Research
Symbiotic germination and development of the
myco-heterotroph Monotropa hypopitys in nature and
its requirement for locally distributed Tricholoma spp.
Blackwell Publishing, Ltd.
J. R. Leake1, S. L. McKendrick1, M. Bidartondo2 and D. J. Read1
1
Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK; 2Department of Plant and Microbial Ecology, University of
California, Berkeley, CA 94720 –3102, USA
Summary
Author for correspondence:
J. R. Leake
Tel: +44 114222 0055
Fax: +44 114222 0002
Email: [email protected]
Received: 13 January 2004
Accepted: 29 March 2004
doi: 10.1111/j.1469-8137.2004.01115.x
• 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.
Key words: Monotropaceae, seedling development, fungal specificity, mycoheterotrophy, in situ germination, Tricholoma.
© New Phytologist (2004) 163: 405–423
Introduction
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.
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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
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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
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 µm 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
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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 grassdominated 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
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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  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  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
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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 *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
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.
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
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)
Mean dimensions of seed or seedling
(± SE) or defining limits
First observation of each stage
(months after sowing)
Stage
Description
Length
Width
( n)
Slack 1
1995
Slack 2
1995
Slack 1
1997
0
Ungerminated seed (Fig. 1a)
72.8 µm ± 0.5
237
–
–
–
1
Rupture 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)
Unbranched seedling with fully developed
mycorrhizal fungal mantle (Fig. 1c,d)
Branched seedling with side roots
a) 1–4 roots (Fig. 1e)
b) 5+ roots (Fig. 1f–h)
Plant with shoot buds (Fig. 1h,i,k)
116.4 µm ± 0.7
max. < 135 µm
124 µm ± 4
max. < 160 µm
> 160 µm
All > 900 µm
2
3
4
84 µm ± 3
8
7
6
2.5
> 80 µm
44
9
6
2.5
96% > 400 µm
51
9
9
14
26
13
13
13
25
2.5
2.5
ND
ND
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.
Fig. 1 Stages in development of Monotropa hypopitys. (a) Stage 0 – ungerminated seed (bar, 100 µm). (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 µm). (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 µm). 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 µm). (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 µm). (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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Fig. 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 R 2 of 0.94
for the full dataset presented in (b).
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.
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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
Fig. 3 The sequence of initiation and
development of root branches in seedlings of
Monotropa hypopitys.
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(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.
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).
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Fig. 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.
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).
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
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Fig. 4 continued
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,
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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
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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
Time after sowing
7 months
− Adults
+ Adults
9 months
− Adults
+ Adults
14 months
− Adults
+ Adults
Mean percentage of packets containing germinating seedlings
in each plot (n = five plots)
46.5 a
98.5 b
13.4 a
98.5 b
13.2 a
87.0 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
n=7
3.6 a
n = 14
1.0 a
n=4
8.7 b
n = 14
3.3 a
n=8
9.1 b
n = 45
Mean percentage of all sown seeds which germinated in each
packet (n = total packets sampled)
0.4 a
n = 15
3.1 b
n = 15
0.1 a
n = 15
7.6 b
n = 15
0.1 a
n = 50
5.8 b
n = 57
Variable
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.
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).
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.
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.
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
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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.
c. 12 800 c. 26 000 c. 35 000
0 (0†)
50 (2†)
815 (> 62†)
40 (26†)
1
246 (79†)
58 (32†)
0
131 (65†)
c. 8200
0
0
0
c. 10 200
57 (23†)
51 (15†)
50 (31†)
c. 6800
0
0
0
7827
600 (nd†)
29 (4†)
20 (2†)
1968
1842
7299
4 (2†)
51 (39†)
35
1 (0†) 126 (34†)
0
0 (0†)
3 (0†)
0
2109
107
0
0
53 a
1186 b
98 b
0a
158 b
0a
643 b
35 a
180 b
5a
107 b
Total number of seedlings
13 a
recovered (live + dead)
Number of seeds sampled
1945
Number of Stage 1 seedlings
13
Number of Stage 2 seedlings
0
Number of branched seedlings (Stages 3– 4)
0
+ Adults
− Adults
Variable
7 months
9 months
− Adults + Adults − Adults + Adults
14 months
− Adults
+ Adults
21 months
− Adults
+ Adults
26 months
− Adults
+ Adults
Total over 26 months
Time after sowing
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
Research
© New Phytologist (2004) 163: 405–423 www.newphytologist.org
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.
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).
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Table 4 Expt 2: effect of Salix repens cover on the mean percentage germination (all stages) of Monotropa hypopitys sown in four replicate 1m2 plots either within dense stands of Salix repens (+ Salix) or in grassy areas without (− Salix) shoots in Oct 1995
8 months
Variable
− Salix
repens
13 months
+ Salix
repens
− Salix
repens
+ Salix
repens
9.1 a1
85.2 b1
41.5 a1
50.0 a1
1.2 a1
n=2
15.9 b1
n=9
4.0 a1
n=5
26.7 b1
n=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 a2
Mean percentage of packets containing germinating
seedlings in each plot (n = 4 plots in each case)
Mean percentage of seeds, which germinated
in each packet excluding packets with no seed
germination (n = the number of packets with
germinating seeds)
Number of seeds sampled
831
147 b2
876
26 a2
922
121 b2
872
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).
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
Fig. 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.
© New Phytologist (2004) 163: 405–423 www.newphytologist.org
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Fig. 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.
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).
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
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Table 5 Molecular characterisation of mycorrhizal fungi associated with seedling and adult Monotropa hypopitys plants of three habitats
Geographic location
and plant community
Type of plant material
(number of samples)
ITS-RFLP
type
MtLSU
ITS
region
Newborough Warren, Anglesey, UK
(Salix repens dune slack)
M. hypopitys seedlings (2)
I
Tricholoma
(1) AF351892
T. cingulatum
(1) AF34698
M. hypopitys adult (6)
S. repens (3)
I
I
Tricholoma (2)
nd
T. cingulatum 1
nd
Millers Dale, Derbyshire, UK
(Salix caprea scrub on limestone)
M. hypopitys adult (2)
I
nd
T. cingulatum
Dalby Forest, North Yorks, UK
(Pinus sylvestris on limestone)
M. hypopitys adult (14)
II
Tricholoma (1)
T. terreum 2
(2) AF377215
As above
As above
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.
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 SalixType 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
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
© New Phytologist (2004) 163: 405–423 www.newphytologist.org
to draw comparisons with those of the largest family of mycoheterotrophic 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
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situation seen in many orchids including the fully mycoheterotrophic 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 mycoheterotrophic 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.
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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
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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 monticola Bidartondo & Bruns (2002), and Monotropsis
421
422 Research
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 mycoheterotrophic 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
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.
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