Glomus achrum and G.bistratum, two new species of arbuscular mycorrhizal fungi (Glomeromycota) found...

Glomus achrum and G. bistratum, two newspecies of arbuscular mycorrhizal fungi(Glomeromycota) found in maritime sand dunes

Janusz Błaszkowski, Przemysław Ryszka, Fritz Oehl, Sally Koegel,Andres Wiemken, Gabor M. Kovacs, and Dirk Redecker

Abstract: Two new arbuscular mycorrhizal fungal species, Glomus achrum sp. nov. and Glomus bistratum sp. nov.(Glomeromycota), are described and illustrated. Both species produce small, hyaline spores in aggregates formed in thesoil and inside roots. Glomus achrum was associated with roots of Ammophila arenaria (L.) Link colonizing maritimedunes of the Vistula Bar in northern Poland, and G. bistratum occurred among vesicular-arbuscular mycorrhiza ofXanthium cf. spinosum growing in dunes of the Mediterranean Sea adjacent to Veriko, Greece. Spores of G. achrum areglobose to subglobose, (25–)43(–55) mm in diameter, rarely egg-shaped, oblong to irregular, 15–45 mm � 55–65 mm. Theirwall consists of three hyaline layers: a mucilaginous, short-lived outermost layer; a laminate middle layer composed ofloose sublayers; and a flexible innermost layer. The outermost and the innermost layers stain deeply red in Melzer’s re-agent. Spores of G. bistratum are globose to subglobose, (20–)29(–50) mm in diameter, and have a wall composed of twopermanent, hyaline layers. The outer layer is unit, smooth, and the inner one laminate. Only the inner layer stains yellowin Melzer’s reagent. Both species formed vesicular-arbuscular mycorrhiza in single-species cultures with Plantagolanceolata L. as the host plant. Phylogenetic analyses of partial 18S rDNA subunit and internal transcribed spacer (ITS)region sequences placed G. achrum and G. bistratum into Glomus group A, but did not reveal any closely related de-scribed species. Environmental sequences from the public databases suggested that G. achrum occurred in at least twoother plant species from geographically distant regions. No such evidence could be obtained for G. bistratum, which iscurrently known only from the type location.

Key words: arbuscular mycorrhizal fungi, Glomeromycota, molecular phylogeny, new species, Glomus, sand dunes.

Resume : Les auteurs decrivent et illustrent deux nouvelles especes de champignons du genre Glomus, les G. achrum etG. bistratum. Les deux especes forment de petites spores hyalines en agregats, dans le sol et dans les racines. On a re-trouve le G. achrum associe aux racines de l’Ammophila arenaria colonisant des dunes maritimes de Vistula Bar dans lenord de la Pologne et le G. bistratum des mycorhizes a vesicules et arbuscules de Xanthium cf. spinosum poussant sur desdunes de la mer Mediterranee adjacentes a Veriko, en Grece. Les spores du G. achrum globulaires a subglobulaires mesu-rent (25–)43(–55) mm en diametre, sont rarement ovoıdes, oblongues a irregulieres, 15–45 mm � 55–65 mm. Leurs paroiscomportent trois couches hyalines; l’une mucilagineuse, l’une exterieure a courte duree de vie suivie d’une couche me-diane laminee composee de sous-couches laches, et une paroi interne flexible. Les parois les plus internes et externes de-viennent rouge fonce avec le reactif de Meltzer. Les spores du G. bistratum, de forme globulaire a subglobulaires,mesurent (20–)29(–50) mm en diametre et possedent une paroi composee de deux couches permanentes hyalines. La paroiexterne lisse comporte une seule unite et la paroi interne est laminee. Seule la paroi interne devient jaune avec le reactifde Meltzer. Les deux especes forment des mycorhizes a vesicules et arbuscules lorsqu’inoculees individuellement sur laplante hote Plantago lanceolata. Les analyses phylogenetiques des sequences partielles de la sous-unite 18S rADN et del’espaceur interne transcrit (ITS) placent le G. achrum et le G. bistratum dans le groupe Glomus A, sans montrer d’etroiterelation avec des especes deja decrites. Les sequences environnementales de bases de donnees publiques suggerent que leG. achrum se retrouve chez au moins deux autres especes de plantes de regions geographiques distantes. On ne retrouveaucune information similaire pour le G. bistratum qui n’est connu que de la localite d’origine du specimen type.

Mots-cles : champignons a arbuscules et vesicules, Glomeromycota, phylogenie moleculaire, nouvelles especes, Glomus,dunes.

[Traduit par la Redaction]

Received 17 June 2008. Published on the NRC Research Press Web site at botany.nrc.ca on 1 April 2009.

J. Błaszkowski.1 Department of Plant Protection, University of Agriculture, Słowackiego 17, PL-71434 Szczecin, Poland.P. Ryszka. Department of Ecological Microbiology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.F. Oehl, S. Koegel, A. Wiemken, and D. Redecker. Botanical Institute, University of Basel, Hebelstrasse 1, Basel 4056, Switzerland.G.M. Kovacs. Department of Plant Anatomy, Institute of Biology, Eotvos Lorand University, Pazmany Peter setany 1/C, 1117 Budapest,Hungary.

1Corresponding author (e-mail: [email protected]).

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Botany 87: 260–271 (2009) doi:10.1139/B08-138 Published by NRC Research Press

IntroductionArbuscular mycorrhizal fungi are considered to occur

commonly in the world and associate with at least 80% ofvascular land plants (Smith and Read 1997). Habitats espe-cially favoring these fungi are maritime sand dunes (Koske1987; Dalpe 1989; Tadych and Błaszkowski 2000), mainlybecause of their low nutrient content and organic compo-nents (Koske 1988; Nicolson and Johnston 1979). The asso-ciation of arbuscular mycorrhizal fungi with maritime duneplants may be of considerable ecological significance fortheir establishment and growth, because these fungi enhanceplant nutrient uptake, increase plant tolerance to drought andsalt stress, and protect against soil pathogens (Koske et al.2004). At least 32 newly described species of arbuscularmycorrhizal fungi have originally been associated with rootsof dune plants and many others have occurred in maritimedunes (Sridhar and Beena 2001; Błaszkowski 2003).

At present, arbuscular mycorrhizal fungi are placed in thephylum Glomeromycota C. Walker & Schuessler, comprising4 orders, 10 families, and 14 genera (Schußler et al. 2001;Błaszkowski 2003; Oehl and Sieverding 2004; Walker andSchußler 2004; Sieverding and Oehl 2006; Walker et al.2007a, 2007b; Palenzuela et al. 2008; Walker 2008). Themost numerous group of fungi in the Glomeromycota is thegenus Glomus Tul. & C. Tul., including ca. 53% of all arbus-cular mycorrhizal fungi described to date, i.e., ca. 200 species(Błaszkowski 2003). However, Morton (2000) hypothesized,when 154 species were known in the literature, that the num-ber of existing species of arbuscular fungi may be at leasttwo-fold higher. This hypothesis is in accordance with our fre-quent observations that several unknown morphospecies occurin the soils with rather low spore numbers. Such morphospe-cies can hardly be formally described until a sufficient numberof spores has been investigated and (or) propagated in single-species arbuscular fungal cultures (e.g., Tchabi et al. 2008).The hypothesis also agrees with the results of recent molecularinvestigations into the diversity of arbuscular fungi, which in-dicated that many revealed sequence types cannot be assignedto named fungi (e.g., Hijri et al. 2006). To formally describe anarbuscular mycorrhizal fungus, its spore morphology has to beknown. However, obtaining spores might be difficult at timesas Stutz and Morton (1996) showed that a lack or rarity ofsporulation is a common phenomenon in some habitats,although this does not indicate the absence of a fungus. An ef-fective method for forcing the production of spores by fungihidden in roots is the cultivation of field-collected rhizospheresoils and roots in successive (Stutz and Morton 1996) or long-term (Oehl et al. 2004) trap cultures.

Examination of trap cultures with rhizosphere soils and rootsof maritime dune plants revealed many aggregates of spores oftwo undescribed species of the genus Glomus that had not beenfound in the field-collected samples used to establish these cul-tures. These fungi are described below as Glomus achrum sp.nov. and Glomus bistratum sp. nov. and, based on the nuclearrDNA sequences, their phylogenetic position is presented.

Materials and methods

Establishment and growth of trap and single-speciescultures, extraction of spores, and staining of mycorrhizae

Spores examined in this study were derived from both

pot trap and single-species cultures. Trap cultures were es-tablished to obtain a large number of living spores and toinitiate sporulation of species that were present but thatwere not detected in the field collections (Stutz and Morton1996). The method used to establish trap cultures, theirgrowing conditions, and the methods of spore extractionand staining of mycorrhizae were as those described previ-ously (Błaszkowski et al. 2006).

Single-species cultures were also established and grownas described by Błaszkowski et al. (2006), with two excep-tions. First, instead of marine sand, their growing mediumwas an autoclaved commercially available coarse-grainedsand (grains 1.0–10.0 mm in diam., 80.50%; grains 0.1–1.0 mm in diam., 17.28%; grains < 0.1 mm in diam.,2.22%) mixed (5:1, v/v) with clinopthilolite (Zeocem,Bystre, Slovakia) with a grain size range of 2.5–5 mm. Cli-nopthilolite is a crystaline hydrated alumosilicate of alkalimetals and alkaline earth metals having, e.g., a high ion ex-change capability and selectivity, as well as a reversible hy-dration and dehydration. The pH of the sand–clinopthilolitemixture was 7.3. Second, the cultures were kept in transpar-ent plastic bags, 15 cm wide and 22 cm high as suggestedby Walker and Vestberg (1994), rather than open pot cul-tures (Gilmore 1968). To prevent contamination of the cul-tures with other AMF but still to allow exchange of gases,we left an opening, about 1 cm wide, in the centre of theupper part of each bag, while the edges on both sides wereclosed with small plastic clips. The cultures were wateredwith tap water once a week and harvested after 5 months,when spores were extracted for study. To reveal mycorrhizalroot structures, root fragments located ca. 1–5 cm below theupper level of the growing medium were cut off with a scal-pel. The host plant used in both trap and single-species cul-tures was Plantago lanceolata L.

Microscopy surveyMorphological properties of spores and their wall struc-

ture were determined based on the examination of at least100 spores mounted in either lactic acid (for spore size) orpolyvinyl alcohol – lactic acid – glycerol (PVLG; Omar etal. 1979) and a mixture of PVLG and Melzer’s reagent(1:1, v/v; for wall structure and the phenotypic and biochem-ical properties of the spore wall components). Spores at alldevelopmental stages were crushed to varying degrees byapplying pressure to the cover slip, and then stored at 65 8Cfor 24 h to clear their contents from oil droplets. They werethen examined under an Olympus BX 50 compound micro-scope equipped with Nomarski differential interference con-trast optics. Microphotographs were recorded on a Sony3CDD color video camera coupled to the microscope.

Terminology of spore structure is that suggested bySturmer and Morton (1997) and Walker (1983). Spore col-our was examined under a dissecting microscope on freshspecimens immersed in water. Colour names are from Kor-nerup and Wanscher (1983). Nomenclature of fungi andplants is that of Walker and Trappe (1993) and Mirek et al.(1995), respectively. The authors of the fungal names arethose presented at the Index Fungorum Web site www.indexfungorum.org/AuthorsOfFungalNames.htm. Voucherspecimens were mounted in PVLG and a mixture of PVLGand Melzer’s reagent (1:1, v/v) on slides and deposited in

Błaszkowski et al. 261

Published by NRC Research Press

the Department of Plant Protection (DPP), University ofAgriculture, Szczecin, Poland, and in the herbarium at Ore-gon State University (OSC) in Corvallis, Oregon, USA.

Colour microphotographs of spores of the new species canbe viewed online at www.agro.ar.szczecin.pl/~jblaszkowski/.

DNA extraction, polymerase chain reaction, and DNAsequencing

DNA crude extracts were produced from spores, as de-scribed by Redecker et al. (1997). The extracts were usedas templates for nested polymerase chain reactions using theprimer pairs NS5–ITS4 and GLOM1310–ITS4i, respectively(Redecker et al. 2003). These primers amplify a region ofthe rDNA comprising the 3’ end of the 18S small subunit,the internal transcribed spacer, and the 5.8S rDNA subunit.The PCR products were cloned into pGEM-T (Catalys,Wallisellen, Switzerland), reamplified from the clones, puri-fied with DNA Pure (Roche, Basel, Switzerland), and se-quenced in both directions using a BigDye TerminatorCycle Sequencing Kit (Version 3.1, Applied Biosystems,Foster City, Calif.) for labeling. Samples were run on anABI 310 capillary sequencer (Applied Biosystems). Foreach species studied, three clones were sequenced to obtainan estimate of rDNA variation within the isolates. Sequenceswere submitted to the EMBL database under the accessionnumbers FM253379–FM253384.

Phylogenetic analysesThe 3’ end of the 18S ribosomal small subunit region

was aligned to previously known sequences from the data-bases in PAUP*4b10 (Swofford 2001). The ITS1/5.8S subu-nit/ITS2 region was included into another datasetcomprising the respective gene regions from reference fungifrom the databases, as well as unpublished sequences fromother projects in our lab. Representatives of the closestBLAST matches (Altschul et al. 1997) of the new sequencesaccording were present in this dataset. Phylogenetic analysesof subclades containing the new sequences and their closestrelatives were conducted after optimizing the alignmentwithin the respective group.

Phylogenetic trees were inferred using distance, parsi-mony, or maximum likelihood criteria as implemented inPAUP*. Neighbor joining or heuristic search algorithmswere applied for the respective criteria. Maximum likelihoodmodels and parameters were estimated using Modeltest 3.5(Posada 2004).

In addition, Bayesian analyses were performed with the18S dataset using MrBayes 3.1.1 for Macintosh (Ronquistand Huelsenbeck 2003).The analysis was run over 1.5 mil-lion generations using four Monte Carlo chains. Every100th tree was retained and 10% of the trees were discardedas a ‘‘burn-in.’’

Taxonomy

Glomus achrum Błaszk., D. Redecker, Koegel, Schutzek,Oehl & Kovacs sp. nov.

MYCOBANK NO.: MB 512539.Sporocarpia ignota. Sporae singulatim vel gregatim in

solo vel in radice efformatae. Fascicula globosa, oblongavel irregulares 70–280 mm � 90–480 mm. Sporae hyalinae;

globosae vel subglobosae; (25–)43(–55) mm diam; raro ovoi-deae, oblongae vel irregulares; 15–45 mm � 55–65 mm.Tunica sporae stratis tribus (strati 1–3); stratum ‘‘1’’ cadu-cum, glabrum, hyalinum, (0.7–)1.0(–1.2) mm crassum, insolutione Melzeri purpurascens vel magenteum; stratum‘‘2’’ laminatum, glabrum, hyalinum, (1.5–)2.5(–3.9) mmcrasso; stratum ‘‘3’’ elasticum, glabrum, hyalinum, (0.5–)0.7(–1.0) mm crassum, in solutione Melzeri rubellum velrubidum. Hypha sporifera hyalina; recta vel recurvata; cy-lindrica vel infundibuliformis; (2.9–)4.3(–5.1) mm lata adbasim sporae; pariete hyalino, (1.0–)1.7(–3.4) mm crasso,stratis 1–3 in parietem sporae continuantibus. Porus hy-phae (0.7–)1.5(–2.5) mm diam. Mycorrhizas vesiculo-arbusculares formans.

TYPUS: Polonia, Sedinum (Szczecin), infra P. lanceolata,10 March 2008, J. Błaszkowski 2885 (DPP, Holotypus).Spores formed in the soil in loose to compact aggregates;70–280 mm � 90–480 mm, occasionally inside roots (Fig. 3(see Figs. 1–8)); develop blastically at the tip of hyphaebranched from a parent hypha continuous with a mycorrhi-zal extraradical hypha or secondary branches of the mainbranches of the parent hypha (Figs. 1–3). SPORES: hyaline;globose to subglobose; (25–)43(–55) mm in diameter; rarelyegg-shaped, prolate to irregular; 15–45 mm � 55–65 mm;with one subtending hypha (Figs. 1, 2, 4, 7, and 8). WALL

STRUCTURE OF SPORES: composed of three hyaline layers (layers1–3; Figs. 4–8). Layer 1, forming the spore surface, mucila-ginous, roughened, (0.7–)1.0(–1.2) mm thick when intact,usually more or less deteriorated in mature spores, almostalways completely sloughed in older specimens. Layer 2laminate, (1.5–)2.5(–3.9) mm thick, easily stratifying intogroups or single laminae in crushed spores. Layer 3 flexible,(0.5–)0.7(–1.0) mm thick. In Melzer’s reagent, layers 1 and 3stain purplish red (14B8) to greyish Magenta (14C6) andreddish white (8A2) to deep red (11C8), respectively(Figs. 4–8). SUBTENDING HYPHA: hyaline; straight or recurved;cylindrical to slightly funnel-shaped; (2.9–)4.3(–5.1) mmwide at the spore base (Figs. 4, 7, and 8). WALL OF SUBTEND-

ING HYPHA: hyaline; (1.0–)1.7(–3.4) mm thick at the sporebase; composed of three layers continuous with spore walllayers 1–3 (Figs. 4 and 8). PORE: (0.7–)1.5(–2.5) mm in diam-eter, occluded by a curved septum continuous with sporewall layer 3 (Fig. 8). GERMINATION: unknown.

MYCORRHIZAL ASSOCIATIONS: in the field, G. achrum occurredamong vesicular-arbuscular mycorrhiza of Ammophila are-naria (L.) Link colonizing maritime dunes of the VistulaBar adjacent to Krynica Morska (54822’N, 19826’E), Poland,but did not sporulate. Spores of this fungus were originallyisolated from a pot trap culture established and grown as de-scribed in the Materials and methods.

In single-species cultures with P. lanceolata as the hostplant, G. achrum formed mycorrhiza consisting of arbus-cules, vesicles, as well as intra- and extra-radical hyphae(Figs. 9–14). Arbuscules were scarce and consisted of atrunk grown from a parent hypha and numerous brancheswith fine tips. Vesicles were rare and widely dispersed ornumerous and evenly distributed along the roots, dependingon the root fragment examined. They were egg-shaped toprolate; 10.0–45.0 mm � 27.5–130.0 mm; rarely globose tosubglobose; (30.0–)40.0(–55.0) mm in diameter. Intraradical

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Figs. 1–8. Glomus achrum. Figs. 1 and 2. Spores in loose aggregates. Fig. 3. Intraradical spores. Figs. 4 and 8. Spore wall layers (swl) 1–3and subtending hyphal wall layers (shwl) 1 and 2. Figs. 5–7. Spore wall layers (swl) 1–3. Fig. 1. Spores in lactic acid. Figs. 2–8. Spores inPVLG+Melzer’s reagent. Figs. 1–8, differential interference microscopy. Scale bars: Figs. 1 and 2 = 20 mm; Fig. 3 = 50 mm; Figs. 4–8 =10 mm.



hyphae grew along the root axis, were generally numerous,(1.0–)4.4(–7.1) mm wide, straight or slightly curved, andsometimes formed H- or Y-shaped branches and coils. Thecoils usually were ellipsoid; 12.5–25.0 mm � 30.0–37.5 mm;rarely circular; 30.0–37.9 mm in diameter, when seen in aplan view; and usually widely dispersed along the root frag-ments. Extraradical hyphae were (2.5–)5.1(–9.6) mm wideand generally occurred in low abundance. All the mycorrhi-zal structures stained intensively blue in 0.1% trypan blue.PHYLOGENETIC POSITION: all conclusions concerning the phylo-genetic positions of G. achrum and G. bistratum (a speciesdescribed below) were supported well by distance, parsi-mony, maximum likelihood, and Bayesian analyses. Thephylogenetic trees (Fig. 15) demonstrate that G. achrum is a

member of Glomus group A in the Glomeraceae (Schwarzottet al. 2001) and has no close relative among described spe-cies of known phylogenetic positions.

The 18S tree in Fig. 15 A provides an overview of thephylogenetic relationships across the family. The ITS treesin Fig. 15 (B and C) focus on the subclades containing theclosest relatives of the two Glomus species studied. A com-plete ITS-based phylogeny of Glomus group A is not pre-sented as it was not possible to unequivocally align the ITSsequences outside these clades.

Environmental sequences obtained from sporophyte of therattlesnake fern, Botrychium virginianum (L.) Sw., sampledin Hungary (Kovacs et al. 2007) grouped among the threedifferent sequence clones from G. achrum, both in the ITS

Figs. 9–14. Mycorrhiza of G. achrum in roots of Plantago lanceolata stained in 0.1% trypan blue. Figs. 9 and 10. Arbuscules (arb) withtrunk (tr) and intraradical hypha (ih). Fig. 11. Vesicle (ves). Fig. 12. H-shaped branch (Hb) of intraradical hyphae. Fig. 13. Y-shaped branch(Yb) of intraradical hypha. Fig. 14. Coil (c) and arbuscule (arb). Figs. 9–14, differential interference microscopy. Scale bars: Figs. 9–14 =10 mm.

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and the 18S tree. An ITS sequence from Prunus africana(Hook f.) Kalkm. from Ethiopia (Wubet et al. 2004) alsoclustered among the clones from G. achrum. A sequencefrom Galium aparine L. from Thuringia, Germany (Renkeret al. 2003), was closely associated with the aforementionedclade.

COLLECTIONS EXAMINED: Poland, Szczecin, under pot-culturedP. lanceolata, 10 March 2008, J. Błaszkowski 2885 (DPP,Holotype); J. Błaszkowski 2814–2884 and 2886–2894 (DPP,Isotypes) and two slides at OSC.

ETYMOLOGY: Latin, achrum, referring to the hyaline sporesformed by the fungus.

DISTRIBUTION AND HABITAT: using traditional methods of find-ing of arbuscular fungi (not molecular), G. achrum has, sofar, only been found associated with vesicular-arbuscularmycorrhizal roots of A. arenaria growing in maritime dunesof the Vistula Bar adjacent to Krynica Morska, Poland.Spores of this fungus were not found in either ca. 3000field-collected soils or ca. 2000 pot trap cultures represent-ing other regions of Europe, as well as Africa, Asia, and theUSA. (J. Błaszkowski, personal observation).

No spores of arbuscular fungi were found in the fieldmixture of rhizosphere soil and roots. The species of arbus-cular fungi co-occurring with G. achrum in the trap culturecontaining the field-collected rhizosphere soil and rootswere Glomus minutum Blaszk., Tadych, & Madej andGlomus lamellosum Dalpe, Koske, & Tews.

However, molecular data provided some evidence thatG. achrum may be geographically widespread. ITS sequen-ces of arbuscular mycorrhizal fungi obtained from Bo-trychium virginianum from Hungary and Prunus africanafrom Ethiopia (Kovacs et al. 2007; Wubet et al. 2004) clus-tered among the clones obtained from G. achrum in the phy-logenetic trees (Fig. 15), falling within the genetic variationof the isolate. Considering that ITS distinguish most mor-phospecies in Glomus group A, we interpret this as evidencefor conspecificity. As rDNA shows considerable variationwithin species and even within single spores of the Glomer-omycota, we cannot exclude that other sequences clusteringclosely with this clade also originate from this morphospe-cies.

NOTES: the distinctive morphological properties of G. achrumare its small spores formed in aggregates and remaining hya-line throughout their entire life cycle, and the three-layeredspore wall with the structural laminae middle layer consist-ing of loose laminae and the other two layers staining inten-sively in Melzer’s reagent (Figs. 1–8).

Other known species of the genus Glomus producing con-glomerations of only hyaline spores of a three-layered sporewall are Glomus diaphanum J.B. Morton & C. Walker andGlomus proliferum Dalpe & Declerck. Our molecular analy-ses revealed that both species are phylogenetically distantfrom G. achrum. For G. proliferum, the 18S and ITS regionswere available in the databases for comparison (18S, seeFig. 15A; ITS accession number, AJ973393), for G. diapha-num only ITS sequences (accession numbers AJ972458,AJ972457) could be compared. Both for G. diaphanum andG. proliferum, the similarity to the ITS was low, thus thesespecies are not included in Fig. 15 (B and C).

Morphologically, G. achrum and G. diaphanum are

mainly separated by the phenotypic and biochemical proper-ties of the components of their spore wall. All three sporewall layers of both species are of the same types (Mortonand Walker 1984; Morton 2002; Błaszkowski 2003). How-ever, the mucilaginous outermost spore wall layer ofG. achrum stains much more intensively in Melzer’s reagent[purplish red (14B8) to greyish Magenta (14C6); Figs. 3, 4,7, and 8] than that of G. diaphanum [pinkish white (7A2);Błaszkowski 2003]. The laminate middle wall layer ofspores of the former species generally disintegrates incrushed spores (Figs. 4–8), whereas it remains compact incrushed spores of the latter fungus. It also is much thinner[(1.5–)2.5(–3.9) mm thick vs. (3.9–)6.0(–8.3) mm thick inG. diaphanum; Błaszkowski 2003]. The flexible innermostspore wall layer of the fungus described here distinguishesits strong reactivity in Melzer’s reagent (Figs. 4, 7, and 8),which does not occur at all in the third wall layer ofG. diaphanum spores (Morton 2002; Błaszkowski 2003).Other properties diverging the two fungi reside in the sub-tending hyphae of their spores. Apart from the differencesin shape (cylindrical to slightly funnel-shaped in G. achrum(Figs. 4, 7, and 8) vs. cylindrical to slightly flared inG. diaphanum), the subtending hyphae of these species alsodiffer in width and thickness of their wall. Even the upperrange of width of the subtending hypha of spores ofG. achrum (5.1 mm) does not attain the lower one of that ofspores of G. diaphanum (70 mm), whose wall generally alsois thicker [(3.0–)3.4(–3.8) mm thick vs. (1.0–)1.7(–3.4) mmthick in G. achrum; Błaszkowski 2003].

Compared with G. achrum spores, those of G. proliferummay be markedly larger [(25–)43(–55) mm diam. vs. (22–)40–66(–76) mm in diameter when globose; Declerck et al.2000; Błaszkowski 2003]. However, the most important dif-ferences between these species reside in the phenotypic andbiochemical properties of components of their spore wall.The only spore wall component sharing the species discussedhere is their mucilaginous outermost one staining intensivelyin Melzer’s reagent (Figs. 4, 7, and 8). Although a laminatelayer occurs in the spore wall of both species, it is a middlestructure in G. achrum (Figs. 4–8), and the innermost one inG. proliferum (Błaszkowski 2003). Most importantly, thelaminate spore wall layer of G. achrum easily stratifies ineven slightly crushed spores (Figs. 4–8), whereas this layergenerally remains intact in vigorously crushed G. proliferumspores. Moreover, the former species does not develop thesemi-flexible and semi-permanent middle spore wall layerof the latter fungus, but synthesizes a flexible innermostlayer reacting in Melzer’s reagent (Figs. 4–8), whose the lat-ter fungus does not develop. Finally, the subtending hypha ofspores of G. achrum is much narrower [(2.9–)4.3(–5.1) mmwide vs. (8.8–)10.0(–11.2) mm wide in G. proliferum], has athinner wall [(1.0–)1.7(–3.4) mm thick vs. (1.7–)3.5(–6.6) mmthick], and slightly differs in shape (cylindrical to slightlyfunnel-shaped (Figs. 4, 7, and 8) vs. cylindrical to slightlyflared; Declerck et al. 2000; Błaszkowski 2003).

Glomus cerebriforme McGee and G. minutum also pro-duce only hyaline spores of a similar size range and appear-ance to those of spores of G. achrum (McGee 1986;Błaszkowski et al. 2000; Błaszkowski 2003). Unfortunately,although considerable effort was made to obtain livingspores or sequences of G. cerebriforme, these were not



Fig. 15. Phylogenetic trees based on rDNA sequences from Glomus achrum, G. bistratum, and other members of the Glomeraceae. Se-quences from spores are labeled with the fungal species name and the database accession number. Environmental sequences are labeled withthe accession number first, the internal sequence code (e.g., ISA273, ZS635), if applicable, and the plant host genus or family. Where mul-tiple species of the same plant genus are present, the species name is provided. (A) Bayesian tree based on the 3’ end region of the 18Ssmall subunit. Numbers on the nodes denote posterior probabilities. Glomus etunicatum and G. claroideum were used as outgroup. Thedataset comprised 369 characters. (B and C) Neighbor joining trees rooted by midpoint rooting based on the 5.8S subunit – ITS2 region ofrDNA from G. bistratum, G. achrum, and their respective close relatives. Numbers denote bootstrap values from 1000 replications. Thedatasets comprised 365 and 420 characters, respectively.

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made available to us. Additionally, DNA extraction fromspores of G. minutum collected from an old single-speciesculture failed. Thus, we have to rely solely on spore mor-phology to explain the differences between G. achrum andthe latter two species. The wall of spores of G. cerebriformeand G. minutum contains only two layers, and not three asthat of G. achrum spores (Figs. 4–8). Additionally, sporesof G. cerebriforme are formed on racemose hyphae in epi-geous sporocarps in the field or in loose hypogeous hyphalmasses in pot cultures (McGee 1986), whereas those of thespecies characterized here form at the tip of straight hyphalbranches being a part of aggregates borne only in the soil(Figs. 1 and 2).

An additional four described Glomus spp. produce con-glomerations of relatively small spores, which are hyalinethroughout a part of their life cycle. These are Glomus ar-borense McGee, Glomus microaggregatum Koske, Gemma,& P.D. Olexia, Glomus segmentatum Trappe, Spooner, &M.H. Ivory, and Glomus viscosum Nicol. (Trappe 1979;Koske et al. 1986; McGee 1986; Walker et al. 1995; Morton2002; Błaszkowski 2003, J. Błaszkowski, personal observa-tion). However, with age, spores of these species darkenfrom yellowish white (1A2; G. segmentatum; Błaszkowski2003) to brownish yellow (G. microaggregatum; Koske etal. 1986). Additionally, the walls of spores of G. arborenseand G. microaggregatum are simpler in structure, containingonly one and two layer(s), respectively (J. Błaszkowski, per-sonal observation; Koske et al. 1986; McGee 1986).Although G. segmentatum and G. viscosum have spores of athree-layered spore wall, none of the components of thiswall reacts in Melzer’s reagent (Trappe 1979; Walker et al.1995; Morton 2002; Błaszkowski 2003; vs. spore wall layers1 and 3 stain intensively in this reagent in G. achrum;Figs. 3, 4, 7, and 8). Moreover, the spore wall of G. visco-sum comprises a semi-flexible middle layer, which is lack-ing in that of G. achrum, but the latter species differentiatesa flexible innermost layer (Figs. 4–8) that is absent in thespore wall of the former fungus.

Glomus bistratum Błaszk., D. Redecker, Koegel, Symanc-zik, Oehl & Kovacs sp. nov.

MYCOBANK NO.: MB 512540.Sporocarpia ignota. Sporae gregatim in solo, raro in rad-

ice efformatae. Fascicula globosa, oblonga vel irregulares,120–650 mm � 160–800 mm. Sporae hyalinae; globosae velsubglobosae; (20–)39(–50) mm diam. Tunica sporae stratisduobus (strata 1 ad 2); stratum ‘‘1’’ diuturnum, glabrum,hyalinum, (1.0–)1.6(–2.0) mm crassum; stratum ‘‘2’’ lamina-tum, glabrum, hyalinum, (0.7–)1.0(–1.5) mm crassum, in sol-utione Melzeri luteolum vel flavescens. Hypha sporiferahyalina; recta vel recurvata; parum infundibuliformis vel in-fundibuliformis; (2.9–)5.7(–8.8) mm lata ad basim sporae;paries hyphae hyalinus, (1.2–)1.8(–2.5) mm crassus, instrata 1–2 sporae continuans. Porus (0.5–)1.9(–2.9) mmdiam., apertus. Mycorrhizas vesiculo-arbusculares formans.

TYPUS: Polonia, Sedinum (Szczecin), infra P. lanceolata,1 April 2008, J. Błaszkowski 2895 (DPP, Holotypus).Spores occur in the soil in loose to compact aggregates,120–650 mm � 160–800 mm, sometimes containing dozensof spores, rarely inside roots; origin blastically at the tip ofhyphae branched from a parent hypha continuous with a my-

corrhizal extraradical hypha or secondary branches of themain branches of the parent hypha (Figs. 16 and 17). SPORES:

hyaline; globose to subglobose; (20–)39(–50) mm in diame-ter; with one subtending hypha (Figs. 16, 17, and 19–23).WALL STRUCTURE OF SPORES: composed of two permanent, hya-line layers (layers 1 and 2; Figs. 18–23). Layer 1, formingthe spore surface, unit sensu Walker (1983), smooth, (1.0–)1.6(–2.0) mm thick. Layer 2 laminate, (0.7–)1.0(–1.5) mmthick. In Melzer’s reagent, only layer 2 stains yellowishwhite (4A2) to butter yellow (4A5; Figs. 16 and 20–23).SUBTENDING HYPHA: hyaline; straight or recurved; flared tofunnel-shaped, (2.9–) 5.7(–8.8) mm wide at the spore base(Figs. 22 and 23). WALL OF SUBTENDING HYPHA: hyaline; (1.2–)1.8(–2.5) mm thick at the spore base; composed of twolayers continuous with spore wall layers 1 and 2 (Figs. 22and 23). PORE: (0.5–)1.9(–2.9) mm in diameter, open(Figs. 22 and 23). GERMINATION: unknown.MYCORRHIZAL ASSOCIATIONS: in the field, G. bistratum occurredamong vesicular-arbuscular mycorrhizae of Xanthium cf.spinosum L. colonizing sand dunes of the MediterraneanSea adjacent to Veriko (40808’N, 22835’E), Greece, butspores were not found in the field samples. Spores of thisfungus were, for the first time, revealed in a trap culture ear-lier inoculated with a mixture of rhizosphere soil and rootssampled from under the plant species listed above.

In single-species pot culture with P. lanceolata as the hostplant, G. bistratum formed mycorrhiza consisting of arbus-cules and vesicles, as well as intra- and extra-radical hyphae(Figs. 24–27). Arbuscules were few to numerous, dependingon the root fragment examined, and usually were widely dis-persed along the roots. They consisted of short trunks grownfrom parent hyphae and numerous branches with fine tips.Vesicles were not numerous and always occurred singly.They were ellipsoid to prolate, 10.0–27.5 mm � 22.5–45.0 mm. Intraradical hyphae grew along the root axis, were(1.2–)3.9(–6.1) mm wide, straight or slightly curved, and oc-casionally formed H- or Y-shaped branches and coils. Thecoils always were ellipsoid; 12.5–25.0 mm � 42.5–82.5 mm;when seen in a plane view; and usually widely dispersedalong the roots. Extraradical hyphae were (1.2–)2.2(–2.9) mmin diameter and generally occurred in low abundances. All themycorrhizal structures stained intensively blue in 0.1% trypanblue.PHYLOGENETIC POSITION: phylogenetic analyses of sequences ofspores of G. bistratum showed it to be a member of Glomusgroup A sensu Schwarzott et al. (2001), but has no closemolecular relative among described species of this group(Fig. 15). No environmental sequences were found to beclosely related to G. bistratum. Both in the 18S and the ITStrees, it was strongly separated from other taxa.COLLECTIONS EXAMINED: Poland, Szczecin, under pot-culturedP. lanceolata, 1 April 2008, J. Błaszkowski 2895 (DPP, Hol-otype); J. Błaszkowski 2896–2911 (DPP, Isotypes) and twoslides at OSC.ETYMOLOGY: Latin, bistratum, referring to the two-layeredspore wall of the fungus.DISTRIBUTION AND HABITAT: to date, G. bistratum was foundonly at the type location characterized in the section ‘‘My-corrhizal associations.’’ Spores of this fungus were notfound either in ca. 3000 field-collected soils or in ca. 2000



Figs. 16–23. Glomus bistratum. Figs. 16 and 17. Spores in loose aggregates. Figs. 18–21. Spore wall layers (swl) 1 and 2. Figs. 22 and 23.Spore wall layers (swl) 1 and 2 and subtending hyphal wall layers (shwl) 1 and 2. Figs. 17–19. Spores in PVLG. Figs. 16 and 20–23. Sporesin PVLG + Melzer’s reagent. Figs. 16–23, differential interference microscopy. Scale bars: Fig. 16 = 20 mm; Figs. 17–23 = 10 mm.

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pot trap cultures representing different other regions of Eur-ope, as well as Africa, Asia, and the USA. (J. Błaszkowski,personal observation). Since there are also no environmentalsequences in the databases that can be assigned to this spe-cies, we conclude that G. bistratum seems to be a relativelyrare species, a finding supported by both morphological andmolecular methods.

In the field, apart from the mycorrhiza of G. bistratum,X. cf. spinosum harboured spores of two undescribed Glo-mus spp. and Scutellospora persica (Koske & C. Walker)C. Walker & F.E. Sanders. The arbuscular mycorrhizal fungiaccompanying G. bistratum in the trap culture with theX. cf. spinosum rhizosphere soil and root mixture were Glo-mus clarum Nicol. & N.C. Schenck, Glomus gibbosumBlaszk., and an undescribed Glomus sp.NOTES: Glomus bistratum is morphologically distinguished by(i) its rather compact aggregates with small, spores (gener-ally smaller than 50 mm in diam.) remaining hyalinethroughout their entire life cycle; (ii) the simple, two-layeredspore wall structure with the permanent, unit outer layerthicker than the laminate inner one; (iii) the lack of an evan-escent spore wall layer forming the spore surface of mostknown Glomus spp. (Bentivenga and Morton 1995); and(iv) the usually funnel-shaped subtending hypha, whosewall layers, continuous with the two spore wall layers, ex-tend far below the spore base (Figs. 16–23).

Other known members of the genus Glomus producingsmall, hyaline spores with bi-layered walls grouped in ag-

gregates are G. cerebriforme (McGee 1986) and G. minutum(Błaszkowski et al. 2000). Similarly as in G. bistratum(Figs. 18–23), one of the two spore wall layers ofG. cerebiforme and G. minutum is laminate (McGee 1986;Błaszkowski et al. 2000; Błaszkowski 2003). However, inG. cerebriforme, the laminate layer forms the surface of itsspores and surrounds a thin flexible layer, whereas the lami-nate spore wall layer of G. bistratum and G. minutum is aninner structure, overlaid with a semi-flexible layer. InG. bistratum, the laminate spore wall layer is thinner thanthe outer layer forming the spore surface (Figs. 18–23), theinverse in G. minutum. Additionally, spores of G. cerebri-forme and G. minutum may be slightly larger (up to 80 mmand 65 mm in diam., respectively; McGee 1986; Błaszkow-ski et al. 2000; Błaszkowski 2003) than the largest sporesof G. bistratum (up to 50 mm in diam.) and only spores ofthe species described here have a funnel-shaped subtendinghypha (Figs. 22 and 23), and not cylindrical or slightlyflared as have spores of G. cerebriforme and G. minutum,respectively. Finally, spores of G. bistratum (Figs. 16 and17) and G. minutum occur in aggregates and originate at thetip of mostly straight hyphal branches (Błaszkowski et al.2000; Błaszkowski 2003), whereas those of G. cerebriformeare grouped in either epigeous sporocarps, 1–5 cm wide, orloose hypogeous hyphal masses and form on racemose hy-phae (McGee 1986).

Glomus achrum (a species described above), G. diapha-num, and G. proliferum also form only hyaline spores. How-

Figs. 24–27. Mycorrhiza of Glomus bistratum in roots of Plantago lanceolata stained in 0.1% trypan blue. Fig. 24. Arbuscule (arb) andintraradical hypha (ih). Fig. 25. Vesicle (ves), intraradical spores (is), and H-shaped branch (Hb) of intraradical hyphae. Fig. 26. H-shapedbranch (Hb) of intraradical hyphae. Fig. 27. Y-shaped branch (Yb) of intraradical hypha and coil (c). Figs. 24–27, differential interferencemicroscopy. Scale bars: Figs. 24–27 = 10 mm.



ever, in contrast to G. bistratum producing spores of a two-layered wall, spores of G. achrum, G. diaphanum, andG. proliferum have a wall with three layers. The only sporewall component linking these fungi is a laminate layer(Morton and Walker 1984; Morton 2002; Błaszkowski2003). Glomus bistratum does not have the outermost muci-laginous and the innermost flexible spore wall layers ofG. achrum and G. diaphanum. Additionally, compared withG. bistratum producing spores only in rather compact aggre-gates, the spore aggregates of G. diaphanum are muchlooser and most spores of this fungus occur singly in thesoil. Moreover, only the smallest G. diaphanum spores[(50–)90(–130) mm diam] overlap in size with the largestones (50 mm in diam.) of the fungus discussed here.

Also when compared with the spore wall of G. prolif-erum, that of G. bistratum lacks the outermost mucilaginouslayer and the semi-flexible, semi-permanent middle layer ofthe former fungus (Declerck et al. 2000; Błaszkowski 2003).Additionally, spores of G. proliferum may be markedlylarger (up to 76 mm in diam.) than those of the species de-scribed here (up to 50 mm in diam.) and only the lowerrange of width (8.8–11.2 mm wide) of its cylindrical toslightly flared subtending hypha overlaps with the upperrange of width (2.9–8.8 mm wide) of the cylindrical tofunnel-shaped subtending hypha of G. bistratum (Błasz-kowski 2003). The subtending hyphal wall of the formerspecies also is much thicker [(1.7-)3.5(–6.6) mm thick] thanthat of the latter fungus [(1.2–)1.8(–2.5) mm thick].

Morphological differences between G. bistratum andG. arborense, G. microaggregatum, G. segmentatum, andG. viscosum are large. The morphological characters of thelatter four fungi were already presented in the last paragraphof NOTES regarding G. achrum (see above).

AcknowledgementsThis study was supported in part by the Polish Committee

of Scientific Research, grant No. 2 PO4C 041 28 and 164/N-COST/2008/0; the Hungarian Research Fund (OTKAK72776); and the Swiss National Science Foundation. Weacknowledge the technical help of Jens Schutzek, Sarah Sy-manczik, and Zuzana Sykorova (Institute of Botany, Univer-sity of Basel). The authors are grateful for the help ofanonymous reviewers for valuable comments on the manu-script.

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