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Submitted 23 May 2017, Accepted 22 June 2017, Published 2 July 2017
Corresponding Author: Dennis E. Desjardin – e mail –ded@sfsu.edu 977
The type species of Tetrapyrgos and Campanella (Basidiomycota,
Agaricales) are redescribed and epitypified
Desjardin DE1*, Perry BA2, Shay JE1, Newman DS1 and Randrianjohany E3
1Department of Biology, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA
2Department of Biology, California State University East Bay, 25800 Carlos Bee Blvd., Hayward, CA 94542, USA
3Centre National de Recherches sur l'Environnement (CNRE), BP 1739, Lab. de Microbiologie de l'Environnement (LME),
Antananarivo, Madagascar
Desjardin DE, Perry BA, Shay JE, Newman DS, Randrianjohany E 2017 – The type species of Tetrapyrgos and
Campanella (Basidiomycota, Agaricales) are redescribed and epitypified. Mycosphere 8(8), 977–985, Doi
10.5943/mycosphere/8/8/1
Abstract
Tetrapyrgos atrocyanea, the type species of Tetrapyrgos, and Campanella buettneri, the type species of Campanella,
are redescribed and epitypified based on recently collected material from Madagascar and Príncipe, respectively,
supported with morphological and LSU, ITS sequences data. Line drawings, colour photographs, and comparisons with
similar taxa are provided.
Key Words – agarics – Campanella buettneri – fungal diversity – taxonomy – Tetrapyrgos atrocyanea
Introduction
There has been renewed interest in clarifying the generic boundaries of Tetrapyrgos E. Horak (1987) and
Campanella Hennings (1895). The morphological distinctions between these two genera and the ways they have been
treated taxonomically by various workers were succinctly documented by Honan et al. (2015). However, they noted
that distinguishing Tetrapyrgos from Campanella was problematical because of the lack of recently collected specimens
of their type species upon which comprehensive morphological and molecular redescriptions can be based. The
holotype specimen of the type species of Tetrapyrgos (Pterospora atrocyanea Métrod) was collected in 1935 in
Madagascar, and is stored in alcohol (PC), rendering it of limited molecular phylogenetic value. The holotype specimen
Mycosphere 8(8): 977–985 (2017) www.mycosphere.org ISSN 2077 7019
Article
Doi 10.5943/mycosphere/8/8/1
Copyright © Guizhou Academy of Agricultural Sciences
978
of the type species of Campanella (C. buettneri Henn.) was collected in 1892 in Togo, is also stored in liquid (B), and
of limited value for molecular analyses. Our recent fieldwork in Madagascar and the West African islands of São Tomé
and Príncipe (approx. 700 miles from Togo) have yielded specimens that we recognize as Tetrapyrgos atrocyanea and
Campanella buettneri, respectively. The purpose of this paper is to redescribe these two important type species, based
on morphological characteristics of these recent specimens which we designate as epitype material, and to provide ITS
and nLSU sequences to aid in distinguishing the species from others, and for future research. Determining whether
Tetrapyrgos and Campanella represent a single, morphologically variable genus is beyond the scope of this
contribution.
Materials and Methods
Morphological observations
Fresh material of Tetrapyrgos atrocyanea was collected in Madagascar in Febuary 2014, and fresh material of
Campanella buettneri was collected in São Tomé and Príncipe April 2006 and 2008. Macromorphological notes and
photographs were documented soon after collection, before drying the basidiomes for transport and herbarium storage.
Colour terms and notations in parentheses are those of Kornerup & Wanscher (1978). Micromorphological analyses
were performed using dried material rehydrated in 100% ethanol followed by Melzer’s reagent, or Congo Red Solution
and 3% KOH, and documented using a Nikon Optiphot-2 compound microscope fitted with a drawing tube. Because
of the unusual tetrahedral shape of basidiospores of Tetrapyrgos, their length and width were determined by measuring
the sides of a quadrilateral into which each spore would fit. Spore statistics were calculated as: xm, the arithmetic mean
of the spore length by the spore width (± standard deviation) from n spores measured in a single specimen; Q, the
quotient of spore length and spore width in any one basidiospore indicated as a range of variation in n spores measured;
Qm, the mean of Q-values in a single specimen; n, the number of spores measured per specimen; s, the number of
specimens studied. Specimens are deposited in the H.D. Thiers Herbarium at San Francisco State University (SFSU).
Molecular methods
Total genomic DNA was extracted from dried material using the Extract-N-Amp Plant PCR Kit (Sigma-
Aldrich, St. Louis, MO) following the manufacturer instructions. PCR protocols followed those outlined in Perry et al.
(2007). The nuclear ribosomal internal transcribed spacer region (ITS) and ribosomal large subunit (nLSU) were
amplified using primer pairs ITS1-F/ITS4 (Gardes and Bruns 1993; White et al. 1990) and LROR/LR7 (Moncalvo et.
al. 2000), respectively. Amplification products were cleaned using the Exo-SAPit kit (Affymetrix, Santa Clara, CA),
and sent to ELIM Biopharmaceuticals (Hayward, CA) for sequencing. Resulting sequencing products were edited,
assembled, aligned and compared to top BLAST hits in Geneious 9.0 (Biomatters Ltd., Auckland, New Zealand). All
sequences generated as part of this study have been deposited in GenBank (accessions MF075136 – MF075139).
Taxonomy
Tetrapyrgos atrocyanea (Métrod) E. Horak, Sydowia 39: 102. 1987 (1986). Figs. 1, 2a–e
Basionym: Pterospora atrocyanea Métrod, Prod. Fl. Mycol. Madag. 3: 140. 1949.
Holotype – MADAGASCAR. Antananarivo (as Tananarive), in a pasture in a government park, 16 Dec. 1934, coll. by
R Decary, No. 1642 (PC).
Epitype hic designari – MADAGASCAR. Region Analamanga, Antananarivo, Parc Botanique de Zoologique, near the
garden of Crops Wild and Relatives, S18˚55.530', E47˚31.350', elevation 1270 m, 8 Feb. 2014, coll. by DS Newman
and JE Shay, JES 216 (MF075139–LSU, MF075137–ITS, SFSU).
MycoBank: MB 131349; Facesoffungi number: 03427
979
Description of epitype material:
Pileus 5–25 mm diam, convex to plano-convex, centrally slightly depressed in age; margin striate; surface dull,
dry, felted; disc initially brownish grey (5C1–3), developing dark bluish grey tones in age, margin initially white,
becoming pinkish buff to orangish white (5A3) in age, staining dark bluish grey (20E3–4) in splotches. Context up to
1 mm thick, pliant, tenacious, concolorous with pileus surface, staining dark bluish grey. Lamellae adnate with a
decurrent tooth to arcuate, close (19–25) with 2–3 series of lamellulae, moderately broad (1–2 mm), white, often
becoming pale orange white (5A2) in age, sometimes staining bluish grey; edges pruinose. Stipe 10–36 1–2 mm,
central, cylindrical or narrowed downward, tough, pliant, hollow, non-insititious, arising from a flattened pad of bluish
black mycelium; surface dull, dry, felted-pruinose; apex white when young, becoming orangish white (5A2) to
brownish grey (5E–F3) in age; base brownish grey (5F3) to dark bluish grey (20D–E3–4) to bluish black (20F4–5).
Fig. 1 – Basidiomes of Tetrapyrgos atrocyanea (Epitype, JES 216). Scale bars = 10 mm
Photographs by DS Newman
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Basidiospores (8–) 10–11.8 (6.5–) 7–9 µm [xm = 11.0 ± 0.63 7.9 ± 0.75 µm, Q = 1.13–1.77, Qm = 1.41 ±
0.27, n = 20, s = 1], tetrahedral, hyaline, inamyloid, thin-walled. Basidia 30–40 7–8.5 µm, clavate, 4-spored, hyaline.
Basidioles subclavate to subfusoid. Pleurocystidia absent. Lamellar edge sterile. Cheilocystidia abundant, 30–50 4–
5 µm, irregularly cylindrical, densely diverticulate, usually with a knob-like apex; apical knob 5–14 3.8–7 µm, broadly
obtuse, smooth; diverticula 1.5–5 1–3.5 µm, cylindrical to knob-like, obtuse; hyaline, inamyloid, non-gelatinous,
thin-walled. Subhymenium non-gelatinous. Pileipellis a well-developed Rameales-structure of hyphae 3.5–7 µm diam,
irregularly cylindrical, sometimes forked, densely diverticulate; terminal cells repent to erect, similar to the
cheilocystidia, typically with a knob-like apex; apical knob 5–16 4.5–9 µm, broadly obtuse, smooth; diverticula 1.5–
5 1.5–3.5 µm, cylindrical to knob-like, obtuse; hyaline, inamyloid, non-gelatinous. Pileus trama of loosely interwoven
hyphae 2–5 µm diam, cylindrical to irregular in outline, hyaline, inamyloid, strongly gelatinous. Lamellar trama regular,
hyphae 2–5 µm diam, cylindrical to irregular in outline, hyaline, inamyloid, strongly gelatinous. Stipitipellis similar to
the pileipellis, a well-developed Rameales-structure of densely diverticulate hyphae with apical knobs; stipe cortical
hyphae 2.5–5 µm diam, parallel, cylindrical, hyaline, inamyloid, non-gelatinous, thin- to thick-walled; stipe medullary
hyphae 3–10 (–12) µm diam, cylindrical, hyaline, inamyloid, non-gelatinous. Clamp connections common in all tissues.
Fig. 2 – Micromorphological features of Tetrapyrgos atrocyanea (Epitype, JES 216). a. Basidiospores. b. Basidium
and basidioles. c. Cheilocystidia. d. Pileipellis terminal cells. e. Stipitipellis cells. Scale bar = 10 µm. Illustration by
DE Desjardin.
Habit, habitat and known distribution – Gregarious, sometimes subcespitose, on woody debris amongst
Casuarina, Panicum, Galinsoga, Commelina and bamboo, in a botanical garden and zoological park. Madagascar.
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Notes – Tetrapyrgos atrocyanea (Bas. Pterospora atrocyanea Métrod) is the type species of the genus
Tetrapyrgos, established by Horak (1987) to replace the invalid Pterospora Métrod (1949) (non Pterospora Nuttall
1818, Pyrolaceae). The holotype specimen was collected in a government park in Antananarivo, Madagascar, and
comprehensively described in the protologue (Métrod 1949). The holotype, preserved in alcohol, was studied by Horak
(1968) who provided more details on micromorphology. As noted by Honan et al (2015), the documentation of
tetrahedral basidiospores, Rameales-type pileipellis and stipitipellis, diverticulate cheilocystidia with apical knobs,
bluish stains, and a stipe with white apex and bluish black base provided enough data to help circumscribe the genus
Tetrapyrgos. However, the poorly preserved holotype and absence of recently collected material prohibited generation
of molecular sequences to help distinguish the taxon from similar species, especially from T. longicystidiata A.H.
Honan, Desjardin & T.J. Baroni, newly described from neotropical populations, from T. subcinerea (Berk. & Broome)
E. Horak, described from Sri Lanka and widely distributed throughout south and southeast Asia, and from the genus
Campanella. During recent fieldwork in Madagascar, co-authors Shay and Newman encountered several specimens of
Tetrapyrgos, and one in particular (JES 216) was collected in a government park in Antananarivo, quite possibly the
same park from which the holotype originated. We herein accept this specimen as epitype material for T. atrocyanea.
The morphology of JES 216 matches quite nicely that described in the protologue (Métrod 1949) and
subsequent type study (Horak 1968), differing only slightly in basidiospore size. Métrod (1949) reported the
basidiospores as "7–8 2.5–3.5 µm, ovoid with two lateral wings 2–2.5 µm long" (collectively 7–8 6.5–8.5 µm),
while Horak (1968) noted the basidiospores as tetrahedral, 7–9 µm broad and long. Our material shows distinctly
"winged", tetrahedral basidiospores 8–11.8 7–9 µm, i.e., up to several microns longer than reported for the holotype
specimen.
Pairwise comparisons of aligned, overlapping ITS sequences of the epitype of T. atrocyanea with the top ten
BLAST results indicate 99.9% similarity to the holotype specimen of T. longicystidiata (EF175544) from Puerto Rico,
and 99.6% similarity to a specimen of T. longicystidiata from Bolivia (EF175533). In the protologue of T. atrocyanea
(Métrod 1949; pg. 129, Fig. 87c) and Horak's type study (Horak 1968; pg. 528, Fig. e) they illustrated very long
cheilocystidia (45–55 µm sensu Horak), the diagnostic feature of T. longicystidiata (Honan et al. 2015; pg. 109, Fig.
2d). These data suggest that the recently described new world taxon is a synonym of T. atrocyanea, described from
Madagascar.
Campanella buettneri Henn., Bot. Jb. 22: 95. 1895. Figs. 3, 4a–d
Holotype – AFRICA, Togo, Bismarckburg, on wood, 1892, coll. by R. Büttner (B).
Epitype hic designari – AFRICA, Príncipe island, Bom Bom resort area, N1˚41.559', E7˚24.171', 20 April 2008, coll.
by D.E Desjardin, DED 8276 (MF075138–LSU, MF075136–ITS, SFSU)
MycoBank: MB 208007; Facesoffungi number: 03428
Description of epitype material:
Pileus 5–12 mm diam, auriform, asymmetrically convex to campanulate in profile, hemispherical to
subchordate in face view, margin undulate, sometimes cleft; surface rugulose to pustulose, tessellate, dull, dry, white
overall, drying white to pale cream, unstaining. Context thin, rubbery-pliant, white. Lamellae adnate to point of
attachment, remote with many cross-lamellae, costate-reticulate, intervenose, relatively narrow, white, drying dark
cream, unstaining. Stipe absent; with or without a poorly developed pseudostipe extending from the margin, usually in
the cleft, or eccentrically from the pileus surface. Odor and taste not distinctive.
Basidiospores 8.6–10.2 6.0–7.2 µm [xm = 9.7 ± 0.46 6.6 ± 0.34 µm, Q = 1.4–1.6, Qm = 1.48 ± 0.04, n = 26, s = 1],
broadly ellipsoid, rarely approaching subglobose, lacking bulges, smooth, hyaline, inamyloid, thin-walled. Basidia 28–
32 7–8 µm, clavate, 4-spored. Basidioles clavate to subclavate, seldom subfusoid. Cheilocystidia not differentiated.
Hymenial cystidia common on sides and edges of lamellae, 38–48 9–12 µm, fusoid to ventricose, hyaline, thin-walled
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or with wall up to 0.5 µm thick (not metuloids). Pileipellis a well-developed Rameales-structure; hyphae 3–5 µm diam,
cylindrical to irregular in outline, dendrohyphidia-like, densely diverticulate, hyaline, inamyloid, thin-walled, non-
gelatinous; diverticula 2–12 1–2 µm, cylindrical to narrowly conical, mostly obtuse, hyaline, thin-walled. Pileus trama
of loosely interwoven hyphae 2–4 (–5) µm diam, cylindrical, non-diverticulate, hyaline, inamyloid, embedded in a
strongly gelatinous matrix. Lamellar trama regular, hyphae 2.5–5 µm diam, hyaline, inamyloid, gelatinous. Clamp
connections common in all tissues.
Fig. 3 – Basidiomes of Campanella buettneri (Epitype, DED 8276). Scale bar = 10 mm
Photograph by BA Perry
Habitat and known distribution – Gregarious on woody debris of undetermined dicotyledonous plants. Africa
(Cameroon, Príncipe, São Tomé, Togo), Hawaii.
Additional material examined – São Tomé, Parque Nacional Obo, along trail from Bom Succeso to Lagoa
Amelia, N0˚17.317', E6˚36.746', 28 April 2006 and 2 May 2006, coll. by D.E. Desjardin, material lost in transit. United
States. Hawaiian Islands, Hawai`i, Lava Tree State Park near Leilani, N19˚28'08", W154˚54'10", 30 July 2005, coll. by
Ed Lickey, TFB 12565 (TENN-F-60782).
Notes – Campanella buettneri is the type species of the genus Campanella, established by Hennings (1895) from
material collected in Togo and Cameroon. We are unaware of recently collected material from these countries upon
which the generic concept of Campanella can be refined, but we have collected material from the nearby island nation
983
of São Tomé and Príncipe that we recognize as representing this important species, and we herein accept it as epitype
material for C. buettneri.
Fig. 4 – Micromorphological features of Campanella buettneri (Epitype, DED 8276). a. Basidiospores. b. Basidium
and basidioles. c. Hymenial cystidia. d. Pileipellis hyphae. Scale bar = 10 µm. Illustration by DE Desjardin.
The Príncipe specimen matches the protologue (Hennings 1895) and differs only slightly from an analysis of
the holotype specimen studied by Singer (1975). Campanella buettneri is characterized by small, rubbery-pliant
basidiomes with a white, ear-shaped, tessellate-verrucose pileus 5–12 mm diam attached to the substrate by a small,
eccentric to lateral pseudostipe, costate-reticulate, intervenose, white hymenophore, broadly ellipsoid basidiospores
with mean 9.7 6.6 µm, fusoid-ventricose hymenial cystidia, a Rameales-type pileipellis, and growth on woody debris.
The protologue reported the basidiospores as subglobose, 7–9 µm, while Singer (1975) reported those in the holotype
specimen as ellipsoid, 8.7 6.3 µm; neither author mentioned lateral bulges on the basidiospores. The basidiospores of
the Príncipe material are mostly broadly ellipsoid with only a few tending towards subglobose, but the range in variation
encompasses the sizes reported by Hennings (1895) and Singer (1975); likewise, they lack lateral bulges. Additional
noteworthy features of the species include the distinct fusoid-ventricose, non-refractive, thin- to firm-walled hymenial
cystidia, strongly gelatinous pileus trama, a non-gelatinous pileipellis of irregular hyphae with relatively long, narrow
diverticula, and white basidiomes that do not discolor greyish green.
Singer (1975) compared C. buettneri to C. alba (Berk. & M.A. Curtis) Singer, a species described from Cuba
and rather widespread in the neotropics, indicating that the latter differed primarily in having basidiospores with lateral
bulges. Campanella alba differs also in forming few, scattered hymenial cystidia that are narrowly cylindrical (2.7–7.5
µm) and subcapitate to capitate, whereas in C. buettneri the hymenial cystidia are fusoid-ventricose, 9–12 µm diam,
and non-capitate. Pairwise comparisons of aligned, overlapping ITS sequences of C. buettneri with the top ten BLAST
results indicate closest similarity (99.5%) to a sequence determined as C. alba (DQ449943) obtained from a specimen
collected on Hawai`i island, USA. Examination of the latter specimen (TENN-F-60782) indicated morphology nearly
identical to that of the epitype specimen designated herein (albeit with fewer hymenial cystidia), and we recognize the
Hawaiian material as representing C. buettneri, not C. alba.
984
Acknowledgments
São Tomé and Príncipe: We thank Dr. Robert C. Drewes (California Academy of Sciences) who continues to
initiate, coordinate and lead multiorganism biotic surveys on São Tomé and Príncipe; Eng. Arlindo de Ceita Carvalho,
Director General of the Ministry of Environment, Victor Bonfim, Salvador Sousa Pontes and Danilo Barbero for
permission to collect and export specimens for study. We are indebted to Société de Conservation et Développement
for logistics and housing support, especially the wonderful staffs of Omali Lodge and Bom Bom Island. We are grateful
for the support and cooperation of Bastien Loloumb of Zuntabawe and Faustino Oliviera, former Director of the
botanical garden at Bom Sucesso. We were assisted in the field by Jose Ramos Maria Vital Pires on Príncipe and by
Quintino Quade Cabral, Martinho Nascimiento and Jose Clara on São Tomé. For continuing support, we are most
grateful to Ned Seligman, Quintino Quade Cabral and Roberta dos Santos of STePUP. We are grateful to the College
of Science and Engineering at San Francisco State University for partial funding to support travel to São Tomé and
Príncipe, and to the G. Lindsay Field Research Fund of the California Academy of Sciences (CAS) for financially
supporting the expedition in 2006 and the Hagey Research Venture Fund (CAS) in 2008.
Madagascar: We are grateful to Rokiman Letsara of the California Academy of Science, Madagascar
Biodiversity Research Station for his invaluable efforts in planning and facilitating field work. We are thankful for the
knowledge of local mycologist Emile Randrianjohany and his colleagues at the Centre National de Recherches sur
l'Environnement (CNRE) for their valuable field assistance.
We thank the Department of Biology, San Francisco State University, Mycological Society of San Francisco, Sonoma
County Mycological Association, and the generous donors from a Kickstarter campaign who partially funded fieldwork.
References
Gardes M, Bruns TD. 1993 – ITS primers with enhanced specificity for basidiomycetes – application to the
identification of mycorrhizae and rusts. Molecular Ecology 2, 113–118.
Hennings P. 1895 – Fungi camerunensis. I. Botanische Jahrbücher fur Systematik, Pflanzengeschichte und
Pflanzengeographie 22, 72–111.
Honan AH, Desjardin DE, Perry BA, Horak E, Baroni TJ. 2015 – Towards a better understanding of Tetrapyrgos
(Basidiomycota, Agaricales): new species, type studies, and phylogenetic inferences. Phytotaxa 231(2), 101–
132.
Horak E. 1968 – Synopsis generum Agaricalium. Beiträge zur Kryptogamenflora der Schweiz 13, 1–741.
Horak E. 1987 (1986) – Tetrapyrgos Horak (nom. et gen. nov.) replacing Pterospora Métrod (1949; nom. preocc.).
Sydowia 39, 101–103.
Kornerup A, Wanscher JH. 1978 – Methuen Handbook of Colour. 3rd ed. Eyre Methuen, London.
Métrod G. 1949 – Les Mycènes de Madagascar. Prodrome à une flore mycologique de Madagascar 3, 1–146.
Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R. 2000 – Phylogenetic relationships of agaric fungi based
on nuclear large subunit ribosomal DNA sequences. Systematic Biology 49, 278–305.
Perry BA, Hansen K, Pfister DH. 2007 – A phylogenetic overview of the family Pyronemataceae (Ascomycota,
Pezizales). Mycological Research 111, 549–571.
Singer R. 1975 – The neotropical species of Campanella and Aphyllotus with notes on some species of Marasmiellus.
Nova Hedwigia 26, 847–896.
White TJ, Bruns T, Lee S, Taylor J. 1990 – Amplification and direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: PCR Protocols: a guide to methods and applications. Innis MA, Gelfand DH, Sninsky JJ,
White TJ. Eds. Academic Press, San Diego. p. 315–322.