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Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana 59717
Gregory M. Mueller
Department of Botany, The Field Museum, 1400 S. Lake Shore Dr., Chicago, Illinois 60605-2496
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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The alpine zone is comprised of habitats at elevations above treeline, and macromycetes play important ecological roles as decomposers and mycorrhizal symbionts here as elsewhere. Laccaria is an important group of ectomycorrhizal basidiomycetes widely used in experimental and applied research. A systematic study of alpine Laccaria species using morphological, cultural and molecular (ribosomal DNA internal transcribed spacer) data revealed five taxa in the Rocky Mountain alpine zone: L. laccata var. pallidifolia, L. nobilis (the first published report for arctic-alpine habitats), L. pumila, L. montana and L. pseudomontana (a newly described taxon similar to L. montana with more ellipsoidal, finely echinulate basidiospores). All occur in the southern Rocky Mountains of Colorado; however, only L. pumila and L. montana were found on the Beartooth Plateau in the northern Rocky Mountains of Montana and Wyoming. All are associated with dwarf and shrub Salix species, with L. laccata var. pallidifolia also associated with Dryas octopetala and Betula glandulosa. Maximum-parsimony phylogenetic analysis of rDNA-ITS sequences for 27 Laccaria accessions supports the morphological species delineations.
Key words: Agaricales, arctic-alpine macromycetes, internal transcribed spacer (ITS) sequences, macrofungi, molecular phylogenetics, Tricholomataceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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). While the macrofungal mycota of arctic-alpine regions such as Greenland (Borgen et al 2000, Kobayasi et al 1971, Lamoure et al 1982, Lange 1948–1957, Petersen 1977, Watling 1977, 1983), Iceland (Christiansen 1941, Hallgrímsson 1981, 1998), Svalbard (Gulden and Torkelsen 1996, Kobayasi et al 1968, Ohenoja 1971, Watling 1983) the Alps (Eynard 1977, Favre 1955, Graf 1994, Lo Bue et al 1994, Senn-Irlet 1987, 1988) and Alaska (Kobayasi et al 1967, Laursen and Chmielewski 1982, Miller 1982a, b, Miller et al 1982, Saccardo et al 1910) have been the subject of previous investigations, that of the North American alpine is virtually unknown (Cripps and Horak 1999). The results presented in this paper were obtained as part of a National Science Foundation-sponsored biotic inventory of Rocky Mountain alpine macromycetes (Cripps 2002, Cripps and Eddington 2005, Cripps and Horak 1999, Cripps et al 2001, 2002), and represents the first detailed systematic study of Laccaria species in the North American alpine zone.
Laccaria Berkeley & Broome (Basidiomycota, Hymenomycetes, Agaricales, Tricholomataceae sensu Singer 1986) is a genus commonly reported from arctic-alpine habitats, and it has been noted that the arctic-alpine taxa are in need of revision (Lamoure et al 1982, Watling 1987). A monographic study of North American taxa by Mueller (1992) did not include alpine specimens. Species of Laccaria typically form ectomycorrhizae with a variety of tree species. Because of the ability of some Laccaria species to grow vegetatively and/or germinate from basidiospores in culture, they are widely used as experimental systems for studies of ectomycorrhizal (EM) basidiomycetes (e.g. Bills et al 1999, Klironomos and Hart 2001, Kropp and Mueller 1999, Lei et al 1991, Podila et al 2002), and alpine species have potential for applied use in high altitude land reclamation (Graf 1997).
Published literature reports of Laccaria in arctic-alpine habitats comprise 27 taxa (TABLE I). However, due to synonomy, nomenclatural misapplications and misidentifications common in Laccaria taxonomy, it is probable that the number of arctic-alpine taxa is considerably lower in reality. TABLE I presents the most comprehensive published summary of arctic-alpine Laccaria to date, providing a starting point for subsequent taxonomic and biogeographic studies.
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, Smaglik 2000). A better understanding of the composition and ecology of soil communities is critical for assessing and responding to these effects. In addition, biotic surveys of poorly studied regions are important for inferring the biogeography and evolution of taxa (Müller and Magnuson 1987) and for addressing questions regarding the higher-level phylogenetic relationships among the agaricoid fungi (Moncalvo et al 2000). This study focuses on the alpine component of the North American arctic-alpine mycota assessing the systematics of Rocky Mountain alpine Laccaria species with a morphological study of 92 collections from Colorado, Montana and Wyoming and a phylogenetic analysis of ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences from selected collections. Morphological and molecular characters support the recognition of four known and one previously undescribed species. Rocky Mountain alpine distributions and EM host associations of these taxa are documented.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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) media. Basidiomes were preserved by warm air-drying on an electric dryer and deposited in the Montana State University-Bozeman fungal herbarium (MONT) or the herbarium of the Eidgenössiche Technische Hochschule Zü rich (ZT). Laccaria specimens collected during the course of this study are listed by taxon (TABLE II).
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and are noted as combinations of plate, column and row numbers (e.g. 8A5). Preparation of hand sections for observation of micromorphological characters and use of descriptive terms follow Largent et al (1977). Sections were mounted in 3% KOH for measurement of basidiospores and other micromorphological features and in Melzer’s reagent to test for amyloidity. Basidiospore measurements were made with the hilar appendix and ornamentation excluded. Length-width ratios (Q) were calculated for each basidiospore, and a mean calculated for each collection (Qm). Basidiospore measurements were made from hymenial tissue to maintain consistency between collections. Measurements were made from a random sampling of 10–20 basidiospores and 5–20 basidia and cystidia for each collection, from multiple basidiomes when possible, to represent intracollection variation. Width measurements of basidia and cystidia were made at the widest point. Drawings of micromorphological features were prepared using a drawing tube attached to a Leica DMLS research microscope. Culture morphology was observed for some collections, noting color and relative growth rate on PDA and/or MMN media. Some taxa did not grow in culture, and successful isolation of L. pumila was rare. Scanning electron microscopy.— – Lamellar fragments (approx. 1 mm2) were removed from dried basidiomes, attached to aluminum mounts using double-stick tape and gold-palladium sputter coated at a nominal coating thickness of 15 nm using a Hummer VII sputtering system (Anatech Ltd., Alexandria, Virginia). Basidiospores were examined at 15 kV using a JEOL JSM-6100 scanning electron microscope.
DNA extraction, PCR amplification and DNA sequencing.— –
DNA was extracted from dried basidiomes using a procedure modified from Edwards et al (1991) and Weiss et al (1998), or using the DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, California). In the former, dried lamellar tissue was ground in an extraction buffer containing 200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS, and treated with 1 µg/µl ribonuclease A. DNA was precipitated with 1/10 volume of 3 M sodium acetate (pH 5.2) and two volumes of ethanol, then resuspended in 1x TE buffer (pH 8.0) or sterile ddH2O.
Preliminary experiments showed that crude extractions of Laccaria genomic DNA failed to PCR amplify, therefore, DNA extractions were purified further using a phenol: chloroform extraction following Sambrook et al (1989). An aliquot of the DNA solution was diluted 1 : 25 to 1 : 625 (determined empirically, depending on sample) with sterile ddH2O prior to PCR amplification.
PCR amplifications of the ribosomal DNA ITS1/5.8S/ITS2 region used the basidiomycete-specific primers ITS-1F and ITS-4B (Gardes and Bruns 1993) in 40 µL mixtures consisting of 8 µL template DNA (approx. 50–100 ng), 8 µL each of forward and reverse primers (20 ng /µl), 4 µL 10x PCR buffer (Fisher Buffer A containing 1.5 mM MgCl2, Fisher Scientific, Pittsburgh, Pennsylvania), 3.2 µL dNTP mixture (2.5 mM each dNTP; Promega Corp., Madison, Wisconsin), 0.3 µl Eppendorf MasterTaq DNA polymerase (Brinkmann Instruments, Westbury, New York), and 8.5 µL sterile ddH2O. Cycling parameters were as follows: 94 C for 2 min, followed by 30 cycles of 94 C for 30 sec, 55 C for 1 min, and 72 C for 1 min; followed by a final elongation step of 72 C for 5 min. PCR products were purified prior to sequencing using the QIAquick PCR purification kit (QIAGEN Inc., Valencia, California).
PCR products were direct sequenced on both strands using the primers ITS-1F or ITS-4B. DNA sequencing reactions were performed using ABI Prism BigDye Terminator chemistry and analyzed using an ABI PRISM 377 automated DNA sequencer (Applied Biosystems, Foster City, California). Initial multiple sequence alignments were performed using ClustalX software (Thompson et al 1997) and edited manually using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Ambiguously aligned sequence regions were unequivocally coded and step matrices were generated for each of these regions using INAASE 2.3b (Lutzoni et al 2000, Miadlikowska et al 2002, software available for download from http://www.lutzonilab.net). For unequally weighted parsimony analyses, symmetric step matrices were constructed for the unambiguously aligned regions using the program STMatrix 2.2 (F. Lutzoni and S. Zoller, Duke Univ.; software available at http://www.lutzonilab.net) as described by Miadlikowska et al (2002). Sequence data are available in GenBank (see TABLE II
for accession numbers), and the phylogenetic data matrix in TreeBase (study accession number S1355; matrix accession number M2395).
Phylogenetic analyses.— –
Phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford 2001), using maximum parsimony as the optimality criterion. Out-group taxa were Tricholoma unifactum (GenBank accession number AF241514
[GenBank]
) and Tricholoma portentosum (GenBank accession number AF349686
[GenBank]
). Outgroups were selected based on shared family classification (Family Tricholomataceae in Singer’s 1986 classification) and trophic type (ectomycorrhizal) with Laccaria. To clarify the identity of specimens within the L. bicolor complex and examine the relationship of the Rocky Mountain alpine collections to subalpine material, the analysis included the type specimens of L. trichodermophora G.M. Mueller, L. nobilis G.M. Mueller, L. amethysteooccidentalis G.M. Mueller, a designated "representative specimen" (Mueller 1992) of L. bicolor (Maire) Orton and non-alpine collections of L. bicolor, L. montana Singer, L. nobilis, L. proxima (Boudier) Patouillard, L. tortilis (Bolton) Cooke, and L. trichodermophora (TABLE II).
A primary analysis was performed on the full data set consisting of the unambiguously aligned positions and INAASE characters. All characters were specified as unordered and equally weighted, with gaps treated as missing data. A branch-and-bound search was performed using 1000 random-addition sequences with one tree held at each step, tree bisection-reconnection branch swapping, Multrees option enabled, and branches collapsed when maximum branch length equaled zero. Parsimony-uninformative characters were excluded. Branch confidence was assessed using 1000 bootstrap replicates with full heuristic searches, one random addition sequence per bootstrap replicate, and saving one tree per random addition sequence. In order to assess the effect on tree topology of weighting nucleotide changes and transitions between INAASE character states unevenly, two additional analyses were conducted. In the first, the unambiguously aligned regions were treated as equally weighted and the INAASE characters were treated as unequally weighted (using step matrices generated by INAASE). For the second, a weighted parsimony analysis was conducted using the full data matrix (unambiguously aligned regions + INAASE characters), using step matrices constructed in STMatrix 2.2 and INAASE 2.3b, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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). The strict consensus tree from this analysis shows several well-supported clades corresponding to morphological species described in the Taxonomy section: L. laccata var. pallidifolia (100% bootstrap support), L. nobilis (92%) and a previously undescribed taxon, described here as L. pseudomontana (99%). The two other species, L. montana and L. pumila, group together with moderate bootstrap support (65%), but relationships within this clade are unresolved. Due to this lack of phylogenetic resolution as well as the observed consistency in the number of spores per basidia (the primary character used to delimit the two species) within collections (Mueller 1992), L. montana and L. pumila are maintained as distinct taxa in this study. The two additional analyses yielded an identical consensus tree topology to that produced in the primary analyses (data not shown).
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KEY TO ROCKY MOUNTAIN ALPINE LACCARIA SPECIES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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Though not found in alpine habitats during the course of the present study, L. proxima (Boud.) Pat. is included in the key because this species was found in Beartooth subalpine areas in mixed EM plant communities that included Salix shrubs, and because its apparent affinity for disturbed sites and mine tailings makes it likely to be found in such sites above treeline.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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, 10, 21–22
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For complete descriptions of this species, see Mueller (1992) and Osmundson (2003). Diagnostic features for the Rocky Mountain alpine collections examined are described below.
Pileus (5–) 10–20 (–30) mm in diam, convex, shallow convex or nearly omphaloid, glabrous, occasionally lubricous, indistinctly translucent-striate or nonstriate; pale orange (5A8 to darker), hygrophanous, drying to paler orange (5B6); margin equal, uneven or crenulate, rimulose in age. Lamellae adnate to subdecurrent or rarely decurrent, broadly attached, adnate, subdistant, thick, pale orange or pink. Stipe 15–50 x 2–5 mm, equal to slightly enlarged toward base, generally appearing long and thin, straight to undulating, glabrous, minutely fibrillose, or finely longitudinally striate, occasionally tough, rubbery; pale orange. Basal tomentum white. Context white or pale orange.
Basidia four-spored. – Basidiospores (5–) 6.2–10 (–11) x (4–) 5.6–9.8 (–10.8) µm (mean = 6.8–8.8 x 6–8.8 µm), Q = 1–1.22 (–1.32) (Qm = 1–1.07 (–1.18), subglobose or broadly ellipsoidal, hyaline, echinulate; echinulae =1.5 (–2) µm in length, 0.4–1 µm wide at base.
Culture morphology.. Culture not obtained.
Rocky Mountain alpine habitat and distribution.. Solitary to scattered, occurring in alpine habitats in the Sawatch Range, 10-mile Range, Front Range and San Juan Mountains in Colorado. Associated with Dryas octopetala, Betula glandulosa, Salix reticulata, Salix glauca and in mixed habitats with D. octopetala + unidentified dwarf Salix and Betula glandulosa + Salix cf. reticulata.
Specimens examined.. U.S.A. COLORADO. Pitkin/Lake Co.: Sawatch Range, Independence Pass, 14 Aug 1999, CLC 1370 (MONT); Summit Co.: 10-mile Range, Blue Lake Dam, near Breckenridge, 2 Aug 2001, CLC 1603 (MONT); 3 Aug 2001, CLC 1633 (MONT); San Juan Co.: San Juan Mountains, Horseshoe Lake, 6 Aug 2001, CLC 1655 (MONT); Gunnison/Chaffee Co.: San Juan Mountains, Cottonwood Pass, 12 Aug 2001, CLC 1724 (MONT).
Comments..
Laccaria laccata var. pallidifolia exhibits a wide range of phenotypic variability (Mueller 1992), and can therefore be difficult to distinguish from several other Laccaria species (see comments under other species descriptions). As such, reports of this species in the literature must be viewed with caution. Characters observed in the Rocky Mountain alpine collections were largely concordant with those described by Mueller (1992), with two minor differences. While the range of mean basidiospore length and width observed in the Rocky Mountain alpine L. laccata var. pallidifolia collections overlaps that noted for the species by Mueller (1992), the range for Rocky Mountain alpine collections has a lower minimum size. Cheilocystidia, reported as absent or scattered to abundant by Mueller (1992), were absent or rare in the Rocky Mountain alpine material. Examination of the reference collection TENN 43090 (TABLE II) showed micromorphological features corresponding to those noted for the Rocky Mountain alpine collections.
Originally described from New York but widely reported from arctic-alpine habitats, L. laccata var. pallidifolia was collected in the Colorado field sites but not on the Beartooth Plateau during the course of this study. Like alpine L. nobilis, L. laccata var. pallidifolia is associated with dwarf willows; however, L. laccata var. pallidifolia occurs with the additional hosts Dryas octopetala and Betula glandulosa, and was collected once with shrub Salix species. Laccaria laccata var. pallidifolia is a cosmopolitan, commonly collected species (Mueller 1992). Rocky Mountain subalpine collections are reported from Colorado and Idaho, and host plant families are reported as Pinaceae, Fagaceae and Betulaceae (Mueller 1992).
Laccaria montana Singer, 1973. Sydowia 7 : 89. FIGS. 5, 11, 23–25
Type: SWITZERLAND: Valais, Borgne de Ferpecle, 1955–1960 m altitude, 11 Jul l971, Singer M5464 (F).
For complete descriptions of this species, see Mueller (1992) and Osmundson (2003). Diagnostic features for the Rocky Mountain alpine collections examined are described below.
Pileus 0.5–2.5 (–3.5) cm in diam, convex becoming plane or occasionally uplifted, with shallow central depression or rarely a low broad umbo, glabrous or minutely scaly, especially when dry; translucent-striate to plicate-sulcate; orange brown (6D7 to 6B5-6C6), red brown or brick red (7D6-7E8), mildly to strongly hygrophanous, drying to pale orange buff (5C6); margin involute to decurved, often uplifted-undulating in age, entire or occasionally crenate, occasionally splitting in age. Lamellae adnate or rarely short decurrent, moderately thick, broad (occasionally narrow or ventricose), subdistant, greyish orange (5B5–7B5) or pinkish orange-brown (6C5–6C7). Stipe 10–28 (–47) x (1–) 2–4 mm, equal, solid becoming hollow in age, glabrous or minutely fibrillose; concolorous with pileus, brownish-orange or red-brown (5C5–7D6). Basal tomentum white, scant to moderately dense. Context thin, white to pale orange-brown.
Basidia four-spored. – Basidiospores 8–11 (–12) x (7–) 8–9.5 (–10.5) µm (mean = 9–10 x 8.6–9 µm), Q = 1–1.29 (Qm = 1.04–1.16), subglobose to broadly ellipsoidal or occasionally globose, hyaline, echinulate; echinulae =1.8 (–2.5) µm in length, 0.4–1 (–1.4) µm wide at base.
Culture morphology.. Culture not obtained.
Rocky Mountain alpine habitat and distribution.. Scattered to gregarious, rarely solitary; usually among mosses; occurring in alpine habitats on the Beartooth Plateau. Only one collection observed from alpine field sites in Colorado, collected in the San Juan Mountains. Associated primarily with the shrub willows Salix planifolia and S. glauca; also found in association with the dwarf willow Salix arctica in Wyoming.
Specimens examined.. USA. COLORADO. Jackson Co.: Colorado State Forest, Cameron Pass, 15 Sep 1981, TENN 42880 (TENN); Larimer Co.: Roosevelt National Forest, Blue Lake Trail, 13 Sep 1981, TENN 42877 (TENN); San Juan Co.: San Juan Mountains, Mineral Basin, 30 Jul 2002, CLC 1853 (MONT); Summit Co.: Front Range, Loveland Pass Lake, 20 Aug 1999, DBGH 20424 (DBGH). MONTANA. Carbon Co.: Beartooth Plateau, near source of Quad Creek, 10 Aug 1999, TWO 264 (MONT); 28 Jul 2001, TWO 441 (MONT); Carbon Co. at Wyoming State Line, Beartooth Plateau, Highline Trailhead, 1 Aug 2000, TWO 319 (MONT); 14 Jul 2001, TWO 369 (MONT); 3 Aug 2001, TWO 504 (MONT); 3 Aug 2001, TWO 505 (MONT); 16 Aug 2001, TWO 540 (MONT); 19 Aug 2001, TWO 559 (MONT); 19 Aug 2001, TWO 561 (MONT); 18 Jul 2002, TWO 613 (MONT); 15 Aug 2002, TWO 710 (MONT). WYOMING. Park Co.: Beartooth Plateau, north of Frozen Lake, 31 Jul 2001, TWO 477 (MONT); 4 Aug 2001, TWO 512 (MONT); 17 Aug 2001, TWO 553 (MONT); 1 Sep 2001, TWO 591 (MONT).
Comments..
Characters observed in Rocky Mountain L. montana collections were largely concordant with those described by Mueller (1992), with two minor differences. While the range of mean basidiospore length and width observed in the Rocky Mountain collections overlaps that noted for the species by Mueller (1992), the range for the Rocky Mountain collections has a lower maximum size. Cheilocystidia, reported as absent except in one collection by Mueller (1992), were absent in some of the Rocky Mountain alpine collections while scattered to abundant in others. Examination of the reference collection TENN 42880 (TABLE II) showed micromorphological features corresponding to those of the Rocky Mountain alpine collections.
Laccaria montana appears similar in the field to L. pumila, L. tortilis and small, striate forms of L. laccata var. pallidifolia (Mueller 1992), as well as to the newly described taxon L. pseudomontana (see comments under that species). Laccaria laccata var. pallidifolia differs by having smaller, globose to subglobose basidiospores. Laccaria tortilis and L. pumila differ by having two-spored basidia (see additional comments under L. pumila). Mueller (1992) states that at least some previous arctic reports of L. tetraspora (e.g. Kobayasi et al 1967, Miller et al 1982) are L. montana. Lahaie (1981) reports L. montana to have been collected near Betula nana and Arctostaphylos alpina from an arctic site on the Tuktoyaktuk Peninsula, Northwest Territories, Canada. Laccaria montana is reported in Rocky Mountain subalpine habitats in Colorado, Idaho, Montana and Wyoming, in association with Betula spp., Salix spp. and species in the Pinaceae.
Laccaria nobilis Smith apud G.M. Mueller. Mycotaxon 20: 105–108, 1984. FIGS. 3, 9, 18–20
Type: USA: Colorado, Larimer Co., Roosevelt National Forest, Rayah Wilderness, Blue Lake Trail, 13 September 1981 G.M. Mueller 1198 (TENN 42527) (TENN).
For a complete description of this species, see Mueller (1992). Diagnostic features for the Rocky Mountain alpine collections examined are described below.
Pileus (10–) 20–50 (–70) mm in diam, convex, occasionally nearly plane with shallow central depression in age; nearly glabrous when young, becoming minutely scaly with concentrically-arranged fine scales in age, not striate; pale pinkish orange or dark orange, occasionally darker at margin, hygrophanous, drying to pale orange; margin involute becoming decurved to uplifted, entire or undulating slightly, occasionally rimulose in age. Lamellae adnate to adnexed, broad, moderately thick, subdistant, pink. Stipe 20–50 x 3–8 mm, equal or more frequently gradually basally enlarged to clavate, robust, tough, solid becoming hollow at least in some collections; surface longitudinally striate, rough-fibrous; concolorous with or paler than pileus, pale whitish orange or pale orange-brown, apex pink in some young specimens. Basal tomentum generally white under field conditions, but noted as violet in one collection. Context white, pale orange, pinkish-white or pale violet.
Basidia four-spored.
Basidiospores 5.4–9.5 x (4–) 5–7.8 (–8.6) µm (mean = 6.2–8 x 5.5–7 µm), Q = 1–1.32 (Qm = 1.06–1.15), subglobose or broadly ellipsoidal, hyaline, echinulate; echinulae =1.5 (–2.5) µm in length, (0.2–) 0.4–1.2 µm wide at base. Cheilocystidia filiform, cylindrical to irregular, hyaline, 29–44 x 1–4 µm; absent to abundant.
Culture morphology.. Dikaryotic mycelia on PDA and MMN moderately fast-growing; pale violet becoming bright violet then fading to red-brown, pale violet or nearly white.
Rocky Mountain alpine habitat and distribution.. Solitary to scattered, occurring in alpine habitats in the Front Range, Sawatch Range and San Juan Mountains in Colorado. Associated with dwarf willows including Salix arctica and S. reticulata and shrub willows including Salix planifolia and S. glauca.
Specimens examined.. USA. COLORADO: Summit/Clear Creek Co.: Front Range, Loveland Pass, 7 Aug 1999, CLC 1304 (MONT); San Juan Co.: San Juan Mountains, Black Bear Basin, 3 Aug 2000, CLC 1445 (MONT); 3 Aug 2000, ZT 9048 (ZT); Mineral Basin, 7 Aug 2001, CLC 1672 (MONT); Stony Pass, 28 Jul 2002, CLC 1825 (MONT); Horseshoe Lake, 6 Aug 2001, CLC 1656 (MONT); Cinnamon Pass, 10 Aug 2001, CLC 1709 (MONT); Pitkin/Chaffee Co.: Sawatch Range, Independence Pass, 10 Aug 1999, ZT 7478 (ZT); 11 Aug 1999, CLC 1347 (MONT); 13 Aug 1999, CLC 1365 (MONT); 14 Aug 1999, ZT 7472 (ZT); 6 Aug 2000, CLC 1469 (MONT); 13 Aug 2001, CLC 1742 (MONT); Pitkin Co.: Sawatch Range, Linkins Lake Valley, 8 Aug 2000, CLC 1482 (MONT).
Comments..
Laccaria nobilis is distinguished by having medium to large-sized, robust, minutely scaly basidiomata, relatively small, subglobose or broadly ellipsoidal basidiospores produced on four-spored basidia, and violet mycelial growth on PDA and MMN media. The ranges of mean basidiospore length and width observed in the Rocky Mountain alpine L. nobilis collections overlap those noted for the species by Mueller (1992), but with lower minima and maxima. Cheilocystidia, noted as absent for the species (Mueller 1992), ranged from absent (in most collections) to abundant in the alpine collections. The presence of a violet basal tomentum was noted in only one collection (CLC 1482); however, molecular analyses indicated that this collection was closely related to otherwise morphologically similar collections that lacked violet coloration, including the collection CLC 1469 that exhibited no ITS sequence divergence from CLC 1482. These findings indicate that violet pigmentation in the basal tomentum, while useful for identification, may be rare under field conditions in the Rocky Mountain alpine zone and that the lack of this character in collections of the L. bicolor species complex has likely led to their inclusion in some cases under L. laccata var. pallidifolia (e.g., Lahaie 1981). Laccaria nobilis can be distinguished by production of violet mycelial mats on PDA and MMN media, underscoring the value of attempting to obtain tissue cultures from field specimens to facilitate identification. Storing fresh basidiomes of subalpine L. bicolor that originally lacked violet basal mycelia in a covered plastic container under refrigeration has resulted in new growth of violet mycelia at the base of the stipe (Osmundson unpubl); this method may represent a simpler alternative to obtaining tissue cultures of members of this species complex for observing mycelial coloration.
Laccaria nobilis and L. laccata var. pallidifolia are the two taxa with medium-sized to large-sized basidiomes collected at alpine field sites during the course of this study. Both have four-spored basidia and globose to subglobose basidiospores; L. laccata var. pallidifolia generally, though not always, has larger basidiospores. Because of these overlaps in basidiospore size and shape, L. nobilis can be difficult to distinguish from L. laccata var. pallidifolia in the absence of a violet basal tomentum and/or mycelial culture. However, in the present study, basidiome stature and surface texture were found to be useful characters for distinguishing the two species: L. nobilis basidiomes were consistently more robust than L. laccata var. pallidifolia, with minutely scaly pilei and basally enlarged to nearly clavate, rough fibrous-striate stipes. Distinctions based on these characters were supported by molecular data. Laccaria nobilis was collected at alpine field sites in Colorado, but was not found on the Beartooth Plateau.
Described previously only from the western and Great Lakes regions of North America, L. nobilis is here reported for the first time from arctic-alpine habitats and for the first time in occurrence with non-Pinaceae hosts.
This phylogenetic analysis of rDNA ITS sequence data provides additional support for distinguishing three species within the L. bicolor complex (L. bicolor, L. nobilis and L. trichodermophora), previously supported by mating studies, morphological differences, and RFLP patterns (Mueller 1992, Mueller and Gardes 1991). In this analysis, the Rocky Mountain alpine collections formed a well-supported clade with the type specimen of L. nobilis, which was collected from a subalpine locality in Colorado. An additional Rocky Mountain subalpine collection (TWO 752) included in this analysis formed a well-supported clade with the designated Representative Specimen of L. bicolor.
Laccaria pseudomontana Osmundson, C. Cripps, et G.M. Muell., sp. nov. FIGS. 4, 12, 16–17
Species insignis basidiomeis parvis et atroaurantiobrunneis vel rubrobrunneis, basidiis tetrasporibus, basidiosporis late ellipsoideis et subtiliter echinulatis. Habitu Laccaria montana Singer similis, differt basidiosporis leviter plus ellipsoideis, spinis brevibus et angustis, basidiomeis saepe minoribus.
HOLOTYPUS: USA. COLORADO. Summit Co.: 10-mile Range, Blue Lake Dam, near Breckenridge, 23 Aug 2001, C.L. Cripps 1625 (MONT).
Pileus 4–10 (–18) mm in diam, convex to plane, glabrous, slightly striate; dark orange or red-brown, hygrophanous; margin decurved, entire or crenate. Lamellae adnate or subdecurrent, thick, subdistant, pink; edges entire; lamellulae present. Stipe 10–15 x 1–2 mm, equal, glabrous or fibrous-striate, dark orange-brown or red-brown. Context thin, white.
Pileipellis of interwoven, inamyloid, cylindrical, mostly repent hyphae with widely scattered fascicles of hyphae oriented nearly perpendicular to pileal surface. Hyphae hyaline or having intracellular pigment appearing pale orange-brown in 3% KOH. Stipitipellis of parallel, cylindrical, repent, inamyloid, hyaline hyphae. Caulocystidia absent. Lamellar trama of subparallel to interwoven, hyaline, inamyloid hyphae. Subhymenium undifferentiated. Clamp connections present in all tissues. Pleurocystidia absent. Cheilocystidia absent. Basidia clavate, hyaline, 32.5–37.5 x 10–12.5 µm, four-spored; sterigmata =8 µm in length. Basidiospores (6.5–) 7.5–10.8 x 6.5–9.5 µm (mean = 8–9.8 x 6.8–8.4 µm), Q = 1.04–1.39 (Qm = 1.16–1.18), subglobose or broadly ellipsoidal, hyaline, echinulate; echinulae =1 (–1.8) µm in length, 0.4–0.6 µm wide at base.
Culture morphology.. Culture not obtained.
Rocky Mountain alpine habitat and distribution.. Scattered, usually among mosses; collected in alpine habitats in the 10-mile Range and San Juan Mountains in Colorado. Associated with Salix glauca, an unidentified shrub Salix sp., and in a mixed stand of Salix planifolia and Betula glandulosa. Not reported from the Beartooth Plateau.
Material examined.. USA. COLORADO. Summit Co.: 10-mile Range, Blue Lake Dam, near Breckenridge, 23 Aug 2001, CLC 1625 (HOLOTYPE; MONT); Pitkin/Lake Co.: San Juan Mountains, Independence Pass, 15 Aug 2001, CLC 1771 (MONT); San Juan Co.: San Juan Mountains, U.S. Basin, 8 Aug 2001, CLC 1682 (MONT).
Comments.. Laccaria pseudomontana is distinguished by having small, dark orange or red-brown basidiomes, four-spored basidia and moderately sized, broadly ellipsoidal, finely echinulate basidiospores. Laccaria pseudomontana closely resembles L. montana in both macro- and micromorphology; however, Laccaria pseudomontana often has smaller basidiomes than L. montana and has basidiospores with shorter and narrower echinulae and a slightly more ellipsoidal shape than does L. montana. Although two of the L. pseudomontana collections examined had smaller (0.5–1.5 cm), red-brown basidiomes, the third collection had a basidiome morphology indistinguishable from that of typical L. montana collections and was distinguishable from L. montana only by basidiospore characters. Though difficult to distinguish from L. montana, collections of L. pseudomontana formed a distinct, well-supported clade in phylogenetic analyses of rDNA-ITS sequence data.
The present study included one alpine and two subalpine herbarium collections previously identified as L. montana and collected in Colorado. The two subalpine collections were included in Mueller’s (1992) monographic study and were used as nomenclatural reference specimens to represent L. montana for the present study. One of these subalpine collections (TENN 42877) corresponds to Beartooth Plateau L. montana collections in terms of basidio-spore shape and echinulae dimensions. Basidiospores of the other subalpine reference collection (TENN 42880) and the alpine reference collection (DBG 20424) appear more similar to L. pseudomontana than to the Beartooth Plateau L. montana collections. Examination of the holotype of L. montana (Singer M5464, F) by Mueller (1992) showed basidiospores having a globose to broadly ellipsoidal shape (Q = 1–1.11 [–1.26]) and uncrowded, 1.5–1.8 mm long echinulae, characters that correspond to those of the Beartooth Plateau L. montana collections. Further morphological and molecular analyses including collections of L. montana sensu lato will be necessary to determine the distribution and abundance of L. pseudomontana.
Laccaria pseudomontana can be distinguished from small L. laccata var. pallidifolia basidiomes by having more ellipsoidal (higher average length to width ratio) basidiospores with shorter, narrower echinulae and often by having a darker basidiome coloration, and from L. pumila by having four-spored basidia and smaller basidiospores with shorter, narrower echinulae.
Laccaria pseudomontana is associated with shrub willows (Salix spp.) in Colorado and is not reported from the Beartooth Plateau.
Laccaria pumila Fayod, Annali Accad. Agric. Torino 35 : 91, 1893. FIGS. 7, 8, 13–15
Type: FRANCE: dept. Alpes maritimes, Col de la Cayolle, 2500 m altitude, 18 Jul 1976, J. Trimbach 1463 (L, neotype fide Mueller and Vellinga, 1986).
For complete descriptions of this species, see Mueller (1992) and Osmundson (2003). Diagnostic features for the Rocky Mountain alpine collections examined are described below.
Pileus 5–15 (–35) mm in diam, convex to plane or nearly omphaloid, often with slight central depression, usually translucent-striate, glabrous or minutely fibrillose, occasionally lubricous; pale orange-brown (6D8) or nearly red-brown, strongly hygrophanous; margin decurved, often becoming uplifted in age, entire, irregular or slightly eroded. Lamellae adnate to short decurrent, narrow or moderately broad, moderately thick or thick, subdistant, greyish-orange or pinkish orange (6B5–6B6), occasionally forked. Stipe (6–) 15–35 (–50) x 1–4 mm, equal, solid, often tough; glabrous or minutely fibrillose, pale pinkish brown, pinkish orange or dark orange-red (6D7); base of stipe often opaque, whitish. Basal tomentum white, moderately dense to lacking entirely. Context thin, white to pale orange.
Basidia two-spored.
Basidiospores (8–) 9–13.5 (–15) x (6.8–) 7.5–10.5 (–14.5) µm (mean = 9.5–11.4 x 8–9.5 (–10.5) µm), Q = 1–1.3 (Qm = 1.08–1.18), subglobose to broadly ellipsoidal or occasionally globose, hyaline, echinulate; echinulae =1.5 (–2) µm in length, 0.4–1 (–1.2) µm wide at base.
Culture morphology.. Dikaryotic mycelia on PDA and MMN slow-growing; white.
Rocky Mountain alpine habitat and distribution.. Solitary to scattered or gregarious, usually among mosses; occurring in alpine habitats in the Sawatch Range and San Juan Mountains in Colorado and the Beartooth Plateau in Montana/Wyoming. Primarily associated with dwarf willows in Colorado, and with the shrub willows Salix planifolia and S. glauca in Montana and Wyoming.
Specimens examined.. USA. COLORADO. Summit/Clear Creek Co.: Front Range, Loveland Pass, 7 Aug 1999, ZT 8068 (ZT); San Juan County: San Juan Mountains, Black Bear Basin, 3 Aug 2000, CLC 1446 (MONT); 3 Aug 2000, ZT 9049 (ZT); Cinnamon Pass, 1 Aug 2000, CLC 1435 (MONT); 10 Aug 2001, CLC 1699 (MONT); Mineral Basin, 30 Jul 2002, CLC 1850 (MONT); CLC 1851 (MONT); Stony Pass, 28 Jul 2002, CLC 1819 (MONT); Emma Lake, 31 Jul 2002, CLC 1872 (MONT); Ouray Co.: San Juan Mountains, Imogene Pass, 29 Jul 2002, CLC 1835 (MONT); 29 Jul 2002, CLC 1837 (MONT); Lake Co.: Sawatch Range, Haggeman’s Pass, 14 Aug 1998, CLC 1252 (MONT); Pitkin/Lake Co.: San Juan Mountains, Independence Pass, 8 Aug 2000, ZT 9083 (ZT). MONTANA. Carbon Co.: Beartooth Plateau, near source of Quad Creek, 10 Aug 1999, TWO 265 (MONT); 10 Aug 1999, TWO 268 (MONT); 31 Jul 2000, TWO 314 (MONT); 21 Aug 2000, TWO 335 (MONT); 21 Aug 2000, TWO 337 (MONT); 21 Aug 2000, TWO 348 (MONT); 28 Jul 2001, TWO 442 (MONT); 30 Jul 2001, TWO 465 (MONT); 5 Aug, 2001, TWO 520 (MONT); 27 Aug 2002, TWO 716 (MONT); 27 Aug 2002, TWO 717 (MONT); 27 Aug 2002, TWO 718 (MONT); At Wyoming state line, Beartooth Plateau, Highline Trailhead, 30 Jul 1997, ZT 6232 (ZT); 7 Aug 1998, CLC 1201 (MONT); 19 Aug 2001, TWO 560 (MONT); 19 Aug 2001, TWO 562 (MONT); 15 Aug 2002, TWO 709 (MONT); 28 Aug 2002, TWO 726 (MONT); 28 Aug 2002, TWO 730 (MONT); Clark Fork Picnic Area (subalpine, with Salix shrubs and possibly conifers), 12 Jul 2001, TWO 362 (MONT); 19 Jul 2001, TWO 374 (MONT); McLaren mine tailings (subalpine, with conifers and Salix shrubs), 21 Jul 2001, TWO 411 (MONT). WYOMING. Park Co.: Beartooth Plateau, north of Frozen Lake, 29 Jul 1997, CLC 1104 (MONT); 21 Aug 1999, CLC 1404 (MONT); 3 Aug 2001, TWO 501 (MONT); 21 Aug 2001, CLC 1777 (MONT); 1 Sep 2001, TWO 589 (MONT); North of Gardner Headwall, 31 Jul 2002, TWO 663 (MONT); Beartooth Highway, near Top of the World Store (subalpine, with Salix shrubs), 21 Jul 2001, TWO 409 (MONT).
Comments..
Characters observed in Rocky Mountain L. pumila collections were largely concordant with those described by Mueller (1992). The ranges of mean basidiospore length and width observed in the Rocky Mountain alpine collections overlap those noted for the species by Mueller (1992), but with lower minimum and maximum sizes. Examination of the reference collection TENN 42553 (TABLE II) showed micromorphological features corresponding to those of the Rocky Mountain alpine collections examined. Laccaria pumila appears to be indistinguishable from L. montana on the basis of macromorphological characteristics alone in Rocky Mountain alpine habitats; however, the two taxa are easily distinguished micromorphologically, with L. pumila having two-spored basidia and slightly larger basidiospores. Biological differences between two-spored and four-spored species may be ecologically relevant: Tommerup et al (1991) suggest that secondary homothallism (i.e. production of single basidiospores containing both mating type nuclei required for formation of a dikaryotic mycelium), as demonstrated in the two-spored species Laccaria fraterna, may represent an advantage in primary successional or disturbed sites. L. pumila and L. montana are distributed sympatrically on the Beartooth Plateau.
Laccaria pumila is reported by Mueller (1992) as appearing similar to L. tortilis and to small, striate forms of L. laccata var. pallidifolia. Laccaria pumila can be distinguished from L. laccata var. pallidifolia by having larger, more broadly ellipsoidal basidiospores and two-spored basidia, and from L. tortilis (Bolton) Cooke by having fewer globose basidiospores with shorter, narrower echinulae. Laccaria tortilis has been reported from subalpine habitats in Colorado, Montana, Oregon and Washington (O.K. Miller Jr. unpubl, Mueller 1992), but was not observed in alpine habitats during the present study. Mueller (1992) states that previous arctic records of L. tortilis (Kobayasi et al. 1967, Lange 1955) are most likely L. pumila and that L. altaica, commonly reported from arctic-alpine habitats, is a synonym of L. pumila; Sivertsen (1993), however, considers L. pumila and L. altaica to be distinct species differing in basidiospore shape and echinulae density. Examination of Kobayashi et al.’s (1967) basidiospore illustrations confirms Mueller’s identification of this taxon as L. pumila. In addition to occurring in alpine habitats, L. pumila was found in subalpine habitats in Montana in association with Salix spp. and Populus tremuloides (Cripps and Osmundson unpublished). Mueller (1992) reports collections from Rocky Mountain subalpine habitats in Wyoming and Colorado, and reports L. pumila to be associated with Salix spp, Betula spp., and species in the Pinaceae.
DISCUSSION – The present study represents the first report on North American alpine Laccaria species. Five species, one previously undescribed and another reported for the first time from arctic-alpine habitats, are reported from the Rocky Mountain alpine zone: L. laccata var. pallidifolia, L. montana, L. nobilis, L. pumila and L. pseudomontana. Phylogenetic analyses of rDNA ITS sequences revealed clades corresponding to circumscribed morphological species and aided in distinguishing morphological traits useful for reliably identifying Laccaria species in the Rocky Mountain alpine zone.
The five taxa described here can be distinguished on the basis of macromorphological and/or micro-morphological characters. Laccaria pumila, L. montana and L. pseudomontana are distinguished by having small basidiomes, often with translucent-striate pilei, and having relatively large, subglobose to broadly ellipsoidal basidiospores. In the phylogenetic analysis of rDNA ITS sequence data, L. montana and L. pumila form a single, unresolved clade in the strict consensus tree, with only moderate bootstrap support. Due to this lack of phylogenetic resolution as well as the observed consistency in the number of spores per basidia (the primary character used to delimit the two species) within collections (Mueller 1992), L. montana and L. pumila are maintained as distinct taxa in this study; additional collections and wider geographic sampling are necessary for more clearly establishing the relationship between these two taxa. An additional taxon, described here as Laccaria pseudomontana and having small, dark orange to red-brown basidiomata, was collected in Colorado alpine habitats. Originally thought to represent a small form of L. laccata var. pallidifolia or L. montana, these specimens formed a distinct, well-supported clade in the phylogenetic analyses.
Laccaria nobilis and L. laccata var. pallidifolia in the Rocky Mountain alpine zone are characterized by producing larger basidiomes (longer stipes and generally broader pilei) than the other species described in the present study. Though an important diagnostic character-violaceous lamellae and violet tomentum at the base of the stipe when young and fresh (Mueller 1992)-was generally lacking in the Rocky Mountain collections, the present study describes other macromorphological and basidiospore characters that distinguish the two species in the North American alpine zone.
Laccaria laccata var. pallidifolia is supported in this analysis as a distinct but morphologically variable taxon including both convex and nearly omphaloid pileal forms and exhibiting a relatively wide range in basidiospore size. This species is highly divergent at the molecular level compared to the other species studied, and is supported by numerous synapomorphic single nucleotide polymorphisms in the ITS region.
In terms of EM associations, Laccaria nobilis occurs with Salix planifolia and Salix glauca (both shrub species) and S. arctica and S. reticulata (both dwarf species). Laccaria laccata var. pallidifolia is associated with Dryas octopetala, Betula glandulosa and Salix reticulata, and was collected only once near a shrub Salix species (S. glauca). Laccaria pumila is associated with both dwarf and shrub Salix species. Laccaria montana is associated predominantly with the shrub willows S. planifolia and S. glauca. Laccaria pseudomontana is associated with Salix shrubs, and was encountered in a mixed stand of Salix shrubs and Betula glandulosa. The three smaller-statured species are nearly always encountered in moss-covered areas in proximity to the EM host plant. The occurrence of sympatric populations of L. pumila and L. montana on the Beartooth Plateau allows the opportunity to observe differences in small-scale distributional and ecological patterns between the two species. Although L. pumila is associated primarily with dwarf Salix species and L. montana primarily with shrub Salix species, the observation that L. pumila is occasionally found with Salix shrubs suggests that the two species are not spatially isolated on the basis of host specificity alone.
Laccaria nobilis, reported in the present study in association with Salix spp., occurs in association with conifer species in subalpine habitats. Host association data therefore suggest that a mycorrhizal host shift has occurred in arctic-alpine populations of this taxon. A similar host shift from gymnosperm (Pinus) to angiosperm (Quercus costaricensis) hosts has been documented for the closely related species L. trichodermophora (Mueller and Strack 1992).
Compared to many arctic-alpine macromycetes, Laccaria species generally appear to be widely distributed and not strictly limited to arctic-alpine habitats. The species found in the present study are reported in the literature to occur in other arctic-alpine localities, with the exception of the newly described taxon L. pseudomontana, which may be found to be more widely distributed after additional collections previously identified as Laccaria montana have been examined, and L. nobilis, which has been previously described only from western North America and the Great Lakes region. This result indicates little or no endemicity in Rocky Mountain alpine Laccaria, and is concordant with the observation that none of the EM host plants associated with Laccaria spp. in this region are endemic to the Rocky Mountain alpine zone. However, smaller-scale differences in distributions were observed between taxa over the geographic area encompassing the central and southern Rocky Mountain alpine zone: The two taxa characterized by more robust basidiomes (L. laccata var. pallidifolia and L. nobilis) and the smaller L. pseudomontana are reported only from the Colorado field sites, Laccaria pumila occurs commonly in both Colorado and Montana/Wyoming, and Laccaria montana is regularly collected on the Beartooth Plateau, but was only collected once in Colorado during the course of this study (FIG. 26). A number of factors, including EM host specificity, climate and soil conditions, ecological community setting and geologic, evolutionary or dispersal events, might contribute to the observed distributions; however, more extensive sampling might reveal fewer differences between regions than were observed in this study.
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, Jumpponen et al 2002) suggests a possible use in reclamation of high altitude habitats; a better understanding of species limits, geographic distributions, host associations and ecological roles of alpine species can provide a foundation for the development of plant-fungal systems for such use. The wide distribution of the genus in arctic-alpine habitats highlights the potential use of Laccaria as a model genus for evolutionary studies in arctic-alpine mycorrhizal macromycetes; a robust taxonomic classification of arctic-alpine taxa is a necessary foundation for such studies. In general, host specificity, successional patterns and species-specific ecological interactions are poorly understood for EM fungi in arctic-alpine habitats, and these subjects offer important avenues for both observational and experimental studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Current address: Institute of Systematic Botany and the Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, New York Botanical Garden, Bronx, NY 10458 and Department of Ecology, Evolution and Environmental Biology, Columbia University, New York, NY 10027. E-mail: tosmundson{at}nybg.org
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO ROCKY MOUNTAIN...TAXONOMYLITERATURE CITED |
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