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Instituto Multidisciplinario de Biología Vegetal (CONICET), C.C. 495, 5000, Córdoba, Argentina
James M. Trappe
Department of Forest Science, Oregon State University, Corvallis, Oregon 97331-5752
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSTAXONOMYRESULTSDISCUSSION AND CONCLUSIONSLITERATURE CITED |
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Field studies in Argentina’s Yunga District revealed Alpova austroalnicola sp. nov., a hypogeous fungus associated with Alnus acuminata ssp. acuminata. Morphological and molecular studies based on amplification and sequencing of the nuclear LSU rDNA gene showed its unique identity within Alpova. Related genera included in the analyses were Boletus edulis, Rhizopogon spp., Suillus luteus and Truncocolumella citrina. Additional observations of animal diggings around the sites and microscopic examination of fecal pellets of the nine-banded armadillo (Dasypus novemcinctus novemcinctus) indicate A. austroalnicola is consumed and its spores dispersed by animals.
Key words: Alnus acuminata, Boletales, Dasypus, molecular systematics, mycophagy, phylogeny
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSTAXONOMYRESULTSDISCUSSION AND CONCLUSIONSLITERATURE CITED |
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. Trappe (1975) discovered it had been described earlier as Rhizopogon diplophloeus Zeller & C.W. Dodge and recombined it as A. diplophloeus (Zeller & C.W. Dodge) Trappe & A.H. Sm. The species is characterized by its hypogeous to sometimes emergent habit; strict association with Alnus spp; solid, gelatinous gleba that darkens when exposed to air; smooth, thin-walled, small, ellipsoid to oblong, hyaline to pale brown spores; presence of clamp connections; lack of a hymenial palisade; and a layer of large, inflated cells in the peridium. Trappe (1975) broadened the concept of Alpova to include taxa with similar macro-morphology that were related to the genus Rhizopogon. Beaton et al (1985) broadened it even further by placing several new Australian Eucalyptus-associated species in Alpova.
Molecular data now have demonstrated that the morphological criteria formerly considered important in defining genera of hypogeous fungi can be inadequate for that purpose. Grubisha et al (2001) have shown that Alpova diplophloeus relates to the Boletaceae, whereas the Rhizopogon-related species placed by Trappe (1975) in Alpova subgen Alpova sec Rhizopogonella belongs in the "suilloid radiation," in the Rhizopogonaceae. Bougher and Lebel (2002) transferred the Australian species earlier assigned to Alpova to their new genus Amarrendia. Accordingly, the concept of Alpova must revert to the strict sense of its original description. This concept would include two, perhaps four, of the morphologically similar species in Trappe’s (1975) Alpova subgen. Alpova sec. Alpova: A. diplophloeus, A. nauseosus (Coker and Couch) Trappe, possibly A. mollis (Lloyd) Trappe and A. trappei. However A. mollis is known only from the type collection and A. trappei differs strongly from the others in its peridial structure; neither is known to be associated with Alnus spp. Grubisha et al (2001) suggested that Alpova might not be mono-phyletic but recognized the need for new studies including more taxa.
A new species of Alpova recently was collected in the Yunga District of Argentina in an Alnus acuminata Kunth ssp. acuminata forest. The distribution of A. acuminata ssp. acuminata ranges from Venezuela to the Andes in Argentina (Furlow 1979, Cabrera and Willink 1980, Aceñolaza 1995). Molecular phylogenetic analysis can be useful for testing whether species with disjunctive ranges are within the same lineage (Koufopanou et al 1977). This is particularly important when morphological characters are few or apparently have converged enough that it is difficult to separate similar taxa (Rizzo et al 2003). The nuc-LSU-rDNA gene has been used previously to investigate phylogenetic relationships, particularly in the Boletales, providing suitable resolution for identifying lineages of fungi with good support for terminal branches (Bridge 2002, Grubisha et al 2001, Humpert et al 2001, Moncalvo et al 2000, Wang et al 2002).
We here describe Alpova austroalnicola based on its unique morphological characters and molecular data obtained from nuc-LSU-rDNA gene analysis. We also obtained data on its use as food and spore dispersal by the Argentine nine-banded armadillo, Dasypus novemcinctus novemcinctus, known by the common name of mulita grande or armadillo de nueve bandas. This subspecies inhabits northern Argentina.
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METHODS |
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Sporocarps were photographed with a Leica M420 stereo microscope. Color reactions and microscopic characters were determined from hand-sectioned mounts in 15% KOH, Melzer’s reagent, cotton blue and FeSO4 and photographed with a Zeiss Axiophot light microscope. Voucher specimens were deposited in the Museo Botánico de Córdoba Herbarium (CORD).
Fecal pellet analysis.— –
Armadillo fecal pellets were collected near animal diggings on the sites where Alpova austroalnicola sp nov. was sampled. Twelve samples of collected pellets were analyzed microscopically (25 fields randomly selected per sample at 600x). Fungal elements (spores and hyphae), plant, animal and mineral material were counted following procedures of McIntire and Carey (1989).
Molecular and phylogenetic analysis.— – A small amount of sporocarp tissue was ground with a drill-driven plastic pestle in an Eppendorf tube containing 200 µL of 2x CTAB lysis buffer. Additional buffer was added up to 500 µL and mixed; the tubes were frozen and thawed twice, alternating between dry ice and a 65 C water bath. The tubes were incubated in the bath 30–60 min. Chloroform was added to the mixture, which was spun 15 min at 13 000 rpm. The aqueous phase was removed and cleaned with a glass-milk solution (GENECLEAN III®, BIO 101); the extracted DNA then was stored in 30 µL dd H2O at –20 C.
The nuclear LSU rDNA locus was amplified via polymerase chain reaction (PCR) with LROR and LR3 primers (Vilgalys and Hester 1990). PCR reactions were performed in 50 µL reaction mixtures containing ddH2O, 1 or 2 µL of DNA template, 2 µL of each primer pair (10 µM), 25 µL buffer E (MasterAmp 2 x PCR PreMixes: 100 mM Tris-HCl, 100 mM KCl, 400 uM each dNTP, 5 uM MgCl2, and 4 x MasterAmp Enhancer, Epicentre Technologies, Madison, Wisconsin) and 0.5 µL of 5 U/µL Taq polymerase. The DNA was amplified with a PTC Programmable Thermal Controller and thermal cycling, as follows: 94 C (2 min), [94 C (30 s), 51 C (30 s), 72 C (45 s)] x 30, [94 C (30 s), 53 C (30 s), 72 C (45 s + 5 s per cycle)] x 5, 72 C (5 min), 4 C (15 min). PCR products were viewed on 1% agarose gels (Gibco-BRL ultra PURE, Life Technologies) in a UV light transilluminator (UVP Laboratory products), stained with ethidium bromide and quantified with a low DNA mass ladder (Gibco-BRL Ultra PURE, Life Technologies). The amplified DNA was purified with a PCR purification kit (QIAquick, QIAGEN Inc.). Purified PCR products were sequenced with LROR primer on a 373 DNA Sequencer (Applied Biosystems).
LSU rDNA sequences were assembled with SeqEditor version 1.0.3 (Applied Biosystems) and visually aligned with PAUP* 4.0b10 (Swofford 1999). Two collections of Alpova austroalnicola and three collections of A. diplophloeus sequences were compared with closely related taxa sequences selected from GenBank: A. diplophloeus, A. trappei Fogel., Boletus edulis Fr, Rhizopogon occidentalis Zeller & C.W. Dodge, R. truncatus Linder, R. villosulus Zeller, Suillus luteus (Fr.) Gray and Truncocolumella citrina Zeller. Herbarium, collector and GenBank accession numbers are provided (TABLE I).
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TAXONOMY |
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–9
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Etymology.— – Latin austro (southern) and alnicola (dweller with alder), in reference to its association with Alnus in the Southern Hemisphere.
Macroscopic characters.— –
Basidiomata subhypogeous to hypogeous, globose to irregular, 4–11 x 6–15 mm at maturity (FIG. 1
). Peridium 2-layered, smooth to slightly velvety or felty, 400–570 µm thick in single specimens depending on sporocarp size, at maturity light brown with some darker areas, the depressions paler, when bruised turning dark brown, off-white in cross section, drying dark brown. Rhizomorphs concolorous with peridium or slightly darker, up to 80 µm broad, appressed at the basidioma base and scattered on its sides. Gleba solid, rubbery to gelatinous, exuding a sticky substance when cut; chambers 0.4–0.65 mm broad, separated by pale brown meandering veins (FIG. 1), maturing from the center of the gleba outward, the content pale brownish yellow, darkening slightly when exposed or aged, drying dark brown to black, hard and waxy when sectioned. Chemical reactions: KOH slightly brown on peridium, not reactive on gleba; FeSO4 pale green on peridium, not reactive on gleba. Columella, stipe and basal mycelium lacking. Odor none.
Microscopic characters.— –
Peridiopellis yellow in KOH in cross-section; suprapellis up to 70 µm thick, with some tangled, cylindrical, obtuse hyphae 11–35 x 4.5–8 µm and some projecting and scattered dermatocystidia 4–5.5 µm broad (FIG. 4) on the surface; inward constituted of ± isodiametric cells 8–19 µm broad with walls up to 1 µm thick and pigmented contents (FIG. 2), embedded in a gelatinous matrix at maturity; subpellis up to 500 µm, most cells inflated to form a textura angularis/textura epidermoidea gradient, cells in the outer part pale yellow, 12–40 x 5–20 µm mixed with isodiametric cells 8–32 µm broad (FIG. 3), toward the gleba the cells hyaline, 9.5–20 µm broad (FIG. 9) and confluent with the tramal tissue, occasional "giant" cells 45–65 µm broad and with walls 1–2 µm thick (FIG. 5) scattered throughout; conductive hyphae infrequent, 3–5 µm broad, yellow in Melzer’s reagent. Rhizomorph hyphae 2–3.5 µm broad, compactly arranged within, on the surface loosely woven, slightly colored and flexuous.
Glebal veins 30–55 µm thick (FIG. 9), of hyaline, parallel to subparallel hyphae 3.2–5 µm broad, with gelatinous-thickened walls in age, at the intersections forming a pseudoparenchyma of cells 5–16 µm broad (FIG. 9). Locules in young sporocarps filled by hyaline hyphae 1.5–4 µm broad, basidia and scattered, large, spherical cells with an attachment (FIG. 6) in a gelatinous matrix (FIG. 9), at maturity a few basidia persisting among spores. Hymenial palisade lacking; basidia abundant, clavate, 25–30 µm long x 2 µm broad at the base and 4–6 µm at the apex, hyaline, thin walled (FIG. 7), autolysing by maturity, 8-spored, the sterigmata less than 0.5 µm long. Mature spores are not attached to basidia although they may be.
Basidiospores hyaline singly, pale yellow in mass, smooth, initially globose, soon becoming ellipsoid to oblong or occasionally allantoid, (5)6–7(8.5) x 2.2–3 µm, the walls initially thin but slightly thickened at maturity, in youth with two guttules giving the appearance of a septum but these usually absent at maturity (FIG. 8), detaching at maturity but often held in the gel in much the same relative position to each other as when attached; spore walls strongly cyanophilic in cotton blue in youth, weak or acyanophilic at maturity, not reactive to Melzer’s reagent.
Clamp connections common in all tissues.
Habit, habitat and season.— – Subhypogeous to hypogeous among Alnus acuminata spp. acuminata roots, probably as a mycorrhizal associate, not abundant, most easily found where emergent in banks; Mar.
Specimens examined.— – ARGENTINA, SALTA PROVINCE, Santa Victoria, Los Toldos, site M28B, 1702 m, 28 Mar 2001, L.S. Domínguez 2290 (PARATY PE, CORD); site M42, 1778 m, 30 Mar 2001, L.S. Domínguez 2291 (HOLOTY PE, CORD).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSTAXONOMYRESULTSDISCUSSION AND CONCLUSIONSLITERATURE CITED |
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).
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). In this topology both Alpova austroalnicola collections (CORD LSD 2290 and CORD LSD 2291) clustered together next to Alpova diplophloeus (four collections) and Alpova trappei, although the bootstrap figure was low in the A. austroalnicola-A. trappei relationship. The grouping of A. austroalnicola collections is well supported with a bootstrap value of 94. The relationship of Alpova species with Boletus edulis is supported by a bootstrap value of 100. The Rhizopogonaceae represented by Rhizopogon occidentalis, R. truncatus and R. villosulus appear as a nonconsistent paraphyletic clade at the base of the tree.
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DISCUSSION AND CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMETHODSTAXONOMYRESULTSDISCUSSION AND CONCLUSIONSLITERATURE CITED |
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, Clemençon 1977, Gross 1980, Molina 1981, Godbout and Fortin 1983, Brunner and Horak 1990).
The lack of support for a close relationship of A. diplophloeus, A. austroalnicola and A. trappei probably reflects the data of Grubisha et al (2001), indicating the nonmonophyletic nature of Alpova, although the low number of specimens analyzed, especially A. trappei (1), precludes a more accurate interpretation. Alpova trappei is associated with North American members of the Pinaceae (Trappe 1975, Fogel 1977), and its peridial structure lacks the peridial layer of inflated cells. The close phylogenetic relationship of Boletus (B. edulis) and Alpova spp is supported strongly, thus representing the boletoid radiation within Boletales (Bruns and Szaro 1992, Bruns et al 1998).
Ours is the first report of mycophagy by the armadillo Dasypus novemcinctus novemcinctus. The relatively low number of A. austroalnicola spores in the feces suggests the armadillo eats these hypogeous fungi opportunistically. Moreover no other hypogeous fungi have been found in the A. acuminata spp. acuminata forests, and the number of epigeous mushroom species known in the area is relatively low (Becerra 2002, Nouhra et al 2003), indicating that relatively few, highly specialized, ectomycorrhizal fungi occur in this ecosystem. Similar data have been reported for other Alnus-dominated communities (Molina 1979, 1981; Brunner and Horak 1990).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: nouhra{at}imbiv.unc.edu.ar
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LITERATURE CITED |
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). 5. Isolated "giant" cells of subpellis. 6. Spherical cells in gleba of young specimens. 7. Eight-spored mature basidia (
) of various fungal and other structures observed within the analyzed fecal pellets samples (N=12). Fungal elements are expressed as numbers (*). Plant (leaf fragments and pollen grains) and mineral material (stones) mean values are based on visual estimated percentages (%). (SE) Standard error