Table Of ContentFungal Diversity
Morphological and ITS identification of Cortinarius species (section Calochroi)
collected in Mediterranean Quercus woodlands
Ortega, A.1, Suárez-Santiago, V.N.1* and Reyes, J.D.2
1Departamento de Botánica, Facultad de Ciencias, Universidad de Granada, 18071 GRANADA, Spain
2Paseo Virgen de Linarejos, 6. 23700 LINARES (Jaén), Spain
Ortega, A., Suárez-Santiago, V.N. and Reyes, J.D. (2008). Morphological and ITS identification of Cortinarius species
(section Calochroi) collected in Mediterranean Quercus woodlands. Fungal Diversity 29: 73-88.
Species of Cortinarius sect. Calochroi show a high grade of morphological variability in the Mediterranean region with
regard to vicariant populations in the Eurosiberian region. This has contributed to confusion on taxonomy and
nomenclature, especially when the variability affects the characters traditionally used for taxonomic delimitation. In this
paper, we have attempted to clarify the taxonomy and nomenclature of several Calochroi taxa in the Mediterranean area
by using macromorphological and spore analyses, as well as phylogenetic analysis of ITS sequences. We have
demonstrated the usefulness of ITS sequences for species rank identification in Cortinarius (barcoding). We have been
able to assign the studied material to eight species using ITS sequences (C. haasii, C. parasuaveolens, C. platypus, C.
rickenianus, C. sancti-felicis, C. selandicus, C. splendidior and C. subgracilis), and to detect misidentified collections
that were used by their authors to describe a new species (C. violaceipes). Moreover, molecular data have been useful to
test the taxonomic value of morphological and spore characters. The morphological analysis has enabled us to detect
morphological variability previously unreported in several taxa. A new variety, C. haasii var. quercus-ilicicola, is
proposed on the basis of morphological and molecular data. Also, the taxonomic status of C. rickenianus is discussed.
Finally, our results increase both the distribution area as well as the ecological preferences of five studied species, and
several important biogeographical disjunctions are reported (C. selandicus and C. subgracilis).
Key words: Cortinarius, FESEM, ITS sequences, morphological and spore diversity, sclerophyllous forests
Article Information
Received 24 March 2007
Accepted 26 September 2007
Published online 31 March 2008
*Corresponding author: V.N. Suárez-Santiago; e-mail: [email protected]
Introduction populations of taxa differing morphologically
with respect to their northern vicariants is
The Mediterranean basin is the largest of frequent. Consequently, many taxonomic
the world’s five Mediterranean-climate regions. proposals have been made in order to explain
It surrounds the Mediterranean sea, stretching Mediterranean fungal variability. This situation
west to east from Portugal to Jordan (Lebanon, is frequent in the case of the genus Cortinarius,
Israel) and north to south from northern Italy and specifically in the taxa of section Calochroi
and Slovenia to Morocco (Médail and Quézel, M.M. Moser & E. Horak (Frøslev et al., 2006,
1999). The Mediterranean region harbours a 2007).
high degree of biological diversity, being Calochroi species show high variability
considered one of the 25 biodiversity hotspots in Mediterranean Quercus woodlands (Q. ilex
of the world (Myers et al., 2000). This high subsp. ballota, Q. ilex subsp. ilex, Q. coccifera,
biodiversity level is closely associated with the etc.) with regard to vicariant populations in the
great ecological heterogeneity of the Mediter- Eurosiberian region growing under broadleaf
ranean region and its complex historical trees and/or conifers. Different interpretations
changes. that mycologists offer for this morphological
Fungal diversity in the Mediterranean variability between the Mediterranean vs.
basin is high, and the presence in this area of central-European populations have resulted in
73
confusion concerning taxonomy and nomen- Gemini, Field Emission Scanning Electron
clature, especially when the variability affects Microscope (FESEM) in the Servicio Técnico
the characters traditionally used for taxonomic de Apoyo a la Investigación of the University
delimitation (e.g. spore size; Clemençon, of Granada. We also analysed a character that
1979). The incorporation of molecular data to could be used to differentiate C. molochinus
the systematics of the genus Cortinarius has Bidaud & Ramm from C. nymphicolor
enabled not only clarification of interspecific Reumaux, based on our own material together
phylogenetic relationships, but also the taxo- with data from the literature (Moser and Jülich,
nomic delimitation of numerous species, and 1986; Bidaud et al., 1993, 2001; Cadiñanos-
has made it possible to evaluate the taxonomic Aguirre, 1995; Soop, 2005). This character is
value of morphological characters used in the the degree of slenderness, defined by three
taxonomy of this genus. In this sense, the parameters: (i) Ratio between pileus diameter
usefulness of the sequences of the internal (P.D.) and stipe length (S.L.); (ii) ratio between
transcribed spacers (ITS) of the nuclear stipe length (S.L.) and stipe width (S.W.); (iii)
ribosomal DNA (nrDNA) have been shown in ratio between stipe width (S.W.) and bulb
many studies (Liu et al., 1995, 1997; Seidl, width (B.W.). Also, we have used the index of
2000; Peintner et al., 2003, Kytövuori et al., slenderness (I.s. = l2/D·d; l = stipe length, D =
2005; Frøslev et al., 2006, 2007), and they have pileus diameter, d = stipe width) (Kuyper,
been proposed as species-identifier sequences 1986). All average values of the morphological
(barcoding) in Cortinarius (Frøslev et al., measurements are represented as underlined
2007). In this paper, we have attempted to values in the TAXONOMY section.
clarify the taxonomy and nomenclature of The molecular analysis included 98 ITS
several taxa of Cortinarius sect. Calochroi in sequences in total, from which 17 were
the Mediterranean area. To this end we generated as part of this study and the
performed macro- and micromorphological remainder taken from GenBank. The selection
analyses, and phylogenetic analysis of ITS of the sequences taken from GenBank was
sequences. based on the morphological analyses carried
out by us as well as on the results of Frøslev et
Materials and methods al. (2007). Thus, we chose 70 sequences of the
/calochroid subclade (to which the species
The studied material has been collected studied here belong) and three sequences
from localities of Granada and Jaén provinces related to this subclade (one of C. aureofulvus
(Andalusia, southern Spain). Vouchers are M.M. Moser and two of C. flavovirens Rob.
deposited in the University of Granada Henry) (Frøslev et al., 2007). Cortinarius
herbarium (GDA-GDAC). We have also caroviolaceus P.D. Orton, C. saporatus
studied material for comparison from the Britzelm., and C. odorifer Britzelm. were
herbaria of CUSSTA, Córdoba (JA-Cussta), chosen as outgroup species. EMBL accession
herbarium of Institute of Microbiology of numbers for the 17 new sequences are shown in
Innsbruck, Austria (IB), and personal herbaria the TAXONOMY part, while the GenBank
of A. Bidaud (AB), J.A. Cadiñanos Aguirre accession number for the remainder are shown
(CAD), Dr. Garcia Bona (MN) and R. in the ITS tree.
Mahiques (MES). For colour definition, we Total genomic DNA was extracted using
relied on Kornerup and Wanscher (1973). KOH the CTAB method (Doyle and Doyle, 1987).
(25-40%) macrochemical reactions were tested The entire ITS region (ITS-1, 5.8S and ITS-2)
on dried material. Microscopic examinations was amplified by PCR, using primers ITS-5
were made with a Zeiss optical microscope (× (White et al., 1990) and C26A (Wen and
2000). Where possible, for each population, we Zimmer, 1996). PCR reactions were performed
measured 20 spores × 3 basidiomata and in a volume of 50 µl under standard conditions
calculated the range of values, including the (Innis et al., 1990). Automated sequencing of
mean, as well as the length:width ratio (Q: the purified PCR products was performed in
L/W). The spores were morphologically both directions using the ITS-5 and C26A
studied using a Leo (Zeiss), model 1539 primers on a 3100-Avant Genetic Analyzer.
74
Fungal Diversity
Nucleotide sequences were edited with the heuristic searches were performed to assess
SEQMAN II v. 3.61 program from the DNA- internal support for nodes. Fig. 1 shows one of
star software package (LASERGEN). the 46 most parsimonious trees obtained from
The 98 ITS sequences were aligned using the phylogenetic analysis.
the CLUSTAL option of the MEGALIN Species identification of the collections
program from the DNAstar software package studied was based on the analysis of the ITS
(LASERGEN), followed by a visual inspect- sequences in combination with the morpho-
tion. The data matrix is available from logical characters. Collections were assigned to
TreeBASE (study accession number S1888, a species when their ITS sequences formed a
matrix accession number M3469). Nine regions strongly supported monophyletic group with
(90 characters) were ambiguously aligned in the sequences of that species, with low internal
the whole matrix data. However, they were genetic variation and good correlation with the
invariable (except for few cases) at intra- morphological characters. The mean genetic
specific level and unambiguously aligned variation (treating the gaps as missing data)
among closely related species. Therefore, these within the clades considered as the same
regions were considered to calculate the species was between 0 and 0.0034 (0-2
intraspecific distance values and the diver- nucleotide changes), while this value was
gence values between closely related species. between 0.02123 and 0.057 (12-32 changes) for
In the phylogenetic analysis the ambiguously the comparisons between most closely related
aligned regions were treated following the species according to the tree shown in Fig. 1.
method of Lutzoni et al. (2000) to account for Thus, collections with the same ITS sequences
the phylogenetic information that these regions or with very low variation between the
give. According to this method, first the sequences were considered as the same species.
homologous regions of the alignment Table 1 shows the main comparative
containing ambiguously aligned sequences characters between the studied species,
were delimited. Secondly, each of these regions including both morphological, ecological and
was coded as a new character, replacing its genetic data.
respective ambiguous region. Thirdly, each of
the coded characters was subjected to a specific Taxonomy
step matrix to account for the differential
number of changes needed to transform one Cortinarius haasii var. quercus-ilicicola A.
sequence into another. We calculated the Ortega, Suár.-Sant. & J.D. Reyes, var. nov.
divergence (p-distance) between the sequences = Cortinarius haasii (M.M. Moser) M.M. Moser,
Kleine Kryptogamen Flora 2: 294 (1967) pro parte.
using PAUP* version 4.0b10 (Swofford, 2003).
= Phlegmacium arquatum var. haasii M.M.
Phylogenetic analysis was performed using the
Moser, Die Gattung Phlegmacium: 353 (1960) pro parte.
maximum-parsimony criterion (MP), as = Cortinarius calochrous var. haasii (M.M.
implemented in PAUP* 4.0b10, and it involved Moser) Brandrud in Brandrud & Melot, Nord. J. Bot. 10
(5): 536 (1990) pro parte.
heuristic searches. The data matrix was
= Cortinarius violaceipes Bidaud & Consiglio
subjected to 1,000 replicates of random
sensu auct., Gal. Hisp. Ital. sensu Atlas des Cortinaires
sequence additions using tree bisection- XI: 615 (2001) pro parte (AB 92.10.387).
reconnection (TBR) branch-swapping under the Icon. Sel. : Consiglio et al., Boll. Ass. Micol. ed
Ecol. Romana 61, Anno XX (1):31 (2004) (as C.
Fitch criterion (unordered states and equal
violaceipes).
weights), and MaxTrees setting set to 5,000.
Etymology: The Latin quercus-ilicicola refers to
Gaps were treated as missing data. Only ten the habitat, Mediterranean forest of Quercus ilex.
trees were allowed to be held at each step, in A typo differt carpophoris minoribus, pilei cutis
colore pallidiore (flavo vel flavo-ochraceo) et pilei
order to minimize the time the algorithms spent
squamulis haud conspicuis. Holotypus in Hispania,
searching for trees on sub-optimal islands. The
Granada, parc natural Sierra de Huétor, 1250 m supra
starting tree was obtained by stepwise addition. mare, solitarius vel gregarius, sub Quercu ilice subsp.
The characters were optimized by accelerated ballota, in humo calcarea, 2.XII.2006, lectus, leg. A.
transformation. Finally, 1,000 bootstrap Ortega, in herbario GDA sub nº 52537 conservatur.
replicates (BS: Felsenstein, 1985) with 10
75
Table 1. Main comparative characters between the studied species. Data for KOH macrochemical
reaction, index of slenderness (I.s.), spore measurements, plant associates, distribution (Distr.), and
genetic distances are shown. Interspecific genetic distances are refered to the most closely related
species according to the Fig. 1.
Species KOH I.s. Spores Plant Distr. Genetic distances
C B (m.v.) (m.v.) associates Intraspecific Interspecific
C. haasii var. - - 4.06 11.3-12.2 × Q. ilex M 0.0000 (0.0009 C. splendidior:
quercus-ilicicola 6.7-7.3 for C. haasii s.l) 0.0212
C. parasuaveolens + + 4.71 9.7-11.2 × Q. ilex, M 0.0018 C. insignibulbus:
5.8-6.8 Q. coccifera, 0.0232;
Q. pubescens C. sancti-felicis:
0.0233
C. platypus - - 3.63 10.3 × Fagus, E-M 0.00316 Cortinarius sp.
5.9-6.1 Quercus AY669563:
0.0422
C. rickenianus + + 4.47 9.9-11.4 × Carpinus, E-M 0.0000 Cortinarius sp.
6-6.7 Fagus, AY669566,
Quercus DQ323974:
0.02573
C. sancti-felicis + + 3.50 10.8-11.2 × Q. ilex M 0.0005 C. insignibulbus:
6.6.-6.7 0.0281
C. selandicus - + 7.10 11.3-12 × Fagus, E-M 0.0000 C. calochrous:
6.7-7.4 Q. ilex 0.0298
C. sodagnitus + + 4.95 9.1-10.4 × broadleaf E-M 0.0013 -
5.2-6.2 trees
C. splendidior - - 5.22 11.5-11.9 × Q. ilex M 0.0000 C. haasii: 0.0212
6.8-7.1
C. subgracilis + + 5.27 11.8-12.6 × Conifers, E-M 0.0011 C. chailluzii:
6.6-7.2 Q. ilex 0.0265
C = cap, B = bulb
+ = red, - = brown or reddish brown
m.v. = mean values
E = Eurosiberian region, M = Mediterranean region
Pileus 35-60(-70) mm, hemispherical, wide, erect, ± sinuous, cylindrical hyphae, with
convex, plano-convex, then plane or slightly slightly clavate terminal cells pigmented with a
depressed at centre, light orange (5A5) or moderate or strong yellowish or ochraceous-
greyish orange (5B4), with whitish yellow or yellowish cellular or encrusted pigment. The
whitish orange (4A3, 5A2), pale yellow (3A3) basal part of the epicutis is formed by loosely
and greyish yellow (3B3) hues, especially at interwoven repent hyphae, that, finally, form a
margin. Lamellae crowded, lilac or lilac-violet thick pseudoparenchymatous lower layer of
(16B4, 16C6). Stipe (35-)50-60 × 10-17 mm, as (sub)parallel, 5-10-13-20 µm wide hyphae with
long as or smaller than cap diameter (D.P./S.L. cellular or parietal yellowish pigment. Basidia
= 0.89-1.47) and little or moderately slender 4-spored, (× 8-10 µm), claviform, hyaline or
(S.L./S.W. = 2.83-5.4), cylindrical, with a yellowish. Marginal cells rare, (× 7-9 µm)
broad, flattened marginate bulb (× 22-36 mm); cylindrical or claviform, hyaline. Spores (Fig.
index of slenderness = 1.93-4.06-6.06 greyish 2a) 10.8-11.3-12.2-14 × 6-6.7-7.3-8 µm,
violet (17B3) or pale violet (17A3), sometimes ellipsoid-amygdaliform, amygdaloid, subcitri-
only in the upper part. Basal mycelium form or citriform (Q: L/W: 1.5-1.66-1.72-1.87),
yellowish (4A4-5). Context white with violet with moderate or coarse ± interconnected
lilac hues in stipe. Smell and taste not warts.
distinctive. KOH reddish brown on the cap and Habitat and known distribution: Cortina-
surface of the bulb (bulbipellis). rius haasii is a well-known species in Europe,
Pileipellis an ixocutis, epicutis thick, the where it usually grows in conifer woodlands
upper-layer made up of gelatinized 1.5-3.5 µm (Brandrud et al, 1996; Bidaud et al., 2001).
76
Fungal Diversity
However, it also grows under broadleaf trees values between the sequences, being null the
(Frøslev et al., 2007), and it is not rare in the intragroup values (p = 0.0000 both populations
Mediterranean forests of Quercus ilex, where under conifers and populations under Quercus)
also fruits another close taxon: C. splendidior while the intergroup mean distance value was p
Bidaud. Therefore, our data confirm that C. = 0.0017. This latter distance value
haasii and C. splendidior share the same corresponds to one nucleotide difference, but
habitat in the Mediterranean region. there is also an indel (of one base pair)
Material examined: SPAIN, Granada, Huétor differentiating the sequences of both groups.
Santillán, natural park of the Sierra de Huétor, forest These results support the existence of two
track of the Pajareras, from the forest house of the
ecological variants of C. haasii with a different
Peñoncillos to the Fuente de la Teja, Km 0.5, 1250 m, in
geographical distri-bution, and which we have
Quercus ilex subsp. ballota and Cistus laurifolius
woodlands, on non-carbonate soil, 2 December 2006, A. recognized at the variety level. The
Ortega (GDA 52537; holotype designated here), EMBL Mediterranean sclero-phyllous forests collec-
accession number: AM709874; Jaén, La Iruela, el tions of C. haasii have some small and stable
Cantalar, natural park of the Sierras de Cazorla, Segura y
morphological differ-ences with respect to the
Las Villas, 810 m, under Quercus ilex subsp. ballota, on
central European ones: (i) smaller cap diameter
calcareous soil, 19 November 2005, J.D. Reyes (GDA
50852); idem (GDA 50854) EMBL accession number: [pileus 35-60(-70) mm vs. 50-90 mm], (ii) paler
AM709873; Siles, la Laguna, Sierra de Segura, 1350 m, yellowish pileus, which colour is preserved in
same habitat, 17 November 2005, J.D. Reyes (GDA dried basidiomata, and (iii) absence of
52538); idem (GDA 52539). FRANCE, Murs
ochraceous or cream-coloured scales at disk,
(Vaucluse), 700 m, sous chênes vertes et buis, in calcaire
which can present whitish veil remnants.
terrain, 30 October 1992, A. Bidaud (herb. AB 92.10.387
as C. violaceipes), EMBL accession number:
AM709872. Cortinarius parasuaveolens (Bon & Trescol)
Notes: The identification of the AB Bidaud, Moënne-Locc. & Reumaux, Bull.
92.10.387, GDA 50854 and GDA 52537 FAMM 18: 23 (2000).
collections as C. haasii was based on = Cortinarius sodagnitus var. parasuaveolens
morphological and molecular analysis. Fig. 1 Bon & Trescol, Doc. mycol. XIX (73): 36 (1988).
shows that their sequences group into a = Cortinarius sodagnitus Rob. Henry sensu
Brandrud, Breitenbach & Kränzlin, Soop etc. pro parte,
strongly supported clade (97% BS) together the
non sensu Frøslev et al. (2005).
other C. haasii sequences. The mean genetic
= Cortinarius violaceipes Bidaud & Consiglio,
distance value within this clade was very low Atlas des Cortinaires XI: 615 (2001). (See Frøslev et al.,
(p = 0.0009), which together with the 2007).
morphological evidence supports the species Habitat and known distribution: This
identification of our collections. Conversely to species is present in Europe, but it is more
the low intraspecific distance value for C. frequent in the Mediterranean region,
haasii, the interspecific genetic distance value especially in sclerophyllous and mesophilous
between C. haasii and its most closely related Quercus spp. forests.
species (C. splendidior; Fig. 1) was high (p = Material examined: SPAIN, Álava, Cantonad, in
mesophilous Quercus faginea and Q. ilex forests, 14
0.0212, a mean of 12.42 ITS differences plus 3
November 2004, J.A. Cadiñanos Aguirre (CAD);
indels of one base pair each), which
Córdoba, La Zarcilla, Zuheros, natural park of Sierras
corroborates their specific segregation. On the subbéticas cordobesas, evergreen oak forest with Arbutus
other hand, a significant datum is the unedo and Viburnum lantana, 15 November 1992, J.
correlation of the intraspecific molecular Gómez (GDAC 36403; as C. sodagnitus); Zuheros, in Q.
ilex subsp. ballota forests, 850 m, 19 November 2002, J.
variation in C. haasii with respect to the
Gómez et al. (JA-Cussta 4868; as C. sodagnitus);
ecological conditions. The ITS sequences from
Girona, coll d’Urra, la Garrotxa, in meso-xerophilous
central European conifer forests populations evergreen oak forests, 19 October 1999, J.A. Cadiñanos
(AY669561, DQ323966, DQ663311, Aguirre (CAD); Granada, Alhama de Granada, puerto
DQ663312, DQ663315, DQ663316) are del Navazo, 1200 m, under Q. ilex subsp. ballota, 10
December 1987, A. Ortega & M.T. Vizoso (GDAC
grouped together, and they form a sister clade
30809; as C. sodagnitus); Cambil, Mata Bejid, evergreen
to Mediterranean Q. ilex-forests populations
oak forest, 2 December 1987, A. Ortega, M.T. Vizoso &
(Fig. 1). This differentiation is supported by the M. Zea (GDAC 30810; as C. sodagnitus); idem, 27
77
Fig. 1. Phylogenetic relationships of species in the /calochroid subclade sensu Frøslev et al., 2007 (Cortinarius sect.
Calochroi s. auct.). Phylogram of one of the 46 most parsimonious trees (length: 516; CI: 0.593; RI: 0.861; HI: 0.407)
obtained from the parsimony analysis. Bootstrap values ≥ 50% are shown above branches. Branches collapsing in the
strict consensus tree are marked with an asterisk. The accession number (for the sequences taken from GenBank) or
herbarium references (for the sequences generated in this study) are shown after each taxon name. Sequences generated
in this study are marked with a dark circle. Species studied are indicated at right.
78
Fungal Diversity
November 1990, A. Ortega & M.T. Vizoso (GDAC mixed with whitish or pale cream hues, after
36773; as C. sodagnitus), EMBL accession number: discolouring and then in older basidiomata
AM709875; Cambil, Gibralberca, same habitat, 5
brownish orange (5B5) or cinnamon brown
December 2006, J.D. Reyes (GDA 52541, 52542,
(8D4); violet (9C3), pinkish vinaceous (12B3),
52543). FRANCE: Mazet de Romanin, bei St. Rëmy de
Provence, under Q. ilex and Q. coccifera, 20 October lilac (15B3) universal veil remnants abundant
1974, M.M. Moser (IB 74/595); Tour du Cardinal, on cap surface, and pale violet (18A2) on bulb;
Alpilles, Provence, same habitat, 24 October 1974, M.M. GDA 52540: golden yellow (5B7) and greyish
Moser (IB 74/621).
orange (5C3), with several reddish-grey or
Notes: In our opinion three characters
purplish-grey hues (7B2); whitish or greyish-
define this species: (i) the variable colour of the
white universal veil remnants on cap. Lamellae
pileus (Cadiñanos-Aguirre, 2004) which
crowded, violet (15C4, 15C4) and purplish
discolours intensely when dried, and then the
grey (14A2). Stipe 50-60 × 13-15 mm, slightly
older basidiomata become yellowish-
slender (S.L./S.W. = 3.8-4.6), cylindrical, with
ochraceous or ochraceous; (ii) KOH reddish
a variable pale violet (19B3) marginate bulb (×
brown, reddish vinaceous or red on cap, pinkish
18-35 mm); whitish, with lavender greyish
red on bulbipellis; (iii) Spores: 8.8-9.7-11.2-12
(19A2), lavender (20A3) or pinkish (15A2)
× 5.6-5.8-6.8-7.2 µm. Two species closely
diffuse hues, but turning golden yellow (4A7)
related to C. parasuaveolens (Fig. 1) are C.
or greyish orange (5C3) in older basidiomata.
insignibulbus Bidaud & Moënne-Locc., which
KOH reddish brown (8D8) in cap and
has a different habitat (i.e. under Carpinus and
bulbipellis.
Fagus) and smaller spores (Frøslev et al.,
Pileipellis an ixocutis, epicutis thick, the
2006), and C. sancti-felicis Frøslev & T.S.
upper-layer made up of gelatinized 2-2.5-3(-4)
Jeppesen, which no presents or it has lesser
µm wide, erect, ± sinuous, cylindrical, some-
presence of violaceous-lilac hues on cap. At the
times slightly clavate at top hyphae, hyaline or
molecular level, the only intraspecific differ-
pigmented, with ± intensely yellowish cellular
ences for C. parasuaveolens were two
pigment, some hyphae with encrusted-parietal
nucleotide changes between the sequence
pigment (older basidiomata). The basal part of
AF503548 and the remaining C. parasua-
the epicutis is formed by loosely interwoven
veolens sequences, while a mean of 13.25
repent hyphae, which, finally, form a pseudo-
changes (p = 0.0232) plus five indels (one of
parenchymatous layer of (sub)parallel, hyaline,
two base pairs and four of one base pair) and
4-8.5 µm wide hyphae. Clamps presents at all
13.33 changes (p = 0.0233) plus five indels
septa. Basidia 4-spored, (× 7.5-9.5 µm),
(two of two base pairs and three of one base
claviform. Lamellae edge with frequent
pair) distinguish the sequences of C.
clustered hyaline sterile cells 22-35 × 6.5-16
parasuaveolens from the those of C. insigni-
µm, cylindrical, claviform, sub-pyriform, etc.
bulbus and C. sancti-felicis, respectively.
Spores (Fig. 2b) 9.6-10.3-11 × 5.6-5.9-6.1-6.6
Sequences of C. insignibulbus and C. sancti-
µm, amygdaloid or subcitriform [Q: L/W: 1.5-
felicis differ by an average of 16.33 nucleotide
1.67-1.73-1.8(-2)], with moderate or rather
changes (p = 0.02813) plus four indels (of one
coarse ± interconnected warts.
base pair each).
Habitat and known distribution: In the
Cortinarius platypus (M.M. Moser) M.M. Iberian Peninsula it is known only from Burgos
Moser, Kleine Kryptogamen Flora, 2: 292 province (northern Spain), growing in Q. ilex
(1967). and Q. faginea forests (Cadiñanos-Aguirre,
= Phlegmacium platypus M.M. Moser, Die 2004); for this reason our records increase its
Gattung Phlegmacium: 353 (1960). distribution area. In Europe it fruits under
Pileus 45-60 mm, convex, plano-convex
deciduous trees and is not a frequent taxon.
then plane or, sometimes, slightly depressed at
Material examined: SPAIN, Granada, Huétor
centre. Diversely coloured, GDA 50853: when Santillán, natural park of the Sierra de Huétor, forest
young pale violet (18A2), pale bluish violet track of the Pajareras (from forest house of the
Peñoncillos to Fuente de la Teja, Km 0.5), 1250 m
(22A3) or pale purplish grey (9B2, 12B2)
Quercus ilex subsp. ballota, on non-carbonate soil, 9
79
Fig. 2. Field emission scanning electron microscopy (FESEM) of spores. a. Cortinarius haasii var. quercus-ilicicola
(holotype). b. C. platypus (GDA 52540). c. C. rickenianus (IB 74/579). d. C. rickenianus (GDA 52546). e. C.
splendidior (GDA 50848). f. C. subgracilis (GDA 50847). Bars = 2 µm.
80
Fungal Diversity
December 2006, A. Ortega (GDA 52540), EMBL = Cortinarius rickenianus Maire sensu M.M.
accession number: AM709877; Jaén, La Iruela, El Moser, herb. IB n. 74/579, 79/672.
Cantalar, natural park of the Sierras de Cazorla, Segura y = Cortinarius molochinus Bidaud & Ramm in
las Villas, 870 m, Q. ilex subsp. ballota, on calcareous Bidaud et al., Atlas des Cortinaires XI (2): 613 (2001).
soil, 24 November 2005, J.D. Reyes (GDA 50853), = Cortinarius rickenianus Maire sensu Ramm &
EMBL accession number: AM709876; idem, 19 Henry, Bull. Soc. Mycol. France 109: pl. 266 (1993).
November 2005, (GDA 50855). = Cortinarius cf. molochinus Ramm, Doc.
Notes: The Andalusian material is Mycol. XXV (97): 65 (1995).
morphologically close to that iconographed by Pileus 37-90 mm, convex, trapezoidal-
Bidaud et al. (2001; pl. 526), since their convex, then plane or sometimes concave-
basidiomata have a similar slenderness index depressed at centre: when young, pale violet
(I.s. = 4.26-4.6 vs. 3.49-4.36-5.89); however, (19A2) and pale bluish (20A3), soon
the iconographic materials by Moser (1960; discolouring and then golden yellow (5B7),
Fig. 111) and Cadiñanos-Aguirre (2004: 42) orange (5B8), yellowish orange (4A7) or
show more stocky basidiomata (I.s. = 1.73 and yellowish (3A3,5, 4A3), older basidiomata pale
2.73-2.88, respectively). Moreover, we have yellowish. Whitish violet-blue (17A2, 18A2)
observed that the relative bulb width is and purplish whitish lilac (14A2) fibrillose
considerable in the Andalusian collections universal veil remnants abundant on cap
(S.W./B.W. = 0.3-0.5), except for the collection surface. Lamellae crowded, pale violet (17B4)
GDA 52540 (S.W./B.W. = 0.72), and this and mauve or pale lilac (16B4), these tinges
character is observable also in the above- persisting even in the dried herbaria material.
mentioned iconographic materials. Similarly, Stipe 50-150 × 10-20 mm, slender (S.L./S.W. =
several differences in the cap and bulb colour 3.8-7.7), cylindrical, with a marginate bulb (×
distinguish the two Andalusian populations. 20-35 mm); whitish, with a bluish lilac tinge
Nevertheless, their spore size and morphology (19A3) under the lamellae, but turning golden
of sterile edge cells are similar. ITS sequences yellow (4A7) or yellowish (4A3) in older
completely resolve the taxonomic ascription of basidiomata. Context white, bluish lilac-violet
both Andalusian collections, since their (19A3) at the top of the stipe and yellow-
sequences come into the C. platypus clade (Fig. ochraceous at the bulb. Smell and taste not
1), and they coincide entirely with the sequence distinctive (these characteristic refer to the
of the type material (DQ083778; p = 0.000). material from Granada, Spain). KOH reddish
Frøslev et al. (2007) demonstrated that C. pink (12 A-6) in cap and in the bulbipellis.
platypus is a genetically polymorphic species, Pileipellis an ixocutis, epicutis thick,
since they found six polymorphic sites in the made up of an upper layer of gelatinized, 2-2.5-
ITS sequences of this species, three of which 3(-4) µm wide, erect, ± sinuous, cylindrical,
were also seen as intragenomic polymorphisms; sometimes with a slightly clavate top hyphae,
and they identified two allelic ITS variants hyaline or pigmented, with ± intensely
(“a”, to which the sequences of our collections yellowish cellular pigment. The basal part of
belong, and “b”, represented by the sequences the epicutis is formed by loosely interwoven
AF503546 and DQ323965). However, as these repent hyphae, which finally form a
authors noted and we have checked in our pseudoparenchymatous layer of (sub) parallel,
collections, the intraspecific molecular varia- hyaline, hyphae 5-8 µm wide. Clamps presents
tion in C. platypus is not correlated with any at all septa. Basidia 4-spored, (× 8-9.5 µm),
other character (morphological or ecological). claviform. Sterile cells 25-35 × 8-12 µm,
Therefore, all the aforementioned evidence hyaline, cylindrical, claviform, subpyriform,
does not distinguish any infraspecific taxa in C. etc. Spores (Figs. 2c,d) 9.2-9.9-11.4-12.2 × 5.6-
platypus. 6-6.7-7.2 µm, amygdaloid or subcitriform (Q:
L/W: 1.5-1.60-1.73-1.9), with moderate or
Cortinarius rickenianus Maire, Fungi rather coarse ± interconnected warts.
Catalaunici II: 111 (1937). Habitat and known distribution: This
= Cortinarius nymphicolor Reumaux, Atlas des species has been collected from several French
Cortinaires V: 151 (1993).
localities (Bidaud et al., 2001), also from
81
Czech Republic and Switzerland (Frøslev et al., it is yellowish with violaceous or lilac hues.
2005), and Slovenia (IB 79/672). For this The KOH reaction is pinkish-red on the cap
reason our record substantially increases its and bulbipellis in C. rickenianus, and of the
distribution area. This species is frequent in the same colour (but inconstant on pileus) in C.
Mediterranean area. parasuaveolens. Bidaud et al. (1993; f. 70)
Material examined: SPAIN, Castelló, Tinença de suggested that the pileipellis structure differs
Benifassá, 650 m, in Quercus ilex subsp. ballota and slightly between the two species, C.
Pinus halepensis forests, on calcareous soil, 1 November
parasuaveolens having a wider and more
2003, R. Mahiques (MES 3968; as C. parasuaveolens),
compact pseudoparenchymatous basal layer.
EMBL accession number: AM709883; Granada, Huétor
Santillán, natural park of the Sierra de Huétor, forest This character was not observed by us.
track of the pajareras (from forest house of the We analysed the ITS sequences of two
Peñoncillos to Fuente de la Teja, Km 0.5), 1250 m, Q. collections of C. rickenianus sensu M.M.
ilex subsp. ballota, on non-carbonate soil, 10 December
Moser (= C. nymphicolor Reumaux) from IB
2005, A. Ortega (GDA 50851), EMBL accession
(IB 74/579; IB 79/672) which agree comple-
number: AM709878; idem, 2 December 2006, A. Ortega
(GDA 52545); Güéjar Sierra, near to Hotel Santa Cruz, tely (p = 0.000; Fig. 1) with the three
1450 m, under Q. ilex subsp. ballota, on non-carbonate sequences considered by Frøslev et al. (2006,
soil, 1 November 1989, Ortega et al. (GDAC 30120; as 2007) as C. nymphicolor (DQ083795, DQ
C. sodagnitus var. parasuaveolens), EMBL accession
083796, DQ323969), one of which coming
number: AM709880; Jaén, Cambil, Gibralberca, under
from the type material of C. molochinus. For
Q. ilex subsp. ballota, 6 December 2006, J.D.Reyes
(GDA 52546), EMBL accession number: AM709879; this reason, we believe that C. rickenianus, C.
idem, (GDA 52547, 52548, 52549). FRANCE, Mazet de nymphicolor and C. molochinus correspond to
Romanin, St. Rémy de Provence, in Quercetum (Q. ilex the same species. In terms of nomenclature,
and Q. coccifera), 20 October 1974, M.M. Moser (IB
Frøslev et al. (2006, 2007) considered C.
74/579; as C. rickenianus), EMBL accession number:
molochinus to be synonymous with C.
AM709881. SLOVENIA, Grcevec, ca 40 Km Südöstl.
Laibach, mistewald (Büche, Carpinus, Eiche, Fich), 10 nymphicolor because the latter name was older;
October 1979, M.M. Moser (IB 79/672; as C. however, according to our results, C.
rickenianus), EMBL accession number: AM709882. rickenianus Maire has priority.
Notes: The Andalusian material corres-
The southern Spain collections respond to
ponds strictly to the morphological concept of
two biotypes of C. rickenianus. One (GDA
C. molochinus (= C. rickenianus sensu Ramm
50851, GDA 52545, GDA 52546, GDA 52549)
& Henry), and it is very close to French
has slender basidiomata [I.s. = 5.6-7.05-8.3(-
mycologists’ concept of C. nymphicolor (= C.
12.3)], while the other (GDAC 30120, GDA
rickenianus sensu auct.) and to C. para-
52547, GDA 52548) has stocky basidiomata
suaveolens (= C. sodagnitus sensu lato, pro
(I.s. = 2.2-2.8-3.5). These latter coincide with
parte). Also it is related to some members of
the traditional concept of this species [I.s. =
the C. parvus group (e.g. C. lilacinovelatus
1.8-3.2-6(-8)] (Bidaud et al., 1993, 2001;
Reumaux & Ramm) according to the data
Cadiñanos-Aguirre, 1995; Soop, 2005, etc.).
published by Frøslev et al. (2005).
Moreover, we noted clear differences in the
In the opinion of French mycologists the
spore size within the Andalusian material, as
presence of pinkish-lilac or violaceous hues on
some collections have larger mean values
the cap surface and the pinkish-red KOH
(11.4-12.8 × 6-6.8 µm) and others have smaller
reaction relate C. molochinus sensu str. to C.
mean values (10-10.8 × 6-6.4 µm). However,
nymphicolor sensu auct. galliae, C. parasua-
slenderness and spore size did not correlate.
veolens, and C. sodagnitus; all these species
These results show that C. rickenianus is a
also fruit in Mediterranean Quercus forests.
highly variable species, in which the
Cortinarius rickenianus sensu lato differs
morphological variability of the basidiomata
from C. parasuaveolens by its paler pileus with
and spore size correspond to the phenotypical
pinkish-lilac hues, while C. parasuaveolens has
plasticity of this species, since the spore
a more yellowish or yellowish-ochraceous
dimensions could differ according to
pileus with lilac-violaceous or violet hues.
environmental parameters (e.g. temperature
Moreover, the C. rickenianus stipe is whitish
flucutations; Clemençon, 1997).
except at the apex, while in C. parasuaveolens
82
