DNA Barcoding Data Reveal Important Overlooked Diversity of Cortinarius sensu lato (Agaricales, Basidiomycota) in the Romanian Carpathians

Abstract

Cortinarius s.l. is a globally distributed agaricoid genus that has been well studied in Europe with over 1000 described species. However, the information about their taxonomy and diversity in eastern Central Europe is still limited. Only 124 species have been reported so far from Romania, based solely on morphological observations. The aim of this study was to re-examine the diversity of the genus Cortinarius s.l. in the Romanian Carpathian area, employing molecular phylogenetic and morphological methods. During intensive field work in the period 2017–2020, a total of 234 Cortinarius s.l. specimens were collected and studied with integrative taxonomic methods. For all the samples, we amplified and sequenced the nrDNA ITS region, which is the widely used official barcode marker of fungi. These sequences were compared to the data found in public databases (GenBank, UNITE, BOLD). Based on phylogenetic analyses, we identified 109 Cortinarius s.l. species, which represent 40 sections and 3 clades. Out of these species, 43 have previously been documented from Romania based on morphological identification methods, while 66 species are reported as new to the country.

Full text

Citation: Szabó, E.; Dima, B.; Dénes,
A.L.; Papp, V.; Keresztes, L. DNA
Barcoding Data Reveal Important
Overlooked Diversity of Cortinarius
sensu lato (Agaricales,Basidiomycota)
in the Romanian Carpathians.
Diversity 2023,15, 553. https://
doi.org/10.3390/d15040553
Academic Editor:
Stephan Koblmüller
Received: 14 March 2023
Revised: 3 April 2023
Accepted: 9 April 2023
Published: 13 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
diversity
Article
DNA Barcoding Data Reveal Important Overlooked Diversity
of Cortinarius sensu lato (Agaricales,Basidiomycota) in the
Romanian Carpathians
Emerencia Szabó1,2,3,† , Bálint Dima 4, *,† , Avar L. Dénes 1, Viktor Papp 5and Lujza Keresztes 1,6
1Centre of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, University of
Babes
,-Bolyai Cluj-Napoca, Clinicilor 5-7, 400006 Cluj-Napoca, Romania; emerenciaszabo@gmail.com (E.S.);
avar.lehel@gmail.com (A.L.D.); keresztes2012@gmail.com (L.K.)
2
Doctoral School of Integrative Biology, Babe¸s-Bolyai University, Republicii 44, 400015 Cluj-Napoca, Romania
3Institute of Interdisciplinary Research in Bio-Nano-Sciences, Babes
,-Bolyai University,
Treboniu Laurian 42, 400271 Cluj-Napoca, Romania
4Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C,
H-1117 Budapest, Hungary
5Department of Botany, Institute of Agronomy, Hungarian University of Agriculture and Life Sciences,
Villányi út 29-43, H-1118 Budapest, Hungary; papp.viktor@uni-mate.hu or agaricum@gmail.com
6Hungarian Department of Biology and Ecology, Faculty of Biology and Geology, University of Babes
,-Bolyai
Cluj-Napoca, Clinicilor 5-7, 400006 Cluj-Napoca, Romania
*Correspondence: cortinarius1@gmail.com
† These authors contributed equally to this work.
Abstract:
Cortinarius s.l. is a globally distributed agaricoid genus that has been well studied in
Europe with over 1000 described species. However, the information about their taxonomy and
diversity in eastern Central Europe is still limited. Only 124 species have been reported so far from
Romania, based solely on morphological observations. The aim of this study was to re-examine
the diversity of the genus Cortinarius s.l. in the Romanian Carpathian area, employing molecular
phylogenetic and morphological methods. During intensive field work in the period 2017–2020, a total
of
234 Cortinarius
s.l. specimens were collected and studied with integrative taxonomic methods. For
all the samples, we amplified and sequenced the nrDNA ITS region, which is the widely used official
barcode marker of fungi. These sequences were compared to the data found in public databases
(GenBank, UNITE, BOLD). Based on phylogenetic analyses, we identified 109 Cortinarius s.l. species,
which represent 40 sections and 3 clades. Out of these species, 43 have previously been documented
from Romania based on morphological identification methods, while 66 species are reported as new
to the country.
Keywords: Eastern Europe; MrBayes; nrDNA ITS; RAxML; phylogeny; revision; taxonomy
1. Introduction
Cortinarius (Pers.) Gray s.l. (sensu lato) has been considered to be the biggest genus of
the order Agaricales [
1
], with a cosmopolitan distribution of over 3000 described species [
2
].
Cortinarius species are important ectomycorrhizal fungi (EcM) associated with differ-
ent trees and shrubs, belonging to the order Fagales, families Caesalpiniaceae,Cistaceae,
Dipterocarpaceae
,Myrtaceae,Pinaceae,Polygonaceae,Rhamnaceae,Rosaceae and Salicaceae as
well as a few herbaceous plants in the Cyperaceae. Because of their EcM nutritional mode,
they also play a key role in carbon cycling, especially in boreal forests. Several groups
have narrow ecological preferences, and they are sensitive to environmental changes; there-
fore, some species have been used as indicators for valuable natural environments [
3
].
Species belonging to Cortinarius have a highly variable appearance, from mycenoid to
tricholomatoid basidiomata. Their color can be uniformly brown or colorful, and the
Diversity 2023,15, 553. https://doi.org/10.3390/d15040553 https://www.mdpi.com/journal/diversity

Diversity 2023,15, 553 2 of 26
surface of the pilei is dry, fibrillose, silky, squamose or viscid [
4
]. Another common feature
of Cortinarius is a cobweb-like partial veil, the usually rusty-brown spore print and the
lack of a germ pore and perisporium [
5
]. In the beginning of the 21st century, molec-
ular phylogenetic studies showed that the genus also contains several species (mainly
extra-European) with sequestrate fruiting body forms [
6
]. According to IndexFungorum
(http://www.indexfungorum.org, accessed on 7 March 2023), 5819 Cortinarius names have
been published worldwide (accessed 2 March 2023). However, this number includes all
infrageneric taxa, and still the number of existing species names are estimated to be over
5000. Many of these names were inconsistently used in the literature, and there is also a
high number of synonyms. One of the most challenging parts of Cortinarius taxonomy is to
examine which species have already been described [3].
Since the early 19th century, many researchers have focused on the genus Cortinarius
in Europe, e.g., [
7
–
9
]. From the 20th century onwards, several fundamental morphological
works were published from Europe, e.g., [
10
–
18
]. From the late 1980s, two taxonomic
schools have spread in Europe, representing the South European (especially French, Italian
and Spanish) and the Scandinavian directions. During this period, the Atlas des Corti-
naires [
19
–
45
] described approximately 1500 new species, while the more conservative
species concept of the Scandinavian school, the Cortinarius, Flora Photographica [
46
–
49
],
dealt with only 300 species. The introduction of molecular tools in addition to the macro-
and micromorphological character-based identification led to a more precise and reliable
classification and helped avoid misunderstandings in the taxonomy and nomenclature
of the genus. Northern Europe became one of the most investigated and well-studied
regions [
4
,
50
–
63
], but in the last few decades, several other studies focused also on the
Central European region [
64
–
70
]; however, the information from this region is still lim-
ited. Similarly, there is a lack of information regarding the southern parts of Europe [
3
],
with only a few papers [
71
–
73
] published from the Mediterranean area so far. Depending
on the identification and classification concepts, the genus Cortinarius s.l. was separated
into several subgenera by different authors. Moser [
15
] named seven subgenera, while
Moënne-Loccoz et al. [
19
,
20
] and Bidaud et al. [
25
,
26
] mentioned six subgenera in their
works. Scandinavian researchers [
46
–
49
] separated the genus into five subgenera. In the
molecular era, phylogenetic analyses showed that these subgenera are mostly artificial,
e.g., [
3
,
70
]. New infrageneric classifications of Cortinarius based on multigene phylogenetic
analyses were proposed by Garnica et al. [
74
] and Soop et al. [
75
]. Recently, Liimatainen
et al. [
76
] has split the genus Cortinarius into 10 genera based on genomic and multi-gene
sequence data. This new classification, however, is not adopted in the current study, due to
practical reasons.
Fungal research in Romania has received increasing interest since 1950, leading to
8727 reported
fungal species [
77
,
78
], including both macro- and microfungi. Although
there are a number of publications that mention the genus Cortinarius and provide scarce
species lists from few areas in the Carpathians [79–89], systematic study that only focuses
on this genus is still lacking. Based on an extensive literature search, we know about
124 reported
Cortinarius species from Romania, identified based only on their morpho-
logical characteristics. This is a relatively low number when compared to the more than
1000 known European Cortinarius species [90].
Using solely morphological characteristics to identify Cortinarius species is very chal-
lenging and needs a lot of experience and solid knowledge. During the development of
the basidiocarps, several characteristics may change significantly and overlap with other
species, i.e., their intraspecific variability is high. Micromorphological characteristics, such
as the size, shape or ornamentation of the basidiospores, also play an important role in
the identification process, e.g., [
4
]. Nonetheless, DNA-sequence-based analyses (e.g., DNA
barcoding) are the most reliable identification methods to date, allowing the investigation
of the differences between species and varieties too. The nrDNA ITS (Internal Transcribed
Spacer) region has been proposed and started to be widely used in molecular taxonomy
as the universal and official barcoding region for fungi [
91
]. ITS is composed of three

Diversity 2023,15, 553 3 of 26
subregions: ITS1, 5.8S and ITS2, of which the ITS1 and ITS2 spacers show higher evolu-
tionary rates than the 5.8S; therefore, these are the best suited for studying interspecific
level differences [
92
]. This region evolved relatively rapidly compared to the mitochondrial
genes; therefore, in the case of Cortinarius too, it is useful and widely used for species
identification purposes [3,56,74,93,94].
To study fresh material, we based our study mainly on sampling in the Apuseni
Mts, which is a prominent forested area of Romania [
95
] and located in northern part of
the Western Romanian Carpathians. The mountain range is regarded as an important
biogeographical area between the Pannonian Plain and the Transylvanian Plateau as well
as in the Southern and Eastern Carpathians. Due to its location, several biogeographical
regions are present in the area (e.g., alpine, arctic, Mediterranean, etc.), which has resulted
in it being one of the regions in Europe with high biodiversity [96]. Several suitable forest
types and hot spots for Cortinarius are represented in the area, both on calcareous and
siliceous bedrocks. The forest vegetation is dominated by oak (Quercus petraea agg.), mixed
hornbeam–beech (Carpinus betulus,Fagus sylvatica), beech (F. syvatica), mixed beech and
spruce (F. sylvatica,Picea abies) and spruce (P. abies) forests [
97
]. Additionally, we also
extended our sampling to some localities in the Eastern Carpathians (
T
,
inutul Sării, Baraolt
and Liban).
Due to the limited knowledge and the lack of molecular genetic data of the genus
Cortinarius in Romania, the aims of this study were to make a pilot revision of the diversity,
taxonomy and distribution of Cortinarius s.l. species, using DNA barcoding combined with
morphological methods, and update the list of species of this important ectomycorrhizal
genus.
2. Materials and Methods
2.1. Taxon Sampling
For our pilot survey, samples were collected from sites including the main habitat types,
characteristic for the Transylvanian part of Romania, such as broad-leaved forests (40%),
coniferous forests (35%), mixed forests (11%) and other habitat types (14%; e.g., transitional
woodland–shrub, pastures, natural grasslands). The collections were made from 2017 to the
autumn of 2020, with the professional collaboration of K. Babos, L. Bartha, G. Bélfenyéri, A.
Dénes, R. Erös, K. Fehér, L. Gál, B. Jancsó, K. Macalik and Cs. Szabó.
A total of 234 Cortinarius samples were collected (Table 1). Every sample has a unique
code (e.g., C001, where the C means the first letter of the genus name), and in every
case the collection coordinates were recorded. The coordinates were registered in the
decimal degrees (DD) format, with six decimals (Table 1). The majority of the samples were
photographed in the field (Figure 1), and gross morphology was noted. All samples were
dried with a dehydrator at 45
◦
C. Samples were deposited at the Faculty of Biology and
Geology, Babes
,-Bolyai University, Cluj-Napoca, Romania.
Table 1.
Metadata of Cortinarius samples studied and identified during this work. All sequences are
newly generated.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. alboviolaceus C213 04.10.2020 Sovata 46.662003 25.215961 OP099671
C. anfractoides C361 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099771
C. anfractoides C363 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099773
C. anomalus C145 30.08.2020 Liban 46.552611 25.525509 OP099625
C. anomalus C160 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099635
C. anomalus C324 07.11.2020 Cheile Somes
,ului Cald 46.67071058 22.81810123 OP099745

Diversity 2023,15, 553 4 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. anomalus C159 * 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP132853
C. aureopulverulentus
C338 07.11.2020 Cheile Somes
,ului Cald 46.62928894 22.7806518 OP099756
C. balteatocumatilis C290 01.11.2020 Chinteni 46.895154 23.520302 OP099723
C. balteatocumatilis C291 01.11.2020 Chinteni 46.895154 23.520302 OP099724
C. balteatus C103 10.07.2020 Zetea 46.4733373 25.3454161 OP099607
C. bergeronii C223 24.10.2020 Cheile Vârghi¸sului 46.183191 25.590322 OP099680
C. brunneus C246A 24.10.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099695
C. bulliardii C359 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099769
C. caesiophylloides C009 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099571
C. calochrous C221 24.10.2020 Cheile Vârghi¸sului 46.205054 25.558222 OP099678
C. calochrous C233 24.10.2020 Cheile Vârghi¸sului 46.203207 25.559007 OP099685
C. camphoratus C097 19.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099603
C. camphoratus C153 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099630
C. camphoratus C202 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099661
C. caninus C040 22.09.2018 Valea Ierii 46.524239 23.275145 OP099579
C. caninus C146 30.08.2020 Liban 46.552103 25.524967 OP099626
C. caninus C149 03.09.2020 Mărtinis
,46.2647 25.355 OP099627
C. caninus C189 04.10.2020 Chiheru de Jos 46.686903 25.040178 OP099648
C. caninus C192 04.10.2020 Pădurea Bunet
,ilor 46.081617 25.034936 OP099651
C. caninus C193 04.10.2020 Pădurea Bunet
,ilor 46.081617 25.034936 OP099652
C. caninus C194 04.10.2020 Pădurea Bunet
,ilor 46.081617 25.034936 OP099653
C. caninus C199 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099658
C. caninus C200 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099659
C. caninus C206 04.10.2020 Chiheru de Jos 46.681697 25.035186 OP099665
C. caninus C207 04.10.2020 Chiheru de Jos 46.681697 25.035186 OP099666
C. caninus C255 01.11.2020 Harghita 46.538506 25.612982 OP099701
C. caperatus C074 13.10.2019 Statiunea Muntele
Baisoara 46.522375 23.273463 OP099594
C. caperatus C092 19.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099598
C. caperatus C093 26.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099599
C. caperatus C120 28.07.2020 Cheile Somesului Cald 46.626862 22.788439 OP099617
C. caperatus C143 * 08.08.2020 Statiunea Muntele
Baisoara 46.53262527 23.28001233 OP132852
C. catharinae C089 02.12.2019 Făgetul Clujului 46.735133 23.539162 OP099596
C. cinereobrunneolus C343 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099759
C. cinnamomeus C105 10.07.2020 Zetea 46.4733373 25.34546 OP099608

Diversity 2023,15, 553 5 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. claricolor C094 26.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099600
C. collinitus C003 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099565
C. collinitus C011 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099573
C. collinitus C045 22.09.2018 Valea Ierii 46.525571 23.274678 OP099580
C. collinitus C048 22.09.2018 Valea Ierii 46.524456 23.274586 OP099583
C. collocandoides C349 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099760
C. colymbadinus C114 23.06.2020 Liban 46.553983 25.520218 OP099613
C. corrosus C263 01.11.2020 Ghimes 46.528277 26.033167 OP099704
C. corrosus C273 01.11.2020 Ghimes 46.528277 26.033167 OP099710
C. croceus C108 * 23.06.2020 Liban 46.553983 25.520218 OP132850
C. croceus C115 23.06.2020 Liban 46.553983 25.520218 OP099614
C. croceus C116 23.06.2020 Liban 46.553983 25.520218 OP099615
C. croceus C124 28.07.2020 Cheile Somesului Cald 46.6342004 22.7476044 OP099618
C. daulnoyae C175 18.10.2020 Făgetul Clujului 46.738306 23.539194 OP099639
C. daulnoyae C176 18.10.2020 Făgetul Clujului 46.738306 23.539194 OP099640
C. daulnoyae C286 01.11.2020 Chinteni 46.895154 23.520302 OP099720
C. daulnoyae C298 * 01.11.2020 Făgetul Clujului 46.732552 23.543583 OP142445
C. daulnoyae C307 01.11.2020 Făgetul Clujului 46.729857 23.254839 OP099735
C. daulnoyae C310 01.11.2020 Făgetul Clujului 46.734696 23.540594 OP099737
C. daulnoyae C360 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099770
C. delibutus C253 01.11.2020 Harghita 46.538506 25.612982 OP099699
C. delibutus C254 01.11.2020 Harghita 46.538506 25.612982 OP099700
C. aff. delibutus C110 23.06.2020 Liban 46.553983 25.520218 OP099612
C. elatior C352 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099762
C. elegantior C322 07.11.2020 Cheile Somes
,ului Cald 46.67071058 22.81810123 OP099743
C. elegantissimus C241 24.10.2020 Cheile Vârghi¸sului 46.201743 25.560039 OP099690
C. eliae C293 01.11.2020 Chinteni 46.895154 23.520302 OP099725
C. eliae C294 01.11.2020 Chinteni 46.895154 23.520302 OP099726
C. eliae C295 01.11.2020 Chinteni 46.895154 23.520302 OP099727
C. fraudulosus C257 01.11.2020 Harghita 46.538506 25.612982 OP099702
C. fulminoides C334 07.11.2020 Cheile Somes
,ului Cald 46.64369044 22.7320642 OP099753
C. gallurae C379 21.10.2020 Bătarci 48.014053 23.146755 OP099780
C. geniculatus C357 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099767
C. gentilis C051 22.09.2018 Valea Ierii 46.524888 23.274323 OP099585
C. glaucopus C012 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099574
C. glaucopus C248 02.11.2020 Mănăstireni 46.792118 23.153491 OP099697
C. glaucopus C328 07.11.2020 Cheile Somes
,ului Cald 46.64377853 22.73244046 OP099748

Diversity 2023,15, 553 6 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. glaucopus C329 07.11.2020 Cheile Somes
,ului Cald 46.64377853 22.73244046 OP099749
C. glaucopus C332 07.11.2020 Cheile Somes
,ului Cald 46.64369044 22.7320642 OP099751
C. glaucopus C335 07.11.2020 Cheile Somes
,ului Cald 46.64420997 22.72738871 OP099754
C. aff. glaucopus C326 07.11.2020 Cheile Somes
,ului Cald 46.6310984 22.76807258 OP099746
C. aff. glaucopus C331 07.11.2020 Cheile Somes
,ului Cald 46.64369044 22.7320642 OP099750
C. aff. glaucopus C341 * 07.11.2020 Cheile Somes
,ului Cald 46.64060986 22.81455206 OP132857
C. hadrocroceus C106 10.07.2020 Zetea 46.4733373 25.34546 OP099609
C. hadrocroceus C140 08.08.2020 Statiunea Muntele
Baisoara 46.51092527 23.27111959 OP099621
C. hillieri C171 18.10.2020 Făgetul Clujului 46.738306 23.539194 OP099637
C. hinnuleus C377 21.10.2020 Bătarci 48.014053 23.146755 OP099779
C. holoxanthus C046 22.09.2018 Valea Ierii 46.524456 23.274586 OP099581
C. holoxanthus C052 22.09.2018 Valea Ierii 46.524888 23.274323 OP099586
C. holoxanthus C095 19.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099601
C. huronensis C016 15.08.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099575
C. huronensis C021 15.08.2018 Statiunea Muntele
Baisoara 46.537253 23.305079 OP099578
C. huronensis C151 03.09.2020 Mărtinis
,46.2647 25.355 OP099629
C.
hydrotelamonioides C205 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099664
C. incognitus C004 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099566
C. lacustris C387 21.10.2020 Bătarci 48.014053 23.146755 OP099783
C. largus C232 24.10.2020 Cheile Vârghi¸sului 46.199804 25.583554 OP099684
C. largus C242 24.10.2020 Cheile Vârghi¸sului 46.630342 25.835442 OP099691
C. largus C385 * 21.10.2020 Bătarci 48.014053 23.146755 OP132858
C. leproleptopus C356 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099766
C. lilacinovelatus C305 01.11.2020 Făgetul Clujului 46.729734 23.548487 OP099734
C. luridus C390 21.10.2020 Bătarci 48.014053 23.146755 OP099785
C. luridus C123 * 28.07.2020 Cheile Somesului Cald 46.643648 22.7281299 OP132851
C. luridus C289 * 01.11.2020 Chinteni 46.895154 23.520302 OP132856
C. masseei C388 21.10.2020 Bătarci 48.014053 23.146755 OP099784
C. aff. magicus C285 01.11.2020 Chinteni 46.895154 23.520302 OP099719
C. aff. magicus C303 01.11.2020 Făgetul Clujului 46.729599 23.548782 OP099732
C. multiformis C001 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099564
C. multiformis C008 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099570
C. multiformis C138 08.08.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099619

Diversity 2023,15, 553 7 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. multiformis C144 08.08.2020 Statiunea Muntele
Baisoara 46.50358372 23.264886235 OP099624
C. multiformis C181 04.10.2020 Sovata 46.699756 25.173806 OP099642
C. multiformis C184 04.10.2020 Sovata 46.699756 25.173806 OP099645
C. multiformis C096 19.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099602
C. multiformis C006 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099568
C. napus C214 04.10.2020 Sovata 46.662003 25.215961 OP099672
C. neofurvolaesus C047 22.09.2018 Valea Ierii 46.524456 23.274586 OP099582
C. neofurvolaesus C049 22.09.2018 Valea Ierii 46.524456 23.274586 OP099584
C. ochraceopallescens C055 22.09.2018 Valea Ierii 46.524076 23.274521 OP099587
C. ochraceopallescens C056 18.11.2018 Făgetul Clujului 46.720612 23.5606 OP099588
C. ochraceopallescens C283 01.11.2020 Făgetul Clujului 46.717317 23.536817 OP099717
C. odoratus C284 01.11.2020 Borsa 46.951797 23.60519 OP099718
C. odoratus C299 01.11.2020 Făgetul Clujului 46.734583 23.543577 OP099730
C. odoratus C304 01.11.2020 Făgetul Clujului 46.729743 23.54856 OP099733
C. olearioides C354 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099764
C. olearioides C355 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099765
C. olidoamarus C288 01.11.2020 Chinteni 46.895154 23.520302 OP099722
C. olidoamarus C296 01.11.2020 Chinteni 46.895154 23.520302 OP099728
C. olidoamarus C381 21.10.2020 Bătarci 48.014053 23.146755 OP099781
C. ominosus C182 04.10.2020 Sovata 46.699756 25.173806 OP099643
C. pallidostriatus C315 06.11.2020 Cheile Somes
,ului Cald 46.67023386 22.81820682 OP099741
C. pelerinii C219 * 24.10.2020 Brădut
,46.201631 25.598088 OP132854
C. persoonianus C174 18.10.2020 Făgetul Clujului 46.738306 23.539194 OP099638
C. pilatii C070 13.10.2019 Statiunea Muntele
Baisoara 46.6776035 23.4564326 OP099591
C. pruinatus C391 21.10.2020 Bătarci 48.014053 23.146755 OP099786
C. pseudodaulnoyae C311 01.11.2020 Almas
,u 46.869822 23.146055 OP099738
C. pseudodaulnoyae C386 21.10.2020 Bătarci 48.014053 23.146755 OP099782
C. pseudofervidus C142 08.08.2020 Statiunea Muntele
Baisoara 46.5113753 23.27106945 OP099623
C. pseudofervidus C141 08.08.2020 Statiunea Muntele
Baisoara 46.51101007 23.277106504 OP099622
C. pseudonaevosus C007 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099569
C. purpurascens C208 04.10.2020 Sovata 46.662003 25.215961 OP099667
C. purpurascens C327 07.11.2020 Cheile Somes
,ului Cald 46.63110059 22.76854548 OP099747
C. purpurascens C367 18.11.2020 Romuli 47.560767 24.530413 OP099775
C. purpurascens C370 19.11.2020 Parva 47.44473 24.64913 OP099777
C. purpurascens C371 20.11.2020 Rebris
,oara 47.44884 24.58896 OP099778

Diversity 2023,15, 553 8 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. radicosissimus C313A 06.11.2020 Cheile Somes
,ului Cald 46.67023386 22.81820682 OP099740
C. renidens C019 15.08.2018 Statiunea Muntele
Baisoara 46.538338 23.305955 OP099577
C. renidens C109 23.06.2020 Liban 46.553983 25.520218 OP099611
C. renidens C119 28.07.2020 Cheile Somesului Cald 46.626862 22.788439 OP099616
C. rubellus C098 05.08.2017 Ponok 46.638228 22.815111 OP099604
C. rubricosus C281 01.11.2020 Făgetul Clujului 46.717317 23.536817 OP099715
C. rubrophyllus C102 10.07.2020 Zetea 46.56182 25.3733821 OP099606
C. rufoallutus C005 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099567
C. saginus C150 03.09.2020 Mărtinis
,46.2647 25.355 OP099628
C. salor C297 01.11.2020 Rediu 46.733722 26.537746 OP099729
C. saporatus C369 19.11.2020 Parva 47.440112 24.650527 OP099776
C. scaurocaninus C222 24.10.2020 Cheile Vârghi¸sului 46.203991 25.558544 OP099679
C. scaurocaninus C230 24.10.2020 Cheile Vârghi¸sului 46.20322 25.559007 OP099683
C. scaurocaninus C239 24.10.2020 Cheile Vârghi¸sului 46.205054 25.558222 OP099688
C. scaurocaninus C240 24.10.2020 Cheile Vârghi¸sului 46.215552 25.5473 OP099689
C. semisanguineus C010 27.07.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099572
C. semivelatus C350 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099761
C. sodagnitus C280 01.11.2020 Făgetul Clujului 46.717317 23.536817 OP099714
C. sodagnitus C282 01.11.2020 Făgetul Clujului 46.717317 23.536817 OP099716
C. spadicellus C154 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099631
C. spadicellus C155 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099632
C. spadicellus C157 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099633
C. spadicellus C158 10.10.2020 Statiunea Stana de Vale 46.697647 22.626014 OP099634
C. spadicellus C211 04.10.2020 Sovata 46.662003 25.215961 OP099670
C. spilomeus C196 04.10.2020 Chiheru de Jos 46.672231 25.051561 OP099655
C. subargyronotus C358 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099768
C. subdecolorans C229 24.10.2020 Cheile Vârghi¸sului 46.205947 25.557701 OP099682
C. subfoetens C333 07.11.2020 Cheile Somes
,ului Cald 46.64369044 22.7320642 OP099752
C. aff. sublilacinopes C309 01.11.2020 Făgetul Clujului 46.738275 23.537126 OP099736
C. subparvannulatus C071 13.10.2019 Statiunea Muntele
Baisoara 46.6776035 23.4564326 OP099592
C. subporphyropus C178 10.10.2020 Gros
,ii T
,ibles
,ului 47.52174 24.15647 OP099641
C. subpurpurascens C091 30.11.2019 Făgetul Clujului 46.698086 23.587791 OP099597
C. subpurpurascens C195 04.10.2020 Chiheru de Jos 46.672231 25.051561 OP099654
C. subpurpurascens C216 19.10.2020 Feleacu 46.695459 23.58806 OP099674
C. subpurpurascens C217 19.10.2020 Feleacu 46.695459 23.58806 OP099675
C. subpurpurascens C237 24.10.2020 Cheile Vârghi¸sului 46.199842 25.583542 OP099686

Diversity 2023,15, 553 9 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. subtortus C017 15.08.2018 Statiunea Muntele
Baisoara 46.5382 23.3071099 OP099610
C. subtortus C075 13.10.2019 Statiunea Muntele
Baisoara 46.522375 23.273463 OP099595
C. subtortus C099 19.08.2017 Statiunea Muntele
Baisoara 46.520139 23.271811 OP099605
C. subtortus C139 08.08.2020 Statiunea Muntele
Baisoara 46.50358372 23.262648623 OP099620
C. subtortus C243 24.10.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099692
C. subtortus C245 24.10.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099694
C. subtortus C247 24.10.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099696
C. subtortus C073 13.10.2019 Statiunea Muntele
Baisoara 46.522375 23.273463 OP099593
C. sulphurinus C318 06.11.2020 Cheile Somes
,ului Cald 46.67071058 22.81810123 OP099742
C. talimultiformis C185 04.10.2020 Sovata 46.699756 25.173806 OP099646
C. talimultiformis C201 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099660
C. talimultiformis C312 06.11.2020 Cheile Somes
,ului Cald 46.67023386 22.81820682 OP099739
C. talimultiformis C323 07.11.2020 Cheile Somes
,ului Cald 46.67071058 22.81810123 OP099744
C. testaceomicaceus C342 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099758
C. tirolianus C218 24.10.2020 Cheile Vârghi¸sului 46.204486 25.558374 OP099676
C. tirolianus C259 * 01.11.2020 Harghita 46.538506 25.612982 OP132855
C. tirolianus C268 01.11.2020 Ghimes 46.528277 26.033167 OP099707
C. tirolianus C269 01.11.2020 Ghimes 46.528277 26.033167 OP099708
C. tirolianus C276 01.11.2020 Ghimes 46.528277 26.033167 OP099712
C. tirolianus C277 01.11.2020 Ghimes 46.528277 26.033167 OP099713
C. torvus C353 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099763
C. traganus C183 04.10.2020 Sovata 46.699756 25.173806 OP099644
C. traganus C210A 04.10.2020 Sovata 46.662003 25.215961 OP099669
C. traganus C244 24.10.2020 Statiunea Muntele
Baisoara 46.50627098 23.26640838 OP099693
C. trivialis C220 24.10.2020 Brădut
,46.201631 25.598088 OP099677
C. trivialis C366 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099774
C. aff. trivialis C225 24.10.2020 Cheile Vârghi¸sului 46.203991 25.5585445 OP099681
C. aff. trivialis C362 07.11.2020 Făgetul Clujului 46.6993848 23.5488839 OP099772
C. turgidus C064 04.10.2019 Valea Bratcutei 46.886256 22.58647 OP099590
C. turgidus C163 19.10.2020 Făgetul Clujului 46.738306 23.539194 OP099636
C. turmalis C203 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099662
C. turmalis C209 04.10.2020 Sovata 46.662003 25.215961 OP099668
C. turmalis C215 19.10.2020 Feleacu 46.695459 23.58806 OP099673
C. ultrodistortus C107 10.07.2020 Zetea 46.4733373 25.34546 OP099610

Diversity 2023,15, 553 10 of 26
Table 1. Cont.
Species DNA-
Codes
Collection
Date Location Latitude Longitude GenBank
acc. no.
C. uraceonemoralis C238 24.10.2020 Cheile Vârghi¸sului 46.199056 25.573537 OP099687
C. variicolor C057 06.09.2019 Demsus 45.5623456 22.700035 OP099589
C. variicolor C186 04.10.2020 Chiheru de Jos 46.686903 25.040178 OP099647
C. variicolor C197 04.10.2020 Chiheru de Jos 46.672231 25.051561 OP099656
C. variicolor C198 04.10.2020 Chiheru de Jos 46.672231 25.051561 OP099657
C. variicolor C204 04.10.2020 Chiheru de Jos 46.679428 25.035528 OP099663
C. varius C275 01.11.2020 Ghimes 46.528277 26.033167 OP099711
C. varius C337 07.11.2020 Cheile Somes
,ului Cald 46.62928894 22.7806518 OP099755
C. varius C340 07.11.2020 Cheile Somes
,ului Cald 46.62928894 22.7806518 OP099757
C. venetus C251 01.11.2020 Harghita 46.538506 25.612982 OP099698
C. venetus C260 01.11.2020 Harghita 46.538506 25.612982 OP099703
C. venetus C265 01.11.2020 Ghimes 46.528277 26.033167 OP099705
C. venetus C266 01.11.2020 Ghimes 46.528277 26.033167 OP099706
C. venetus C271 01.11.2020 Ghimes 46.528277 26.033167 OP099709
C. aff. vibratilis C188 04.10.2020 Chiheru de Jos 46.686903 25.040178 ON832643
C. violaceus C190 04.10.2020 Chiheru de Jos 46.686903 25.040178 OP099649
C. violaceus C191A 04.10.2020 Pădurea Bunet
,ilor 46.081617 25.034936 OP099650
C. xanthochlorus C287 01.11.2020 Chinteni 46.895154 23.520302 OP099721
C. xanthochlorus C301 01.11.2020 Făgetul Clujului 46.729576 23.548556 OP099731
* Short sequences excluded from the phylogenetic analyses.
Diversity 2023, 15, x FOR PEER REVIEW 9 of 24
Figure 1. Basidiomata of some Cortinarius s.l. species new to Romania: (A,B) C. catharinae C089 (sect.
Calochroi), (C) C. daulnoyae C298 (sect. Phlegmacioides), (D) C. fulminoides C334 (sect. Aureocistophili),
(E) C. hadrocroceus C140 (sect. Dermocybe), (F) C. lacustris C387 (sect. Hinnulei), (G,H) C. masseei C388
(sect. Obtusi), (I) C. subdecolorans C229 (singleton), (J) C. subfoetens C333 (sect. Glaucopodes), Photos.
(A,C,E,F,I,J) E. Szabó; (B,D) A. Dénes, (G,H) K. Babos and K. Fehér.
Table 2. Cortinarius species discovered new to Romania. Infrageneric classification, habitat types as
well as basidiospore measurements and Q values are given for each species. BLf–broad-leaved for-
est; Cf–coniferous forest; Mf–mixed forest; P–pastures; TWS–transitional woodland–shrub; NG–
natural grasslands.
Species Section/Clade Habitat
No. of Collected
Samples Basidiospores
Cortinarius anfractoides Rob. Henry and Trescol 1987 Infracti BLf 2 8.8–9.5 × 7.2–7.4 µm
Q = 1.2–1.3
Cortinarius aureopulverulentus M.M. Moser 1952 Calochroi Cf 1 12.4–13.1 × 8.0–8.7 µm
Q = 1.5–1.6
Cortinarius balteatocumatilis Rob. Henry 1939 Phlegmacioides P 2 8.4–9.2 × 5.2–5.9 µm
Q = 1.5–1.7
Cortinarius brunneus (Pers.) Fr. 1838 Brunnei Cf 1 8.2–9.5 × 6.1–6.8 µm
Q = 1.2–1.5
Cortinarius caesiophylloides Kytöv., Liimat., Niskanen,
Brandrud and Frøslev 2014 Multiformes Cf 1 10.4–11.0 × 6.2–6.6 µm
Q = 1.57–1.73
Cortinarius catharinae Consiglio 1997 Calochroi BLf 1 9.7–10.5 × 5.8–6.3 µm
Q = 1.6–1.8
Cortinarius cinereobrunneolus Chevassut and Rob.
Henry 1982 Urbici BLf 1 7.7–8.4 × 5.0–5.2 µm
Q = 1.5–1.7
Figure 1.
Basidiomata of some Cortinarius s.l. species new to Romania: (
A
,
B
)C. catharinae C089 (sect.
Calochroi), (
C
)C. daulnoyae C298 (sect. Phlegmacioides), (
D
)C. fulminoides C334 (sect. Aureocistophili),
(
E
)C. hadrocroceus C140 (sect. Dermocybe), (
F
)C. lacustris C387 (sect. Hinnulei), (
G
,
H
)C. masseei C388
(sect. Obtusi), (
I
)C. subdecolorans C229 (singleton), (
J
)C. subfoetens C333 (sect. Glaucopodes), Photos.
(A,C,E,F,I,J) E. Szabó; (B,D) A. Dénes, (G,H) K. Babos and K. Fehér.

Diversity 2023,15, 553 11 of 26
2.2. Micromorphological Study
We studied the basidiospores of our collections (Figure 2). In general, the size of the
spores are 5–15
×
3–8
µ
m. Basidiospores were studied in 3% KOH or Melzer’s reagent,
the latter to observe the dextrinoid reaction of the spore wall in some groups [
98
]. The
analyses were performed with an Olympus CX23 microscope with 1000
×
magnification
using immersion oil and an oil immersion lens. The photographs were taken with a
Canon 700D camera attached to the microscope. Photos were stacked with Zerene Stacker
(zerenesystems.com/cms/home), and the size of the spores was measured with Piximètre
(http://ach.log.free.fr/Piximetre/) software. In addition to the basidiospore sizes, col-
oration and the ornamentation were also noted. From the measured parameters, the Q value
(length/width ratio) was calculated, which indicates the shape of the spores (
Q = 1.01–1.05
:
globose, Q = 1.05–1.15: subglobose, Q = 1.15–1.30: broadly ellipsoid,
Q = 1.30–1.60
: ellip-
soid, Q = 1.60–2.0: oblong, Q = 2.0–3.0: cylindrical). Basidiospore ranges for the species
new to Romania are given in Table 2. To exclude aberrant spores, the values are based on
spores within the 0.75 confidence interval.
Diversity 2023, 15, x FOR PEER REVIEW 12 of 24
Cortinarius aff. sublilacinopes Calochroi BLf 1 11.7–12.2 × 7.0–7.4 µm
Q = 1.6–1.7
Cortinarius subporphyropus Pilát 1954 Purpurascentes Mf 1 11.1–12.1 × 6.6–7.3 µm
Q = 1.6–1.7
Cortinarius subpurpurascens (Batsch) J. Kickx f. 1867 Purpurascentes BLf 5 10.6–11.2 × 6.0–6.5 µm
Q = 1.7–1.8
Cortinarius testaceomicaceus Bidaud 2014 Exsulares BLf 1 9.5–11.2 × 6.8–7.5 µm
Q = 1.3–1.6
Cortinarius tirolianus Bidaud, Moënne-Locc. and
Reumaux 2005 Glaucopodes Cf 6 7.5–7.9 × 5.0–5.2 µm
Q = 1.5–1.6
Cortinarius aff. trivialis Myxacium BLf 2 13.4–14.7 × 8.0–8.8 µm
Q = 1.6–1.7
Cortinarius ultrodistortus Rob. Henry and Vagnet
1992
/Ultrodistortu
s Cf 1 7.4–8.8 × 4.5–5.0 µm
Q = 1.5–1.9
Cortinarius uraceonemoralis Niskanen, Liimat., Dima,
Kytöv., Bojantchev and H. Lindstr. 2014 Uracei BLf 1 9.1–10.3 × 5.5–6.0 µm
Q = 1.58–1.87
Cortinarius aff. vibratilis Vibratiles BLf 1 8.4–8.9 × 5.2–5.6 µm
Q = 1.6–1.7
Figure 2. Basidiospores of some Cortinarius s.l. species new to Romania: (A) C. catharinae C089 (sect.
Calochroi), (B) C. daulnoyae C298 (sect. Phlegmacioides), (C) C. fulminoides C334 (sect. Aureocistophili),
(D) C. subdecolorans C229 (singleton), (E) C. subfoetens C333 (sect. Glaucopodes), (F) C. hadrocroceus
C140 (sect. Dermocybe), (G) C. masseei C388 (sect. Obtusi), (H) C. lacustris C387 (sect. Hinnulei). Scale
bar: 10 µm. Photos: E. Szabó.
2.3. Molecular Genetic Analysis
DNA extractions, PCR amplifications, gel electrophoreses, and the purification of the
PCR products were performed in the molecular laboratory at the Interdisciplinary Re-
search Institute on Bio-Nano-Sciences of Babeș-Bolyai University, Cluj-Napoca, Romania.
Genomic DNA was extracted from a small piece of the lamella (10–15 mg) under
sterile conditions using the ISOLATE II Genomic DNA Kit (Bioline Meridian Bioscience,
Inc. Cincinnati, OH, USA) following a modified protocol: (1) After the crushing the dried
samples, 180 µL of Lysis Buffer GL and 25 µL of Proteinase K solution were added to the
sample and were mixed using Thermoblock for 3 h at 56 °C for better amalgamation; (2)
Figure 2.
Basidiospores of some Cortinarius s.l. species new to Romania: (
A
)C. catharinae C089 (sect.
Calochroi), (
B
)C. daulnoyae C298 (sect. Phlegmacioides), (
C
)C. fulminoides C334 (sect. Aureocistophili),
(
D
)C. subdecolorans C229 (singleton), (
E
)C. subfoetens C333 (sect. Glaucopodes), (
F
)C. hadrocroceus
C140 (sect. Dermocybe), (
G
)C. masseei C388 (sect. Obtusi), (
H
)C. lacustris C387 (sect. Hinnulei). Scale
bar: 10 µm. Photos: E. Szabó.
Table 2.
Cortinarius species discovered new to Romania. Infrageneric classification, habitat types as
well as basidiospore measurements and Q values are given for each species. BLf–broad-leaved
forest; Cf–coniferous forest; Mf–mixed forest; P–pastures; TWS–transitional woodland–shrub;
NG–natural grasslands.
Species Section/Clade Habitat
No. of
Collected
Samples
Basidiospores
Cortinarius anfractoides Rob. Henry and
Trescol 1987 Infracti BLf 2 8.8–9.5 ×7.2–7.4 µm
Q = 1.2–1.3
Cortinarius aureopulverulentus M.M. Moser 1952 Calochroi Cf 1 12.4–13.1 ×8.0–8.7 µm
Q = 1.5–1.6

Diversity 2023,15, 553 12 of 26
Table 2. Cont.
Species Section/Clade Habitat
No. of
Collected
Samples
Basidiospores
Cortinarius balteatocumatilis Rob. Henry 1939 Phlegmacioides P 2 8.4–9.2 ×5.2–5.9 µm
Q = 1.5–1.7
Cortinarius brunneus (Pers.) Fr. 1838 Brunnei Cf 1 8.2–9.5 ×6.1–6.8 µm
Q = 1.2–1.5
Cortinarius caesiophylloides Kytöv., Liimat.,
Niskanen, Brandrud and Frøslev 2014 Multiformes Cf 1 10.4–11.0 ×6.2–6.6 µm
Q = 1.57–1.73
Cortinarius catharinae Consiglio 1997 Calochroi BLf 1 9.7–10.5 ×5.8–6.3 µm
Q = 1.6–1.8
Cortinarius cinereobrunneolus Chevassut and Rob.
Henry 1982 Urbici BLf 1 7.7–8.4 ×5.0–5.2 µm
Q = 1.5–1.7
Cortinarius claricolor (Fr.) Fr. 1838 Claricolores Cf 1 6.4–7.2 ×3.5–3.7 µm
Q = 1.8–1.9
Cortinarius colymbadinus Fr. 1838 Uracei Mf 1 8.5–9.4 ×5.4–5.8 µm
Q = 1.5–1.7
Cortinarius corrosus Fr. 1838 Calochroi Cf 1 12.5–14.1 ×7.5–7.9 µm
Q = 1.6–1.9
Cortinarius daulnoyae (Quél.) Sacc. 1910 Phlegmacioides Cf 7 12.4–13.2 ×7.3–7.7 µm
Q = 1.7–1.8
Cortinarius aff. delibutus Delibuti Mf 1 8.5–8.9 ×7.0–7.4 µm
Q = 1.2–1.3
Cortinarius eliae Bidaud, Moënne-Locc. and
Reumaux 1996 Phlegmacioides P 3 11.5–12.5 ×6.8–7.1 µm
Q = 1.7–1.8
Cortinarius fraudulosus Britzelm. 1885 Arguti Cf 1 15.9–16.6 ×8.6–9.0 µm
Q = 1.8–1.9
Cortinarius fulminoides (M.M. Moser)
M.M. Moser 1967 Aureocistophili Mf 1 9.4–10.2 ×5.9–6. 5 µm
Q = 1.5–1.6
Cortinarius gallurae D. Antonini, M. Antonini and
Consiglio 2005 /Gallurae BLf 1 8.6–9.4 ×5.7–6.5 µm
Q = 1.4–1.6
Cortinarius geniculatus Bidaud 2014 Bovini BLf 1 10.7–12.0 ×6.4–6.7 µm
Q = 1.6–1.8
Cortinarius glaucopus aff. Glaucopodes Cf 2 8.5–9.3 ×5.4–5.7 µm
Q = 1.6–1.7
Cortinarius hadrocroceus Ammirati, Niskanen,
Liimat. and Bojantchev 2014 Dermocybe Cf 2 7.3–7.8 ×4.3–4.7 µm
Q = 1.6–1.8
Cortinarius hillieri Rob. Henry 1938 Bovini BLf 1 10.7–11.8 ×6.1–6.7 µm
Q = 1.7–1.8
Cortinarius holoxanthus (M.M. Moser and
I. Gruber) Nezdojm. 1980 Dermocybe NG 3 9.3–9.9 ×4.8–5.2 µm
Q = 1.8–2.1
Cortinarius huronensis Ammirati and
A.H. Sm. 1972 Dermocybe Cf 3 8.0–8.5 ×4.9–5.4 µm
Q = 1.47–1.65
Cortinarius hydrotelamonioides Rob. Henry 1970 Firmiores BLf 1 9.3–10.3 ×5.1–5.8 µm
Q = 1.7–1.9
Cortinarius incognitus Ammirati and
A.H. Sm. 1972 Dermocybe Cf 1 7.4–8.0 ×5.2–5.4 µm
Q = 1.3–1.6
Cortinarius lacustris Moënne-Locc. and
Reumaux 1997 Hinnulei BLf 1 9.6–11.1 ×5.9–6.6 µm
Q = 1.5–1.8

Diversity 2023,15, 553 13 of 26
Table 2. Cont.
Species Section/Clade Habitat
No. of
Collected
Samples
Basidiospores
Cortinarius leproleptopus Chevassut and
Rob. Henry 1988 Leprocybe BLf 1 7.9–8.5 ×6.8–7.3 µm
Q = 1.1–1.2
Cortinarius lilacinovelatus Reumaux and
Ramm 2001 Calochroi BLf 1 11.1–11.7 ×6.1–6.5 µm
Q = 1.7–1.8
Cortinarius luridus Rob. Henry 1969 Hinnulei BLf 3 8.9–9.7 ×6.3–6.7 µm
Q = 1.4–1.5
Cortinarius masseei Bidaud, Moënne-Locc. and
Reumaux 1993 Obtusi BLf 1 7.0–7.8 ×4.9–5.3 µm
Q = 1.4–1. 6
Cortinarius aff. magicus Glaucopodes Cf 2 7.6–8.0 ×5.0–5.3 µm
Q = 1.5–1.6
Cortinarius neofurvolaesus Kytöv., Niskanen,
Liimat. and H. Lindstr. 2005 Bovini NG 2 8.5–9.5 ×5.1–5.9 µm
Q = 1.5–1.8
Cortinarius ochraceopallescens Moënne-Locc. and
Reumaux 2001 Calochroi BLf 3 12.4–13.2 ×6.4–6.9 µm
Q = 1.8–2.0
Cortinarius odoratus (Joguet ex M.M. Moser) M.M.
Moser 1967 Calochroi BLf 3 11.6–12.5 ×6.8–7.2 µm
Q = 1.7–1.8
Cortinarius olidoamarus A. Favre 1986 Glaucopodes P 3 8.4–9.2 ×5.2–5.9 µm
Q = 1.5–1.7
Cortinarius ominosus Bidaud 1994 Dermocybe TWS 1 6.8–7.7 ×4.4–4.7 µm
Q = 1.5–1.6
Cortinarius pallidostriatus Rob. Henry 1968 Hydrocybe Cf 1 8.6–9.6 ×5.5–6.0 µm
Q = 1.5–1.7
Cortinarius pelerinii Bellanger, Carteret and
Reumaux 2013 Anomali BLf 1 8.8–9.4 ×6.3–7.0 µm
Q = 1.3–1.4
Cortinarius persoonianus Bidaud 2009 Infracti BLf 1 8.7–9.5 ×7.2–7.6 µm
Q = 1.2–1.3
Cortinarius pilatii Svrˇcek 1968 Flexipedes Cf 1 9.0–9.7 ×6.0–6.5 µm
Q = 1.4–1.6
Cortinarius pruinatus Bidaud, Moënne-Locc. and
Reumaux 1993 Obtusi BLf 1 10.5–11.7 ×6.2–7.3 µm
Q = 1.5–1.7
Cortinarius pseudodaulnoyae Rob. Henry and
Ramm 1991 Phlegmacioides BLf 2 12.7–13.2 ×7.2–7.7 µm
Q = 1.7–1.8
Cortinarius pseudofervidus Niskanen, Liimat.,
Ammirati and Kytöv. 2014 Dermocybe NG 2 6.9–7.6 ×4.3–4.9 µm
Q = 1.4–1.7
Cortinarius pseudonaevosus Rob. Henry 1957 Phlegmacioides Cf 1 12.9–14.4 ×7.5–8.3 µm
Q = 1.7–1.8
Cortinarius radicosissimus Moënne-Locc. 1997 Hinnulei Cf 1 8.6–9.4 ×6.8–8.0 µm
Q = 1.2–1.3
Cortinarius renidens Fr. 1838 Renidentes Cf 3 7.8–8.1 ×5.6–6.0 µm
Q = 1.3–1.4
Cortinarius rubricosus (Fr.) Fr. 1838 Rubricosi BLf 1 10.0–10.9 ×7.0–7.5 µm
Q = 1.4–1.6
Cortinarius rubrophyllus (Moënne-Locc.) Liimat.,
Niskanen, Ammirati and Dima 2014 Dermocybe Cf 1 6.0–6.3 ×3.8–4.3 µm
Q = 1.5–1.6
Cortinarius rufoallutus Rob. Henry ex Bidaud and
Reumaux 2006 Multiformes Cf 1 9.7–10.3 ×5.7–5.93 µm
Q = 1.7–1.8

Diversity 2023,15, 553 14 of 26
Table 2. Cont.
Species Section/Clade Habitat
No. of
Collected
Samples
Basidiospores
Cortinarius saginus (Fr.) Fr. 1838 Phlegmacium TWS 1 10.5–10.8 ×6.0–6.5 µm
Q = 1.7–1.8
Cortinarius saporatus Britzelm. 1897 Calochroi Mf 1 10.5–11.7 ×6.4–7.6 µm
Q = 1.5–1.8
Cortinarius scaurocaninus Chevassut and Rob.
Henry 1982 Glaucopodes Cf 4 8.7–9.3 ×5.2–5.4 µm
Q = 1.6–1.8
Cortinarius semivelatus Rob. Henry 1970 Squalidi BLf 1 8.0–8.7 ×4.9–5.7 µm
Q = 1.4–1.7
Cortinarius sodagnitus Rob. Henry 1935 Calochroi BLf 2 11.5–12.5 ×6.3–7.1 µm
Q = 1.6–1.9
Cortinarius spadicellus Brandrud 1997 Phlegmacioides Mf 5 10.9–11.5 ×6.9–7.1 µm
Q = 1.5–1.7
Cortinarius subargyronotus Niskanen, Liimat. and
Kytöv. 2014 Uracei BLf 1 9.5–10.3 ×5.9–6.4 µm
Q = 1.5–1.8
Cortinarius subdecolorans M. Langl. and
Reumaux 2000 /Dionysae BLf 1 9.8–10.6 ×6.2–6.6 µm
Q = 1.5–1.6
Cortinarius subfoetens M.M. Moser and
McKnight 1995 Glaucopodes Mf 1 8.3–8.8 ×5.4–5.7 µm
Q = 1.5–1.6
Cortinarius aff. sublilacinopes Calochroi BLf 1 11.7–12.2 ×7.0–7.4 µm
Q = 1.6–1.7
Cortinarius subporphyropus Pilát 1954 Purpurascentes Mf 1 11.1–12.1 ×6.6–7.3 µm
Q = 1.6–1.7
Cortinarius subpurpurascens (Batsch)
J. Kickx f. 1867 Purpurascentes BLf 5 10.6–11.2 ×6.0–6.5 µm
Q = 1.7–1.8
Cortinarius testaceomicaceus Bidaud 2014 Exsulares BLf 1 9.5–11.2 ×6.8–7.5 µm
Q = 1.3–1.6
Cortinarius tirolianus Bidaud, Moënne-Locc. and
Reumaux 2005 Glaucopodes Cf 6 7.5–7.9 ×5.0–5.2 µm
Q = 1.5–1.6
Cortinarius aff. trivialis Myxacium BLf 2 13.4–14.7 ×8.0–8.8 µm
Q = 1.6–1.7
Cortinarius ultrodistortus Rob. Henry and
Vagnet 1992 /Ultrodistortus Cf 1 7.4–8.8 ×4.5–5.0 µm
Q = 1.5–1.9
Cortinarius uraceonemoralis Niskanen, Liimat.,
Dima, Kytöv., Bojantchev and H. Lindstr. 2014 Uracei BLf 1 9.1–10.3 ×5.5–6.0 µm
Q = 1.58–1.87
Cortinarius aff. vibratilis Vibratiles BLf 1 8.4–8.9 ×5.2–5.6 µm
Q = 1.6–1.7
2.3. Molecular Genetic Analysis
DNA extractions, PCR amplifications, gel electrophoreses, and the purification of the
PCR products were performed in the molecular laboratory at the Interdisciplinary Research
Institute on Bio-Nano-Sciences of Babes
,-Bolyai University, Cluj-Napoca, Romania.
Genomic DNA was extracted from a small piece of the lamella (10–15 mg) under
sterile conditions using the ISOLATE II Genomic DNA Kit (Bioline Meridian Bioscience,
Inc. Cincinnati, OH, USA) following a modified protocol: (1) After the crushing the dried
samples, 180
µ
L of Lysis Buffer GL and 25
µ
L of Proteinase K solution were added to the
sample and were mixed using Thermoblock for 3 h at 56
◦
C for better amalgamation; (2) the
elution of the DNA was performed in two steps with 30–30
µ
L of Elution Buffer G solution

Diversity 2023,15, 553 15 of 26
(before spinning, the elution buffer stayed in the column for 3 min). The concentration of
the DNA solutions was measured with a NanoDrop ND-1000 Spectrophotometer (Nano-
Drop Technologies, Wilmington, DE, USA). All DNA concentrations fell between 30 and
100 ng/µL.
The target region was amplified using the following primers: ITS1F (5
0
-CTTGGTCATT-
TAGAGGAAGTAA-3
0
) and ITS4 (5
0
-TCCTCCGCTTATTGATATGC-3
0
) [
94
]. The PCR was
performed in a volume of 50
µ
L of the reaction mixture, which contained 0.5
µ
L of MyTaq
™
DNA Polymerase (Bioline Reagents Ltd., London, UK), 10
µ
L of 5
×
MyTaq
™
Reaction
Buffer, 1
µ
L of the primer pairs (20
µ
M each, from the following primer mix: 10
µ
L ITS1F
+ 10
µ
L ITS4 + 80
µ
L PCR-grade water), 1
µ
L of DNA solution and 37.5
µ
L of PCR-grade
water. The PCR conditions included an initial 5 min denaturation step at 95
◦
C, followed
by 35 cycles of denaturation of 1 min at 95
◦
C, 1 min of annealing at 52
◦
C and 1 min of
elongation at 72 ◦C. Finally, there was a 10 min final extension at 72 ◦C.
The success of the PCR was confirmed with a 1% agarose gel electrophoresis of 2
µ
L
from each sample, performed at 120 V for 20 min. The gel was dyed with ethidium-bromide
and was examined under UV light. After the run, the positive samples were purified using
Wizard SV Gel and a PCR Clean-Up System (Promega, Madison, Wisconsin, USA USA)
following the provided protocol, with one modification: for a more concentrated solution of
the PCR product, the elution of the PCR product was performed in two steps with
20–20 µL
of nuclease free water (before spinning, the buffer stayed in the column for 3 min). The
purity of the PCR product and the concentration of the solution was measured with a
spectrophotometer.
The PCR products were sent to Macrogen Europe (Amsterdam, The Netherlands) for
Sanger sequencing with ITS1F and ITS4 primers.
2.4. Phylogenetic Analysis
The results of the sequencing were downloaded from the Macrogen Europe website.
The quality of the sequences was analyzed using Trev from the Staden Program Pack-
age [
99
], and the primers’ connection region was cut manually. Homologous sequences
were searched by using the BLASTn algorithm [100] both in public, non-curated database
(GenBank; http://www.ncbi.nlm.nih.gov/, accessed on 12 November 2021), and in curated
public databases (UNITE; http://unite.ut.ee/, BOLD; http://www.boldsystems.org/).
Reference and type sequences (Table S1) were downloaded and added to our own dataset.
The sequences were aligned in BioEdit [
101
] using MAFFT with the E-INS-I strat-
egy [
102
]. To refine the nucleotide alignment, the phylogenetically informative indel
positions were coded in FastGap 1.2 [
103
], and the binary matrix was added to the nu-
cleotide alignment (ITS1, 5.8S, ITS2) in SeaView 4 [
104
]. Our dataset was analyzed based
on Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The ML phyloge-
netic reconstruction was performed in raxmlGUI [
105
] using rapid bootstrap analysis with
1000 replicates
. Three nucleotide partitions (ITS1, 5.8S, ITS2) were set to the GTRGAMMA
substitution model in addition to one binary partition (indel characters) that was set to
default. The BI analysis was inferred in MrBayes 3.2.6 [
106
], with a MCMC (Markov
chain Monte Carlo) algorithm, and the GTR +
Γ
substitution model with gamma distri-
bution. The nucleotide partitions and the binary matrix from gap coding were treated as
a mixed data type. The analysis ran for 10,000,000 generations with 25% burn-in. The
resulting phylogenetic tree was visualized in FigTree v1.4.2 [
107
], MEGA 7 [
108
] and Adobe
Illustrator CS4.

Diversity 2023,15, 553 16 of 26
3. Results
In total, 234 sequences were analyzed (Table 1). BI and ML analyses of the nrDNA
ITS region revealed topologically similar phylograms. A preliminary survey was con-
ducted to see whether the 10 different genera of Cortinariaceae established by Liimatainen
et al. [
76
] could be reconstructed based only on single gene analysis, but ITS was not found
to be suitable for recognizing these genera. Therefore, we decided to treat Cortinarius in
a classical sense. In order to achieve a better overview of the morphological similarities
of the studied species, the dataset was divided into two parts. The first alignment was
composed of
760 characters
. After gap coding, a binary set of 267 characters was added
to the nucleotide alignment, leading to a mixed data matrix containing 1027 characters.
The alignment contained ITS sequences of leprocyboid, dermocyboid, rozitoid, telamo-
nioid, and myxacioid species, i.e., from the classical morphological groups such as subgen.
Cortinarius, subgen. Telamonia, and subgen. Myxacium. Our results showed (Figure 3) that
the studied species belonged to sections Anomali,Bovini,Brunnei,Camphorati,Cortinarius,
Defibulati,Delibuti,Dermocybe,Exulares,Firmiores,Flexipedes,Hinnulei,Hydrocybe,Lepro-
cybe,Myxacium,Obtusi,Orellani,Renidentes,Rozites,Rubricosi,Spilomei,Squalidi,Telamonia,
Tragani
,Uracei,Urbici and Vibratiles, as well as to the clades /Gallurae and /Ultrodistortus.
The second alignment included ITS sequences of species from the morphological subgen.
Phlegmacium and was composed of 712 characters. In this case, 185 binary characters from
the gap coding were added to the nucleotide characters, resulting in a final alignment of
897 characters. Our results showed (Figure 4) that the studied species belonged to sections
Arguti,Aureocistophili,Calochroi,Claricolores,Glaucopodes,Infracti,Multiformes,Phlegma-
cioides,Phlegmacium,
Purpurascentes
,Subtorti,Turmales and Varii, and the clade /Dionysae
(= Phlegmacium sect. Dionysae). Most sections and clades are well-supported with ML
bootstrap support values between 80–100% and BI posterior probabilities between 0.92–1.
For the sectional names, the taxonomic work of Liimatainen et al. [3,56,76] were followed.
Based on our study, we were able to recognize 109 Cortinarius s.l. species from Romania,
belonging to 40 sections and 3 clades. Out of these, 43 species have been previously
documented, but their identifications were based only on morphology (Table S2). The
remaining 66 species are reported here as new to Romania (Table 2). Among these, we
were not able to link any of the described Cortinarius names to six phylogenetically well-
separated species; therefore, we used the ‘aff.’ prefix before the epithets which link them
to their closest phylogenetic or morphological species (Figures 3and 4). These are C. aff.
delibutus in sect. Delibuti,C. aff. glaucopus and C. aff. magicus in sect. Glaucopodes,C. aff.
sublilacinopes in sect. Calochroi,C. aff. trivialis in sect. Mycaxium and C. aff. vibratilis in sect.
Vibratiles. Concerning the ongoing Cortinarius studies in Europe, our unpublished results
(data not shown) indicate that these species are likely taxonomic novelties, but unveiling
their taxonomy and nomenclature needs further analyses and the results will be included
in different publications.

Diversity 2023,15, 553 17 of 26
Diversity 2023, 15, x FOR PEER REVIEW 15 of 24
Figure 3. Cont.

Diversity 2023,15, 553 18 of 26
Diversity 2023, 15, x FOR PEER REVIEW 16 of 24
Figure 3. Maximum likelihood phylogenetic tree of the dermocyboid, leprocyboid, rozitoid, tela-
monioid, and myxacioid sections/clades of the genus Cortinarius s.l. based on nrDNA ITS sequence
analyses with gap coding. Sequences produced in this study are in blue and boldface and labelled
with their voucher numbers. Sequences from public repositories are marked with their Gen-
Bank/UNITE accession numbers. ML bootstrap values of >70% as well as Bayesian posterior proba-
bilities of >0.9 are placed above or below branches. The scale bar indicates 0.03 expected change per
site per branch.
Figure 3.
Maximum likelihood phylogenetic tree of the dermocyboid, leprocyboid, rozitoid, telam-
onioid, and myxacioid sections/clades of the genus Cortinarius s.l. based on nrDNA ITS sequence
analyses with gap coding. Sequences produced in this study are in blue and boldface and labelled with
their voucher numbers. Sequences from public repositories are marked with their GenBank/UNITE
accession numbers. ML bootstrap values of >70% as well as Bayesian posterior probabilities of
>0.9 are
placed above or below branches. The scale bar indicates 0.03 expected change per site
per branch.

Diversity 2023,15, 553 19 of 26
Diversity 2023, 15, x FOR PEER REVIEW 17 of 24
Figure 4. Cont.

Diversity 2023,15, 553 20 of 26
Diversity 2023, 15, x FOR PEER REVIEW 18 of 24
Figure 4. Maximum likelihood phylogenetic tree of the phlegmacioid sections/clades of the genus
Cortinarius s.l. based on nrDNA ITS sequence analyses with gap coding. Sequences produced in this
study are in blue and boldface and labelled with their voucher numbers. Sequences from public
repositories are marked with their GenBank/UNITE accession numbers. ML bootstrap values of
>70% as well as Bayesian posterior probabilities of >0.9 are placed above or below branches. The
scale bar indicates 0.03 expected change per site per branch.
Figure 4.
Maximum likelihood phylogenetic tree of the phlegmacioid sections/clades of the genus
Cortinarius s.l. based on nrDNA ITS sequence analyses with gap coding. Sequences produced in
this study are in blue and boldface and labelled with their voucher numbers. Sequences from public
repositories are marked with their GenBank/UNITE accession numbers. ML bootstrap values of
>70% as well as Bayesian posterior probabilities of >0.9 are placed above or below branches. The
scale bar indicates 0.03 expected change per site per branch.

Diversity 2023,15, 553 21 of 26
4. Discussion
In this study, we updated the checklist of the largest agaric genus, Cortinarius s.l.,
in Romania, employing nrDNA ITS barcoding. Macrofungi, including Cortinarius, were
only studied using classical morphological methods in Romania to date; thus, our work
presents the first biodiversity study using the combination of molecular phylogenetic and
morphological methods in the country. Similar to the results of other national barcoding
projects in Europe, e.g., in Norway [
109
–
111
], Finland [
112
], the Netherlands [
113
] or in
Austria (https://www.abol.ac.at/en/project/higher_fungi/, accessed on 1 July 2022), our
results showed a high number of previously not reported taxa at the country level. From
the 109 Cortinarius s.l. species identified in the course of this study, 66 species are reported
here as new to Romania, raising the known number of species up to ca. 190. This number
is, however, lagging far behind that from Western and Northern European countries, where
species observation activities and DNA barcoding campaigns and databases are in a more
advanced stage compared to Romania or its neighboring countries, e.g., in Bulgaria or
Hungary. For example, in Norway, the listed number of Cortinarius species is over 550 [
114
],
while based on data from the older literature, the number of species in Bulgaria is 105 [
115
],
and in Hungary, there are 169 species [
116
]; however, these numbers are hardly comparable
with each other due to the different methods used in species identification (i.e., integrative
taxonomy vs. morphological species recognition).
The majority of the species discovered as new to Romania belongs to phlegmacioid
lineages/sections, especially to sect. Calochroi (nine species) as well as sect. Glaucopodes
and sect. Phlegmacioides (six species in either section). Sect. Dermocybe is represented with
7 new species to the country, whereas 29 other sections/clades are represented by only
1–3 species in our dataset. Altogether, the 109 identified species in this study belong to
43 sections/clades. After actualizing the species list of genus Cortinarius s.l. with our new
data, we assessed ca. 190 species now known in Romania. However, we are aware of
the fact that among the previously published data, there could be wrongly identified and
named collections; thus, without taxonomic revision of these materials, the correct number
of Cortinarius species cannot be accurately established. Based on the various valuable
habitats and nature types in the Carpathian Mountains and adjacent regions in Romania
(out of which we only conducted samplings from the Apuseni Mts, and few sites in the
Eastern Carpathians), we anticipate discovering an even larger diversity of Cortinarius
s.l. species than that established in this work, when the sampling is extended to all areas
with suitable habitats for Cortinarius in Romania. We also believe that other groups of
macrofungi will be shown to be more species-rich when accurate investigations combining
molecular techniques (e.g., DNA barcoding) together with morphological identification
methods will be applied in the future.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/d15040553/s1, Table S1: Cortinarius sequences from public databases
used in this study, Table S2: Cortinarius taxa published to date from Romania and identified with
morphological methods. References [53,79,81,85,87,117–129] are cited in Supplementary Materials.
Author Contributions:
Conceptualization, E.S., B.D. and L.K.; methodology, E.S. and B.D.; molecular
analysis, E.S., B.D. and A.L.D., resources, L.K. and V.P., data curation, E.S. and B.D., writing—original
draft preparation, E.S. and B.D., writing—review and editing, E.S., B.D., A.L.D., L.K. and V.P.,
supervision B.D. and L.K., funding acquisition L.K. and V.P. All authors have read and agreed to the
published version of the manuscript.
Funding:
This research was partially funded by the Collegium Talentum Program of Hungary. The
work of B.D. was funded by the János Bolyai Research Scholarship of the Hungarian Academy of
Sciences; the National Research, Development and Innovation Office of Hungary (OTKA FK-143061);
and the ELTE Thematic Excellence Programme 2020 (TKP2020-IKA-05), financed by the National
Research, Development and Innovation Office of Hungary. The support of the János Bolyai Research
Scholarship of the Hungarian Academy of Sciences to V.P. is highly appreciated.
Institutional Review Board Statement: Not applicable.

Diversity 2023,15, 553 22 of 26
Data Availability Statement:
Data are contained within the article and Supplementary Materi-
als. Some data can also be found in publicly available datasets: https://www.ncbi.nlm.nih.gov/;
http://www.mycobank.org/;http://www.indexfungorum.org/, accessed on 2 March 2023.
Acknowledgments:
We would like to thank Krisztina Babos, LászlóBartha, Gábor Bélfenyéri, Anna
Dénes, Réka Erös, Kinga Fehér, LászlóGál, Boróka Jancsó, Kunigunda Macalik and Csilla Szabó
for their help with the sample collection, Boróka Jancsófor her help with the microscopical spore
photos, and Ágota Szabófor her help in recording habitat composition. We are grateful to the
three anonymous reviewers (especially to one of them) for their valuable comments which greatly
improved our manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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