researchnature.berkeley.edu/brunslab/papers/bidartondo2004c.pdfchronology of germination and...

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Blackwell Publishing, Ltd.

Symbiotic germination and development of the myco-heterotroph

Monotropa hypopitys

in nature and

its requirement for locally distributed

Tricholoma

spp.

J. R. Leake

1

, S. L. McKendrick

1

, M. Bidartondo

2

and D. J. Read

1

1

Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK;

2

Department of Plant and Microbial Ecology, University of

California, Berkeley, CA 94720–3102, USA

Summary

• Germination and symbiotic development of the myco-heterotrophic plant

Monotropa hypopitys

were studied by sequential recovery of packets of seed buriedin dune slacks in relation to distance from mature

M. hypopitys

and presence andabsence of shoots of its autotrophic coassociate

Salix repens

.• Fungal associates of

M. hypopitys

growing under

S. repens

in the dune slacks, andunder

S. caprea

and

Pinus sylvestris

at two other locations in the UK, were identifiedby molecular analysis.• While the earliest stage of germination could be found in the absence both ofmature

M. hypopitys

, and

S. repens

, further development was dependent uponmycorrhizal colonisation, which was most common close to these plants. Molecularanalysis 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, myco-heterotrophy,

in situ

germination,

Tricholoma

.

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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

Introduction

The subfamily Monotropoideae (Ericaceae) consists of tengenera (Wallace, 1975). All the species lack chlorophyll andhence can be characterised as myco-heterotrophs (Leake,1994). The most widely distributed species of this subfamily,

Monotropa hypopitys

, has fascinated biologists for well overa century and a half (Leake, 1994). It was Kamienski (1881)who made the major conceptual advance in understandingthe nutrition of the plant. In providing the first detaileddescription of the fungal sheath on the roots of

M. hypopitys

,he realized that virtually all the nutrients taken up by the plantmust be acquired through its fungus, and that connectionsbetween the fungus and adjacent autotrophic plants mightenable the myco-heterotoph to indirectly parasitise anautotroph.

However, none of the early workers were able to do muchmore than speculate on the nature of the relationship betweenthe plant and its fungal partner and unfortunately, whileinterest in the plant has been unabated to the present time,speculation has continued to characterise many of the asser-tions made about its biology, and the nature of its nutrient,particularly carbon, supply and on the identity of its fungalassociate(s). Most remarkably, for all species in the Mono-tropoideae, virtually nothing is known of germination anddevelopmental stages up to point flowering, since their mainphases of growth are subterranean and the plants are onlyobserved when their inflorescences emerge above ground.

Two recent advances have facilitated progress in these areas.The first is the development of procedures enabling sowingand sequential recovery of the minute seeds and seedlings ofplants like

Monotropa

so that the chronology of their symbiotic


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germination and growth can be accurately determined inthe 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 formmycorrhizal associations with their roots (Bidartondo &Bruns, 2001, 2002). Here we describe the application of boththese approaches to the study of

M. hypopitys

. The first detailedanalyses of the factors determining germination and of thechronology of seedling development are provided, and theidentities of the fungi forming the mycorrhizas are established.

Materials and Methods

Studies of germination and development of

M. hypopitys

werecarried out at Newborough Warren National Nature Reserve,Anglesey, North Wales (National Grid Ref: SH 413632). Thisis an area of coastal dunes and dune slacks, the latter supportingareas of

Salix repens

scrub within which several large popu-lations 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 processesof distances from naturally occurring mature plants of thisspecies (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 associatedwith seedlings and mature plants of

M. hypopitys

at New-borough. For comparative purposes, this analysis was repeatedusing roots collected from mature

M. hypopitys

plants growingunder

Pinus sylvestris

on Jurassic limestone in Dalby Forest,North Yorkshire, UK (NGR SE 874876) and under

Salixcaprea

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 anumber of flowering plants growing in a calcareous dune-slack(Slack 1 – see Expt 1) at Newborough Warren NNR on17 August 1995. They were dried over calcium chloride atroom temperature for 4 wk then stored in air-tight glass vialsat 4

°

C until needed. A subsample of seed was tested forviability 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 intoseed packets constructed from 40

×

60 mm rectangles of53 µm nylon plankton netting (Plastock Associates, Birken-head, UK). The nylon was folded once and clipped into2 mm

×

2 mm

×

36 mm plastic slide mounts (Rasmussen &Whigham, 1993). A length of coloured nylon line, which wasattached to each mount, extended above the soil surface afterburial of the packets to facilitate their recovery.

Using strung quadrats as templates, seed packets wereinserted into

c.

10 cm deep slots cut in the turf with a sharp

chisel as described in McKendrick

et al

. (2000a). There were100 packets inserted in a grid pattern in each 1 m

2

replicateplot. The positions of the corners of each plot were mappedusing co-ordinates to nearby fence-posts before the quadratswere removed. Seed packets were inserted in two separate duneslacks, 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 populationof

M. hypopitys

. Ten 1 m

2

plots were established at this site andthese contained a total of 1000 seed packets. The plots werearranged so that five contained mature plants of

M. hypopitys

,while the other five were placed so that there were noobservable plants of

M. hypopitys

within five metres of the plotboundary (Expt 1).

A small supplementary experiment was established in Slack1 in 1997 to examine the possibility of germination occurringin the autumn immediately following seed ripening.

In Expt 1 seed packets were inserted between 18 Septemberand 3 October 1995. The grid co-ordinate position of eachpacket within the quadrats was written on the plastic slidemount with a permanent marker to facilitate mapping of thespatial distribution of seedling germination following harvests.

Harvests were taken 7, 9, 14, 21, and 26 months aftersowing (on 30 April 1996, 25 June 1996, 27 November 1996,25 June 1997 and 27 November 1997, respectively). At the firsttwo harvests, when it was not known whether germinationhad occurred, only three replicate samples were removed fromeach 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 perplot. At the later harvests more samples were taken from theplots that contained adult

M. hypopitys

than from thosewithout mature individuals to enable targeting of packetscontaining the highest frequency of germinated seeds. At thefinal harvest, 12–20 packets were taken per plot. A randomnumber table was used to select grid locations from whichpackets were sampled at all harvests.

In the supplementary experiment, 15 packets were sown inSeptember 1997, there being three sets of five each planted within50 cm of a flowering spike of

M. hypopitys

. These packets were allharvested 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 seconddune slack (Slack 2). Whereas in the previous decade Slack 2had supported a large population of

M. hypopitys

, this had

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now declined so that plants were scarce and much lessabundant than in Slack 1. A further distinguishing feature ofSlack 2 was that within the

Salix

scrub there occurred, as aresult of earlier activities of the

Salix

-specific ring die-backpathogen

Roselinia desmazieresii

near-circular patches of grass-dominated vegetation. In this slack, eight 1 m

2

plots were set up,each containing 100 packets. Four of the plots were locatedwithin the

Salix

-dominated vegetation, while four were placedin the grassy areas and did not contain

Salix

shoots. It isnecessary here to recognise that while the occurrence of

Salix

roots was reduced in these plots, some of them almostcertainly entered the grassy areas from surrounding thickets,and so were present under both circumstances. Again, the eightplots were dispersed across the slack.

Packets were sown in the plots between the 3 and 12 October1995, using the approaches described in Expt 1. Harvestswere carried out 6, 8, 13, 20, and 25 months after sowing, on17 April 1996, 26 June 1996, 28 November 1996, 26 June1997 and 28 November 1997, respectively.

Post-harvest analysis of seedlings in Expts 1 and 2

Immediately following each harvest, packets were returned tothe laboratory where they were stored moist at 4

°

C overnight.Over the course of the following 3–4 d, the packets wereopened and examined microscopically to detect the extentof germination and of seedling development. The time takento process the large number of samples made it necessary topreserve the contents of each packet by mounting the seedlingson a glass slide in a drop of 50% glycerol, placing a coverslip over the specimens and sealing with clear nail varnish.These slides were stored at 4

°

C until the seedlings could bemeasured. Seedlings too large to be mounted in this way weremeasured fresh.

In Expt 1, the total number of seeds in each seed packet andnumbers of seedlings that were live and dead were countedand the seedlings measured. At the first two harvests thelength and breadth of representative seedlings at all stages ofdevelopment were measured and the extent of fungal infec-tion and seedling development were recorded. At the harveststaken over the first 21 months, measurements, from allsampled packets, were made of total seedling length (includingall branches) of all seedlings that were greater than 0.135 mmlong, and had reached the second stage of development (seelater for definition of developmental stages). At the laterharvests (26 months onwards) the intermingling of rootsof live and dead seedlings made measurements impractical andrecords were made only of the stage of development andnumbers of branches produced by seedlings.

At the 14-month harvest a very large number of seedlingshad germinated and were at the first stage of developmentand, in this case, it was too time consuming to distinguishthose seedlings that were alive from those that were dead. Atthe 21-month and 26-month harvests the total numbers of

seeds sampled were estimated from the average numbers of seedsper packet at the earlier harvests, since ungerminated seedswere in too advanced a state of decay to reliably be counted.

Differences between the percentage of seed packets inwhich germination occurred in plots with and without adult

M. hypopitys

were assessed at each harvest by

of arc-sinetransformed percentages. Similarly, the effect of adults on themean percentage of seeds that germinated in each packet,excluding packets with no germination, and the percentage ofall sown seeds that germinated in each packet were analysedby

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 aftersowing). At the later harvests 10–12 packets were sampledfrom each plot. The presence and absence of germinatingseedlings were noted for each seed packet and the germinatedseedlings that were found in the plots containing

Salix repens

were measured and their stages of development and whetherthey were alive or dead were recorded. At the first (6 month)harvest we did not make full counts of the numbers of seedsand seedlings, and at the harvests taken at 21 and 25 monthsafter sowing we only recorded seedlings that had reachedStage 2 because of the increasing difficulty of distinguishingdead seeds and small seedlings.

Molecular identification of fungi

DNA analyses were carried out on 27 samples from threelocations (Newborough, Dalby Forest and Miller’s Dale)with a view to determining the identities of the fungi formingmycorrhiza on

M. hypopitys

growing with three differentautotrophic 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 theseplants) were analysed from Newborough Warren. Twosamples, each representing a different adult

M. hypopitys

plantgrowing under

S. caprea

were analysed from Millers Dale.Fourteen separate root samples from three individual adultplants of

M. hypopitys

growing under

P. sylvestris

were exam-ined from Dalby Forest. The methods used for the analyseswere those of Gardes & Bruns (1996). DNA was extractedfrom individual roots and the ITS region of the nuclear ribo-somal 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 productswere then screened by restriction fragment length polymor-phism (RFLP) using the endonucleases

Alu-l

and

Hinf-l

(NewEngland Bio Labs, Beverley, MA, USA). In the cases of thoseITS-RFLP’s that exhibited a unique pattern the followingfungal DNA regions were amplified and sequenced: first afragment of the mitochondrial large subunit (mtLSU) (Bruns

et al

., 1998) and second the nuclear ITS region. Sequencingof 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 CoreKit (Perkin Elmer Co., Foster City, CA, USA). The raw datawere processed using DNA Sequencing Analysis v.2.1.1 andSequence Navigator v.1.0.1 (Applied Biosystems Co., FosterCity, CA, USA) software. Sequences from the mtLSU weremanually aligned to the database of Bruns et al. (1998). Toinfer relationships, the neighbour-joining algorithm imple-mented in the program *d64 (Swofford, 1993) wasemployed. Sequences from the ITS region were used to queryGenBank via BLAST (http://www.ncbi.nlm.nih.gov/blast).

Results

The results of the observations made on packets harvestedfrom both Expts 1 and 2 were first pooled to provide anoverall view of the chronology of germination of M. hypopitys

and to enable description of the distinctive stages throughwhich 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 distinctstages (Table 1). Ungerminated seeds (Stage 0) were opaque(Fig. 1a) and light microscope observations indicated that theycontained abundant lipid droplets. After burial in the field for7 months those seeds that had not germinated had a meanlength of 116 microns.

By this time in the main experiments, and within 10 wk ofseed sowing in the supplement to Expt 1, changes in theappearance of some seeds were recognisable (Table 1). Germi-nation (Stage 1) was indicated by the rupturing, at one end ofthe 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)

Stage Description

Mean dimensions of seed or seedling (± SE) or defining limits

First observation of each stage (months after sowing)

Length Width (n)Slack 11995

Slack 21995

Slack 11997

0 Ungerminated seed (Fig. 1a) 116.4 µm ± 0.7 72.8 µm ± 0.5 237 – – –max. < 135 µm

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)

124 µm ± 4 84 µm ± 3 8 7 6 2.5max. < 160 µm

2 Unbranched seedling with fully developed mycorrhizal fungal mantle (Fig. 1c,d)

> 160 µm > 80 µm 44 9 6 2.5

3 Branched seedling with side roots a) 1–4 roots (Fig. 1e) b) 5+ roots (Fig. 1f–h)

All > 900 µm 96% > 400 µm 51 9 13 2.59 13 2.5

14 13 ND4 Plant with shoot buds (Fig. 1h,i,k) 26 25 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).

http://www.ncbi.nlm.nih.gov/blast

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the outer faces of the endosperm cells. These events were asso-ciated with an increase in translucence of the embryo, and theemergence of a small portion of its tissue through the seedcoat (Fig. 1b). The majority, but not all Stage1 seedlings werevisibly colonised by fungal hyphae (Fig. 1b).

The increases in length and breadth of seedlings at Stage 1were 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 seedsindicated that 95% of them had lengths less than 135 microns,

many Stage 1 seedlings were also in this size category. Thedetermination of development to Stage 1 therefore requiredthe extremely labour intensive microscopic examination ofeach individual seedling. While intensive analysis of this kindwas possible at the early harvests when numbers of germinat-ing seeds were relatively small it was not feasible later whennumbers of packets and of seedlings being processed rose toin excess of 100 s and then 1000 s at the succeeding harvests.On these later occasions all seeds greater than 135 micronslong were counted as having germinated.

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 R2 of 0.94 for the full dataset presented in (b).


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Development of seedlings beyond Stage 1 was dependentupon colonisation by a symbiotic fungal partner and was asso-ciated with the initiation of a mycelial mantle around part ofthe seedling axis (Fig. 1c). By the time seedlings had achievedlengths and breadths of 0.16 × 0.08 mm, a complete fungalmantle was normally present (Figs 1d,e and 2a). This level ofdevelopment characterises Stage 2 (Table 1) and is the first atwhich, on the basis of size alone, it is possible to define theseedlings as being symbiotic. At this stage the seedling is stillunbranched. Plants were observed to have achieved this stage ofdevelopment only 2.5 months after sowing in the supplementto Expt 1, while in the main Expts 1 and 2, Stage 2 wasreached, respectively, after 9 and 6 months (Table 1).

The third stage of development involved the emergence ofside roots from the main axis of the seedling. This branchingwas first observed to have occurred only 2.5 months after theautumn 1997 sowing in the supplementary experiment. InExpts 1 and 2 it was first seen in harvests taken, respectively,after 9 and 13 months (Table 1). Because Stage 3 seedlingsshowed a large range in size and underwent considerablegrowth before reaching the next stage of development, thiscategory was divided into two substages that were demarcatedon the basis of numbers of these roots. Seedlings with betweenone and four side roots were placed in Stage 3a, while thosewith five or more were included in Stage 3b (Table 1).

The next developmental stage (Stage 4) was defined by theappearance of shoot buds that were first observed after 25 and26 months, respectively, in the two experiments (Table 1).In the most advanced buds, overlapping scale leaves weredeveloping at the shoot apex. The shoot buds normallyemerged 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 mostungerminated seeds and only after the seedlings had developeda complete fungal mantle (Stage 2) were they unambigu-ously larger than ungerminated seed (Fig. 2a). Their lengthsand breadths continue to increase in proportion until seed-lings achieve about 1 mm in length and begin to produceside branches (Fig. 2a,b). In seedlings larger than this, thewidth of the main root ranged from 0.45 to 0.75 mm andchanged little with increasing total root length (Figs 2e–hand 3b). The change in the length-breadth relationshipcoincided with the initiation of branching (Stage 3) in mostseedlings. The development of branches introduces con-siderable additional variation in seedling widths, as themain axis swells immediately before the emergence of eachnew root apex.

Patterns of seedling growth and root branching Germinatingseedlings soon establish a single apical root meristem fromwhich growth proceeds, leaving the original testa attached tothe base (Fig. 3). Unipolar growth proceeds until a lateral rootmeristem emerges at a position on the main axis, typicallywithin 0.5 mm of the original embryonic cells (Fig. 3). Asecond branch is formed simultaneously, or soon afterwards,diametrically opposite the first. This gives rise to a seedling ofcruciate form. The orientation of the seedling in the packetappears to have little influence on the morphogenetic pattern.The third and fourth branches subsequently emerge fromthe main axis closer to the primary meristem than the firstbranches (Figs 1e and 3). Growth proceeds from this point ina variety of ways. In most cases further branching continuesto occur from the main axis (Figs 1f and 3) but eventuallyextension of the lateral roots exceeds the length of the mainaxis, and secondary lateral roots are developed (Fig. 1g,h). Bythis stage the seedlings are structurally complex, and wheremany of them are growing together in a packet, it can bedifficult to separate them (Fig. 1f,g).

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


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The progression of seedling growth and mortality with timeThe percentage of live and dead seedlings occurring in each ofa series of length-based size class categories were plotted for allplants of Stage 2 or later recovered from both dune slacksduring the harvests of June and November 1996 and June1997 (Fig. 4a,b). At the first of these harvests over 80% of theStage 2 seedlings in Slack 1 had lengths > 0.367 mm, whilenone of the symbiotic seedlings in Slack 2 had reached thislength. At this time over 30% of the seedlings of the smallestsize class in Slack 1 had died (Fig. 4a). Mortality in Slack 2 was70% (Fig. 4b).

Over the ensuing 5 months to November 1996, consider-able growth had occurred in seedlings recovered from bothdune slacks. While the numbers and proportions of seedlingsin the smallest size class decreased, the sizes of many indivi-duals had increased substantially, the largest now attaininglengths up to 55 mm in Slack 1 (Fig. 4a) and up to 208 mmin Slack 2 (Fig. 4b).

The modal size class of seedling lengths in the first slack was0.368–0.999 mm and it reached the even higher value of 7.4–20.1 mm in Slack 2. Dead seedlings were no longer observedin the smallest size category in either slack, indicating that

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.


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those of the type that had been recorded as being dead in theprevious harvest had by now decomposed sufficiently to beunrecognisable. Mortality was now seen in the larger seedlingcategories in Slack 1 (Fig. 4a) and in the very largest seedlingsobtained from Slack 2 (Fig. 4b).

By the next harvest, in June 1997, the proportions of seed-lings in the smallest size-classes, and of those that were deadin all size classes, had increased (Fig. 4a,b). The increase inproportion of small seedlings, coupled with the observationthat some of these new seedlings were found in packets con-taining plantlets that had reached Stages 2 and 3 and died,

suggests that a new cohort of seedlings had germinated sincethe previous harvest, that is between 14 and 21 months aftersowing.

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 seedgermination The presence of adult M. hypopitys in the 1 m2

plots was consistently associated with enhanced seed germinationover the first 14 months after planting (Table 2). In the plots

Fig. 4 continued


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supporting adults 87–99% of the packets sampled over thisperiod contained germinating seeds, whereas in their absencethe 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) wasalso generally higher in the plots with, relative to those with-out, adult M. hypopitys plants. However, this effect was onlysignificant (P < 0.05) at the 9- and 14-month harvests.

The percentage of all seeds that germinated was increasedsignificantly in the plots supporting adults at all three harvests(P < 0.05) (Table 2). The numbers of seedlings were up to76 times higher in plots with than in those without adultspikes of M. hypopitys.

Effects of the presence of mature plants on seedling develop-ment The extent of seedling development was stronglyinfluenced by the presence of adult M. hypopitys in Slack 1. Inthe plots containing adults nearly 1200 seedlings germinatedout of a total of 35 000 sampled (Table 3). Of the 1200, over350 developed a full mycorrhizal mantle, and over 130 reachedthe branching stage or beyond (Stages 3–4). By contrast, ofan estimated total of 26 000 seeds sampled from plots withoutadults, only 53 had germinated (Table 3) and only one ofthese reached Stage 2.

Over the 26-month sampling period there were markedchanges in the proportions of seedlings recorded in the dif-ferent developmental stages in both sets of plots (Table 3).However, these changes were more marked in the presence ofadults where both the numbers and proportions of seedlingsin Stage 1 were greater, as was the extent of development beyondStage 1. In these plots Stage 1 seedlings were found up to andincluding 21 months after sowing. Progressive development

was indicated by the observation that while the numbers andproportions of Stage 1 seedlings decreased after 14 months,those in Stages 2 and 3 increased (Table 3). This situation wasin marked contrast to that seen in plots lacking mature indi-viduals. Here the presence of Stage 1 seedlings was observedonly in the first 14 months, and with the exception of the oneseedling that reached Stage 2 by 9 months, no developmentbeyond Stage 1 was observed. The complete absence of seed-lings at the 21 and 26 month harvests indicated that anyearlier germinants had both died and decomposed in theintervening period of time. Death of seedlings was commonin both sets of plots and appeared to be caused largely bydesiccation.

The effects of proximity to adults on the spatial distributionof germination Analyses of the spatial distribution of seedpackets in which germination had occurred, and of the mostadvanced developmental stages attained by seedlings in eachsampled packet showed strong relationships with the presenceof adult M. hypopitys (Fig. 5). In all the plots containing adultssome seedlings reached Stage 3 of development, and in threeof the five plots, plants had produced shoot buds (Stage 4) bythe final harvest at 33 months. By contrast, none of the plotslacking mature spikes of M. hypopitys yielded seedlings beyondStage 1 (Fig. 5). The absence of any sign of mycorrhizal colon-isation in these seedlings strongly indicated that it was thepresence or absence of the appropriate fungus in these plotsthat determined the fate of seeds arriving in them.

Despite the marked stimulation of germination andgrowth of seedlings in the presence of adults at the 1 m2 plotscale, there was no evidence at smaller spatial scales that ger-mination was enhanced in the immediate vicinity of the adultplants. When the germination data from all plots were pooled,and the numbers of packets with and without germinating

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

Variable

Time after sowing

7 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 3.6 a 1.0 a 8.7 b 3.3 a 9.1 bn = 7 n = 14 n = 4 n = 14 n = 8 n = 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 bn = 15 n = 15 n = 15 n = 15 n = 50 n = 57

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.


Research 415

seeds were compared for samples taken within 20 cm of flower-ing shoots of M. hypopitys and samples more than 35 cm fromthe nearest known adults, there were no significant differences(χ2 = 0.53, d.f. = 1, P > 0.05). Similarly, despite the fact thatthe numbers of adults of M. hypopitys varied considerablybetween plots (in the range 4–18 flowering spikes) and thattheir occupancy of 10 × 10 cm square subdivisions of thequadrat area (range 4–14 quadrat squares) also varied greatlybetween plots, there were no significant differences betweenplots in the proportion of sampled packets that containedgerminating 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 andindividual seed levels At 8 months after sowing, the percent-age of packets containing germinated seeds was significantlyhigher (P < 0.05) in the plots with dense Salix cover than inthose from which its shoots were absent and its rootingdensity reduced (Table 4). By 13 months, however, the numberof packets containing germinating seeds in the plots withoutSalix cover had increased to the extent that there was nolonger a statistically significant effect of Salix cover. It wasnoticeable that both germination and seedling developmentwere much more patchy than seen in Expt 1. In retrospect werecognised that because of the high observed variability, arigorous test of plot effects in this experiment would haverequired an increase in the sampling intensity beyond the24 packets that were recovered at each of the harvests overthe first 13 months.

Analyses of the percentage germination occurring in indi-vidual packets revealed a consistent and significant positiveeffect of Salix cover at both harvests (Table 4). Thus, althoughat 13 months there was no significant difference between thepresence and absence of Salix cover in numbers of packetssupporting germinating seedlings, the numbers of seedlingsrecovered from each packet were significantly lower (P < 0.05)in plots without Salix shoots. Numbers of seedlings per packetwere an order of magnitude greater in plots covered with Salixthan in those without Salix shoots, reaching 27% in theformer treatment after 13 months. The total number of seed-lings obtained was from 4–50 times greater in the plots withSalix cover, than in those without it (Table 4).

In addition to effects upon seedling numbers, Salix coveralso influenced the extent of development and longevityof seedlings in the packets. In the plots without Salix shoots,although germination was occasionally recorded, thedevelopment of seedlings never progressed beyond Stage1. Further, by 20 months after sowing ( June 1997), all seed-lings from these plots had died. By contrast, in several of theplots with dense Salix cover not only did developmentprogress beyond Stage 1, but in several cases it reached Stage4 (see Table 1, Fig. 6).Ta

ble

3Ex

pt 1

: eff

ect

of p

rese

nce

and

abse

nce

of a

dults

on

tota

l num

bers

of

seed

lings

of

Mon

otro

pa h

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at

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t de

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5 r

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m f

rom

adu

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− A

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) or

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flow

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ults

(+

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whe

n th

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ere

burie

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Sep

t 19

95

Tim

e af

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Tota

l ove

r 26

mon

ths

Varia

ble

7 m

onth

s−

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lts+

Adu

lts9

mon

ths

− A

dults

+ A

dults

14 m

onth

s−

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lts+

Adu

lts21

mon

ths

− A

dults

+ A

dults

26 m

onth

s −

Adu

lts+

Adu

lts−

Adu

lts+

Adu

lts

Tota

l num

ber

of s

eedl

ings

13 a

107

b5

a18

0 b

35 a

643

b0

a15

8 b

0 a

98 b

53 a

1186

bre

cove

red

(live

+ d

ead)

Num

ber

of s

eeds

sam

pled

1945

2109

1968

1842

7299

7827

c. 6

800

c. 1

0 20

0c.

820

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12

800

c. 2

6 00

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35

000

Num

ber

of S

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1 s

eedl

ings

1310

74

(2†)

51 (

39†)

3560

0 (n

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057

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00

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the

first

14

mon

ths.


Research416


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Relationships between seedling development and presenceand absence of mature M. hypopitys in plots with dense Salixcover While mature spikes of M. hypopitys were observed in oneof the dense Salix plots (Plot 3a, Fig. 6), their presence was nota prerequisite for growth of seedlings since equivalent amountsof development occurred in plots, for example 5a (Fig. 6), lack-ing adults. A further indication of the inherent patchiness ofthe germination environment is provided by the observation that,while fully mycorrhizal seedlings were found in six packets within20 cm of adult M. hypopitys plants in Plot 3a, of the 18 otherpackets sampled within the same distance, 12 contained no seed-lings while six supported seedlings that had reached only Stage 1.

The results of this Experiment appear to be at some variancewith those of Expt 1, which suggested that the presence of adultM. hypopitys was a prerequisite for development of seedlingsbeyond Stage 1 (Fig. 4). Clearly, the possibility exists that non-emergent immature individuals of M. hypopitys were present inthose plots (1a, 5a, Fig. 6) of Slack 2 that had been presumedto lack the plant. Alternatively, pockets of appropriate ino-culum not supporting growth of M. hypopitys may have beenmore 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 youngplants was unpigmented, its mycelium being normally ofbright white appearance. Where a single hypha colonised aseedling 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 ofmycelial development in its immediate vicinity. The result wasthat in packets that supported a number of seedlings, extensivewefts of the white mycelium were visible surrounding seedlings(Fig. 1d–h). With the aid of a dissecting microscope rhizomorphscould be seen to extend from these wefts through the walls ofseed packets to the ectomycorrhizal mantles of Salix repensroots. Woody roots of Salix were occasionally observed toenter seed packets that had become partly opened followingburial. Under these circumstances ectomycorrhizal short rootsemerging from the woody axes were colonised by the whitefungus, and rhizomorphs could again be seen to form directconnections to the fungal mantles on nearby developing M.hypopitys seedlings (Fig. 1j). Mature M. hypopitys were alsofound to be consistently associated with what appeared to bethe same white fungus (Fig. 1k). Ectomycorrhizal roots of S.repens were often observed to proliferate against the outerwalls of the seed packets. Here, they could be colonised by avariety of ectomycorrhizal fungi amongst which that with thewhite mycelial mantle was normally present. (Fig. 1L). Again,mycelia of this fungus could, under some circumstances, betraced into packets containing germinated seeds.

Molecular identification of fungal symbionts

Two M. hypopitys seedlings obtained from packets, togetherwith samples of roots taken from six adult plants from

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

Variable

8 months 13 months

− Salix repens

+ Salix repens

− Salix repens

+ Salix repens

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 = 2 n = 9 n = 5 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 147 b2 26 a2 121 b2

Number of seeds sampled 831 876 922 872

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).

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.


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Newborough Warren and two from Derbyshire, all produceda single combination of ITS-RFLP fragments. The same ITSpattern was also found in the three samples of roots of S. repensthat had been collected from positions adjacent to plants ofM. hypopitys and which, according to visual observation, sharedthe same fungus. We refer to this Salix-type ITS pattern asType 1 (Table 5).

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

Sequences from the fungal mtLSU of roots of both Types Iand II indicated that the fungi were members of the genusTricholoma. The ITS sequence of the Salix type producedmatches lower than 92% with those available in databases.However, based upon information gained from analysis ofTricholoma–Monotropa associations (Bidartondo & Bruns, 2001)it was decided to investigate the possibility that the fungusinvolved in this case was T. cingulatum. When the ITS regionsof two basidiocarp collections (Leiden Herbarium: Nordeloos95210 and Bas 8966-Accession Numbers AF349697 and

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.


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AF377197, respectively) of this European Salix-specificectomycorrhizal fungus were examined, they were found toproduce 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 mycor-rhizal roots of both adults and seedling of M. hypopitys thatyielded the ITS-RFLP Type I sequences were colonised by T.cingulatum. Circumstantial evidence in favour of this conclu-sion was provided by the observation that T. cingulatum is theonly member of this genus regularly to produce carpophoresunder S. repens in the Newborough dune system (A.F.S. Taylorpersonal 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 and99%. The closest match was to 576 of 579 base pairs from T.terreum accession AF062614. It was therefore inferred that allmycorrhizal roots that produced ITS-RFLP Type II sequenceswere colonised by T. terreum (Table 5), which is a widespreadectomycorrhizal fungus of the Pinaceae across Eurasia.

Discussion

This study provides the first definitive chronology of germina-tion and development of M. hypopitys. This is the first completerecord of growth from seedling to initiation of shoot buds forany species in the Monotropoideae. We establish the criticaldependence of the developmental processes upon a narrow cladeof ectomycorrhizal fungi in the genus Tricholoma and upon thespecific autotrophic coassociates of these fungi, which, in ourstudy 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 ofsymbiotic 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 mostother monotropes produce ‘dust seeds’ that show remarkableconvergent evolution in their form and anatomy to those ofthe very distantly related family Orchidaceae (Koch, 1882;Francke, 1934; Leake, 1994; Arditti & Ghani, 2000). Themost striking similarity to orchid seed is the elongated andinflated testa that very loosely encloses the seeds. Even finedetails of the testa of M. hypopitys seed correspond closely tothose in orchids. The cells have raised anticlinal and periclinalwalls, deep brown pigmentation and they curve to forma twist down the long-axis of the testa, these featurespresumably selected, as in orchids, to enhance air-bouyancyand dispersal by wind (Leake, 1994). In M. hypopitys, as inorchids, 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 arenormally regarded as extreme examples of morphologicalreduction and arrested postfertilisation cell division, theembryos of Monotropa are exceptionally simple. Whereas inthe most extremely simplified orchid embryo, seen in the fullymyco-heterotrophic Epipogium aphyllum, embyogenesis iscompleted in three mitotic cycles yielding eight cells (Geitler,1956), in M. hypopitys the embryo consists of only four cellsproduced by two mitotic divisions (Koch, 1882). It is likelythat the requirement for early fungal colonisation of this minuteembryo arises from the fact that the endogenous nutritionalresource for the support of its development consists of onlynine endosperm cells. Because of the early cessation of celldivision in the seeds, and the lack of differentiation of theembryo the ontogeny of germinating seedlings of these plantsis of considerable interest.

The present study reveals that following fungal colonisa-tion, embryo development in M. hypopitys is distinct fromthat in orchids since it leads to a unipolar axis comprising ahistologically differentiated radicle. This contrasts with the

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 M. hypopitys seedlings (2) I Tricholoma T. cingulatum(Salix repens dune slack) (1) AF351892 (1) AF34698

As above M. hypopitys adult (6) I Tricholoma (2) T. cingulatum 1As above S. repens (3) I nd nd

Millers Dale, Derbyshire, UK M. hypopitys adult (2) I nd T. cingulatum(Salix caprea scrub on limestone)

Dalby Forest, North Yorks, UK M. hypopitys adult (14) II Tricholoma (1) T. terreum 2(Pinus sylvestris on limestone) (2) AF377215

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.


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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), inwhich the apical meristem forms a shoot initial and roots areeither not formed, or develop later from one or more basalmeristems. In M. hypopitys, according to our observations,c. 2 yr of development are required before the stage of budformation is reached and buds are produced adventitiouslyand not from the apical meristem. The delay in production ofshoot meristems in M. hypopitys may reflect the need to accu-mulate the large amounts of carbon required to sustain exten-sion of the flowering spike and seed set, and may be regardedas an advanced feature in fully myco-heterotrophic plantswhose shoots never photosynthesise. In the orchids, after aninitially myco-heterotrophic phase of growth, most of the17 500 species produce photosynthetic green shoots so earlyinvestment in shoot production is likely to be advantageous inall but the c. 100 species that remain fully myco-heterotrophic.Furthermore, in the orchids, extensive intracellular fungal col-onisation provides a large internal surface area for the transferof carbon from fungus to plant, whereas in monotropoidmycorrhizas fungal penetration is confined exclusively to thesingle epidermal cell layer of the root. The haustorial pegs ofthe unique monotropoid mycorrhizas (Lutz & Sjolund, 1973;Duddridge & Read, 1982; Robertson & Robertson, 1982)provide a smaller area of interface between fungus and plantfor carbon transfer. This would explain the need to increase,by root growth, the extent both of plant–fungal interface andstorage volume before shoot buds can be initiated in themonotropoid plant. The abrupt change in length-breadthrelationship of seedlings of Monotropa on reaching only 1 mmin length contrasts with the much more gradual transitionin length: breadth ratios with growth seen in representativefully myco-heterotrophic orchids (McKendrick et al. 2000a,2002), reflecting the low surface area to volume ratios ofthe orchids in which there is extensive internal fungalcolonisation.

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 solidmedia in the laboratory. In the first study to employ mesh bagsto facilitate burial and recovery of ‘dust seeds’ in nature,Francke, 1934) mixed seed of M. hypopitys with small amountsof soil collected from different depths in the natural habitatsof the plant, placed the mixtures in ‘fine-meshed gauze’ bagsand returned them, again at a range of depths as well as atdifferent distances from mature M. hypopitys plants, to thefield. Bags planted in Oct were harvested the following May,June and July No germination was observed on the first twooccasions, but at the July harvest around 0.3% of seeds showedevidence of cell division and were recorded as having germinated.

Francke reported that neither depth of sowing nor distancefrom mature M. hypopitys had any impact upon the pattern ofgermination. From the descriptions of the seedlings recordedby Francke as having ‘germinated’ it appears they haddeveloped to Stage 1, but had not been colonised by mycor-rhizal fungi. His observations are consistent with thoseof the present study, which showed that in plots where nomycorrhizal colonisation occurred, up to 0.7% of seedsreached this stage in the first 13–14 months but developedno further.

Whilst our studies also support the suggestion that theprocess of germination can begin in the absence of fungal infec-tion, we cannot exclude possible fungal involvement in theinitiation of the germination process since chemical signalsfrom specific fungi in the vicinity of the seed may providethe trigger for these events. We found that in plots where theTricholoma was present there was an up to 10-fold increasein percentage germination (Tables 2 and 4). In other mono-tropes fungal stimulation of germination by the specific fun-gal partners of the plants, and by closely related (but possiblyincompatible) fungi has been demonstrated (Bruns & Read,2000). Similarly, there is evidence from field studies of myco-heterotrophic orchids that germination can be initiated byspecific fungal partners before they penetrate the seeds(McKendrick et al., 2000a, 2002). The present study providesonly indirect information on this aspect of M. hypopitysgermination biology. Of the c. 26 000 seeds harvested fromplots containing no adults plants of M. hypopitys (Expt 1) only53 plants (Table 3) achieved Stage 1 of germination and onlyone became colonised by fungus, so being enabled to progressbeyond this stage. This latter development, singular though itis, suggests that T. cingulatum occurred in plots lacking adultM. hypopitys. While, evidently, its occurrence was scarce thepossibility remains that the fungal symbiont was presentin sufficient amounts to trigger the small fraction of Stage 1germinations observed, or that other fungi can, at least toa limited extent, initiate germination. The similarly low levelsof Stage 1 germination observed in Expt 2, in the presence ofreduced density of Salix roots, could be explained on the samebasis, but detailed molecular analysis of the fungal commu-nity of roots of the autotroph would be necessary to determinewhether the occurrence of T. cingulatum was a prerequisite forStage 1 germination. In the absence of definitive evidence foror against the dependence of seed germination on the prox-imity of a fungal symbiont it would be inappropriate to clas-sify the initiation of the germination process as an asymbioticor a symbiotic event. However, this study makes it very clearthat any seedling development beyond the few cell divisionsthat define Stage 1 has an absolute dependence upon coloni-sation by a specific fungal partner. Since seedlings that reachStage 1 yet fail to become so colonised die, whereas those thatform mycorrhizal associations develop normally, at least toStage 4, there seems to be every reason for referring to thegermination 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 andtemporal variability both within and between packets. At thewithin packet level, particularly in the harvests taken at 20and 21 months, large branched seedlings could be foundadjacent to others that were at Stage 1. Since the failure of thelatter to develop further is unlikely under these circumstancesto be due to absence of a compatible inoculum, it is more likelyto indicate that a dormancy mechanism facilitates staggeredgermination in this plant. Indeed, analyses based upon sizeclass distributions (Fig. 4a,b) suggest that germination may bestaggered over several years. In this context it was of interestthat at 26.7%, the highest mean percentage germinationwithin packets (see Table 4) was much lower than the 60–70% viability indicated by the tetrazolium test conducted onfresh seed.

Rates of development of seedlings within the packets alsovaried greatly between years. Thus packets planted in September1997 and harvested in November the same year yieldedmore germination and much faster seedling development thanin 7 months from the September 1995 sowings. Whereas theseeds sown in 1995 showed their main phase of germinationand development in the spring of the following year, taking9–13 months to achieve Stage 4, some of those sown inSeptember 1997 had reached this stage within the 10-wk autumnperiod to November of that year. It is not clear whether suchmarked temporal variability is attributable to interyear differ-ences in climatic or biotic conditions. Clearly availability ofmoisture could directly affect the potential for seedling growthor indirectly influence seedling development through itseffects upon activities of the fungal symbiont.

The likelihood that factors other than climatic wereinvolved in determining the observed variability was indicatedby the large amount of small-scale inter packet heterogeneity.Thus in seed packets located only 10 cm apart in the samedune slack large branched seedlings could be found in onecase and zero germination in the other. The most likely expla-nation of these small-scale effects is that the packets support-ing no germination were located too far from a source of theessential inoculum of T. cingulatum. If this is the case theresult provides a graphic demonstration both of the patchinessof 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 ofbeing alive were found in some packets even at the finalharvest (33 months) but their viability was not confirmed.Seedling mortality was high throughout the experiment andoccurred at all stages of development. The high death rate,combined with the low rates of germination meant that onlya 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 outof an estimated total of 35 000 seeds sown. While desiccationof packets during summer months appeared to contributesignificantly to the high mortalities, it is possible they arose,in part, as an artefact of our experimental method. The nylonmesh bags could be seen to be constraining the growth of thelarger seedlings. In addition, elimination of direct contactbetween seedlings and the soil surrounding the packets,combined with the relatively shallow planting position, mayhave increased their susceptibility to drought.

Identity of the fungal symbionts of M. hypopitys

Over the long history of curiosity about the biology ofmonotropaceous plants numerous assertions have beenmade concerning the identity of their fungal symbionts(Bidartondo & Bruns, 2001, 2002). Because these have nor-mally been based upon circumstantial evidence, in particularthe observed proximities between plants and fungal fruitbodies, most of these are likely to have been spurious. OnlyMartin (1985) successfully combined observations of fungalfruiting patterns with meticulous morphological examinationof mycorrhizal roots of M. hypopitys to provide what hasproved subsequently to be an accurate identification of thefungal symbiont as a species of Tricholoma.

The application of molecular methods enabling definitiveidentification of the fungi forming mycorrhizal structures hasgreatly advanced our understanding of the biology of theseassociations and has confirmed that a high degree of speci-ficity exists between Tricholoma species and M. hypopitys inEurope, North America and Japan (Bidartondo & Bruns,2001). This specificity operates both in geographical mosaics,which may be linked to the distributions of their fungal andautotrophic hosts, and in phylogenetic control within Mono-tropa. Phylogenetically distinct Eurasian, Swedish and NorthAmerican lineages of the plant are associated with differentclades within the genus Tricholoma (Bidartondo & Bruns,2002).

Other members of the Monotropoideae are also associatedwith Tricholoma species including Pityopus californicus andAllotropa virgata, which is exclusively associated with T. mag-nivelare (Bidartondo & Bruns, 2001). Another group of speciesin the subfamily, comprising Monotropa uniflora (Cullingset al., 1996; Bidartondo & Bruns, 2001), Monotropastrumhumile (Bidartondo & Bruns, 2001) and Cheilotheca malay-ana (MI Bidartondo, unpublished) are exclusively associatedwith members of the Russulales, including species of Russulaand Lactarius. By contrast, two other monotropes, Pterosporaandromeda and Sarcodes sanguinea are specifically associatedwith species of Rhizopogon (section Amylopogon) throughoutmost of their geographic range (Kretzer et al., 2000; Bidartondo& Bruns, 2001). Pleuricospora fimbriolata associates withGautieria monticola Bidartondo & Bruns (2002), and Monotropsis


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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. Itis a constraint that will be further exacerbated by the restric-tion of the two Tricholoma species identified as symbionts inthe present study to cohosts in the Salicaceae and Pinaceae.This level of specialisation might be partly explained if theTricholoma species in question were quantitatively importantcomponents of the ectomycorrrhizal communities of whichthey are a part. However, records based upon the occurrenceof carpophores of T. cingulatum and T. terreum, at the nationalscale in the UK (Phillips, 1981) or in the localised habitatsexamined in this study, suggest that these fungi are occasionalrather than dominant members of the mycoflora. Clearlyfragmentation and isolation of these plant communities byhuman activities in countries like the UK will have increasedthe threats to plants with such specialised requirements overrecent centuries. Tricholoma species appear to be particularlysensitive to pollutant N deposition and changes in forestmanagement, both of which are implicated in the recentmarked decline in abundance of these species in many partsof Europe (Arnolds, 1991).

Recent studies have confirmed that exceptionally high levelsof 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 ectomycorrhizalassociations with autotrophic trees. High fungal specificity hasalso recently been confirmed for other fully myco-heterotophicplants that exploit arbuscular mycorrhizal fungi of tropicaland subtropical trees including the orchid-like Arachnitisuniflora and achlorophylous Gentianaceae (Bidartondo et al.,2002). In an evolutionary context, the selective advantages ofspecialisation on a restricted number of partners remain unclear.However, it is noteworthy that exceptionally high levels ofspecificity are a widely acknowledged feature of parasiticorganisms (Price, 1980; Thompson, 1994) and since Björkman(1960), it has been recognised that the removal, by mono-tropes, of carbon from the symbiotic partners of autotrophsmight constitute a specialised form of epiparasitism (Cullingset al., 1996). This is further supported by recent evidencefrom stable isotope analyses that indicates exceptional enrich-ment in heavy carbon and nitrogen isotopes in these plants,which is related to, but higher than, the heavy isotope enrich-ments seen in their specific fungal partners (Trudell et al., 2003).

The status of the fungal partner in such epiparasitic associ-ations is also unclear. While it has been recognised that M.hypopitys must constitute a net carbon drain on its fungal asso-ciate, there is little evidence that the fitness of the fungus isreduced. On the contrary, there was evidence in the present

study that in the presence of seedlings of M. hypopitys thevigour of T. cingulatum mycelium was considerably increased(Fig. 1). Based upon a similar observation in the case of theassociation between another monotrope, Sarcodes sanguinea,and its fungal symbiont, Rhizopogon ellenae, Bidartondo et al.(2000) referred to the epiparasite as ‘a cheater that stimulatesits victims’. The nature of the mechanism involved in thisstimulation remains unknown but the potential advantageto the epiparasite in the form of improved carbon supplyseems clear.

Issues concerning the balance between the partners in thetri–partite association between M. hypopitys-Tricholoma spp.and the autotrophs should not be allowed to cloud the factthat this is a relationship which is sustained by the transfer ofcarbon through a shared mycorrhizal mycelium from anautotrophic to a fully myco-heterotrophic plant. It followsthat the erroneous popular conception of M. hypopitys asbeing ‘a saprophyte feeding on decaying organic matter’(Fitter et al., 1996) or ‘saprophytic’ (Preston, Pearman & Dines,2002) should now be corrected. The recent confirmation thatthe same transfer processes can sustain phylogenetically dis-tinct myco-heterotrophs colonised by arbuscular (Bidartondoet al., 2002) and orchid-ectomycorrhizal fungi (McKendricket al., 2000b, 2002; Selosse et al., 2002; Selosse, Bayer &Moyerson, 2002; Taylor et al., 2002), indicates that this directpathway for net carbon transfer between plants has been inde-pendently selected on numerous occasions in nature in all themain types of mycorrhizal association.

Acknowledgements

We thank the NERC for financial support (GR3/10062 toJ.R. Leake & D.J. Read), and Welsh Natural Heritage forpermission to sample the Newborough Warren Monotropapopulation. We especially thank Irene Johnson who assistedwith the assembly of seed packets, their burial, harvestingand analysis. We gratefully acknowledge Else Vellinga for T.cingulatum herbarium material, Ryan Bowman for laboratoryassistance, and the USDA for grant 9600479 to Prof. T.D. Bruns.

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