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Keywords:

  • Monotropaceae;
  • seedling development;
  • fungal specificity;
  • myco-heterotrophy;
  • in situ germination;
  • Tricholoma

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Germination and symbiotic development of the myco-heterotrophic plant Monotropa hypopitys were studied by sequential recovery of packets of seed buried in dune slacks in relation to distance from mature M. hypopitys and presence and absence of shoots of its autotrophic coassociate Salix repens.
  • • 
    Fungal associates of M. hypopitys growing under S. repens in the dune slacks, and under S. caprea and Pinus sylvestris at two other locations in the UK, were identified by molecular analysis.
  • • 
    While the earliest stage of germination could be found in the absence both of mature M. hypopitys, and S. repens, further development was dependent upon mycorrhizal colonisation, which was most common close to these plants. Molecular analysis showed that when growing with Salix, M. hypopitys associated with the Salix-specific ectomycorrhizal fungus Tricholoma cingulatum, whereas under Pinus it was colonised by the closely related, Pinaceae-specific, T. terreum.
  • • 
    We establish the first definitive chronology of development of M. hypopitys and highlight its critical dependence upon, and specificity for, locally distributed Tricholoma species that link the myco-heterotroph to its autotrophic coassociates.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The subfamily Monotropoideae (Ericaceae) consists of ten genera (Wallace, 1975). All the species lack chlorophyll and hence can be characterised as myco-heterotrophs (Leake, 1994). The most widely distributed species of this subfamily, Monotropa hypopitys, has fascinated biologists for well over a century and a half (Leake, 1994). It was Kamienski (1881) who made the major conceptual advance in understanding the nutrition of the plant. In providing the first detailed description of the fungal sheath on the roots of M. hypopitys, he realized that virtually all the nutrients taken up by the plant must be acquired through its fungus, and that connections between the fungus and adjacent autotrophic plants might enable the myco-heterotoph to indirectly parasitise an autotroph.

However, none of the early workers were able to do much more than speculate on the nature of the relationship between the plant and its fungal partner and unfortunately, while interest in the plant has been unabated to the present time, speculation has continued to characterise many of the assertions made about its biology, and the nature of its nutrient, particularly carbon, supply and on the identity of its fungal associate(s). Most remarkably, for all species in the Monotropoideae, virtually nothing is known of germination and developmental stages up to point flowering, since their main phases of growth are subterranean and the plants are only observed when their inflorescences emerge above ground.

Two recent advances have facilitated progress in these areas. The first is the development of procedures enabling sowing and sequential recovery of the minute seeds and seedlings of plants like Monotropa so that the chronology of their symbiotic germination and growth can be accurately determined in the field (Rasmussen & Whigham, 1993; McKendrick et al., 2000a), and the second is the availability of molecular tools, which make it possible to identify the fungal species that form mycorrhizal associations with their roots (Bidartondo & Bruns, 2001, 2002). Here we describe the application of both these approaches to the study of M. hypopitys. The first detailed analyses of the factors determining germination and of the chronology of seedling development are provided, and the identities of the fungi forming the mycorrhizas are established.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Studies of germination and development of M. hypopitys were carried out at Newborough Warren National Nature Reserve, Anglesey, North Wales (National Grid Ref: SH 413632). This is an area of coastal dunes and dune slacks, the latter supporting areas of Salix repens scrub within which several large populations of M. hypopitys occur.

The aim of the experimental work was to determine the chronology of germination and seedling development in M. hypopitys, and to evaluate the influence upon these processes of distances from naturally occurring mature plants of this species (Expt 1), and from its autotrophic partner, Salix repens (Expt 2). An analysis was carried out, using molecular methods, of the identity of the mycorrhizal fungal symbionts associated with seedlings and mature plants of M. hypopitys at Newborough. For comparative purposes, this analysis was repeated using roots collected from mature M. hypopitys plants growing under Pinus sylvestris on Jurassic limestone in Dalby Forest, North Yorkshire, UK (NGR SE 874876) and under Salix caprea on Carboniferous limestone at Millers Dale, Derbyshire, UK (NGR SK 152728).

Construction and deployment of seed packets

Seeds of M. hypopitys were collected from ripe capsules of a number of flowering plants growing in a calcareous dune-slack (Slack 1 – see Expt 1) at Newborough Warren NNR on 17 August 1995. They were dried over calcium chloride at room temperature for 4 wk then stored in air-tight glass vials at 4°C until needed. A subsample of seed was tested for viability with tetrazolium chloride (Van Waes & Debergh, 1986) and a positive staining reaction was obtained in 60–70% of seeds. Approximately 100–200 seeds were placed into seed packets constructed from 40 × 60 mm rectangles of 53 m nylon plankton netting (Plastock Associates, Birkenhead, UK). The nylon was folded once and clipped into 2 mm × 2 mm × 36 mm plastic slide mounts (Rasmussen & Whigham, 1993). A length of coloured nylon line, which was attached to each mount, extended above the soil surface after burial of the packets to facilitate their recovery.

Using strung quadrats as templates, seed packets were inserted into c. 10 cm deep slots cut in the turf with a sharp chisel as described in McKendrick et al. (2000a). There were 100 packets inserted in a grid pattern in each 1 m2 replicate plot. The positions of the corners of each plot were mapped using co-ordinates to nearby fence-posts before the quadrats were removed. Seed packets were inserted in two separate dune slacks, both of which contained populations of M. hypopitys.

Expt 1. The effect of the presence and absence of adult plants on germination and development of M. hypopitys

The first site (Slack 1) supported a low growing population of S. repens in which there was a patchily distributed population of M. hypopitys. Ten 1 m2 plots were established at this site and these contained a total of 1000 seed packets. The plots were arranged so that five contained mature plants of M. hypopitys, while the other five were placed so that there were no observable plants of M. hypopitys within five metres of the plot boundary (Expt 1).

A small supplementary experiment was established in Slack 1 in 1997 to examine the possibility of germination occurring in the autumn immediately following seed ripening.

In Expt 1 seed packets were inserted between 18 September and 3 October 1995. The grid co-ordinate position of each packet within the quadrats was written on the plastic slide mount with a permanent marker to facilitate mapping of the spatial distribution of seedling germination following harvests.

Harvests were taken 7, 9, 14, 21, and 26 months after sowing (on 30 April 1996, 25 June 1996, 27 November 1996, 25 June 1997 and 27 November 1997, respectively). At the first two harvests, when it was not known whether germination had occurred, only three replicate samples were removed from each plot in order to conserve packets. From the third harvest, by which time it was known that germination had occurred, the number of packets sampled was increased to 10–15 per plot. At the later harvests more samples were taken from the plots that contained adult M. hypopitys than from those without mature individuals to enable targeting of packets containing the highest frequency of germinated seeds. At the final harvest, 12–20 packets were taken per plot. A random number table was used to select grid locations from which packets were sampled at all harvests.

In the supplementary experiment, 15 packets were sown in September 1997, there being three sets of five each planted within 50 cm of a flowering spike of M. hypopitys. These packets were all harvested after two and a half months on 28 November 1997.

Expt 2. The effects of presence and absence of S. repens cover on germination and development of M. hypopitys seedlings

The second experiment (Expt 2) was carried out in the second dune slack (Slack 2). Whereas in the previous decade Slack 2 had supported a large population of M. hypopitys, this had now declined so that plants were scarce and much less abundant than in Slack 1. A further distinguishing feature of Slack 2 was that within the Salix scrub there occurred, as a result of earlier activities of the Salix-specific ring die-back pathogen Roselinia desmazieresii near-circular patches of grass-dominated vegetation. In this slack, eight 1 m2 plots were set up, each containing 100 packets. Four of the plots were located within the Salix-dominated vegetation, while four were placed in the grassy areas and did not contain Salix shoots. It is necessary here to recognise that while the occurrence of Salix roots was reduced in these plots, some of them almost certainly entered the grassy areas from surrounding thickets, and so were present under both circumstances. Again, the eight plots were dispersed across the slack.

Packets were sown in the plots between the 3 and 12 October 1995, using the approaches described in Expt 1. Harvests were carried out 6, 8, 13, 20, and 25 months after sowing, on 17 April 1996, 26 June 1996, 28 November 1996, 26 June 1997 and 28 November 1997, respectively.

Post-harvest analysis of seedlings in Expts 1 and 2

Immediately following each harvest, packets were returned to the laboratory where they were stored moist at 4°C overnight. Over the course of the following 3–4 d, the packets were opened and examined microscopically to detect the extent of germination and of seedling development. The time taken to process the large number of samples made it necessary to preserve the contents of each packet by mounting the seedlings on a glass slide in a drop of 50% glycerol, placing a cover slip over the specimens and sealing with clear nail varnish. These slides were stored at 4°C until the seedlings could be measured. Seedlings too large to be mounted in this way were measured fresh.

In Expt 1, the total number of seeds in each seed packet and numbers of seedlings that were live and dead were counted and the seedlings measured. At the first two harvests the length and breadth of representative seedlings at all stages of development were measured and the extent of fungal infection and seedling development were recorded. At the harvests taken over the first 21 months, measurements, from all sampled packets, were made of total seedling length (including all branches) of all seedlings that were greater than 0.135 mm long, and had reached the second stage of development (see later for definition of developmental stages). At the later harvests (26 months onwards) the intermingling of roots of live and dead seedlings made measurements impractical and records were made only of the stage of development and numbers of branches produced by seedlings.

At the 14-month harvest a very large number of seedlings had germinated and were at the first stage of development and, in this case, it was too time consuming to distinguish those seedlings that were alive from those that were dead. At the 21-month and 26-month harvests the total numbers of seeds sampled were estimated from the average numbers of seeds per packet at the earlier harvests, since ungerminated seeds were in too advanced a state of decay to reliably be counted.

Differences between the percentage of seed packets in which germination occurred in plots with and without adult M. hypopitys were assessed at each harvest by anova of arc-sine transformed percentages. Similarly, the effect of adults on the mean percentage of seeds that germinated in each packet, excluding packets with no germination, and the percentage of all sown seeds that germinated in each packet were analysed by anova following arc-sine transformation.

In Expt 2, three seed packets were sampled from each plot at each of the first three harvests (6, 8 and 13 months after sowing). At the later harvests 10–12 packets were sampled from each plot. The presence and absence of germinating seedlings were noted for each seed packet and the germinated seedlings that were found in the plots containing Salix repens were measured and their stages of development and whether they were alive or dead were recorded. At the first (6 month) harvest we did not make full counts of the numbers of seeds and seedlings, and at the harvests taken at 21 and 25 months after sowing we only recorded seedlings that had reached Stage 2 because of the increasing difficulty of distinguishing dead seeds and small seedlings.

Molecular identification of fungi

DNA analyses were carried out on 27 samples from three locations (Newborough, Dalby Forest and Miller's Dale) with a view to determining the identities of the fungi forming mycorrhiza on M. hypopitys growing with three different autotrophic associates, namely Salix repens, Salix caprea, and Pinus sylvestris.

Eleven samples (two seedlings and six adults of M. hypopitys, and three groups of S. repens roots associated with these plants) were analysed from Newborough Warren. Two samples, each representing a different adult M. hypopitys plant growing under S. caprea were analysed from Millers Dale. Fourteen separate root samples from three individual adult plants of M. hypopitys growing under P. sylvestris were examined from Dalby Forest. The methods used for the analyses were those of Gardes & Bruns (1996). DNA was extracted from individual roots and the ITS region of the nuclear ribosomal repeat was amplified by the polymerase chain reaction (PCR) using the fungus – specific primers ITSIF and ITS4 (White et al., 1990; Gardes & Bruns, 1993). PCR products were then screened by restriction fragment length polymorphism (RFLP) using the endonucleases Alu-l and Hinf-l (New England Bio Labs, Beverley, MA, USA). In the cases of those ITS-RFLP's that exhibited a unique pattern the following fungal DNA regions were amplified and sequenced: first a fragment of the mitochondrial large subunit (mtLSU) (Bruns et al., 1998) and second the nuclear ITS region. Sequencing of both strands was performed with an ABI model 377 Sequencer (Applied Biosystems Co., Foster City, CA, USA) using an ABI Prism Dye Terminator Cycle Sequencing Core Kit (Perkin Elmer Co., Foster City, CA, USA). The raw data were processed using DNA Sequencing Analysis v.2.1.1 and Sequence Navigator v.1.0.1 (Applied Biosystems Co., Foster City, CA, USA) software. Sequences from the mtLSU were manually aligned to the database of Bruns et al. (1998). To infer relationships, the neighbour-joining algorithm implemented in the program paup*d64 (Swofford, 1993) was employed. Sequences from the ITS region were used to query GenBank via BLAST (http://www.ncbi.nlm.nih.gov/blast).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results of the observations made on packets harvested from both Expts 1 and 2 were first pooled to provide an overall view of the chronology of germination of M. hypopitys and to enable description of the distinctive stages through which the seedlings passed in the course of their development.

Chronology of germination and stages of seedling development

The development of M. hypopitys seedlings has five distinct stages (Table 1). Ungerminated seeds (Stage 0) were opaque (Fig. 1a) and light microscope observations indicated that they contained abundant lipid droplets. After burial in the field for 7 months those seeds that had not germinated had a mean length of 116 microns.

Table 1.  Definition of stages of development of seedlings of Monotropa hypopitys and the chronological sequence of these developmental stages in the two dune slacks studied (Expts 1 and 2)
StageDescriptionMean dimensions of seed or seedling (± SE) or defining limitsFirst observation of each stage (months after sowing)
LengthWidth(n)Slack 1 1995Slack 2 1995Slack 1 1997
  1. Note that seeds planted in the first slack in 1997 were all harvested within 2.5 months of sowing so the time required to develop the more advanced stages were not determined (ND). The number (n) indicates the size of sample used to derive the mean dimensions and their ranges.

0Ungerminated seed (Fig. 1a)116.4 m ± 0.7 max. < 135 m72.8 m ± 0.5237
1Rupture of seed coat and emergence of tissue, usually at one end of the embryo. Some, but not all, Stage 1 seedlings are visibly colonised by fungus (Fig. 1b)124 m ± 4 max. < 160 m84 m ± 3  8 7 62.5
2Unbranched seedling with fully developed mycorrhizal fungal mantle (Fig. 1c,d)> 160 m> 80 m 44 9 62.5
3Branched seedling with side roots a) 1–4 roots (Fig. 1e) b) 5+ roots (Fig. 1f–h)All > 900 m96% > 400 m 51 9132.5
    9132.5
   1413ND
4Plant with shoot buds (Fig. 1h,i,k)   2625ND
image

Figure 1. Stages in development of Monotropa hypopitys. (a) Stage 0 – ungerminated seed (bar, 100 m). (b) Stage 1 – seedling with new tissue emerging on the right side breaking through the brown outer wall of the seed inside the testa. Note the colonisation by the hyaline fungus (bar, 100 m). (c) Stage 2 seedling with the early development of a full mycorrhizal mantle formed by the hyaline fungus. Expansion of the seedling has ruptured the testa (bar, 100 m). Note the increasing density and diameters of the fungal hyphae compared with (b). (d) Stage 2 seedlings (black arrows) surrounded by hyaline mycelium interconnected by mycelial cords, together with Stage 1 seedlings (white arrows). The growth of the fungus is apparently stimulated around the seedlings. Note the mycelial cord growing to the Stage 1 seedling at the bottom left (double white arrow). (Bar, 500 m). (e) Stage 3a branched seedling with between one and four branches. Note the testa still attached to the base (white arrow) and the complete mycelial mantle. The slight patterning on the surface is due to the nylon mesh packet (bar, 500 m). (f) Stage 3b seedling with more than four branches (on the left) and a mass of mainly Stage 3 seedlings on the right. Note again the extensive masses of white mycelium just around the M. hypopitys plants (bar, 1 mm). (g) Stage 3b seedlings forming a tangled mass within a packet. (h) Stage 4 seedling with shoot bud (white arrow) produced adventitiously (bar, 1 mm). (i) Detail of shoot bud showing overlapping unpigmented scale leaves (bar, 1 mm). (j) Detail of the outside of a seed packet with strongly adhering roots and soil. M. hypopitys seedlings (black arrows) and Salix repens ectomycorrhizal roots (white arrowheads) are interlinked by the white mycelial cords of their shared fungal symbiont. Note the high density of the white fungus around the M. hypopitys seedlings. The main woody roots of Salix repens are indicated by long white arrows (bar, 1 mm). (k) Shoot buds on an established M. hypopitys plant in the dune slacks showing the same white fungal associate (bar, 1 mm). (l) Ectomycorrhizal root tips of Salix repens colonised by the Tricholoma cingulatum interlinking to M. hypopitys (j). Note the increased width of the root-tips colonised by the Tricholoma (bar, 1 mm).

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By this time in the main experiments, and within 10 wk of seed sowing in the supplement to Expt 1, changes in the appearance of some seeds were recognisable (Table 1). Germination (Stage 1) was indicated by the rupturing, at one end of the embryo, of the carapace-like thickened cell walls enclosing the outer faces of the endosperm cells. These events were associated with an increase in translucence of the embryo, and the emergence of a small portion of its tissue through the seed coat (Fig. 1b). The majority, but not all Stage1 seedlings were visibly colonised by fungal hyphae (Fig. 1b).

The increases in length and breadth of seedlings at Stage 1 were so small that it was not possible, on the basis of size alone, to distinguish between germinated and ungerminated seeds (Fig. 2a). While measurements of the ungerminated seeds indicated that 95% of them had lengths less than 135 microns, many Stage 1 seedlings were also in this size category. The determination of development to Stage 1 therefore required the extremely labour intensive microscopic examination of each individual seedling. While intensive analysis of this kind was possible at the early harvests when numbers of germinating seeds were relatively small it was not feasible later when numbers of packets and of seedlings being processed rose to in excess of 100 s and then 1000 s at the succeeding harvests. On these later occasions all seeds greater than 135 microns long were counted as having germinated.

image

Figure 2. The relationships between length and breadth of seeds and seedlings of Monotropa hypopitys at different stages of development and in relation to fungal colonisation. (a) Stages 0–2. The length and breadth of ungerminated seeds (Stage 0); of newly germinated seedlings, with or without visible fungal association (Stage 1); and of unbranched seedlings with a mycorrhizal mantle (Stage 2). (b) Stages 0–3, showing the effect of root branching on the relationship between length and breadth of seedlings. The dotted line denotes the data ranges for stages 0–2 presented in (a). The fitted curve is a polynomial regression line, with an R2 of 0.94 for the full dataset presented in (b).

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Development of seedlings beyond Stage 1 was dependent upon colonisation by a symbiotic fungal partner and was associated with the initiation of a mycelial mantle around part of the seedling axis (Fig. 1c). By the time seedlings had achieved lengths and breadths of 0.16 × 0.08 mm, a complete fungal mantle was normally present (Figs 1d,e and 2a). This level of development characterises Stage 2 (Table 1) and is the first at which, on the basis of size alone, it is possible to define the seedlings as being symbiotic. At this stage the seedling is still unbranched. Plants were observed to have achieved this stage of development only 2.5 months after sowing in the supplement to Expt 1, while in the main Expts 1 and 2, Stage 2 was reached, respectively, after 9 and 6 months (Table 1).

The third stage of development involved the emergence of side roots from the main axis of the seedling. This branching was first observed to have occurred only 2.5 months after the autumn 1997 sowing in the supplementary experiment. In Expts 1 and 2 it was first seen in harvests taken, respectively, after 9 and 13 months (Table 1). Because Stage 3 seedlings showed a large range in size and underwent considerable growth before reaching the next stage of development, this category was divided into two substages that were demarcated on the basis of numbers of these roots. Seedlings with between one and four side roots were placed in Stage 3a, while those with five or more were included in Stage 3b (Table 1).

The next developmental stage (Stage 4) was defined by the appearance of shoot buds that were first observed after 25 and 26 months, respectively, in the two experiments (Table 1). In the most advanced buds, overlapping scale leaves were developing at the shoot apex. The shoot buds normally emerged from the side of the main seedling axis (Fig. 1h–i).

Seedling growth and development

The relationship between seedling length and breadth at different stages of development  Newly germinated seedlings (Stage 1) were only slightly longer and wider than most ungerminated seeds and only after the seedlings had developed a complete fungal mantle (Stage 2) were they unambiguously larger than ungerminated seed (Fig. 2a). Their lengths and breadths continue to increase in proportion until seedlings achieve about 1 mm in length and begin to produce side branches (Fig. 2a,b). In seedlings larger than this, the width of the main root ranged from 0.45 to 0.75 mm and changed little with increasing total root length (Figs 2e–h and 3b). The change in the length-breadth relationship coincided with the initiation of branching (Stage 3) in most seedlings. The development of branches introduces considerable additional variation in seedling widths, as the main axis swells immediately before the emergence of each new root apex.

image

Figure 3. The sequence of initiation and development of root branches in seedlings of Monotropa hypopitys.

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Patterns of seedling growth and root branching  Germinating seedlings soon establish a single apical root meristem from which growth proceeds, leaving the original testa attached to the base (Fig. 3). Unipolar growth proceeds until a lateral root meristem emerges at a position on the main axis, typically within 0.5 mm of the original embryonic cells (Fig. 3). A second branch is formed simultaneously, or soon afterwards, diametrically opposite the first. This gives rise to a seedling of cruciate form. The orientation of the seedling in the packet appears to have little influence on the morphogenetic pattern. The third and fourth branches subsequently emerge from the main axis closer to the primary meristem than the first branches (Figs 1e and 3). Growth proceeds from this point in a variety of ways. In most cases further branching continues to occur from the main axis (Figs 1f and 3) but eventually extension of the lateral roots exceeds the length of the main axis, and secondary lateral roots are developed (Fig. 1g,h). By this stage the seedlings are structurally complex, and where many of them are growing together in a packet, it can be difficult to separate them (Fig. 1f,g).

The progression of seedling growth and mortality with time   The percentage of live and dead seedlings occurring in each of a series of length-based size class categories were plotted for all plants of Stage 2 or later recovered from both dune slacks during the harvests of June and November 1996 and June 1997 (Fig. 4a,b). At the first of these harvests over 80% of the Stage 2 seedlings in Slack 1 had lengths > 0.367 mm, while none of the symbiotic seedlings in Slack 2 had reached this length. At this time over 30% of the seedlings of the smallest size class in Slack 1 had died (Fig. 4a). Mortality in Slack 2 was 70% (Fig. 4b).

imageimage

Figure 4. The percentage of live and dead seedlings of Monotropa hypopitys in different size-classes with time after sowing (a) within 1 m of adult M. hypopitys plants in Slack 1 (Expt 1) and (b) in plots containing Salix repens in Slack 2 (Expt 2). Only seedlings longer than 0.135 mm are recorded.

Over the ensuing 5 months to November 1996, considerable growth had occurred in seedlings recovered from both dune slacks. While the numbers and proportions of seedlings in the smallest size class decreased, the sizes of many individuals had increased substantially, the largest now attaining lengths up to 55 mm in Slack 1 (Fig. 4a) and up to 208 mm in Slack 2 (Fig. 4b).

The modal size class of seedling lengths in the first slack was 0.368–0.999 mm and it reached the even higher value of 7.4–20.1 mm in Slack 2. Dead seedlings were no longer observed in the smallest size category in either slack, indicating that those of the type that had been recorded as being dead in the previous harvest had by now decomposed sufficiently to be unrecognisable. Mortality was now seen in the larger seedling categories in Slack 1 (Fig. 4a) and in the very largest seedlings obtained from Slack 2 (Fig. 4b).

By the next harvest, in June 1997, the proportions of seedlings in the smallest size-classes, and of those that were dead in all size classes, had increased (Fig. 4a,b). The increase in proportion of small seedlings, coupled with the observation that some of these new seedlings were found in packets containing plantlets that had reached Stages 2 and 3 and died, suggests that a new cohort of seedlings had germinated since the previous harvest, that is between 14 and 21 months after sowing.

Expt 1. The effect of distance from adult plants on germination and growth of M. hypopitys

Effects of the presence of mature plants on percentage seed germination  The presence of adult M. hypopitys in the 1 m2 plots was consistently associated with enhanced seed germination over the first 14 months after planting (Table 2). In the plots supporting adults 87–99% of the packets sampled over this period contained germinating seeds, whereas in their absence the percentage of packets yielding seedlings was substantially, and significantly (P < 0.05) lower at each of the three harvests (Table 2).

Table 2.  Expt 1: effect of presence and absence of adults on germination (all stages) of Monotropa hypopitys in 5 replicate 1-m2 plots which were either > 5 m from adults (− Adults) or contained 4–18 flowering adults (+ Adults) when the packets were buried in Sept 1995
VariableTime after sowing
7 months − Adults+ Adults9 months − Adults+ Adults14 months − Adults+ Adults
  1. Where mean values at each harvest share the same letter they are not significantly different (ANOVA, P > 0.05). The mean values in each case are arcsine back-transformed.

Mean percentage of packets containing germinating seedlings in each plot (n = five plots)46.5 a98.5 b13.4 a98.5 b13.2 a87.0 b
Mean percentage of seeds which germinated in each packet excluding packets with no seed germination (n = packets in which germination occurred) 1.6 a 3.6 a 1.0 a 8.7 b 3.3 a 9.1 b
n = 7n = 14n = 4n = 14n = 8n = 45
Mean percentage of all sown seeds which germinated in each packet (n = total packets sampled) 0.4 a 3.1 b 0.1 a 7.6 b 0.1 a 5.8 b
n = 15n = 15n = 15n = 15n = 50n = 57

The percentage germination within each seed packet (excluding packets in which no germination occurred) was also generally higher in the plots with, relative to those without, adult M. hypopitys plants. However, this effect was only significant (P < 0.05) at the 9- and 14-month harvests.

The percentage of all seeds that germinated was increased significantly in the plots supporting adults at all three harvests (P < 0.05) (Table 2). The numbers of seedlings were up to 76 times higher in plots with than in those without adult spikes of M. hypopitys.

Effects of the presence of mature plants on seedling development  The extent of seedling development was strongly influenced by the presence of adult M. hypopitys in Slack 1. In the plots containing adults nearly 1200 seedlings germinated out of a total of 35 000 sampled (Table 3). Of the 1200, over 350 developed a full mycorrhizal mantle, and over 130 reached the branching stage or beyond (Stages 3–4). By contrast, of an estimated total of 26 000 seeds sampled from plots without adults, only 53 had germinated (Table 3) and only one of these reached Stage 2.

Table 3.  Expt 1: effect of presence and absence of adults on total numbers of seedlings of Monotropa hypopitys at different developmental stages in 5 replicate 1-m2 plots which were either > 5 m from adults (− Adults) or contained 4–18 flowering adults (+ Adults) when the packets were buried in Sept 1995
VariableTime after sowingTotal over 26 months
7 months − Adults+ Adults9 months − Adults+ Adults14 months − Adults+ Adults21 months − Adults+ Adults26 months − Adults+ Adults− Adults+ Adults
  1. The numbers of dead seedlings are indicated (†), but were not counted for Stage 1 seedlings at 14 months (nd). Differences between the proportion of seeds sampled that germinated in plots with and without adults were determined by χ2 tests at each harvest. Values sharing the same letter at the same harvest are not significantly different (P < 0.05). For the samples taken from 21 months onwards the total numbers of seeds in the sampled packets was estimated from the mean number of seeds per packet (n= 167 packets) sampled over the first 14 months.

Total number of seedlings recovered (live + dead)  13 a 107 b   5 a 180 b  35 a 643 b0 a158 b0 a98 b53 a1186 b
Number of seeds sampled194521091968184272997827c. 6800c. 10 200c. 8200c. 12 800c. 26 000c. 35 000
Number of Stage 1 seedlings  13 107   4 (2)  51 (39)  35 600 (nd)0 57 (23)0 0 (0)50 (2) 815 (> 62)
Number of Stage 2 seedlings   0   0   1 (0) 126 (34)   0  29 (4)0 51 (15)040 (26) 1 246 (79)
Number of branched seedlings (Stages 3–4)   0   0   0 (0)   3 (0)   0  20 (2)0 50 (31)058 (32) 0 131 (65)

Over the 26-month sampling period there were marked changes in the proportions of seedlings recorded in the different developmental stages in both sets of plots (Table 3). However, these changes were more marked in the presence of adults where both the numbers and proportions of seedlings in Stage 1 were greater, as was the extent of development beyond Stage 1. In these plots Stage 1 seedlings were found up to and including 21 months after sowing. Progressive development was indicated by the observation that while the numbers and proportions of Stage 1 seedlings decreased after 14 months, those in Stages 2 and 3 increased (Table 3). This situation was in marked contrast to that seen in plots lacking mature individuals. Here the presence of Stage 1 seedlings was observed only in the first 14 months, and with the exception of the one seedling that reached Stage 2 by 9 months, no development beyond Stage 1 was observed. The complete absence of seedlings at the 21 and 26 month harvests indicated that any earlier germinants had both died and decomposed in the intervening period of time. Death of seedlings was common in both sets of plots and appeared to be caused largely by desiccation.

The effects of proximity to adults on the spatial distribution of germination  Analyses of the spatial distribution of seed packets in which germination had occurred, and of the most advanced developmental stages attained by seedlings in each sampled packet showed strong relationships with the presence of adult M. hypopitys (Fig. 5). In all the plots containing adults some seedlings reached Stage 3 of development, and in three of the five plots, plants had produced shoot buds (Stage 4) by the final harvest at 33 months. By contrast, none of the plots lacking mature spikes of M. hypopitys yielded seedlings beyond Stage 1 (Fig. 5). The absence of any sign of mycorrhizal colonisation in these seedlings strongly indicated that it was the presence or absence of the appropriate fungus in these plots that determined the fate of seeds arriving in them.

image

Figure 5. The spatial distribution of flowering shoots of the established population of Monotropa hypopitys and of seedling germination and the most advanced stages of development recorded for M. hypopitys seeds in packets within the 1 m2 plots in Expt 1. Packets were planted in plots supporting adult M. hypopitys (with M. hypopitys) and > 5 m from the nearest known M. hypopitys (without M. hypopitys). Data are from all harvests. The coordinate locations were unreadable on eight packets and these are not plotted. These included four samples from plots without M. hypopitys in which germination to Stage 1 was observed. Note that the figure does not indicate the spatial locations of the plots relative to each other.

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Despite the marked stimulation of germination and growth of seedlings in the presence of adults at the 1 m2 plot scale, there was no evidence at smaller spatial scales that germination was enhanced in the immediate vicinity of the adult plants. When the germination data from all plots were pooled, and the numbers of packets with and without germinating seeds were compared for samples taken within 20 cm of flowering shoots of M. hypopitys and samples more than 35 cm from the nearest known adults, there were no significant differences (χ2 = 0.53, d.f. = 1, P > 0.05). Similarly, despite the fact that the numbers of adults of M. hypopitys varied considerably between plots (in the range 4–18 flowering spikes) and that their occupancy of 10 × 10 cm square subdivisions of the quadrat area (range 4–14 quadrat squares) also varied greatly between plots, there were no significant differences between plots in the proportion of sampled packets that contained germinating seeds (χ2 = 8.99, d.f. = 4, P > 0.05).

Expt 2. The effects presence and absence of S. repens cover on germination and growth of M. hypopitys

Effects of Salix repens  cover on germination at the packet and individual seed levels  At 8 months after sowing, the percentage of packets containing germinated seeds was significantly higher (P < 0.05) in the plots with dense Salix cover than in those from which its shoots were absent and its rooting density reduced (Table 4). By 13 months, however, the number of packets containing germinating seeds in the plots without Salix cover had increased to the extent that there was no longer a statistically significant effect of Salix cover. It was noticeable that both germination and seedling development were much more patchy than seen in Expt 1. In retrospect we recognised that because of the high observed variability, a rigorous test of plot effects in this experiment would have required an increase in the sampling intensity beyond the 24 packets that were recovered at each of the harvests over the first 13 months.

Table 4.  Expt 2: effect of Salix repens cover on the mean percentage germination (all stages) of Monotropa hypopitys sown in four replicate 1-m2 plots either within dense stands of Salix repens (+ Salix) or in grassy areas without (−Salix) shoots in Oct 1995
Variable8 months13 months
− Salix repens+ Salix repens− Salix repens+ Salix repens
  1. The mean percentage values in each case are arcsine back-transformed. Where the values at each harvest share the same letter they are not significantly different (P > 0.05) following Kruskal-Wallis test (1) or χ2 contingency test (2).

Mean percentage of packets containing germinating seedlings in each plot (n = 4 plots in each case)  9.1 a1 85.2 b1 41.5 a1 50.0 a1
Mean percentage of seeds, which germinated in each packet excluding packets with no seed germination (n = the number of packets with germinating seeds)  1.2 a1 15.9 b1  4.0 a1 26.7 b1
n = 2n = 9n = 5n = 6
Mean percentage of all sown seeds, which germinated in each packet (n = 12 packets sampled in each case)  0.0 a1  5.9 b1  0.7 a1  7.2 a1
Total seedlings (live + dead)  3 a2147 b2 26 a2121 b2
Number of seeds sampled831876922872

Analyses of the percentage germination occurring in individual packets revealed a consistent and significant positive effect of Salix cover at both harvests (Table 4). Thus, although at 13 months there was no significant difference between the presence and absence of Salix cover in numbers of packets supporting germinating seedlings, the numbers of seedlings recovered from each packet were significantly lower (P < 0.05) in plots without Salix shoots. Numbers of seedlings per packet were an order of magnitude greater in plots covered with Salix than in those without Salix shoots, reaching 27% in the former treatment after 13 months. The total number of seedlings obtained was from 4–50 times greater in the plots with Salix cover, than in those without it (Table 4).

In addition to effects upon seedling numbers, Salix cover also influenced the extent of development and longevity of seedlings in the packets. In the plots without Salix shoots, although germination was occasionally recorded, the development of seedlings never progressed beyond Stage 1. Further, by 20 months after sowing (June 1997), all seedlings from these plots had died. By contrast, in several of the plots with dense Salix cover not only did development progress beyond Stage 1, but in several cases it reached Stage 4 (see Table 1, Fig. 6).

image

Figure 6. The spatial distribution of seedling germination and the most advanced stages of development recorded for Monotropa hypopitys seeds in packets within the 1 m2 plots in Expt 2. Packets with planted under Salix repens (with Salix repens) or in grassy areas devoid of Salix shoots (without Salix repens). Data are from all harvests. Note that the figure does not indicate the spatial locations of the plots relative to each other.

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Relationships between seedling development and presence and absence of mature M. hypopitys in plots with dense Salix cover  While mature spikes of M. hypopitys were observed in one of the dense Salix plots (Plot 3a, Fig. 6), their presence was not a prerequisite for growth of seedlings since equivalent amounts of development occurred in plots, for example 5a (Fig. 6), lacking adults. A further indication of the inherent patchiness of the germination environment is provided by the observation that, while fully mycorrhizal seedlings were found in six packets within 20 cm of adult M. hypopitys plants in Plot 3a, of the 18 other packets sampled within the same distance, 12 contained no seedlings while six supported seedlings that had reached only Stage 1.

The results of this Experiment appear to be at some variance with those of Expt 1, which suggested that the presence of adult M. hypopitys was a prerequisite for development of seedlings beyond Stage 1 (Fig. 4). Clearly, the possibility exists that nonemergent immature individuals of M. hypopitys were present in those plots (1a, 5a, Fig. 6) of Slack 2 that had been presumed to lack the plant. Alternatively, pockets of appropriate inoculum not supporting growth of M. hypopitys may have been more widely present on Salix roots in this slack than in Slack 2.

Characteristics of the fungal symbiont

The fungus associated with M. hypopitys seedlings and young plants was unpigmented, its mycelium being normally of bright white appearance. Where a single hypha colonised a seedling it frequently took on a pale green colouration, when viewed under transmitted light at high (× 400) magnification. Colonisation of a seedling led to a distinct stimulation of mycelial development in its immediate vicinity. The result was that in packets that supported a number of seedlings, extensive wefts of the white mycelium were visible surrounding seedlings (Fig. 1d–h). With the aid of a dissecting microscope rhizomorphs could be seen to extend from these wefts through the walls of seed packets to the ectomycorrhizal mantles of Salix repens roots. Woody roots of Salix were occasionally observed to enter seed packets that had become partly opened following burial. Under these circumstances ectomycorrhizal short roots emerging from the woody axes were colonised by the white fungus, and rhizomorphs could again be seen to form direct connections to the fungal mantles on nearby developing M. hypopitys seedlings (Fig. 1j). Mature M. hypopitys were also found to be consistently associated with what appeared to be the same white fungus (Fig. 1k). Ectomycorrhizal roots of S. repens were often observed to proliferate against the outer walls of the seed packets. Here, they could be colonised by a variety of ectomycorrhizal fungi amongst which that with the white mycelial mantle was normally present. (Fig. 1L). Again, mycelia of this fungus could, under some circumstances, be traced into packets containing germinated seeds.

Molecular identification of fungal symbionts

Two M. hypopitys seedlings obtained from packets, together with samples of roots taken from six adult plants from Newborough Warren and two from Derbyshire, all produced a single combination of ITS-RFLP fragments. The same ITS pattern was also found in the three samples of roots of S. repens that had been collected from positions adjacent to plants of M. hypopitys and which, according to visual observation, shared the same fungus. We refer to this Salix-type ITS pattern as Type 1 (Table 5).

Table 5.  Molecular characterisation of mycorrhizal fungi associated with seedling and adult Monotropa hypopitys plants of three habitats
Geographic location and plant communityType of plant material (number of samples)ITS-RFLP typeMtLSUITS region
  1. For each mycorrhizal sample, the number of sequences obtained from different roots is noted in parentheses together with the GenBank Accession number for each unique sequence submitted. nd, not determine.

Newborough Warren, Anglesey, UK (Salix repens dune slack)M. hypopitys seedlings (2)ITricholoma (1) AF351892T. cingulatum (1) AF34698
 As aboveM. hypopitys adult (6)ITricholoma (2)T. cingulatum 1
 As aboveS. repens (3)Indnd
Millers Dale, Derbyshire, UK (Salix caprea scrub on limestone)M. hypopitys adult (2)IndT. cingulatum
Dalby Forest, North Yorks, UK (Pinus sylvestris on limestone)M. hypopitys adult (14)IITricholoma (1)T. terreum 2 (2) AF377215

Similarly, the roots of 14 pine-associated M. hypopitys plants all exhibited a unique set of ITS-RFLP fragments. This Pinus-type pattern is referred to as Type II (Table 5).

Sequences from the fungal mtLSU of roots of both Types I and II indicated that the fungi were members of the genus Tricholoma. The ITS sequence of the Salix type produced matches lower than 92% with those available in databases. However, based upon information gained from analysis of Tricholoma–Monotropa associations (Bidartondo & Bruns, 2001) it was decided to investigate the possibility that the fungus involved in this case was T. cingulatum. When the ITS regions of two basidiocarp collections (Leiden Herbarium: Nordeloos 95210 and Bas 8966-Accession Numbers AF349697 and AF377197, respectively) of this European Salix-specific ectomycorrhizal fungus were examined, they were found to produce identical sequences both to each other and to the Salix-Type I mtLSU and ITS accessions (AF351892 and AF34698, respectively, –Table 5). It is therefore inferred that all mycorrhizal roots of both adults and seedling of M. hypopitys that yielded the ITS-RFLP Type I sequences were colonised by T. cingulatum. Circumstantial evidence in favour of this conclusion was provided by the observation that T. cingulatum is the only member of this genus regularly to produce carpophores under S. repens in the Newborough dune system (A.F.S. Taylor personal communication).

The nrITS sequence of Type II (AF377215 –Table 5) obtained from adult M. hypopitys matched six accessions (AF062613,14,16,18,21) of T. terreum to between 96 and 99%. The closest match was to 576 of 579 base pairs from T. terreum accession AF062614. It was therefore inferred that all mycorrhizal roots that produced ITS-RFLP Type II sequences were colonised by T. terreum (Table 5), which is a widespread ectomycorrhizal fungus of the Pinaceae across Eurasia.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study provides the first definitive chronology of germination and development of M. hypopitys. This is the first complete record of growth from seedling to initiation of shoot buds for any species in the Monotropoideae. We establish the critical dependence of the developmental processes upon a narrow clade of ectomycorrhizal fungi in the genus Tricholoma and upon the specific autotrophic coassociates of these fungi, which, in our study areas, were Salix repens, S. caprea and Pinus sylvestris.

Comparisons between myco-heterotrophic growth in Monotropa and orchids

The detailed descriptions of the ontogeny and chronology of symbiotic growth and development in Monotropa, enable us to draw comparisons with those of the largest family of myco-heterotrophic plants, the Orchidaceae. M. hypopitys, and most other monotropes produce ‘dust seeds’ that show remarkable convergent evolution in their form and anatomy to those of the very distantly related family Orchidaceae (Koch, 1882; Francke, 1934; Leake, 1994; Arditti & Ghani, 2000). The most striking similarity to orchid seed is the elongated and inflated testa that very loosely encloses the seeds. Even fine details of the testa of M. hypopitys seed correspond closely to those in orchids. The cells have raised anticlinal and periclinal walls, deep brown pigmentation and they curve to form a twist down the long-axis of the testa, these features presumably selected, as in orchids, to enhance air-bouyancy and dispersal by wind (Leake, 1994). In M. hypopitys, as in orchids, each flower produces many thousands of tiny seeds, and a single shoot can support 10 or more seed capsules (Copeland, 1941).

However, even by comparison with orchid seeds, which are normally regarded as extreme examples of morphological reduction and arrested postfertilisation cell division, the embryos of Monotropa are exceptionally simple. Whereas in the most extremely simplified orchid embryo, seen in the fully myco-heterotrophic Epipogium aphyllum, embyogenesis is completed in three mitotic cycles yielding eight cells (Geitler, 1956), in M. hypopitys the embryo consists of only four cells produced by two mitotic divisions (Koch, 1882). It is likely that the requirement for early fungal colonisation of this minute embryo arises from the fact that the endogenous nutritional resource for the support of its development consists of only nine endosperm cells. Because of the early cessation of cell division in the seeds, and the lack of differentiation of the embryo the ontogeny of germinating seedlings of these plants is of considerable interest.

The present study reveals that following fungal colonisation, embryo development in M. hypopitys is distinct from that in orchids since it leads to a unipolar axis comprising a histologically differentiated radicle. This contrasts with the situation seen in many orchids including the fully myco-heterotrophic species Neottia nidus-avis (McKendrick et al., 2002) and Corallorhiza trifida (McKendrick et al., 2000a), in which the apical meristem forms a shoot initial and roots are either not formed, or develop later from one or more basal meristems. In M. hypopitys, according to our observations, c. 2 yr of development are required before the stage of bud formation is reached and buds are produced adventitiously and not from the apical meristem. The delay in production of shoot meristems in M. hypopitys may reflect the need to accumulate the large amounts of carbon required to sustain extension of the flowering spike and seed set, and may be regarded as an advanced feature in fully myco-heterotrophic plants whose shoots never photosynthesise. In the orchids, after an initially myco-heterotrophic phase of growth, most of the 17 500 species produce photosynthetic green shoots so early investment in shoot production is likely to be advantageous in all but the c. 100 species that remain fully myco-heterotrophic. Furthermore, in the orchids, extensive intracellular fungal colonisation provides a large internal surface area for the transfer of carbon from fungus to plant, whereas in monotropoid mycorrhizas fungal penetration is confined exclusively to the single epidermal cell layer of the root. The haustorial pegs of the unique monotropoid mycorrhizas (Lutz & Sjolund, 1973; Duddridge & Read, 1982; Robertson & Robertson, 1982) provide a smaller area of interface between fungus and plant for carbon transfer. This would explain the need to increase, by root growth, the extent both of plant–fungal interface and storage volume before shoot buds can be initiated in the monotropoid plant. The abrupt change in length-breadth relationship of seedlings of Monotropa on reaching only 1 mm in length contrasts with the much more gradual transition in length: breadth ratios with growth seen in representative fully myco-heterotrophic orchids (McKendrick et al. 2000a, 2002), reflecting the low surface area to volume ratios of the orchids in which there is extensive internal fungal colonisation.

Asymbiotic vs symbiotic germination in M. hypopitys

Using media and methods previously employed by Burgeff (1932) in studies of orchid seed germination, Francke (1934) unsuccessfully tried to germinate seeds asymbiotically on solid media in the laboratory. In the first study to employ mesh bags to facilitate burial and recovery of ‘dust seeds’ in nature, Francke, 1934) mixed seed of M. hypopitys with small amounts of soil collected from different depths in the natural habitats of the plant, placed the mixtures in ‘fine-meshed gauze’ bags and returned them, again at a range of depths as well as at different distances from mature M. hypopitys plants, to the field. Bags planted in Oct were harvested the following May, June and July No germination was observed on the first two occasions, but at the July harvest around 0.3% of seeds showed evidence of cell division and were recorded as having germinated. Francke reported that neither depth of sowing nor distance from mature M. hypopitys had any impact upon the pattern of germination. From the descriptions of the seedlings recorded by Francke as having ‘germinated’ it appears they had developed to Stage 1, but had not been colonised by mycorrhizal fungi. His observations are consistent with those of the present study, which showed that in plots where no mycorrhizal colonisation occurred, up to 0.7% of seeds reached this stage in the first 13–14 months but developed no further.

Whilst our studies also support the suggestion that the process of germination can begin in the absence of fungal infection, we cannot exclude possible fungal involvement in the initiation of the germination process since chemical signals from specific fungi in the vicinity of the seed may provide the trigger for these events. We found that in plots where the Tricholoma was present there was an up to 10-fold increase in percentage germination (Tables 2 and 4). In other monotropes fungal stimulation of germination by the specific fungal partners of the plants, and by closely related (but possibly incompatible) fungi has been demonstrated (Bruns & Read, 2000). Similarly, there is evidence from field studies of myco-heterotrophic orchids that germination can be initiated by specific fungal partners before they penetrate the seeds (McKendrick et al., 2000a, 2002). The present study provides only indirect information on this aspect of M. hypopitys germination biology. Of the c. 26 000 seeds harvested from plots containing no adults plants of M. hypopitys (Expt 1) only 53 plants (Table 3) achieved Stage 1 of germination and only one became colonised by fungus, so being enabled to progress beyond this stage. This latter development, singular though it is, suggests that T. cingulatum occurred in plots lacking adult M. hypopitys. While, evidently, its occurrence was scarce the possibility remains that the fungal symbiont was present in sufficient amounts to trigger the small fraction of Stage 1 germinations observed, or that other fungi can, at least to a limited extent, initiate germination. The similarly low levels of Stage 1 germination observed in Expt 2, in the presence of reduced density of Salix roots, could be explained on the same basis, but detailed molecular analysis of the fungal community of roots of the autotroph would be necessary to determine whether the occurrence of T. cingulatum was a prerequisite for Stage 1 germination. In the absence of definitive evidence for or against the dependence of seed germination on the proximity of a fungal symbiont it would be inappropriate to classify the initiation of the germination process as an asymbiotic or a symbiotic event. However, this study makes it very clear that any seedling development beyond the few cell divisions that define Stage 1 has an absolute dependence upon colonisation by a specific fungal partner. Since seedlings that reach Stage 1 yet fail to become so colonised die, whereas those that form mycorrhizal associations develop normally, at least to Stage 4, there seems to be every reason for referring to the germination process overall as being symbiotic.

Temporal and spatial heterogeneity of germination in M. hypopitys

Seed germination and development showed high spatial and temporal variability both within and between packets. At the within packet level, particularly in the harvests taken at 20 and 21 months, large branched seedlings could be found adjacent to others that were at Stage 1. Since the failure of the latter to develop further is unlikely under these circumstances to be due to absence of a compatible inoculum, it is more likely to indicate that a dormancy mechanism facilitates staggered germination in this plant. Indeed, analyses based upon size class distributions (Fig. 4a,b) suggest that germination may be staggered over several years. In this context it was of interest that at 26.7%, the highest mean percentage germination within packets (see Table 4) was much lower than the 60–70% viability indicated by the tetrazolium test conducted on fresh seed.

Rates of development of seedlings within the packets also varied greatly between years. Thus packets planted in September 1997 and harvested in November the same year yielded more germination and much faster seedling development than in 7 months from the September 1995 sowings. Whereas the seeds sown in 1995 showed their main phase of germination and development in the spring of the following year, taking 9–13 months to achieve Stage 4, some of those sown in September 1997 had reached this stage within the 10-wk autumn period to November of that year. It is not clear whether such marked temporal variability is attributable to interyear differences in climatic or biotic conditions. Clearly availability of moisture could directly affect the potential for seedling growth or indirectly influence seedling development through its effects upon activities of the fungal symbiont.

The likelihood that factors other than climatic were involved in determining the observed variability was indicated by the large amount of small-scale inter packet heterogeneity. Thus in seed packets located only 10 cm apart in the same dune slack large branched seedlings could be found in one case and zero germination in the other. The most likely explanation of these small-scale effects is that the packets supporting no germination were located too far from a source of the essential inoculum of T. cingulatum. If this is the case the result provides a graphic demonstration both of the patchiness of distribution of the inoculum and of its slow rate of spread.

Seedling longevity and mortality

Seeds that had not germinated but that had the appearance of being alive were found in some packets even at the final harvest (33 months) but their viability was not confirmed. Seedling mortality was high throughout the experiment and occurred at all stages of development. The high death rate, combined with the low rates of germination meant that only a very small proportion of seedlings achieved advanced stages of development. In the presence of adult M. hypopitys plants (Expt 1), only 66 seedlings of Stage 4 were recovered alive out of an estimated total of 35 000 seeds sown. While desiccation of packets during summer months appeared to contribute significantly to the high mortalities, it is possible they arose, in part, as an artefact of our experimental method. The nylon mesh bags could be seen to be constraining the growth of the larger seedlings. In addition, elimination of direct contact between seedlings and the soil surrounding the packets, combined with the relatively shallow planting position, may have increased their susceptibility to drought.

Identity of the fungal symbionts of M. hypopitys

Over the long history of curiosity about the biology of monotropaceous plants numerous assertions have been made concerning the identity of their fungal symbionts (Bidartondo & Bruns, 2001, 2002). Because these have normally been based upon circumstantial evidence, in particular the observed proximities between plants and fungal fruit bodies, most of these are likely to have been spurious. Only Martin (1985) successfully combined observations of fungal fruiting patterns with meticulous morphological examination of mycorrhizal roots of M. hypopitys to provide what has proved subsequently to be an accurate identification of the fungal symbiont as a species of Tricholoma.

The application of molecular methods enabling definitive identification of the fungi forming mycorrhizal structures has greatly advanced our understanding of the biology of these associations and has confirmed that a high degree of specificity exists between Tricholoma species and M. hypopitys in Europe, North America and Japan (Bidartondo & Bruns, 2001). This specificity operates both in geographical mosaics, which may be linked to the distributions of their fungal and autotrophic hosts, and in phylogenetic control within Monotropa. Phylogenetically distinct Eurasian, Swedish and North American lineages of the plant are associated with different clades within the genus Tricholoma (Bidartondo & Bruns, 2002).

Other members of the Monotropoideae are also associated with Tricholoma species including Pityopus californicus and Allotropa virgata, which is exclusively associated with T. magnivelare (Bidartondo & Bruns, 2001). Another group of species in the subfamily, comprising Monotropa uniflora (Cullings et al., 1996; Bidartondo & Bruns, 2001), Monotropastrum humile (Bidartondo & Bruns, 2001) and Cheilotheca malayana (MI Bidartondo, unpublished) are exclusively associated with members of the Russulales, including species of Russula and Lactarius. By contrast, two other monotropes, Pterospora andromeda and Sarcodes sanguinea are specifically associated with species of Rhizopogon (section Amylopogon) throughout most of their geographic range (Kretzer et al., 2000; Bidartondo & Bruns, 2001). Pleuricospora fimbriolata associates with Gautieria monticolaBidartondo & Bruns (2002), and Monotropsis odorata and Hemitomes congestum both use Hydnellum spp. (Bidartondo & Bruns, 2001).

Fungal specificity and epiparasitism

The exceptionally high level of fungal specificity seen in M. hypopitys must place a major constraint on its distribution. It is a constraint that will be further exacerbated by the restriction of the two Tricholoma species identified as symbionts in the present study to cohosts in the Salicaceae and Pinaceae. This level of specialisation might be partly explained if the Tricholoma species in question were quantitatively important components of the ectomycorrrhizal communities of which they are a part. However, records based upon the occurrence of carpophores of T. cingulatum and T. terreum, at the national scale in the UK (Phillips, 1981) or in the localised habitats examined in this study, suggest that these fungi are occasional rather than dominant members of the mycoflora. Clearly fragmentation and isolation of these plant communities by human activities in countries like the UK will have increased the threats to plants with such specialised requirements over recent centuries. Tricholoma species appear to be particularly sensitive to pollutant N deposition and changes in forest management, both of which are implicated in the recent marked decline in abundance of these species in many parts of Europe (Arnolds, 1991).

Recent studies have confirmed that exceptionally high levels of fungal specificity are a feature not only of Monotropoideae (Bidartondo & Bruns, 2001) but also of most fully myco-heterotrophic orchids studied to date (Taylor et al., 2002), most of which associate with fungi that form ectomycorrhizal associations with autotrophic trees. High fungal specificity has also recently been confirmed for other fully myco-heterotophic plants that exploit arbuscular mycorrhizal fungi of tropical and subtropical trees including the orchid-like Arachnitis uniflora and achlorophylous Gentianaceae (Bidartondo et al., 2002). In an evolutionary context, the selective advantages of specialisation on a restricted number of partners remain unclear. However, it is noteworthy that exceptionally high levels of specificity are a widely acknowledged feature of parasitic organisms (Price, 1980; Thompson, 1994) and since Björkman (1960), it has been recognised that the removal, by monotropes, of carbon from the symbiotic partners of autotrophs might constitute a specialised form of epiparasitism (Cullings et al., 1996). This is further supported by recent evidence from stable isotope analyses that indicates exceptional enrichment in heavy carbon and nitrogen isotopes in these plants, which is related to, but higher than, the heavy isotope enrichments seen in their specific fungal partners (Trudell et al., 2003).

The status of the fungal partner in such epiparasitic associations is also unclear. While it has been recognised that M. hypopitys must constitute a net carbon drain on its fungal associate, there is little evidence that the fitness of the fungus is reduced. On the contrary, there was evidence in the present study that in the presence of seedlings of M. hypopitys the vigour of T. cingulatum mycelium was considerably increased (Fig. 1). Based upon a similar observation in the case of the association between another monotrope, Sarcodes sanguinea, and its fungal symbiont, Rhizopogon ellenae, Bidartondo et al. (2000) referred to the epiparasite as ‘a cheater that stimulates its victims’. The nature of the mechanism involved in this stimulation remains unknown but the potential advantage to the epiparasite in the form of improved carbon supply seems clear.

Issues concerning the balance between the partners in the tri–partite association between M. hypopitys-Tricholoma spp. and the autotrophs should not be allowed to cloud the fact that this is a relationship which is sustained by the transfer of carbon through a shared mycorrhizal mycelium from an autotrophic to a fully myco-heterotrophic plant. It follows that the erroneous popular conception of M. hypopitys as being ‘a saprophyte feeding on decaying organic matter’ (Fitter et al., 1996) or ‘saprophytic’ (Preston, Pearman & Dines, 2002) should now be corrected. The recent confirmation that the same transfer processes can sustain phylogenetically distinct myco-heterotrophs colonised by arbuscular (Bidartondo et al., 2002) and orchid-ectomycorrhizal fungi (McKendrick et al., 2000b, 2002; Selosse et al., 2002; Selosse, Bayer & Moyerson, 2002; Taylor et al., 2002), indicates that this direct pathway for net carbon transfer between plants has been independently selected on numerous occasions in nature in all the main types of mycorrhizal association.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the NERC for financial support (GR3/10062 to J.R. Leake & D.J. Read), and Welsh Natural Heritage for permission to sample the Newborough Warren Monotropa population. We especially thank Irene Johnson who assisted with the assembly of seed packets, their burial, harvesting and analysis. We gratefully acknowledge Else Vellinga for T. cingulatum herbarium material, Ryan Bowman for laboratory assistance, and the USDA for grant 9600479 to Prof. T.D. Bruns.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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