inter- and intra-isolate rrna large subunit variation in glomus coronatum spores

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(2001)

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Research

Blackwell Science Ltd

Inter- and intra-isolate rRNA large subunit variation in

Glomus coronatum

spores

J. P. Clapp

1

, A. Rodriguez

1

and J. C. Dodd

2

1

Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK;

2

International Institute of Biotechnology/Research School of Biosciences

(UKC), 1/13 Innovation Buildings, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8HL, UK

Summary

•

High levels of variation are reported in the large subunit (LSU) rRNA gene, D2region of

Glomus coronatum

, a well characterized species of arbuscular mycorrhizalfungus (AMF).

•

Clones (435) containing the D2 regions from 7 isolates of

G. coronatum

wereinvestigated for intra- and inter-isolate sequence variation using PCR-single-strandconformational polymorphism (PCR-SSCP) as a prescreen before sequencing. Isol

-

ates of

G. mosseae

,

G. constrictum

and

G. geosporum

, three species of AMF withsimilar spore ontogeny and morphology, were also analysed.

•

Analysis of 138 representative sequences indicated that most were unique; thisvariation could not be attributed to DNA polymerase or cloning artefacts. Only 13sequences were found in more than one isolate. Neighbour-joining analysis showedthat most sequences from

G. coronatum

formed a main group although severalsequences from

G. mosseae

and

G. constrictum

clustered with

G. coronatum

.

•

There was greater than expected variation in the LSU D2 region sequences from

G. coronatum

. The four

Glomus

species, closely related by spore morphology, mightrepresent part of a genetic continuum. Implications for the concept of species inAMF, the use of rRNA sequences to estimate biodiversity and

in situ

detection infield ecology are discussed.

Key words

:

Glomus coronatum

,

rRNA large subunit

,

genetic variation

,

SSCP

.

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Author for correspondence:

J. P. ClappTel: +01227 764 000Fax: +01227 463 482Email: [email protected]

Received:

21 July 2000

Accepted:

18 October 2000

Introduction

Arbuscular mycorrhizal fungi (AMF) are globally distributedsoil fungi, associated with > 80% of vascular plant families.Their importance in phosphate nutrition and water economyhas been well documented (reviewed Smith & Read,1997). More recently they have been considered to have amultifunctional role in natural plant communities (Read,1989; Allen, 1992; Newsham

et al.

, 1995), maintenance ofplant community biodiversity (Gange

et al.

, 1993; Moora &Zobel, 1996; van der Heijden

et al.

, 1998) and global phos-phorus cycling (Schlesinger, 1991). More than 150 specieshave been described ( Walker & Trappe, 1993) but there isaccumulating evidence that the low morphological diversityof AMF spores, may not reflect their physiological and geneticplasticity. Functional differences exist between closely related

taxa which may reflect genetic differences (Van der Heijden

et al.

,1998) and indicate that identification of morphospecies maynot be sufficient or appropriate for ecological studies (Bachmann,1998). Morphotyping requires considerable experience of bothculturing and spore morphology of a wide range of AMF. Sporecounts however, may not reflect the

in planta

composition ofan AMF community at the time of sampling (Clapp

et al.

,1995). AMF

in planta,

however, cannot be reliably identifiedto species using intraradical hyphal morphology, althoughdiscrimination of fungi belonging to different families ispossible in particular host plant species (Abbott, 1982;Merryweather & Fitter, 1998; Dodd

et al.

, 2000).In recent years biochemical and molecular techniques,

e.g. isozyme profiles (Dodd

et al.

, 1996) or sequencing ofribosomal genes (Helgason

et al.

, 1998) have been extensivelyexploited in attempts to identify (Simon

et al.

, 1992; Simon

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et al.

, 1993), determine phylogenetic relationships (e.g.Redecker

et al.

, 2000) and genetic heterogeneity (Hijri

et al.

,1999; Hosny

et al.

, 1999; Lanfranco

et al.

, 1999; Pringle

et al.

, 2000) of AMF. Heterogeneity of these genes withinsingle spores and isolates of AMF has been known for severalyears (Sanders

et al.

, 1995; Lloyd-MacGilp

et al.

, 1996) fromPCR-RFLP and sequencing studies and has also beenreported for the 18S rRNA genes (Clapp

et al.

, 1999; Hosny

et al.

, 1999; Schüßler, 1999). Several of these studies(Antoniolli

et al.

, 2000; Pringle

et al.

, 2000) have indicatedthat the level of genetic diversity in AMF rRNA genes is veryhigh and this is supported by studies of the whole genomeusing other techniques including RAPD ( Wyss & Bonfante,1993; Abbas

et al.

, 1996), M13-primed minisatellites (Zézé

et al.

, 1997), microsatellite-primed PCR ( Vandenkoornhuyse& Leyval, 1998), PCR-generated microsatellite loci (Longato& Bonfante, 1997) and AFLP (Rosendahl & Taylor, 1997).

An important question that needs to be addressed is howsequence diversity equates to biodiversity in AMF as this hasimplications for the interpretation of field investigations(Helgason

et al.

, 1998) and the species concept in AMF.Spores of AMF can contain thousands of nuclei which havebeen hypothesized to represent the true individual in theGlomales (Sanders, 1999; Pringle

et al.

, 2000) but attemptsto determine whether the sequence diversity found in singlespores originates in polymorphic genes within single nuclei orwithin different nuclei have not been successful (Hijri

et al.

,1999; Hosny

et al.

, 1999; Redecker

et al.

, 1999; Schüßler,1999). It is clear however, that nuclear exchange can be medi-ated in some groups through anastomosis (Giovannetti

et al.

,1999), which may allow traditional species boundaries, basedon the morphology of spores to become blurred. This paperreports the first investigation of genetic diversity in AMFwhere variation has been assessed using sample sizes of severalhundred sequences. This was possible through the applicationof PCR-single-strand conformational polymorphism (PCR-SSCP) (Orita

et al.

, 1989) to screening cloned PCR productsfor differences in sequence. This technique was originally con-ceived to detect point mutations and has a sensitivity on parwith DNA sequencing over a defined length of DNA (Hayashi& Yandell, 1993). Thus, inserts screened by SSCP could becategorized on the basis of their sequence and representativessequenced for phylogenetic analysis.

The objective of this study was to determine the magnitudeand structure of inter- and intra-isolate sequence variation ofthe large subunit (LSU) rRNA gene, D2 region, by samplingseveral hundred sequences from a well characterized species ofAMF,

Glomus

coronatum

. This was intended to provide datathat would allow a clear interpretation of sequence diversity,its relationship to biodiversity and the species concept inAMF. The study concentrated on 7 isolates of

G. coronatum

but included three other

Glomus

species with similar sporemorphologies, following on from an earlier investigation(Dodd

et al.

, 1996). These species together have been closely

linked on the basis of their morphological similarity (Dodd

et al.

, 1996) and ITS sequences (Lloyd-MacGilp

et al.

, 1996).We report the findings of the largest sequence analysis of anygroup of AMF to date.

Materials and Methods

Origins and maintenance of the fungi

A complete survey of morphological characters of isolates inthe

G. coronatum

group has been reported elsewhere (Dodd

et al.

, 1996) and these are not repeated in this paper. Sevenisolates of

G. coronatum

and three other species of

Glomus

were investigated in this study (Table 1). This representsapprox. 80% of

G. coronatum

isolates currently availableand maintained in culture world-wide. The fungi weremaintained in an attapulgite clay product (Agsorb 8/16 fromOil-Dri Ltd, Wisbech, UK) and a durite sand (a particulateby-product of calcined flint pebbles heated in a furnace andconsisting of 97% silica with small amounts of iron oxides,calcium oxides and alumina and a mean pH of 8.3) in Kent(UK), or a variety of other sands or sandy soils in Pisa (Italy).The culturing conditions and isolation history of the pot culturesvaried. All were open cultures made from initial single-sporeor multispore inocula. Isolate

G.

coronatum

BEG139 wasincluded as it had a similar occlusion of the hyphal attachmentas that found in the

G. mosseae

/

G. coronatum

grouping andclustered with the BEG28 holotype (BEG28T) using isozymeand SDS-PAGE screening (Dodd

et al.

, 1996). Differencesin wall structure and hyphal attachment were observed forspores of

G. coronatum

BEG139, which was found to have amore persistent outer hyaline unit wall (after Walker, 1983),often a straighter hyphal attachment and infrequently aninner membranous-like layer, which differentiated it from theholotype (BEG28T), WUM2 and BEG49 (Dodd

et al.

, 1996).Examples were found where these characters overlapped inthe isolates studied and BEG139 was also found to differfrom the other isolates in that it did not produce sporocarps,although this was concluded to be neither a consistent noruseful taxonomic character (Dodd

et al.

, 1996, INVAM:http://invam.caf.wvu.edu/). The expanding wall, which wasdeemed to be an important trait in defining the species (Giovannetti

et al.

, 1991), was also shown to be an inconsistent character(Dodd

et al.

, 1996, INVAM: http://invam.caf.wvu.edu/) sinceit was absent in BEG49 and BEG139.

G. coronatum

BEG28Twas subcultured by both Dr Chris Walker and Dr John Doddin 1996. The isolate sent to Dr Chris Walker was maintainedby him in Edinburgh (UK) for 2 yr before being passed toDr Dodd in Kent (UK) and is called BEG28W in this paper.The isolate maintained continuously at Kent for 4 yr after itsreceipt in 1996 is called BEG28K for this paper. A furtherisolate (

G.

coronatum

FO97) was isolated and cultured usingmultiple spores from the same field site as the holotype,BEG28(T) in 1997 by Dr L. Avio and subsequently


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Research 541

subcultured again using multiple spores. Species with similarspore morphologies,

G. constrictum

BEG130,

G. geosporum

BEG11 and

G. mosseae

BEG25 were also included in thestudy.

Isozyme analysis

Pot cultured material from each isolate was carefully passedthrough a 710-

µ

m sieve and collected on a 45-

µ

m sievebefore back washing into Petri dishes. The pot cultures werecarefully checked for purity and at the initiation of thestudies in 1998 were found to contain only the expected taxa.Spores from each isolate were collected on clean filter paper( Whatman No. 1) and transferred to Eppendorf tubescontaining Sucrose-Triton-Extraction-Buffer (STEB: 20 mMTris, 10 mM NaHCO

3

, 10 mM MgCl

2

6H

2

O, 0.1 mMNa

2

EDTA, 10 mM

β

-mercaptoethanol, 10% (w/v) Sucrose,0.001% (v/v) Triton X100). One hundred spores werecrushed on ice for 2 min using a glass pestle and thencentrifuged for 20 min at 4

°

C (20 000

g

). The supernatantwas transferred to another Eppendorf and stored at

−

80

°

Cuntil required. A Hoefer Mighty Small (AP-Biotech) verticalgel electrophoresis system (0.75 mm gel thickness) wasused with the electrode buffer containing 25 mM Tris and192 mM glycine at pH 8.3. Proteins were separated at pH 8.8in a 375-mM Tris-HCL buffer and stacked at pH 6.8 in a125-mM Tris-HCl buffer. The nondenaturing resolvinggel contained 7.5% acrylamide and the stacking gel 5%acrylamide. Gels were stained for esterase (EST, EC 3.1.1.1.)or malate dehydrogenase (MDH, EC 1.1.1.37.). Gels werereplicated three times for each enzyme. The following seven

G. coronatum

isolates were analysed for both enzymes: BEG28T,BEG28W, BEG28K, FO97, BEG49, WUM2 and BEG139as were

G. geosporum

BEG11 and

G. constrictum

BEG130.

DNA extraction

One-hundred and fifty healthy AMF spores were selected forDNA isolation from each isolate. The spores were surfacesterilized using 0.2% (w/v) Chloramine T, 0.02% (w/v)Streptomycin Sulphate, 0.01% (w/v) Gentamicin Sulphateand 0.002% (v/v) Tween 20 for 8 min and then thoroughlyrinsed with sterile water. Spores were placed in 1.5 mlEppendorf tubes, centrifuged to the base and crushed witha sterile microhomogenizer in 200

µ

l of cell lysis solution(Puregene). The mixture was incubated at 65

°

C for 90 minand protein removed by two phenol-chloroform extractions.DNA was precipitated using 0.8 volumes isopropanol andresuspended in 25

µ

l TE buffer pH 8 (10 mM Tris-HCl,1 mM EDTA). Samples were stored at

−

20

°

C until use.

PCR

A primer was designed to amplify a 460-bp region ofthe variable D2 region of the 23S rRNA gene: ALF01(5

′

-GGAAAGATGAAAAGAACTTTGAAAAGAG) withthe primer NDL22 (Van Tuinen

et al.

, 1998). The PCRproduct size was within the optimal range of SSCP fordetecting point differences between PCR products.PCR reaction mixtures contained 1

µ

l (30 ng) DNA tem-plate, 2

µ

l 10x Super

Tth

buffer (HT Biotechnology Ltd,Cambridge, UK), 1.2

µ

l MgCl

2

(25 mM stock solution),1.75

µ

l dNTP (4 mM stock solution), 30 pmols of eachprimer, 1.5 units of Super

Tth

(HT Biotechnology Ltd), and11.85

µ

l PCR-grade water (Sigma-Aldrich Chemic Gmbh,Steinheim, Germany). The amplification was carried out usinga PTC-200 thermocycler (MJ Research, Watertown, MA, USA)with the following programme: 36 cycles @ denaturation95

°

C for 0.75 min, annealing 50

°

C for 1 min and extension

Table 1 Morphological data and culturing history of the isolates of arbuscular mycorrhizal fungi (AMF) included in this study

SpeciesBEGcode

Local code

Country oforigin

StartingInoculum

VoucherNo.

Spore diameter/hyphal attachment width (µm)

Wall thickness( µm)

Date collected(day/month/year) Source

G. coronatum* BEG28 BEG28T Italy MS JCDAR1 226 ± 7/45 ± 2 4.8 ± 0.4 30–06–1980 M. GiovannettiG. coronatum BEG28 BEG28W Scotland MS JCDAR2 259 ± 5/35 ± 2 7.8 ± 0.6 30–06–1980 C. Walker/

subcult./96 J.C. DoddG. coronatum BEG28 BEG28K England MS JCDAR3 221 ± 2/41 ± 3 4.8 ± 0.8 30–06–1980 J.C. Dodd

subcult./96G. coronatum – FO97 Italy MS JCDAR4 271 ± 6/52 ± 2 4.8 ± 0.2 1997 M. GiovannettiG. coronatum – WUM2 Australia MS JCDAR5 225 ± 7/40 ± 2 5.5 ± 0.3 1977 J.C. DoddG. coronatum BEG49 ALM-1 Spain MS JCDAR6 222 ± 5/34 ± 2 4.9 ± 0.2 02–10–1993 J.C. DoddG. coronatum BEG139 AD-1 Abu-Dhabi MS BEG139 220 ± 5/24 ± 1 8.5 ± 0.3 No data J.C. DoddG. constrictum BEG130 ALM-4 Spain MS BEG130 286 ± 3/37 ± 10 29 ± 0.8 02–10–1993 J.C. DoddG. mosseae BEG25 – England SS JCDAR7 Dodd et al. (1996) 1982 J.C. DoddG. geosporum BEG11 – England SS JCDAR8 Dodd et al. (1996) 10–01–1982 J.C. Dodd

The holotype isolate of Glomus coronatum BEG28 is indicated by a *. MS, multispore; SS, single spore starting inoculum for cultures. BEG, La Banque Européenne des Glomales.


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at 72°C for 3 min. PCR products were checked by agarose gelelectrophoresis and quantified using an UV spectrophotometer(AP-Biotech).

Cloning

Amplified products were purified using Wizard PCR Preps(Promega, Madison, WI, USA), ligated into pGEM-T EasyVector (Promega) and transformed into competent Escherichiacoli (DH5α) according to the manufacturer’s protocols.Recombinant colonies were selected by blue/white screeningand the presence of the correct sized inserts detected by PCRamplification directly from white colonies. Typically, between100 and 150 recombinant clones were obtained per plate.Frequencies of each sequence were estimated using the num-ber of times a clone containing a particular sequence was foundas a percentage of clones analysed per isolate and over allG. coronatum isolates.

PCR-SSCP

Between 35 and 59 cloned inserts from each isolate werescreened for sequence variation using PCR-SSCP and thefrequencies of each SSCP type recorded. Two microlitres of eachPCR product were added to 18 µl of denaturing loading buffer(95% formamide, 10 mM NaOH, 0.25% (w/v) BromophenolBlue and 0.25% (w/v) Xylene Cyanol (FF), denatured for 2 minat 94°C and snap cooled on ice before loading 2 µl onto a0.5X MDE (mutation determination enhancement) gel(Flowgen, Staffordshire, UK) following the manufacturer’sinstructions. The electrophoresis was run at 5 W for 10 kVhrin a Hoefer SQ3 sequencing system (Hoefer Pharmacia BiotechInc., San Francisco, CA, USA). Addition of urea (up to 5% w/v) and differing gel concentrations did not improve band resolu-tion or separation. The SSCP bands were stained with silverfollowing standard procedures (Flowgen) and the gel dried inthe oven at 70°C for 2–3 h. A flat bed scanner (EPSON GT-9600, EPSON, Singapore) was used to obtain images of thegels. Profiles were compared by eye and grouped according tosimilarity. An alternative system for SSCP was also tried, theHoefer Mighty Small system, however, this resulted in lessresolution and sensitivity, inferior screening capacity andproved to be more time consuming than the Hoefer SQ3system that was preferred in this study.

Phylogenetic analysis

Recombinant plasmids were extracted using Wizard Minipreps(Promega) from bacterial clones representing each PCR-SSCPpattern according to the manufacturer’s instructions. Anyrecombinant clone that produced an SSCP pattern thatwas almost identical to another but that could not be con-sidered so with 100% certainty (due to, for example, a gelartefact) was also sequenced. The sequencing was carried out

using plasmid-based forward and reverse sequencing primersby a commercial company (ABC, Imperial College Schoolof Medicine, London, UK). Traces were manually checkedfor base-calling errors using Chromas. Any sequence withuncertain nucleotide calling, was re-sequenced. All sequenceswere compared with the GenBank sequence database todetermine phylogenetic affiliation using BLAST at NCBI(http://www.ncbi.nlm.nih.gov/index.html) before proceedingfurther with the analysis. Sequences that did not clusterwith Glomalean sequences were excluded from subsequentanalyses.

The D2 region sequences (excluding primer sites) werealigned using ClustalW (Thompson et al., 1997) at EBI(http://www2.ebi.ac.uk /clustalw/) and edited in JalView(http://www2.ebi.ac.uk /~michele/jalview/). A Neighbour-Joining tree (Saitou and Nei, 1987) was constructed usingPAUP 4.06 (Swofford, 1999). A second method, MaximumParsimony (PAUP version 4.0, Swofford, 1999) was also usedto produce a more conservative tree where only phylo-genetically informative sites were analysed. Maximum par-simony analysis was performed using heuristic search optionswith 100 bootstrap replications (Felsenstein, 1985) branchswapping by tree-bisection-reconnection (TBR), branchesable to collapse to yield polytomies and with parsimonyuninformative characters excluded. Single, unambiguouslyaligned gaps were treated as fifth character states. Trees wererooted using a basidiomycete outgroup. Trees from bothmethods were displayed using Treeview (Page, 1996).

Secondary structure analysis

The secondary structure of each unique sequence wasanalysed and compared with those of other closely relatedsequences deposited in GenBank. The secondary structurewas based on that of the Saccharomyces cerevisiae LSU obtainedfrom RNAviz (http://rrna.uia.ac.be/rnaviz/). Counts ofnucleotides that did not have complementary bases in theopposing sequence were made in stem structures. Basesthat were excluded from the Maximum Parsimony analysis(i.e. those that were phylogenetically uninformative) wereanalysed for their presence and frequency in loop and stemstructures and whether they were compensatory in thelatter. Secondary structure information was obtained frommfold (http://mfold2.wustl.edu/~mfold/) and visualized inRNAviz.

DNA polymerase error control experiments

Two experiments were carried out on BEG28W to excludethe possibility that the high degree of variation detected wascaused by DNA polymerase nucleotide incorporation orcloning artefacts. A mathematical assessment of the numberof expected incorporation errors for Tth DNA polymerasewas also undertaken.


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Enzyme comparison In the first practical investigation ofthe variation encountered, a comparison was made betweenthe DNA polymerase used in this study (Tth) and a proof-reading enzyme (Pfu). Forty-eight clones were screened byPCR-SSCP from PCR reactions using each of these enzymes.The Null hypothesis was that the frequencies and types ofPCR-SSCP patterns should not be similar if the variation wasdue to random incorporation errors.

Replicated cloning The second experiment involved threeindependent PCR amplifications of a single multispore DNAextraction using Tth DNA polymerase. The products of eachamplification were independently cloned and 50 clones fromeach were compared using PCR-SSCP analysis. The expectationwas that the majority of observed PCR-SSCP patterns wouldbe re-isolated between replicates but a small proportion ofadditional patterns would be encountered as expected in aPoission series.

Results

Morphological analysis

The colour range for spores of all G. coronatum isolatesincluded here fell into the ochre-sienne-rust range (Anon,1969) including that of G. constrictum BEG130 as recordedpreviously (Dodd et al., 1996). Table 1 shows various measure-ments of the spores of each isolate. The mean spore sizeranged between 220 and 272 µm, which is larger than thatreported by INVAM (http://invam.caf.wvu.edu/myc_info/taxonomy/glomaceae/glomus) for G. coronatum AU202(mean of 154ìm). Interestingly BEG139 had the lowest meanspore size and a funnel-shaped hyphal attachment but thelargest mean pigmented wall thickness placing these valuesin an intermediate position between G. coronatum and G.constrictum BEG130. A further character which distinguishedBEG139 from the other G. coronatum isolates was that, likeG. constrictum, it did not produce sporocarps (Dodd et al.,1996). All cultures used in this investigation were initiallyobserved to contain spores of the prescribed morphotype.However, when BEG28K was re-examined in March 2000after sequencing studies, a G. geosporum morph was seen tobe present along with the expected G. coronatum spores.

Isozyme analysis

The esterase and MDH profiles of spore extracts showed thatall the G. coronatum isolates formed a group which includedG. coronatum BEG139 and G. constrictum BEG130 (Fig. 1).These isolates all had two putative MDH loci, a slow movingmonomorphic locus and a faster moving polymorphic locus.G. geosporum-specific MDH and esterase isozymes were detectedin the isolate G. coronatum BEG28K. The esterase isozymeprofiles were also identical for all isolates of G. coronatum and

G. constrictum with the exception of G. coronatum BEG28K(Fig. 1) and FO97 (result not shown). In these cases an additionalband was present which was subsequently identified as having anidentical mobility to that of G. geosporum BEG11 (Fig. 1). Asnoted above, re-examination of the BEG28K isolate indicatedthat both G. coronatum and G. geosporum spores were present.However G. geosporum spores were not in evidence in theFO97 culture, which had been isolated as a trap and takeninto multispore culture in 1998, and comprised the firstgeneration of spores produced.

D2 amplification and sequencing

The variable D2 LSU domain was successfully amplified frommultispore extracts of each isolate, using the ALF01 andNDL22 primers and resulted in a product 460 bp in length.In total 435 clones from 10 AMF isolates were analysed bySSCP. Of these 378 were identified as Glomalean after cloneswith representative SSCP patterns were sequenced. Theremaining 13% contained inserts identified as non-glomaleansequences. The primers showed reasonably high specificity forGlomales, with at most nine (range 2 – 9, mean 4.1) PCR-SSCP patterns out of the total number of patterns for each

Fig. 1 Esterase (upper) and malate dehydrogenase (lower) isozyme profiles of protein extracts from spores of arbuscular mycorrhizal fungus (AMF). Lane 1, Glomus coronatum BEG28T; Lane 2, Glomus coronatum BEG28W; Lane 3, Glomus coronatum BEG28K; Lane 4, Glomus coronatum BEG49; Lane 5, Glomus coronatum WUM2; Lane 6, Glomus coronatum BEG139. Arrows in Lane 3 indicate Glomus geosporum isozyme. BEG, La Banque Européenne des Glomales.

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isolate (range 16 –27, mean 20.4) attributed to nonglomaleanfungi. In total, the 10 isolates in this study yielded 138 glomaleansequences of which 100 were obtained from the seven G.coronatum isolates (Accession Numbers: AF304871-AF304970),13 from G. mosseae BEG25 (Accession Numbers: AF304982-AF304994), 11 from G. constrictum BEG130 (AccessionNumbers: AF304971-AF304981) and 14 from G. geosporumBEG11 (Accession Numbers: AF304995-AF305008).

Thirteen sequences in total were found in more than oneisolate (Table 2, Fig. 2, (Accession Numbers: AF304873-AF304875,AF304878, AF304880-AF304883, AF304900, AF304910,AF304926, AF304938 and AF304945) ) but each was notnecessarily found in every isolate. G. coronatum, G. coronatumBEG28K and BEG28T itself, were each found to have onlytwo sequences in common with other G. coronatum isolates.These isolates had no sequences in common with each other,however G. coronatum BEG28W had all four. The Spanishisolate BEG49 had six sequences also found in other G.coronatum isolates and, of these, four were common to theAustralian isolate WUM2. However G. coronatum BEG139,from Abu Dhabi, had no sequences in common with any ofthe other G. coronatum isolates although it did share onesequence with G. constrictum (BEG130). Analysis of thesequences common to the Italian G. coronatum isolates, indic-ated that several sequences were not detected in the holotypeculture G. coronatum BEG28T and its subculture BEG28K

but were detected in the subculture BEG28W. Interestingly,the most recently isolated culture FO97 had 5 sequences incommon with BEG28W and one that was also detected in G.constrictum BEG130. Five of the seven shared sequencesfound in FO97were also present in BEG28W.

Analysis of secondary structure

Analysis of the sequences from the seven G. coronatum isolatesindicated that the variation seen was supported by similarvariation in other Glomalean sequences available fromGenBank (Table 3a). This table shows the relative numbers ofnon-compensatory bases found in the secondary structureanalyses of sequences obtained from the seven G. coronatumisolates, those of the G. coronatum BEG28 sequence AF145739and the combined values from 11 other Glomaceae sequences.The results indicate that the magnitude and range of non-compensatory variation seen in the stem regions are broadlysimilar. Overall in the sequences classified as G. coronatumfrom the seven isolates, only 52 bases represented variationthat was not supported by identical variation in at least oneother sequence (parsimony uninformative positions). Withone exception these were all point mutations. Table 3b showsthe occurrence of these in stem and loop structures. Themajority (54%, 28 bases) were found to occur in loops,33% (17 bases) were found in stems but had compensatory

Table 2 Sequences found in more than one isolate of Glomus coronatum

Percentagerange of frequenciesper isolate

Percentageof G.coronatumclones

Isolates

Sequence name BEG28T BEG28W BEG28K FO97 WUM2 BEG49 BEG139 BEG130 BEG25 BEG11

BEG28W-3 – + – – – + – – – – 2.4–22.2 3.2BEG28W-4 – + – – – + – – – – 2.8–4.9 1.1BEG28W-5 + + – + – – – – – – 2.3–8.3 1.8BEG28W-8 – + + + + + – – – – 1.8–25.0 8.2BEG28W-10 + + – + – – – – – – 3.1–11.1 2.9BEG28W-11 – + – + – – – – – – 2.3–2.8 0.7BEG28W-12 – + – + + – – – – – 1.8–5.6 1.4BEG28W-13 – + + – + + – – – – 2.8–7.3 3.2FO97–1 – – – + – – – + – – n/a 0.7BEG49–5 – – – – + + – – – – 2.4–5.5 1.4BEG49–15 – – – – + + – – – – 4.9–5.5 1.8WUM2–6 – – – + + – – – – – 2.3–12.7 2.5BEG139–12 – – – – – – + + – – n/a 0.4A 1 3 3 2 4 2 2 1 2 3B 19* 14 3** 14 17 23 18 13 13 14

The presence of a sequence in a particular isolate is indicated by + and absence by −. Row A, indicates the number of sequences that represented > 10% of the total number of clones analysed from each isolate. Row B, indicates the total number of sequences obtained from each isolate. *, this value is exclusive of the Gigasporaceae sequence obtained from BEG28T; and **, this value is exclusive of G. geosporum-like sequences and those sequences that formed an outlying cluster with a sequence from an isolate of G. intraradices. BEG, La Banque Européenne des Glomales.



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Fig. 2 Charts showing the frequencies of each sequence type per isolate of arbuscular mycorrhizal Fungi (AMF). Bars in red indicate sequences found in more than one isolate. BEG, La Banque Européenne des Glomales.



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mutations in the opposite strand. Only 13% (7 bases) werefound in stem regions without compensatory base changesrepresenting 0.02% of the bases analysed.

Sequence frequencies

Intra-isolate frequencies The frequencies of the 11 (13 ifthe sequences BEG139 –12 and FO97 –1 which were com-mon to G. constrictum are included, see Fig. 2) sequences thatwere common to more than one G. coronatum isolate areshown in Table 2 and Fig. 2. The range of frequencies ofthese sequences was between 0.4% and 8.2% of G. coronatumclones (n = 279). Sequence BEG28W-8 was the mostabundant with 8.2% of G. coronatum clones containing this

sequence (Table 2). All the G. coronatum isolates exceptBEG139 and, surprisingly, BEG28T had this sequence.The GenBank sequence AF145739 differed from sequenceBEG28W-8 by a single deletion that was not found in anyother BEG28 sequence.

The number of different sequences obtained from the G.coronatum isolates varied from 13 to 23 and of these between2 and 8 were shared with other G. coronatum isolates. How-ever, many sequences were unique to each isolate (Fig. 2,Fig. 3). In most cases each isolate had between 1 and 4sequences that comprised a higher proportion of the clones.The frequencies of these more abundant sequences rangedbetween 10 and 35% of the total number and were often notthe same as those that were common to more than one isolate

Isolate nMean number of non-compensatory positions

Range of non-compensatory positions

BEG28T* 19* 24.1 20–29BEG28W 14 24.2 20–29BEG28K** 3** 27.0 25–31FO97 14 23.7 20–26WUM2 17 24.4 23–26BEG49 23 24.7 21–27BEG139 18 21.8 14–24G. coronatum BEG28 (AF145739) 25.0 n/aGlomaceae (11 seqs#) 11 27.8 23–37

*, this value is exclusive of the Gigasporaceae sequence obtained from BEG28T; and **, thisvalue is exclusive of Glomus geosporum-like sequences and those sequences that formed anoutlying cluster with Glomus intraradices. # Glomus mosseae (Y07656), G. mosseae BEG84(AF145738), G. mosseae BEG85 (AF145736), Glomus constrictum BEG130 (AF145741), G. geosporum BEG90 (AF145742), Glomus fragilistratum BEG5 (AF145747), Glomuscaledonium BEG20 (AF145745), Glomus intraradices (X99640), G. coronatum BEG28(AF145739), G. coronatum BEG49 (AF145740). Sequence AF145740 clusters with Glomusconstrictum BEG130 NOT G. coronatum. BEG, La Banque Européenne des Glomales.

No. of parsimony uniformative bases in Glomus coronatum isolates

Isolate n LOOPS

STEMS

Noncompensatory Compensatory

BEG28T* 19* 5 3 3BEG28W 14 6 0 3BEG28K** 3** 0 0 1FO97 14 4 1 0WUM2 17 2 1 0BEG49 23 4 1 3BEG139 18 7 1 7TOTAL 28 7 17

*, this value is exclusive of the Gigasporaceae sequence obtained from BEG28T; and **, this value is exclusive of Glomus geosporum-like sequences and those sequences that formed an outlying cluster with a sequence from an isolate of Glomus intraradices. BEG, La Banque Européenne des Glomales.

Table 3b Analysis of the positions in a secondary structure model of polymorphic bases that were not supported by an identical base change in another sequence

Table 3a Comparisons of the number of noncompensatory bases found in an analysis of the secondary structure of the variable rRNA LSU D2 regions of seven isolates of Glomus coronatum



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(Fig. 2, Fig. 3). The effect of this was that most isolatesformed a distinct cluster of sequences, some of which werefound in other isolates, but these were not necessarily the mostcommon (Fig. 3).

Sequence relationships

In total 151 sequences (including representatives fromother genera and species obtained from GenBank) were

Fig. 3 Chart (a) overall frequencies of each Glomus coronatum sequence obtained during this study expressed as a percentage of the total number of Glomales clones obtained from the G. coronatum isolates. Each bar represents the sum of the contributions from each Glomus coronatum isolate. Sequences obtained from different isolates are given individual colours (see charts b–h). Charts (b–h) frequencies of sequences from each G. coronatum isolate expressed as a percentage of the total number of clones obtained from each G. coronatum isolate, indicating the proportion contributed by each isolate to the totals shown in chart (a). Charts (i–k) frequencies of sequences obtained from Glomus constrictum BEG130, Glomus mosseae BEG25 and Glomus geosporum BEG11, respectively, expressed as a percentage of all clones containing glomalean sequences analysed. Bars in red indicate sequences found in more than one isolate. Individual sequence names are not indicated, refer to Fig. 2. BEG, La Banque Européenne des Glomales.



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Fig. 4 Neighbour-joining tree indicating the relationships of the sequences obtained in this study. Cluster 1 represents the main groups of Glomus coronatum sequences, Cluster 2 represents a basal group of sequences obtained from G. coronatum isolates that did not cluster with the main group. Cluster 3 shows six sequences from G. coronatum BEG28K that cluster with a Glomus intraradices sequence X99640. Cluster 4, 5 and 6 represent sequences obtained from Glomus constrictum BEG130, Glomus geosporum BEG11 and G. mosseae BEG25, respectively. BEG, La Banque Européenne des Glomales.



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assessed over 483 characters (including spaces); 337 (73%)base positions were variable and, of these, 103 (30%) werephylogenetically uninformative.

Neighbour-joining analysis The neighbour joining tree(Fig. 4) gave a clearer impression of the clustering of sequencesin relation to their different origins than Maximum Parsimonyanalysis. The majority of G. coronatum sequences (85 of 89)formed a main G. coronatum group (cluster 1) which includedthe GenBank sequence of G. coronatum BEG28 AF145739obtained from a subculture of BEG28T and maintainedin Copenhagen, Denmark since 1996. This group alsocontained five sequences from G. mosseae BEG25 and threefrom G. constrictum BEG130. Cluster two contained onesequence that originated from the BEG28T isolate, two fromBEG28W and 1 from BEG49. The third cluster containedsix sequences that came from BEG28K but clustered moststrongly with a G. intraradices sequence, X99640. No evid-ence of G. intraradices spores or mycelium were found inthe BEG28K culture. A single sequence (BEG28T-18, Acces-sion Number: AF304970), found in 10% of the clones ofBEG28T, clustered with Gigasporaceae sequences, but inspec-tion of the culture revealed no evidence of Scutellosporaand/or Gigaspora structures. PCR contamination was ruled outsince no work was being carried out on gigasporaean spores.

In addition to the G. coronatum clusters there were threethat corresponded to the sequences obtained from the isolatesof G. mosseae, G. constrictum and G. geosporum. The sequencesfrom the former two isolates formed discrete clusters, how-ever, in both cases, some were obtained which clustered withthe G. coronatum groups. With the exception of BEG28K, nosequences from the G. coronatum isolates fell into the clustersof these 3 species. Sequences originating from other isolatesof G. mosseae available in GenBank (Y07656, AF145738,AF143735 and AF145736) clustered with the sequences fromBEG25, although none were identical. The G. geosporumBEG11 cluster was also distinct and included the G. geospo-rum BEG90 sequence AF145742, but several sequences fromthe BEG28K isolate also clustered with this group. This wasinvestigated further and spores with morphology correspond-ing to that of G. geosporum were found in the culture. Thisculture had been checked several times since its initiation in1996 and until this point no G. geosporum spores had beenfound. Retrospective examination of isozyme profiles (seeprevious) indicated that bands previously considered to bepolymorphic isozymes, in fact had identical mobility to thosefrom pure cultures of G. geosporum BEG11. These bands werefound in isolates BEG28K and the first generation of sporesfrom FO97. This implies that the original site where theholotype of G. coronatum (BEG28T) was isolated may alsobe colonized by G. geosporum.

The G. constrictum BEG130 cluster was also discrete andtwo GenBank sequences of G. caledonium (AF145745) andG. fragilistratum (AF145747) clustered basally to it indicating

their close taxonomic affiliation. No other sequences from theG. coronatum, G. mosseae or G. geosporum isolates clusteredwith this group. However, a GenBank sequence AF145740(G. coronatum BEG49) did so. All sequences from the BEG49isolate used in this investigation clustered with the other G.coronatum isolates and sequence AF145740 should in ouropinion be viewed with caution.

Maximum parsimony analysis Analysis, excluding unin-formative sequence variation, allowed a more conservativeinterpretation of the sequence variation. The results largelysupported the tree topology found by Neighbour-Joiningwith one exception. Seven of the 14 sequences isolated fromG. geosporum BEG11 and the G. geosporum BEG90 sequence(AF145742) clustered with the G. coronatum main group.The single sequence from BEG28T that clustered with theGigasporaceae in the Neighbour-Joining tree, in this casewas indistinguishable from the sequence of Gigaspora rosea( Y12075). Once again the G. coronatum BEG49 sequenceAF145740 clustered with the G. constrictum BEG130 group.

DNA polymerase error control experiments

Enzyme comparison The enzyme Tth DNA polymerase hasan error rate per base of 7.7 × 10–5 whereas Pfu has an errorrate of 1.6 × 10–6 per base (Lundberg et al., 1991). Theenzyme Pfu was found to generate 13 distinct PCR-SSCPpatterns, whereas the Tth enzyme generated 14. Of these 12were identical between the two enzymes. There was thereforevery little apparent effect of the proof reading enzyme and theNull hypothesis was therefore rejected.

Replicated cloning The results were as follows: ampli-fication 1 had 12 patterns, amplification 2 had the same 12patterns plus two additional patterns, the third amplificationhad the original 12 patterns and similarly three additionalpatterns. The cloned PCR products from which the 12identical PCR-SSCP patterns were obtained and the firsttwo additional patterns were sequenced (Accession Numbers:AF304871-AF304884). In total there were 70 variable sitesfound representing 16.9% of the total sequence length(414 bp). Of these 25 bp (6%) were represented by parsimonyuninformative characters which would normally be excludedfrom phylogenetic analysis.

Mathematical analysis The expected error due to nucleotidemisincorporation for the enzyme Tth DNA polymerase was7.7 × 10–5 per base (Arakawa et al., 1996). The chance of anerror occurring in a single molecule is given by: np(1 − p)n–1.In our case this indicated that 0.034 or 3.4 clones in every 100could be expected to include a single error, when 50 clonesare investigated this drops to an expectation of approximatelytwo clones. The probability of getting an identical base mis-incorporation error in a second PCR product, independent



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of the first (i.e. in a different culture or independent PCRamplification) is given by: [np(1 − p)n–1]2 · (1/n)(n – 1/n) · (1/b) ·(3/b). where n is the base length of the product, p is theprobability of getting an error and b is the number ofcharacter states (4). For a 460-bp product this approximatesto 4.75 × 10–7 per clone, or 2.375 × 10–5 per 50 clones. Thisvalue is negligible and this situation is unlikely to occur bychance.

Discussion

Sequence variation

The objective of this study was to determine the magnitudeand structure of sequence variation in the D2 region of thelarge subunit rRNA gene of G. coronatum following on frompreviously obtained data (Dodd et al., 1996). The D2 regionhad previously been reported as containing sufficient inter-species polymorphism to discriminate different AMF ( VanTuinen et al., 1998). Based on earlier studies of other regions,the analysis expected to identify a major group, of perhaps 10sof sequences that would be shared throughout the majority ofthe isolates, but the number of new sequences found witheach additional isolate studied was expected to fall. However,this was not the case. The screening and phylogenetic analysisindicated that the level of variation was higher than expectedwith the majority of sequences being unique to their isolateof origin. However, with the exception of G. coronatumBEG139, all G. coronatum isolates contained a few sequencesthat could be found in other G. coronatum isolates, althoughthese were in the minority. The failure to reach a plateau in theacquisition of novel sequences after 100 from the seven G.coronatum isolates in this study, indicated that the variationdetected here is unlikely to represent the full range andconsiderably more could be expected. The possibility thatthe high level of variation seen was introduced by DNApolymerase or cloning artefacts was addressed in three experi-ments and an analysis of secondary structure. The math-ematical arguments indicated that the sequences were likelyto represent real variation since they occurred too frequentlyto be explained by chance, although in some cases theyvaried by only a single base. Similarly the use of a proofreading enzyme failed to reduce the number of differentsequences obtained and in fact replicated the majority ofPCR-SSCP patterns. Finally, the replicate cloning experimentshowed beyond doubt that the differences were real since 12PCR-SSCP patterns were obtained in triplicate from a singleisolate. The secondary structure analysis indicated that mostof the variation that was unsupported by similar variation inanother sequence was found in loops and as compensatoryvariation in stems (87%). These clearly demonstrated thatmost of the variation seen was real and only 0.02% of variablebases overall were not supported by identical variation foundin another different sequence.

This investigation has attempted to explore the extent ofgenetic variation of AMF previously reported in smallerstudies (Lloyd-MacGilp et al., 1996; Antoniolloi et al., 2000;Pringle et al., 2000), by screening significantly greater num-bers of sequences from a larger number of isolates. There havebeen no other investigations that have analysed comparableamounts of data and none with which to directly compare themagnitude of the variation encountered in this study. Thevariation encountered here however, has precedent in theresults of other studies (Antoniolli et al., 2000; Pringle et al.,2000). An analysis of ITS region sequences from 3 fieldcollected spores of G. mosseae, found 23 sequences, no two ofwhich were the same (Antoniolli et al., 2000). These authorsalso indicated that the level of variation they encountered wasnot consistent with DNA polymerase-induced errors. Asimilar investigation by Pringle et al. (2000) obtained 39 ITSsequences from 16 spores of Acaulospora colossica taken froma field soil and analysis of type 1 sequences only (the remain-ing types are likely to be contaminating fungi), indicatedthat only two were identical (n = 24). Within that data set,7 type 1 sequences originated from a single field spore andwere all different. In our study, the discovery of only 11sequences in more than one G. coronatum isolate seemedinitially to be rather low, but this seems to fit the patterndemonstrated above. A rough calculation showed that asequence could be expected to be found more than onceonly after approx. 25 clones had been sequenced. Thus themagnitude of variation obtained in this study was corrobor-ated by existing data from other regions of the rRNA genes ofAMF.

The species concept in AMF

It is not possible to determine AMF biodiversity, using aLinnean concept of species, through the analysis of rRNAsequence-dependent data until the range and frequency ofsequence variation can be obtained for each discrete taxon,assuming such a thing exists (Bachmann, 1998). It is how-ever, becoming increasingly apparent that the species as adiscrete entity is a flawed concept in many microorganisms(Bachmann, 1998). Although species are widely accepted asbasic units of biodiversity, it is surprisingly difficult to definethe category of species and to devise objective methods for itsrecognition. This is particularly true of the AMF, where themagnitude of genetic variation as shown in this study, is verylarge and the boundaries between taxonomic units are oftenoverlapping using both morphological (Dodd et al., 1996)and molecular criteria (Lloyd-MacGilp et al., 1996). Theanalyses carried out in this study indicated that each isolateof G. coronatum formed a discrete unit which had a minor-ity of its ribosomal RNA genes in common with othermorphologically similar isolates. The phylogenetic analysisindicated that the sequences obtained from each isolate inthis study were divided between those that represented



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the respective main cluster for each species and those thatclustered with others from unrelated origins. G. geosporumsequences formed a discrete cluster and no sequences felloutside, although when these sequences were analysed byMaximum Parsimony, half clustered with the main group ofG. coronatum, as did those from G. coronatum BEG139 (datanot presented). Sequences from both G. mosseae BEG25 (5out of 13) and G. constrictum BEG130 (3 out of 12) werefound to cluster in the main group of G. coronatum sequences.Spores typical of different genera (Morton et al., 1997) havebeen found with the same 18S sequences (Sawaki et al., 1998)and it is no less possible that morphological similarity betweenthese 4 species belies genetic similarities or even a geneticcontinuum. This is perhaps borne out by the phylogeneticanalysis of the BEG28, FO97 and G. geosporum BEG11isolates. Similar findings have been reported by Antoniolliet al. (2000), where ITS sequences more closely related toG. coronatum were isolated from a single G. mosseae spore.However, Antoniolli et al. (2000) did not suggest that thisfinding was the result of both sequence types existing togetherin single spores, but rather that it was a result of difficulties incorrectly distinguishing between spore morphotypes withinthe G. mosseae complex from the field. The process of nuclearexchange via anastomosis (Giovannetti et al., 1999) mayenable exchange of different nuclei between isolates of closelyrelated species, which could result in each spore containinga mixture of nuclei from other taxa, but at lower frequencies.If this is correct it raises the intriguing possibility ofmorphological switching mediated by differing proportionsof ‘nonmain group’ nuclei and could contribute to thecontinuum seen in spore morphology of other Glomus species( J. C. Dodd et al., unpublished). Thus, the reliance on sporemorphology alone to infer species may not be sufficient, andin light of the magnitude of sequence variation currentlybeing reported from a range of AMF species (Lloyd-MacGilpet al., 1996; Antoniolli et al., 2000; Pringle et al., 2000), theacceptability of submitting single sequences to databases asrepresentatives of a species is also questionable, as demon-strated by the G. coronatum BEG49 sequence (AF145740).The evidence from our study clearly indicated that thissequence was most probably not from G. coronatum BEG49but G. constrictum, since no other G. coronatum sequencesclustered with G. constrictum BEG130 sequences. Similarly,a sequence obtained from BEG28T was clearly affiliated tothe Gigasporaceae. Since this sequence made up a signific-ant proportion of the clones investigated from BEG28T andno evidence of contamination of this culture could befound, we believe this is not likely to be an artefact. This isthe first reported occasion where a Gigasporaceae sequencehas been isolated from Glomus spp. spores, however, thereverse situation has been reported on two earlier occasions(Clapp et al., 1999; Hosny et al., 1999). This perhapsadds credence to a genetic association between these twogenera.

Implications for the assessment of biodiversity

The difficulty in delimiting the genetic diversity of a singletaxon of AMF has been demonstrated in this investigationusing well-defined cultures. Investigation of natural AMFcommunities using rRNA genes as indicators of biodiversityis likely to be a very difficult proposition indeed until therange and magnitude of variation across several currentlyaccepted AMF taxa have been determined. In an analysisof 62 PCR products obtained from field root material,Helgason et al., 1999, reported 38 that were determined tobe Glomalean in origin of which 36 were unique. Theseclustered into 8 groups on phylogenetic analysis and theimplication was that this reflected the presence of 8 dis-tinct taxa identified from studies of intraradical hyphalmorphology (Merryweather & Fitter, 1998). This may notbe a correct assumption since the magnitude of sequencevariation indicated by our study and colleagues highlights thedifficulties of trying to use sequence diversity as an indicatorof biodiversity (Helgason et al., 1999). It is quite possible thatsequences originating from a single spore can cluster withdifferent taxa resulting in misleading information if singlesequences only are considered. The range of sequence diver-sity in different AMF taxa indicates that the use of specificprimers for the study of community diversity, where fungalsequence diversity is not well characterized, is limited. Theinterpretation of sequence data obtained from studies ofnatural AMF communities can only be carried out if themagnitude of sequence variation of the fungi have beenpreviously established. Specific primers will remain usefulhowever, where sequences characteristic of an isolate aretargeted, for example in microcosm experiments (Van Tuinenet al., 1998) or where well characterized isolates of AMF areto be followed under field conditions. The presence of suchsequences, in common to most G. coronatum isolates, offersthe possibility of targeting these for specific detection, but thisis only possible after a large-scale genetic, such as presentedhere. Similarly, the scale of this investigation has allowedthe identification of sequences that are most abundant inparticular isolates. If these can be targeted by PCR, itshould be possible to follow particular isolates in mixedcommunities.

Maintenance of AMF pot cultures

Cultures initiated from small numbers of spores, with similarspore morphologies, obtained from trap cultures or directlyfrom the field, may include misidentified spores or otherextraneous propagules. This can lead to cultures, thought tobe derived from a single morphotype, in fact comprisingmore than a single taxon. Since the conditions that inducesporulation for different species of AMF are diverse and largelyunexplored ( J. Morton, pers. comm), such contaminationcould remain undetected for considerable periods of time



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before environmental conditions arise where they are able tosporulate. A subsequent discovery of unexpected spore types islikely to be dismissed as pot-to-pot contamination. Subculturing(via selected multispore or crude inoculum transfer) mayalso cause the selection and loss of different morphotypes.In this investigation of intra- and inter-isolate variation inG. coronatum we have apparently detected a cryptic contaminantin a culture derived from the G. coronatum holotype (BEG28T-DNA sequences and isozymes profile) and from G. coronatumFO97, isolated from the same site as the holotype (isozymesonly). Sequence analysis indicated the presence of G. geosporumsequences in BEG28K, in addition to G. coronatum-likesequences that clustered with those from other G. coronatumisolates but not in FO97. Subsequent observation of sporemorphologies and retrospective analysis of isozyme profilesconfirmed the apparent presence of a G. geosporum morphin BEG28K. This provided evidence that G. geosporum islikely to be found at the locality where BEG28T was isolated.Both FO97, maintained in Pisa and the isotype, BEG28Kwere kept under conditions where cross contamination by aG. geosporum isolate was very unlikely and the most likely routefor the appearance of this taxon in a ‘pure’ open pot culturewas coisolation. However, the presence or absence of G.geosporum in the BEG28 cultures overall may reflect differencesbetween the environmental conditions under which the cultureswere maintained and which affected sporulation ( J. Morton& J. C. Dodd, pers. comm). The absence of G. geosporumsequences during the molecular analysis of FO97 may simplyhave been because G. geosporum was not sporulating in theculture at the time when the spores were selected.

The segregation of the sequences ITS T2 and ITS T4 intoseparate nuclei, as demonstrated by Hijri et al. (1999), indic-ated that nuclei can be expected to be frequently lost fromhyphal networks through random processes. The differencesfound between BEG28T – its subcultures – BEG28W andBEG28K and FO97 (isolated from the same collection site inItaly), with respect to the sequences common to more thanone isolate were interesting. Several sequences appeared tohave been lost from BEG28T and its subculture BEG28Kbut retained by the subculture BEG28W. Whilst the mostrecently isolated culture FO97, had five sequences in com-mon with BEG28W. The loss of sequences may reflect differ-ences in culturing conditions or selection via subsequentsubculturing. When spores are used as propagules it suggeststhat genetic diversity can change rapidly under differentculturing conditions and supports the genetic drift reportedby Wyss & Bonfante (1993) for G. mosseae BEG12 heldlong-term in different European laboratories, using RAPDanalysis.

Conclusion

The analysis of LSU D2 region sequences from G. coronatumin this study showed that the magnitude of variation was

considerably higher than expected. Each isolate of G.coronatum, with the exception of BEG139, had some D2sequences that were also found in other isolates, althoughthese were in the minority. In all cases the majority ofsequences detected in each isolate were unique, but thisvariation could not be attributed to DNA polymerase orcloning artefacts. The four species of AMF in the investigationwere closely related on the basis of spore morphology andappeared to represent parts of a genetic continuum since somesequences were more closely related to the other species in thegroup. The use of rRNA sequences to estimate biodiversity isnot considered possible until the taxon under investigationhas been adequately characterized. In this study somesequences were identified that were found in most of the G.coronatum isolates and that could be targeted to detect thepresence of G. coronatum. It is clear that the magnitude ofvariation found in other taxa needs to be determined as datafrom other Glomus spp., Acaulospora spp. and Scutellosporaspp. indicate the phenomenon of high levels of geneticvariability is wide spread in the Glomales. G. geosporum-likespores were found in a subculture of the holotype isolate ofG. coronatum BEG28T and inferred by isozyme analysis ofG. coronatum FO97. The most likely route for these to haveentered the culture was considered to be coisolation andthe subsequent maintenance of a cryptic contaminant. It isby no means certain that ‘pure’ cultures of AMF are composedof homogeneous genotypes nor free of cryptic contamina-tion. This work is being continued with other specieswithin the Glomales and will provide further urgently neededdata to elucidate the genetic variation found within thesesymbionts.

Acknowledgements

We would like to thank Professor Manuela Giovannetti andDr Luciano Avio for several of the G. coronatum isolates usedin this investigation. This work was partly supported by fundscontributed from the following sources:, Dr J. C. Dodd,International Institute of Biotechnology (IIB) andBEGNET project EU Framework IV, contract numberBIO4-CT97–2225. Alia Rodriguez wishes to express herthanks to COLFUTURO, Carrera 15 no. 37–15, Santafede Bogota (Colombia), Research School of Biosciences,University of Kent and IIB for financial support of herPhD studies.

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inter- and intra-isolate rrna large subunit variation in glomus coronatum spores

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