to that aim. As usual, the Trust is offering a number of bursaries for those research students and postdoctoral scientists who are presenting posters. The first of this series of three, and the 8th New Phytologist Symposium, will be ‘Impacts of soil microbes on plant population dynamics and productivity’, to be held at the Viikki Biocenter (Infocenter), University of Helsinki, Finland on 10 –14 June 2002. If you are interested in attending, please get in touch with Robin Sen ([email protected]). If you have any other queries about the symposia, do not hesitate to get in touch with Central Office ([email protected]).

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New Phytologist Tansley reviews 2000–2001 FREE MATERIALS To mark New Phytologist’s 100th Anniversary Year, articles from the prestigious Tansley review series are being made available free of charge to download. Go to www.newphytologist.com and follow the links to download your copy.

2000 • The structure of photosynthetic complexes in bacteria and plants: an illustration of the importance of protein structure to the future development of plant science Cogdell RJ, Lindsay JG New Phytologist 145: 167–196 • Numerical and physical properties of orchid seeds and their biological implications Arditti J, Ghani AKA New Phytologist 145: 367– 421 • Possible roles of zinc in protecting plant cells from damage by reactive oxygen species Cakmak I New Phytologist 146: 185 –205 • Oxygen processing in photosynthesis: regulation and signalling Foyer CH, Noctor G New Phytologist 146: 359 –388 • Mechanisms of caesium uptake by plants White PJ, Broadley MR New Phytologist 147: 241–256 • Ecological hazards of oceanic environments Crawford RMM New Phytologist 147: 257–281 • Impact of ozone on the reproductive development of plants Black VJ, Black CR, Roberts JA, Stewart CA New Phytologist 147: 421– 447 • Cyanobacterium–plant symbioses Rai AN, Söderbäck E, Bergman B New Phytologist 147: 449 – 481 • Carbon economy in lichens Palmqvist K New Phytologist 148: 11–36

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• Post-ingestion metabolism of fresh forage Kingston-Smith AH, Theodorou MK New Phytologist 148: 37–55 • Theoretical considerations of optimal conduit length for water transport in vascular plants Comstock JP, Sperry JS New Phytologist 148: 195 –218 • Pathways to abscisic acid-regulated gene expression Rock CD New Phytologist 148: 357–396

2001 • Scaling ozone effects from seedlings to forest trees Samuelson LJ, Kelly JM New Phytologist 149: 21– 42 • The apoplast and its significance for plant mineral nutrition Sattelmacher B New Phytologist 149: 167–192 • Tree and forest functioning in response to global warming Saxe H, Cannell MGR, Johnsen Ø, Ryan MG, Vourlitis G New Phytologist 149: 369 – 400 • Unravelling response-specificity in Ca2+ signalling pathways in plant cells Rudd, JJ, Franklin-Tong, VE New Phytologist 151: 7–34 • Calmodulin as a versatile calcium signal transducer in plants Snedden WA, Fromm, H New Phytologist 151: 35 – 66 • MAP kinase signal transduction pathways in plants Morris PC New Phytologist 151: 67– 89

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