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Short title: Phylogeny of Acantholichen
From one to six: Unrecognized species diversity in the genus Acantholichen (lichenized
Basidiomycota: Hygrophoraceae)
Manuela Dal-Forno
Department of Environmental Science and Policy, George Mason University, Fairfax, Virginia
22030-4444
Robert Lücking
Science & Education, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois
60605;
Botanischer Garten und Botanisches Museum, Königin-Luise-Straße 6-8, D-14195 Berlin,
Germany
Frank Bungartz
Biodiversity Assessment, Charles Darwin Foundation (AISBL), Puerto Ayora, Santa Cruz,
Galápagos, Ecuador
Alba Yánez-Ayabaca
Universidad Central del Ecuador, Quito, Ecuador
Marcelo P. Marcelli
Instituto de Botânica, Núcleo de Pesquisa em Micologia, Av. Miguel Stéfano 3687, São
Paulo/SP, CEP 04301-902, Brazil
Adriano A. Spielmann
Laboratório de Botânica/Liquenologia, Centro de Ciências Biológicas e da Saúde, Universidade
Federal de Mato Grosso do Sul, Caixa Postal 549, CEP 79070-900, Campo Grande, Mato
Grosso do Sul, Brazil
In Press at Mycologia, preliminary version published on November 17, 2015 as doi:10.3852/15-060
Copyright 2015 by The Mycological Society of America.
Luis Fernando Coca
Herbario Universidad de Caldas, Edificio Bicentenario, Manizales, A.A. 275, Caldas, Colombia
José Luis Chaves
Laboratorio de Hongos, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa
Rica
Andre Aptroot
ABL Herbarium, G.v.d.Veenstraat 107, NL-3762 XK Soest, the Netherlands
Harrie J. M. Sipman
Botanischer Garten und Botanisches Museum, Freie Universität, Königin-Luise-Straße 6-8, D-
14195 Berlin, Germany
Masoumeh Sikaroodi
Patrick Gillevet
Department of Environmental Science and Policy, George Mason University, Fairfax, Virginia
22030-4444
James D. Lawrey
Department of Biology, George Mason University, Fairfax, Virginia 22030-4444
Abstract: We present a taxonomic revision of the lichenized basidiomycete genus Acantholichen,
species of which produce a characteristic blue-gray, microsquamulose thallus with spiny apical
hyphal cells known as acanthohyphidia. Since its discovery, the genus was thought to be
monospecific, only including the generic type, A. pannarioides. However, a detailed
morphological and anatomical study of recently collected specimens from the Galápagos, Costa
Rica, Brazil and Colombia, combined with a molecular phylogenetic analysis of the internal
transcribed spacer (ITS1-5.8S-ITS2) region and 28S of the nuc rDNA and RPB2 sequences,
revealed a much more diverse and widespread species assemblage. Based on the results of these
analyses, we describe five new species in the genus: A. albomarginatus, A. campestris, A.
galapagoensis, A. sorediatus and A. variabilis. We also provide an identification key to all
species, anatomical and morphological descriptions, photographs and a table comparing main
characters of each species.
Key words: Basidiolichens, lichenized fungi, montane forest, phylogeny, taxonomy,
tropical diversity
INTRODUCTION
The genus Acantholichen was established by Jørgensen (1998) for A. pannarioides, based on a
material originally set aside as a possible sterile ascolichen in the genus Parmeliella. The species
is characterized by a microsquamulose thallus, dark blue to slightly gray, with spiny apical cells
on both thallus surfaces, giving it a white-pruinose appearance. Jørgensen (1998) recognized
these as acanthohyphidia, structures known to be produced by basidiomycetes. Because no
sexual structures were observed in the specimen and no other lichen species are known to
produce acanthohyphidia, the species was described as a new basidiolichen. At the time of its
description, A. pannarioides appeared to be tropical, with specimens collected in the moist
montane forests of South and Central America (Jørgensen 1998).
In a molecular phylogenetic study, Lawrey et al. (2009) confirmed the identity of A.
pannarioides as a lichenized basidiomycete and placed it in the Dictyonema clade in the family
Hygrophoraceae of the Agaricales. Dictyonema had never before been considered an agaric
genus, having been assigned to several families, including Atheliaceae, Aphyllophoraceae,
Corticiaceae, Thelephoraceae (Parmasto 1978) and more recently Phanerochaetaceae
(Zmitrovich et al. 2006). Members of the Dictyonema clade are entirely lichenized and have a
cyanobacterial Rhizonema photobiont that is known only from lichens (Lücking et al. 2009,
Lücking et al. 2014a). Acantholichen shares some morphological characters, such as the
paraplectenchymatous cortex, with other lineages in the clade, most notably those in the foliose
genus Corella, to which Acantholichen appears to be most closely related (Dal-Forno et al.
2013).
Recent investigations indicate that the Dictyonema clade might be a source of a remarkable
number of undescribed species (Lücking et al. 2013a, 2014b). Studies have revealed a previously
unrecognized diversity of basidiolichens in a variety of phylogenetic groups, not only in the
Dictyonema clade (Chaves et al. 2004; Yánez et al. 2012; Dal-Forno et al. 2013; Lücking et al.
2013a, b, 2014b) but also in the chlorolichen genera Lichenomphalia (Redhead et al. 2002),
Lepidostromatales (Fisher et al. 2007; Ertz et al. 2008; Hodkinson et al. 2012, 2014; Sulzbacher
et al. 2013; Yanaga et al. 2015) and Multiclavula (Nelsen et al. 2007). Our hypothesis is that
Acantholichen also may represent a potentially important source of new species that warrants
further investigation.
For this study we obtained several distinctive new specimens of Acantholichen from the
Galápagos Islands, Costa Rica, Brazil and Colombia. These specimens at first glance similar are
in color and outward appearance to A. pannarioides but exhibit phenotypic differences indicating
they might represent separate species. We combined a detailed morphological and anatomical
study of the specimens with a molecular phylogeny obtained with three commonly used markers,
the internal transcribed spacer regions (ITS1-5.8S-ITS2 =ITS) and the 28S subunit gene of nuc
rDNA and the DNA-directed RNA polymerase II second largest subunit (RPB2) gene to: (i) test
the hypothesis that the specimens represent new species of Acantholichen in the
Hygrophoraceae; (ii) provide a preliminary assessment of the ecology, distribution and degree of
endemism of the known species; and (iii) provide descriptions, diagnoses and a key to all
species.
MATERIALS AND METHODS
Taxon sampling.—Our Acantholichen dataset includes 17 samples (SUPPLEMENTARY TABLE I), most collected
during field trips 2010–2014 throughout South and Central America.
Light microscopy.—All specimens were examined with a LEICA MS5 (Wetzlar, Germany), an OLYMPUS SZX12
(Shinjuku, Japan) and a Stereostar Zoom stereoscopic (Reichert, Austria) dissecting microscopes and with a ZEISS
Axioskop 2 (Jena, Germany), an OLYMPUS BH-2 (Shinjuku, Japan) and a NIKON Optiphot (Japan) compound
microscopes. Thallus sections were studied in water only, and microphotographs of them were taken with DAGE
MTI DC-330 3CCD (Michigan City, Indiana) and JENOPTIK ProgRes C3 and C5 (Jena, Germany) digital
microscope cameras attached to the aforementioned microscopes. Macrophotos were taken in situ with CANON
Powershot SX20IS (Ota, Japan), NIKON F301 (Tokyo, Japan) and Sony Alpha 33 DSLR (Thailand) digital
cameras.
DNA extraction, polymerase chain reaction and DNA sequencing.—Genomic DNA was extracted from lichenized
thalli (ca. 2 mm
2
squamules) with the Bio 101 Fast DNA Spin kit for tissue (Qbiogene, Illkirch, France) following
the manufacturer's protocol with slight modifications. About 10 ng extracted DNA were subjected to a standard PCR
in a 20 μL reaction volume using either Taq Gold polymerase (Applied Biosystems, Foster City, California) or Bio-
X-Act Long Mix (Bioline USA, Taunton, Massachusetts) following the manufacturer's protocols. Sequence data
were obtained from the ITS barcode (approx. 700 bp), nuc 28S rDNA (approx. 1470 bp) and RPB2, between the 6
and 7 conserved domains (approx. 1000 bp). PCR products were visualized on a 1% agarose gel with ethidium
bromide and purified with magnetic beads (Agencourt Bioscience, Beverly, Masachusetts). The purified PCR
products were used in standard sequencing reactions with BigDye Terminator Ready Reaction Mix 3.1 (Applied
Biosystems). Primers used were: ITS5 and ITS4 for ITS (White et al. 1990); LR0R, LR3R, LR5, LR7 and LR16 for
28S (http://www.biology.duke.edu/fungi/mycolab/primers.htm); and bRPB2-6F or bRBP2-5F and bRBP2-7R,
bRBP2-7R2 or bRPB2-7.1R for RPB2 (Denton et al. 1998, Liu et al. 1999, Matheny 2005). The sequencing
reactions were purified with Sephadex G-50 (Sigma-Aldrich, St Louis, Missouri), dried in a SpeedVac, denatured in
HiDi Formamide (Applied Biosystems) and run on an ABI3130-xl capillary sequencer (Applied Biosystems). The
data collected were analyzed with ABI software, and 500–700 bases were collected for each primer used. For one
sample (Acantholichen sorediatus R. Lücking s.n.) we were unable to get a sequence through Sanger sequencing and
therefore applied pyrosequencing techniques following the protocols of Lücking et al. (2014c).
Sequence alignment and phylogenetic analysis.—New sequences were assembled with the software Sequencher 5.0
(Gene Codes Corp., Ann Arbor, Michigan) for manual corrections in base calling and to make contigs of
overlapping fragments. This resulted in 35 new sequences that were assembled with three sequences from GenBank
using BIOEDIT 7.09 (Hall 1999) and automatically aligned with the program MAFFT with the −
AUTO option
(Katoh and Toh 2005). For phylogenetic analyses Corella brasiliensis was used as outgroup because Corella was
shown to be the sister genus of Acantholichen by Dal-Forno et al. (2013). The concatenated dataset alignment was
subjected to analysis of ambiguously aligned regions using the GUIDANCE web server (Penn et al. 2010a, 2010b)
and because the alignments were highly scored (overall 0.97) no regions were removed. The alignments were
subjected to maximum likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al. 2005, Stamatakis 2006), with
parametric bootstrapping using 500 replicates under the
GTRGAMMA model. Each gene first was analyzed separately,
and all data eventually were combined because no conflict was detected (Mason-Gamer and Kellogg 1996). For the
concatenated dataset, when sequences were missing for determined sample, we used the missing data approach. The
concatenated dataset also was analyzed under a Bayesian Inference (BI) framework with MrBAYES 3.1.2
(Huelsenbeck and Ronquist 2001), with two independent runs, a total chain length of five million generations, and
four separate chains each, resampling every 200 trees and generating a 50% majority-rule consensus tree from the
sampled trees after discarding 25% burn-in to obtain posterior probability estimates. We also ran an additional BI
analysis under the same parameters but partitioning the dataset according to the three loci. All trees were visualized
in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Photoshop CS5.1.
To test whether Acantholichen would remain monophyletic with the addition of multiple specimens, we
downloaded from GenBank sequences from the Dictyonema clade corresponding to species published by Dal-Forno
et al. (2013) and Lücking et al. (2013a, b). For Acantholichen, only species in which all three loci are available were
used for this analysis, with a single specimen representing the species was selected when many were available. We
performed a MAFFT alignment (Katoh and Toh 2005), which was subjected to ML search in RAxML under the
same settings described above.
R
ESULTS
Alignment lengths were 713bp for the ITS region, 1472bp for the 28S, and 1017bp for the RPB2,
for a total of 3202 sites in the combined dataset, with 250 variable sites. No regions in the
alignments were deleted. The final ML optimization likelihood was -5648.546362. For the
analysis in MrBayes for the partitioned data, the average standard deviation of split frequencies
reached a number inferior of 0.01 after 110000 generations. The average arithmetic mean and
harmonic means between chains were -5697.91 and -5722.99, respectively. The 50% majority
rule consensus tree was generated from 1102 trees in 2 files, sampling 828 of them.
The phylogenetic tree inferred by ML only, including representative taxa from other genera
in the Dictyonema clade, recovered Acantholichen as monophyletic (FIG. 1a) and shows Corella
to be a genus sister to Acantholichen. Phylogenetic trees inferred by ML and BI (FIG. 1b) were
congruent in topology. Analysis of the partitioned and non-partitioned datasets recovered the
same tree topology with similar support values, and we show only the results from the analysis
of partitioned data (FIG. 1b). Analysis of the combined three-marker dataset suggests that there
are six distinct species, a result that agrees with our detailed morphological and anatomical
observations. With the moderate to high support for species relationships within the genus, it is
now possible to define distinct species based on phylogenetic, morphological and geographic
structure.
Our phylogeny indicates that Acantholichen sorediatus seems to be sister of the rest of the
species in the genus (FIG. 1b). The relationship of A. albomarginatus to A. pannarioides, A.
variabilis, A. campestris and A. galapagoensis does not seem strongly supported in ML (70%) or
BI (pp = 0.79). Two species newly described in this paper (A. variabilis and A. campestris)
appear to be closely related and together form a clade sister to A. galapagoensis.
TAXONOMY
Acantholichen P.M. Jørg., Bryologist 101:444. 1998.
Thallus morphology: Microsquamulose, squamules with a coarsely pruinose appearance,
individual squamules scattered or growing closely together and sometimes even merging; their
shape either round and broad or elongated and thin, each squamule basally attached to the
substrate, rarely centrally (one species), squamules, if distinctly elongated, typically proliferating
from their tips; typically distinctly swollen, rarely ± flattened (one species); ca. 0.1–2(–3) mm
wide and 0.1–2(–3) mm long; not, sparsely, moderately or highly branching; their color ranging
from mostly blue and green hues when fresh, to dark grey and blue when dry, rarely distinctly
whitened along their margin, with or without soredia, which can give the thallus an additional
granulose appearance.
Thallus anatomy: Heteromerous, squamules 100–240 µm thick in section, with a distinct,
paraplectenchymatous (5–)10–30 µm thick upper cortex, formed by 2–3 layers of cells, a 70–150
µm thick photobiont layer, and a 10–100 µm thick medulla of more or less dense, irregular to
rounded or elongated hyphae; the lower side lacking a cortex. Upper cortex and lower surface
with acanthohyphidia, i.e., a modified hypha terminal cells with pin-like outgrowths (Kirk et al.
2008), that can be small to large, 4–50 µm long, 4–10(–12) µm wide, and are always
conspicuously spiny, these cells that can be irregular, subglobose, pyriform, elongate, subclavate
or clavate, they spread across the entire lamina, or can be more abundant along the margin of the
squamules; no clamp-connections observed among hyphae. Typically, acanthohyphidia can be
more abundant in the lower surface. Photobiont Rhizonema, forming clusters of 8–17 µm diam.
of coiled cyanobacterial filaments (“chroococcoid”), wrapped within a dense hyphal sheath
formed by jigsaw puzzle-shaped cells (Cora-type). Sexual reproduction structures unknown.
Chemistry: All spot tests negative (P−, K−, KC−, C−), UV–, no secondary metabolites
detected.
Distribution and ecology: So far, the genus is known only from South and Central
America, inhabiting mostly habitats at high altitudes (only in Galápagos as low as 500 m), in
moist and cloudy forests, in areas with high light intensity. Species of Acantholichen have been
found on a wide range of different substrates, but typically accompanied by and/or overgrowing
bryophytes and other lichens. Acantholichen campestris is the only known species that grows on
rock while the other species grow on bark and wood, often overgrowing mosses and liverworts.
One specimen in Galápagos (A. Aptroot 63214) was collected on liverworts growing on soil. All
species show high levels of endemism. Jørgensen (1998) had already commented that the genus
is most likely to occur in moist montane regions of South and Central America.
Comments: Because of its unique microsquamulose thallus with coarsely pruinose
appearance, Acantholichen is morphologically very different from other members of the
Dictyonema clade. As originally pointed out by Jørgensen (1998), members of the genus
resemble species of the Pannariaceae, but all species of the lichenized basidiomycete
Acantholichen are easily distinguished from these lichenized ascomycetes by the presence of
acanthohyphidia. Phylogenetically, Acantholichen is sister to Corella¸ both in turn forming a
sister clade to Cora (Dal-Forno et al. 2013). The three genera form a clade characterized by a
heteromerous thallus, with chroococcoid shaped cyanobacterial cells of the genus Rhizonema.
Corella is a genus that is morphologically very similar to Cora, but it shares with Acantholichen
the same paraplectenchymatous cortex. The “unshaven chin” appearance described by Jorgenson
(1998) is typical of this genus.
A summary of the morpho-anatomical characters most useful in distinguishing the
different species is provided (TABLE I). For most species no single character can be used to
differentiate them from all others. However, each species exhibits a unique combination of
characters. Five new species are recognized and described here.
K
EY TO SPECIES OF ACANTHOLICHEN
1.1 Squamules granular to flattened, with conspicuous white margins; acanthohyphidia may
be sparse to absent in some parts of the lamina but are always abundant along the margins A. albomargin
a
1.2 Squamules not granular to flattened but distinctly swollen (inflated), margins not
conspicuously white but in some species more abundantly “pruinose” than the squamule
surface ...................................................................................................................................2
2.1 Acanthohyphidia up to 50 μm long, elongate to clavate .......................................................3
2.2 Acanthohyphidia up to 20 μm long, pyriform, subglobose, subclavate and/or irregularly
shaped ...................................................................................................................................4
3.1 Squamules dark blue when fresh (without greenish tinge), acquiring a grayish tinge when
dried, acanthohyphidia size variable (10–50 μm) .............................................. A. variabilis
3.2 Squamules blue-green when fresh (i.e. with greenish tinge), turning dark blue when dried,
acanthohyphidia typically large (30–50 μm) ............................................... A. pannarioides
4.1. Squamules sparsely branched, forming soredia along the margins .................. A. sorediatus
4.2. Squamules moderately to abundantly branched, lacking soredia .........................................5
5.1 Squamules intricated and tiled (growing over each other) but not appearing fruticose when
fresh with a distinct olivaceous hue; endemic to the Galápagos Islands ................................
..................................................................................................................... A. galapagoensis
5.2 Squamules abundantly branched, but not intricated as above, with a microfruticose
appearance, not olivaceous ............................................................................... A. campestris
Acantholichen albomarginatus Dal-Forno, Marcelli & Lücking, sp. nov. FIG. 2
MycoBank MB814549
Typification: BRAZIL. MINAS GERAIS: Itamonte, Parque Nacional do Itatiaia, Estrada das
Prateleiras, 22°21′39.7″S, 44°43′57.1″W, 2190 m. Cloud forest, on the edge of forest, by the
road, dense vegetation on road side banks, growing on bryophytes and liverworts, 6 Jan 2013,
Dal-Forno, M. & M.P. Marcelli no. 2043 (holotype CGMS). Isotypes GMUF, B. GenBank
accessions: KT429797 = ITS; KT429809 = 28S.
Etymology: albo (Latin) = white, marginatus (Latin) = edged, referring to the
characteristic white margins of well developed squamules.
Diagnosis: Differs from other species by mostly flat squamules with acanthohyphidia
abundant along their margin, looking as if it has a prothallus.
Thallus granular to microsquamulose; when not completely granulose (soredia),
squamules are broad, attached centrally to the substrate, flattened, ca. 0.1–1 mm across, little to
moderately branched, often merging and closely adhering, and thus forming an almost
continuous crust up to 5 mm across, blue green when fresh, dark bluish green and gray when dry,
the closely adhered parts typically rimmed with a white margin (hypha only, no photobiont).
Pruinose appearance present frequently but not always on all lamina (certain squamules have
scattered acanthohyphidia, which also lack soredia). Soredia present and abundant across the
thallus. Thallus in cross section 100–120 µm thick, dominated by a thick photobiont layer, and
with a thin cortex and medulla. Photobiont Rhizonema clusters of densely coiled cyanobacterial
filaments wrapped within a hyphal sheath formed by jigsaw puzzle-shaped cells (Cora-type).
Acanthohyphidia small, 4–14 µm long and 4–7 µm wide, mostly irregular but sometimes
subglobose; present on the upper and lower surfaces but most abundant along the thallus
margins, where they sometimes become pronounced and then resemble a prothallus; generally
profuse across the upper surface of squamules that have not merged into a crust (granulose-
sorediate portion).
Distribution and ecology: Known from a single, large collection growing on mosses and
hepatics in a high-elevation cloud forest of the renowned Itatiaia National Park, Brazil, a part of
the Atlantic Forest biome. With the high number of lichens and bryophytes present, the location
is known locally as “the lichenologist and bryologist heaven”. Due to the unique morphology of
A. albomarginatus it is easily overlooked, even by the most careful lichenologists, because from
a distance it looks like a mass of bluish gray hyphae resembling a non-lichenized
cyanobacterium or just developing hyphae.
Comments: The difference in color from the lamina and margins reflects location and
abundance of the acanthohyphidia across the thallus surface. The larger squamules overall have
few acanthohyphidia on the thallus lamina; mostly they are found along the margins, which is
particularly apparent if viewed in cross section under the microscope. Acantholichen
albomarginatus therefore also morphologically differs distinctly from all other species by this
white margin contrasting with the blue-green lamina. Overall it has a far less pruinose
appearance, which easily can be observed under the stereoscope or with a good hand lens. This
character may be well developed only in large thalli, however. Our single collection is a large,
well developed specimen and it is difficult to assess if smaller developmental stages may
resemble other Acantholichen species. Nevertheless the holotype of this species is clearly distinct
from all other Acantholichen species. Only A. sorediatus also has acanthohyphidia similarly
concentrated along the squamule margin, but it distinctly differs by the way its soredia are
formed. In A. albomarginatus the whole thallus is microsquamulose to granular and thus in parts
it irregularly dissolves into sorediate granules. That is not the case for A. sorediatus, where
soredia are restricted to the margin of its squamules. Acantholichen sorediatus further differs by
its vivid green color and the way each squamule is attached at the base not the center of the
squamule. A. albomarginatus is blue green, and it is the only species that has centrally attached
squamules.
Acantholichen campestris Dal-Forno, Spielmann & Lucking, sp. nov. FIG. 3
MycoBank MB814550
Typification: BRAZIL. SANTA CATARINA: Campo Alegre, Campos do Quiriri, 26°01′35″S,
48°58′57.4″W, 1380 m. On exposed rocky outcrops, on top of the mountain, growing on
liverwort and lichens on rock, 3 Feb 2012, Spielmann, A. A.; L.S. Canês & E.L. Gumboski
10243b (holotype CGMS). Isotype GMUF. GenBank accessions: KT429798 = ITS; KT429810
= 28S; KT429818 = RPB2.
Etymology: campis (Latin) = plains, fields, a reference to the ecosystem where the type
species is found.
Diagnosis: Differs from other species by its continuous, highly branching squamulose to
almost microfruticose thallus.
Thallus microsquamulose; squamules slightly broad to mostly elongated, attached basally
to the substrate and proliferating from the tips, swollen, 0.1–1 × 0.1–1 mm, richly branching,
thus becoming almost microfruticose, gray when dry (fresh material not seen but bluish gray
when rehydrated). Overall uniform white pruinose appearance. Soredia absent. Thallus in cross
section 120–140 µm thick, dominated by a thick photobiont layer and with a thin cortex and
medulla. Photobiont Rhizonema, clusters of densely coiled cyanobacterial filaments wrapped
within a hyphal sheath formed by jigsaw puzzle-shaped cells (Cora-type). Acanthohyphidia
small, 10–15 × 6–8 µm, pyriform.
Distribution and ecology: This is the only species of Acantholichen presently known to
overgrow lichens and liverworts that inhabit rock, not bark. The Campos do Quiriri is part of the
Brazilian Atlantic Forest biome, under a subcategory classified as high-altitude fields (“Campos
de Altitude”), featuring a dominance of low vegetation, shrubs and small trees. This species was
found on the top of the mountain, in an area with many rocky outcrops.
Comments: This species Acantholichen shares many characters with the other species
described here, such as a small acanthohyphidia and an overall swollen appearance of its
squamules. However, the species differs from all others in forming a continuous thallus, where
individual squamules repeatedly branch and proliferate, giving the thallus an overall
microfruticose aspect, the squamules often growing into almost erect, vertical structures. All
other species grow more or less prostrate on the substrate. Acantholichen campestris is also the
only species so far known from rocks growing outside densely forested humid habitat.
Unfortunately it was not possible to document this species with photographs in the field because
the material was discovered only by accident among a herbarium specimen of Cora.
Nevertheless this collection was relatively recent and sufficiently fresh for the molecular analysis
and it is therefore possible that the color of rehydrated specimens does not differ significantly
from that of fresh specimens.
Acantholichen galapagoensis Dal-Forno, Bungartz & Lücking, sp. nov. F
IG. 4
MycoBank MB814551
Typification: ECUADOR. GALÁPAGOS ISLANDS: Isla Santa Cruz, along trail from Bellavista
to El Puntudo, upper Cinchona forest, 0°39′002″S, 90°20′42″W, 684 m. Dense forest of
Cinchona pubescens, some live trees but mostly dead trees due to management control of the
invasive trees, on bryophyte, growing over Frullania sp., 23 Jun 2010, Dal-Forno, M. 1205
(holotype CDS 44756). Isotypes GMUF, F, B. GenBank accessions: KT429785 = ITS;
KT429800 = 28S; KT429812 = RPB2.
Etymology: The epithet refers to the type locality.
Diagnosis: Differs from other species by its elongated, light bluish gray to olivaceous,
occasionally necrotic beige squamules, which can grow into large thalli.
Thallus microsquamulose; squamules elongated, attached basally to the substrate and
proliferating from the tips, and moderately swollen, 0.1–0.2(–0.3) mm broad, up to 2(–3) mm
long, abundantly branched and typically intricately tiled, many growing together, occasionally
thus shading one another, light blue gray to olivaceous when fresh, darker olivaceous gray when
dry, the shaded parts becoming necrotic and pale beige to orange. Overall uniform pruinose
appearance. Soredia absent. Thallus in cross section 130–160 µm thick, dominated by a thick
photobiont layer, and with a thin cortex and medulla. Photobiont Rhizonema, clusters of densely
coiled cyanobacterial filaments wrapped within a hyphal sheath formed by jigsaw puzzle-shaped
cells (Cora-type). Acanthohyphidia typically small, rarely of moderate size, 12–16(–20) × 6–10
µm, subglobose to pyriform.
Distribution and ecology: All material of Acantholichen collected in Galápagos belongs
to the same species, and it is considered here to be endemic to this archipelago. In general
specimens do not grow directly on trees or shrubs but typically establish on epiphytic liverworts
and mosses, which in turn are common on a variety of substrates, including introduced trees
(Cinchona pubescens, Psidium guava), native shrub (Zanthoxylon fagara) and endemic trees
(Scalesia pedunculata, Psychotria spp.). One specimen was collected on a soil-inhabiting
bryophyte (Campylopus sp.). The populations overgrowing Frullania (Jubulaceae,
Marchantiophyta) on the introduced tree Cinchona in Santa Cruz represent the best developed
material. The type specimen was collected in this particular habitat, where the endemic A.
galapagoensis is surprisingly abundant. At the type locality individual thalli are among the
largest and best developed specimens known, covering dead tree trunks and up to 1 m long,
indicating that the species thrives particularly well in these humid highlands around El Puntudo
and Cerro Crocker. In contrast collections from all other islands are mostly minute and not well
developed, with the notable exception of collections from Cerro Azúl, one of the highest and the
southernmost volcano of Isabela, where specimens grow exuberantly and abundantly on the dead
basal sheaths of fronds of the endemic Galápagos tree fern (Cyathea weatherbyana). Because
Cinchona is introduced to the archipelago, one can assume that the endemic, now threatened tree
ferns represent the original, natural habitat of this endemic Acantholichen.
Other specimens examined: ECUADOR. GALÁPAGOS ISLANDS: Isla Isabela, Volcán Alcedo, 1100 m,
on bryophytes, growing over hepatics on Zanthoxylum fagara, 7 Mar 2006, A. Aptroot No. 65187 (CDS 31771,
GMUF); 1066 m, on bark of Zanthoxylum fagara, 6 Mar 2006, F. Bungartz No. 4125 (CDS 28152, GMUF); −Isla
San Cristóbal, Cerro San Joaquín, 771 m, on bark, branches and twigs of Miconia robinsoniana, 24 Aug 2008, F.
Bungartz No.8577 (CDS 41223, GMUF, F). −Isla Santa Cruz, along trail from Bellavista to El Puntudo, 684 m, on
bryophyte, growing over Frullania sp., 23 Jun 2010, M. Dal-Forno No. 1202 (CDS 44753, GMUF), M. Dal-Forno
No. 1204 (CDS 44755, GMUF, F); 733 m, on bark, trunk of Cinchona pubescens, 8 Feb 2007, F. Bungartz No. 5593
(CDS 33035, GMUF, F, B); close to El Puntudo, 735 m, on bark of Scalesia pedunculata, 23 Feb 2007, F. Nugra
No. 400 (CDS35155, GMUF, F); eastern slope below the summit of El Puntudo, 780 m, on bark of Cinchona, 28
Feb 2006, A. Aptroot No. 64679 (CDS 31253, GMUF, F); NE slope of El Puntudo, 813 m, on bark of Cinchona
pubescens, 10 Aug 2008, F. Bungartz No.8152 (CDS 40798, GMUF, F); behind El Puntudo, previously the farm of
Don Benito, 732 m, on bark of Cinchona pubescens, 3 Feb 2007, F. Nugra No. 379 (CDS 35134, GMUF, F); Isla
Santiago, permanent plot No. 11 Pampa dentro, 870 m, on bark of Zanthoxylum fagara, 24 Mar 2006, A. Aptroot No.
65554 (CDS 32142, GMUF, F).
Comments: Our phylogenetic studies demonstrate that this species, as most other
basidiolichens in the Galápagos, are endemic to the archipelago (Dal-Forno et al. in prep). The
phylogeny places the species as a sister of the clade containing A. campestris and A. variabilis.
Its most diagnostic characters are the olivaceous thalli of elongated and intricate, often
overlapping or “tiled”, squamules; this structural arrangement immediately differentiates A.
galapagoenis from other Acantholichen species. Another characteristic of this taxon is that, when
well developed, the thallus can get unusually large, occasionally covering bryophytes on tree
trunks up to 1 m long. In addition, in large specimens the thallus center squamules often become
necrotic. Because of the tiled, overlapping growth of the squamules, squamules in the thallus
center regularly become shaded and are unable to photosynthesize; these areas then lose the
characteristic pigmentation of the photobiont and become beige (FIG. 6a, b). Squamules of other
Acantholichen species are mostly broader and rarely overlap each other, thus typically do not
become necrotic. The species first was reported from the archipelago as Acantholichen
pannarioides (Jørgensen 1998, Yánez et al. 2012), but the molecular data presented here and a
thorough morphological and anatomical analysis of all material clearly indicates that all reports
are based on one and the same species, which is endemic to the Galápagos. Throughout the
archipelago no other lichen closely resembles A. galapagoensis (Bungartz et al. 2013).
Acantholichen pannarioides P.M. Jørg., Bryologist 101(3):444 (1998) F
IG. 5
Type: COSTA RICA. HEREDIA: NE of Heredia, S slope of Barva volcano, “Calle Cienaga’,
Concepcion, 1580–1700 m, 17 Feb 1990, Döbbeler, P. & J. Poelt s.n. (GZU).
Thallus microsquamulose; squamules broad to slightly elongated, basally attached to the
substrate, swollen, up to 2 × 1.5–1.8 mm, moderately branched, grayish blue green with slightly
paler margins when fresh, dark blue when dry. Overall uniform, white pruinose appearance.
Soredia absent. Thallus in cross section 200–240 µm thick, with a thick photobiont layer, a thin
cortex and an unusually well developed thick medulla with abundant acanthohyphidia.
Photobiont Rhizonema, clusters of densely coiled cyanobacterial filaments wrapped within a
hyphal sheath formed by jigsaw puzzle-shaped cells (Cora-type). Acanthohyphidia large, 28–50
× 6.5–9.5 µm, elongate to clavate.
Distribution and ecology: Our single recent collection of this taxon, identified by
comparison with the description and images (light and SEM microscope) of the type material,
corroborates the distribution pattern because our specimen was found in a wet montane forest in
the Cordillera Talamanca of Costa Rica, where the material grows on an old fence post along
with species of Heterodermia and Frullania. Jørgensen (1998) also considered material from
Venezuela (BG), continental Ecuador (GB) and the Galápagos (COLO) as part of A.
pannarioides. The Galápagos specimens here are described as new and we have had no access to
the other material because it was not found in the cited herbaria. Therefore it presently is not
possible to confirm that this species occurs outside Costa Rica; most likely the specimens from
Venezuela and Ecuador represent one of the other species distinguished here or are entirely novel
taxa.
Comments: The acanthohyphidia in Acantholichen pannarioides are considerably longer
than in the other taxa, except for A. variabilis, in which they can be as large but are overall much
more variable in size. Morphologically the two species have a very different colors when fresh;
A. pannarioides is gray blue green with a lighter greenish hue toward the tips, while A. variabilis
is dark blue uniformly. Acantholichen campestris also resembles A. pannarioides, but the
squamules of the latter species are always sparse, growing separate and are considerably broader.
Acantholichen campestris in comparison develops a much more continuous thallus with richly
branching squamules that look almost microfruticose.
Other specimens examined: COSTA RICA. PUNTARENAS: Las Alturas Biological Station, near the
Panama border on the western slopes of the Talamancan range, montane rain forest, 8°56′43″N, 82°50′00″W, 1500
m. Growing mostly on wood from fence but also on mosses, 25 May 2012, M. Dal-Forno No. 1752 (GMUF,
INBio).
Acantholichen sorediatus Dal-Forno, Sipman & Lücking, sp. nov.
FIG. 6
MycoBank MB814552
Typification: COSTA RICA. PUNTARENAS: San Vito de Coto Brus, Las Cruces
Biological Station; 82°58′W, 8°47′N, 1200 m. On ridge beyond Río Java, undergrowth of
disturbed primary forest; growing on trunk, with other lichens (Hypotrachyna, Normandina,
Leptogium), Oct 2004, Lücking, R. s.n. (holotype CR). Isotype GMUF. GenBank accessions:
KT429794 = ITS; KT429806 = 28S.
Etymology: The epithet refers to the marginal soredia found in this species.
Diagnosis: Differs from other species by the green granular to microsquamulose thallus
with the formation of soredia along the squamule margin.
Thallus granular to microsquamulose; granules initially 0.1 mm diam, eventually forming
larger, broad squamules, basally attached to the substrate, swollen, 0.25–1.5 × 0.3–0.9 mm, not
to moderately branched, green when fresh, but becoming dark bluish gray when dry. White
pruinose appearance due to acanthohyphidia on both upper and lower surface, although these
spiny hyphae are more abundant along the margin of the squamules, where granular, ecorticate
soredia are formed. Soredia present, marginal and frequent. Thallus in cross section 130–190 µm
thick, dominated by a thick photobiont layer and with a thin cortex and medulla. Photobiont
Rhizonema, clusters of densely coiled cyanobacterial filaments wrapped within a hyphal sheath
formed by jigsaw puzzle-shaped cells (Cora-type). Acanthohyphidia of moderate size, 15–20 ×
5–8 µm, irregular, pyriform to subclavate.
Distribution and ecology: The type locality is part of the Cordillera Central in Costa
Rica, an area with active volcanos. This ecology appears similar to areas where specimens of A.
galapagoensis grow on Santa Cruz Island in the Galápagos. Nevertheless specimens of A.
sorediatus are not only molecularly well separated they are also morphologically distinct. Both
specimens examined grow directly on the bark, while other species tend to grow on bryophytes.
Other specimen examined: COSTA RICA. CARTAGO: Irazú Volcano National Park, part of the Cordillera
Volcanica Central Conservation Area, summit of the Irazu Volcano, 25 km ENE of San Jose, access road to crater
and surroundings, 9°59′N, 83°51′W, 3300 m. Alpine paramo zone, disturbed paramo vegetation, on bark (stem), 6
Jul 2002, H.J.M. Sipman No. 48329 (B, GMUF, F).
Comments: The most remarkable feature of A. sorediatus is the formation of true soredia
along the margin of its squamules. The species has acanthohyphidia that are mostly irregular and
of relatively uniform, moderate size, in that regard somewhat similar to those of A.
albomarginatus, although typically slightly longer. These two species also share a granulose-
sorediate thallus morphology, but the color (green vs. blue green) and squamules (swollen vs.
flattened) are easily distinguishable. Acantholichen campestris also resembles A. sorediatus, but
this species lacks soredia, although its highly pruinose squamules can be densely branched along
their rim, a character that could be mistaken for the formation of soredia.
Acantholichen variabilis Dal-Forno, Coca & Lücking, sp. nov. F
IG. 7
MycoBank MB814553
Typification: COLOMBIA. VALLE DEL CAUCA: Cerro San Antonio (= Cerro de la Horqueta),
Dagua, 10 km by via El Mar, 03°29′40″N, 76°37′25″W, 1946 m. Lower montane wet forest, in
forest edge, high light intensity, on moss, 08 April 2014, Coca, L. F. 5209 (holotype FAUC).
Isotypes CUVC, GMUF, F. GenBank accessions: KT429796 = ITS; KT429808 = 28S.
Etymology: The epithet refers to the variable size of the acanthohyphidia in this species.
Diagnosis: Differs from other species by dark blue, much inflated, rounded squamules
with abundant acanthohyphidia on both sides that are highly variable in structure and size.
Thallus microsquamulose; squamules broad to elongated, basally attached to the
substrate, swollen, 0.1–1 mm × 0.1–0.5 mm, moderately to abundantly branched, dark blue when
fresh but becoming dark bluish gray when dry. Overall uniform pruinose appearance. Soredia
absent. Thallus in cross section 110–130 µm thick, dominated by a thick photobiont layer, and
with a thin cortex and medulla. Photobiont Rhizonema, clusters of densely coiled cyanobacterial
filaments wrapped within a hyphal sheath formed by jigsaw puzzle-shaped cells (Cora-type).
Acanthohyphidia highly variable in size, small, medium or large, 10–50 × 8–10(–12) µm,
subglobose, pyriform to subclavate when short and elongate to clavate when large.
Distribution and ecology: This species so far is known only from a single collection from
lower montane wet forest in Colombia. It shares a similar ecology with A. albomarginatus, in
which both species are found in forests of high altitude, in habitats that are wet but nevertheless
exposed to high light intensity, such as the edge of the forest.
Comments: This species much resembles the type species Acantholichen pannarioides
overall in morphology and anatomy. However, when fresh the material is more swollen, rounder
and much darker than that of A. pannarioides, which remains distinctly more greenish when
rehydrated. The dried squamules get a little flattened, thus to some extent resembling A.
albomarginatus, but differing by the location of acanthohyphidia on the squamules (in A.
albomarginatus these are not as evenly distributed across the surface, especially where
individual squamules fuse into a crust and have a white margin). Another distinct characteristic
of A. variabilis is the highly variable size of its acanthohyphidia. Even across one single
squamule these cells vary considerably from very small to large.
DISCUSSION
Our multilocus phylogeny (ITS, 28S, RPB2), combined with anatomical and morphological
characters, indicates that Acantholichen as currently defined is monophyletic and that at least six
distinct species in the genus can be distinguished, one from the Galápagos Islands, two from
Brazil, one from Colombia and two from Costa Rica, including the type species, A. pannarioides
(Jørgensen 1998). The Acantholichen clade is monophyletic and sister to the genus Corella in a
large, diverse and entirely lichenized clade, Dictyonema sensu lato an important element within
the agaric family Hygrophoraceae (Dal-Forno et al. 2013, Lodge et al. 2014).
Before studies by our working group beginning with Chaves et al. (2004), the taxonomy of
basidiolichens assigned to Dictyonema received little attention, with the exception of
Oberwinkler (1970, 2001, 2012) and Parmasto (1978). The monotypic lichen Acantholichen
pannarioides, when first described (Jørgensen 1998), was not known to be a member of this
group until the molecular phylogenetic study of Lawrey et al. (2009) established its position in
the Dictyonema clade. Apart from Acantholichen the genera now recognized in this clade (Dal-
Forno et al. 2013) are Cora, Dictyonema sensu stricto, Corella and Cyphellostereum, all
containing lichenized basidiomycetes with a cyanobacterial Rhizonema photobiont.
All presently recognized Acantholichen species share the same overall aspect, with
microsquamulose thalli and acanthohyphidia that are not found elsewhere in the Dictyonema
clade. However, they share the paraplectenchymatous cortex as a synapomorphy with Corella.
Acantholichen species differ mainly in color, size, arrangement and overall appearance of the
squamules, and shape, size and location of the acanthohyphidia. Thallus colors include various
hues of blue, green and gray, all typical colors of Dictyonema s.l. caused by the Rhizonema
photobiont (Lücking et al. 2009, Lücking et al. 2014a). In the Cora-clade, which includes
Acantholichen, Corella and Cora, the cyanobacterium Rhizonema form clusters of more or less
individual, angular-rounded cells that are surrounded by a jigsaw puzzle-shaped hyphal sheath.
The characteristic spiny hyphae, acanthohyphidia, found in Acantholichen species are not
exclusive to this genus. Although not found in other lichenized fungi, acanthohyphidia are
reported for many nonlichenized basidiomycetes, and it was the presence of these characteristic
hyphae that led Jørgensen (1998) to propose Acantholichen as a new genus of basidiolichens.
Morphologically they are similar to the spiny periphysoids and/or periphyses in Fissurina and
Acanthothecis (Graphidaceae, Ascomycota) (Staiger 2002), but the latter are much smaller and
often difficult to discern. At present the function of acanthohyphidia in Acantholichen is
unknown. However, we can hypothesize that they may serve as water repellants or regulators.
When rehydrating specimens with acanthohyphidia on the entire surface, water is not absorbed
immediately but only after adding extra pressure with forceps. Specimens with acanthohyphidia
mostly on squamule margins absorb water quickly, as usually happens in other genera within the
Dictyonema s.l. clade. Obviously this hypothesis requires further testing.
The precise phylogenetic position of Acantholichen within the Dictyonema clade was not
well established until Dal-Forno et al. (2013) showed that Corella is sister of Acantholichen and
not Cora, as had been hypothesized based on similar morphology and anatomy. The close
relationship of Acantholichen and Corella is supported by similar cortical structures in these
genera. Acantholichen and Corella species have an upper cortex formed by multilayers of
rounded to angular and densely packed hyphae, while Cora species have a cortex formed by
loosely interwoven elongated hyphae. It is open to question whether the foliose or
microsquamulose growth form is ancestral in the Acantholichen-Corella clade. However,
because the sister clade Cora is entirely foliose the most parsimonious hypothesis would be for
Acantholichen to be derived from a foliose or macrosquamulose form similar to Corella and
some Cora species.
Notably until now neither Corella nor Acantholichen have ever been found fertile whereas
Cora species frequently produce basidiocarps. However, this does not necessarily mean that they
have permanently lost the ability to reproduce sexually. With our current collections, including
several yet undescribed species in the genus Corella, we are not abandoning the possibility that
fertile structures will be found in known or new species in either genus.
Although there is good support for the monophyly of four of the five groups we recognize
as genera in the Dictyonema clade, one genus, Dictyonema s.s., appears to be paraphyletic
(Lawrey et al. 2009, Dal-Forno et al. 2013). Lawrey et al. (2009) demonstrated that the
monophyly of the genus could not be rejected based on Simodaira-Hasegawa tests whereas
Bayes Factor for topology hypothesis testing based on an expanded dataset (Dal-Forno unpubl)
rejects the monophyly of Dictyonema s.s. Because this issue remains unresolved, we are taking a
conservative approach and retain Dictyonema s.s. as a transitional grade linking the basal
mushroom-like Cyphellostereum species to the more derived lichenized foliose species of the
genus Cora. An alternative solution would be to use a single genus, Dictyonema, for the entire
clade or else split Dictyonema s.s. further into smaller genera. We do not favor either solution at
this point but think that a large number of morphoanatomical characters separate the four
monophyletic clades and the single, paraphyletic grade we are recognizing as genera, showing a
clear pattern of increased thallus complexity from primitive to more derived clades (Dal-Forno et
al. 2013).
Our discovery of several new species of Acantholichen, combined with reports of newly
described basidiolichens in other clades, supports the notion that lichenized species may
represent an important source of undescribed biodiversity in the Basidiomycota. An example is
the discovery by Lücking et al. (2014b) of a remarkable number of unrecognized species in what
Parmasto (1978) originally considered to be a single species, Cora pavonia (= Dictyonema
glabratum). Large Cora species are hardly overlooked, but the minute Acantholichen squamules
are so tiny and easily mistaken for something else that the low known diversity probably results
from specimens escaping notice in the field. Similarly, as shown in the genus Cora in Lücking et
al. (2014b), preliminary data suggest there may be high levels of local endemism in
Acantholichen species, such as A. galapagoensis, so far known only from the Galápagos Islands.
However, more collections are needed for all species to clarify their actual distributions and
ecologies.
Our study emphasizes the importance of examining species using molecular tools but also
studying their morphology and anatomy in the field. Many important characters present in fresh
material, especially color and texture, are lost in herbarium specimens over time. This may
explain why previous investigators were unable to recognize species variability with dried
specimens. Our results thus suggest that these lichens must be observed, photographed and
measured when fresh, in much the same way that other mushroom-forming agarics are treated.
ACKNOWLEDGMENTS
The study was supported by NSF grant DEB 0841405, “Phylogenetic diversity of mycobionts and photobionts in the
cyanolichen genus Dictyonema, with emphasis on the Neotropics and the Galápagos Islands” (PI J. Lawrey, Co-PIs
R. Lücking, P. Gillevet, local Galápagos coordinator F. Bungartz). We acknowledge help from participants in a
course organized by the Organization for Tropical Studies (OTS), during which we were permitted to collect in
several areas of Costa Rica. We want to thank L.S. Canêz and E. Gumboski for their help during a collection trip
that lead to the discovery of A. campestris. Similarly we thank Bianca Regina da Hora and Agnes Elisete Luchi, for
their field help in the Itatiaia National Park. Morgan Gostel is thanked for providing the SEM image of A.
galapagoensis. The Charles Darwin Foundation continues to support the Galápagos Lichen Inventory, which
permitted access to Acantholichen specimens freshly collected in the archipelago. For this we are especially
indebted to Galápagos National Park, particularly Washington Tapia and Galo Quedaza for granting specimen
export permits. The lichen inventory is part of the Census of Galápagos Biodiversity by Charles Darwin Foundation
(donors cited at http://www.darwinfoundation.org/datazone/checklists/). Taxonomic research on Galápagos species,
with the goal of establishing the first IUCN red list of endemic Galápagos lichens, is supported by the Mohamed bin
Zayed Species Conservation Fund, project 152510692. This study is contribution number 2122 of the Charles
Darwin Foundation for the Galápagos Islands.
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LEGENDS
FIG. 1. Phylogenetic trees inferred by maximum likelihood (RAxML) using three loci (ITS, 28S, RPB2). A.
Dictyonema clade: 22 specimens plus Eonema pyriformis as outgroup, genera belonging to the Cora-clade
highlighted. B. Acantholichen: 17 specimens with Corella brasiliensis as outgroup, distinct species highlighted.
Support values are below the branches for ML for both trees and Bayesian Inference (BI) with partitioned data for
1b only. Branches were thickened for moderate to strong values (ML bootstrap values above 70 and BI posterior
probabilities above 0.95).
F
IG. 2. Acantholichen albomarginatus (holotype). A. Habitat. B, C. Growth aspect and color in fresh material. D, E.
Growth aspect and color in dry material. F. Acanthohyphidia. Bars: B = 3 mm, C = 1 mm, D = 1 mm, E = 0,1 mm, F
= 5 μm.
F
IG. 3. Acantholichen campestris (isotype). A. Habitat. B, C. Growth aspect and color in rehydrated material. D, E.
Growth aspect and color in dry material. F. Acanthohyphidia. Bars: B = 1 mm; C = 1 mm; D = 1 mm; E = 1 mm; F
= 15 μm.
F
IG. 4. Acantholichen galapagoensis. A. Growth aspect and color in fresh material (F. Bungartz 5593), with
yellowish brown bases in old parts of the thallus. B. Growth aspect and color in dry material but wetted (holotype).
C, D. Growth aspect and color in dry material (F. Bungartz 5593). E. Heteromerous thallus cross section, showing
cortex, photobiont layer and medulla (F. Bungartz 5593). F. Acanthohyphidia, inlay SEM image of the
acanthohyphidia (isotype GMUF). Bars: A = 1 mm, B = 1 mm, C = 1 mm, D = 0.1 mm, E = 50 μm, F = 10 μm.
F
IG. 5. Acantholichen pannarioides (M. Dal-Forno 1752). A, B. Growth aspect and color in fresh material. C.
Growth aspect and color in dry material. D. Paraplectenchymatous cortex. E, F. Acanthohyphidia. Bars: A = 2 mm,
B = 2 mm, C = 1 mm, D = 10 μm, E = 10 μm, F = 10 μm.
F
IG. 6. Acantholichen sorediatus (holotype). A−C. Growth aspect and color in fresh material. D. Growth aspect and
color in dry material. E. Acanthohyphidia. F. Paraplectenchymatous cortex. Bars: A = 1 mm, B = 1 mm, C = 1 mm,
D = 1 mm, E = 10 μm, F = 20 μm.
F
IG. 7. Acantholichen variabilis (holotype). A, B. Growth aspect and color in fresh material. C, D. Growth aspect
and color in dry material. E. Close up of acanthohyphidia. F. Acanthohyphidia. Bars: A = 1 mm, B = 1 mm, C = 0.1
mm, D = 0.1 mm, E = 0,1 mm, F = 10 μm.
FOOTNOTES
Submitted 5 Mar 2015; accepted for publication 2 Oct 2015.
1
Corresponding author. E-mail: manudalforno@hotmail.com
T
ABLE I. Characters distinguishing species in Acantholichen
Appearance
of squamules
Attachment
of the
squamules
Growth of
the
squamules
Branching
pattern
Soredia Color fresh Color dried Acanthohyphidia
location
Acanthohyphidia
size and shape
Geographical
location
A.
albomarginatus
Mostly flat;
partly broad,
partly
granulose
Squamules
attached in
the center
Scattered to
many
growing
together
Sparsely to
moderately
branched
Present,
across the
entire
thallus
Blue green
with white
margins
Dark bluish
green and
gray
Predominantly
marginal, but also
across the lamina
Small (≤ 15 μm),
mostly irregular,
rarely subglobose
Brazil, southeast
(this study)
A. campestris Swollen;
slightly broad
to mostly
elongated
Squamules
attached at
the base and
proliferating
from their
tips
Growing
together
Abundantly
branched to
microfruticulose
Absent Not
available
(bluish gray
when
rehydrated)
Gray Laminal (evenly
across the
surface)
Small (≤ 15 μm),
pyriform
Brazil, south (this
study)
A.
galapagoensis
Swollen;
elongated
Squamules
attached at
the base and
proliferating
from their
tips
Many
growing
together
Abundantly
branched
Absent Light blue-
gray to
olivaceous
Dark
olivaceous
gray
Laminal (evenly
across the
surface)
Small (≤ 15 μm,
rarely up to 20 μm),
subglobose to
pyriform
Galápagos Islands
(this study)
A. pannarioides Swollen;
broad to
elongated
Squamules
attached at
the base
Scattered to
slightly
growing
together
Moderately
branched
Absent Grayish
blue green
Dark blue Laminal (evenly
across the
surface)
Large (up to 50 μm),
elongate to clavate
Costa Rica (this
study, Jørgensen
1998); Venezuela
and Ecuador
(Jørgensen 1998)
A. sorediatus Swollen;
broad
Squamules
attached at
the base
Scattered Not or very
sparsely
branched
Present,
marginal
Green Dark bluish
gray
Predominantly
marginal, but also
across the lamina
Medium (15–20
μm), irregular,
pyriform to
subclavate
Costa Rica (this
study)
A. variabilis Swollen;
broad to
elongated
Squamules
attached at
the base
Scattered to
slightly
growing
together
Moderately to
abundantly
branched
Absent Dark blue Dark bluish
gray
Laminal (evenly
across the
surface)
Small, medium and
large (10–50 μm),
subglobose,
pyriform to
subclavate when
short, elongate to
clavate when large
Colombia (this
study)
