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New insights in Russula subsect. Rubrinae: phylogeny and the quest for synapomorphic characters

Authors:
  • Institute of Botany, Plant Science and Biodiversity Centre, Slovak Academy of Sciences

Abstract

Russula is one of the most speciose genera of mushroom-forming fungi, but phylogenetic relationships among species and subgeneric groupings are poorly understood. Our multi-locus phylogenetic reconstruction places R. firmula, R. rubra, R. rutila and R. veternosa in a well-supported Rubrinae clade, belonging to the Integrae clade of the Crown clade of the genus Russula. Traditional morphology-based classifications placed these four species in two different subsections based on the presence or absence of incrustations on pileocystidia. The Integrae clade also contains R. integra and related species that are traditionally placed in other groups based on their mild taste. Ancestral state reconstruction suggests that the common ancestor of the Crown clade and the Integrae clade probably did not have any incrustations in the pileipellis, had a mild taste, yellow spore print and were associated with angiosperms. All four species of the Rubrinae clade are defined by a darker yellow or ochre spore print, acrid taste and incrustations on pileocystidia. This last character contradicts the former splitting of the group because incrustations were apparently overlooked in R. firmula and R. veternosa. Incrustation type is now highlighted as being important for the delimitation of species and groups within the Crown clade. Pink or red staining of the incrustations in sulphovanillin is present in all species of the Rubrinae clade and a majority of the analysed species of the Integrae clade. The delimitation of the Rubrinae clade and its species circumscriptions are summarised here in a new diagnostic key.
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Mycological Progress
ISSN 1617-416X
Mycol Progress
DOI 10.1007/s11557-017-1322-0
New insights in Russula subsect. Rubrinae:
phylogeny and the quest for synapomorphic
characters
Miroslav Caboň, Ursula Eberhardt,
Brian Looney, Felix Hampe, Miroslav
Kolařík, Soňa Jančovičová, Annemieke
Verbeken & Slavomír Adamčík
1 23
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ORIGINAL ARTICLE
New insights in Russula subsect. Rubrinae: phylogeny
and the quest for synapomorphic characters
Miroslav Caboň
1
&Ursula Eberhardt
2
&Brian Looney
3
&Felix Hampe
2
&
Miroslav Kolařík
4
&SoňaJančovičová
5
&Annemieke Verbeken
6
&Slavomír Adamčík
1
Received: 21 March 2017 /Revised: 6 July 2017 / Accepted: 10 July 2017
#German Mycological Society and Springer-Verlag GmbH Germany 2017
Abstract Russula is one of the most speciose genera of
mushroom-forming fungi, but phylogenetic relationships
among species and subgeneric groupings are poorly under-
stood. Our multi-locus phylogenetic reconstruction places
R. firmula,R. rubra,R. rutila and R. veternosa in a well-
supported Rubrinae clade, belonging to the Integrae clade of
the Crown clade of the genus Russula. Traditional
morphology-based classifications placed these four species
in two different subsections based on the presence or absence
of incrustations on pileocystidia. The Integrae clade also con-
tains R. integra and related species that are traditionally placed
in other groups based on their mild taste. Ancestral state re-
construction suggests that the common ancestor of the Crown
clade and the Integrae clade probably did not have any incrus-
tations in the pileipellis, had a mild taste, yellow spore print
and were associated with angiosperms. All four species of the
Rubrinae clade are defined by a darker yellow or ochre spore
print, acrid taste and incrustations on pileocystidia. This last
character contradicts the former splitting of the group because
incrustations were apparently overlooked in R. firmula and
R. veternosa. Incrustation type is now highlighted as being
important for the delimitation of species and groups within
the Crown clade. Pink or red staining of the incrustations in
sulphovanillin is present in all species of the Rubrinae clade
and a majority of the analysed species of the Integrae clade.
The delimitation of the Rubrinae clade and its species circum-
scriptions are summarised here in a new diagnostic key.
Keywords Sulphovanillin .Incrustations .Agarics .
Multi-locus phylogeny .Ancestral state reconstruction
Introduction
Russula Pers. is a species-rich genus of ectomycorrhizal fungi
with more than 265 morphological species recognised in
Europe (Sarnari 1998,2005), corresponding to 295 molecular
operational taxonomic units (MOTUs) defined by ITS nrDNA
sequence data retrieved from GenBank (Looney et al. 2016).
The majority of Russula species and infrageneric taxa were
described based on phenotype (Romagnesi 1967;Sarnari
2005) and only a few studies reconsidered existing
morphology-based concepts using molecular data (e.g. Liu
et al. 2015). Recent studies suggest that some morphologically
defined groups correspond to well-defined phylogenetic line-
ages (Russula subsect. Xerampelinae Singer in Adamčík et al.
2016a); however, many morphological groups appear to be
polyphyletic (Russula subsect. Maculatinae Romagn. in
Adamčík et al. 2016b). Adamčík et al. (2016b)demonstrated
a high similarity of ITS nrDNA sequences among specimens
Section Editor: Zhu-Liang Yang
Electronic supplementary material The online version of this article
(doi:10.1007/s11557-017-1322-0) contains supplementary material,
which is available to authorized users.
*Miroslav Caboň
miroslav.cabon@savba.sk
1
Department of Cryptogams, Institute of Botany, Plant Science and
Biodiversity Centre, Slovak Academy of Sciences,
Bratislava, Slovakia
2
Staatliches Museum für Naturkunde Stuttgart, Stuttgart, Germany
3
Department of Ecology and Evolution Biology, University of
Tennessee, Knoxville, TN, USA
4
Laboratory of Fungal Genetics and Metabolism, Institute of
Microbiology, Czech Academy of Sciences, Praha, Czech Republic
5
Department of Botany, Faculty of Natural Sciences, Comenius
University, Bratislava, Slovakia
6
Department of Biology, Ghent University, Ghent, Belgium
Mycol Progress
DOI 10.1007/s11557-017-1322-0
Author's personal copy
referred on a morphological basis to either R. firmula Jul.
Schäff., R. rubra (Lam.) Fr., R. rutila Romagn. or
R. veternosa Fr. These species share several similar morpho-
logical characters (yellow spore print, acrid taste of the flesh,
amyloid suprahilar spot), but they are traditionally classified
in various groups. Russula rubra and R. rutila are placed
within Russula subsect. Rubrinae (Melzer & Zvára) Singer
based on the presence of incrusted pileocystidia, while
R. firmula and R. veternosa are described as lacking such
incrustations (Romagnesi 1967;Sarnari1998). Singer
(1986) and Sarnari (1998) placed them in a single group, R.
subsect. Urentes Maire, but Romagnesi (1967) classified
R. veternosa in R. subsect. Maculatinae and R. firmula in R.
subsect. Urentinae Maire.
The most comprehensive multi-locus Russula phylogeny,
published by Looney et al. (2016), includes only one of the
four above-mentioned species, R. firmula. According to the
authors, the species is part of a large phylogenetic lineage
called the Crown clade. Based on ITS sequence similarity
(Adamčík et al. 2016b), we expect that all four species will
be members of the Crown clade. In this study, we present a
multi-locus phylogeny of the Crown clade of Russula as de-
fined by Looney et al. (2016). Our phylogenetic sampling is
based on representation of the majority of morphologically
defined sections and subsections recognised by Romagnesi
(1967), Singer (1986) and Sarnari (1998) that are, according
to Looney et al. (2016), part of this Crown clade. We are
exploring the phylogenetic support for a lineage that includes
R. rubra, the type species of subsection Rubrinae.Weseekto
determine whether R. firmula,R. rutila and R. veternosa in-
deed belong to a single lineage and whether this group can be
morphologically recognised. In addition, it is our objective to
test the species limits by including material from distant areas
of Europe and from a variety of habitats. We will also analyse
if traditional morphological classification coincides with de-
fined molecular lineages of the Crown clade.
Materials and methods
Sampling
Samples of the four target species, R. firmula (11 collections),
R. rubra (8 collections, including the type), R. rutila (15 col-
lections, including the type) and R. veternosa (9 collections),
were identified based on morphology following Knudsen
et al. (2012). Type collections of the target species were se-
quenced when possible. The neotypes of R. firmula designat-
ed by Romagnesi (1967) and Sarnari (1998) were not studied
because the actual types are illustrations cited in the
protologue, making the neotypes invalid. We also included
the type and two recent collections of R. quercilicis Sarnari,
a Mediterranean species classified in Rubrinae based on its
incrusted pileocystidia (Sarnari 1998). All sequences from the
public databases GenBank (https://www.ncbi.nlm.nih.gov/
genbank) and UNITE (https://unite.ut.ee) matching 97% and
higher similarity with any of the target species clade were
included in the phylogenetic analysis.
For determining the phylogenetic placement of the four
target species, we used a selection of European species
placed in the Crown clade by Looney et al. (2016). We
also sought to sample the type species of every section
and subsection suggested to be a part of the Crown clade.
In addition, we included some well-known and common
European species. All collections sequenced in this study
originate from various countries of Europe, but a major
part of sampling came from Central Europe (Slovakia and
Germany) and a majority of them were morphologically
identified and sequenced by the authors of this study. This
sampling is supplemented by a few sequences published
by Looney et al. (2016). Three species of Russula subge-
nus Russula are used as an outgroup. All sequences used
in our study are listed in Supplementary Material 1.
Molecular analyses
Total genomic DNA was extracted from dried material using
the methods previously described by Adamčík et al. (2016b).
We amplified three molecular markers: (1) the internal tran-
scribed spacer regions of the ribosomal DNA (ITS); (2) partial
mitochondrial small subunit ribosomal DNA (mtSSU); (3) the
region between domains six and seven of the nuclear gene
encoding the second largest subunit of RNA polymerase II
(rpb2). The ITS region was amplified using the primers
ITS1FITS4 (White et al. 1990; Gardes and Bruns 1993).
The mtSSU region was amplified using the primer pair MS1
and MS2 (White et al. 1990). Both molecular markers were
amplified with Hot Start Firepol Polymerase (Solis BioDyne,
Tartu, Estonia) using the same cycling protocol: 95 °C/15 min;
35 repeats (95 °C/30 s, 50 °C/30 s, 72 °C/1 min); 72 °C/
10 min; cooling to 4 °C. For amplification of rpb2,weused
a new forward primer A-Russ-F (5-TGTC
GGGTCCCATNATYGAA-3), designed using Primer-
BLAST (Ye et al. 2012) and a reverse primer frpb2-7CR
(Matheny 2005). The rpb2 was amplified with Hot Start
Firepol Polymerase, using the following cycling protocol:
95 °C/15 min; 35 repeats (95 °C/1 min; 58 °C/1 min; increas-
ing temperature 59 °C/10 s; 60 °C/10 s ... up to 71 °C/10 s;
72 °C/1 min); 72 °C/10 min; cooling to 4 °C. The polymerase
chain reaction (PCR) products were purified using ExoSAP
enzymes (Thermo Fisher Scientific, Wilmington, DE, USA)
or QIAquick PCR Purification Kit (Qiagen, Hilden,
Germany). Samples were sequenced directly with BigDye
3.1 technology (Applied Biosystems, now Thermo Fisher
Scientific, Wilmington, USA) or sent to Macrogen Europe
(Amsterdam, the Netherlands).
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Phylogenetic analysis
Sequences were edited in the BioEdit Sequence Alignment
Editor version 7.2.5 (Hall 2013) or Geneious version R10
(Kearse et al. 2012). Intra-individual polymorphic sites having
more than one signal were marked with NC-IUPAC ambiguity
codes. Final datasets were aligned by MAFFT version 7 using
the strategy E-INS-i (Katoh and Standley 2013) and further
edited in AliView version 1.17.1 (Larsson 2014). All three
single-locus datasets were concatenated into one multi-loci
dataset using SeaView v.4.5.1 (Gouy et al. 2010). The
concatenated final alignment has been deposited at
TreeBASE (20799). The multi-locus dataset was analysed
using two different methods: Bayesian inference (BI) and
the maximum likelihood method (ML). For ML analysis, the
concatenated alignment was loaded as a PHYLIP file into the
RAxML GUI version 1.2 (Silvestro and Michalak 2012)and
analysed as a partitioned dataset under the GAMMA + I + G
model with 1000 bootstrap iterations. For BI, the dataset was
divided into six partitions: ITS, mtSSU, intronic region 7 of
rpb2 and the 1st, 2nd and 3rd codon positions of rpb2.The
best substitution model for each partition was computed joint-
ly in PartitionFinder v.1.1.1 (Lanfear et al. 2012). BI was
computed independently twice in MrBayes version 3.2.6
(Ronquist et al. 2012) with four Markov chain Monte Carlo
(MCMC) chains for 10,000,000 iterations until the standard
deviation of split frequencies reached below the 0.01 thresh-
old. The convergence of runs was visually assessed using the
Trace function in Tracer version 1.6 (Rambaut et al. 2014).
Ancestral state reconstruction
To assess the character evolution of the most important mor-
phological characteristics used for infrageneric classification
within the genus Russula, we performed ancestral state recon-
struction on the dataset used for the phylogenetic analysis.
Only species recognised morphologically by authors of this
study are included, and every species is represented by a sin-
gle collection. A choice of three morphological characters
traditionally used for the grouping of species into infrageneric
taxa of the genus Russula and the host preference are
analysed. The coding of all three morphological characters is
based on our own observations. Visibility and red staining of
incrustations on pileocystidia and primordial hyphae were
scored using both carbolfuchsin and sulphovanillin. Spore
print colour is scored in three classes: pale up to IIb accord-
ing to Romagnesi (1967), intermediate IIc to III and yellow
IIId to IVe. The taste of the context is classified in three
categories: mild, weakly acrid and acrid. The preference of
coniferous or angiosperm host was assigned based on pub-
lished Russula monographs (Romagnesi 1967; Einhellinger
1994;Sarnari1998,2005; Knudsen et al. 2012) and data from
the DEEMY database of ectomycorrhizae (http://www.
deemy.de).
A species tree was inferred using the same procedure for
the ML analysis, only with species clades pruned to a single
representative. Character history was traced across the tree
topology using the Trace Character History function using
an ML approach with stored probability models implemented
in Mesquite v. 2.74 (Maddison and Maddison 2010).
Significant support for character states was assessed using a
cut-off value of a difference of 2.0 between log-likelihoods of
states. The resulting trees were ladderised at the root and
displayed as balls and sticks graphs. We chose to perform
these analyses on the full clade even though our choice of
molecular loci result in poorly resolved relationships through-
out most of the Crown clade. We feel this is justified as these
outgroup taxa inform the likelihood model with how prevalent
these traits are across the relevant clade and how frequent
transitions have occurred in the character states. Our interpre-
tation will focus on well-resolved clades associated with the
species of interest.
Morphological analysis
The morphological circumscription of the group of R. rubra
and related species is based on comparisons of our morpho-
logical observations of three collections selected for each of
the four target species. To narrow the number of micro-
morphological characters efficiently, we compared the full
detailed descriptions of one collection per species with similar
published descriptions (Adamčík and Jančovičová 2012,
2013) to other species of R. subsect. Maculatinae and R.
subsect. Urentinae, characterised according to Romagnesi
(1967) by acrid taste, yellow spore print, spores with an am-
yloid suprahilar spot and presence of non-incrusted
pileocystidia. Characters selected based on this first-step com-
parison were further tested for their taxonomic significance to
define the R. rubra group from other species of the Crown
clade and for differences between species within the lineage.
Micro-morphological characteristics were observed using
an Olympus CX41 microscope equipped with an Artray
ARTCAM-300MI camera at a magnification of 1000×.
Spores were measured using QuickPHOTO MICRO version
2.1 software and spore dimensions exclude ornamentation.
All drawings of microscopic structures, with the exception
of spores, were made with a camera lucidausing an
Olympus U-DA drawing attachment at a projection scale of
2000×. The Q value was used to indicate the length/width
ratio of the spores. The spore ornamentation density was esti-
mated following Adamčík and Marhold (2000). The cystidia
density estimates follow Buyck (1991). The contents of
hymenial cystidia and pileocystidia were illustrated as ob-
served in Congo red preparations from dried material, with
the exception of some pileocystidia, for which the contents
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are indicated schematically (dotted). Spores were observed on
the lamellae with Melzers reagent. All other microscopic ob-
servations were made in ammoniacal Congo red, after a short
treatment in warm, aqueous KOH solution to dissolve the
gelatinous matrix and improve tissue dissociation. Statistics
for the measurements of microscopic characteristics were
based on 30 measurements per specimen and expressed as
the mean ± standard deviation. The classification of spore
print colour follows Romagnesi (1967).
Incrustation of pileocystidia is the distinguishing char-
acter of R. subsect. Rubrinae (Romagnesi 1967;Sarnari
1998). For this reason, we paid special attention to the
observation of incrustations and contents of pileocystidia
using various reagents: Congo red solution, Cresyl blue
(Buyck 1989), carbolfuchsin (Romagnesi 1967)and
sulphovanillin. In Cresyl blue, the presence of ortho- or
metachromatic reactions as explained by Buyck (1989)
was examined. Acid-resistant incrustations of the primor-
dial hyphae or pileocystidia were stained with
carbolfuchsin and observed in distilled water after incu-
bation for a few seconds in a 10% solution of HCl
(following Romagnesi 1967). For observations in
sulphovanillin, two techniques were tested: pre-prepared
freshsolutionofvanillininsulphuricacid(Moser1978;
Kreisel and Schauer 1987;Adamčík and Knudsen 2004)
or crystals of vanillin dissolved in sulphuric acid just be-
fore preparation of the object (http://www.deemy.de).
Because the index of refraction of sulphuric acid is
different from that of water and the strong acid quickly
causes dissolution of cell walls, we used less concentrated
sulphuric acid. We dissolved a few crystals of vanillin in
one drop of concentrated acid and added one additional
drop of distilled water or used a pre-prepared 50% solu-
tion of sulphuric acid.
Results
Phylogenetic analysis
The final dataset was composed of 77 specimens belong-
ing to the four studied species and samples with a high
sequence similarity, 37 samples of 35 different species of
theCrowncladeandthreeoftheoutgroup.Insummary,
117 specimens were included in the analysis. Of these, 46
encompassed only ITS (mainly sequences retrieved from
the public databases) and 20 encompassed only two of the
threemarkers.Wewereabletoobtainsequencesfromthe
types of R. rubra and R. rutila, but DNA amplification of
the R. veternosa type failed. The final topologies of the
ML and BI analyses were not congruent, as we expected,
given the high sequence variation found between mem-
bers of the Crown clade. There is no significant clustering
across the backbone of the Crown clade. The clade we
call the Integrae clade, referring to one of the oldest
names of an infrageneric Russula taxon typified by
R. integra (L.) Fr., received moderate statistical support
(85/1) (Fig. 1). Inside this clade, the four strongly sup-
ported subclades concordant with morphologically de-
fined species (called further by the species name),
R. firmula,R. rubra,R. rutila and R. veternosa, are placed
in one monophyletic group (100/1), called here the
Rubrinae clade. The R. rutila subclade does not include
any non-European collections, shows little sequence var-
iation and contains 15 collections sequenced in this study
(including the type specimen), a collection TU101893
originally identified as R. decipiens (Singer) Svrček re-
trieved from the UNITE database, and an unidentified
Russula sequence LM5409, originating from an environ-
mental sample. The remaining three subclades of the
Rubrinae clade contain some sequences of Asian, or in
the case of R. firmula subclade, also of North American
origin. In the R. veternosa subclade, there is a little se-
quence variability and two environmental samples from
Iran are nearly identical with one other from Europe. All
samples (11 European and two from the Middle East)
clusteredintheR. veternosa subclade probably represent
a single species. Our six European collections identified
as R. rubra and the type collection of the species are
clustered in a strongly supported subclade together with
another three European collections originating from envi-
ronmental samples. Sister to this subclade are three se-
quences from Papua New Guinea that probably represent
a closely related undescribed species.
Ten sequences from the collections morphologically
assigned to R. firmula cluster with 17 other European
collections (73/0.99) and form a moderately supported
group (80/1) together with unidentified Asian and North
American environmental samples for which conspecificity
with this species is unclear. Russula quercilicis, the other
species classified in the subsect. Rubrinae based on
incrusted pileocystidia, is placed outside the Rubrinae
clade.
Ancestral state reconstruction
After reducing the four species in the Rubrinae clade each to a
single representative, the topology of the resulting ML tree is
similar to results of the phylogeny using the complete dataset.
The analysis shows good support for the Rubrinae clade, the
Integrae clade and the Crown clade (Fig. 2).
The absence of any incrustations on pileocystidia or
primordial hyphae is the most probable ancestral state
for both the Crown clade and the Integrae clade, but the
analysis suggests that, within the Integrae clade, most an-
cestors possessed pink incrustations in sulphovanillin and
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some members lost this colour change (R. quercilicis and
R. cremeoavellanea Singer) or had no incrustation at all
(R. badia Quél. and R. tinctipes J. Blum ex Bon). There is
not enough support to resolve relationships for the other
Fig. 1 Maximum likelihood phylogeny inferred from three loci (ITS,
mtSSU and rpb2) with four target species-level clades highlighted, as
well as the known superclades comprising them. Collection labels are
updated with appropriate taxon names, except where collector
identifications disagree. Basidiomata samples are labelled by herbarium
code and collections number in parentheses, sequences of environmental
samples or collections without reference to a herbarium specimen are
labelled with accession numbers in italics (sequences from the UNITE
database start with UDB, others are from GenBank). Countries of origin
are included for species in the Rubrinae clade. Bootstrap values followed
by Bayesian posterior probabilities are indicated at the nodes
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members of the Crown clade outside of the Integrae clade,
but the analysis suggests that the incrustation evolved
several times from non-incrusted ancestors.
The analysis suggests mild taste as the ancestral state for
both the Crown clade and the Integrae clade. The acrid or
weakly acrid taste probably evolved more recently and in
several lineages within the Crown clade. The Rubrinae clade
together with the clade of R. badia and R. quercilicis represent
two of the independent lineages with acrid taste. Acrid species
are also found out of the Integrae clade, but the analysis on the
current dataset does not allow enough resolution to determine
if they share a single origin of acridity with the Rubrinae
clade.
The spore print analysis suggests that most of the ancestors in
the Crown clade possessed a darker yellow spore print and the
paler spore print evolved multiple times. This is especially well
demonstrated in the Integrae clade, showing support for isolated
positions of R. decolorans,R. rubra and R. velenovskyi Melzer
& Zvára with intermediate spore print colour among yellow
spore print species. The strong support for a relationship of
pale- and dark-spored species is also shown in the species pairs
of R. nauseosa (Pers.) Fr. R. nitida (Pers.) Fr. and R. odorata
Romagn. R. versicolor Jul. Schäff.
Ancestral state reconstruction of the host preference dem-
onstrates a similar pattern as the one of the spore print. The
majority of ancestors on higher ranks of the phylogeny were
probably associated with angiosperms and the high probabil-
ity for such ancestors is demonstrated for both the Integrae
clade and the Rubrinae clade. There is high support for isolat-
ed positions of some species preferring coniferous trees that
probably evolved from ancestors with preference for angio-
sperm hosts, including R. firmula,R. badia and R. integra.
In conclusion, the common ancestors of the Crown clade
and the Integrae clade probably did nothave any incrustations,
had a mild taste, a yellow spore print and were associated with
an angiosperm host. Taste, spore print and host preference
showed only one unidirectional switch in the Crown clade:
from mild to acrid, from yellow to white and from angiosperm
to conifer. The only specific change in evolution of the
Integrae clade is a dominance of species with pink incrusta-
tions in sulphovanillin that probably evolved early in the clade
history and some species lost such incrustations later.
Morphological delimitation of the Rubrinae clade
Selected morphological characters further tested for their
significance to define the Rubrinae clade are presented in
Tab le 1. The spores (Figs. 3and 4), basidia and
pleurocystidia of R. veternosa are the smallest among
the four compared species, but the values of the other
three species of the R. rubra lineage fall within the vari-
ation of R. maculata Quél. and R. vinosopurpurea Jul.
Schäff. (Adamčík and Jančovičová 2013). The spore or-
namentation shows high variability among the species
compared here and cannot define the whole Rubrinae
clade, but it may, rather, serve as a good distinguishing
character at the species rank. Hyphal terminations in the
pileipellis near the pileus marginshowamoreorless
uniform pattern in all four species: relatively narrow ter-
minal cells that are cylindrical or subulate and usually
apically constricted or attenuated and are usually followed
by one unbranched subterminal cell or are directly arising
from branched hyphae of the subpellis. The terminal cells
of R. rubra and R. rutila are usually more distinctly and
frequently attenuated (Fig. 4). The only character that de-
fines the Rubrinae clade perfectly is the presence of in-
crustations on pileocystidia. The presence of such incrus-
tations is a new discovery of this study for R. firmula and
R. veternosa. These incrustations are sometimes weakly
acid-resistant and difficult to see after a carbolfuchsin
treatment, but after ca. 30 min, they stain bright pink in
sulphovanillin.
Analysis of incrustations
Because our morphological analysis revealed incrustations
for unexpected species of the Rubrinae clade (Table 1,
Fig. 5), we analysed their presence for all species accept-
ed in this study. We observed the presence and colouring
of incrustations using the carbolfuchsin treatment, Cresyl
blue and sulphovanillin.
Our observations with carbolfuchsin treatment confirmed,
in most cases, the presence or absence of acid-resistant incrus-
tations in agreement with the literature, the exceptions being
incrustations observed in R. firmula and R. veternosa, when
none have been mentioned previously. The species with
incrusted primordial hyphae, traditionally classified within
R. subgenus Incrustatula Romagn. (Sarnari 1998), all have
abundant acid-resistant incrustations: R. caerulea Fr.,
R. claroflava Grove, R. risigallina (Batsch) Sacc., R. turci
Bres., R. velutipes Ve l e n . , R. vinosa Lindblad and R. zvarae
Velen. Acid-resistant incrustations were also observed in some
species with incrusted pileocystidia, which have, in addition
to incrustations, contents that turn grey in sulphovanillin. The
red staining of incrustations on pileocystidia after
carbolfuchsin treatment was often weak, instable and restrict-
ed to the bases of the pileocystidia, and were sometimes un-
convincing in R. cremeoavellanea,R. firmula,R. integra,
R. laeta F.H. Møller & Jul. Schäff., R. quercilicis,R. rubra,
R. rutila,R. velenovskyi and R. veternosa. The remaining
Fig. 2 Maximum likelihood phylogeny of the Crown clade Russula
species using three loci (ITS, mtSSU and rpb2). Bootstrap labels are
displayed for all nodes. Trees are displayed as balls and sticks graphs
with proportional log-likelihoods of ancestral character states displayed
as pie graphs at the given nodes
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Tab l e 1 Comparison of selected characters among four species of the Russula rubra lineage. All values except for the height of spore ornamentation are averages of 20 measurements
R. veternosa
PC0124979 T!
SAV F-2588
SAV F-3391
R. rubra
SAV F-914
SAV (HK14300c)
SAV F-3229
R. rutila
SAV F-1564
GENT (2007 BT103)
SAV (HK14028)
R. firmula
GENT (2010 BT85) T!
SAV F-2655
SAV (HK14300c)
Spore size 7.2 × 5.8 μm
7.5 × 6 μm
7.2 × 5.7 μm
8.1 × 6.8 μm
8.2 × 6.9 μm
8.4 × 6.9 μm
8.9 × 7.2 μm
8.5 × 6.8 μm
8.7 × 7 μm
8.6 × 7.1 μm
8.3 × 6.9 μm
8.4 × 7 μm
Spore ornamentation Spines 0.71.1
μm high,
mainly isolated
War t s 0.4 0.6 μm high,
merged in chains,
occasionally connected
by fine lines
War t s 0.6 0.9 μm high,
merged in chains,
occasionallyconnected
by short, fine lines
Spines 0.81.2 μm high,
mainly isolated
TC near the pileus margin 29.4 × 2.5 μm
31.1 × 2.6 μm
28.6 × 2.6 μm
Cylindrical, apically
obtuse or constricted to attenuated
33.5 × 3.4 μm
30.1 × 3.1 μm
28.4 × 3.1 μm
Subulate, occasionally
cylindrical, apically
usually attenuated
or constricted
31.7 × 2.9 μm
24.5 × 2.9 μm
28.1 × 3 μm
Subulate and apically
attenuated
31.1 × 3.1 μm
27.7 × 2.9 μm
26.7 × 3 μm
Subulate or cylindrical,
apically usually
attenuated or constricted
Incrustations on pileocystidia Weakly acid-resistant after carbolfuchsin treatment, turning slowly to bright pink in sulphovanillin
Basidia size 33.2 × 11 μm
36.1 × 11.2 μm
35.5 × 10.8 μm
50.3 × 11 μm
54.9 × 11.3 μm
47.8 × 10.9 μm
48 × 11.7 μm
50.4 × 11.9 μm
44.7 × 12 μm
46.5 × 13.1 μm
48.8 × 12.9 μm
41.9 × 12.1 μm
Pleurocystidia size 58.9 × 10.5 μm
59.8 × 10.6 μm
56.5 × 10.6 μm
86 × 11.7 μm
82.5 × 11.5 μm
78.5 × 11.3 μm
65.8 × 9.2 μm
63.1 × 9.3 μm
58.6 × 9.9 μm
72.4 × 10.1 μm
76.1 × 10.3 μm
68.9 × 10.6 μm
TC terminal cells of hyphae in the pileipellis; T! type specimen
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species of the Crown clade included in this study, as well as
representatives of R. subgenus Russula,havenoacid-resistant
incrustations on any cells of the pileipellis.
Russula lepida Fr. is the only species with distinct meta-
chromatic incrustations in pileipellis in Cresyl blue, showing
purple incrustations especially in the deeper part of the
suprapellis and occurring on both pileocystidia and other un-
differentiated hyphae in the pileipellis. We do not think that
the presence of a weak metachromatic reaction in the
pileipellis of some species of the Integrae clade (e.g.
R. laeta) has taxonomic significance.
There is a very quick colour change of incrustations to
bright pink or red in R. caerulea,R. claroflava,R. velutipes
and R. vinosa (Fig. 5). However, the distribution and type of
incrustations are different, and these differences probably cor-
respond also with their different positions in the phylogenetic
tree. Russula velutipes has very abundant incrustations in the
form of droplets but the deep red colouring is usually restrict-
ed only to the basal parts of primordial hyphae, the tips of
which often bear only hyaline droplets. Large droplets turning
quickly to deep and bright red can be found in R. caerulea,but
the tips of primordial hyphae are usually not incrusted.
Primordial hyphae of R. claroflava and R. vinosa are incrusted
on the whole surface with very fine, granulose incrustations
that turn quickly to bright pink in sulphovanillin. The majority
of species of the Integrae clade tend to have incrustations that
turn slowly, after ca. 30 min, to bright pink: R. firmula,
R. integra,R. laeta,R. rubra,R. rutila,R. velenovskyi and
R. veternosa. These incrustations are in the shape of irreg-
ular droplets or patches on the surface of pileocystidia,
usually only near their basal part, but they sometimes
cover the whole surface. The colour change is slow and
not always convincing, but, usually, the pink colour
shows a very striking contrast, especially near the basal
septa, where the grey or black colouring of contents is
lacking. The intensity of the colour change and the visi-
bility of the incrustations are weak and less conspicuous
in R. rubra (Fig. 5b). Incrustations on primordial hyphae
of R. risigallina,R. turci,R. velutipes and R. zvarae do
not change colour (remain hyaline) in sulphovanillin.
Incrustations on pileocystidia of R. cremeoavellanea do
not change colour in sulphovanillin, and only the
pileocystidia contents slightly turn grey. The pileocystidia
contents of R. quercilicis turn dark grey to black, whereas
the incrustations are first yellowish and, after ca. 60 min,
turn black.
Type studies and species concepts within the Rubrinae
clade
Our preliminary morphological identifications were in
agreement with the results of phylogenetic analysis. To
confirm that the results of this study are in agreement with
the original concepts of the studied species, the available
type material was included. Information on types of all
four species of the Rubrinae clade is summarised in
Tab le 2. The species concepts of R. rubra and R. rutila
have also been confirmed by successfully sequenced type
specimens. The DNA extraction of the R. veternosa
epitype failed, but the morphological investigation is
clearly in agreement with the morphology of recently
studied and sequenced collections of this species
Fig. 3 Russula veternosa (epitype, PC0124979). aPileocystidia near the
pileus margin. bPileocystidia near the pileus centre. cSpores in Melzers
reagent. dHyphal terminations in the pileus margin. eHyphal
terminations near the pileus centre. fBasidia. gBasidiola. hMarginal
cells. iPleurocystidia. jCheilocystidia. Contents of cystidia are
represented as observed in Congo red for some elements only, while
others with plus sign indicated their contents schematically. Scale bar
equals 10 μm, but only 5 μm for spores. Drawings by: S. Jančovičová
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(Supplementary Material 2). We did not study any of the
R. firmula neotypes proposed by Romagnesi (1967)or
Sarnari (1998) since they were not available for loan dur-
ing our study. In our opinion, both R. firmula neotypes are
superfluous because the original description (Schaeffer
1940) clearly refers to Schaeffersdescriptionof
R. nitida (Pers.) Fr.that cites several illustrations avail-
able for the type designation. For the stability of the spe-
cies concept, we are designating here Bresadolas plate
458 (Bresadola 1929) that is cited by Schaeffer (1933)
as the lectotype of the species and our collection GENT
(2010 BT85) as the epitype of R. firmula (Supplementary
Material 3).
Key to species of the Rubrinae clade in Europe
Circumscription of the Rubrinae clade: (1) spores with
amyloid suprahilar spot; (2) taste of the context strongly
acrid; (3) spore print ochre or yellow; (4) pileocystidia
turning dark grey to black in sulphovanillin and with in-
crustations staining pink in sulphovanillin after ca.
30 min, that are especially visible at the base.
Fig. 4 Microscopic structure of
pileipellis and spores of three
studied species. Russula firmula
(SAV F-2655). aPileocystidia
near the pileus margin. bSpores
in Melzers reagent. cHyphal
terminations in the pileus margin.
Russula rubra (SAV F-914). d
Pileocystidia near the pileus
margin. eSpores in Melzers
reagent. fHyphal terminations in
the pileus margin. Russula rutila
(SAV F-1564). gPileocystidia
near the pileus margin. hSpores
in Melzers reagent. iHyphal
terminations in the pileus margin.
Contents of cystidia are
represented as observed in Congo
red for some elements only, while
others with plus sign indicated
their contents schematically.
Scale bar equals 10 μm, but only
5μm for spores. Drawings by: S.
Jančovičová
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&1 Spores with isolated prominent spines
&2 Pileus with predominantly pale red, pink, ochre and
cream colours; spores mainly up to 8.5 μm long; usually
associated with Fagus ....................R. veternosa
&2* Pileus with predominantly wine-red, blue-red, purple
and red-brown colours; spores mainly longer than 8.5 μm;
usually associated with coniferous trees . . . . . . .R. firmula
&1* Spores with warts merged in chains and connected by
occasional lines
&3 Basidiomata medium sized to large (60100 mm), with
thick context turning slowly grey; pileus cuticle velutinous
or matt; spore print ochre (IIIbIIIc)..........R. rubra
&3* Basidiomata small to medium sized (3070 mm); con-
text soon becoming fragile, thin and not turning grey;
pileus cuticle shiny at least near the pileus margin; spore
print yellow (IVbIVd).......................R. rutila
Discussion
Congruence of morphology-based classifications
with the Crown clade phylogeny
Our phylogeny is based on, altogether, 39 species of the
Crown clade. The four target species are grouped in the
Rubrinae clade, which is nested in the larger Integrae clade.
In general, the phylogenetic tree often shows strong support
for relationships of pairs or triplets of species, but the other
nodes on higher ranks usually received weak support, partly
explained by incomplete species representation in the Crown
clade and a limited number of gene markers.
Looney et al. (2016) analysed a large dataset of ITS
Russula sequences retrieved from GenBank and identified
1064 MOTUs worldwide, with 295 represented in Europe.
Fig. 5 Pink incrustations on
pileocystidia observed in
sulphovanillin. aRussula firmula
(SAV F-2137). bRussula rubra
(SAV F-4216). cRussula rutila
(SAV F-1564). dRussula
veternosa (SAV F-1403). e
Russula caerulea (SAV F-2151). f
Russula claroflava (SAV F-1791)
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The four-locus phylogeny presented by Looney et al. (2016)
recognised eight major clades within the genus Russula.The
clades Farinipes, Nigricans, Archaea, Heterophylla and
Compacta are represented by species with a non-amyloid
suprahilar spot on their spores. The Delica clade is represented
by species with an amyloid suprahilar spot but have frequent
short lamellulae. A majority of species with an amyloid
suprahilar spot on spores are in the Russula clade and the
Crown clade, but the morphological distinction of these two
groups will require further analysis. The Crown clade was
represented by nearly 50% of the MOTUs in the total dataset.
Some Russula groups with a non-amyloid suprahilar spot on
the spores, which fall outside the Crown clade, are probably
less diverse in Europe than in other continents and, therefore,
we expect that more than half of European MOTUs belong to
the Crown clade. The total species number of this clade will
certainly be higher than 100 in Europe.
In our opinion, of all traditional classifications (Singer
1986; Romagnesi 1987; Bon 1988; Sarnari 1998), the one
presented by Romagnesi (1987) corresponds best to the cur-
rently known phylogeny. Romagnesi defines nine subgenera,
of which R. subg. Compacta (Fr.) Bon, R. subg.
Heterophyllidia Romagn., R. subg. Ingratula Romagn. and
R. subg. Russula, are typified by species out of the Crown
clade (Looney et al. 2016). Types of R. subg. Incrustatula
(R. lilacea Quél.), R. subg. Tenellula Romagn. (R. puellaris
Fr.), R. subg. Polychromidia Romagn. (R. integra), R. subg.
Coccinula Romagn. (R. paludosa Britzelm.) and R. subg.
Insidiosula Romagn. (R. firmula) all belong in the Crown
clade. The other classifications define groups mixed of species
of the Crown clade and members of other Russula clades. In
the following, we discuss the morphological delimitation of
clades recognised in this study and their nomenclature.
Polyphyletic origin of analysed morphological charac-
ters in this study suggests that delimitation of larger phy-
logenetic lineages within the Crown clade based on mor-
phology will be very hard and, in some cases, likely im-
possible. Morphological recognition of smaller phyloge-
netic groups of closely related taxa seems to be more
realistic, less difficult and particularly important as a start
for species identification. A good example of smaller
groups well-supported by both molecular and morpholog-
ical characters is the Rubrinae clade defined in our study,
where we discovered that the pink incrustation of
pileocystidia in sulphovanillin is a good synapomorphic
character, combined with strongly acrid taste of the con-
text. Other similar examples within the Crown clade are
the Maculatinae clade (Adamčík et al. 2016b)a
ndthe
Xerampelinae clade (Adamčík et al. 2016a).
Species circumscription within the Rubrinae clade
Identification of Russula species may be very challenging
because of a considerable number of nomenclatural and
taxonomic problems: published names that are currently
not accepted, missing types or a lack of recent type stud-
ies (Buyck and Adamčík 2013). In this study, we adopted
the concept and names of widely accepted species in the
recent literature.
Among the four European species of the Rubrinae
clade, only R. rubra has been previously reported to have
acid-resistant incrustations (Romagnesi 1967). Apart from
this, the species is also recognisable by field characters
(large, thick-fleshed basidiomata, flesh turning grey, pale
ochre spore print, bright red cap cuticle with velvety as-
pect). A similar species, R. rutila, has smaller and thin-
Tabl e 2 Overview of the information about types of species within the R. rubra lineage
Species Original description Type status Designating publication Country of origin Type condition
R. firmula Schaeffer (1940), cited full
description in Schaeffer
(1933)asR. nitida
Lectotype Designated here Not specified Illustration, Bresadola (1929),
pl. 458 (as R. badia)
Epitype Designated here Germany Herbarium specimen GENT (2010
BT85), sequences ITS KU928142,
mtSSU KY471568, rpb2 KY616675
R. rubra Fries (1821)LectotypeSarnari(1998) France Illustration, Bulliard (1781), pl. 42,
fig. B (as Agaric sanguin)
Epitype Sarnari (1998) France Herbarium specimen PC0723456
(Romagnesi n°52241), sequence
ITS (GenBank KY582680)
R. rutila Romagnesi (1952) Holotype Romagnesi (1952) France Herbarium specimen PC0723457
(Romagnesi n°12-IX-45), sequence
ITS (GenBank KY582681)
R. veternosa Fries (1838)LectotypeSarnari(1998) Not specified Illustration, Paulet (1855), pl. 74,
f. 3 (as Agaricus vaternosus)
Epitype Sarnari (1998) France Herbarium specimen PC0124979
(Romagnesi n°53206), no
sequence data
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fleshed basidiomata with discoloured pileus cuticle at the
disc and a yellow spore print (Fig. 6). Russula veternosa
has a yellow spore print and larger basidiomata with a
pale pink-yellowish pileus. The pileus colour of
R. firmula is usually dark purple, lilac or with brownish
tints towards the disc. What makes the field identification
easy is ecology. Russula firmula is always associated with
conifers and probably has a preference for Picea
(Supplementary Material 1). Russula veternosa is consid-
ered to be exclusively associated with Fagus,R. rutila
with Quercus and while R. rubra does not show any pref-
erence for a tree genus, it only occurs in deciduous for-
ests. In case the field aspect of basidiomata and host as-
sociation are ambiguous (e.g. occurrence in mixed for-
ests), the spore size and ornamentation are the most useful
characters for recognising species within the Rubrinae
clade. Russula rubra and R. rutila have spores with rela-
tively low warts that are merged and chained (Fig. 4).
Russula firmula and R. veternosa have prominent isolated
spines and differ by smaller spores of the latter (Table 1).
Sometimes, species identification might fail because of
not recognising a proper group and, for this reason, we
provided (see above) a key that defines not only species
circumscription but also delimitation of the Rubrinae
clade as a whole.
Host preference and geographical distribution
The results of our ancestral state reconstruction suggest
that the host plant of the Crown clade ancestors were
angiosperms, which is in agreement with the results of
Looney et al. (2016). The possible driver for species di-
versification within the genus Russula might not be only
host or climate switching, but also geographical distance
or disjunction. The phylogeny of the Rubrinae clade
shows at least one Asian species sister to each European
species R. firmula and R. rubra. The Indian (Kashmir,
Himalaya) collection placed in the R. firmula clade prob-
ably represents a closely related species to R. firmula and
was also collected under conifers (Itoo et al. 2013).
Sequences in the R. rubra clade originating from Papua
New Guinea (UDB013117, UDB013204, UDB013239)
probably represent a separate closely related species to
R. rubra, which is associated with deciduous trees of the
genera Castanopsis and Nothofagus (Tedersoo and Põlme
2012). We think that the placement of the Iranian collec-
tions associated with Fagaceae in the R. veternosa clade
but sister to the majority of European collections of
R. veternosa is probably due to limited sequence data
(only ITS) and they probably represent the same species.
It would be interesting to study the phylogenetic relation-
ship of European R. veternosa with the material reported
from Japan (Tsujino et al. 2009), eastern North America
(Burlingham 1913) and western North America (Woo
1989). We also hypothesise that host switching is a pos-
sible driver for speciation in the Crown clade. For exam-
ple, sister to the European species clade of R. maculata
and R. nympharum F. Hampe & Marxm., associated with
deciduous trees, are Asian collections from Pakistan and
China, all associated with conifers (Adamčík et al.
2016b).
Incrustations on the hyphae in the pileipellis
Our analysis revealed an important synapomorphic character
for R. subsect. Rubrinae. Besides the acid-resistant incrusta-
tions known in R. rubra and R. rutila,wehavedemonstrated
their presence in R. firmula and R. veternosa as well. The
staining of the incrustations after carbolfuchsin treatment is
weak and might be easily overlooked. Staining of incrusta-
tions in sulphovanillin is, in some species, more conspicuous,
and this is very convincing in R. vinosa,R. claroflava and
R. caerulea. The pink incrustations of Rubrinae clade mem-
bers and also of some other mild-tasted species of the Integrae
clade (e.g. R. integra) might be easily overlooked, because it
usually becomes visible after ca. 30 min. Some publications
(e.g. Moser 1978) recommend using concentrated sulphuric
acid to prepare sulphovanillin, which is good for observing the
dark grey or black colour change of the pileocystidia contents,
but this may cause quick dissolution of incrustations, as well
as rupturing of the cell walls. The presence of pink incrusta-
tions in sulphovanillin is a character so far reported just re-
cently in the North American taxon R. vinosa var. occidentalis
Singer (Adamčík et al. 2015).
In our opinion, the pink incrustations of pileipellis hy-
phae in sulphovanillin may become a powerful tool for
classification in the genus Russula. They were observed
on both primordial hyphae and pileocystidia with contents
darkening in sulphovanillin, but some Russula members
have acid-resistant incrustations not turning pink, with
both primordial hyphae (e.g. R. turci,R. risigallina)and
pileocystidia (e.g. R. quercilicis). The intensity and veloc-
ity of the pink reaction in sulphovanillin is very different
among species, but these differences do not correspond to
how they were reported in the literature. The acid-
resistant incrustations in the pileipellis were introduced
by Melzer and Zvára (1927), but the general utilisation
of the character started after the publication of
Romagnesis monograph of the genus Russula
(Romagnesi 1967). Since then, R. rubra has been con-
stantly reported as bearing incrusted pileocystidia and,
for quite a long time, it remains the only member of the
R. subsection Rubrinae defined by incrusted pileocystidia,
a yellow spore print and acrid taste. Romagnesi (1967)
also mentioned incrusted pileocystidia in R. rutila, but
he classified this species in the subsection Maculatinae.
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Bon (1988)placedR. rutila in Rubrinae and Sarnari
(1998) expanded the group by including R. quercilicis
and R. blumiana Blum ex Bon. Contrary to the published
information, R. veternosa and R. firmula traditionally re-
ported as having no incrustations, but they have more
conspicuous and abundant pink incrustations in
sulphovanillin than the other two species of the
Rubrinae clade (Fig. 5). Russula quercilicis is not a close-
ly related species to the Rubrinae clade, and its incrusta-
tions in sulphovanillin do not turn pink. During this study,
we were not able to access material of R. blumiana or its
type designated by Bon (1986). There is no mentioning of
incrustations for R. veternosa in the literature, but it was
possibly not checked because this is a well-defined spe-
cies. It is often in the search for unravelling species com-
plexes that additional detailed morphological observations
are made. For example, when the taxonomic concept of
R. firmula was challenged, the authors described incrus-
tations on pileocystidia for related or similar taxa.
Marxmüller (2014) described no incrustations in
R. firmula but reported incrusted pileocystidia for the
closely related R. transiens (Singer) Romagn. and Singer
(1962) described a new species R. piceetorum Singer
(invalid name, no type designated) bearing incrusted
pileocystidia and other morphological characters identical
to R. firmula.
Among 14 species that form the Integrae clade in our phy-
logeny, nine have the pink incrustations in sulphovanillin, two
have only acid-resistant incrustations and three have no in-
crustations. The absence of visible incrustations in some spe-
cies might be caused by a very thick gelatinous-slimy matter
in which the hyphal terminations in the pileipellis are
embedded and which may form a barrier for contact of the
chemical reagent with incrustations. For example, Romagnesi
(1967) described weak acid-resistant incrustations on the
pileocystidia of R. paludosa and R. tinctipes, but the authors
of this study as well as Sarnari (2005) were not able to see any
incrustations.
Conclusions
The multi-locus phylogeny of the Crown clade shows strong
support for a Rubrinae clade that comprises four European
species. This group is morphologically defined by a yellow
spore print, acrid taste of the context and pileocystidia with
pink incrustations in sulphovanillin. We propose to call the
group Russula subsect. Rubrinae.Russula firmula and
R. veternosa both have, contrary to traditional opinion, pink
incrustations on pileocystidia in sulphovanillin and are new
for the subsection, whereas incrustations of R. quercilicis do
not stain pink in sulphovanillin and, accordingly, the species is
not placed as a close relative of the group in the phylogeny.
The Rubrinae clade is placed in the Integrae clade, which is
dominated by species with pink incrustations in
sulphovanillin. Further classification of the Crown clade re-
quires better sampling and more genetic markers. Colour
changes not only of the context, but also of the incrustations,
are demonstrated to have importance for delimitations of
Russula taxa on various ranks.
Acknowledgements We very much appreciated the help of H.
Kaufmann, L. Michelin and any collectors we may have forgotten to
mention by accident for supplying us with interesting collections. P.
Fig. 6 Field appearance of four
Russula species included in this
study. aRussula firmula
(TU106603), photo by Velo Liiv.
bRussula rubra (GENT
FH12/227), photo by Felix
Hampe. cRussula rutila (SAV
F-1497), photo by Per Marstad. d
Russula veternosa (SAV F-1403),
photo by Per Marstad. Scale bar
equals 30 mm
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Marstad and V. Liiv are acknowledged for providing photos of the field
aspect of some studies species. The curator and staff of the herbarium PC
are acknowledged for the loan of the type material. The studies of S.
Adamčík, M. Caboňand S. Jančovičová were funded by the Slovak
Research and Development Agency APVV-15-0210. The studies of M.
Caboňand M. Kolařík were supported by bilateral scientific mobility
project SAV-AV ČR 16-06 and Intra-Visegrad scholarship no. 51400484.
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... Several recent molecular phylogenetic studies have revised groups of species across Russula, revealing patterns of geographic distribution, unique suites of morphological features, and ecological specialization (Adamčík et al. 2016a(Adamčík et al. , 2016bCaboň et al. 2017Caboň et al. , 2019. Traditional species complexes have been revised using phylogenetics combined with an analysis of micromorphological features and ecological data (Adamčík et al. 2016a). ...
... Closely related Russula species have been found to possess conserved plant host preferences across climatic and geographic barriers, or they can occur across broadly different ecosystems (Caboň et al. 2019). Traditional morphological characters used to circumscribe infrageneric groups and species complexes have been questioned because available evidence indicates that many are a result of convergent evolution, including spore pigmentation and the presence of incrustations in the cap cuticle (Caboň et al. 2017). Morphometrics combined with a multivariate analysis of morphological components of the pileipellis has been a useful approach for delimiting species in closely related species complexes where the internal transcribed spacer (ITS) region alone has failed to resolve lineages (Adamčík et al. 2016b). ...
Article
The Roseinae clade is a lineage of the genus Russula primarily composed of species of Russula subsect. Roseinae. Species in this morphologically distinct clade possess a white to pale cream spore print, mild taste, positive reaction to sulfovanillin, and primordial hyphae with acid-resistant crystals in the pileipellis. Here, we present a morphological and phylogenetic assessment that distinguishes seven eastern North American species of the core Roseinae lineage and a new subsection, Russula subsection Albidinae, to accommodate members of the Albida clade. We assign the previously described species R. peckii, R. rubellipes, and R. pseudopeckii to three species-level clades, and three other species, R. cardinalis, R. cordata, and R. rheubarbarina, are described as new. Comparative morphological analyses reveal differences in the conformation of terminal elements in the pileipellis, spore size, hymenial cystidia contents, and pigmentation on the stipe surface as key features to recognize species in the group. Based on the analysis of publicly available data, we recognize a potential total of nine temperate North American species within R. subsect. Roseinae, in addition to four from Central America, two from Europe, and 14 from Asia.
... We targeted five DNA regions: (1) the internal transcribed spacer regions of nuclear ribosomal DNA (ITS); (2) partial large subunit of nuclear ribosomal DNA (LSU); (3) partial mitochondrial small subunit of ribosomal DNA (mtSSU); (4) the region between domains six and seven of the nuclear gene encoding the second largest subunit of RNA polymerase II (rpb2); (5) part of the translation elongation factor 1-alpha (tef1α). The following primer pairs were used for the DNA amplifications: ITS1F-ITS4 for ITS [22,23], LR0R-LR5 for LSU [24], MS1-MS2 for mtSSU [22], A-Russ-F-frpb2-7CR for rpb2 [25,26], EF1-983F-EF1-1567R for tef1α [27]. All molecular markers were amplified with 5× HOT FIREPol ® Blend Master Mix (Solis BioDyne, Tartu, Estonia) applying PCR conditions of Caboň et al. [26]. ...
... The following primer pairs were used for the DNA amplifications: ITS1F-ITS4 for ITS [22,23], LR0R-LR5 for LSU [24], MS1-MS2 for mtSSU [22], A-Russ-F-frpb2-7CR for rpb2 [25,26], EF1-983F-EF1-1567R for tef1α [27]. All molecular markers were amplified with 5× HOT FIREPol ® Blend Master Mix (Solis BioDyne, Tartu, Estonia) applying PCR conditions of Caboň et al. [26]. The PCR products were purified using Exo-Sap enzymes (Thermo Fisher Scientific, Wilmington, Germany) and sequenced at SeqMe sequencing company (Dobříš, Czech Republic). ...
Article
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Russula subsect. Maculatinae is morphologically and phylogenetically well-defined lineage of ectomycorrhizal fungi associated with arctic, boreal, temperate and Mediterranean habitats of Northern Hemisphere. Based on phylogenetic distance among species, it seems that this group diversified relatively recently. Russula ayubiana sp. nov., described in this study, is the fifth in the group known from relatively small area of northern Pakistan situated in southwestern Himalayas. This is the highest known number of agaric lineage members from a single area in the world. This study uses available data about phylogeny, ecology, and climate to trace phylogenetic origin and ecological preferences of Maculatinae in southwestern Himalayas. Our results suggest that the area has been recently colonised by Maculatinae members migrating from various geographical areas and adapting to local conditions. We also discuss the perspectives and obstacles in research of biogeography and ecology, and we propose improvements that would facilitate the integration of ecological and biogeographical metadata from the future taxonomic studies of fungi in the region.
... The pileipellis ortho-or metachromatic reactions were examined in Cresyl Blue after Buyck (1989). Sulfovanillin (SV) was used to observe color changes of cystidia contents (Caboň et al. 2017). Observations and measurements of microscopic features were made in 1 % Congo Red, 5 % KOH, or Melzer's reagent. ...
Article
Russula hainanensis (Russulaceae, Russulales), a member of Russula subgenus Heterophyllidia section Ingratae, from Hainan, tropical China, is described as new based on morphological and phylogenetic evidence. An illustrated morphological description as well as comparisons with related species are presented.
... Pileipellis was examined in Cresyl blue to verify the presence of ortho-or metachromatic reactions [20]. Sulfovanillin (SV) was used to observe the color changes of cystidia contents [21]. The ornamentation and structure of basidiospores were illustrated under a scanning electron microscope (SEM-JEOL JSM-6510). ...
Article
Full-text available
Three new species are described and illustrated here based on morphological evidence and phylogenetic analysis from China. Russula leucomarginata is recognized by a yellowish red to reddish brown pileus center, a yellowish white to reddish white and sometimes cracked margin, and a reddish white to pastel pink stipe. Russula roseola is characterized by its reddish white to ruby red pileus center, pink to rose margin, adnate to slightly decurrent lamellae with unequal-length lamellulae, reddish white to pink stipe, and occasionally three-celled pileocystidia. Russula subsan-guinaria is morphologically characterized by a reddish brown to dark brown pileus center, a reddish orange to brownish red margin with striation, a reddish white to pink stipe with an expanded base, basidiospores with moderately distant to dense amyloid warts, and hymenial cystidia turning to reddish black in SV. In this study, we performed phylogenetic analysis based on ITS sequence and 28S-RPB1-RPB2-mtSSU datasets. Detailed morphological features and phylogenetic analysis indicate that these three new species belong to Russula subg. Russula.
... Microscopic characters were studied on dried material mainly in Congo Red 53 , except for spore morphology, which was observed in Melzer reagent. Chemical tests were applied with Cresyl Blue 54 , sulfovanillin 55 , and carbolfuchsin 56 to observe colour changes, incrustations, and cystidia contents. Line drawings and measurements of all microscopic characters were done using Nikon Eclipse Ni (Nikon Instrument Inc., Japan), with the aid of the software NIS-element BR 5.02.03 at a projection scale of 2000 ×. ...
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Dry dipterocarp forests are among the most common habitat types in Thailand. Russulaceae are known as common ectomycorrhizal symbionts of Dipterocarpaceae trees in this type of habitat. The present study aims to identify collections of Russula subsection Amoeninae Buyck from dry dipterocarp forests in Thailand. A multi-locus phylogenetic analysis placed Thai Amoeninae collections in two novel lineages, and they are described here as R. bellissima sp. nov. and R. luteonana sp. nov. The closest identified relatives of both species were sequestrate species suggesting that they may belong to drought-adapted lineages. An analysis of publicly available ITS sequences in R. subsect. Amoeninae did not confirm evidence of any of the new species occurring in other Asian regions, indicating that dry dipterocarp forests might harbor a novel community of ectomycorrhizal fungi. Macromorphological characters are variable and are not totally reliable for distinguishing the new species from other previously described Asian Amoeninae species. Both new species are defined by a combination of differentiated micromorphological characteristics in spore ornamentation, hymenial cystidia and hyphal terminations in the pileipellis. The new Amoeninae species may correspond to some Russula species collected for consumption in Thailand, and the detailed description of the new species can be used for better identification of edible species and food safety in the region.
... Pileipellis were examined in Cresyl blue to verify the presence of ortho-or metachromatic reactions (Buyck 1989). Sulfovanillin (SV) was used to observe colour changes of cystidia contents (Caboň et al. 2017). Spores measurements are expressed as: (Min-)AV-SD-AV-AV+SD(-Max), Min = the minimum value, Max = the maximum value, AV = Average, SD = standard deviation, Q value indicates length/width ratio of the spores. ...
Article
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Two new species of Russula are described and illustrated in this paper. Russula clavulus is recognised by a pale yellow pileus centre, white margin with tuberculate striation, white to pale lamellae with small pale yellow spots, white to light yellow spore print, subglobose to broadly ellipsoid spores with short or long ridges and hymenial cystidia on lamellae sides that are mainly subclavate or fusiform. Russula multilamellula is morphologically characterised by the brownish orange to hazel pileus centre and satin white to yellowish-white margin with brownish tinge, lamellulae that are usually irregular in length and often anastomosing with lamellae, subglobose to broadly ellipsoid spores with short or long ridges and clavate hymenial cystidia. The combination of morphological features and multigene phylogenetic analyses of ITS-nrLSU-RPB2-mtSSU data indicated that these two new taxa belong to Russula subg. Heterophyllidia sect. Ingratae.
... Phylogenies based on multiple unlinked loci provide independent estimates of the organismal phylogeny, and congruence among these estimates furnishes strong evidence of species divergence (Taylor et al. 2000). In Russulaceae, the following markers are often used alongside ITS: LSU, rpb1, rpb2 (De Crop et al. 2014, tef1 (De Lange et al. 2021), and more rarely gpd, atp6, mtSSU, and Mcm7 (Nuytinck et al. 2007; Van de Putte et al. 2012;Caboň et al. 2017Caboň et al. , 2019Looney et al. 2020). ...
Article
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Species delimitation is one of the most fundamental processes in biology. Biodiversity undertakings, for instance, require explicit species concepts and criteria for species delimitation in order to be relevant and translatable. However, a perfect species concept does not exist for Fungi. Here, we review the species concepts commonly used in Basidiomycota, the second largest phylum of Fungi that contains some of the best known species of mushrooms, rusts, smuts, and jelly fungi. In general, best practice is to delimitate species, publish new taxa, and conduct taxonomic revisions based on as many independent lines of evidence as possible, that is, by applying a so-called unifying (or integrative) conceptual framework. However, the types of data used vary considerably from group to group. For this reason we discuss the different classes of Basidiomycota, and for each provide: (i) a general introduction with difficulties faced in species recognition, (ii) species concepts and methods for species delimitation, and (iii) community recommendations and conclusions.
... After pretreatment of dried specimens in 5% potassium hydroxide (KOH), other micromorphological characters were identified and measured in Congo red. The coloring of the cystidia contents was observed in a sulfovanillin (SV) solution (Caboň et al. 2017). The pileipellis were examined in cresyl blue to verify the presence of ortho-or metachromatic reactions (Buyck 1989). ...
Article
Full-text available
2021) Morphological and phylogenetic evidence for two new species of Russula subg. Heterophyllidia from Guangdong Province of China. MycoKeys 82: 139-157. https://doi. Abstract Two new species of Russula subg. Heterophyllidia from Guangdong Province of China were described and illustrated based on morphological characters, and their identity supported by molecular phylogeny. R. luofuensis is morphologically characterized by a grayish yellow to brownish orange pileus center with a purplish gray to grayish magenta margin, a surface that is cracked and broken into small golden-brown patches, subglobose to broadly ellipsoid basidiospores with warts fused in short or long chains and a su-prapellis composed of hyphal extremities with inflated, ellipsoid or globose cells and attenuated terminal cell. R. subbubalina is distinguished by the blanched almond to dark salmon pileus that is cracked with age, subglobose to broadly ellipsoid basidiospores with wart fused in short or long chains and frequently connected by line connections, a suprapellis with hyphal ends composed of inflated or ellipsoid cells and attenuated terminal cell, and pileocystidia that are mainly clavate and sometimes with round or ellipsoid appendage. The phylogenetic analyses based on ITS-nrLSU-mtSSU-TEF1 dataset were performed using maximum likelihood and Bayesian analysis. In terms of morphological features and molecular data, the former species belongs to subsect. Virescentinae, whereas the latter comes under subsect. Heterophyllinae.
... Measurements of other elements were made using an eyepiece micrometer and line drawings were prepared with the aid of a camera lucida (Olympus U-DA) on an Olympus CX21 or BX43 microscope, at original magnifications of 1500× or 2000×. Tissues were mounted in Cresyl Blue (Buyck 1989), sulfovanillin (Cabon et al. 2017) and treated with carbolfuchsin (Romagnesi 1967) to observe the presence and colour changes of incrustations and cystidium contents. All cited collections in the species descriptions have been sequenced, at least for ITS. ...
Article
Full-text available
Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R . albonigra s. lat. represents a species complex consisting of at least five European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit R . albonigra are not always reliable. Therefore, a new delimitation of the R . albonigra complex is proposed and a key to the described European species of R . subgen. Compactae is presented. A lectotype and an epitype are designated for R . albonigra and three new European species are described: R . ambusta , R . nigrifacta, and R . ustulata . Different thresholds of UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality sequences.
Preprint
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Dry dipterocarp forests are among the most common habitat types in Thailand. Russulaceae are known as common ectomycorrhizal symbionts of Dipterocarpaceae trees in this type of habitat. The present study aims to identify collections of Russula subsection Amoeninae Buyck from dry dipterocarp forests in Thailand. A multi-locus phylogenetic analysis placed Thai Amoeninae collections in two novel lineages, and they are described here as Russula bellissima sp. nov. and R. luteonana sp. nov. The closest identified relatives of both species were sequestrate species suggesting that they may belong to drought-adapted lineages. The analysis of publicly available ITS sequences in R. subsect. Amoeninae did not confirm evidence of any of the new species occurring in other Asian regions, indicating that dry dipterocarp forests might harbor a novel community of ectomycorrhizal fungi. Macromorphological characters are variable and are not totally reliable for distinguishing the new species from other previously described Asian Amoeninae species. Both new species are defined by a combination of differentiated micromorphological characteristics in spore ornamentation, hymenial cystidia and hyphal terminations in the pileipellis. The new Amoeninae species may correspond to some Russula species collected for consumption in Thailand, and the detailed description of the new species can be used for better identification of edible species and food safety in the region.
Article
Full-text available
The current generally accepted concept of Russula maculata defines the species by yellow-brownish spots on the basidi-omata, an acrid taste, a yellow spore print and a red pileus. This concept was tested using collections originating from various geographical areas mainly in Europe. Analyses of the ITS region suggested that there were three species within this broad concept. One of them, R. maculata, was identified based on the sequence from the epitype. Two other species, R. nympharum and R. sp., are described here as newly identified species. The European species R. maculata and R. nympharum grow in deciduous forests, are similar in their field aspect and are distinctly different in micro-morphological characteristics of spores, pleurocystidia and pileipellis. An Asian species, R. sp., is associated with pine and has smaller basidiomata and spores. These three species form the R. maculata complex and represent the sister clade to the R. globispora complex. This clade consists of species also characterized by a yellow-brownish context discolouration but with a different type of spore ornamentation. All of the other tested species had an acrid taste and yellow spore print but did not have a conspicuous yellow-brownish context discolouration and were placed in various unrelated clades.
Article
Full-text available
Ectomycorrhizal (ECM) fungi, symbiotic mutualists of many dominant tree and shrub species, exhibit a biogeographic pattern counter to the established latitudinal diversity gradient of most macroflora and fauna. However, an evolutionary basis for this pattern has not been explicitly tested in a diverse lineage. In this study, we reconstructed a mega-phylogeny of a cosmopolitan and hyper-diverse genus of ECM fungi, Russula, sampling from annotated collections and utilizing publically available sequences deposited in GenBank. Metadata from molecular operational taxonomic unit cluster sets were examined to infer the distribution and plant association of the genus. This allowed us to test for differences in patterns of diversification between tropical and extratropical taxa, as well as how their associations with different plant lineages may be a driver of diversification. Results show that Russula is most species-rich at temperate latitudes and ancestral state reconstruction shows that the genus initially diversified in temperate areas. Migration into and out of the tropics characterizes the early evolution of the genus, and these transitions have been frequent since this time. We propose the 'generalized diversification rate' hypothesis to explain the reversed latitudinal diversity gradient pattern in Russula as we detect a higher net diversification rate in extratropical lineages. Patterns of diversification with plant associates support host switching and host expansion as driving diversification, with a higher diversification rate in lineages associated with Pinaceae and frequent transitions to association with angiosperms. This article is protected by copyright. All rights reserved.
Article
Species ofRussulasubsect.Xerampelinaeare notoriously difficult to identify and name and have not been subject to molecular study. A group of species, referred to here as theR. clavipescomplex, growing in association withSalix,BetulaandPopulusas well as coniferous tree species from temperate to arctic and alpine habitats, were examined. Analyses of the nuc rDNA internal transcribed spacer (ITS) region and a numerical analysis of morphological characters were used. TheR. clavipescomplex is a monophyletic group withinRussulasubsect.Xerampelinae, according to molecular results. The complex includes three species:R. nuoljaeis a phylogenetically and morphologically well-supported species while the other two,R. clavipesandR. pascua, are similar based on ITS data and morphology but separate based on their ecology.Russula pseudoölivascensis conspecific withR. clavipes Several combinations of characters traditionally used in the taxonomy ofR.subsect.Xerampelinaeare inappropriate for species delimitation in this group and the adequacy of the ITS for species identification in this group is discussed. Detailed microscopic observations on the type collection ofR. nuoljaeare presented and illustrated, along with a key to the European members ofR.subsect.Xerampelinae.