Tricholoma punctatum sp. nov. from Serbia

Abstract

A new species of Tricholoma recorded from a temperate forest in central Serbia is described and illustrated. It is characterized by the presence of punctate, often elevated, almost black spots arranged in one to three circular zones on the pileus. The new species showed morphological differences from closely related taxa and molecular phylogenetic analysis of ITS and LSU regions confirm the distinct taxonomic status of the finding.

Full text

MYCOTAXON
ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022
October–December 2022—Volume 137(4), pp. 737–749
https://doi.org/10.5248/137.737
Tricholoma punctatum sp. nov. from Serbia
N L, E Č, M K
1 Mycological Society “Šumadija”
Daničićeva 120/36,34000 Kragujevac, Serbia
2 University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology
Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia
*C : eleonora.capelja@dbe.uns.ac.rs
A–A new species of Tricholoma recorded from a temperate forest in central Serbia
is described and illustrated. It is characterized by the presence of punctate, oen elevated,
almost black spots arranged in one to three circular zones on the pileus. e new species
showed morphological dierences from closely related taxa and molecular phylogenetic
analysis of ITS and LSU regions conrm the distinct taxonomic status of the nding.
K : Atrosquamosa, Tricholomataceae, taxonomy, fungal diversity
Introduction
Tricholoma (Fr.) Staude (Tricholomataceae, Agaricales) is a cosmopolitan
genus, mainly distributed in temperate and subtropical zones of both
hemispheres (Tedersoo & al. 2010). e majority of Tricholoma species are
described from North America where more than one hundred species have
been reported so far, and this region appears to be the centre of species richness
(Bessette & al. 2013). ere are about ninety species listed from Europe (Bon
1991, Riva 1988, 1998, 2003; Kibby 2010), but the overall diversity of Tricholoma
taxa in Europe is still poorly resolved.
Serbia presents an exceptional place for mycological research due to the
presence of various habitats: thermophilous oak forest at lower altitudes
(usually Quercus cerris L.); succeeded by mesophilous beech and coniferous
forests (usually Fagus sylvatica L.; Picea abies (L.) H. Karst.; Abies alba Mill.) in

738 ... Lukić, Čapelja, Karaman
higher altitudes. Pine forests can be sporadically found at all altitudes. Although
they are some published data about diversity of Agaricomycetes in Serbia (Čolić
1967, Karaman & al. 2005, Karadžić & Milijašević 2008, Lukić 2009, 2013;
Ivančević & Davidović 2011, Sadiković & al. 2012), a thorough and systematic
investigation of this group of fungi is still lacking. Tricholoma is quite frequent
in Serbia (Uzelac 2009), with basidiomes oen appear in late October and
November in thermophilous deciduous forests. Since Tricholoma species show
limited microscopic variation, and are characterized by hyaline, subglobose to
oblong spores, simple pileipellis structures, and lack of well-dierentiated sterile
elements (e.g., cystidia), species identication and partly also the infrageneric
classication have mainly been based on macromorphology. In a recent work,
the combination of morphological and molecular data showed that characters
such as pileus color, pileipellis structure, presence of clamp connections and
spore size seem to be rather conserved within accepted sections, while the
presence of a distinct ring, and especially host specicity were highly variable
(Heilmann-Clausen & al. 2017).
e rst comprehensive molecular work on European Tricholoma (Heilman-
Clausen & al. 2017) revealed ten clades/sections which were not completely in
agreement with Singer’s classication. Also, they considered European species of
sections Caligata, Atrosquamosa, and Terre a to be well evaluated taxonomically,
while for all other sections their sampling was limited and are in need for further
phylogenetic studies. Heilman-Clausen & al. (2017) discovered several clades
with unassigned names, and one of them included two European species T.
josserandii Bon and T. f ucat um (Fr.) P. Kumm., taxa that were grouped together
with North and Central American species described and mentioned in study
by Ovrebo & al. (2019). A phylogenetic analysis by Reschke & al. (2018) of
Tricholoma collections from Asia, Europe, and North America revealed major
clades that were similar to those presented by Christensen & Heilman-Clausen
(2013). Also, sequences of T. imbricatum (Fr.) P. Kumm. specimens from USA
formed a highly supported clade that was separated from the Asian/European
T. imbricatum clade. Additionally, T. aurantiipes Hongo, T. davisiae Peck, T.
muscarium Kawam. ex Hongo, and T. muscarioides Reschke & al. comprised
a clade that was formally described as a new section Muscaria. All of the
preceding analyses were based only on ITS sequences, which has proved to be
a stable marker for species delimitations; but this region cannot resolve higher
taxonomic relationships at a suciently detailed level (Frøslev & al. 2005).
Infrageneric classication proposed in previous studies should be viewed as
preliminary and further studies including multiple molecular markers should

Tricholoma punctatum sp. nov. (Serbia) ... 739
be conducted. Here we present Serbian specimens with unique morphological
characters diering from previous Tricholoma species recorded from Europe
(Riva 1988, 1998, 2003); and phylogenetic analyses based on ITS and LSU
conrm these specimens as a new species, proposed here as T. punctatum.
Material and methods
Studied material
Specimens studied for this paper were collected by the rst author during eld trips
in central Serbia during October–November 2020. Nearly one hundred basidiomes
were recorded during three visits at the dened location (close to the city of
Kragujevac, 44.0181°N 20.8839°E, altitude 240 m). e habitat of the newly proposed
Tricholoma species (widely observed) is an old oak forest (mostly Quercus robur L. and
Q. cerris) with several other interspersed trees (Pinus nigra J.F. Arnold, P. sylvestris L.,
Carpinus betulus L., Tilia cordata Mill., Populus alba L., Betula pendula Roth) within
the Memorial area of Šumarice near the city of Kragujevac with an approximate area
of 350 ha. By virtue of the forest age and absence of economic exploitation the dened
area represents a very valuable mycological habitat. During twenty years of research in
this area twenty-one Tricholoma species (and several varieties and forms) have been
found mostly within sections Genuina, Tricholoma, Atrosquamosa, and Ter r ea (Lukić
& al., unpublished results).
Basidiomes were photographed in their natural habitat, at the area of their
appearance. Morphological data were recorded from the fresh specimens, which were
collected within a period of two months, on three occasions. Color codes follow the
Munsell soil color charts (Munsell 1975). Two collections (about ten specimens in
each collection) were dried by natural ventilation in the shadow and were preserved
in sealed glass containers. Microscopic characters were observed under a Motic SFC
28 microscope (Germany), measured using an ocular micrometer, and recorded by a
BCAM3 camera (Germany). e fresh sections of basidiomes were stained by Congo
red. Basidiospore dimensions were recorded without 5% of the largest and 5% of
the smallest measured values and Q indicates the length/width ratio of the spores.
Measurements of the other microscopic characters are given as ranges. e voucher
specimens have been deposited in the Fungarium at BUNS Herbaria, Department of
Biology and Ecology, University of Novi Sad, Novi Sad, Serbia (BUNS).
Molecular & phylogenetic analysis
Total DNA was extracted from dry specimens employing a modied protocol based
on Murray & ompson (1980). Amplication of ITS and LSU regions was performed
under following conditions: 2 min denaturation at 95°C, followed by 35 amplication
cycles, each consisting of 30 s denaturation at 95°C, a 30 s annealing step at 54°C,
and 1 min extension at 72°C; the nal step included 10 min incubation at 72°C. e
primers ITS1F and ITS4B (White & al. 1990, Gardes & Bruns 1993) were employed
toamplify the ITS rDNA region, while LR0R and LR5 (Gardes & Bruns 1993, Vilgalys

740 ... Lukić, Čapelja, Karaman
& Hester 1990) were used for the 28S rDNA region. PCR products were checked in 1%
agarose gels, and amplicons weresequenced with one or both PCR primers. Sequence
chromatograms were inspected and manually edited with FinchTV 1.5.0 (Geospiza,
Inc.; Seattle, WA, USA; http://www.geospiza.com). Newly generated ITS and LSU
sequences were deposited in GenBank.
Additional sequences were retrieved from GenBank or UNITE database: for ITS,
26 Tricholoma sequences from 15 taxa, with a Clitocybe outgroup sequence; and for
LSU, 19 Tricholoma sequences from 13 taxa, with a Ganoderma outgroup sequence. A
separate phylogenetic analysis was performed on each of the gene regions. Alignments
for both analyses were carried out with MUSCLE . (Edgar 2004) using the default
settings. Substitution models were selected using the ModelTest-NG v0.1.6 (Darriba
& al. 2020). GTR + I + G (General time reversible model + Proportion of invariant
+ Gamma) model was chosen for ITS datasets while K80+I+G (Kimura 2-parameter
model + Proportion of invariant + Gamma) was chosen for LSU dataset. Maximum
Likelihood (ML) analyses were performed in MEGA X (Kumar & al. 2018) with
the number of bootstraps set to 1000. MUSCLE .andModelTest-NG v0.1.6 were
used through CIPRES Science Gateway (Miller & al. 2010). e alignments and
phylogenetic trees were deposited in Treebase.
Taxonomy
Tricholoma punctatum Lukić, sp. nov. F 1–3
MB 
Basidiomes of T. punctatum dier by the presence of dark colored, elevated, circularly
arranged spots on the lobed gray pileus.
T: Serbia, Šumadija, Kragujevac, “Memorial complex of Sumarice”, 240 m a.s.l.,
on soil in mixed thermophilous deciduous forest (Quercus robur, Q. cerris, Carpinus
betulus), October 2020, Nebojša Lukić (Holotype, BUNS 12-00766; GenBank
OK631797, OL310859).
E: e specic epithet refers to the distinctly spotted surface of pilei.
P: 50–110 mm, at rst convex, becoming attened with broad, low umbo.
Fully developed pileus is oen irregularly circular, lobed, with wavy margin
(F. 1). Surface is densely felt, then breaking up in small ne scales. Pileipellis
can be separated easily by half of the pileus radius. Color is gray [N7/0-N4/0],
grey-brown [5YR6/4-5YR7/3] with lighter margin zone (F. 1 A–C). it is
characterized by punctate, almost black spots (more or less pronounced) placed
in one, usually two or even three circular zones at half and at 3/4 of the pileus
radius (F. 1 A, C, D). ese punctate black-colored spots are oen elevated
and can be removed by nail quite easily. L strongly sinuate (clearly
expressed, F. 1 B, C), rather broad, quite thick and deep, whitish [N9/0], then
pale grey [N7/0], not spotted. Intermediate lamellae numerous. e number of

Tricholoma punctatum sp. nov. (Serbia) ... 741
F. 1. Tricholoma punctatum: basidiocarps in situ.

742 ... Lukić, Čapelja, Karaman
those half-length lamellae is nearly equal to the number of ordinary lamellae.
Edges are entire and wavy. Color changes are not observed. S: 40–90 ×
10–20 mm, robust and rm, cylindrical but not so rare attened or with
vertical grooves, widened at apex, tapering at base, white on ends, rough and
covered for the most part with gray [N6/0] or gray-brown bers (F. 1, B, C).
No cobweb ring zone observed. Basal mycelium whitish. F: white then
faintly gray. O: clearly farinaceous, strong aer cutting. T: farinaceous
without bitter component.
B: 4.6–5.1 × 3.6–4.1 µm (60 spores, from 3 basidiomes),
subglobose to ellipsoid, Q = 1.20–1.35 (F. 2 A). B: 25–35 × 3.5–6µm,
mainly 4-spored (F. 2 B). P: a cutis, breaking upon trichoderm
scales (F. 2 C); individual hyphal elements generally 33–75 × 7–14µm;
subpellis poorly dierentiated; mainly globose inated cells 20-30 µm in
diameter (fully developed), present as separated clusters in punctate colored
elevated bers within pileipellis (F 2 and 3). C-: absent.
F. 2. Tricholoma punctatum (holotype BUNS 12-00766): A. basidiospores; B. basidia; C. pileipellis.

Tricholoma punctatum sp. nov. (Serbia) ... 743
F. 3. Tricholoma punctatum (holotype BUNS 12-00766): globose inated cells in punctate colored
areas within pileipellis.
A  : SERBIA, Š, Kragujevac, “Memorial
complex of Sumarice”, 240 m a.s.l., on soil in mixed thermophilous deciduous forest
(Quercus robur, Q. cerris, Carpinus betulus), October 2020, Nebojša Lukić (BUNS 12-
00782, GenBank ON598607)
H  : Basidiomes solitary or oen in small groups. Habitat is
a mixed thermophilous deciduous forest (Quercus robur, Q. cerris, Carpinus
betulus) overgrown with Crataegus monogyna, Prunus spinosa, and P. cerasifera,
on neutral or slightly acidic clayey soil.
Key to Tricholoma sect. Atrosquamosa Kühner & Romagn.
Cap greyish to almost black but also greyish-brown, densely squamulose-
brillose, stem oen squamulose too, smell dierent but farinaceous aer
cutting, spores medium sized.
1. Stem white, more or less shiny, oen with weakly expressed, scattered blackish
scales, smell of uncut fruit bodies sweetish ..................................................................... 2
1. Stem white to greyish, with a dense covering of darker brils (or scales), smell of
uncut fruit bodies spicy or farinaceous ............................................................................ 3
2. Basal mycelium yellow, cap mostly brillose, especially gills and cap margin
reddening when old, in deciduous forests, mostly Fagus and Quercus .... T. orirubens
2. Basal mycelium white; cap mostly with dark (black) scales, with conifers, but also
with Tilia and Corylus ........................................................................ T. atrosquamosum

744 ... Lukić, Čapelja, Karaman
3. In Picea forests (Fennoscandia), cap oen with an olivaceous tinge, especially at
margin .................................................................................................... T. olivaceotinctum
3 In thermophilous deciduous forests on clayey or calcareous soil, cap without
olivaceous tinge .................................................................................................................... 4
4. Stem less brillose or squamulose, widened stem base strongly reddening
............................................................................................................................ T. basirubens
4.Stem base not reddening ................................................................................................. 5
5. Cap margin woolly in young fruit bodies, cap strongly squamulose
...................................................................................................................... T. squarrulosum
5 Cap with conspicuous darker spots mainly arranged in two circular zones and
oen lobed, cap margin paler, spores mostly subglobose ........................ T. punctatum
Phylogenetic results
Newly generated ITS and LSU sequences of Tricholoma punctatum were
approximately 780 bp and 750 bp long, respectively. BLAST sequence analysis
showed maximum similarity with T. orirubens AMB 17410 (MT462641) for ITS
sequence, while LSU sequence of T. punctatum showed maximum sequence
match with T. sulphurescens G0243 (MK278617) and T. orirubens (MK278611)
with exactly the same scores. Phylogenetic analysis of ITS (F. 4) and LSU
(F. 5) datasets shows that ITS and LSU sequences of T. punctatum generated
in this study formed their own lineage within the clade corresponding to sect.
Atrosquamosa. In the analysis of ITS sequences T. punctatum was shown as
sister clade of T. orirubens, while in the LSU tree T. punctatum is placed within
the T. orirubens clade.
Discussion
Morphologically, the main distinguishing feature of Tricholoma punctatum is
the pileus appearance and the presence of the globose inated cells in punctate
black-colored are as in pileipellis (F. 1). ese characters are similar to those
of Tricholoma rufenum P. Donati which possesses drop-like spots on the pileus.
However, the pileus of T. rufenum is smooth, shiny and slightly greasy (similar
to T. portentosum (Fr.) Quél.) (Riva 1998). In addition, T. rufenum has bigger
spores (5.5–7.2 × 4.6–5.2 m; Riva 2003) than T. punctatum and a white stipe.
e drop-like spots are as smooth as the whole pileus surface, in contrast to
the elevated spots of T. punctatum. Similar smooth drop-like spots appear on
the pileus of T. viridilutescens M.M. Moser native to Europe. Similar pileus
appearance can be also found in the T. scalpturatum var. atrocinctum Romagn.
but the aforementioned feature is not constant (Heap 2020).

Tricholoma punctatum sp. nov. (Serbia) ... 745
F. 4. Maximum likelihood tree inferred from the ITS sequences of Tricholoma species, with a
Clitocybe avellaneoalba outgroup. Bootstrap values ≥50%, based on 1000 replicates are shown at
the branch nodes. e tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. is analysis involved 28 Tricholoma nucleotide sequences. ere were 914
positions in the nal dataset.
Besides the particular pileus appearance, T. punctatum shows
morphologically similar features with T. orirubens Quél., T. atrosquamosum
Sacc., T. squarrulosum Bres. and T. scalpturatum (Fr.) Quél.
Unlike T. punctatum, T. orirubens has greenish yellow basal mycelium
(Christensen & Heilmann-Clausen 2013), reddening lamellae (mainly in
mature stage), white stipe and slightly bigger (and more elongated) spores.
When compared with T. punctatum, T. atrosquamosum has dark gray to black
pileus (no brown shade; Riva 1988), blackish spotted gills, white, shiny stem
with blackish occules or green (or red) spots at base, dierent smell of pepper
(Baccardo & al. 2008), as well as distinct habitat and mycorrhizal partners
and bigger and more elongated spores (Courtecuisse 1999). Tricholoma
atrosquamosum is found in North America and Europe, but is generally

746 ... Lukić, Čapelja, Karaman
recognized as rare in Europe and in danger of extinction in the Netherlands
(Noordeloos & Christensen 1999). Basidiomes appear under deciduous and
coniferous trees, particularly beech and spruce on calcareous soils.
Tricholoma squarrulosum is smaller (Moser 1983, Phillips 2006), with
dense black squamules on pileus with whitish woolly margin and strongly
blackish squamulose stipe (Kibby 2010) while T. punctatum has grey brillose
stem. Tricholoma squarrulosum also has dierent smell and bigger and more
elongated spores (Q =1.35–1.72) (Christensen & Heilmann-Clausen 2013).
Tricholoma squarrulosum is also rare (though more widely distributed in
southern Europe), and associated with oak, pine, and spruce on chalk soils,
with its basidiomes appearing from September to November.
Furthermore, comparing to T. punctatum, the species T. scalpturatum is
smaller in size (Courtecuisse & Duhem 1995), with yellowing lamellae (Galli
2003), white or grey smooth stipe (sometimes with cobweb ring zone) and
more elongated spores (Q = 1.4–1.54) (Christensen & Heilmann-Clausen
2013). Likewise, it should be emphasized that new species T. punctatum usually
have lobed pileus and deep, strongly sinuate lamellae.
F. 5. Maximum likelihood tree inferred from the LSU sequences of Tricholoma taxa, with a
Ganoderma adspersum outgroup. Bootstrap values ≥50%, based on 1000 replicates are shown at
the branch nodes. e tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. is analysis involved 20 Tricholoma nucleotide sequences. ere were 762
positions in the nal dataset.

Tricholoma punctatum sp. nov. (Serbia) ... 747
Beside the morphological dierences between T. punctatum and related
species, the former species showed dierences on molecular level as well,
which can be clearly seen from the phylogenetic tree obtained in analysis
of ITS sequences (F. 4). T. punctatum sequence was grouped within the
Atrosquamosa clade and placed as sister clade of T. orirubens. Contrary to
this, in the LSU tree (F. 5) T. punctatum was placed within the T. orirubens
clade. ese results may be due to the fact that LSU is more conserved among
species and have less clearly dened barcode gap than ITS (Schoch & al. 2012).
Grouping of species in clades/sections in both analyses were in accordance
with data of Heilmann-Clausen & al. (2017).
Conclusion
According to the data presented, Tricholoma punctatum belongs to section
Atrosquamosa, which is one of ten proposed Tricholoma sections based on
molecular and morphological data (Christensen & Heilmann-Clausen 2013).
It is characterized by constant morphological features such are pileus color,
pileipellis structure, absence of clamp connections and spores size and shape. is
section is amongst those (sections Tricholoma, Atrosquamosa, Pardinicutis, and
Terrea ) which include species with a greyish, radially brillose, squamulose to
felty pileus (Christensen & Heilmann-Clausen 2013). e main distinguishing
characteristic of our new species is the presence of globose inated cells in
punctate areas of the pileus, and of the dark colored, elevated, circularly placed
(in one to three tiers) spots on the cap. In addition, other characteristics that
separate T. punctatum from similar species, T. orirubens, T. atrosquamosum, T.
squarrulosum, and T. scalpturatum, are the size and shape of spores, brillose
stipe, lobed pileus, strongly sinuate and deep gills. Furthermore, molecular
data based on newly generated ITS sequences provided evidence supporting
the distinct taxonomic status of T. punctatum.
Acknowledgments
e authors thank George Zervakis (Agricultural University of Athens, Greece)
and Katerina Rusevska (Faculty of Natural Science and Mathematics, Ss. Cyril and
Methodius University in Skopje, Macedonia) for presubmission review.
Literature cited
Baccardo F, Traverso M, Vizzini A, Zotti M. 2008. Funghi d’Italia. Bologna, Zanichelli.
Bessette AE, Bessette AR, Roody WC. 2013. Tricholomas of North America. University of Texas
Press, US.
Bon M. 1991. Flore Mycologique d’Europe, vol. 2: les Tricholomes & ressemblants. St Valerysur-
Somme, France.

748 ... Lukić, Čapelja, Karaman
Christensen M, Heilmann-Clausen J 2013. e genus Tricholoma. Fungi of Northern Europe, vol.
4. Copenhagen, Danish Mycological Society.
Čolić D. 1967. Sinekološka analiza ore gljiva u rezervatu sa pančićevom omorikom na Mitrovcu
(planina Tara). Zaštita Prirode 34: 389–505.
Courtecuisse R. 1999. Mushrooms of Britain & Europe. London, Harper Collins.
Courtecuisse R, Duhem B. 1995. Mushrooms & toadstools of Britain & Europe. London, Harper
Collins.
Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T. 2020. ModelTest-NG: a new and
scalable tool for the selection of DNA and protein evolutionary models. Molecular Biology and
Evolution, 37(1): 291–294. https://doi.org/10.1093/molbev/msz189
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Research 32(5): 1792–1797. https://doi.org/10.1093/nar/gkh340
Frøslev TG, Matheny PB, Hibbett D. 2005. Lower level relationships in the mushroom
genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS
phylogenies. Molecular Phylogenetics and Evolution 37: 602–618. https://doi.org/10.1016/j.
ympev.2005.06.016
Galli R. 2003. I Tricolomi, 2nd ed. Milano, Edinatura.
Gardes M, Bruns TD. 1993. ITS primers with enhanced specicity for Basidiomycetes —application
to the identication of mycorrhizae and rusts. Molecular Ecology 2: 113–118. https://doi.
org/10.1111/j.1365-294X.1993.tb00005.x
Heap J. 2020. An unusual variety of Tricholoma scalpturatum. Field Mycology 21(1): 24–25. https://
doi.org/10.1016/j.dmyc.2020.01.008
Heilmann-Clausen J, Christensen M, Frøslev TG, Kjøller R. 2017. Taxonomy of Tricholoma in
northern Europe based on ITS sequence data and morphological characters. Persoonia 38:
38–57. https://doi.org/10.3767/003158517X693174
Ivančević B, Davidović M. 2011. Makromicete na području Bojčinske šume i okvir zanjihovo
očuvanje: Zaštita Prirode 61(2): 21–33.
Karadžić D, Milijašević T. 2008.he most important parasitic and saprophytic fungi in austrian
pine and scots pine plantations in Serbia. Bulletin of the Faculty of Forestry 97: 147–170.
https://doi.org/10.2298/GSF0897147K
Karaman M, Matavulj M, Novaković M, Savić D. 2005. Gljive Fruške gore. 8. Simpozijum o Flori
Jugoistočne Srbije i Susednih Regiona, Niš, Jun. 2005. Zbornik Apstrakata: 22.
Kibby G. 2010. e genus Tricholoma in Britain. Field Mycology 11(4): 113–140. https://doi.
org/10.1016/j.dmyc.2010.10.004
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics
Analysis across computing platforms. Molecular Biology and Evolution 35: 1543–1549. https://
doi.org/10.1093/molbev/msy096
Lukić N. 2009. e distribution and diversity of Boletus genus in central Serbia. Kragujevac Journal
of Science, 31:59-68.
Lukić N. 2013. Rod Amanita u Srbiji. Gljivarsko društvo Šumadije, Kragujevac, Serbia
Moser M. 1983. Keys to agarics and boleti. Tonbridge, White Friars Press.
Munsell. 1975. Munsell soil color charts. Baltimore, Munsell Color.
Murray MG, ompson WF. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic
Acids Research 8(19): 4321-4325.https://doi.org/10.1093/nar/8.19.4321
Noordeloos ME, Christensen M. 1999. Tricholoma (Fr.) Staude. 107–148, in: C. Bas & al. (eds).
Flora Agaricina Neerlandica, vol. 4.

Tricholoma punctatum sp. nov. (Serbia) ... 749
Ovrebo CL, Hughes KW, Halling RE. 2019. ree new species of Tricholoma from Costa Rica.
Phytotaxa 392: 33–44. https://doi.org/10.11646/phytotaxa.392.1.3
Phillips R. 2006. Mushrooms. London, Macmillan.
Reschke K, Popa F, Yang ZL, Kost G. 2018. Diversity and taxonomy of Tricholoma species from
Yunnan, China, and notes on species from Europe and North America. Mycologia 110: 1081–
1109. https://doi.org/10.1080/00275514.2018.1512295
Riva A. 1988. Tricholoma (Fr.) Staude. Fungi Europaei, vol. 3. Alassio, Edizioni Candusso.
Riva A. 1998. Tricholoma (Fr.) Staude. Fungi Non Delineati, vol. 5. Alassio, Mykoora.
Riva A. 2003. Tricholoma (Fr.) Staude, supplemento. Fungi Europaei, vol. 3a. Alassio, Edizioni
Candusso.
Sadiković D, Čapelja E, Dašić M. 2012. Basidiomycetes of Temska village area (Eastern Serbia, Mt
Stara Planina). Biologica Nyssana 3: 91–96.
Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W & al. 2012.
Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker
for fungi. Proceedings of the National Academy of Sciences of the United States of America.
109: 6241–6246. https://doi.org/10.1073/pnas.1117018109
Tedersoo L, May TW, Smith ME. 2010. Ectomycorrhizal lifestyle in fungi: global diversity,
distribution, and evolution of phylogenetic lineages. Mycorrhiza 20: 217–263. https://doi.
org/10.1007/s00572-009-0274-x
Uzelac B. 2009. Gljive Srbije i zapadnog Balkana. Beograd, BGV Logik.
Vilgalys R, Hester M. 1990. Rapid genetic identication and mapping of enzymatically amplied
ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246.
https://doi.org/10.1128/jb.172.8.4238-4246.1990
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplication and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. 315–322, in: MA Innis & al. (eds). PCR protocols: a guide to
methods and applications. Academic Press, London. https://doi.org/10.1016/B978-0-12-
372180-8.50042-1