Abstract and Figures

Based on a combination of morphological and molecular investigations, a critical revision of the widely distributed myxomycete Tubifera ferruginosa is presented. A phylogeny of the morphospecies, based on partial 18S nuc rDNA sequences, displays several clearly distinct clades, all differing by a genetic distance (p distance) of at least 0.15, with the distance within the clades below 0.11. These molecular differences correlate with morphological characters, such as the shape of sporothecal tips, the color of immature fructifications and the ultrastructure of the inner surface of the peridium. The combination of morphological and molecular data provides evidence that T. ferruginosa is actually a species complex, representing at least seven species. These are T. ferruginosa sensu stricto, T. applanata, T. corymbosa, T. dudkae, T. magna, T. montana and T. pseudomicrosperma. Among these T. applanata and T. dudkae (as Reticularia dudkae) were described recently based on morphological characters and the 18S nuc rDNA phylogeny confirmed their separation. Another four species, T. corymbosa, T. magna, T. montana and T. pseudomicrosperma, are described here. We propose an epitype for T. ferruginosa sensu stricto and recognize subsp. ferruginosa and subsp. acutissima within this species. All studied taxa of the T. ferruginosa complex are shown to lack a capillitium. Structures formerly described as capillitium represent the hyphae of fungi occurring within the fructifications. Copyright © 2015, Mycologia.
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A critical revision of the
Tubifera ferruginosa
Dmitry V. Leontyev
Department of Biotechnology, Kharkiv State Zoovet-
erinary Academy, Akademichna Str. 1, Kharkiv,
Ukraine 62341
Martin Schnittler
Institute of Botany and Landscape Ecology, Ernst
Moritz Arndt University Greifswald, Soldmannstr. 15,
Griefswald, Germany D-17487
Steven L. Stephenson
Department of Biological Sciences, University of
Arkansas, Fayetteville, Arkansas 72701
:Based on a combination of morphological
and molecular investigations, a critical revision of
the widely distributed myxomycete
Tubifera ferrugi-
is presented. A phylogeny of the morphospecies,
based on partial 18S nuc rDNA sequences, displays
several clearly distinct clades, all differing by a genetic
distance (
distance) of at least 0.15, with the distance
within the clades below 0.11. These molecular
differences correlate with morphological characters,
such as the shape of sporothecal tips, the color of
immature fructifications and the ultrastructure of the
inner surface of the peridium. The combination of
morphological and molecular data provides evidence
T. ferruginosa
is actually a species complex, repres-
enting at least seven species. These are
T. ferruginosa
sensu stricto,
T. applanata
T. corymbosa, T. dudkae
magna, T. montana
T. pseudomicrosperma
. Among
T. applanata
T. dudkae
) were described recently based on morpho-
logical characters and the 18S nuc rDNA phylogeny
confirmed their separation. Another four species,
T. corymbosa, T. magna, T. montana
T. pseudomi-
, are described here. We propose an epitype
T. ferruginosa
sensu stricto and recognize subsp.
and subsp.
within this species.
All studied taxa of the
T. ferruginosa
complex are
shown to lack a capillitium. Structures formerly
described as capillitium represent the hyphae of fungi
occurring within the fructifications.
Key words:
18S rRNA gene, capillitium, phyloge-
ny, Reticulariaceae, species description
The myxomycete genus
J.F. Gmel. is charac-
terized by the formation of pseudoaethalia, dense
clusters of sporothecae that may extend several
centimeters. In
, the sporothecae mostly are
cylindrical, are covered with a membranous peridium
and filled with banded-reticulate spores. The most
common species in the genus,
T. ferruginosa
J.F. Gmel., is easy to recognize by its prostrate spongy
hypothallus, rusty brown spore mass and sparsely
occurring columella, which never reaches the apex
of a sporotheca (Martin and Alexopoulos 1969,
Nannenga-Bremekamp 1991, Neubert et al. 1993,
Ing 1999). Because of its wide distribution and
conspicuous fructifications,
T. ferruginosa
has been
reported in nearly all field surveys that have focused on
wood-inhabiting myxomycetes (www.discoverlife.org).
As is the case for most lignicolous myxomycetes,
there are no reports of the successful culture of
T. ferruginosa
under laboratory conditions (Clark and
Haskins 2010).
During the nearly 200 y that
T. ferruginosa
been studied, the taxonomic status of the species has
never been seriously questioned, although Lado
(2005–2015) listed no less than 19 heterotypic
synonyms of this name. A detailed analysis provided
by Nannenga-Bremekamp (1961) led to the conclu-
sion that there is “no sufficient reason” to divide
T. ferruginosa
into several species. However, more than
40 y later, Leontyev and Fefelov (2005) postulated
T. ferruginosa
might represent a complex of
cryptic species. Later two morphotypes of the species
were described as distinct taxa:
Tubifera applanata
Leontyev & Fefelov (Leontyev and Fefelov 2009,
2012) and
Reticularia dudkae
Leontyev & G. Moreno
(Leontyev and Moreno 2011). The primary character
used to separate these two taxa from the species
complex was the shape of the sporothecae, which
were cylindrical and convex in typical
T. ferruginosa
cylindrical and flat in
T. applanata
and spherical in
R. dudkae
. A second distinguishing character was the
ultrastructure of the inner surface of the peridium,
which was smooth in typical
T. ferruginosa
, incrusted
by rings in
T. applanata
and covered by wave-like
folds in
R. dudkae
. For the latter the spherical
sporothecae was the reason for assigning the species
to the closely related genus
(Leontyev and
Fefelov 2009, 2012; Leontyev and Moreno 2011) and
not to
. However, as we will describe herein
this placement was incorrect. Nevertheless, both new
Submitted 15 Oct 2014; accepted for publication 25 May 2015.
Corresponding author. E-mail: protista@mail.ru
107(5), 2015, pp. 959–985. DOI: 10.3852/14-271
#2015 by The Mycological Society of America, Lawrence, KS 66044-8897
species appear quite similar to
T. ferruginosa
and the
taxonomic value of the characters used in their
circumscription remained questionable.
Even after the exclusion of
T. applanata
T. ferruginosa
complex remains rather
polymorphic. Both field observations and morpho-
logical comparisons of herbarium specimens revealed
differences in the color of immature fructifications,
the shape of the sporothecal tips, the color and
iridescence of the peridium, the overall size of the
pseudoaethalium and spore diameter. These observa-
tions inspired the present study, which was undertak-
en to disentangle the various taxonomic entities that
make up this intricate species complex.
In an effort to determine the correlation between
phenotypic and genotypic variability, we sequenced
the first part of the 18S nuc rDNA in
T. ferruginosa
and a number of related species. This gene seems
to be a promising marker for barcoding of protists in
general (Pawlowski et al. 2012) and is especially
variable in myxomycetes (Fiore-Donno et al. 2012,
2013; Nandipati et al. 2012). However, the 10 known
insertion sites for group I introns (Lundblad et al.
2004, Fiore-Donno et al. 2013) often make it difficult
to obtain the entire 18S gene sequence in myxomy-
cetes. Fortunately a portion consisting of the first ca.
700 bp of this gene (most of the 59domain) is free of
introns, possesses three extremely variable helices and
can be obtained in one amplification step (Novozhi-
lov et al. 2013).
We examined a total of 152 specimens, 111 of
which corresponded to the traditional concept of
sensu Martin & Alexopoulos (1969), Nannenga-
Bremekamp (1991) and Ing (1999). Using the morpholog-
ical characters mentioned above,
T. applanata
(18 speci-
mens) and
Reticularia dudkae
(6) were separated. The
remaining 87 specimens were preliminarily considered to
T. ferruginosa.
To reconstruct the phylogeny of the
T. ferruginosa
complex, we examined several related species. These were
T. microsperma
(Berk. & M.A. Curtis) G.W. Martin (five
Lindbladia tubulina
Fr. (two),
Lycogala epiden-
(L.) Fr. (five),
L. flavofuscum
(Ehrenb.) Rostaf. (one),
sp. (four),
Reticularia jurana
Meyl. (seven),
R. splendens
Morgan (eight),
R. lycoperdon
Bull. (one),
R. olivacea
(Ehrenb.) Fr. (two) and four species of the recently
revalidated genus
(see Leontyev et al. 2014a, b, c),
A. bombarda
Berk. & Broome (one),
A. lloydiae
S.L. Stephenson & Schnittler (three),
A. morula
G. Moreno,
Leontyev, D.W. Mitchell, S.L. Stephenson, C. Rojas &
Schnittler (one) and
A. repens
Leontyev, Schnittler, G.
Moreno, S.L. Stephenson, D.W. Mitchell & C. Rojas (two).
Together with nine already published sequences (Fiore-
Donno et al. 2013) labeled as
Cribraria cancellata
Nann.-Bremek. (one specimen),
C. violacea
Rex (one),
Schrad. (one),
T. ferruginosa
T. dimor-
Reticularia lycoperdon
R. jurana
Lycogala epidendrum
(two), a total of 161 partial 18S
rDNA sequences were used in the present study, of which
118 belong to the genus
TION 1). The majority (156) of the sequences represent the
family Reticulariaceae and remaining five (
Lindbladia tubulina
) belong to the Cribrariaceae,
a family that forms a basal clade within the bright-spored
myxomycetes (Fiore-Donno et al. 2013). The material
examined was collected in 15 countries, mostly from the
temperate zone of Eurasia and North America but also from
other regions of the world (SUPPLEMENTARY INFORMATION 1).
Morphological studies.—
Specimens were studied with dis-
secting microscopes (Leica MSV226, Stemi 2000) and light
microscopes (Zeiss Axioscop 2 plus, Zeiss Axio Imager A1),
equipped with differential interference contrast (DIC). The
freeware program CombineZP was used to create a compos-
ite digital image from several stacked images. Microscopic
measurements were made with the program Axio Vision (Carl Zeiss Imaging Solutions GmbH, Germany).
Alphanumeric codes for colors in descriptions are given
according to the Munsell color scale (Munsell 1912).
In all studied specimens, the length, width and height of
pseudoaethalia were determined. Twenty-five spores, peri-
dial rings and warts were measured for each specimen to
derive an estimate for the range of variation. The range
of variation for all the structures is given as (minimum –)
mean minus standard deviation – mean plus standard
deviation (– maximum).
Scanning electron microscopy (SEM) was carried out at
the M. G. Kholodny Botanical Institute, Ukraine, with a Jeol
JSM-6060 microscope and at the University of Arkansas,
using a FEI Nova Nanolab 200 FIB/SEM microscope. In
both cases, sporocarps were sputter-coated with gold-
palladium to form a 5 nm cover and then studied at 5–15 kV.
DNA sequencing and deposition.—
As noted above the first
ca. 700 bp of the nuc 18S rDNA was chosen as a molecular
marker. DNA was extracted from 5 to 6 adjacent sporothe-
cae using the Invitek Spin Food Kit II and Invitec Spin Plant
Mini Kit (Stratec Molecular GmbH, Germany). Sporothecae
were cooled to 280 C in a 2 mL safe-lock Eppendorf tube
along with acid-washed glass beads 0.7–1.1 mm diam (Sigma
Chemicals, Washington) and disrupted 1 min at 30 Hz with
a Wig-L-Bug grinding mill (Reflex Analytical Corporation,
New Jersey). We followed the manufacturer’s protocol
except for the final step, where DNA was eluted in 50 mL
of an elution buffer instead of 200 mL.
DNA was amplified with five primers reported by Fiore-
Donno et al. (2013). These were S1 (59–AACCTGGTT
forward), SR4 Bright (TGCTGGCACCAGACTTGT, reverse)
and SR4Lyc (CCGGACTTGTCCTCCAGT, reverse). About
80%of sequences were obtained with the primer combina-
tion S1/SR4Bright. However, this combination was in-
effective for several species, for which other combinations
were successful. These were
Alwisia lloydiae
SR4Bright, SFATri/SR4Lyc),
Lycogala epidendrum
SR4Lyc, SFATri/SR4Lyc, SF1Tri/SR4Lyc) and
T. magna
The PCR reaction was carried out in 40 cycles (95 C, 2.5
min; 52 C, 30 s; 72 C, 1 min). Results were verified by
electrophoresis in an agarose gel in TA buffer, stained with
a Safe Red (NBS Biologicals Ltd, UK) or Rothi Stain (Carl
Roth GmbH, Germany). The product was purified with MSB
Spin PCRapace (Stratec Molecular GmbH, Germany). In
the purification process, we followed the manufacturer’s
protocols except we added 20 mL elution buffer instead of
the recommended 10 mL when preparing the final solution.
Cycle sequencing was carried out in 40 cycles (96 C, 70 s;
53 C, 5 s; 60 C, 4 min); for fragment detection an ABI
3130xl BigDye 3.1 capillary sequencer was used. All unique
sequences among the 149 obtained were deposited in
Sequence alignment.—
Alignments were made with the online
version of multiple alignment program MAFFT (mafft.
cbrc.jp/alignment/software/), using the E-INS-option (Ka-
toh et al. 2005) and default gap penalties. The alignment is
available on TreeBASE, submission no. 17296 (http://
Sequence comparisons.—
To identify a barcode gap between
18S rDNA sequences that let us consider a pair of specimens
as separate species (Schoch et al. 2012), we checked the
frequency of each value of
-distance index between any two
studied sequences (1890 combinations in total). This index
is calculated as the proportion (
) of nucleotide sites
at which two sequences being compared are different
and varies from 0 (sequences identical) to 1 (no matches)
(Hall 2011, Tamura et al. 2011). The frequencies of all
-values were visualized with a histogram (SUPPLEMENTARY
Phylogenetic analyses.—
The 1219 positions of the alignment
(48–1266) out of the total of 1540 were used to construct
the phylogeny. Analyses were carried out with both
maximum likelihood (ML) and Bayesian inference (BI).
ML was run on MEGA 5.1 (Hall 2011, Tamura et al. 2011),
using bootstrap with 1000 replicates and the NNJ heuristic
search method. BI was computed with MRBAYES 3.2.1
(Huelsenbeck and Ronquist 2001, 2012) using one cold
and three heated Monte Carlo Markov chains in two
simultaneous runs with a temperature increment of 0.2.
The number of generations, sample frequencies and burn-
in ratio were set to 10 million, 10 and 0.25, respectively.
Branch support was estimated by ML bootstrap replicates
and Bayesian posterior probabilities. In both analyses, the
evolutionary model was set to GTR with gamma-distributed
rate variation across sites with a proportion of invariable
sites. The evolutionary model was selected using AICs and
BIC tests in MEGA 5.1.
Generic diversity.—
In both ML and BI phylogenies,
the genera
form rather
well supported clades (FIG. 1), united in one large
monophyletic branch of the Reticulariaceae, with the
same topology as presented by Fiore-Donno et al.
(2013) and Leontyev et al. (2014c). Within the genus
, three species (
R. jurana, R. lycoperdon,
R. splendens
) form a cluster sister to
, while
two other species belong to other clades. According
to our phylogeny,
R. dudkae
appears to be the part of
the cluster of
. This led us to transfer
to the latter. Another species of
R. olivacea
) was found to belong to the cluster of the
Cribrariaceae, instead of the Reticulariaceae.
Species diversity.—
Among the 161 sequences from
members of the Reticulariaceae and Cribrariaceae, we
recognized 62 genotypes, 33 belonging to the 118
specimens of
considered in this study. A
pairwise comparison of all possible combinations
between these genotypes using
distance revealed
a continuum of values, 0.001–0.51, with an average
value of 0.37. However,
levels 0.11–0.15 did not
occur for any of these combinations (SUPPLEMENTARY
INFORMATION 2). We consider this barcode gap as the
natural threshold between the genetic diversity within
a species and between species of the studied family.
A less pronounced gap, at
value 0.02–0.09, separates
the group of specimens that differ from the other
closest genotypes at the
-level of 0.09–0.11. This
difference, found in only a two genotypic clusters,
Tubifera ferruginosa
sensu strictu (s. str.) and
, we consider to be a subspecies threshold
(see below). Finally, all genotypes differing from the
closest genotype at a
level below 0.09 (most of them
even below 0.02) are considered to be elements of
genetic diversity within a particular species and is
indicated herein as sequentially numbered genotypes
(gt1, gt2, gt3 etc.) of this species (SUPPLEMENTARY
values 0.11–0.15 were used as a species
threshold, we united our 61 individual genotypes into
30 clusters, which we consider to be genotypic species.
As a result, in
Alwisia lloydiae, Reticularia jurana
R. lycoperdon
R. splendens
Tubifera microsperma
one morphospecies appeared to be represented by
a single genotypic species, sometimes with several
genotypes differing by
values below 0.11, thus
providing additional evidence of the validity of our
T. ferruginosa, Lycogala epidendrum
R. olivacea
more than one genotypic species was
found within the morphospecies.
L. epidendrum
, four of the ten 18S genotypes
were found to represent four different species not yet
formally described. These species were indicated by
FIG. 1. Phylogeny of the
Tubifera ferruginosa
complex and related species of the Reticulariaceae based on partial 18S rDNA
sequences (1219 positions). The tree was constructed by Bayesian inference. ML bootstrap replicates (above 50%) and
Bayesian posterior probabilities (above 0.5) are shown for each branch. A hyphen indicates a conflicting topology. Bar
indicates the fraction of substitutions per site. Clusters separated by
-distance values $0.15 are colored and labeled with
taxon names, except for the genotypes
sp. 1–3 and
sp. 1–4 representing putatively new species not
characterized in this study. * The genotype displayed by the type specimen of the respective taxon.
More material is needed to study these potentially
new taxa.
The two studied specimens of
R. olivacea
a high
difference, suggesting their separation into
two genotypic species. One of them has a close
relationship with
Lindbladia tubulina
, while another
is distant from
but still more distant from
all other species of
and from Reticular-
iaceae in general.
T. ferruginosa
sensu lato (s.l.), the 31 genotypes
formed 10 clusters with the species level of difference
between them (
$0.15), suggesting that the
complex is represented by at least 10
species (FIGS. 2–9). Two of them, with 18 and six
investigated specimens belonging to a single geno-
type, respectively, correspond to the recently de-
T. applanata
(FIG. 4) and
R. dudkae
(FIG. 5).
Our 18S rDNA phylogeny substantially supports the
separation of both species.
Among the eight remaining genotypic clusters
within the
T. ferruginosa
complex, we selected the
one represented by the greatest number of specimens
(42%of all investigated specimens for this complex)
that correspond most closely to the descriptions and
illustrations of
T. ferruginosa
(Batsch 1786, Lister
1925, Martin and Alexopoulos 1969, Emoto 1977,
Nannenga-Bremekamp 1991, Poulain et al. 2011) to
T. ferruginosa
s. str. (FIGS. 2, 3). For this,
now more narrowly circumscribed taxon, we present
an emended description.
The original holotype of the
T. ferruginosa
s.l. was
described by Batsch (1786) who kept his herbarium in
the University of Jena (Germany). At the present
time, none of Batsch’s collection of
survived (J. Zu¨ndorf pers comm). Therefore, we
consider the holotype of the species as lost and
designate the original illustration as the lectotype.
One of our collections was selected to designate an
Among the seven genotypic clusters that remained
T. ferruginosa
complex, both the number and
condition of the herbarium specimens available
enable us to describe four new species under the
T. magna, T. montana, T. corymbosa
(FIGS. 4–9). Three other clusters,
sp. 1,
sp. 2,
sp. 3) are not
characterized herein because the limited material
available for study is insufficient to let us draw any
conclusions about the diagnostic features of these
putative new species.
Infraspecific level of diversity.—
The cluster represent-
T. ferruginosa
s. str. comprises nine 18S geno-
types, differing from each other at a
value level
below 0.11 (SUPPLEMENTARY INFORMATION 2). At least
some of these genotypes appear to be limited to
certain geographical regions: western Europe (gt1: 28
specimens), eastern Europe (gt2: eight specimens),
Asia (gt3: one specimen), North America (gt4–5: five
specimens) and Central America (gt6–7: three speci-
mens). Taking into consideration the fact that
was described originally from Germany
(Batsch 1786, Gmelin 1792) and that in this region
the western European genotype (gt1) seems to be
most common, we used a specimen of this genotype
for the neotypification of the species.
Fructifications corresponding to the two most
distant genotypes of
T. ferruginosa
s. str. display one
prominent peculiarity—the elongated, acute conical
tips of the sporothecae. Field observations have
revealed that the color of young fructifications in
these two 18S genotypes differs from that of other
specimens of
T. ferruginosa
s. str., being pink vs
scarlet red. The 18S rDNA sequences of this genotype
differ from other genotypes in the group at the
“subspecies” level of 0.09–0.11 (see above). Therefore
we separate this peculiar genotypic group as a separate
T. ferruginosa
and consider the remaining genotypes 1–7 as the
autotype subspecies
T. ferruginosa
(FIG. 2). These are the first subspecies to be proposed
for myxomycetes and the first intraspecific taxa of this
group based on molecular genetic data.
Among the new taxa, only
T. montana
has some
infraspecific genetic variability because it is represent-
ed by 11 very similar (
values 0.002–0.020) but
different genotypes (FIG.1,SUPPLEMENTARY INFORMA-
TION 2). Like
T. ferruginosa
s. str. the genotypes of
seem to be restricted to certain geographic
regions: eastern Europe (gt1–3, four specimens);
western Europe (gt4, four specimens), Asia (gt5–6,
two specimens) and North America (gt7–11, eight
specimens). One of the genotypes of
T. montana
(gt11) was found to be heterozygous, with hetero-
geneities at six positions of 18S sequence.
For all descriptions, additional specimens examined
Tubifera ferruginosa (Batsch) J.F. Gmel. Syst. nat.
2:1472 (1792) s. str., emend. Leontyev, Schnittler &
S.L. Stephenson FIGS. 2–3
Stemonitis ferruginosa
Batsch, Elenchus fungorum,
Continuatio prima: 31, tab. 30, fig. 175 (1786).
Lycoperdon ferruginosum
(Batsch) Timm, Flora
megapolitanae Prodomus exhibeus plantas ductatus
Megapolitano: 276 (1788).
MycoBank MBT 201303 (epitype)
Tubifera ferruginosa
s. str. subsp.
. a. Immature pseudoaethalium at the “bright” stage. b. Immature
pseudoaethalia showing the “bright” and “dark” stages. c. Mature pseudoaethalium. d–f. Individual sporothecae as observed
from above. g. Individual sporothecae in lateral view. h. Iridescent peridium of individual sporothecae in cross section. i. Tips
of individual sporothecae. j. Sclerified apex of a sporotheca. k. Outer surface of the peridium, covered with granules. l. Smooth
Batsch, Elenchus fungorum, Conti-
nuatio prima, tab. 30, fig. 175b. 1786 (lectotype,
POMMERN: Western Pomerania, in the vicinity of
Greifswald, Weitenhagen, 54u029550N, 13u259130E,
47 m, dead wood of
Fagus sylvatica
(?), 19 Jul 2011,
M. Schnittler
(epitype, designated here, GFW22067).
Pseudoaethalia solitary or grouped, (3–)4–25(–60)
mm long, (3–)4–15(–25) mm wide, (1–)2–6(–14) mm
high, rounded, short ovoid or drop-shaped as
observed from above, pulvinate to hemispherical,
often forming moniliform complexes, yellowish
brown (5YR2–4/6; FIGS. 2c, 3e–g); the surface formed
by the free tips of sporothecae. Sporothecae cylindri-
cal, rounded in cross section, straight, directed from
the base to the external surface of the fructification,
0.3–0.5 mm diam (FIG. 2g, h). Tips of sporothecae
uniform in diameter, not accreted, hemispherical to
conical, with blunt, papillate or subulate apices
(FIGS. 2d–f, i, 3g–k); apices often sclerified and in this
case black (FIG. 2f). Hypothallus spongy, white when
fresh, sandy-yellow when mature (5Y8/4). Peridium
semitransparent, light brown in reflected light, mod-
erately shining, dull or iridescent with blue, green and
purple tints (FIGS. 2f, h, 3i). External surface of the
peridium verrucose as observed in SEM (FIGS. 2k, 3i).
Internal surface of peridium smooth as observed in
SEM (FIGS. 2l, 3m), with wavy folds (FIGS. 2m, 3n, o)
and extremely rarely with rings 0.4–1.1 mm diam
(FIG. 2n). Columella mostly absent but may appear in
some sporothecae of a fructification, never reaching
the top of the sporocarp, irregularly cylindrical with
a conical tip, composed of loose material and de-
graded spores, ochraceous brown. Capillitium and
pseudocapillitium absent (see FIG. 13 and DISCUSSION
below). Spores in mass rust-brown (2.5YR4–5/10–12),
pale brownish in transmitted light, globose, (5.6–)
6.4–7.3(–8.9) mm in diam, banded-reticulate (FIGS. 2o,
p, 3q, r). Immature fructifications with different tints
of pink and red but never bright orange, later turning
dark-brown or black (FIGS. 2a, b, 3a, b, d).
Habit, habitat and distribution:
Found in temperate
zones of Europe, Asia and North America, in different
types of forests, on the strongly decomposed wood,
covered with bryophytes.
The main diagnostic characters of
s. str. are the cylindrical sporothecae with
a smoothly rounded, hemispherical or conical tip,
which may have blunt or subulate apices, depending
on the subspecies (see below). In all other species of
the genus, except
T. dudkae
, there is a clearly visible
“shoulder” between the vertical wall and the flattened
tip of the sporothecae (see FIGS. 4g, 7k, 9h). In
, the upper surfaces of the sporothecae lack
this feature but it is the only species where the
sporothecae are not cylindrical.
The upper parts of sporothecae in
T. ferruginosa
str. are free, never tightly attached to each other and
thus are not prismatic from mutual pressure. There is
only one other species of the genus,
T. corymbosa
which the upper parts of the sporothecae are free but
in this species they are mostly truncate and brightly
iridescent. In addition,
T. corymbosa
possesses spher-
ical sporothecae near the hypothallus (see below).
The pseudoaethalia in
T. ferruginosa
s. str. are
considerably smaller than those of
T. applanata, T.
T. magna
but larger than those of
T. corymbosa
(FIG. 10). The average spore size in
s. str. also differs from those of the other
species. In
T. montana
T. dudkae
the spores are
larger but in
T. pseudomicrosperma
they are somewhat
smaller (FIG. 14).
Tubifera ferruginosa (Batsch) J.F. Gmel. subsp.
ferruginosa Leontyev, Schnittler & S.L. Stephenson,
subsp. nov. FIG.2
MycoBank MB809786
Tips of the sporothecae hemispherical to obtusely
conical, with blunt or papillate apices (FIG. 2d–g).
Young fructifications pale salmon (5YR8/6) to scarlet
red (7.5R5/16; FIG. 2a, b).
Habit, habitat and distribution.
Temperate zones of
Europe, Asia and North America, in several kinds of
forests, where it tends to develop in wet depressions,
on the strongly decomposed wood of conifers (
abies, Abies alba, Pinus sylvestris
) and deciduous trees
Fagus sylvatica, Carpinus betulus
), which may be
entirely covered with bryophytes.
Tubifera ferruginosa (Batsch) J.F. Gmel. subsp.
acutissima Leontyev, Schnittler & S.L. Stephenson,
subsp. nov. FIG.3
MycoBank MB809787
USA. VIRGINIA: mixed mesophytic
forest, 37u219550N, 80u329110W, 1189 m asl, decaying
wood covered with bryophytes, 23 Jun 2013,
(holotype UARK 50546).
inner surface of the peridium. m. Wavy folds on the inner surface of the peridium. n. Ring ornamentation on the inner surface
of the peridium. o, p. Spores. Specimens: a. not collected; b. CWU 2183; c, d. sc22067 (holotype); e. sc22282; f, h, i. sc22099;
g. sc22102; j, l, o. sc22230; k, m, p. CWU MR-0071; n. CWU 2925. Bars: a 510 mm; b 55 mm; c 52mm;dh51mm;i5200 mm;
j550 mm; k 55mm; l 52mm; m 510 mm; n–p 51mm.
Tubifera ferruginosa
a, b. Immature pseudoaethalia at the “bright” stage. c. Immature
pseudoaethalium at the ‘dark’ stage. d. Tips of immature sporothecae at the “bright” stage. e. Almost mature pseudoaethalium
in the field. f, g. A mature pseudoaethalium. h–j. Tips of individual sporothecae. k. Acute apex of sporotheca. l. Outer surface
of peridium, covered with granules. m. Smooth inner surface of peridium. n, o. Wavy folds on the
. Acutissimus (Latin), the most acute, for
the shape of sporothecal apices.
Tips of the sporothecae acute conical, with subulate
apices (FIG. 3g–k). Young fructifications deep pink
(5R5/12–16; FIG. 3a, b, d).
Habit, habitat and distribution.
Temperate zones of
Europe and North America in deciduous and mixed
forests, usually in wet depressions and on strongly
decomposed wood of deciduous trees (
Quercus robur
which may be entirely covered with bryophytes. In
western and central Europe and also in the western
United States, this subspecies is much less common
than subsp.
but in some localities in eastern
Europe, such as the Gomolsha Forests in Ukraine, it is
much more abundant than the type subspecies.
Tubifera dudkae (Leontyev & G. Moreno) Leontyev,
Moreno & Schnittler, comb. nov. FIG.5
Reticularia dudkae
Leontyev & G. Mor-
eno, Bol. Soc. Micol. Madrid 35:86 (2011).
MycoBank MB809788
molsha Forests National Park, 49u379370N, 36u199380
E, 118 m asl, dead wood, 1 Jul 2003,
D.V. Leontyev
(holotype CWU MR-0040[2]).
In honor of Irina O. Dudka.
solitary or in small groups, (18–)25–
28(–45) mm long, (16–)20–22(–30) mm wide, (5–)7–
11(–12) mm high, pulvinate, rounded to short ovate as
observed from above, saturated rust-brown (2.5–5YR2/
6–8; FIG. 5d, e); the surface bullate, formed by in-
dividual sporothecae with dried slime bands between
them (FIG. 5g–i). Sporothecae spherical, ovoid, bag-
like, sinuous, (0.1–)0.4–0.6(–1.0) mm diam, never
elongated and not directed from the base to the top of
the pseudoaethalium (FIG. 5f). Hypothallus spongy,
white when fresh, brownish (10YR5/6) when mature,
forming long strands at the baseof the pseudoaethalium
(FIG. 5a–c). Peridium smooth, translucent, light brown
in reflected light, shining and somewhat iridescent with
blue, green and purple tints. External surface of the
peridium verrucose as observed in SEM (FIG. 5j).
Internal surface of the peridium as observed in SEM,
covered by a reticulum consisting of wavy folds (FIG.5k,
l); rings 0.4–1.0 mm diam occasionally observed (FIG.5
m, n). Columella absent. Capillitium absent. Pseudo-
capillitium may be present in the central part of the
pseudoaethalium, where some individual sporothecae
can lose their individuality and their peridium is
reduced to perforated plates and bands. Spores in mass
rust brown (2.5YR4–5/10-12), pale brownish in trans-
mitted light, globose, (6.2–)6.9–7.6(–8.1) mm diam,
banded-reticulate (FIG. 5o, p). Immature fructifica-
tions at first pale pink (7.5R8/2–4), then coral red
(7.5R6/10–14), then dirty grayish brown (7.5R2–3/2;
FIG. 5a–c).
Habit, habitat and distribution.
Europe and Asia,
both coniferous and mixed forests, mostly on
decorticated but not strongly decomposed wood of
conifers (
The most prominent character of this
species is a pseudoaethalium composed of spherical,
ovate or sinuous sporothecae. These sporothecae never
take the form of a tuft of parallel cylinders, stretching
from the bottom to the top of the fructification, as is the
case in all other species in the
T. ferruginosa
In several species of
, e.g.
R. splendens
, one may observe the deep accretion of
spherical sporothecae, which partially lose their in-
dividual walls and form an aethalium (Lister 1925,
Martin and Alexopoulos 1969). Therefore the spheri-
cal sporothecae in
T. dudkae
was considered as the
primary argument to assign this species to the genus
(Leontyev and Moreno 2011). However, as
indicated above, the 18S rDNA phylogeny contradicts
this placement. Although a pseudoaethalium com-
posed of only spherical sporothecae is unique among
the species of the
, their shape per se is not an
exclusive feature of
T. dudkae
. At least two other
T. dimorphotheca
T. corymbosa
, also have
a number of spherical sporothecae, situated at the base
of the fructification.
The surface of the pseudoaethalium in
T. dudkae
formed by irregular, more or less convex sporothecal
tips. In this respect,
T. dudkae
T. montana
Moreover, the spores of both species are similar in
size (FIG. 14). However,
T. montana
lacks the dried
slime bands between the sporothecal tips and the
surface of the peridium in this species is characterized
by a golden and pinkish iridescence.
Tubifera montana Leontyev, Schnittler & S.L. Ste-
phenson, sp. nov. FIG.6
MycoBank MB809789
Gorgany State Reserve, 48u279230N, 24u149520E, 842
m asl, dead wood of
Picea abies
(?), 13 Aug 2011, D.V.
Leontyev (holotype CWU 2912).
inner surface of peridium. p, q. Spores. Specimens: a–f. CWU MR-0006(2); g–q. UARK 50546 (holotype). Bars: a 510 mm;
b, c, e 55 mm; d, h 5500 mm; f 52 mm; g 51 mm; i 5200 mm; j 5100 mm; k 550 mm; l, p, q 51mm; m 510 mm; n 55mm;
Tubifera applanata
. a. Immature pseudoaethalium (Courtesy of Renato Cainelli). b, c. Mature pseudoaethalium
on the dead wood (Courtesy of Rene´e Lebeuf). c. Mature pseudoaethalium on the litter (Courtesy of Renato Cainelli).
d–f. Individual sporothecae from above. g, h. Cross section of pseudoaethalia. i. Opened sporothecae with columellae visible.
j, k. Tips of individual sporothecae. l. Outer surface of peridium, covered with granules. m, n. Ring ornamentation on inner
: Montanus (Latin), occurring in moun-
Pseudoaethalia solitary or grouped, (10–)13–27(–
32) mm long, (9–)10–16(–19) mm wide, (2–)3–7(–8)
mm high, pulvinate, elongated, curved, crescent-
shaped or irregular as observed from above, ocher
brown to golden brown (7.5YR2–4/6; FIG. 6b–i); the
surface bullate, formed by the tips of the sporothecae.
Sporothecae cylindrical, irregularly curved, directed
mostly from the base to the external surface of the
fructification. Tips of the sporothecae variable in size,
tightly accreted to each other, ovoid, wormlike to
rounded as observed from above, lenticular-convex as
viewed from the side, (0.4–)0.6–0.8(–1.0) mm diam
(FIG. 6g–i). Hypothallus scanty. Peridium semitrans-
parent, light brown in reflected light, iridescent with
golden, bronze, pink or sparsely bluish tints (FIG. 6g–
i). External surface of the peridium verrucose as
observed in SEM (FIG. 6j), sometimes bullate inside
the pseudoaethalium (FIG. 6k). Internal surface of the
peridium, as observed in SEM, covered by a reticulum
consisting of wavy folds (FIG. 6l–n), rings 0.4–1.0 mm
diam occurring only sparsely (FIG. 6n, o). Columella
absent. Capillitium and pseudocapillitium absent.
Spores in mass rust-brown (2.5YR4–5/10-12), brown-
ish in transmitted light, globose, (6.6–)7.1–8.1(–8.9)
mm diam, banded-reticulate (FIG. 6o, p). Immature
fructifications bright orange (2.5YR6/14), then dark
brown (5YR1/2; FIG. 6a, b).
Habit, habitat and distribution.
Temperate zone of
Eurasia and North America, in the forests occurring
in ravines and on the slopes of mountains, on strongly
decomposed wood of conifers (
Picea abies, Pinus
pallasiana, P. sibirica
), often covered with bryophytes.
Field photographs show that it also may occur on leaf
litter. At least in central Europe, the peak period of
fruiting seems to be summer and not autumn as is the
case for
s. str.
This is a polymorphous species with
several slightly different 18S genotypes.
is close to
T. ferruginosa
with respect to
both the phenotype and genotype (FIG. 1). Both
species are characterized by small, grouped pseu-
doaethalia, often occurring on bryophytes. However,
T. montana
never displays the free, hemispherical or
conical sporothecal tips characteristic of
T. ferrugi-
and its spores are considerably larger (FIG. 14).
The difference in plasmodial color is remarkable in
that young fructifications of
T. montana
are bright
orange but never scarlet or pink like those of
Tubifera montana
also resembles
but the latter species is clearly distinguished
by its spherical sporothecae (see comments under
Tubifera magna Leontyev, Schnittler, S.L. Stephen-
son & T. Kryvomaz, sp. nov. FIG.7
MycoBank MB809790
Mountains National Park, woodlands and camping
area near Cades Cove ATBI residence, 35u439400N,
83u119090W, 1480 m asl, decaying wood, 28 Jul 2003,
T.I. Kryvomaz
(holotype UARK 20405).
: Magnus (Latin), large, reflecting the size
of the pseudoaethalia.
Pseudoaethalia solitary, (12–)24–61(–127) mm
long, (8–)20–32(–51) mm wide, (3–)4–5(–6) mm
high, flat pulvinate or less commonly almost hemi-
spherical, rounded or ovate as observed from above,
rust-brown with a delicate copper tint (10R4/10-12;
FIG. 7b–e); the surface formed by the accreted tips of
the sporothecae. Sporothecae straight, prismatic,
relatively short, directed from the base to the external
surface of the fructification (FIG. 7j). Tips of the
sporothecae polygonal, somewhat elongated as ob-
served from above, separated from each other by
fragmentary patches of the white dried slime,
lenticular-convex as viewed from the side, (0.2–)0.4–
0.6(–0.8) mm long, (0.1–)0.3–0.4(–0.5) mm wide
(FIG. 7f–h, k). Hypothallus scanty, at first white and
then beige (5Y8/4), sometimes forming an inter-
rupted rim around the pseudoaethalium (FIG. 7e).
Peridium semitransparent, bright yellow-brown in
reflected light, shining but not iridescent except for
the internal surface of the sporothecae (FIG. 7j).
External surface of the peridium verrucose as
observed in SEM (FIG. 7l). Internal surface of the
peridium, as observed in SEM, covered by a reticulum
consisting of wavy folds (FIG. 7m, n); irregular, curved
rings 0.2–0.7 mm diam only occasionally present
(FIG. 7o). Columella absent. Capillitium and pseudo-
capillitium absent. Spores in mass rust-brown
(2.5YR4–5/10–12), brownish in transmitted light,
globose, (5.7–)6.3–7.2(–8.2) mm diam, banded re-
ticulate (FIG. 7p, q). Immature fructifications bright
lilac-pink (2.5R5-6/12-14; FIG. 7a).
Habit, habitat and distribution.
Temperate zones of
North America, one collection from Europe, in
surface of peridium. o. Spores. Specimens: a, c. RC 8061002; b. RL Myxo30; d. CWU MR-0038; e. CWU MR-0126; f. CWU
MR-0125; g. CWU MR-0136; h. CWU MR-0121; i, k, m, n, o. CWU MR-0038; j, l. CWU MR-0039 (holotype). Bars: a 520 mm;
b, c 510 mm; d–h 51 mm; i, j 5500 mm; k 5100 mm; l–n 52mm; o 51mm.
Tubifera dudkae
. a–c. Immature pseudoaethalia at the “bright” stage. d, e. Mature pseudoaethalia. f. Cross section of
a mature pseudoaethalium with spherical sporothecae visible. g–i. Individual sporothecae observed from above. j. Outer
surface of peridium, covered with granules. k, l. Smooth inner surface of peridium, covered by wavy folds. m. Wavy folds and
coniferous (
Picea rubra
) and deciduous (
spp.) forests, on moderately decomposed
wood and rarely on leaf litter.
Comments. Tubifera magna
is one of two species
of the
T. ferruginosa
complex forming very large
pseudoaethalia, 3–12 cm across, with flattened spor-
othecal tips, which coalesce to produce a nearly
smooth surface of the fructification. The second
T. applanata,
occurs mostly in Eurasia,
T. magna,
except for one known specimen,
appears to be found mostly in North America. In
the pseudoaethalia are rounded from
above and the sporothecal tips are isodiametric,
roughly hexagonal, similar in size and seated in
regular rows, whereas in
T. magna
the pseudoaethalia
are elongated (see FIG. 10), the sporothecal tips are
prolate, variable in size and have no regular position
(FIG. 11). The peridium in
T. applanata
appears dull,
whereas in
T. magna
it is shiny. Only
T. magna
have a white hypothallic rim around the base of the
pseudoaethalium. Large rings that ornament the
peridium in
T. applanata
are not found in
T. magna.
Finally, the color of immature fructifications is
light pink in
T. magna
and flesh to dirty salmon in
T. applanata.
Tubifera pseudomicrosperma Leontyev, Schnittler &
S.L. Stephenson, sp. nov. FIG.8
MycoBank MB809791
USA, MICHIGAN: North Lake Lan-
sing Trail, Lake Lansing Park, hardwood forest,
42u459140N, 84u259590W, 267 m asl, decorticated
wood, 20 Jun 2004,
G.C. Adams
(holotype UARK
Pseudo (Greek), false; Microspermus
(Greek), having small spores; indicating its similarity
Tubifera microsperma
Pseudoaethalia solitary or grouped, (7–)9–18(–23)
mm long, (5–)6–12(–15) mm wide, (2–)3–5(–6) mm
high without the hypothallus, rounded, ovoid or
vermicular as observed from above, often forming
a moniliform complexes, pulvinate to hemispherical
as in a side view, beige brownish to cinnamon-brown
(7.5YR4-6/4; FIG. 8b–f); the surface formed by the
adherent tips of the sporothecae. Sporothecae cylin-
drical to prismatic, rounded or smoothed polygonal
in cross section, straight, directed from the base to the
external surface of the fruit body (FIG. 8i). Tips of the
sporothecae of uniform diam, penta- to hexagonal as
observed from above, flat or inconspicuously convex,
accreted to each other, 0.3–0.5 mm diam (FIG. 8g, h).
Hypothallus forming a wide stub-like structure, on
which the pseudoaethalium is seated, 2–5 mm high,
firm, glossy, white when fresh, brownish-black when
mature (FIG. 8a–e). Peridium beige brownish in
reflected light, dull, not iridescent; pale yellow to
almost hyaline in transmitted light (FIG. 8j). External
surface of peridium verrucose as observed in SEM
(FIG. 8l). Internal surface of peridium, as observed in
SEM, covered with rimmed craters 0.2–0.6 mm diam,
up to 0.5 mm high (FIG.8mo); no wavy folds
observed. Columella absent. Capillitium and pseudo-
capillitium absent. Spores in mass rust-brown
(2.5YR4–5/10-12), pale brownish in transmitted light,
globose, (4.4–)4.8–5.4(–6.1) mm diam, banded-retic-
ulate (FIG. 8p, q). Immature fructifications are pale
pinkish cream (2.5YR8/2-4; FIG. 8a).
Habit, habitat and distribution.
Europe and North
America, in deciduous and mixed forests, on strongly
decomposed wood of deciduous trees, with no moss
The species differs from all representa-
tives of the complex by its black stub-like hypothallus.
Tubifera ferruginosa
s. str. is superficially similar but
has sporothecae with free hemispherical or conical
tips (in contrast to the flat ones in
T. pseudomicros-
), a rust brown, shining and somewhat irides-
cent color of the peridium (which is beige brownish
and dull in
T. pseudomicrosperma
), and much larger
spores (6.4–7.3 mm vs. 4.8–5.4 mmin
T. pseudomicros-
). Another species very similar to
T. pseudomi-
T. microsperma
. However, the latter has
small pseudoaethalia less than 1 cm diam and its
hypothallic basement looks like a stalk, with the
height exceeding the diameter. Spores of
T. micro-
are somewhat larger (FIG. 14).
In both
T. microsperma
T. pseudomicrosperma,
peridium is ornamented with so-called rimmed craters,
covering its whole inner surface (TABLE I). However, in
T. pseudomicrosperma
these craters are much smaller
and therefore indistinguishable in DIC, whereas in
T. microsperma
they are clearly visible (FIG. 12).
Tubifera corymbosa Leontyev, Schnittler, S.L. Ste-
phenson & L.M. Walker sp. nov. FIG.9
MycoBank MB809792
´N: La Selva Biol.
Station, 10u259520N, 84u009120W, 48 m asl, rotting
wood, 1 Jul 2011,
L.M. Walker
(holotype UARK
rings (arrows) on the inner surface of the peridium. n. Ring ornamentation on the inner surface of peridium. o, p. Spores.
Specimens: a, d, f, h, i, k, l, p. CWU MR-0040 (holotype); b, c. CWU 3074; e, g. CWU MR-0120; j, m–o. CWU 2410. Bars: a 520
mm; b, c 510 mm; d–f 52 mm; g, h 51 mm; i 5500 mm; j, m, n 52mm; k, l 55mm; o, p 51mm.
Tubifera montana.
a. Immature pseudoaethalium at the “bright” stage. b. Immature pseudoaethalium at the “dark”
stage. c–f. Mature pseudoaethalia. g–i. Tips of individual sporothecae as observed from above. j. Outer surface of a sporotheca,
covered with granules. k. Wavy folds on the outer surface of the peridium. l, m. Wavy folds on the inner surface of the
peridium. n. Wavy folds and rings (arrows) on the inner surface of the peridium. o, p. Spores. Specimens: a. sc 27541;
b. sc 27530; c. UARK 6348; d, g. CWU 2912; e, f, i, j, n, p. UARK 3587 (holotype); h. UARK 9062; k, o. MM38970; l, m. CWU
MR-0072. Bars: a 55 mm; b–f, j 52 mm; g–i 51 mm; k 52mm; l, m 55mm; n–p 51mm.
Tubifera magna.
a. Immature pseudoaethalium at the “bright” stage. b–e. Mature pseudoaethalia. f–h, k. Tips of
individual sporothecae observed from above. i. Opened sporothecae observed from above. j. Cross section through
a pseudoaethalium with the iridescent peridium visible. l. Outer surface of peridium. m, n. Inner surface of peridium, covered
by wavy folds. o. Ring ornamentation on the inner surface of peridium. p, q. Spores. Specimens: a. Not collected; b. UARK
50645; c. UARK 20393; d, l, m, p. UARK 34118; e–k, n, o, q. UARK 20405 (holotype). Bars: a, b, d 510 mm; c, e 55 mm; f 52
mm; g, i, j 51 mm; h 5500 mm; k 5200 mm; l 510 mm; m, n 55mm; o 50.5 mm; p, q 51mm.
Tubifera pseudomicrosperma.
a. Immature pseudoaethalium at the “bright” stage (Courtesy of Mushroom Observer).
b–e. Mature pseudoaethalia. f–h, k. Tips of individual sporothecae observed from above. i. Hypothallus and basal parts of
sporothecae. j. Peridium observed under DIC. l. Outer surface of peridium. m–o. Inner surface of peridium, covered by
craters. p, q. Spores. Specimens: a. Not collected; b, c, f, g, l, n–q. UARK 20730 (holotype); d, e, j. UARK 25096; h, i, k, m. CWU
MR-0008(2). Bars: a 55 mm; b, d–f 52 mm; c, g 51 mm; f 52 mm, h 5200 mm; i 5500 mm; j 520 mm; k 5100 mm;
l55mm; m 510 mm; n, o, q 51mm; p 52mm.
Tubifera corymbosa.
a–g. Mature pseudoaethalia, spherical sporothecae marked with arrows. h. Tips of individual
sporothecae observed from above. i. Hypothallus, impregnated with spherical sporothecae. j, k. Spherical sporothecae observed
under LM, with spores visible inside. l. Outer surface of peridium in the lateral part of the sporotheca. m. Outer surface of peridium
at the border of the side part of a sporotheca (left) and the tip of a sporotheca (right). n. Inner surface of peridium, covered by
wavy folds. o. Inner surface of the peridium with ring ornamentation. p, q. Spores. Specimens: a–f, h–q. UARK 47853 (holotype);
g. AMFD 251. Bars: a, c 52mm;b,dg51 mm; h, i 5200 mm; j, k 550 mm; l, n 55mm; m, o–q = 2 mm.
. Corymbus (Latin), bunch, corymb, in-
dicating the shape of the pseudoaethalium, which
resembles a corymbose inflorescence.
Pseudoaethalia solitary or grouped, (2.3–)2.5–4.2(–
4.8) mm diam, (2.8–)3.0–4.4(–4.6) mm high, occur-
ring as a dense bunch-liketuftofsporothecae,
narrowed at the base, bright rust-brown (2.5YR2–4/
6; FIG. 9a–g); the surface formed by the free tips of
the sporothecae. Sporothecae are of two types, the
first large and cylindrical, the second small and
spherical. Cylindrical sporothecae make up the bulk
of the pseudoaethalium, directed from the base to the
upper surface of the fruit body with peripheral
sporothecae usually deflected outward; smooth, nar-
rowed at the base, expanded and truncate at the top,
(0.33–)0.37–0.45(–0.49) mm diam at the upper part
(FIG. 9b, d, f). Spherical sporothecae are less numer-
ous, situated at the base of pseudoaethalium, sub-
merged in hypothallic slime, (0.10–)0.12–0.20(–0.31)
mm diam (FIG. 9d, g, i–k). Tips of the cylindrical
sporothecae uniform in diameter, more or less
convex to truncate, rounded as observed from above,
distant from each other, 0.3–0.5 mm diam (FIG. 9g,
h). Hypothallus forming the narrowed base of the
pseudoethalium, loose, spongy, dirty white, incrusted
with spheroid sporothecae (FIG. 9i). Peridium for the
most part of the sporothecae bright rust brown in
reflected light, shining, weakly iridescent in blue,
green and purple tints, yellowish in transmitted light
(FIG. 9f, g); at the sporothecal tips the peridium is
much lighter, silvery white or pale golden ocher,
sometimes with a metallic luster (FIG. 9h). External
surface of peridium covered with warts as observed in
SEM (FIG. 9l) but absolutely smooth at the tips of
sporothecae (FIG.9m, right). Internal surface of
peridium as observed in SEM, covered by a faint
reticulum of wavy folds (FIG. 9n); with sparse rings
of 0.3–0.9 mm diam (FIG. 9o). Columella absent.
Capillitium and pseudocapillitium absent. Spores in
mass rust brown (2.5YR4–5/10-12), pale brownish in
FIG. 10. Size and shape of pseudoaethalia observed from above in species of the
Tubifera ferruginosa
complex. All figures
are to the same scale. Contours are drawn from specimens used in this study. Dotted lines indicate adjacent fructifications that
are likely to have been formed from the same plasmodium. Numbers in brackets indicate the relative proportion of
pseudoaethalium width in relation to length.
transmitted light, globose, (5.3–)5.7–6.3(–6.8) mm
diam, banded-reticulate (FIG. 9p, q). Immature fruc-
tifications unknown.
Habit, habitat and distribution.
Tropical forests of
Central America, on decaying wood.
Comments. Tubifera corymbosa
is a peculiar species,
which might or might not considered as a member of
T. ferruginosa
complex. The two available collec-
tions of this species were given different preliminary
identifications: UARK 47853 as
T. ferruginosa
AFMD 251 as
T. dimorphotheca.
Indeed, these two
species are most similar to
T. corymbosa
ically. However, in our phylogeny,
T. corymbosa
are situated on distant branches (FIG. 1).
The phylogenetic relationship between
T. corymbosa
T. dimorphotheca
remains unclear.
Tubifera corymbosa
differs from all representatives
of the
T. ferruginosa
complex by the presence of the
small spherical sporothecae at the base of the
pseudoaethalium. On the other hand, this suggests
a close relationship with
T. dimorphotheca
, previously
considered to be the only species with this character
(Nannenga-Bremekamp and Loerakker 1981). How-
ever, in
T. dimorphotheca
the spherical sporothecae
are seated on a prominent hypothallic stalk, which is
absent in
T. corymbosa.
The limited material available
from herbaria lets us conclude that the peridium in
T. dimorphotheca
is dull and not iridescent; however,
the authors of the latter species noted that it might be
glossy (Nannenga-Bremekamp and Loerakker 1981).
T. corymbosa
the lateral surface of the sporothecae
the peridium is shining and iridescent with blue,
green and purple tints but at the sporothecal tips the
peridium has a metallic, silvery or golden luster.
Phylogeny vs. morphology.—
The topology of our 18S
rDNA tree generally correlates with the morpholog-
ical characters of the organisms being considered.
This can be demonstrated by the example of
Reticularia olivacea
, a species traditionally considered
to fall within the Reticulariaceae but attributed in our
phylogeny to the Cribrariaceae. Together with the
closely related species
R. simulans
(Rostaf.) D.W.
Mitch. and
R. liceoides
(Lister) Nann.-Bremek.,
differs from all other members of the
Reticulariaceae by having verrucose spores, an oliva-
ceous spore mass and black plasmodium and young
fructifications (Neubert et al. 1993, Poulain et al.
2011). All these characters unite the olive-spored
species of
with members of the Cribrar-
iaceae, especially with
Lindbladia tubulina
similarity first was noted by Rostafin´ sky, who estab-
lished the monotypic genus
Rostaf. and
proposed the new combination
Licaethalium oliva-
(Ehrenb.) Rostaf. for
R. olivacea
(other olive-
spored species were not yet known; Rostafin´ sky 1875),
thus constructing a hypothetical evolutionary se-
quence from the sporangiate
via pseu-
to the aethaliate
. Our 18S rDNA phylogeny provides
a preliminary basis for the possible re-erection of
Rostafin´sky’s genus
FIG. 11. Typical structure of the sporothecal tips in the
Tubifera ferruginosa complex
Tubifera ferruginosa
T. ferruginosa
T. montana
T. dudkae
T. applanata
T. magna
T. corymbosa
All other studied species of the Reticulariaceae
separate into three basal branches that agree with the
genera accepted in the family. These are (i)
and (iii)
Among these branches,
appears to be closest
to the hypothetical last common ancestor of the
family, forming the cluster with the shortest distance
from the basal node. This correlates with morpho-
logical data because the genus is characterized by
a number of presumably plesiomorphic features, such
as individual sporothecae, fibrous stalks and a tubular
capillitium (Leontyev et al. 2014a, b, c). The cluster
that includes
is divided into
two subclusters that correspond to these genera, thus
supporting previous data on their monophyly (Leon-
tyev et al. 2014a, c).
All studied species of
form a monophyletic
cluster. However, the present investigation did not
consider four known species currently assigned to the
genus, including the morphologically deviating
(Rostaf.) T. Macbr. and
T. dictyoderma
Bremek. & Loer. For these reasons, we currently are
unable to draw a final conclusion about the mono-
phyly of the genus
as traditionally circum-
It is noteworthy that the
clade is divided
into two groups, the first consisting of
T. ferruginosa,
T. dudkae
T. magna
T. montana
and the second
FIG. 12. Rimmed craters on the inner surface of the peridium in
Tubifera pseudomicrosperma
(a, SEM; c, DIC) and
T. microsperma
(b, SEM; d, DIC). Note that both SEM and both DIC images are the same scale. Specimens: a, c. UARK 20730;
b, d. UARK 32758. Bars: a–b 55mm; c–d 510 mm.
T. applanata
T. corymbosa
T. microsperma
T. pseudomicrosperma
(FIG. 1). These two groups
unite species with different pseudoaethalia dimen-
sions, sporothecal tips and different kinds of peridial
ornamentation. The only morphological character
that clearly corresponds to this subdivision is spore
size. The first group unites species with larger spores
(6.0–8.5 mm), whereas the second includes taxa with
smaller spores (4.5–6.5 mm, FIG. 14).
The color of immature fructifications.—
observations, often in the same localities (20 y in
Germany, 17 y in the Ukraine), have revealed that the
color of the fructifications in
passes through
two main stages before they are mature. In the first,
brightly colored stage, when the plasmodium leaves
the substrate, almost every taxon has its own tint:
salmon to scarlet in
T. ferruginosa
deep pink in
T. ferruginosa
, coral
red in
T. dudkae
, orange in
T. montana
, pink in
T. magna
, pinkish cream in
T. pseudomicrosperma
dirty salmon to flesh in
T. applanata
(Leontyev and
Fefelov 2009). In the second dark stage, the fructifica-
tions in most taxa become dark grayish or brownish
but in
T. ferruginosa
they turn
almost black.
Because the color of the initial bright stage is so
distinctive, it is important to observe both immature
and mature stages of the same pseudoaethalium, or at
least in the same group of pseudoaethalia. Unfortu-
nately immature fructifications of
become sclerified and lose their color when collected.
FIG. 13. False capillitium, represented by fungal hyphae. a. Illustration from Nannenga-Bremekamp (1991: 54), showing
the “capillitium” (
) covered with spores (
Tubifera ferruginosa
s.l. b. False capillitium inside sporothecae of
T. ferruginosa
. c. False capillitium inside sporothecae of
T. applanata
. d–f. False capillitium of
T. ferruginosa
under a light microscope. g. False capillitium on the outer surface of the peridium of
T. montana
. h. False
capillitium (white arrow) and true capillitium (black arrow) in
T. dictyoderma
. i. Details of the false capillitium in
T. dictyoderma
UARK 23966: septa (white arrows) and a portion with broken wall and a cavity visible (black arrow). Specimens:
b, d–f. CWU 2183; c. CWU MR-0122; g. CWU MR-0072; h, i. UARK 23966. Bars: b 5500 mm; c 51 mm; d, g, h 550 mm; e, f 5
20 mm; i 52mm.
As such, wherever possible, images of immature
pseudoaethalia should be obtained as part of a voucher.
Size and shape of pseudoaethalia.—
The pseudaethalia
dimensions in
species are variable, even in
a group of fructifications appearing on one log.
Nevertheless, the species have informative differences
in this character. Within the
T. ferruginosa
the smallest pseudoaethalia were recorded for
, then the average size tends to increase
T. ferruginosa
T. pseudomicrosperma, T.
T. applanata
and finally,
T. magna
. The
relative proportion of pseudoaethalium width to
length changes from species to species, varying from
FIG. 14. Spore dimensions in the
Tubifera ferruginosa
complex and related species. Bubbles show the relative proportion of
spores of a definite size in each species. Numbers indicate the numbers of measured spores/studied specimens.
TABLE I. Peridium ornamentation in the
Tubifera ferruginosa
Outer surface of the peridium Inner surface of the peridium
Type of granular
ornamentation Granules (mm) Rings or craters (mm)
Density of ring
Density of
wavy folds
Tubifera applanata
A (0.3–) 0.6–0.7 (–1.0) (0.4–) 1.5–2.0 (–2.9) ++
T. dudkae
A (0.3–) 0.4–0.5 (–0.7) (0.4–) 0.6–0.8 (–1.0) ++ +++
T. ferruginosa
A (0.5–) 0.6–0.8 (–1.0) (0.6–) 0.8–0.9 (–1.1) ++
T. montana
B (0.4–) 0.6–0.8 (–1.0) + +++
T. magna
B (0.2–) 0.3–0.6 (–0.8) ++ ++
T. pseudomicrosperma
B (0.2–) 0.3–0.7 (–1.0) +++
T. corymbosa
A (0.6–) 0.8–0.9 (–1.2) (0.3–) 0.4–0.8 (–0.9) + +++
Notes: A, separate rounded granules; B, irregularly verrucose surface; +++, ornamentation dense and prevalent; ++,
ornamentation not dense but widespread; +, ornamentation widely spaced and sparse; –, ornamentation absent.
low values in the elongated pseudoaethalia of
T. magna, T. montana
T. pseudomicrosperma
high in the almost isodiametric fructifications of
T. applanata
T. corymbosa
T. dudkae
coefficients on FIG. 10).
The shapes of the pseudoaethalia are more
distinctive (FIG. 10). In both subspecies of
s. str., they are mostly elongated and
moniliform, divided into segments by numerable
transverse fissures. The fructifications of
T. pseudomi-
are similar. In
T. applanata
T. dudkae
the pseudoaethalia are mostly short ovate, whereas in
T. magna
they are somewhat angular. In
T. montana
the pseudoaethalia are strongly irregular and tend to
be crescent-shaped.
Shape of the sporothecae and their tips.—
We think the
shape of the sporothecae in general and especially the
shape of the sporothecal tips to be as the most reliable
morphological character to identify species in the
complex. Sporothecae can be straight and
arranged in a single layer (cylindrical in
T. ferrugi-
nosa, T. pseudomicrosperma
T. corymbosa
; pris-
matic because of mutual pressure in
T. applanata
T. magna
), or stacked in several layers in a way that
not all sporocarps reach both the top and the base of
the fructification (elongated and strongly curved in
T. montana
; spherical or irregularly bag-shaped in
Each taxon of the
T. ferruginosa
complex displays
an easily recognizable pattern of sporothecal tips
situated at the upper surface of the pseudoaethalium
(FIG. 11). In freshly matured, undamaged fructifica-
tions, these structures are clearly visible with a hand
lens and thus useful to identify species in the field.
Because of the very fragile peridia in the
complex, an imaging of the fresh material
with a macro lens is recommended to document the
shape of sporothecal tips. In
T. ferruginosa
T. corymbosa
tips are free and not accreted, whereas
in all other species tips are tightly accreted, forming
a relatively even, flat or bullate surface. The shape
of the tips can be hemispherical, papillate or obtuse
conical (
T. ferruginosa
), subulate
T. ferruginosa
), truncate (
), lenticular (
T. dudkae, T. montana
almost flat and polygonal (
T. applanata
T. pseudo-
). In
T. dudkae
T. magna
, dried slime bands often are observed
between the sporothecae; these bands are shiny in
the first two species but dull in the latter.
Ornamentation of the peridium.—
This character was
introduced into the taxonomy of
a peculiar ornamentation, referred to as “rimmed
craters”, was described for the inner surface of the
peridium of
T. microsperma
(Nelson et al. 1982). In
T. ferruginosa
was shown to have a smooth
inner peridial surface. Later, annular ornamentations
of the inner surface of the peridium were observed in
T. applanata
and ornamentation consisting of a re-
ticulum of wavy folds was found in
T. dudkae
(Leontyev and Fefelov 2009, Leontyev and Moreno
In this study weanalyzed both the inner and the outer
surface of the peridium in an effort to provide useful
characters for distinguishing species in the
T. ferrugi-
complex (TABLE I). The outer surface of the
peridium in
T. ferruginosa, T. applanata, T. dudkae
T. corymbosa
was found to be covered with granules
(0.3–1.0 mmdiam);in
T. magna, T. montana
T. pseudomicrosperma
the outer surface of peridium is
rough but individual granules are not apparent.
In general the diagnostic significance of the outer
peridial surface is low. However, the ornamentation
of the inner peridial surface is of much higher
diagnostic value (FIGS. 3–7; cf. Leontyev and Fefelov
2009, 2012; Leontyev and Moreno 2011). Contrary to
an opinion expressed by Leontyev and Fefelov (2009)
and Leontyev and Moreno (2011), both rings and
wavy folds were found in all species of the
T. ferruginosa
complex except for
T. applanata
T. pseudomicrosperma
. Nevertheless, these structures
are variable in density and morphology. As shown
previously, peridial rings are frequent and large in
(FIG. 4m, n) but also common in
T. dudkae
T. montana
(FIG.6n) and
T. magna
(FIG. 7o), albeit they are much smaller and less
prominent. In
T. ferruginosa
(FIG.2n) and
(FIG. 9o), rings are extremely rare. In
, the circular ornamentation con-
sists of rimmed craters, which are similar to those of
T. microsperma
(Nelson et al. 1982) and much taller
than the rings present in another species. In both
T. pseudomicrosperma
, these craters
are numerous, covering the surface of the peridium.
However, in
T. pseudomicrosperma
they are much
smaller than those found in
T. microsperma
, where
they sometimes reach 2.5 mm diam (FIG. 12a, b).
Therefore peridial craters of
T. pseudomicrosperma
cannot by identified by DIC (FIG. 12c) whereas in
they are clearly visible (FIG. 12d).
A reticulum of wavy folds covers the inner peridium
surface not only in
T. dudkae
but also in
T. montana,
T. magna
T. corymbosa.
T. montana
ornamentations are reflected in the presence of
furrows in the outer surface of the peridium (FIG. 6k).
T. dudkae
T. montana
folds and rings may be
found on the same portion of the peridium (FIGS. 6n,
7n). In
T. ferruginosa
wavy folds occupy only small
areas and the rest of the peridial surface is smooth
(FIGS. 2l, m, 3m–o). In general wavy folds are most
abundant in species with iridescent peridia. We
suspect that these folds might represent the source
of the iridescence, forming microscopic prisms that
split the light with different wave lengths.
Pseudocapillitium, true capillitium and false capilli-
The absence of capillitium was thought to be
one of the important characters of the Reticularia-
ceae. Any thread-like structures found in members of
the family usually were called a pseudocapillitium
(Martin and Alexopoulos 1969, Nannenga-Breme-
kamp 1991, Ing 1999). However, in a strict sense
pseudocapillitium refers to a structure formed by the
remains of the peridium and columellae of confluent
sporothecae within an aethalium or pseudoaethalium
(Lister 1925). This was never shown convincingly for
Adans. and obviously is not the case in
Alwisia bombarda
A. lloydiae
. Therefore the
thread-like structures in these taxa are likely to
represent a true capillitium (see Leontyev et al.
2014c). In
T. casparyi
the columella has the shape
of a bottle brush as a result of the numerous
perpendicular branches. These branches demon-
strate all features of a typical capillitium but usually
have not been considered as such (Lister 1925, Martin
and Alexopoulos 1969). On the other hand a true
pseudocapillitium, formed by peridial remains, is
known for
T. dudkae
(Leontyev and Moreno 2011)
and all species of
Nannenga-Bremekamp was the first to observe thin,
branched threads, covered with adhering spores, in
many specimens of
T. ferruginosa
s.l. and provided
good illustrations (Nannenga-Bremekamp 1961: 58;
1991: 54). Later, the same structures were reported by
Neubert et al. (1993: 142). Nannenga-Bremekamp
first considered these threads to be a pseudocapilli-
tium (1961) but later (1991) she referred to them as
a true capillitium (FIG. 13a). She also indicated, that
this capillitium may or may not be present in
. Such an inconstancy of this character is
unusual for any species of myxomycete.
Our study of herbarium material shows that the
structures described by Nannenga-Bremekamp occur
sporadically in several species, namely
T. applanata,
T. dictyoderma, T. ferruginosa, T. magna
. In some specimens of each species these
threads are abundant, whereas in others they are
absent. Within one pseudoaethalium they may be
present in only a few sporothecae. As a general
estimate, we observed such structures in 18%of
studied specimens of
. The respective struc-
tures are thin (ca. 0.5–1.0 mm diam) but readily visible
under a dissecting microscope (FIG. 13b, c) because
numerous spores adhere to their surface (FIG. 13d–f,
h). This adhesion is strong; touching the spores with
a needle or rinsing with water does not release them.
Such a strong connection of capillitium and spores
seemingly contradicts the biological function of
a capillitium, which usually is thought to facilitate
spore release. The threads do not show a regular
connection to either the peridium or the columella;
they may be found even at the outer surface of
a sporotheca (FIG. 13g). SEM micrographs reveal
these threads to be hollow and divided by internal
septa (FIG. 13i). All these features indicate that these
“capillitial” structures found in members of the
complex are hyphae of fungi that use the
spores as food. This explains the occasional distribu-
tion of the threads in different species and the strong
adherence of the spores to them. If this explanation is
accepted, all species of the
T. ferruginosa
lack a true capillitium. With respect to a pseudocapil-
T. dudkae
is the only representative of the
complex reported to have this structure present
(Leontyev and Moreno 2011).
Spore size.—Tubifera ferruginosa
s.l. was described
originally as having spores 6–8 mm diam. Spore size
was used to distinguish it from
T. casparyi
with spores
7.5–9 mm and
T. microsperma
with 5–6 mm diam spores
(Lister 1925, Martin and Alexopoulos 1969). Our
study, based on measurements of 25 spores for each
of the 118 studied specimens of the various species of
, revealed noticeable differences in spore size
(FIG. 14). The data indicate that
T. ferruginosa
s. str. is
characterized by more narrow limits of spore size
(6.4–7.3 mm) and that the difference between sub-
is insignificant.
Tubifera pseudomicrosperma, T. corymbosa
have smaller spores than
T. ferruginosa
str., whereas in
T. dudkae
T. montana
the spores
are considerably larger. However, spore dimensions
are not entirely suitable as an identification tool
within the
T. ferruginosa
complex, because their
ranges of variation overlap for most species. Mean-
while, spore size helps to distinguish species that are
morphologically similar, such as
T. applanata
(spores 5.4–6.1 vs. 6.9–7.6 mm),
T. applanata
T. magna
(5.4–6.1 vs. 6.3–7.2 mm),
T. ferruginosa
T. montana
(6.4–7.3 vs. 7.1–8.1 mm),
T. micro-
T. pseudomicrosperma
(5.3–6.4 mm vs. 4.8–
5.4 mm),
T. corymbosa
T. dimorphotheca
vs. 5.7–6.3 mm).
Geographical distribution.—
Most of the new taxa in
Tubifera ferruginosa
complex occur in the
temperate zone of the northern hemisphere. Excep-
tions are the apparently tropical
T. corymbosa
, found
in Mexico and Costa Rica and some undescribed
species, such as
sp. 2 from the Seyshelles and
sp. 3 from New Zealand. Besides,
s.l. has been reported from several
countries of South America (gwannon.com). Howev-
er, it is still impossible to attribute these findings to
the genotypic species of
T. ferruginosa
described in this study or to undescribed taxa.
At least some species in the complex,
T. ferruginosa
T. montana
and probably
T. pseudomicrosperma
, are
widely distributed in all studied regions in Europe,
Asia and North America. However, the geographical
distribution of their 18S rDNA genotypes is much
narrower, suggesting dispersal barriers that limit gene
Other species seem to have a more restricted
distribution. For example,
T. applanata
is common
in southeastern Europe and northern Asia, including
Italy and Croatia (Renato Cainelli pers comm), the
European part of Russia (Inna Zemlyanskaya pers
comm), the Ural Mountains and Siberia (Leontyev
and Fefelov 2009) but rare in eastern North America
(only one collection known thus far).
Tubifera dudkae
is known only from Europe (France, Ukraine) and
northern Asia (Russia) and is still unknown from the
New World. Finally,
T. magna
is widespread in North
America but only a single collection with a different
18S rDNA genotype is known from Europe.
1a. Columella always present, thread-like, shin-
ing, reaching the tip of a sporotheca and
adherent to its peridium; upper peridium
very tough, fructifications durable and re-
sistant to the touch; spore mass ocher, umber
or grayish . . . . . . . . . . . . . . . . . . . . . . . 2
1b. Columella absent or only occasionally present,
in this case thick, irregularly conical, dull,
never reaching the tip of a sporotheca; peri-
dium delicate and fragile, fructifications easily
destroyed upon contact; spore mass rust-
brown ................................. 3
2a. Columella hollow, tubular, shaped like a bottle-
brush because of numerous perpendicular
branches; upper peridium umber brown, spore
mass umber gray; spores 7.5–8 mm...
T. casparyi
2b. Columella solid, with irregular branches; upper
peridium dark red brown, spore mass cinnamon,
hazel brown when fresh, ochraceous in old
collections; spores 4.5–6 mm .....
T. dictyoderma
3a (1). Spherical sporothecae aggregated around the
base of the pseudoaethalium . ........... 4
3b. Spherical sporothecae absent or not distinctly
aggregated around the base of the pseu-
doaethalium ........................ 5
4a. Spherical sporothecae numerous, seated on
a cylindrical stub-like stalk; sporothecal tips
hemispherical, the peridium of the latter with
the same color as the remaining part of the
sporotheca ..............
T. dimorphotheca
4b. Spherical sporothecae few, arranged around
the constricted base of the pseudoaethalium,
distinct stalk absent; sporothecae tips mostly
truncate, the peridium of the latter iridescent
with rainbow colors, sometimes with a silvery
white apex .................
T. corymbosa
5a (3). Hypothallus prominent, smooth, dark brown
to black, forming a thick basal structure or
a vertical cylindrical stalk, which is at least as
tall as the sporothecal layer ............. 6
5b. Hypothallus amorphous, thinner, never form-
ing a basal structure or stalk ............. 8
6a. Sporothecal tips acutely conical; hypothallic
stalk strongly constricted at the base; spores
6.5–7.5 mm ..................
T. papillata
6b. Sporothecal tips hemispherical to almost flat;
hypothallus flat; spores 4.5–6.5 mm ........ 7
7a. Pseudoaethalia 1–2 cm in extent, hypothallus
forming a wide basal structure, sometimes
common for a group of pseudoaethalia;
internal surface of the peridium covered with
rimmed craters 0.2–0.6 mm diam that are
visible only with SEM; occurring in temperate
regions . . ...........
T. pseudomicrosperma
7b. Pseudoaethalia 0.3–0.7 cm in extent, hypothal-
lus forming a stub-like stalk, the height of the
latter structure equal to or exceeding its
diameter; internal surface of the peridium
covered with rimmed craters 0.5–2.5 mm diam,
these clearly visible in DIC; occurring mostly
in the tropics ..............
T. microsperma
8a (5). Most of the sporothecae spherical, distributed
all over the pseudoaethalium (check a cross
section) . . ...................
T. dudkae
8b. Nearly all of the sporothecae elongated,
cylindrical or prismatic ................. 9
9a. Pseudoaethalia large, mostly 3–12 cm in
extent; sporothecal tips flat, angular ...... 10
9b. Pseudoaethalia usually less than 3 cm in
extent; sporothecal tips elevated: lenticular,
hemispherical or conical .............. 11
10a. Sporothecal tips isodiametric, dull; inner side
of the peridium ornamented with rings up to
3mm diam visible in DIC, immature fructifica-
tions dirty flesh to light brownish-salmon;
spores 5.4–6.1 mm; occurring mostly in Eur-
asia ......................
T. applanata
10b. Sporothecal tips elongated, glossy, shining;
peridial rings ,1mm in diam observed only in
SEM, immature fructifications pink;
spores 6.3–7.2 mm; occurring mostly in North
America . . ...................
T. magna
11a (9). Immature fructifications bright orange, spor-
othecal tips adhering to each other, lenticular;
peridium weakly iridescent with golden,
bronze or pinkish tints; spores 7.1–8.1
mm .......................
T. montana
11b. Immature fructifications salmon, red or pink,
sporothecal tips free, strongly convex; peridi-
um weakly iridescent with bluish and greenish
tints; spores 6.4–7.3 mm .............
Tubigera ferruginosa
s. str.)
12a. Tips of sporothecae hemispherical, papillate
to obtusely conical; immature fructifications
palesalmontoscarletred .................
T. ferruginosa
12b. Tips of sporothecae bluntly conical, with
spine-like apices; immature fructifications
deep pink ....
T. ferruginosa
The research was supported by a grant from the German
Academic Exchange Service (DAAD; A/12/04515) and
a scholarship from the Fulbright Scholar Program (grant
68130017) to the first author.Additional support was provided
by the Slime Mold Project at the University of Arkansas. We
thank Laura Walker and Bobbie Okimoto (University of
Arkansas), Anja Klahr and Eva Heinrich (University of
Greifswald, Germany) for help with laboratory work. For
loans of specimens we are indebted to Yuri Novozhilov (St.
Petersburg, Russia), Anna-Maria Fiore-Donno (Cologne,
Germany), Marianne Meyer (Rognaix, France) and Rene´e
Lebeuf (Montre´al, Canada). Thanks in particular are extend-
ed to Renato Cainelli (Italy), Rene´e Lebeuf and adminis-
trators of the Mushroom Observer (United States) for their
kind permission to publish several of their field photographs.
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Supplementary resources (33)

... На момент опису у 2011 році його знахідки (4 зразки) були відомі лише в Україні [LEONTYEV, MORENO, 2011]. Під час критичної ревізії комплексу у 2015 р. до наявних даних було додано знахідки з Франції (Канни) та Росії (Новосибірськ) [LEONTYEV et al., 2015]. Іще одну знахідку в Україні, з тієї ж Харківської області, де розташований типовий локалітет, було наведено у 2017 році [YATSIUK et al., 2017]. ...
... Однак інформації про точні координати знахідок у визначнику немає; сподіваємось, що ці знахідки зрештою будуть опубліковані. Нарешті, вид також присутній у чеклисту міксоміцетів Росії, опублікованому у 2020 році [BORTNIKOV et al., 2020], але у переліку наведено лише вищезгадану знахідку у Новосибірську [LEONTYEV et al., 2015]. ...
... On the one hand, such patterns of multiple cryptic species within morphospecies seem to be common in myxomycetes and may represent a general mode of evolution in this group: all hitherto investigated morphospecies turned out to be complexes of several cryptic species (Aguilar et al., 2014;Dagamac et al., 2017;Feng & Schnittler 2015;Janik et al., 2020;Leontyev, Schnittler, & Stephenson, 2015;Shchepin et al., 2016). This means that we should expect the real number of myxomycete species to be several times bigger than ca. ...
Full-text available
Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1–3 genetic markers (SSU, EF1A, COI) and analyzed 98 specimens with Genotyping by Sequencing (GBS). The structure of the three‐gene phylogeny, SNP‐based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well‐studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes.
... If occurring regionally, such units are expected to suffer less from outbreeding depression, which may facilitate local genetic adaptation. The respective biospecies may be cryptic (Trichia varia, 3 putative biospecies; Feng and Schnittler 2015), partially discernible by subtle morphological characters (Hemitrichia serpula, 4; Dagamac et al., 2017b), or well discernible if novel traits can be found (Tubifera ferruginosa s.l., >5; Leontyev et al., 2015). Although the number of records included was very limited, two studies modeling the distribution of biospecies showed different geographical ranges for those (Badhamia melanospora, Aguilar et al., 2014; Hemitrichia serpula, Dagamac et al., 2017b). ...
To identify potentially suitable areas for the mostly alpine ecological guild of nivicolous (snowbank) myxomycetes, the worldwide distribution of a distinct morphospecies, Physarum albescens, was modelled with a correlative spatial approach using the software MaxEnt from 537 unique occurrence points. Three models were developed, first with only the 19 bioclimatic variables plus elevation from the WorldClim database, second with regularization to correct for pseudo-absence, and third with additional categorical environmental layer on snow cover. All three models showed high mean AUC (area under the curve) values (>0.970). Output maps were comparable, with the third model perhaps the most realistic. For this model, snow cover, precipitation of the coldest quarter (of the year), and elevation predicted best the distribution of Ph. albescens. Elevation alone is a good predictor only in some regions, since (i) elevation of the occurrence points decreases with increasing latitude, and (ii) elevation wrongly predicts the species' occurrence in arid mountain ranges. The model showed mountains in humid climates with highest incidence, which confirmed field studies: a long-lasting snow cover fluctuating with comparatively mild summers is the decisive factor. As such, the model can serve as a predictive map where fructifications of nivicolous myxomycetes can be expected. Limitations of the model are discussed: cryptic speciation within a morphospecies, including the evolution of reproductively isolated units which may lead to local adaptation and niche differentiation, and wider ranges for myxamoebal populations.
... alpestre) and capillitium (Diderma niveum and Lamproderma ovoideum), the size, color and ornamentation of spores (all studied species). The number of specimens investigated does not allow us to draw any taxonomic conclusions, but this fits into a pattern observed for several morphological species of myxomycetes, which show an internal structure of groups of ribotypes, corresponding to putative biological species (Leontyev et al. 2012, Leontyev et al. 2015, Leontyev et al. 2019b. For some of the abovementioned nivicolous taxa similar conclusions have already been made, albeit without taxonomic consequences. ...
Abstract: During a field survey in April 2019 fourteen species of nivicolous myxomycetes were recorded from the Carpathian National Nature Park, East Carpathians, Ukraine. Most abundant were Diderma alpinum, Lepidoderma chailletii, and Lamproderma echinosporum. Five species (Meriderma echinulatum, Lamproderma arcyrioides, L. album, Diderma europaeum and D. microcarpum) are new to the Ukraine. All 81 collected specimens were barcoded for a fragment of the 18S rDNA, resulting in 26 ribotypes. Half of the species (51 specimens) were represented by two to four ribotypes, the remaining seven species (30 specimens) by only one (average: 1.85 ribotypes per species). Intraspecific differences in ribotypes were often accompanied by subtle morphological differences between specimens assigned to different ribotypes, including shape of the fruiting body, its coloration, the structure of the peridium and capillitium, or size, color and ornamentation of spores. Key words: 18S rRNA; biospecies; DNA barcoding; morphospecies; ribosomal small subunit
Myxomycete research in China is mostly focused on abundance-based diversity assessments of natural habitats, or species checklists of local regions, but studies of myxomycete diversity over broad geographical scales are limited. This study reports diversity from four mountainous areas in central China based on a total of 3212 specimens representing 155 species of 38 genera. The most abundant species were Arcyria cinerea, Colloderma oculatum, and Clastoderma debaryanum. Species richness and Shannon-Wiener diversity were significantly different among the study areas, with highest values found for the Tiantangzhai Forest Park for specimens from moist chamber culture and Houhe Reserve from field collections. Species richness and diversity in mixed broadleaf-conifer forests were higher than in broadleaf and coniferous forests. Myxomycetes on substrates cultivated in moist chamber cultures had a more comparable species composition, implying that some species show a higher fruiting propensity in such culture than others. There were significant differences in myxomycete communities of various substrates between study areas and forest types, with the former explaining a higher part of the variation. This first exploration on a large spatial scale in China suggested that the effects of geographical separation on myxomycete communities were greater than separation in forest types.
Early phylogenetic studies refuted most previous assumptions concerning the evolution of the morphological traits in the fruiting bodies of the order Trichiales and did not detect discernible evolutionary patterns, yet they were based on a limited number of species. We infer a new Trichiales phylogeny based on three independently inherited genetic regions (nuclear and mitochondrial), with a fair taxonomic sampling encompassing its broad diversity. Besides, we study the evolutionary history of some key morphological characters. According to the new phylogeny, most fruiting body traits in Trichiales systematics do not represent exclusive synapomorphies or autapomorphies for most monophyletic groups. Instead, the evolution of the features derived from the peridium, stalk, capillitium, and spores showed intricate patterns, and character state transitions occurred rather within- than between clades. Thus, we should consider other evolutionary scenarios instead of assuming the homology of some characters. According to these results, we propose a new classification of Trichiales, including the creation of a new genus, Gulielmina, the resurrection of the family Dictydiaethaliaceae and the genus Ophiotheca, and the proporsal of 13 new combinations for species of the genera Arcyria (1), Hemitrichia (2), Ophiotheca (2), Oligonema (4), Gulielmina (3), and Perichaena (1).
Spore size enables dispersal in plasmodial slime molds (Myxomycetes) and is an important taxonomic character. We recorded size and the number of nuclei per spore for 39 specimens (colonies of 50–1000 sporocarps) of the nivicolous myxomycete Physarum albescens, a morphologically defined taxon with several biological species. For each colony, three sporocarps were analyzed from the same spore mount under brightfield and DAPI-fluorescence, recording ca. 14,000 spores per item. Diagrams for spore size distribution showed narrow peaks of mostly uninucleate spores. Size was highly variable within morphospecies (10.6–13.5 µm, 11–13%), biospecies (3–13%), even within spatially separated colonies of one clone (ca. 8%); but fairly constant for a colony (mean variation 0.4 µm, ca. 1.5%). ANOVA explains most of this variation by the factor locality (within all colonies: 32.7%; within a region: 21.4%), less by biospecies (13.5%), whereas the contribution of intra-colony variation was negligible (<0.1%). Two rare aberrations occur: 1) multinucleate spores and 2) oversized spores with a double or triple volume of normal spores. Both are not related to each other or limited to certain biospecies. Spore size shows high phenotypic plasticity, but the low variation within a colony points to a strong genetic background.
This chapter discusses the adaptations of myxomycetes relating to spore dispersal, which is the key to understanding distribution patterns in this group of protists. Although several groups of protists form spores, myxomycetes are the most successful group, judged by the number of species. Fruiting bodies in myxomycetes are primarily stalked to allow spores to dry out and become airborne. Compound fruiting bodies are a second evolutionary tendency to achieve spore release by means of animal vectors and have appeared parallel in several taxa. Since fruiting bodies are formed only under optimum conditions, species often have larger distribution ranges than indicated by fruiting bodies. In contrast, many morphospecies may be complexes of cryptic biological species, and these may have narrower ecological niches and thus narrower distribution ranges. In addition, molecular studies of widely distributed morphospecies provide evidence for limited gene flow within regional populations. As such, myxomycetes seem to follow the moderate endemicity model more than the ubiquist model of microbial distribution. Molecular markers and barcoding provide novel tools to differentiate species and may link the two separate species concepts in the group, the morphospecies concept and the biospecies concept. Most likely, the number of described morphospecies of myxomycetes will increase steadily. Although field studies in myxomycetes have been carried out for more than 200 years, survey intensity is still very different for different regions of the world and the methods used (direct observations vs moist chamber cultures). The existing data indicate that species diversity patterns in myxomycetes do not follow the “decreasing latitude—increasing diversity” trend that holds true for most macroscopic organisms. Instead, hot spots for myxomycetes seem to be in southern temperate zones, especially broadleaf deciduous forests. The surprisingly distinct and diverse assemblages of myxomycetes in deserts point to precipitation as one of the major factors to explain these patterns.
Within the myxomycetes, phylogenetic research is still at a relatively early stage, and available phylogenies are often based on only a single or a few marker genes. According to the available data, the “true” myxomycetes (exosporous, myxogastric) form a monophyletic clade within the phylum Eumycetozoa with two basal subclades, a dark-spored (treated as the Columellomycetidae) and a bright-spored (the subclass Lucisporomycetidae). Within the first subclade the main evolutionary branches are recognized as the orders Echinosteliales, Echinosteliopsidales, Clastodermatales, Meridermatales, Stemonitidales, and Physarales; for the bright-spored subclade, these are the orders Cribrariales, Reticulariales, Liceales, and Trichiales. In the light of molecular data the traditional criteria used in myxomycete taxonomy have to be reevaluated. The morphology of capillitia and peridia, and especially how these two structures are connected to each other in a particular taxon, seems to be highly informative, whereas their presence or absence is not. Similarly, single versus compound fruiting bodies, a character often used to delimit genera, seems to deserve a lower weight, as these structures have evolved several times in parallel. Many, especially species rich, genera seem not to represent monophyletic units, and further research will most likely change their delimitation.
Unlike fungi, which have a universally accepted barcode marker, universal primers still lack in myxomycetes. Typically, DNA barcode primers were designed based on comparing existing myxomycetes sequences and targeting the conserved regions. However, the extreme genetic diversity within major myxomycetes groups and the frequent occurrence of group I introns have made the development of universal DNA barcode a severe challenge. The emergence of next-generation sequencing provides an opportunity to address this problem. We sequenced the mixed genomic DNA of 81 myxomycetes and extracted the SSU gene's reads using next-generation sequencing. After alignment and assembly, we designed a set of SSU primers that matched all potential SNPs, avoided all known group I intron insertion sites, and were highly conserved between major myxomycetes orders. This set of SSU primers has the potential to become one of the universal primer combinations. Due to the high genetic divergence caused by long and complicated evolutionary histories, the lack of universal barcode primers is common in protists. Our research provides a new method to solve this problem.
Full-text available
It is an online nomenclatural information system of the Eumycetozoans (Myxomycetes, Dictyostelids and Protostelids) of the world, providing information on more than 4,000 names employed in this group.
Full-text available
Tubifera applanata sp. nov. is proposed to validate "Tubulifera applanata" nom. inval. The species diagnosis and some notes on its morphology are provided. At 0.40-0.65 mm diam., individual sporothecae are somewhat larger than in T. ferruginosa and T. microsperma and smaller than those in T. casparyi. Circular ornamentations on the inner peridial surface in T. applanata are larger than previously noted, reaching a size up to 2.9 mu m.
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We studied the capillitial structures produced in Alwisia bombarda (Reticulariaceae, Myxomycetes) by light and electron microscopy. This species develops tubular threads inside of clustered but still separate sporocarps with intact walls forming a cup. These threads have been considered to be a pseudocapillitium for a long time, but they do not represent remains of confluent peridia and thus do not correspond to the classical definition of a pseudocapillitium. Instead, they should be considered as a true capillitium. The structure of the capillitium in A. bombarda is very similar to that found in the genera Dianema and Lycogala, thus providing a new argument for a close relationship between the Reticulariaceae and Dianemataceae and also for considering the tubular threads of Lycogala as representing a true capillitium.
Full-text available
Based on a morphological investigation of a series of specimens collected in New South Wales and Tasmania and a phylogeny constructed with partial 18S ribosomal RNA gene sequences, we describe a new species Alwisia lloydiae; the fourth species within the recently re-validated genus Alwisia. This new species is characterized by short ovate sporothecae with mostly free stalks, morphologically resembling the recently described A. morula. However, the new species possesses a tubular capillitium that suggests an affinity with A. bombarda. The capillitium of the new species is ornamented with globular warts, and this feature separates it from all other members of the genus.
The introductory chapters cover life history, structure, ecology and distribution, how to find and collect material, bark culture techniques, microscopic examination and herbarium storage. The main section of the book is devoted to identification and includes keys, descriptions and illustrations showing the diagnostic features. In the account for each species are notes on differences from other species, reference to other published illustrations, comments on ecology and distribution and useful tips on collection and identification. The book includes all those species of true myxcomycetes, plus one ceratiomyxomycete and one acrasian, known to have occurred in Great Britain and Ireland. There are over 500 line illustrations, a useful bibliography and a comprehensive index.
The inner peridial surface of Tubifera microsperma is covered with numerous crater-like markings, and thus is distinct from that of other species of Tubifera. Ornamentation of the inner peridium, or lack of it, provides useful information with regard to collections appearing to be intermediate between T. microsperma and T. ferruginosa. Spores of all Tubifera species are at least partially reticulate. A new key to the species is provided.