A molecular analysis reveals hidden species diversity within the current concept of Russula maculata (Russulaceae, Basidiomycota)

Abstract

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.

Full text

Phytotaxa 270 (2): 071–088
http://www.mapress.com/j/pt/
Copyright © 2016 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Samantha Karunarathna: 7 Jul. 2016; published: 17 Aug. 2016
http://dx.doi.org/10.11646/phytotaxa.270.2.1
71
A molecular analysis reveals hidden species diversity within the current concept of
Russula maculata (Russulaceae, Basidiomycota)
SLAVOMÍR ADAMČÍK1*, MIROSLAV CABOŇ1, URSULA EBERHARDT2, MALKA SABA3,4, FELIX HAMPE5,
MAREK SLOVÁK1, JESKO KLEINE6, HELGA MARXMÜLLER7, SOŇA JANČOVIČOVÁ8, DONALD H.
PFISTER3, ABDUL N. KHALID4, MIROSLAV KOLAŘÍK9, KAROL MARHOLD1 & ANNEMIEKE VERBEKEN5
1 Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 23, Bratislava, Slovakia
2 Staatliches Museum für Naturkunde Stuttgart, Rosenstein 1, D-70191 Stuttgart, Germany
3 Farlow Herbarium of Cryptogamic Botany, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA
02138, USA.
4 Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
5 Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
6 Körnerstraße 34, D-04107 Leipzig, Germany
7 Zehentbauernstr. 15, D-81539 München, Germany
8 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Botany,
Révová 39, SK-811 02 Bratislava, Slovakia
9 Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20 Praha 4, Czech republic
* Corresponding author’s e-mail: slavomir.adamcik@savba.sk
Abstract
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 de-
ciduous 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.
Key words: DNA barcode, ectomycorrhizal fungi, morphology, Pakistan
Introduction
The genus Russula Persoon (1796: 100) has more than 200 generally accepted species in Europe (Sarnari 1998,
2005) and more than 750 species worldwide (Kirk et al. 2010). Because of this huge taxonomic diversity, current
keys to Russula frequently use intricate combinations of macro- and micro-morphological characteristics and habitat
preferences (e.g., Knudsen et al. 2012). Within this complex framework, Russula maculata Quélet (1878: 323) is
currently considered a well-defined species with striking macro-morphological characteristics, such as its reddish cap,
yellow lamellae due to the yellow spore print, the presence of rusty or yellow-brownish spots on the cap and stipe
surface and its fairly firm, acrid context. These characteristics are believed to allow for field recognition of this species
(Sarnari 1998).
Russula maculata belongs to the generation of early post-Friesian Russula names. The original diagnosis (Quélet
1878) is brief and contains only a rudimentary description of the micro-morphological characteristics, as was typical
for descriptions of agaricoid fungi in that period. However, the conspicuous spots on the pileus surface, at which the
epithet hints, apparently made the species well recognizable, and it became widely accepted. Later interpretations of

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72 • Phytotaxa 270 (2) © 2016 Magnolia Press
Russula maculata were expanded to include some non-spotted russulas with a greyish context discolouration. These
are now considered to be separate species but were originally proposed at an infraspecific rank, e.g., R. maculata
var. decipiens Singer (1931: 212) and R. maculata f. paradecipiens Favre (1992: 19). The introduction of refined
microscopic techniques in the first half of the 20th century allowed for the recognition of two, macro-morphologically
very similar taxa that were first proposed at a varietal rank. These are currently regarded as separate species, Russula
dryadicola Fellner & Landa (1993: 34) and Russula globispora (Blum 1952: 232) Bon (1986: 55). Both differ from R.
maculata and have larger, subglobose spores with isolated warts.
As a well-known and almost ‘classical’ species, Russula maculata was selected as a type of Russula section
Maculatinae Romagnesi (1962: 173). This section of Romagnesi’s influential systematic framework contains all acrid
species with a yellow spore print and reddish pilei (Romagnesi 1967). Sarnari (1998) reconsidered the importance
of the pileus colour for infrageneric classification and re-grouped the bulk of the Maculatinae species in the broader
concept of the Russula subsection Urentes Maire (1910: 122), which is defined by an acrid taste, yellow spore print,
non-incrusted and sulfovanillin positive pileocystidia and the presence of an amyloid suprahilar spot on the spores.
Sarnari was well aware of the heterogeneity of this large group of species and further subdivided it, provisionally
proposing seven morphological groups ranked as a series. According to this concept, the series Maculata n. inval. (incl.
R. maculata) is characterized by a yellow-brownish context discolouration (and a spotted pileus surface), a fairly dark
spore print, large spores and pileocystidia with a few septa.
Russula maculata in its current sense has a wide range of habitats and a wide distribution. It has been reported in
various deciduous forest types e.g., in Scandinavia and Germany (Gminder et al. 2000, Kauffman 2004), preferring
warm, sunny, base rich sites with oak and beech in Germany and Switzerland (Gminder et al. 2000, Kränzlin 2005), the
Mediterranean Quercus ilex forests (Sarnari 1998), and the oak mountain forests of Algeria (Bertault 1978). Gminder
and co-workers also reported occurrences in conifer plantations. The species is also known from Asia, but there,
it is found in association with conifers as follows: with pines in Pakistan (Sultana et al. 2011) and with Keteleeria
evelyniana (Pinaceae) in China (Ge et al. 2012). Das et al. (2006) described an Indian species associated with Quercus,
R. mayawatiana Das, Miller & Sharma (2006: 206), and they placed it close to R. maculata according to ITS data (the
nrDNA internal transcribed spacer region) despite its lack of yellow-brownish spots.
The aim of this study was to determine whether the widely accepted morphological delimitation of R. maculata
corresponds to a single species. For this purpose, a phylogenetic analysis of the ITS region and morphological studies
were performed using material from different geographical areas and habitats.
Materials and methods
Sampling.—Our morphological observations were based on 13 herbarium collections identified as R. maculata
originating from Europe and one collection from Pakistan. All these collections were also sampled for a phylogenetic
analysis of ITS data and were supplemented with other GenBank and UNITE (Kõljalg et al. 2013) sequences from
Europe and Asia that were retrieved in BLAST searches with a high similarity to the target group. To assess the
taxonomic importance of the yellow-brownish context discolouration (i.e., the presence of yellow-brownish to rusty
spots on the stipe and pileus surface), we sampled representatives of the Russula section Maculatinae as defined by
Romagnesi (1967) and of the Russula subsection Urentes as defined by Sarnari (1998) and Knudsen et al. (2012).
Moreover, Russula subsection Rubrinae (Melzer & Zvára 1927: 45) Singer (1932: 242) as emended by Sarnari (1998)
has also been included because one of its representatives, R. rutila Romagnesi (1952: 112), was formerly classified in
the section Maculatinae (Romagnesi 1967). These groups comprise taxa characterized by medium to large basidiomata
with a fairly firm, acrid context, a yellow (or, more rarely, dark ochre) spore print, spores with an amyloid suprahilar
spot and pileocystidia with greying contents in sulfovanillin. This definition covers the following species that were
included in this study: R. aurantioflammans Ruots., Sarnari & Vauras in Sarnari (1998: 717), R. badia Quélet (1881:
668), R. cuprea (Krombholz 1845: 11) Lange (1926: 47), R. decipiens (Singer 1931: 211) Svrček (1967: 228), R.
dryadicola, R. firmula J. Schäffer (1940: 111), R. globispora, R. intermedia Karsten (1888: 38), R. juniperina Ubaldi
(1985: 25), R. rubra (Fries 1821: 58) Fries (1838: 354), R. rutila and R. veternosa Fries (1838: 354). Our sampling
provided a high coverage of the commonly accepted European species in the subsections Urentes and Rubrinae.
Collections sequenced in this study were identified to the species level according to Sarnari (1998) and Knudsen et al.
(2012). In addition, the phylogenetic analysis included some species with a mild taste or paler spore print that appeared
to be closely related to one or more of the 12 target species based on BLAST search results of against GenBank and
UNITE or based on a comparison with the ITS Russula phylogeny published by Looney et al. (2016).

RUSSULA MACULATA (RUSSULACEAE) Phytotaxa 270 (2) © 2016 Magnolia Press • 73
We selected three sequences for each species from reliable sources, preferably from material that had been
identified by us and that originated from geographically distant areas. Three sequences of R. emetica (Schaeffer 1774:
9) Persoon (1796: 100) were used as an outgroup. All specimens and sequences are listed in the Supplementary Tab. 1.
Herbarium material collected by the authors is deposited in the following herbaria: SAV, GENT, STU, M and LAH.
Molecular analysis.—The total genomic DNA was extracted using a variety of protocols (Eberhardt 2012, Nuytinck
& Verbeken 2003, Van de Putte et al. 2010). In addition to these cited methods and consumables, the following procedures
were used: The ArchivePure DNA yeast and Gram-+kit (5 PRIME, Inc. Hilden, Germany) with a longer incubation
time of 0.5 to 12 hours after the addition of Lytic Enzyme Solution or the High Pure PCR Template Preparation Kit for
the Isolation of Nucleic Acids from Mammalian Tissue (Roche Applied Science, Indianapolis, USA). The ITS region
was amplified using the primers ITS1F–ITS4 or alternatively ITS1F–ITS2 and ITS3–ITS4 (White et al. 1990, Gardes
& Bruns 1993) and with polymerase PerfectTAQ (5 PRIME, Hilden, Germany) in accordance with the manufacturers
recommendation. The PCR amplification followed the protocols of Eberhardt (2012) or Knebelsberger & Stöger (2012)
with longer denaturation and annealing times of up to 1 min instead of 30 sec.
The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany) or Exo-Sap
enzymes (Thermo Fisher Scientific, Wilmington, Delaware, USA) and directly sequenced with BigDye 3.1 technology
(Applied Biosystems, now Thermo Fisher Scientific, Wilmington, USA).
Raw sequences were edited in the BioEdit Sequence Alignment Editor version 7.2.5 (Hall 2013) or Sequencher
version 4.8 (Gene Codes Corporation). Intra-individual polymorphic sites having more than one signal were marked
with NC-IUPAC ambiguity codes. Edited sequences were aligned by MAFFT version 7 using the strategy E-INS-i
(Katoh & Standley 2013).
Phylogenetic analysis.—The final alignment included 78 ITS sequences altogether. Twenty-one sequences
corresponded to the current concept of R. maculata. Of these, 14 were obtained from herbarium material, while seven
additional sequences were retrieved from GenBank or UNITE. Only the ITS1 and partial 5.8S rRNA were obtained
from the epitype of R. maculata. The aligned data were analysed using Bayesian inference (BI) and the Maximum
Likelihood method (ML).
For BI, the dataset was divided into three partitions: ITS1, 5.8S and ITS2. The best substitution model for each
partition was computed separately in MEGA (Tamura et al. 2013). The following models were chosen for the analysed
partitions: K2+G (ITS1), JC (5.8S) and HKY+G+I (ITS2). The Bayesian inference was computed independently twice
in MrBayes 3.2.6 (Huelsenbeck et al. 2015) in two simultaneous runs with four MCMC chains each run for 10 000
000 iterations. The convergence of runs was visually assessed using Trace function in Tracer version 1.6 (Rambaut et
al. 2013).
Maximum Likelihood was computed in PhyML using SeaView (Gouy et al. 2010) using GTR+GAMMA
substitution model with 4 rate classes and 1000 bootstrap replications.
Morphological observations.—Micromorphological characteristics were observed using Olympus CX-41 with
oil-immersion lenses at a magnification of 1000×. All drawings of microscopic structures, with the exception of spores,
were made with a ‘camera lucida’ using an Olympus U-DA drawing attachment at a projection scale of 2000×. The
contents of hymenial cystidia and pileocystidia were illustrated as observed in Congo red preparations from dried
material, with the exception of some pileocystidia for which the contents are indicated schematically (dotted). Spores
were observed on the lamellae with Melzer’s reagent. All other microscopic observations were made in ammoniacal
Congo red, after a short treatment in warm, aqueous KOH solution to dissolve the gelatinous matrix and improve
tissue dissociation. All tissues were also examined in Cresyl blue to verify the presence of ortho- or metachromatic
reactions as explained in Buyck (1989). Trama and cystidia were examined in a sulfovanillin solution. Acidoresistant
incrustations of the primordial hyphae were stained with carbolfuchsin and observed in distilled water after incubation
for a few seconds in a 10% solution of HCl (cf. Romagnesi 1967). Spores were scanned with an Artray Artcam
300MI camera and measured by the Quick Micro Photo version 2.1 software. Measurements and line drawings were
made using enlarged, scanned pictures of spores with an accuracy of 0.1 μm. The Q value was used to indicate the
length/width ratio of the spores. Spore measurements excluded ornamentation. The spore ornamentation density was
estimated following Adamčík & Marhold (2000). The cystidia density estimates follow Buyck (1991). Statistics for
the measurements of microscopic characteristics were based on 30 measurements per specimen and expressed as the
mean ± standard deviation. The species descriptions are based on all studied specimens.
To support the morphological delimitation of a species, we compared the full-detail descriptions (similar to those
presented by Adamčík & Jančovičová 2013) of one representative specimen for each clade retrieved in the molecular
analysis. The comparison of these representative specimens allowed us to reduce the number of observed characteristics
to those showing significant differences in the Tab. 1 (see the three-step procedure in Adamčík et al. 2016).

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TABLE 1. Comparison of selected characters observed on the studied material of R. maculata, R. nympharum and R. sp. All values of micro-morphological characters are averages of 30 measurements.
TC = terminal cells of hyphae in pileipellis. Distinguishing characters are in shaded boxes.
R. maculata R. nypharum R. sp.
Herbarium number PC84521 FH
2011BT005 SAV F-933 2009BT05 2010BT112 2010BT184 SAV F-2130 FH
RUS11121505 R-9702 hjb10019 R-0898 LAH
MAM0077
Spores
Size [µm] 9.1 × 7.5 9.3 × 7.8 9.2 × 7.6 9×7.6 9.1×7.6 9.2×7.6 9.3×7.5 8.7 × 7.3 9.6 × 7.9 9×7.6 9.3 × 7.7 8.2 × 6.8
Q1.22 1.19 1.21 1.19 1.2 1.21 1.23 1.19 1.23 1.19 1.21 1.21
ornamentation [µm] 0.6–0.7 0.8–1.1 0.6–0.9
(–1.1) 0.6–1 0.7–1 0.7–0.9 (–1) 0.7 – 1.1 0.3–0.6 0.4–0.6 0.3–0.6 0.4–0.6 0.4–0.6
TC near the
pileus margin
TC size [µm] 28.3 × 3.7 25 × 3.4 21.1 × 3.6 18.2×2.9 21.7×3.2 26.4×3.4 24.4×3.3 21.8 × 2.9 26.6 × 2.9 21.2×2.7 23.8 × 2.5 28.7 × 3
attenuated TC [%] 80 72 50 61 75 85 68 40 37 0 32 66
difference in width of TC 1.85 0.95 1.23 1.02 1.14 1.21 1.03 0.87 0.87 0.35 0.25 1.5
Pileocystidia near the
pileus margin
size [µm] 61.4 × 4.9 51.9 × 5.1 43 × 6.7 63.9×5.1 59×5.5 72×5.9 61.5×5.5 78 × 10.1 64 × 9.1 79×8.8 62.4 × 7.2 45 × 6.5
cell number 1.3 1.3 2.5 1.6 1.3 1.5 1.8 1.7 1.2 1.5 1.3 2.7
Pleurocystidia density 500–600 500–700 400–500 400–500 500–700 400–600 400–500 1000–1100 900–1100 900–1100 800–900 300–400

RUSSULA MACULATA (RUSSULACEAE) Phytotaxa 270 (2) © 2016 Magnolia Press • 75
Results
Molecular analysis.—The BI majority rule consensus and the ML result did not entirely agree in their gross topology,
but corresponded with respect to the clades that include the target species. Figure 1 shows the ML results with both
bootstrap and posterior probability support. The disagreement between the results concerns the placement of R.
intermedia, which is included in a clade with R. vinososordida and R. decipiens in the BI result. This clade, as well
as its sister clade (corresponding topologically to the ML result, but without R. intermedia) had borderline posterior
probability support. The corresponding clades did not have ML bootstrap support (Fig. 1).
All species with yellow-brownish spots included in this study were grouped in a moderately supported clade,
referred to here as the Maculatinae clade, that includes two well-supported subclades referred to as the R. globispora
complex (including R. globispora and R. dryadicola) and the R. maculata complex. The latter subclade contains all
sequences corresponding to the current morphological concept of R. maculata. Within the R. maculata complex, all
sequences originating from European specimens are clustered in a well-supported subclade that contains two well-
supported terminal clades. The first was identified as R. maculata because the sequence from the epitype was included
there. The second terminal clade represents a new species described below as R. nympharum F. Hampe & Marxm.
Our collection LAH MAM077 from Pakistan was tentatively identified as R. maculata and was placed together with
other sequences, one each from China and Papua New Guinea, in a well-supported subclade. Compared to the other
subclades of the Maculatinae clade, these sequences are on long terminal branches, similar in length to the internal
branches of the R. maculata or R. nympharum clades. This extra-European subclade, together with another singleton
sequence of Asian origin, forms a sister cluster to the R. maculata / R. nympharum subclade.
All other species currently classified in the section Maculatinae and the subsection Urentes, but not characterized by
conspicuous yellow-brownish context discolouration and yellow-brownish spots, are placed in various unrelated clades
and sometimes even grouped with mild-tasting species, e.g., R. decipiens with R. vinososordida Ruots. & Vauras in
Vauras & Ruotsalainen (2001: 558), R. intermedia with R. vinosa Lindblad (1901: 57) or R. aurantioflammans with R.
font-queri Singer (1947: 215).
Morphological differences among the species studied.—The group defined here as the R. maculata complex
contains three distinct terminal clades. Two of them seem to correspond to only one phylogenetic species each. The
third clade contains three sequences of Asian origin and shows a relatively high variability in the ITS region. Of
the sequenced collections, only the Pakistani collection LAH MAM077 has been available to us for morphological
analysis and is described in full detail as Russula sp. in the Taxonomy section below. Selected micro-morphological
characteristics of R. maculata and the two new species are compared in Tab. 1. We have not found any macro-
morphological characteristic that distinguishes R. maculata from the second European species R. nympharum, but
there are four distinct microscopic differences. The latter species has a lower spore ornamentation (up to 0.6 μm),
the terminal cells of the hyphae in the pileipellis near the pileus margin are less frequently attenuated and apically
constricted, the pileocystidia are wider than 7 μm on average (the individual pileocystidia are occasionally wider than
10 μm) and the pleurocystidia are more densely arranged. On the other hand, the Pakistani species is distinct even in its
field aspects (Figs. 2–4). It has smaller and less robust basidiomata. Under the microscope, it is similar to R. maculata,
but differs from it by its smaller spores and its pileocystidia composed of more cells and more septae.
Discussion
Delimitation of members of Russula section Maculatinae with yellow-brownish spots.—Our phylogenetic analysis
(Fig. 1) suggests that all infrageneric taxa currently used to accommodate R. maculata based on the acrid taste of the
context and the yellow spore print (i.e., Russula section Maculatinae and R. subsection Urentes) are polyphyletic.
A posterior probability of 0.94 (Fig. 1) in backbone of the tree is the only statistical support given to any clade
retrieved by both BI and ML, and this clade includes both, species that combine an acrid taste of the context and
yellow spore print and species with other characteristics (i.e., mild taste or ochre or paler spore print). In spite of its
inconsistent support, this result is in line with earlier studies of the genus (Eberhardt 2002, Miller & Buyck 2002).
These also suggested that a number of infrageneric taxa including species with a mild-tasting context or species with
yellow spore prints irrespective of taste, are unlikely to have phylogenetic support in taxonomically more inclusive
studies.

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FIGURE 1. Maximum Likelihood phylogeny based on ITS nrDNA region with species-level clades highlighted as well supported clades
of species with yellow-brownish context discoloration. Species belonging to R. sect. Maculatinae in sense of Romagnesi (1967) are
indicated by red brackets and species with mild taste by green brackets. The * indicates a clade which was not retrieved in the BI analyses.
Origin of sequences is listed in the Supplementary table 1. Labels of type collections and country of origin are indicated for members from
the R. maculata complex. Bootstrap values followed by Bayesian posterior probabilities are indicated. Support values are only indicated
for nodes with either a bootstrap value above 70 or a posterior probability at or above 0.90.

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A core group around R. maculata had some bootstrap support and good posterior probability support. It consists of
species that have an acrid taste, a yellow spore print and a yellow-brownish context discolouration. This discolouration,
results in the appearance of yellow-brownish spots on the basidiomata surfaces in early stages of their development,
and seems to be a rather good and stable characteristic. Combined with other traditionally used characteristics, it
allows the identification of a group of fungi that includes both the R. maculata and R. globispora species complexes
(referred here as the Maculatinae clade). Sarnari (1998) was the first author who fully recognized the major taxonomic
importance of this characteristic and consequently considered R. maculata, R. dryadicola and R. globispora to form
a fairly homogeneous, morphological group within the subsection Urentes, which he provisionally referred to as the
“series Maculata” (nom. inval.). The only validly published name with a defined taxonomic rank for an infrageneric
taxon typified by an element contained in the Maculatinae clade is the Russula section Maculatinae. We have not
decided on the taxonomic rank of the Maculatinae clade primarily because no phylogenetic framework exists upon
which a new overall systematic concept of this large and widely distributed genus can be built. Additionally, our
sampling for this study was too limited among potentially related infrageneric taxa.
There are two distinct groups distinguishable by their spore ornamentation in the Maculatinae clade that correspond
to the subclades identified in our molecular analyses. The first, the Russula maculata complex, is defined by moderately
prominent spore ornamentation to low warts with frequent connectives. The second, the Russula globispora complex,
has spores with large isolated spines. It is represented in our analysis by R. dryadicola and R. globispora.
Species nomenclature of the R. maculata complex.—Russula maculata is the only species that was recognized
in the R. maculata complex prior to our study. Recent studies have shown that clearly distinct species are often
overlooked, especially in widely recognized species, with conspicuous macro-morphological characteristics. Such
assumptions can be a reason for false confidence about a species concept (e.g., Morgado et al. 2013, Ainsworth et al.
2013). A good example within the family Russulaceae is the European Lactifluus volemus (Fries 1821: 69) Kuntze
(1891: 857), which was revealed to be a complex of three previously unrecognized species (Van de Putte et al. 2016).
Russula maculata seems to be a similar example.
Most of the characteristics identified here as useful for distinguishing among R. maculata, R. nympharum and
R. sp. from Pakistan have not been used previously for species delimitation and identification. For this reason, it is
difficult to judge if European authors have treated R. maculata in a broad sense or if their descriptions can be referred
to one of the two European species recognized here. The pileocystidia width is the distinguishing characteristic for
R. maculata and R. nympharum (Tab. 1) that is also often included in literature descriptions of R. maculata. The
measurements given by Romagnesi (1967) for R. maculata pileocystidia generally correspond to the typical form (5.2–
8.7 μm wide), whereas Einhellinger (1987) and Sarnari (1998) described and illustrated pileocystidia width ranges that
may correspond to either species (3–11 μm or 4.2–10 μm, respectively). Marxmüller (2014) provided descriptions and
illustrations of two collections: the first (R-8102) probably refers to R. maculata sensu stricto (pileocystidia up to 8
μm), but the sequence of the second (collection R-0898) clearly places it in the R. nympharum clade (Fig. 1). Lejeune
(2004) described a discoloured form of R. maculata (referred to as the “straminoid form”) and his illustration of the
pileocystidia suggests that his description is based on a collection of R. nympharum.
Our study suggests that at least one species of the R. maculata complex occurs in Asia and is associated with
conifers; R. sp. from Pakistan (KU886598) was associated with Pinus roxburghii. Two sequences from outside Europe
(UDB013256 from a Papua New Guinea, from a fruitbody collection, recorded with Castanopsis acuminatissima,
and KR082870 from China with no published ecological details) have the same monophyletic lineage, but sufficient
phylogenetic support does not exist to conclude whether they are conspecific with the Pakistani species. The other
Asian sequence (JN129407 from China) was generated from a mycorrhizal root tip of Keteleeria (Pinaceae) (Ge
et al. 2012) and possibly represents a distinct, undescribed species. Because of geographical distance, ecological
difference, ITS variation and paraphyly (Fig. 1), it is possible that the three other sequences retrieved from GenBank
(Supplementary Tab. 1) represent species different from R. sp. from Pakistan. A previous report of R. maculata from
Pakistan associated with pines (Sultana et al. 2011) probably represents the same species as described here, but we
were not able to obtain the corresponding material for confirmation.
Some taxa, either recently described or with a dubious concept, might represent nomenclatural challenges to these
new species within the R. maculata complex or the wider subsection Urentes. The following two names contain a direct
reference to the epithet “maculata” and should be especially mentioned: R. maculata f. paradecipiens Favre (1992: 19)
and R. globispora var. submaculata Sarnari (1998: 704). The first, now combined at the species rank [R. paradecipiens
(Favre) Favre (1999: 30)], was described as having a distinctly greying or even a blackening context (Favre 1992). The
second might be distinguished by isolated amyloid spines on spores that clearly matched the R. globispora complex as
defined above.

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78 • Phytotaxa 270 (2) © 2016 Magnolia Press
Several other species previously classified in the section Maculatinae were not included in our study. Although
validly published and legitimate, they have not been accepted in most recent studies, e.g., R. britzelmayrii Romell
in Britzelmayr (1893: 12), R. cerasina Martín (1894: 187), R. poetae Reumaux et al. (1999: 425), R. papavericolor
Reumaux in Reumaux et al. (1996: 286). In our opinion, they do not represent close relatives of R. maculata because
no conspicuous yellow-brownish spots were mentioned in the descriptions. For the same reason, we are confident that
the Indian species R. mayawatiana is not a close relative of R. maculata. It has no yellow-brownish spots, moreover,
the spore ornamentation corresponds more to the R. globispora complex. The authors of the Indian species, Das et al.
(2006), placed it close to R. maculata based on ITS data, but the position of both species suggests that the sequence
of R. maculata used was misidentified (unfortunately the authors do not cite sequence vouchers and the sequences are
probably not deposited in public databases).
Distribution and ecology of R. maculata and R. nympharum.—Our assessment of the ecological preferences of
the two European species is exclusively based on the limited material used in this study. The species descriptions in
the literature are difficult to interpret, and therefore, we cannot reliably associate hosts. Our R. maculata collections
originated from areas as far north as Estonia and as far south as Northern Italy. They were found in association
with Quercus, Fagus, Carpinus and Tilia in mostly temperate broadleaf forests, but the Slovak collection SAV F-933
originated from a dry, thermophilous, steppe-like habitat with Quercus cerris. Our sampling of R. nympharum suggests
a certain preference for Mediterranean evergreen oak forests (collections from Southern France from Val des Nymphes
and Mallorca Island), but the collection from Belgium near Brussels is from outside the Mediterranean area. Thus, our
data seem to indicate at least a partly overlapping distribution range because the epitype of R. maculata originated from
Oise region north of Paris and R. nympharum was collected in Belgium, less than 200 km to the north. It is also very
possible that they may share the same type of habitat and even co-occur at a single spot.
Taxonomy
Russula maculata Quél., Bull. Soc. Bot. France 24: 323. 1878. Figs. 2–3
The epitype of the species (PC0084521) was designated, described and illustrated by Adamčík & Jančovičová (2013), and its description
is supplemented here by statistical values of micro-morphological characteristics (Tab. 1).
Examined material.—DENMARK. Vejle: Staksrode Skov, N of Vejle Fjord, Fagus, 10 August 1999, P.B. Hansen,
J. Vesterholt JV99-196 (C); Lolland: Kristianssædevej, S of Kristianssæde Skov, Tilia, 6 July 2001, P.B. Hansen
& J. Vesterholt JV01-061 (C); Sjælland island: Vejlø Skov S of Næstved, Fagus, Quercus, 4 September 2001,
P.B. Hansen JV01-304 (C); ESTONIA. Saarnaki island: 13 km SEE of Käina, 58°48’6”N 23° 03’88”E, Tilia, 23
September 2008, S. Adamčík (SAV F-2130); FRANCE. Oise Dept.: Boran-sur-Oise, Betula, Corylus, Quercus, 14
July 1954, H. Romagnesi 54.43 (PC0084521, epitype); GERMANY. Thüringen: Gustav-Freytag-Weg (Schlossleite)
near Arnstadt, 1 June 2009, J. Girwert FH 2009 BT05 (GENT); Rüdigsdorfer Schweiz, near Nordhausen, Quercus,
18 September 2010, F. Hampe FH 2010 BT184 (GENT); Gotha palace garden, Carpinus, Fagus, Quercus, Tilia, 22
August 2010, F. Hampe FH 2010 BT112 (GENT); Ibid., Tilia, Carpinus, Quercus, 5 July 2011, F. Hampe FH 2011
BT005 (GENT); SLOVAKIA. W Carpathians: Cerová vrchovina Mts., 1.5 km NW of Obručná village, Manický
potok, 48°12’15”N 19°51’59”E, Quercus cerris, 9 June 2004, S. Adamčík (SAV F-933).
Russula nympharum F. Hampe & Marxm., sp. nov. Figs. 4, 6–15
MycoBank no.:—MB 816289.
Etymology.—The species epithet refers to the collection site (Val des Nymphes) of two of the paratypes one of which
was illustrated in Marxmüller (2014).
Holotype (designated here).—SPAIN. Mallorca: Bunyola, associated with Quercus ilex and Arbutus unedo, 15
December 2011, FH11121505 (GENT).
Short diagnosis.—Basidiomata relatively large and with firm, thick context, surface of stipe, pileus and lamellae
with yellow-brownish spots, pileus cuticle red or orange and discolouring to cream, taste acrid, spore print yellow,
spore ornamentation with low (up to 0.6 μm), amyloid warts often merged or connected by line connections, hymenial
cystidia relatively numerous, hyphal terminations in pileipellis near the pileus margin mainly cylindrical, pileocystidia
near the pileus margin 6‒12 μm wide (on average wider than 7 μm).

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FIGURES 2–5. Field appearance of three Russula species included in this study. 2. R. maculata (GENT 2011 BT 005). 3. R. maculata
(GENT 2010 BT 184). 4. Holotype of R. nympharum (GENT RUS11121505). 5. Russula sp. (LAH MAM 0077). Scale bar equals 30 mm,
but only 10 mm for Russula sp. Photos by: F. Hampe (Figs. 2–4) and M. Saba (Fig. 5).
Pileus up to 100 mm broad, fleshy, firm, first hemispherical, then expanded with depressed centre, extreme margin
becoming somewhat sulcate with age, cuticle peeling at margin to 1/3 of pileus radius, glabrous, shining, cream with
pale rose to pale orange zones or reddish orange to pinkish red or deep red, often paler to cream at centre, mostly more
or less covered with small brownish or reddish spots. Lamellae adnate, crowded, more or less furcate towards the stem,
slightly anastomosing, fragile, cream, then yellowish, with yellow-brownish spots at age, edge smooth, concolourous
or reddish especially towards the pileus margin. Stem cylindrical to slightly clavate with rounded base, firm, surface
minutely wrinkled, whitish, staining yellow-brownish to brownish when handled or with age, especially towards the
base. Context firm, whitish, staining yellow-brownish to brownish, taste acrid especially in lamellae, smell fruity or
like cedar wood, FeSO4 and Guaiac very weak or negative. Spore print IV d, IV d–e (coded according to Romagnesi
1967).
Spores broadly ellipsoid, (8–)8.6–9.8(–10.8) × (6.5–)6.9–7.6(–7.9) μm, average 9.2 × 7.3 μm, Q=(1.14–)1.16–
1.23(–1.27), average Q=1.19, ornamentation of moderately large and distant: 4–6(–7) amyloid warts in the circle of
diameter of 3 μm on spore surface, warts 0.3–0.6 μm high, connected with occasional to frequent short or longer fine
line connections [0–3 (–4) line connections in the circle], occasionally fused in short chains or crests [0–3(–4) fusions
in the circle], chains and crests often branched, but rarely forming a reticulate structure, isolated warts rare. Suprahilar
plage amyloid and very distinct. Basidia (39–)45–55.5(–62) × (9–)10.5–13(–14) μm, average 50.3 × 11.6 μm, 4-
spored, clavate, pedicellate; basidiola first cylindrical or ellipsoid, then clavate, ca. 5–10 µm wide. Subhymenium
pseudoparenchymatic. Lamellar trama mainly composed of large sphaerocytes. Hymenial cystidia on lamellar sides
moderately numerous, 800–1100/mm2, fusiform or rarely clavate, pedicellate, acute to acute-pointed on tips and with
2–7(–11) µm long appendage, thin–walled, measuring (60–)75–108.5(–140) × (8–)10.5–13.5(–15) μm, average 91.7 ×
11.9 μm, contents heteromorphous, mostly granular-crystalline, turning brownish-red to almost black in sulfovanilin.
Lamellar edges covered with marginal cells, cheilocystidia and dispersed basidia; marginal cells on lamellar edges

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80 • Phytotaxa 270 (2) © 2016 Magnolia Press
FIGURES 6–9. Russula nympharum (holotype). 6. Pileocystidia near the pileus centre. 7. Pileocystidia near the pileus margin. 8. Hyphal
terminations in the pileus centre. 9. Hyphal terminations near the pileus margin. Contents of cystidia are represented as observed in Congo
Red for some elements only, the others are simply filled with dots to indicate their cystidial nature. Scale bar equals 10 μm. Drawings by:
S. Jančovičová.

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FIGURES 10–15. Russula nympharum (holotype). 10. Basidia. 11. Basidiola. 12. Cheilocystidia. 13. Marginal cells. 14. Basidiospores
in Melzer’s reagent. 15. Pleurocystidia. Contents of cystidia are represented as observed in Congo red. Scale bar equals 10 μm, but only 5
μm for spores. Drawings by: S. Jančovičová.

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82 • Phytotaxa 270 (2) © 2016 Magnolia Press
not well differentiated, narrower than the basidiola on lamellar sides, mainly narrowly clavate to subcylindrical, often
somewhat flexuous or moniliform, with obtuse tips, measuring (15–)20–31.5(–33) × (3–)4–7(–9) µm, average 25.9
× 5.4 μm; cheilocystidia less voluminous than pleurocystidia, clavate or fusiform, pedicellate, mainly with acute
tips and usually with 1–5(–10) µm long appendage, thin-walled, measuring (37–)47–79(–102) × (7–)8–10.5(–12)
µm, average 63.1 × 9.3 μm, contents similar as in pleurocystidia. Pileipellis orthochromatic in Cresyl blue, 140–
250 μm deep, not sharply delimited from the underlying spherocytes of the context; vaguely divided in a 70–120
μm deep, strongly gelatinized suprapellis composed of dense, ascending or erect hyphal ends (trichoderm type) and
protruding and near surface repent pileocystidia, sometimes covered with an additional, up to 40 µm deep transparent,
gelatinous matter that does not colour in Congo red; and a 70–165 μm deep subpellis of less gelatinized, dense,
strongly intricate, horizontally oriented, often branched, 2–5(–7) µm wide hyphae. Acidoresistant incrustations absent.
Hyphal terminations in pileipellis near the pileus margin with gelatinous coating (not an acidoresistant incrustation)
dissolving in KOH, but visible as a hyaline hue in Congo red, terminal cells very variable in size, some very short and
comparatively shorter than subterminal cells, mainly cylindrical, sometimes attenuated towards apices, often flexuous,
partly moniliform, measuring (10.5–)17.5–30.5(–44) × (2–)2.5–3.5(–4.5) µm, average 20.1 × 2.8 μm; subterminal cells
branched or not, often with lateral branches or nodules, more or less equally wide as terminal cells. Pileipellis near the
pileus centre composed of hyphal terminations of one or two cells arising from a dense pseudoparenchymatic subpellis
layer, terminal cells not very conspicuous among very numerous pileocystidia and sometimes completely suppressed,
cylindrical or occasionally subulate, often flexuous-moniliform, measuring (11–)16.5–32.5(–44) × (2–)2.5–3.5(–4.5)
µm, average 24.5 × 2.8 μm. Pileocystidia near the pileus margin numerous and often very voluminous, narrowly to
broadly clavate or fusiform, mainly one-celled, occasionally two or three celled, rarely with more cells, thin-walled
or occasionally with slightly (up to 0.5 µm) thickened walls, obtuse to subacute, usually inflated near apical part, with
terminal cells measuring (26–)41.5–94.5(–132) × (4–)6–11.5(–15.5) µm, average 68.1 × 8.8 μm, contents in Congo red
heteromorphous, usually granulose, but sometimes partly crystalline or banded, in sulfovanilin turning dark brownish
to blackish. Pileocystidia near the pileus centre very abundant, often clustered and in some spots completely replacing
the undifferentiated hyphal terminations, very variable in shape and size, partly narrow and cylindrical, partly similar
to those near the pileus margin (clavate or fusiform), but usually smaller, often flexuous and occasionally moniliform,
terminal cells measuring (18–)28.5–77(–119) × (3–)4.5–8.5(–10.5) µm, average 52.9 × 6.5 μm, contents similar to
those near the pileus margin, but often with additional yellowish pigments visible in KOH or water. Cystidioid hyphae
with heteromorphous contents in subpellis occasional and in the pileus trama dispersed. Clamp connections absent in
all parts.
Examined material.—BELGIUM. Forêt de Soignes, 31 August 2000, H.J. Beker HJB10019 (STU); FRANCE.
Drôme Dept.: Gigors, les Chaux, 5 July 1997, H. Marxmüller R-9702 (M); La Garde-Adhémar, Val des Nymphes,
Quercus ilex, 30 September 2008, H. Marxmüller R-0898 (M); SPAIN. Mallorca island: Bunyola, Quercus ilex,
Arbutus unedo, 15 December 2011, F. Hampe & J. Kleine RUS11121505 (GENT, holotype).
Russula sp. Figs. 5, 16–25
Short diagnosis.—Basidiomata medium to small, pileus with relatively thin context, surface of stipe, pileus and lamellae with yellow-
brownish spots, pileus cuticle red and not discolouring distinctly to cream, spore ornamentation with low (up to 0.6 μm), amyloid
warts often merged or connected by line connections, hymenial cystidia dispersed, hyphal terminations in pileipellis near the pileus
margin sub-cylindrical or subulate, pileocystidia near the pileus margin frequently with more than two cells and 5.5‒7.5 μm wide.
Basidiomata small to medium sized, 40–45 mm tall. Pileus 27‒34 mm in diameter, convex, centrally slightly depressed,
surface dry, smooth, matt, vivid red or strong red with centre reddish orange and rusty spotted with spots sometimes
concentrically arranged; margin even, or slightly involute, without striations. Lamellae regular, adnate, crowded,
light yellow, pale yellow or light orange yellow, brittle, edge entire, concolourous. Stipe 35‒40 × 8‒10 mm, central,
cylindrical to subcylindrical, stuffed, slightly longitudinally wrinkled, white, towards base with light yellow-brownish
or moderate yellow-brownish spots, without pinkish shades. Context compact, not firm, smell and taste not recorded.
Spores (7.4–)7.8–8.6(–9.4) × (6.2–)6.4–7.2(–7.7) μm, average 8.2 × 6.8 μm, Q = (1.13–)1.17–1.24(–1.29), average
1.21, ornamentation consisting of (4–)5–7, moderately large and distant amyloid warts in the circle of diameter of 3 μm
on spore surface, warts 0.4–0.6 μm high, connected with occasional to frequent short or longer fine line connections [(0–
)1–3(–5) line connections in the circle], occasionally fused in short or longer chains [(0–)1–3(–4) fusions in the circle],
chains and crests often branched, but rarely forming a reticulate structure, isolated warts rare. Suprahilar plage amyloid,
large. Basidia (30–)33–37.5(–40) × (10–)11–13 μm, average 35.4 × 12.1 μm, 4-spored, clavate, sometimes pedicellate;

RUSSULA MACULATA (RUSSULACEAE) Phytotaxa 270 (2) © 2016 Magnolia Press • 83
FIGURES 16–19. Russula sp. from Pakistan (LAH MAM 0077). 16. Pileocystidia near the pileus centre. 17. Pileocystidia near the pileus
margin. 18. Hyphal terminations in the pileus centre. 19. Hyphal terminations near the pileus margin. Contents of cystidia are represented
as observed in Congo Red for some elements only, the others are simply filled with dots to indicate their cystidial nature. Scale bar equals
10 μm. Drawings by: S. Jančovičová.

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84 • Phytotaxa 270 (2) © 2016 Magnolia Press
FIGURES 20–25. Russula sp. from Pakistan (LAH MAM 0077). 20. Basidia. 21. Basidiola. 22. Cheilocystidia. 23. Marginal cells. 24.
Basidiospores in Melzer’s reagent. 25. Pleurocystidia. Contents of cystidia are represented as observed in Congo red. Scale bar equals 10
μm, but only 5 μm for spores. Drawings by: S. Jančovičová.

RUSSULA MACULATA (RUSSULACEAE) Phytotaxa 270 (2) © 2016 Magnolia Press • 85
basidiola first cylindrical or ellipsoid, then clavate, ca. 5–10(–12.5) µm wide. Subhymenium pseudoparenchymatic.
Lamellar trama mainly composed of large sphaerocytes. Hymenial cystidia on lamellar sides widely dispersed to
dispersed, 300–400/mm2, fusiform or rarely clavate, pedicellate, thin-walled, measuring (49–)52–65(–72) × (10–
)10.5–14(–16) μm, average 58.6 × 12.3 μm, apically acute to acute-pointed and with (1–)2–7 µm long appendage,
contents heteromorphous, granular-banded, yellowish, turning brownish-red to almost black in sulfovanillin. Lamellar
edges covered with abundant marginal cells, occasional cheilocystidia and dispersed basidia; marginal cells not well
differentiated, similar to the basidiola on lamellar sides, but smaller, mainly clavate to subcylindrical, sometimes
constricted in half length, with obtuse tips, measuring (9–)12–17.5(–19) × (4–)4.5–7(–7.5) µm, average 15 × 5.8
μm; cheilocystidia narrower than pleurocystidia, clavate or fusiform, pedicellate, thin-walled, measuring (42–)48–
64.5(–73) × 8–10.5(–12.5) µm, average 56.5 × 9.3 μm, apically with mainly acute tips and usually with 1–6 µm long
appendages, contents similar as in pleurocystidia. Pileipellis orthochromatic in Cresyl blue, 115–135 μm deep, sharply
delimited from the underlying spherocytes of the context; distinctly divided in a 60–75 μm deep, strongly gelatinized
suprapellis of loose, erect or ascending hyphal terminations and, near surface, with some repent, longer pileocystidia;
and a 55–65 μm deep subpellis of less gelatinized, dense, irregularly, but near the trama horizontally oriented, intricate,
branched, 2–5 µm wide hyphae. Acidoresistant incrustations absent. Hyphal terminations in pileipellis near the pileus
margin slender and branched, thin-walled, with terminal cells measuring (14–)20.5–37(–48) × 2.5–3.5 µm, average
28.7 × 3 μm, mainly narrowly subulate or fusiform, partly subcylindrical, usually apically attenuated or constricted,
often moniliform; near the pileus centre with mainly cylindrical, often flexuous terminal cells, measuring (13–)16.5–
27(–31) × (2–)2.5–4(–4.5) µm, average 21.7 × 3.2 μm, apically obtuse; subterminal cells mainly branched or not,
often with lateral branches or nodules, equally wide as terminal cells. Pileocystidia near the pileus margin numerous,
narrowly clavate or fusiform, mainly two or more-celled [1–5(–6) celled], thin-walled, with terminal cells measuring
(18–)33.5–57(–67) × (4–)5.5–7.5(–8.5) µm, average 45 × 6.5 μm, apically obtuse, subterminal cells equally wide or
narrower, often shorter, contents in Congo red heteromorphous, granulose or crystalline, turning dark reddish-brown to
black in sulfovanillin; near the pileus centre smaller and narrower, with terminal cells measuring (30–)34.5–59.5(–81)
× (3.5–)4–6(–7) µm, average 47.1 × 5.1 μm, more frequently one-celled, with more granular and yellow-coloured
contents. Cystidioid hyphae in subpellis and pileus trama dispersed, with heteromorphous-granulose, yellowish, often
oleiferous contents. Clamp connections absent in all parts.
Examined material.—PAKISTAN. Khyber Pakhtoon Khaw, Mansehra, Batrasi, under Pinus roxburghii, 3 August
2014, Malka Saba MAM 0077 (LAH, duplicate FH00304559).
Acknowledgements
We very much appreciated the help of H.J. Beker, P. Boisen Hansen, M. Della Maggiora, A. Gminder, H. Kaufmann,
U. Pera, A.F.S. Taylor, J. Vesterholt and any collectors we may have forgotten to mention by accident for supplying
us with interesting and exciting collections. The studies of S. Adamčík, M. Caboň, S. Jančovičová, K. Marhold
and M. Slovák were funded by the Slovak national projects VEGA 02/0075/14, APVV-15-0210, and framework
of the project Centre of excellence for protection and use of landscape and for biodiversity ITMS 26240120014
financed by European Fund for Regional Development under the Operational programme Research and Development
(002/2009/4.1/OPVaV). The studies of M. Caboň and M. Kolařík were supported by bilateral scientific mobility project
SAV-AV ČR 16-06 and Intra-Visegrad scholarship no. 51400484. The work of Ursula Eberhardt was supported by the
Swedish Species Project; of Malka Saba by Higher Education Commission (HEC) Pakistan, under Phase II, Batch I,
Indigenous PhD fellowships Program for 5000 scholars and of Miroslav Kolařík by Biotechnology and Biomedicine
Centre of the Academy of Sciences and Charles University from the European Regional Development Fund (BIOCEV
CZ.1.05/1.1.00/02.0109).
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