Mycologia, 98(3), 2006, pp. 468–478. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897
Rediscovery of Alnicola cholea (Cortinariaceae): taxonomic revision and description of its mycorrhiza with Polygonum viviparum (Polygonaceae) Pierre-Arthur Moreau1
Moreau 2005), A. cholea Ku¨hner (Ku¨hner 1981) and A. bohemica (Ku¨hner 1981, Breitenbach and Kra¨nzlin 2000). They all are suspected to be mycorrhizal with dwarf alpine willows (Salix subgen. Chamaetia (Dumort.) Nasarov, especially S. herbacea L. and S. reticulata L.). A. cholea has been documented so far only by the protolog (Ku¨hner 1981) and two bibliographic citations (Bon 1992:9, Ludwig 2001:402, 415). Ku¨hner (1981) himself reported this unusual species only twice: from an abundant type collection (G, No. 6236, holotype, 18 specimens) and from a doubtful and not well documented specimen (G, No. B-12bis, 1 specimen). As part of a taxonomic revision of the genus Alnicola (Moreau 2005), the first present author (P.-A. M.) examined numerous specimens of Alnicola species in various European herbaria, including Ku¨hner’s original collections of A. cholea (G). Additional fresh specimens, collected in the French and Austrian Alps, helped define the species variability. This rare species also was found independently in the alpine zone of the Carpathians. The latter record of Alnicola cholea in a grassland with Polygonum viviparum as the only putative ectomycorrhizal host further encouraged a study of possible mycorrhizal symbiosis between these two species. Polygonum viviparum had never been reported in literature as a host for any Alnicola species. The presence of ectomycorrhiza in Polygonum viviparum was observed in arctic and alpine sites in many works (e.g. Hesselman 1900, Peyronel 1937, Fontana 1977, Read and Haselwandter 1981, Blaschke 1991, Treu et al 1996, Massicotte et al 1998, Ronikier and Mleczko in press). In some investigations the estimated diversity of fungal symbionts was high and several ectomycorrhizal morphotypes were recognized, however only scarce data on identification of fungal symbionts are available (e.g. Fontana 1977, Treu et al 1996, Massicotte et al 1998, Ronikier and Mleczko in press). In papers published to date mostly basic morphological characteristics of ectomycorrhizae were presented. Massicotte et al (1998) analyzed the anatomy of an unidentified P. viviparum mycorrhiza, but it was based on longitudinal and cross sections only. Data on structural details required for thorough comparative studies were lacking. The complex data on Alnicola cholea given below provide new, complementary information on this enigmatic species, confirming its morphological
Laboratoire de Botanique, Faculte´ des Sciences pharmaceutiques et biologiques, Universite´ Lille2, BP83, 3 rue du Professeur Laguesse, F-59006 Lille, France
Piotr Mleczko Institute of Botany, Jagiellonian University, Lubicz 46, PL-31-512 Krako´w, Poland
Michał Ronikier Anna Ronikier Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krako´w, Poland
Abstract: Alnicola cholea, a little-known species so far reported only from the two original localities in the French Alps, is redefined here based on revision of herbarium materials and studies of recent field collections. A detailed morphological and anatomical description of fruit bodies of Alnicola cholea, including a discussion on its taxonomic status and distribution data is provided. Due to the unique combination of characters of Alnicola cholea within the genus, a new monospecific section is introduced for this species: Alnicola sect. Cholea, sect. nov. Mycorrhizal symbiosis of A. cholea with an arcticalpine plant Polygonum viviparum was observed in the Tatra Mountains (Poland). A description of these mycorrhizae is given, providing first detailed data on an identified herbaceous plant mycorrhiza. Key words: Arctic-alpine fungi, Cortinariales, ectomycorrhizae, taxonomy INTRODUCTION
Alnicola Ku¨hner (5Naucoria [Fr. : Fr.] P. Kumm. pro parte, Cortinariaceae, Hebelomeae) is a fairly small genus (about 30 taxa in Europe, Moreau in Horak 2005), mostly represented by Alnus-associated ectomycorrhizal species. From arctic-alpine environments to which the present authors devoted a more general study, only four species are reported: A. tantilla ( J. Favre) Romagn. (Favre 1955; Gulden in Gulden and Jenssen 1988, Breitenbach and Kra¨nzlin 2000), A. chamiteae Ku¨hner (Ku¨hner 1981, Senn-Irlet 1986; a synonym of A. tantilla according to de Haan 2000, Accepted for publication 15 Mar 2006. 1 Corresponding author. E-mail:
[email protected]
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MOREAU ET AL: REDISCOVERY OF ALNICOLA CHOLEA TABLE I.
469
Sequences of Alnicola cholea deposited in the GenBank (see Moreau et al 2006)
Species Alnicola cholea Alnicola cholea Alnicola cholea
Plant host
Geographic origin
Collection no. and herbarium
¨ rtfjo¨llmoen, Ranaa¨lv (N) 19760259 (IB) Salix sp. O Salix herbacea Bourg-Saint-Maurice (F) PAM03_14 (LIP) Polygonum viviparum Tatry (PL) Ronikier01 (LIP)
variability and systematic position. The findings let the present authors give the detailed morphological and anatomical description of the P. viviparum ectomycorrhiza; this paper also contributes to the knowledge of mycorrhizae of Alnicola species, the least known representative of the Cortinariaceae. MATERIALS AND METHODS
Morphological studies of fruit bodies.—Dry fruit bodies were studied in keeping with micromorphological methods routinely used in Basidiomycota: hymenium, pileus and stipe covering (radial cuts) in Congo red after reviving in 5% KOH. Spores measurements were taken from natural spore deposit on the stipe surface (spore print not obtained), in 5% KOH and Melzer’s reagent, completed with observations on hymenium. Mycorrhizal collections and structural analysis.—Several blocks of soil with Polygonum viviparum roots were collected in the patch of alpine grassland in the vicinity of Alnicola cholea fruit bodies in the Tatra Mountains (see information below for details of the locality). All mycorrhizae isolated from soil were grouped according to morphological characters. Putatively identical mycorrhizae from different soil blocks were not put together but analyzed as separate samples. Altogether 23 mycorrhizal samples were analyzed by the PCR-RFLP method to identify the mycorrhiza of Alnicola cholea. Macroscopic and microscopic features of mycorrhizae were examined. The shape of the system, its color, surface texture and presence of extramatrical structures were analyzed on fresh specimens under a dissecting microscope. The structure of respective fungal mantle layers and extramatrical hyphae was observed in ‘‘plan views’’ with the light microscope using the Nomarski interference contrast; preparations were mounted both in water and lactic acid. The methodology of ectomycorrhiza characterization and definitions of feature categories are according to Agerer (1987–2002, 1991). Voucher mycorrhiza specimens are preserved in FAA (formaldehyde, 70% ethanol, acetic acid; 5 : 90 : 5) and deposited in the mycological collection of the Institute of Botany, Jagiellonian University (KRA-F PM 292). DNA extraction and PCR-RFLP analysis.—Mycorrhizal roots of Polygonum viviparum and fruit body tissue (fragments of hymenophore) were stored in CTAB buffer. In the case of fruit bodies, DNA was extracted from the Polish collection (KRAM-F 53043). In addition to the fungal material, DNA from leaves of Polygonum
Accession Accession no. ITS1-5,8S-ITS2 no. 28S rLSU AY900092 AY900091 AY900093
AY900113
viviparum was extracted to check whether primers applied amplify the plant ITS region. The DNA isolation procedure, PCR amplification and PCR-RFLP analysis followed Gardes and Bruns (1993) and Agerer et al (1996). DNA isolation from the mycelium was performed with the modified CTAB method including mechanical grinding of tissue in the CTAB buffer, incubation at 65 C (cell membrane lysis), mixing with cold chlorophorm and extracting DNA from the upper phase, DNA precipitation with isopropanol, washing the pellet with sodium acetate-ethanol solution followed by 80% ethanol, resuspending in 50 mL of deionized water. The amplification was performed with ITS1/ITS4 universal primers for the ITS region of the ribosomal nuclear DNA (e.g. Gardes et al 1991). The reaction mix (final volume 20 mL) contained PCR buffer (103, QIAGEN), 2 mL; MgCl2 (25 mM), 1.8 mL dNTP mix (10 mM, Sigma), (1.59) 0.5 mL; primers (10 mM), 1.0 mL (of each ITS1 and ITS4); Taq polymerase (5 U/mL, QIAGEN), 0.2 mL; DNA extract, 2 mL; ddH2O, up to 20 mL. The PCR profile was 59 at 94 C; (300 at 94 C, 19 at 52 C, 1.59 at 72 C) 332; 59 at 72 C; storage at 4 C. For the restriction analysis, enzymes HapII and EcoRI (Amersham Biosciences) were used. The amplified DNA was incubated overnight at 37 C with single enzymes; reaction mix included 103 enzyme-specific buffer, 1/10 of the final volume; restriction enzyme (10 U/mL or 15 U/mL), 3 U; BSA (10 ng/mL), 0.6 mL; amplified DNA solution, 3 mL; ddH2O, up to 17 mL. Products of restriction were separated by electrophoresis in a 8% polyacrylamide gel at 4 C. Restriction profiles were compared with GeneTools software (Syngene); molecular weight of band was assigned by comparison with GeneRuler 100 bp DNA Ladder (Fermentas). RESULTS
Morphology of fruit bodies and taxonomy.—Morphological description is a synthesis of observations made on Ku ¨ hner’s collections (herb. R. Ku ¨ hner, G) and five recent ones from Austria, France, Poland and Norway. An arctic Norwegian collection in M. Moser’s herbarium (IB, under the name ‘‘Naucoria tantilla’’) was also identified as Alnicola cholea on revision. The conspecificity of most collections was established by DNA sequence analysis (TABLE I, see also Moreau et al 2006). Because the original description is hardly accessi-
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FIG. 1. Alnicola cholea, fruit bodies. A. Coll. PAM00-14 (LIP), picture G. Eyssartier. B. Coll. PAM04080701 (LIP), picture P.-A. Moreau. C. Coll. 4-VIII-2002 (KRAM-F), with Polygonum viviparum, picture M. Ronikier. Bar 5 10 mm.
ble, the Latin diagnosis of the protolog (Ku¨hner 1981) is reproduced below. Alnicola cholea Ku¨hner, Trav sci Parc natl Vanoise 11:133. 1981. FIGS. 1–3 Diagnosis (Ku¨hner 1981). Velum nullum et pileo glabro vel subtiliter pruinoso (sub lente). Pileo 5–14 mm lato, conico campanulato, obscure rufo brunneo, hygrophano. Stipite 11– 22 3 1–2 mm, subaequali, intense vel etiam obscure e rufo brunneo, sursum pruinoso, fistuloso. Lamellis maxime ventricosis, alte sinuatis, adnatis, e lateritiis brunneis. Sporis
11.5–13 3 5.7–6.7 mm, phialiformibus sursum subcuspidatis, 1.2–3.7 mm crassis. Alnicolae amarescenti admodum affinis, sed sporis majoribus, papilla apicali manifestiore et habitatione alpina. Holotypus in herb. Ku¨hner : K. 62–36 (21-8-62).
Pileus 1–2.2 cm, conical-obtuse to campanulate with rounded +/2 prominent umbo, not striate, hygrophanous when old, at first very faintly micaceous-velvety all over, soon eroded and smooth, silky to fibrillose-silky, liver red-brown somewhat purplishtinged, uniform, fading from center to uniformly straw ocher, without visible veil. Lamellae 11–26 at
MOREAU ET AL: REDISCOVERY OF ALNICOLA CHOLEA
FIG. 2. A–E. Alnicola cholea (coll. Ronikier 53036, LIP). A. Spores. B. Basidia. C. Cheilocystidia. D. Pileipellis, radial cut. E. Caulocystidia. Bar 5 10 mm.
stipe, 1–2 series of lamellules, adnexed to almost free, first pale grayish ocher slightly pink-tinged, then bright rusty ocher, at the end dirty rust-brown; edge whitish, pruinose then eroded. Stipe 3–5 3 0.1– 0.2 cm, flexuose, slightly enlarged to almost tapering at basis, pruinose at apex (one-third of the length), sometimes below midlength on young and well developed specimens, slightly silky-fibrillose to the base on hyaline dirty-ocher ground, soon dark gray to dark brown from the base; mycelium white, abundant, visible up to 0.5 cm on the stipe. Context grayish when wet, fading to pale yellowish ocher, more lemon yellow in the stipe before blackening. Odor fungoid to earthy when cut, rather strong and unpleasant, not Pelargonium-like. Flavor extremely bitter. When young and fresh the fungus mimics Macrocystidia cucumis (Ku¨hner 1981) or Collybia fuscopurpurea Konrad & Maubl. Spores (FIGS. 2A; 3A, C) (9)9.8–13(14.5) 3 (6.5)7– 8(8.5) mm, indextrinoid, reddish ocher in water and KOH, polymorphic even on spore print, amygdaliform to almost limoniform, with 6 elongate apex,
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FIG. 3. A, B. Alnicola cholea (coll. PAM 00-14, LIP). A. Spores. B. Cheilocystidia. C–E. Alnicola cholea (coll. GE-03036, LIP). C. Spores. D. Basidia. E. Cheilocystidia. Bar 5 10 mm.
sometimes strikingly umbonate; ornamentation thick (0.8–1.2 mm), punctate to verrucose, with slightly loosening perispore. Basidia (FIGS. 2B, 3D) 28–36 3 7–12.5 mm, (2–)4-spored, distinctly protruding when mature, cylindrical with somewhat attenuate to tapering base. Gill edge sterile; cheilocystidia (FIGS. 2C, 3B, E) 30–48 3 7.5–12 mm, spindle-shaped with long acute neck but also sparsely obtuse to +/2 cylindrical, sometimes yellowish at apex but not significantly thickened. Pleurocystidia rare around the edge, slenderer than cheilocystidia, 38–45 3 6– 7 mm, otherwise absent. Subhymenium 8–12 mm thick, pseudoparenchymatous, well developed. Gill trama regular, of slender hyphae 3 3–8 mm, pale, not incrusted. Pileipellis (FIG. 2D) a cutis of slender hyphae 3 2.5–4.5 mm, coarsely incrusted bright yellow pigment (in water and KOH), on young specimens with sparse terminal 6 erected articles with smooth wall, soon disappearing by erosion. Subpellis reddish brown in water and KOH, pseudoparenchymatous with 2–3 layers of short,
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MYCOLOGIA
incrusted articles 3 6–10 mm. Stipitipellis with abundant clusters of caulocystidia (FIG. 2E) on the upper one-third, 35–80 3 7.5–15 mm, club-shaped to spindle-shaped with long appendage 6 enlarged at apex, often thickened and yellowish, usually articulate with thicker short articles at basis; superficial hyphae slender 3 2–3.5 mm, incrusted, often with hyaline globular inclusions in KOH. Clamp connections present at all septa.
TABLE II. Results of the PCR-RFLP analysis of the ITS region. Bands of the ITS from P. viviparum detected in the mycorrhizae are given in square brackets. * Remaining bands of the P. viviparum ITS/HapII profile were not observed in the gel
¨ DTYROL: OberCollections examined. AUSTRIA. SU gurgl, alt. 2250 m, under Salix foetida along a mossy watercourse, 7-VIII-2004, leg. U. Peintner & P.-A. Moreau, herb. P.-A. Moreau No. 04080701 (LIP). FRANCE. SAVOIE: Pralognan-la-Vanoise, cirque du Ge´ne´py, alt. 2250 m, on ground amongst Salix shrubs (S. foetida, S. hastata etc.) with mixed S. herbacea, 21-VIII-1962, herb. R. Ku¨hner No. 62–36 (G, HOLOTYPE); Bonneval-sur-Arc, refuge des Evettes, alt. 2500 m, among Salix herbacea, 17-VIII-1971, herb. R. Ku¨hner No. B-12bis (G); Bourg-Saint-Maurice, Arc 2000, lake Marloup, alt. 2500 m, Salicetum herbaceae among pebbles along lake, leg. G. Eyssartier, 28-VIII-2003, herb. P.-A. Moreau No. 00-14 (LIP); same locality, on a mineral slope with Salix herbacea, leg. G. Eyssartier, 23-VIII-2003, herb. P.-A. Moreau No. GE-03-036 (LIP). NORWAY. NORD¨ rtfjo¨llmoen, 1976, leg. M. Moser, herb. M. LAND: Ranaelv, O Moser No. 19760259 (IB); HORDALAND: Ulvik, Finse, peneplain of Nordnut-Lille Finsenut, numerous specimens on wet mossy slope with Salix herbacea, S. lanata and Polygonum viviparum, leg. C. Cripps, S. Elborne and U. Peintner during the 7th International Symposium of ArcticAlpine Mycology (ISAM) at Finse, 10-VIII-2005, herb. P.-A. Moreau No. NO05-23 (LIP). POLAND. THE CARPATHIANS: Western Tatra Mountains, Kasprowy Wierch Massif, at the top, between upper station of cable car and meteorological office, alpine meadows with Polygonum viviparum, alt. 1984 m., 29-VII-2002, leg. H. Knudsen & A. Ronikier, No. 53036 (KRAM-F, duplicates in C and LIP); 4VIII-2002, leg. A. Ronikier & M. Ronikier, No. 53043 (KRAM-F).
Weight of bands in respective restriction profiles (in base pairs) [679] 687 377 376 351 351
Validation of a new section.—Morphological characters of A. cholea justify the creation of a new section for this species, here validated. See Discussion below. Section Cholea P.-A. Moreau, sect. nov. Diagnosis: Alnicola sect. Cholea sect. nov. Sporae latae, crassitunicatae, rugoso-verrucosae, amygdaliformes vel citriformes, saepe mammilatae. Pileipellis hyphae filamentosae, incrustatae ; subpellis hyphae polygonales, incrustatae. Cortina nulla. Cheilocystidia caulocystidiaque acicularia, generis typica. Numquam cum Alnis, sed Salicibus Polygonisque associata.
HOLOTYPUS. Alnicola cholea Ku¨hner, Trav. sci. Parc natl Vanoise 11:133. 1981 (so far only known species in this section). Molecular recognition of the mycorrhiza using the PCRRFLP analysis of the ITS rDNA region.—The ITS region was amplified successfully in all DNA
Samples
POLYGONUM VIVIPARUM Enzymes CARPOPHORES MYCORRHIZAE (LEAVES)
EcoRI
ALNICOLA CHOLEA
HapII 315 228 180
[413] 318 229 180 [106]
416
106*
isolates from two fruit bodies of Alnicola cholea and from mycorrhizal samples. The ITS primers also amplified the ITS region in the DNA isolated from leaves of Polygonum viviparum, therefore the plant ITS region also was included in the restriction analysis to recognize plant-derived bands in mycorrhizal profiles. Both the EcoRI and HapII enzymes generated restriction profiles in the ITS region from Alnicola cholea fruit bodies (2- and 3band patterns, respectively). Restriction profiles of all mycorrhizal samples isolated from the patch were analyzed in single gels per profile, together with profiles of Alnicola cholea (fruit bodies) and Polygonum viviparum (leaves). One mycorrhizal sample was characterized by a restriction profile identical to that of Alnicola cholea (TABLE II). The profile also contained traces of bands belonging to the ITS of Polygonum viviparum; however they could be excluded on the basis of direct comparison with the plant profile and calculation of total band weights. Restriction with both enzymes clearly discriminated the mycorrhiza of Alnicola cholea, especially in the case of the particularly distinguishable HapII three-band profile (the profiles of other mycorrhizae were markedly different). Morphology and anatomy of the Alnicola cholea 3 Polygonum viviparum mycorrhiza.— Mycorrhizae (FIG. 4A, B) not branched, 0.5–1.5 mm long, 0.15–0.25 mm diam, mostly straight, rarely bent or flexuous, constricted in proximal part and often also tapering in distal part; growth segments, marked by constriction zones, sometimes present (beaded mycorrhiza); mycorrhizal tip rounded; surface stringy and woolly, loose hyphal strands
MOREAU ET AL: REDISCOVERY OF ALNICOLA CHOLEA
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FIG. 4. A, B. Ectomycorrhizae of Alnicola cholea and Polygonum viviparum; note ectomycorrhizal tip with weakly developed rhizomorphs (arrow). C. Longitudinal section through ectomycorrhizal tip with elongated cells surrounded by Hartig net hyphae. D. Loosely organized rhizomorph. E. Extramatrical hyphae. F, G. Extramatrical hyphae details; note reversely oriented clamp (arrow, F) and thin side branch (arrow, G). All figures from the collection PM 292.
present associated with some mycorrhizae; root cells not shining through; color of mycorrhizal tips white or whitish-cream, older parts with reddish tint. Mantle plectenchymatous to pseudoparenchyma-
tous, without matrix material, hyphal walls hyaline or slightly yellowish up to 0.5 mm thick, with smooth surface; clamps semicircular in side view, as thick as hyphae or slightly thinner, hyphae often constricted
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FIG. 5. A, B. Hyphal net on mantle surface; note anastomoses formed as short bridges (arrows). C, D. Middle mantle layer as compactly arranged plectenchyma. E. Middle mantle layer as transition type to plectenchyma. F. Inner mantle layer. All figures from the collection PM 292.
at septa. Outer mantle layer (FIG. 5A, B) a loose net of branched and anastomosing hyphae 2.5–7 mm diam, with septa 14–130 mm distant; hyphal branches mostly T-shaped, short side branches rarely present; anastomoses between hyphae as short open bridges; re-
versely oriented clamps present but infrequent. Middle mantle layer developed either as compactly arranged plectenchyma (FIG. 5C, D) or a transition type to pseudoparenchyma, with irregular or rectangular hyphal segments (FIG. 5E); hyphal cells
MOREAU ET AL: REDISCOVERY OF ALNICOLA CHOLEA (2.5)4.5–26 mm diam, 8–65(95) mm long; clamps occasionally present but simple septa prevail. Inner mantle layer (FIG. 5F) plectenchymatous, hyphae (1.5)2.5–9 mm diam, with septa 6.5–65 mm distant; clamps rare, mostly simple septa formed. Extramatrical hyphae (FIG. 4E–G) 2.5–7 mm diam, clamps similar to those of outer mantle hyphae, distance between septa from 20 mm to more than 150 mm, secondary simple septa not observed, reversely oriented clamps present but not frequent; walls hyaline or slightly yellowish, smooth, up to 0.5 mm thick; side branches and anastomoses more distantly arranged than in outer mantle hyphae; hyphal junctions as short open bridges. Rhizomorphs (FIG. 4D) occasionally present, formed as rather loosely arranged, undifferentiated bundles of hyphae, reversely growing hyphae present; hyphal diameter 3–8 mm, distance between septa from 13 to more than 170 mm; intrahyphal hyphae not observed. Cystidia and sclerotia not observed. Mantle in longitudinal section (FIG. 4C) plectenchymatous, hyphae thin-walled, hyphal cells 1.5–4 mm radially, 1.5–14 mm tangentially; Hartig net paraepidermal, hyphae in 1–2 rows; Hartig net in plan view a palmetti type, lobes 1–2 mm diam; root cells surrounded by hyphae elongated radially, 54–71 mm long, 20–30 mm broad, the average ratio between tangential and radial measurement of these cells 0.42. DISCUSSION
Taxonomy and systematic position.—Although undoubtedly its member, A. cholea occupies quite a unique position in the genus Alnicola. Ku ¨ hner (1981) created the stirps Amarescens (inval.) for species with spindle-shaped cystidia, not associated with Alnus, grouping A. amarescens, A. chamitae, A. tantilla and A. cholea. While the first three have ellipsoidal, thin-walled and faintly verrucose spores, A. cholea is characterized clearly by limoniform, thick-walled and coarsely ornamented spores (FIGS. 2A, 3A, C). In addition, while ecological and some morphological characters (pileus smooth or nearly so, subpellis pseudoparenchymatous, cystidia along the entire stipe) show affinities with sect. Amarescens, spores exhibit more analogies to other groups of Hebelomeae such as Hebeloma or Hymenogaster. Molecular phylogenetic data confirm the singular position of Alnicola cholea in the genus. The analysis of ribosomal DNA sequences (5.8S + ITS and 28S) conducted by Moreau et al (2006) shows that the collections analyzed morphologically identified as A. cholea form a monophyletic sister clade of all other species of Alnicola. Spindle-shaped cystidia on the gill
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edge (FIGS. 2C, 3B, E) are a synapomorphic character of the genus Alnicola ss. str., including A. cholea. A. cholea resembles A. amarescens and related species with its smooth pileic covering (F IG . 2D) with pseudoparenchymatous subpellis (but thick and coarsely incrusted without superficial hyphae, while having filamentous suprapellis in sect. Amarescens) and by the arctic-alpine distribution with plants other than Alnus. Other Alnicola species are differentiated by ecology (strictly associated with Alnus) and a smooth pileic surface of different structures (no well structured filamentous suprapellis). Therefore A. cholea is here isolated in a monospecific section. Spores of A. cholea are larger and much wider than in any other species of Alnicola with spindle-shaped cystidia, with spores never exceeding 6.5 mm in width (Moreau in Horak 2005). The ornamentation is comparable to that of A. bohemica and some Hymenogaster species, but in the case of A. cholea a certain variability in spore shape and ornamentation can be observed. This sporal variability confused Ku¨hner (1981), who hesitated to put together his two collections, one being less well developed and with somewhat aborted ornamentation and narrower spores. Polish collections (FIG. 2) are more consistent with this second specimen, but many intermediates can be observed (FIG. 3) and a separate grouping of more long-spored specimens is not seen at this time. Alnicola cholea, compared by Ku¨hner to ‘‘un petit Macrocystidia’’ (1981:131), is probably also variable according to alpine climatic conditions, and the characteristic purplish tones on the cap and gills, observed on some Austrian, French and Norwegian collections, may be absent in older specimens. Mycorrhizae.—Mycorrhizae of Alnicola have not been researched in depth. More comprehensive descriptions of mycorrhizae of only two species have been published so far, namely (i) Alnicola escharoides with Alnus glutinosa (Germany, Pritsch et al 1997) and (doubtful identification!) with Alnus acuminata (Argentina, Becerra et al 2002), and (ii) Alnicola subconspersa with Alnus glutinosa (Germany, Pritsch et al 1997). Pritsch and her coworkers (1997) found no differences in morphology and anatomical structure between the mycorrhizae described. These mycorrhizae are white to whitish and hyaline (with root cells shining through in the case of the German collections) or white to yellowish (the South American collection) when young, but brownish or reddish with age. The surface is described as stringy or felty, with abundant emanating hyphae (woolly), and with loose hyphal bundles associated; presence of
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FIG. 6.
Distribution of known localities of Alnicola cholea.
rhizomorphs however does not constitute a constant feature of all ectomycorrhizal tips. Mantles were plectenchymatous in the outer part and pseudoparenchymatous in the inner part with rectangular to irregular hyphal cells (Becerra et al 2002) or a transitional type between plectenchyma and pseudoparenchyma (Pritsch et al 1997). A netlike arrangement of surface hyphae was mentioned by Pritsch et al (1997). In both cases outer mantle hyphae were found to be arranged parallel to root surface. Simple bridges without septa were the only types of anastomoses reported in both papers. The dimensions of emanating and outer mantle hyphae noted for both the Alnicola escharoides and A. subconspersa mycorrhizae were similar and their diameter was about 2–6 mm, whereas the diameter of inner mantle hyphae was up to 11 mm (the South American mycorrhizae).
Emanating hyphae had clamps and smooth, hyaline walls. They were branched and formed anastomoses similar to those in outer mantle hyphae. The color and mantle surface features of the mycorrhizae of Alnicola cholea with Polygonum viviparum, described in the present contribution, are similar to those of the Alnicola mycorrhizae described previously. The differentiation of the mantle into the outer layer (loosely plectenchymatous) and the inner layer (more compact and with enlarged hyphal cells) is shared by all the Alnicola mycorrhizae. Loosely organized undifferentiated rhizomorphs and the type of anastomoses (open, short bridges) are also their common features. Some differences can be identified however. The characteristic parallel arrangement of outer mantle hyphae is absent in the mycorrhizae of Alnicola cholea with Polygonum viviparum. Although the diameter of extramatrical and outer mantle
MOREAU ET AL: REDISCOVERY OF ALNICOLA CHOLEA hyphae are quite similar in all Alnicola mycorrhizae, the dimensions of middle mantle hyphae are considerably bigger in A. cholea. The presence of reversely oriented clamps (FIG. 4F) was not given in the descriptions published previously. A. escharoides and A. subconspersa share more similarities with each other than with A. cholea as regards mycorrhizal anatomical characteristics. This could be expected because these two species are closely related within section Alnicola (Moreau et al 2006). Thus the differences in the mantle structure of A. cholea can reflect the distinct phylogenetic position of this species within the genus Alnicola. However many more species have to be investigated to test the value of mycelial characteristics of mycorrhizae for the systematics of this genus. The mycorrhizae of P. viviparum with A. cholea were simple (not ramified) and cylindrical; these general aspects of morphology are concordant with other descriptions of mycorrhizae formed by this plant (e.g. Blaschke 1991, Treu et al 1996, Massicotte et al 1998, Ronikier and Mleczko in press). Also the characteristic growth modification of rhizodermal cells surrounded by the Hartig net (Massicotte et al 1998) were present in the case of the mycorrhiza described here. Biogeographical distribution and ecology.—Alnicola cholea was observed exclusively in alpine zones of the central and northern European mountains or in the arctic tundra (FIG. 6). It mostly occurred among various dwarf species of Salix in typical communities rich in arctic-alpine mycorrhizal fungi. Arctic-alpine species of Salix are certainly main hosts of this fungus, although no direct investigations were conducted to prove it. The specimen found in the Austrian Alps grew with Salix foetida, the only ectomycorrhizal plant around. Salix herbacea is the most likely host in the case of Ku ¨ hner’s original collection (Ku ¨ hner 1981). In the Tatra Mountains however a local appearance of Alnicola cholea fruit bodies was observed in a patch of alpine grassland where only Polygonum viviparum was present as a potential mycorrhizal host. This finding proves that Alnicola cholea is probably well established as a component of Polygonum-associated ectomycorrhizal community in the studied area in the Tatras. The presence of several mycorrhizal morphotypes in a population of Polygonum viviparum free from ectomycorrhizal shrubs also was observed in another locality in the Tatras (Ronikier and Mleczko in press). These observations show that this herbaceous plant can maintain persistent populations of fungal hosts with the
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local absence of shrubs, although it probably can be treated mostly as a substitutive host for ectomycorrhizal fungi in arctic and alpine areas. ACKNOWLEDGMENTS
The authors warmly thank Dr Philippe Clerc (Jardin et Conservatoire botaniques de Gene`ve, G) and Dr Ursula Peintner (University of Innsbruck, IB) for loan of R. Ku¨ hner’s and M. Moser’s collections respectively, Dr Guillaume Eyssartier (Paris) for the specimens and pictures of his collections of A. cholea, and two anonymous reviewers for useful comments improving the manuscript. Michał Ronikier greatly acknowledges the scholarship for young scientists granted by the Foundation for Polish Science (2003–2004). This study was partly done in a framework of the project financed by the Polish Ministry of Education and Science, grant no. 2P04C 086 30.
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