Xerocomus cisalpinus sp. nov., and the delimitation of species in the X. chrysenteron complex based on morphology and rDNA-LSU sequences*

Mycol. Res. 107 (6): 659–679 (June 2003). f The British Mycological Society

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DOI: 10.1017/S0953756203007901 Printed in the United Kingdom.

Xerocomus cisalpinus sp. nov., and the delimitation of species in the X. chrysenteron complex based on morphology and rDNA-LSU sequences*

Ursula PEINTNER1#, Heidi LADURNER1 and Giampaolo SIMONINI2 1

Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria. Via Bellaria 8, 42100 Reggio Emilia, Italy. E-mail : [email protected]

2

Received 6 June 2002; accepted 31 March 2003.

Species delimitation is still controversial in the Xerocomus chrysenteron complex. We have therefore established comprehensible and reliable species concepts based on statistical evaluation of morphological and ecological characters. We examined many collections from different geographical regions and different developmental stages within collections. Quantitative micromorphological characters (basidiospores, pileipellis end cells) were measured in statistically relevant numbers. The same material was used to generate 24 rDNA-LSU sequences, and the results of phylogenetic analyses clearly confirmed our species concepts : spore size and ornamentation, length of the pileipellis end cells and ‘ pruinatus-hyphae’ are most valuable characters for the delimitation of species in this complex. Molecular data demonstrated that the X. chrysenteron complex is a monophyletic group. All the examined species (X. chrysenteron, X. cisalpinus, X. pruinatus, X. ripariellus, X. dryophilus, X. fennicus, X. porosporus, and X. rubellus) represent independent lineages. The faintly striate spores, a key character characterising species of section Striatulispori, probably evolved independently. In addition, the ‘pruinatus-hyphae ’ have multiple origins, and truncate spore apices are derived at least twice. Xerocomus cisalpinus sp. nov. is characterised by striate spores, the presence of ‘pruinatus-hyphae ’ and a pileipellis strongly reminiscent of X. chrysenteron. For reasons of discussion, microscopical data are presented on Boletellus episcopalis for the first time. Xerocomus fennicus (Boletellus) comb. nov. is proposed. We provide descriptions to all included taxa. Our results once more demonstrate that reliably identified and characterised voucher collections are the basic requirement for meaningful phylogenetic studies.

INTRODUCTION Distinction of species in the genus Xerocomus has always been critical. Due to the high number of newly described taxa (Bresinsky & Schwarzer 1969, Heinemann, Rammeloo & Rullier 1988, Oolbekkink 1991, Klofac & Krisai-Greilhu¨ber 1992) a reliable identification of species within this group of boletes became a comparison between various mycological schools and philosophies. These mycological schools used empirically weighted morphological characters based on personal experience, and due to different weighting of different characters there was little agreement on broadly acceptable species concepts. During our earlier studies (Simonini 1996, 1998a, Ladurner 2001a, b), we developed species concepts for taxa of the X. chrysenteron group based on a ‘ fuzzy ’ set of morphological and ecological characters. Subsequent to this, a small * We dedicate this paper to Reinhold Po¨der on the occasion of his 55th birthday. # Corresponding author.

bolete macroscopically strongly recalling X. chrysenteron was repeatedly collected in Italy during our studies of Xerocomus in southern Europe. Microscopically, these collections exhibited intermediate characters between X. pruinatus and X. chrysenteron and called into question our earlier species concepts. The spores of these new collections were finely longitudinally striate and the typical, ‘pruinatus-hyphae’ (Fig. 1), thick-walled, amyloid hyphae in the stipe context (Ladurner & Po¨der 2000), were constantly observed. Both characters were not compatible with our interpretation of X. chrysenteron. In addition, the pileipellis structure of these collections did not match the species concept of X. pruinatus in Europe, which was broadly accepted only recently (Klofac & KrisaiGreilhuber 1992, Engel et al. 1996, Simonini 1998b). The striking combination of characters exhibited by these new collections was not compatible with any European taxon with striate spores. In order to verify our species concepts and to resolve the puzzling question of whether or not this taxon was

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According to phylogenetic analyses of our molecular data, the intermediate Italian collections represent a new, independent lineage within the X. chrysenteron complex. These collections can also be separated morphologically, we therefore describe Xerocomus cisalpinus sp. nov., and discuss its phylogenetic position. In addition, the distinguishing morphological and ecological characters of taxa belonging to the X. chrysenteron complex are described and discussed.

MATERIALS AND METHODS Macro- and microscopical characterisation

Fig. 1. ‘ Pruinatus-hyphae’ are thick walled, amyloid hyphae in the stipe context as typically found in Xerocomus pruinatus. ‘ Pruinatus-hyphae’ extend more or less abruptly to strikingly thick-walled, to 30 mm broad, irregularly formed hyphae of variable length and show a weak to strong amyloid reaction in the wall depending on their developmental stage. The inner surface has crater-like ornamentation. Nomarski. Bar=10 mm.

a variety of X. pruinatus, to which it seemed closest, its taxonomic position was investigated using molecular techniques. Nuclear ribosomal DNA sequences of the large subunit (nLSU-rDNA) have proven to be most useful for molecular systematics of agarics and boletes at order, genus and species level (Binder & Fischer 1998, Drehmel, Moncalvo & Vilgalys 1999, Moncalvo et al. 2000a, b, 2002, Grubisha et al. 2001, Humpert et al. 2001, Jarosch & Besl 2001, Binder & Hibbett 2001, 2002). For this phylogenetic study we generated numerous nLSU-rDNA sequences from the same collections that we had studied previously to establish our morphological and ecological species concepts. Modern species delimitation in Xerocomus is now, in addition to the classical macro-morphological characters, based on measurable microscopical characters such as spore morphology and pileipellis structure. Currently, the X. chrysenteron group circumscribes six species, three of them (X. fennicus, X. pruinatus, X. ripariellus) with striate spores. For these species the new section Striatulispori was recently erected (Redeuilh 1998). We therefore also wanted to determine if spore striation is a valuable, synapomorphic character for the delimitation of groups within Xerocomus.

Comprehensible and reliable species concepts were developed based on all available traditional methods (including statistics). Our concepts are based on the examination of numerous collections from different geographical regions and on the study of different developmental stages within one collection. Quantitative micromorphological characters (dimensions of the terminal pileipellis elements, spores, cystidia) were measured in statistically relevant numbers (n=31). All collections were carefully and consistently screened for qualitative characters such as the type of the pileipellis, pigmentation, spore ornamentation, microchemical reactions, etc. These data were combined with the respective ecological data (associated trees, fruiting period, edaphic factors, etc.). Colour descriptions of the basidiomes were usually made according to Kornerup & Wanscher (1978). Additionally, the colour code Seguy (1936) was used ; in this case, the letter ‘S ’ precedes the colour codes. The specimens were examined with standard microscopic techniques. Microscopic data were documented by video prints produced with a Sony video camera SASC-C350P and a Sony video printer UP 910. Spores and hymenophoral elements were mounted in 3 % KOH and measured on prints at 2900r magnification. Pileipellis samples were studied in Congo red. Stipe tissue was first treated with Melzer’s then washed in a concentrated chlorale-hydrate solution for the examination of the ‘pruinatus-hyphae ’ (Ladurner & Po¨der 2000). For statistical evaluation, 31 spores and 31 terminal elements of the pileipellis were measured for each collection. Measurements of spores and terminal elements of the pileipellis are given as (minimum) average¡standard deviation (maximum). The spore quotient (Q) is the ratio of spore length to width (Q=l/w). The spore volume is calculated as follows : V=4/3((l/2) * (w * w/2)) * 3.14/2. For scanning electron microscopy (SEM), dried specimens were rehydrated in 25% ammonia solution for 1 h, dehydrated in 70 % aqueous ethanol for 1 h, fixed in pure 1,2-dimethoxy-methane for 1.5 h and then immersed in pure acetone for at least 2 h. After critical point drying, samples were mounted on aluminium porters and sputtered with gold. Micrographs were prepared using a Zeiss DSM-950 SEM.

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Table 1. Material included in the phylogenetic analysis of the Xerocomus chrysenteron complex with the respective GenBank accession numbers for rDNA-LSU sequences, the country of provenance and the potential host trees. When available, herbarium numbers are given after the respective species epithet. Some sequences were retrieved from GenBank. Species and herbarium number

GenBank accession no.

Country

Potential host trees

X. chrysenteron IB19990951 X. chrysenteron IB19990952 X. chrysenteron IB20000405 X. chrysenteron X. ‘chrysenteron’ X. ‘chrysenteron’ X. cisalpinus IB19900700 X. cisalpinus IB19980850 X. cisalpinus IB20000700 X. cisalpinus IB20000701 X. dryophilus IB19990901 X. dryophilus IB19991057 X. fennicus Hel X. fennicus OULU X. porosporus X. porosporus IB19880304 X. porosporus IB19990957 X. pruinatus X. pruinatus X. pruinatus IB19920266 X. pruinatus IB19950970 X. pruinatus IB19961055 X. pruinatus IB19980366 X. ‘pruinatus’ X. ripariellus GR21189 X. ripariellus GR930920 X. ripariellus IB19980360 X. ripariellus GR22465 X. ‘rubellus’ X. rubellus GS1044 X. rubellus GS961 X. rubellus IB19990917 X. subtomentosus X. subtomentosus IB19980452 X. subtomentosus IB19991000

AF514808 AF514807 AF514809 AF347103 AF071537 AF050647 AF514814 AF514815 AF514813 AF514812 AF514823 AF514822 AF514820 AF514821 AF050645 AF514810 AF514811 AF050645 AF050644 AF514824 AF514826 AF514825 AF514827 AF402140 AF514817 AF514819 AF514816 AF514818 AF050649 AF514830 AF514829 AF514828 AF139716 AF514831 AF514832

Austria Austria Austria Switzerland USA Germany Italy Italy Italy Italy Croatia Spain Finland Finland Germany Spain Italy Germany Germany Ukraine Austria Italy Austria Switzerland France France Spain France Germany Italy Italy Croatia Germany Finland Italy

Picea Picea Pinus, Picea Mixed forest Unknown Picea, Fagus Quercus ilex Juniperus, Fagus Quercus suber Quercus suber Quercus ilex Pinus halepensis Betula, Picea Unknown Unknown Cedrus, Fraxinus Quercus Quercus, Acer Mixed forest Fagus Fagus, Picea Quercus, Castanea Fagus, Picea Mixed forest Unknown Unknown Mixed hardwoods Unknown Mixed forest Quercus suber Quercus cerris Quercus Quercus Pinus, Betula Quercus pubescens

Statistical analyses Statistics were carried out with the program SYSTAT10 (SPSS, Chicago, IL). 28 collections of Xerocomus chrysenteron, 18 collections of X. cisalpinus, and 25 collections of X. pruinatus were studied. Collections were chosen in order to cover the widest possible geographic range of these taxa. Four biometrical variables were considered : spore length (SPOLE) and width (SPOWI) as well as length and width of the pileipellis end cells (ENDLE, ENDWI). Mean values of each variable were calculated for each collection, respectively. Two sample t tests were performed with two samples belonging to two different taxa in order to test the null hypothesis that these biometrical variables are not significantly different. Biometric variables can be considered as useful to separate taxa when the null hypothesis is rejected (P<0.005). A discriminant analysis using complete estimation of spore length, spore width, end cells length and end cells width has been performed. The aim of this analysis was to firstly find a linear combination of the four variables that best discriminate among the three taxa, and secondly to check if these three species groups could be

separated, and to thirdly find which variables are most significant for distinction of taxa. Molecular techniques In order to establish the phylogenetic relationships of Xerocomus cisalpinus, nLSU-rDNA sequences were produced from the type material and three collections from other localities. All together, 34 sequences were used for the phylogenetic analyses: 26 sequences were generated from the same material used to establish our morphology- and ecology-based species concepts in the X. chrysenteron species complex. Eight additional sequences were retrieved from GenBank. The material used is listed in Table 1, together with the respective GenBank accession numbers, collection numbers, and information concerning the geographical origin and ecology. DNA was isolated from dried herbarium material following standard protocols (Zolan & Pukkila 1986). Primers used for PCR amplification and sequencing of the nLSU-rDNA were LROR, LR3, LR5, LR3R (Moncalvo et al. 2000b). Amplifications were carried out in a 25 ml reaction mix under standard conditions

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex 70 X. chrysenteron 60

RESULTS Statistical analyses of biometric variables The morphometric variables spore length and width (SPOLE, SPOWI) and pileipellis end cells length and width (ENDLE, ENDWI) of Xerocomus cisalpinus,

50

40

30

Phylogenetic analyses

20

0

10 20 End cells width (µm)

30

Fig. 2. Pileipellis end cells size distribution (length and width) of Xerocomus chrysenteron, X. cisalpinus and X. pruinatus with Gauss’ confidence ellipse (P=0.683). 16 X. chrysenteron X. cisalpinus 15

Spore length (µm)

Phylogenetic analyses of 34 nLSU-rDNA sequences belonging to eight taxa of the Xerocomus chrysenteron complex were performed with PAUP* 4.0b8 (Swofford 1998). Maximum parsimony was used to search for optimal trees, employing the default PAUP* settings (i.e. mulpars=on, steepest descent not in effect, and maxtrees=20000). Maximum parsimony was conducted with a standard step matrix giving transitions twice the weight of transversions. Analyses were carried out with tree-bisection-connection (TBR) branch swapping, and with gaps treated as ‘ missing ’. Most parsimonious trees (MPT) were found using 100 random sequence addition replicates followed by a second heuristic search using the trees from the previous search as starting trees but applying a reconnection limit of eight. To evaluate branch robustness of trees generated by parsimony-based methods, bootstrap analyses (Felsenstein 1985) were conducted using 500 replications, each consisting of ten heuristic searches with random addition sequences and TBR branch swapping. The program Modeltest version 3.06 (Posada & Crandall 1998) was used to test the model of DNA substitution. The ML analysis under the GTR+G model was performed with six substitution types and a user-specified substitution rate matrix, nucleotide frequencies A=0.25450, C=0.21190, G=0.30560, T=0.22800, proportion of invariable sites=0.5965, rate heterogeneity following the discrete gamma approximation with four categories and a shape parameter a=0.7347. The heuristic search with TBR branch swapping used one MPT as starting tree. In addition, two hundred bootstrap replicates were run with ML (starting trees obtained via neighbour-joining, no TBR).

X. cisalpinus X. pruinatus

End cells length (µm)

(Vilgalys & Hester 1990) in a PerkinElmer 9600 thermocycler (Perkin Elmer, Foster City, CA). Sequencing was performed using fluorescent dye terminator chemistries following the manufacturer’s instructions (PerkinElmer) on automated sequencers (ABI 373A, ABI 377, PerkinElmer, Norwalk, CN). Sequence chromatograms were compiled with Sequencher software version 2.0 (Gene Codes, Ann Arbor, MI) or Sequence Navigator (Perkin Elmer). Sequences were submitted to the GenBank under the accession nos. AF514807–AF514832; alignments are deposited in TreeBASE under study accession nos. S878 and matrix no. M1425.

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X. pruinatus

14

13

12

11 4.0

4.5

5.0 Spore width (µm)

5.5

6.0

Fig. 3. Spore size distribution (length and width) of Xerocomus chrysenteron, X. cisalpinus and X. pruinatus with Gauss’ confidence ellipse (P=0.683).

X. pruinatus and X. chrysenteron were measured and statistically analysed. Pileipellis end cells size distribution (length and width) and spore size distribution with Gauss’ bivariate confidence ellipse of X. chrysenteron, X. cisalpinus and X. pruinatus are shown in Figs 2–3. Two-sample t tests of mean values were carried out (Table 2). Each variable of taxon A is compared to variable of taxon B. Mean values of variables are significantly different at P<0.005, and can therefore be considered as useful morphological character for the distinction of these two taxa. X. cisalpinus can easily be separated from both X. chrysenteron and X. pruinatus by the shorter, thinner spores and the longer end cells of the pileipellis (particularly with respect to X. pruinatus). End cells width is not useful to separate

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Table 2. Morphometric variables of Xerocomus cisalpinus, X. pruinatus and X. chrysenteron are tested for their usefulness to distinguish two taxa: two-sample t tests of mean values (MW) of the morphometric variables spore length (SPOLE), spore width (SPOWI), pileipellis end cells length (ENDLE) and end cells width (ENDWI) were carried out. Each variable of taxon A is compared to variable of taxon B. They are significantly different at P<0.005, and can therefore be considered as a useful morphological character for the distinction of these two taxa. Variable

MW of taxon A

MW of taxon B

P-values

ENDLE ENDLE ENDLE ENDWI ENDWI ENDWI SPOLE SPOLE SPOLE SPOWI SPOWI SPOWI

X. cisalp. (51¡7.6 mm) X. cisalp. (51¡7.6 mm) X. chrys. (37.7¡6.6 mm) X. cisalp. (13.2¡2.3 mm) X. cisalp. (13.2¡2.3 mm) X. chrys. (13.8¡2.9 mm) X. cisalp. (12.9¡0.6 mm) X. cisalp. (12.9¡0.6 mm) X. chrys. (13.9¡0.7 mm) X. cisalp. (4.7¡0.1 mm) X. cisalp. (4.7¡0.1 mm) X. chrys. (5.1¡0.3 mm)

X. pruin. (24.6¡2.6 mm) X. chrys. (37.7¡6.6 mm) X. pruin. (24.6¡2.6 mm) X. pruin. (11.1¡3.3 mm) X. chrys. (13.8¡2.9 mm) X. pruin. (11.1¡3.3 mm) X. pruin. (14.1¡0.5 mm) X. chrys. (13.9¡0.7 mm) X. pruin. (14.1¡0.5 mm) X. pruin. (5.2¡0.2 mm) X. chrys. (5.1¡0.3 mm) X. pruin. (5.2¡0.2 mm)

0.000 0.000 0.000 0.024 2.196 0.003 0.000 0.000 0.190 0.000 0.000 0.118

Table 3. Canonical discriminant functions (CDF) standardised by within variances, eigenvalues and cumulative proportion of total dispersion (Cum. prop. tot. disp.).

4

FACTOR 2

2 SPOLE SPOWI ENDLE ENDWI Eigenvalues Cum. prop. tot. disp.

0

–2

–4

X. chrysenteron X. cisalpinus X. pruinatus

–6 –6

–4

–2 0 FACTOR 1

2

4

Fig. 4. Canonical score plot for Xerocomus cisalpinus, X. chrysenteron and X. pruinatus. The axes of this plot are the first two canonical discriminant functions, and the points are the canonical variables scores. Confidence ellipses are centred around the centroid of each group. X. cisalpinus, X. chrysenteron and X. pruinatus are strongly differentiated from each other: there is no overlap between X. cisalpinus and the others, and a small overlap only between X. chrysenteron and X. pruinatus.

X. cisalpinus from the two other taxa. X. chrysenteron can be separated from X. pruinatus by end cell length and end cell width, but not by spore length or spore width. All three species can clearly be separated from each other by their end cell length. The discriminant analysis also demonstrated that end cell length is a significant variable : especially in combination with spore width, end cell length enables significant separations. Combinations of other morphometric characters gave poorly significant separations. The canonical variables are evaluated at the group means. In the canonical variable plot (Fig. 4) the centroid for X. chrysenteron is at x=0.157, y=0.582, the

CDF 1

CDF 2

0.276 0.378 x0.982 0.264 4.793 0.954

0.313 0.457 0.277 0.542 0.231 1.000

centroid for X. cisalpinus is at x=x3.341, y=x0.337, and the centroid of X. pruinatus is at x=2.230, y=x0.409. Equality of group means was tested with the F statistics values. They are proportional to distance measures of the centroids shown in the canonical scores plot. The closer a case is to a particular group’s location, the higher the probability that it belongs to that group. Based on morphometric values, X. cisalpinus, X. chrysenteron and X. pruinatus can easily differentiated from each other : there is no overlap between X. cisalpinus and the others, and only a small overlap between X. chrysenteron and X. pruinatus. X. chrysenteron and X. pruinatus are the two most similar taxa (16.665). X. cisalpinus is more similar to X. chrysenteron (34.240) than to X. pruinatus (77.627). In the classification matrix each case is classified into the group where the value of its classification function appears to be the highest. For X. chrysenteron, 23 cases are classified correctly (82 %) and 5 cases are misclassified (as X. pruinatus). For X. cisalpinus, 17 cases are classified correctly (94 %) and 1 case is misclassified (as X. chrysenteron). For X. pruinatus, 24 cases are classified correctly (96 %) and 1 case is misclassified as X. chrysenteron. The first canonical discriminant function (Table 3) is the linear combination of the variables that best discriminate among groups, the second canonical discriminant function is orthogonal to the first and next best combination of variables. The first discriminant

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex Table 4. Multivariate test statistics. F is the ratio of variation ‘between’ the three groups Xerocomus chrysenteron, X. cisalpinus, X. pruinatus and ‘within’ the three groups. D.F=degree of freedom. Multivariate test

F

D .F

P-tail

Wilks’ lambda=0.140 Pillai’s trace=1.015 Lawley-Hotelling trace=5.024

27.143 17.002 40.191

8,130 8,132 8,128

0.0000 0.0000 0.0000

function depends strongly on end cells length, and in the second place on spore width. The first Eigenvalue (4.793) is high when compared to the second Eigenvalue (0.231). This indicates that the first canonical discriminant function captures most of the differences among the groups (cumulative proportion of total dispersion is 95.4 % with the first canonical discriminant function only). Thus, end cells length and spore width are the most useful biometric features useful to separate the three taxa in question. Multivariate test statistics were applied to test the null hypothesis of identity of the three groups X. chrysenteron, X. cisalpinus and X. pruinatus. The null hypothesis was clearly rejected for all three taxa (Table 4).

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addition, the other three lineages of X. cisalpinus, X. dryophilus and X. pruinatus are closely related and have sister group relationships. The analysed sequences of X. ripariellus include material of X. catalaunicus (IB19980360). The results from our phylogenetic analysis confirm the synonymy of these two species epithets, as already shown in an earlier study based on morphological characters (Ladurner et al. 2001). The phylogenetic analyses confirm our species concepts, as all sequences belonging to one morphospecies fell into the same clade. However, four sequences retrieved from GenBank (X. chrysenteron AF050547, AF071537, X. pruinatus AF402140 and X. rubellus AF050649) clustered in different clades, demonstrating the present taxonomic confusion around these taxa. The collections used for these four sequences are misidentified. This further emphasises the urgent need of broadly accepted morphological species concepts. TAXONOMY Xerocomus cisalpinus Simonini, Ladurner & Peintner, sp. nov. (Figs 6, 14–17) Etym.: Occurring south of the Alps.

Phylogeny of the Xerocomus chrysenteron species complex Phylogenetic analyses were performed with PAUP* 4.0b8 (Swofford 1998). 34 rDNA-LSU sequences of nine Xerocomus spp. treated in this study were aligned manually in the data editor of PAUP*. After exclusion of gaps and areas with ambiguous alignment, 975 characters were analysed: 730 characters were constant, 51 variable characters were parsimony-uninformative and 94 characters were parsimony-informative. Heuristic searches resulted in 20 000 most parsimonious trees (MPT) with tree length of 234 steps, a consistency index (CI) of 0.739, a retention index (RI) of 0.845 and a rescaled consistency index (RC) of 0.625. The phylogenetic analysis with maximum likelihood resulted in 11 ML trees (xln likelihood=2602.04007), one of them is presented in Fig. 5. The X. chrysenteron species complex can clearly be separated from the outgroup X. subtomentosus (L. : Fr.) Que´l. (BS 100 %). Within the X. chrysenteron complex, X. rubellus sequences fall into a distinct clade, which is sister to the other seven included taxa X. chrysenteron, X. porosporus, X. cisalpinus, X. dryophilus, X. pruinatus, X. ripariellus and X. fennicus. Five of these seven species, namely X. porosporus, X. cisalpinus, X. dryophilus and the species pair X. ripariellus/X. fennicus represent independent lineages with high bootstrap support (BS >70 %). A clade of X. chrysenteron species is indicated by tree topologies, but with low bootstrap support (BS 53 %). Basal relationships within the seven core species of the X. chrysenteron complex are not resolved, but tree topologies indicate X. chrysenteron as a basal taxon, and an intermediate position of X. porosporus. X. ripariellus and X. fennicus are sister groups. In

Habitus X. chrysenteronis similis ; statura minuta ; pileus 35–80 mm diam, convexus, pallide ochraceogriseus, olivaceo vel roseo colore suffusus, vulgo obscurior in maturitate, cito minutis rimis ubique notatus; subpellis roseorubra saepe inter cuticulae rimas conspicitur. Pori et tubuli flavi. Stipes 45– 80r4–9 mm, sursum flavus, ad basim ruber. Caro pallide flava, exalbescens, lente sed valde ad caeruleum colorem varians ad stipitis basim. Sporae leviter striatae ut in X. pruinato, mediae, (10.5–) 13¡1 (16)r(4–) 4.5¡0.2 (–5.5) mm ; Q=(2.18) 2.77¡0.18 (3.40); cuticulae partes plerumque cylindricae, super membranam incrustatae, mediae, (13) 5¡17 (123)r(4.5–) 13¡4 (–28.5) mm ; Q=(1.51) 4.06¡1.50 (14.00). Hyphae amyloideae ad stipitis basim ut in X. pruinato. Typus: Italia : Reggio Emilia: prope Villaminozzo in loco dicto Resgadore, alt. 1100 m supra mare, in radura Fagus sylvaticae nemore, 25 Sept. 1998, Franca Franceschetti (IB19980850 – holotypus).

Pileus 35–80 mm broad, fleshy, convex to pulvinate to almost plane, sometimes also slightly depressed at the centre ; surface dry, tomentose, independently from the weather conditions always very soon areolate (cracking into minute scabs), the pink-reddish subpellis usually visible in the fissures, but exceptionally (old basidiomes, dry weather conditions) also only the whitish flesh visible in the fissures ; very variable in colour : in very young basidiomes with uncracked pileus cream-greyish, pale ochre-brown with an olive shade (1/2D2/3, 3B/C2, 4/5B/C2/3, 5/6D2 to 5/6D4), often with a pale pink line at the extreme margin (6/7A2/3), sometimes with a dispersed rosey shade (7D3 to 6, 8E3/4, 8 to 11D2 to 4) or in wet weather conditions even entirely fuchsia-pink, these tinges more intense at the pileus margin (9/10A to C5 to 7) ; when cracked, the pileipellis scabs darker greyishbrown-olive (6/7E 4 to 6), flesh-coloured to rosey in the

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Key to the species of the Xerocomus chrysenteron complex 1

Spores truncate Spores not truncate

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2(1) Spores smooth, basidiomes with dull colours, greyish brown, red tinges only weakly developed, often with a reddish ring zone at the stipe apex, thermophil species associated with Quercus, Carpinus, Fagus, etc. . . . . . . . X. porosporus Spores distinctly striate, young basidiomes vivid red, fading to olivaceous grey when older, with Betula, Alnus, etc., preferring nitrogenrich habitats . . . . . . . . . . . . . . . . . . X. fennicus 3(1) Spore quotient on average <2.5, mainly associated with deciduous trees Spore quotient on average >2.5, with both deciduous trees and conifers

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4(3) Spores on average less than 5.5 mm broad, honey-coloured; context in the base of the stipe with small red dots formed by a striking flamered pigment, or a thin red line delimiting the extreme base of the stipe; often associated with Tilia (but also with Quercus and other deciduous trees and shrubs), preferring ‘disturbed’ sites, e.g. gardens, borders of trails, etc. . . . . . X. rubellus Spores on average broader than 5.5 mm, dark brown to intense yellow-brown; stipe context intensely red coloured in the lower part (reminding Boletus luridus), mainly under Quercus (and Pinus?) . . . . . . . . X. dryophilus 5(3) Spore surface smooth; usually associated with conifers or with Fagus in southern Europe, pileipellis a palisadoderm of moderately long, cylindrical, parallel running hyphae, the terminal cells¡shortly cylindrical to broadly ovoid, acorn-shaped to cystidioid, exceptionally subspherical . . . . . . . . . . . . . . . . . X. chrysenteron Spore surface striate (striation difficult to see with light microscopy) ; pileipellis a palisadoderm of extremely variably shaped elements, the terminal elements slender cylindrical to ¡broadly clavate to pear shaped, ovoid to spherical . . . . . . 6 6(5) Typical ‘pruinatus-hyphae’ lacking, associated with Alnus, Populus, Salix, etc., in humid, nitrogen rich places, context whitish to yellow, spores on average less than 5 mm broad . . . . . . . . . . . . X. ripariellus With ‘pruinatus-hyphae’ (thick-walled, amyloid hyphae in stipe context) . . . . . . . . . 7 7(6) Spores on average broader than 5 mm, associated with Castanea, Fagus, Picea, Pinus, etc., similar to X. chrysenteron, context bright yellow and only weakly bluing when cut. Cap cuticle usually not areolate, few radial fissures start from the pileus margin. X. pruinatus Spores on average less than 5 mm broad, associated with Quercus, Pinus, etc., similar to X. chrysenteron, context pale yellow, whitish when cut, strongly bluing at least in the lower stipe. Cap cuticle always and rapidly areolate. . . . . . X. cisalpinus

cracks (6/7A2 to 4, 8A2 to 5, 7 to 9A3 to 5, S199, 200) ; dull cream-whitish (3/4A2) cracks appear beneath the brownish (6/7F5 to 7) pileipellis layer in dry weather. The pileipellis is often so minutely cracked that the intense pink colour of the subpellis beneath dominates and the pileus colour seems pinkish red. Tubes up to 5–10 mm long, separable, depressed to sinuate around the stipe, sometimes slightly decurrent ; initially bright yellow with olive shades (1/2A7/8, 3A/ B7/8), later dull greenish yellow (2C/D7/8, 3/4C/D6), slowly bluing when bruised. Pores angular, uneven, in mature basidiomes 0.7–2 mm diam, concolorous to the tubes, slowly and only weakly bluing (S351, 352, 366, 367) when bruised. Stipe 45–80 mm long, 4–9 mm broad, cylindrical, sometimes with somewhat widened apex, usually tapering towards the base but also weakly clavate or bulbose; vivid yellow at the apex (3A4 to 8), vivid red or dull red in the lower half (8–10A to C8, 8–11C/D7/8, S121, 136, 151, 166), more rarely red at the extreme base only or entirely yellow with some vague reddish striae only ; in older, overmature specimens the reddish tinges turn into a dull red (10/11E/F7/8, S101, 102) ; the stipe surface becomes dull ochre-brown (S336 to S338) when injured ; sometimes the stipe appears orange due to red dots or red striae in the yellow parts of the stipe; the stipe surface finely floccose or fibrillose, without reticulum ; at the extreme base pale ochre-brown (S336 to 338) ; basal mycelium white. Context soft in the pileus, fibrillose and brittle in the stipe ; in the pileus pale yellow (3A3) soon fading to dirty whitish when exposed (29/30A2), below the pileipellis showing a fine red line, particularly distinct when the pileus shows no red shade, otherwise

inconspicuous; in the stipe vivid yellow (3A4/5) or yellow with dull reddish tinges that are concolorous to the cortex (8 to 11C/D7/8), slowly but strongly bluing (S446 to 448) in the whole stipe or only in its lower part when cut, the red areas in the stipe base darkening, the stipe context at the extreme base dull ochre-brownish (S336 to 339) ; in the area of the stipe/cap connection vivid yellow and only slightly turning green (2A/B7/8) or more or less pale aquamarine (S331 to 335) when cut ; smell not distinctive, somewhat like wet iron ; taste not distinctive, slightly sour. Spore deposit brownish with some olive tinges. Macrochemical reactions weak fleeting-amyloid reaction in stipe context and hymenophoral trama of dry specimen. Basidiospores (10.5–)13¡1(16)r(4–)4.5¡0.5 (–5.5) mm, Q=(2.2) 2.8¡0.2 (3.4), V=(92) 149¡37 (465) mm3 (n=558), subfusiform, slender, with a weakly developed but distinct supra-apicular depression, spore wall up to 0.5 mm thick, intensely honey-coloured and with one to two guttules when mature, the spore surface finely longitudinally striate. Basidia 30–45r 10–13 mm (n=15), inconspicuous, clavate, hyaline to yellowish, mainly 4-spored. Pleurocystidia scarce, 50–90r7–15 mm (n=15), ventricose-fusiform, with hyaline to slightly yellowish content, cheilocystidia similar, scattered. Pileipellis a palisadoderm strongly reminiscent of the pileipellis of X. chrysenteron, terminal elements mostly cylindrical and somewhat tapering towards the apex, often papillate, also acornshaped to exceptionally subspherical, (13–)51¡17 (–123)r(4.5–)13¡4(–28.5) mm, Q=(1.5) 4.1¡1.5 (–14) in Congo red, n=(588), terminal and subterminal elements moderately to heavily incrusted, only

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X. chrysenteron AF347103 X. chrysenteron IB19990952

X. chrysenteron

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X. chrysenteron IB20000405 X. chrysenteron IB19990951 X. porosporus AF050645

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X. cisalpinus IB19980850 X. chrysenteron AF071537 X. dryophilus IB19991057

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X. pruinatus IB19980366

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X. pruinatus AF050644 X. chrysenteron AF050647 X. ripariellus IB19980360 X. ripariellus GR21189 57 69

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X. ripariellus GR930920 93

X. ripariellus GR22465

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X. fennicus RJ126

X. pruinatus AF402140 X. rubellus IB19990917 100 100

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X. rubellus GS961

X. rubellus

X. rubellus GS1044 100

X. subtomentosus IB19980452 X. subtomentosus IB19991000

X. subtomentosus AF139716 –0.005 substitutions/site

Fig. 5. One of 11 ML trees with xln likelihood of 2602.04007. MP bootstrap values are given above branches, ML bootstrap values >60 % are given below branches. Branches not present in the ML strict consensus tree are grey. Branches present in both strict consensus trees (MP and ML) are bold. The spores of a taxon are shown right of the respective clade. Xerocomus subtomentosus was used as outgroup.

exceptionally smooth. Stipitipellis consisting of loosely interwoven, 3–5 mm broad hyphae with hyaline to yellowish content. A well-developed caulohymenium with spore-forming caulobasidia and caulocystidia is present in the upper half of the stipe. Stipe texture formed of parallel running, hyaline, 2–4 mm broad hyphae; in the lower half of the stipe the hyphae are

differentiated into a special hyphal type (‘ pruinatushyphae’) in irregular intervals : inconspicuous, thinwalled and moderately broad hyphae extend more or less abruptly to strikingly thick-walled, up to 30 mm broad, irregularly formed hyphae of variable length, which, depending on their individual developmental stage, show a weak to strong amyloid reaction of the

U. Peintner, H. Ladurner and G. Simonini hyphal wall. The inner surface of these hyphae often shows a particular crater-like ornamented surface. No clamp connections observed. Microchemical reactions : with the exception of the amyloid reacting hyphae in the stipe, no particular reproducible macrochemical reactions were observed. Habitat and distribution : Xerocomus cisalpinus was repeatedly collected in Italy, south of the Alps, in Mediterranean areas : in the regions Lazio, Emilia Romagna, Sardinia, Calabria ; Trentino Alto Adige (South Tyrol) ; it fruits in the autumn and late autumn from sea level to 1150 m, usually scattered, in groups. X. cisalpinus is associated with broad-leaved trees, mainly Quercus spp. (Q. cerris, Q. robur, Q. pubescens, Q. frainetto, Q. suber, Q. ilex), but also with Fagus sylvatica ; the occurrence under Pinus has to be confirmed. Like many other mushroom species, Xerocomus cisalpinus is difficult to recognise in the field based on macromorphological features alone. Because of its obvious cracked cap cuticle, X. cisalpinus is somewhat similar to X. chrysenteron. However, X. cisalpinus can easily be distinguished from both X. chrysenteron and X. pruinatus based on microscopic features : it has a pileipellis structure consisting of substantial cylindrical hyphae similar to X. chrysenteron, but the faintly striate spores and amyloid ‘pruinatus- hyphae ’ are typical for X. pruinatus. In addition, the spores of X. cisalpinus are significantly smaller than those of both X. chrysenteron and X. pruinatus. No similar taxa could be found in an extensive literature screening of publications dealing with Boletus, Boletellus or Xerocomus. Only Boletellus episcopalis could have some affinities with our collections : This bolete from Madagascar is described as smallsized, with a slender red stipe slightly inflated at the base, the cap cuticle is dark brown, cracking in big scabs and the spores are described as faintly ridged (Heim & Perreau 1963). We examined the typus of Boletellus episcopalis (Fig. 18), which is conserved in ethanol in PC(RH128). B. episcopalis is undoubtedly different from X. cisalpinus : the spores of B. episcopalis have a pronouncedly striate surface, which is easily visible with a light microscope ; the spores are (12–) 13.5¡1 (15)r(5.5–)6¡0.5(–6.5) mm ; Q=(2) 2.3¡0.1 (2.6) ; V=(199) 252¡27 (322) mm3 (n=31), and thus clearly wider than spores of X. cisalpinus. ‘Pruinatushyphae’ are lacking. The pileipellis is in bad condition, but it appears more like a trichodermium than like a palisadoderm ; the weakly incrusted pileipellis end cells are (26.5–)82¡39(–154.5)r(5.5–)10¡2.5 (–15.5) mm ; Q=(2) 8.7¡4.9 (22.1) (n=31) and much more elongated and usually tapering towards the tip (Fig. 18). Additional collections examined : Italy: Sardegna : Nuoro, Urzulei, Codula di Luna, alt. 50 m, with Quercus ilex, 2 Nov. 1990, G. Simonini (IB19900700). Calabria : Cosenza, Acri, Cugnale di Falcone, alt. 1000 m, with Q. cerris, Pinus calabrica, 8 Sept. 1995, C. Lavorato (IB19950800); Reggio Calabria, Caulonia, Monte Gremi, alt. 1050 m, with Q. ilex

667 and P. nigra, 29 Oct. 1995, G. Simonini (IB19950801); Emilia Romagna, Reggio Emilia, Villaminozzo, La Magolese, alt. 1150 m, with Fagus sylvatica, 31 Aug. 1997, G. Simonini (IB19970995). Sardegna : Sassari, S. Teresa di Gallura, Rena Maiore, alt. 5 m, with P. pinea, Quercus sp., 29 Oct. 2000, G. Consiglio (IB20000702); Sassari, Tempio Pausania, Aggius, alt. 500 m, with Q. suber, 3 Nov. 2000, G. Redeuilh (IB20000704, GS2310a). Lazio: Latina, Sabaudia, Selva del Circeo, alt. 17 m, with Q. suber, 29 Nov. 2001, A. Riva (GS2405) ; loc. cit., with Quercus spp., 30 Nov. 2001, M. Manavella (GS 2400, GS2403); loc. cit., P. Signorello (GS2404) ; loc. cit., M. Manavella (GS2406) ; loc. cit., G. Simonini (GS2407, IB20010605) ; loc. cit., 1 Dec. 2001, G. Simonini (GS2401) ; loc. cit., M. Manavella (GS2402). Trentino Alto Adige: Bolzano, Naturns, Ladurnhof, alt. 800 m, with Quercus pubescens, 16 May 1999, H. Ladurner (IB19991019).

Xerocomus chrysenteron (Bull.) Que´l. 1888 (Figs 7, 19–20) Basionym : Boletus chrysenteron Bull., Hist. Champ. : 328 (1791). Pileus 30–120 (–200) mm broad, convex to pulvinate to almost hemispherical when young, more convex to applanate when old, sometimes slightly depressed at the centre, initially blackish-brown to chestnut-brown, exceptionally more beige or clay-coloured, often with an olive tinge, also honey-brown, towards the margin more yellowish, in older basidiomes often fading from the centre to a pale clay-colour or greyish brown ; the pileus margin often more olive-yellow or even reddish coloured and then clearly contrasting to the rest of the pileus in older basidiomes (in dry periods already in young specimens). Pileus surface dry, young tomentose, becoming ¡velutinous-tomentose, never viscid, often cracking deeply into coarse clods, typically areolate with the reddish subpellis visible in the fissures. Tubes up to 11 mm long, slightly depressed, sinuate around the stipe, in overmature basidiomes sometimes like decurrent, initially pale yellow, then dull greenishyellow, soon more yellowish-green. Pores large, 0.7–1.5 mm diam, angular, uneven, concolorous to or more olive-yellow, sometimes with rust-red spots, weakly to distinctly bluing when bruised. Stipe 30–90r5–25 mm, cylindrical, solid, somewhat tapering towards the base or also slightly widened. Surface dry, smooth to ingrown fibrillose, yellow at the apex, towards the base or completely or partially red coloured by fine reddish scales or fibrils. Context whitish-yellow to yellow in the pileus, below the pileipellis showing a fine, but distinct red line, more intensely yellow in the stipe, below red coloured parts of the stipitipellis a pink to reddish coloration of the context might be observed. Context in the whole basidiome ¡bluing when bruised. Basal mycelium whitish to pale yellow. Smell and taste inconspicuous to slightly sour. Colour of dried specimen inconspicuous. Spore deposit brownish. Basidiospores (9–)14¡1(–17)r(4–)5¡0.5(–6.5) mm, Q=(2.0) 2.7¡0.2 (3.5), V=(97) 194¡42 (349) mm3 in

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Fig. 14. Basidiomes of Xerocomus cisalpinus (holotype). Bar=1 cm.

Fig. 15. Basidia, basidiospores, two cheilocystidia (left) and two pleurocystidia (right) of Xerocomus cisalpinus (IB19980850 – holotype). Bar=10 mm.

KOH 3% (n=930), subfusiform, with well-pronounced supra-apicular depression, moderately thick-walled (wall up to 0.5 mm thick), intensely honey-coloured and with one to two guttules when mature, smooth, inamyloid, not dextrinoid. Spores from fresh basidiomes sometimes staining intensely dark greyish-blue when tested for amyloidity, but spores of the same basidiomes react completely negative in dry condition. Basidia 30–40(–50)r10–15 mm (n=15), clavate, hyaline to yellowish in KOH 3 %; mainly 4-spored. Pleurocystidia numerous, 50–100r10–18 mm (n=15), usually ventricose-fusiform with slender elongated neck and rounded tip in KOH 3 %. Cheilocystidia similar,

669

Fig. 16. Pileipellis end cells and basidiospores of Xerocomus cisalpinus (IB19980850 – holotype). Bars=20 mm for pileipellis, and 10 mm for basidiospores.

numerous. Pileipellis a palisadoderm of cylindrical, mostly parallel, often heavily incrusted hyphae. Terminal elements versiform, cylindrical, cystidioid, bullet-shaped, acorn-shaped, exceptionally subspherical, sometimes with elongated flexuous neck, irregular, (7.5–)37¡13.5(–104.5)r(5–)13.5¡4(–57.5) mm, Q= (0.8) 2.9¡1.0 (9.7) in Congo red (n=930), subterminal elements as broad as the terminal element, exceptionally also distinctly widened. Terminal elements smooth to heavily incrusted, the pigment often forming pronounced yellowish-brown scabs (KOH 3 %). Stipitipellis a texture of loosely interwoven, richly ramified, mostly hyaline, 3–7 mm broad hyphae. A well-developed caulohymenium of basidia, basidioles and caulocystidia covers the entire stipe; in the upper third of the stipe a continuous layer of caulohymenium is observed, towards the base it turns into ¡isolated bundles of caulohymenial elements. Stipe texture consisting of thin-walled, hyaline, 6–15 mm broad hyphae (KOH 3%). No clamp connections observed. Neither true amyloid, dextrinoid, nor other microchemical reactions were observed in any part of the basidiomes. Habitat and distribution : X. chrysenteron is widespread and common all over Europe, and usually occurs under conifers. This taxon seems to prefer climatically moderate zones and undisturbed habitats. X. chrysenteron is only exceptionally found under deciduous trees, e.g. Fagus, but then preferably in colder, mountainous habitats in southern Europe. The main fruiting period is July–September. Collections examined. Austria : Tyrol : Innsbruck, Hungerburg, with Picea abies, 8 Aug. 1999, H. Ladurner (IB19990951); Afling, with Picea abies and Corylus avellana, 13 Aug. 1999, R. Kuhnert-Finkernagel (IB19950800); Tulfes,

Figs 6–13. Basidiomes of the eight species of the X. chrysenteron species complex in their natural habitat. Fig. 6. Xerocomus cisalpinus (IB20010605). Fig. 7. X. chrysenteron (IB19970995). Fig. 8. X. dryophilus (GS2004). Fig. 9. X. fennicus (JV7150F); photograph by Jukka Vauras. Fig. 10. X. porosporus (GS2106). Fig. 11. X. pruinatus (GS2017). Fig. 12. X. ripariellus (GR22541P – holotype p.p.) ; photograph by Guy Redeuilh. Fig. 13. X. rubellus (GS1894). Photographs where not otherwise stated by G. S.

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Fig. 18. Pileipellis end cells and basidiospores of Boletellus episcopalis (holotype). Bar=20 mm for pileipellis, and 10 mm for basidiospores.

Fig. 17. Scanning electron microscopy (SEM) pictures of basidiospores of Xerocomus cisalpinus (IB19980850 – holotype) with finely longitudinally striate spore surface. Bar=2 mm. Hasental, with Pinus, Picea, 28 July 2000, H. Ladurner (IB20000405). – Italy : Bozen : Partschins, Greithof, with Picea, H. Ladurner (IB19970898); for additional material see Ladurner (2001a). – Switzerland : Zu¨rich : with Picea abies and Fagus sylvatica, 20 Aug. 1985, C. Lavorato (GS1541).

Xerocomus dryophilus (Thiers) Singer 1986 (Figs 8, 21) Basionym : Boletus dryophilus Thiers, Calif. Mush. : 82 (1975). Pileus 40–100 mm broad, young convex to pulvinate, with age often more flattened, then often also with an irregularly bent margin ; young brownish-red to red, sometimes the red cap covered by an olive-brownish tomentum, with maturity at least in certain cases dark red, in other cases the red tinges fade and the brownolive colour predominates. Young basidiomes often with a rosy hue caused by the pale tomentum ; sometimes the still involute margin appears entirely rosered ; pileus surface dry to slightly viscid, in young basidiomes strongly tomentose, in older ones often nearly glabrous, occasionally wrinkled or cracked into fine to coarse scabs.

Tubes to 10 mm long, weakly depressed to narrowly adnexed around the stipe, in old basidiomes sometimes appearing decurrent, olive-yellow, ¡bluing when injured. Pores large, 0.5–1 mm diam, angular, uneven, concolorous with the tubes, bluing when bruised. Stipe 50–80r10–20 mm, solid, cylindrical or apically slightly enlarged. Stipe surface dry to somewhat viscid, smooth. Stipe apex yellow, towards the base reddish, the red tinges more pronounced in older basidiomes. The red coloured parts of the stipe darkening on pressure. Context in the stipe concolorous with its surface, intensely red in the lower half of the stipe (reminiscent of B. luridus) and turning – with the exception of the stipe base – everywhere blue when cut. Context in the pileus yellow, weakly reddish under the pileipellis, only weakly bluing. Basal mycelium yellowish-white. Taste and smell not significant. Colour of exsiccata not significant. Spore deposit brown. Basidiospores (11–)13¡1(–17.5)r(5–)6¡0.5 (–7) mm, Q=(1.7) 2.2¡0.2 (3), V=(151) 252¡46 (414) mm3 in KOH 3% (n=465), elliptical, with a mostly inconspicuous supra-apicular depression, moderately to distinctly thick-walled (wall up to 0.9 mm thick), mature intensely honey-coloured to brown with one to two guttules, smooth, not amyloid, not dextrinoid. Basidia 35–45(–50)r11–14 mm (n=15), clavate, hyaline to yellowish in KOH 3% ; mainly 4-spored. Pleurocystidia numerous, 60–85r10–15 mm (n=15), somewhat irregular fusoid-ventricose with rounded apex, thin-walled, hyaline to yellowish in KOH 3%. Cheilocystidia similar, numerous. Pileipellis a trichoderm consisting of long, slender, partially interwoven, occasionally also branched, septate hyphae. Terminal elements cylindrical, slender, elongated, also ovoid or cystidiod, sometimes branched at the apex or diverticulate, usually tapering, but also with rounded ends, sometimes anastomosing, (8.5–)54.5¡25(–194.5) r(5–)9¡3.5(–14) mm, Q=(1.3) 6.2¡3.4 (23.1) in Congo red (n=465), often smooth, otherwise finely incrusted, sometimes also showing a granular yellowish

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19

20

21

22

23

24

25

26

27

28

Figs 19–28. Pileipellis and basidiospores of species of the Xerocomus chrysenteron group. Fig. 19. X. chrysenteron (GS1541). Fig. 20. X. chrysenteron (IB19970898). Fig. 21. X. dryophilus (GS1941). Fig. 22. X. fennicus (Typus). Fig. 23. X. porosporus (IB1988304). Fig. 24. X. ripariellus (GR21189). Fig. 25. X. pruinatus (IB19991024). Fig. 26. X. pruinatus (IB19950970). Fig. 27. X. rubellus (GS961). Fig. 28. X. rubellus (IB19990917). Bar=20 mm for pileipellis, and 10 mm for basidiospores.

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex content in KOH 3 %. Hyphae of the lower pileipellis moderately to distinctly incrusted, subterminal elements only exceptionally widened. Stipitipellis a loose texture of slender, loosely interwoven, abundantly ramified, hyaline hyphae of 3–5 mm diam with slightly granulose content. Bundles of clavate, hyaline elements occur over the entire length of the stipe. Stipe texture reminding X. subtomentosus, consisting of thin- to thick-walled (wall to 1.5 mm thick) 6–10 mm broad hyphae, the hyphal content in KOH 3 % partly finelygranulose. Without clamp connections. No particular microchemical reactions observed in any parts of the basidiomes. Habitat and distribution : X. dryophilus probably occurs in most thermophilic mixed deciduous forests with a preference for Quercus spp. It is known from Northand Central America and from Europe (Mediterranean area), where it fruits solitarily, to gregarious in deciduous forests. In Europe X. dryophilus was found under Q. ilex, Q. pubescens, Q. robur and Fagus silvatica. On Mallorca the species was collected also in the dune zone under Pinus halepensis. The identity of American and European material was confirmed by Harry Thiers based on morphological characters (Simonini 1994). Collections examined. Croatia : Loscinji, airport, with Pistacia lentiscus, Erica arborea, Quercus ilex, Juniperus oxycedrus, 6 Oct. 1999, Anonymous (IB19990901). – Spain : Alcolea del Pinar, 5 Sept. 1999, Anonymous (IB19991057); for additional material see Ladurner (2001a). – Italy : Reggio Emilia: near Villaminozzo, Calizzo, with Q. pubescens, 21 Sept. 1999, G. Simonini (GS1941) ; Emilia Romagna, Quattrocastella, Parco di Roncolo, alt. 320 m, with Q. pubescens, 3 Oct. 2000, G. Simonini (GS2004). – Mexico : Baja California, with Quercus agrifolia, 13 Feb.1993, A. Montecchi, det. G. Simonini (GS 0933); Canon de las Animas, with Q. agrifolia, 28 Jan. 1998, G. Moreno (IB19980827); for additional material see Simonini (1994).

Xerocomus fennicus (Harmaja) Ladurner & Simonini, comb. nov. (Figs 9, 22) Basionym : Boletellus fennicus Harmaja, Karstenia 39(2) : 37 (1999). Pileus 10–70 mm broad, convex to plane, initially bright red, when older the colour changes to brown from the centre, but some red tinges usually remain persistent, particularly at the pileus margin. Pileus surface dry, ¡finely tomentose, often finely cracking from the margin towards the centre and showing the yellowish context in the fissures. Tubes 2–8 mm long, adnate to depressed around the stipe, bright yellow, intensely bluing when injured. Pores usually rather large, roundish to angular, concolorous with the tubes, intensely bluing when bruised. Stipe 20–70r4–15 mm, solid, cylindrical, concolorous with the pileus, the apex usually paler or yellowish, often ¡brown in old basidiomes, finely floccose or fibrillose.

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Context yellowish white, strongly bluing. Taste and smell not significant. Basal mycelium pale, whitish. Colour of dried specimen not significant. Spore deposit brown. Macrochemical reactions exsiccata with a weak ‘fleeting-amyloid ’ reaction in stipe context and hymenophoral trama. Spores (10–)12.5¡1(–15.5)r(4–)5¡0.5(–6.5) mm, Q=(2) 2.6¡0.2 (3.4), V=(85) 152¡27 (278) mm3 in KOH 3% (n=2635), some slender and nearly cylindrical, others elliptic to broadly subfusiform, rather variable in shape, size and differentiation of the spore apex, mature spores usually with a distinctly truncate apex, but, in some specimens only few truncate spores are observed. Depending on their shape, the spores show a distinct supra-apicular depression; they are usually moderately thick-walled (wall to 0.6 mm thick), mainly honey-coloured and mono- to biguttulate when mature, distinctly longitudinally striate (striation even stronger developed than in X. ripariellus), inamyloid, not dextrinoid. Basidia 30–45r9–12 (n=15), clavate, hyaline to yellowish in KOH 3%, mainly 4-spored. Pleurocystidia scarce, 35–80r10–18 mm (n=15), ventricose-fusiform, often tapering at the apex or with elongated neck, most hyaline, rarely with brownish content in KOH 3%. Cheilocystidia similar, scattered. Pileipellis a physalo-palisadoderm consisting of moderately long, cylindrical, septate hyphae. Terminal elements versiform, rather variable in shape and size, the shape of the terminal elements ranging from cylindrical with rounded or tapering apex to bullet-shaped, broadly pear-shaped, ovoid, elliptical, subspherical or almost spherical, sometimes with elongated outgrows, (10–)32¡11(–77)r(4–)15.5¡5(–43) mm, Q=(0.8) 2.2¡0.8 (7.8) in Congo red (n=685), the penultimate element only exceptionally widened. Pileipellis elements smooth to heavily incrusted, in some collections with large, refractive plaques (KOH 3%) not reacting with Congo red. Stipitipellis consisting of slender, loosely interwoven, mainly hyaline, 3–5 mm broad hyphae with slightly granulose content. In the upper half of the stipe a closed layer of well-developed caulohymenium consists of bundles of caulocystidia and spore-forming caulobasidia. Stipe texture in most collections inconspicuous, formed of parallel running, ¡hyaline, 3–10 mm broad hyphae. Without clamp connections. In seven out of 94 collections of X. fennicus amyloid ‘pruinatus-hyphae’ were found in the stipe context ; no other particular microchemical reactions were observed. Habitat and distribution : Up to now Xerocomux fennicus is known only from Finland (hemiboreal and southern boreal zone) and Austria (one collection near Salzburg), possibly also from Belgium (Schreiner 2000). The fungus occurs in rich deciduous or mixed forests, also in alder thickets on lakeshores and seashores, pastures, parks, yards, and meadows, favouring nitrogen-rich soils, (nearly) always associated with Betula, often accompanied by Alnus sp. and Urtica.

U. Peintner, H. Ladurner and G. Simonini Collections examined: Finland: Varsinais-Suomi : Nauvo commune, Berghamn, Boska¨r, with Betula spp., Alnus glutinosa, Picea abies, 3 Sep. 1992, J. Vauras JV7150F. Etela¨Ha¨me (Tavastia australis) : Ylo¨ja¨rvi, Soppeenma¨ki, park, beneath ash (Fraxinus) trees, five fruit bodies, 5 Aug. 1993, R. Ja¨rvenpa¨a¨ (OULU F46399). Uusimaa (Nylandia): Helsinki, Myllypuro-Puotinharju, herb-rich mixed forest (Betula, Picea, Populus tremula, Prunus padus, Salix caprea, Sorbus, Aegopodium, Filipendula, Rubus idaeus, Urtica dioica), on detritus on soft humus, 19 Aug. 1988, R. Saarenoksa (H). ; Lammi, Pappila, biological Station of the University of Helsinki, in park under Betula, 9 Sept. 1968, H. Harmaja (H – holotype of Boletallus fennicus).

Xerocomus porosporus Imler, Bull. Soc. mycol. Fr. 74: 97 (1958). (Figs 10, 23) Pileus 20–60(–139) mm broad, young pulvinate, slightly convex, then applanate, sometimes irregularly bent, olive-ochre, olive-brown, sometimes nearly blackishbrown when young, paler when old, also with greyish tinges, dull. Pileus margin slightly paler when young, often more greyish. Pileus surface dry, ¡finely tomentose, often finely to distinctly areolate and showing the pale yellowish to whitish context in the fissures. Tubes 13–20 mm long, adnate to adnexed to the stipe, initially pale yellow, later yellowish-green to dull olive-green, bluing when injured. Pores rather narrow, 0.2–0.5 mm in diameter, roundish to angular, concolorous to the tubes or somewhat rust-red in older basidiomes, bluing when bruised. Stipe 30–80(–110)r4–20 mm, solid, cylindrical, sometimes with widened apex, usually tapering towards the base, concolorous with the pileus, in old basidiomes even darker, only at the apex with a narrow yellow(-ish) zone, downwards dull greyish-brown, olive-brown or blackish brown, often with a narrow reddish zone in the upper part or with inconspicuous reddish patches in the lower part, finely floccose or fibrillose (in older basidiomes). Context whitish to cream-coloured in the pileus, in the stipe also pale yellow, there sometimes with reddish tinges, brownish to dark brown in the stipe base, bluing throughout the whole basidiomes. Taste and smell inconspicuous. Basal mycelium whitish to greyish-white. Colour of exsiccata inconspicuous. Spore deposit brownish. Macrochemical reactions not observed. Basidiospores (11–)14¡1(–16.5)r(5–)6¡0.5 (–6.5) mm, Q=(1.8) 2.5¡0.3 (3.1), V=(144) 241¡40 (355) mm3 in KOH 3% (n=217), elliptical to subfusiform with a distinctly truncate apex in at least 80% of mature spores, the spore apex thick-walled at the two apical angles and often markedly thin-walled between these angles, with no distinct germ pore but apically only moderately thick-walled (wall up to 0.7 mm thick), with distinct supra-apicular depression, intensely honey-coloured to yellow-brown, and with one to two guttules when mature, smooth, inamyloid, not dextrinoid. Basidia 30–40r9–13 (n=15), with 3–5 mm

673 long sterigmata inconspicuous, clavate, hyaline to yellowish in KOH 3 % mainly 4-spored. Pleurocystidia scarce, 30–90r9–16 mm (n=15), slender, mainly ventricose-fusiform, hyaline, rarely with brownish content in KOH 3%. Cheilocystidia similar. Pileipellis a physalo-palisadoderm consisting of cylindrical, mostly parallel running hyphae. Terminal elements versiform, cylindrical, cystidioid, acorn-shaped, bullet-shaped, exceptionally subspherical, irregular, (8–)33¡11.5(–68) r(4.5–)7.5¡2(–15) mm, Q=(0.8) 2.6¡1.2 (7.6) in Congo red (n=217). Pileipellis elements smooth to heavily incrusted (KOH 3 %). Stipitipellis consisting of slender, loosely interwoven, mainly hyaline, 3–5 mm broad hyphae with slightly granulose content. In the upper half of stipe a closed layer of well-developed caulohymenium consists of partly spore-bearing caulobasidia (basidioles) and caulocystidia . Stipe texture inconspicuous, formed of parallel, ¡hyaline, 2–4 mm broad hyphae. Without clamp connections. No particular microchemical reactions observed. Neither true amyloid nor dextrinoid reactions observed in any parts of the basidiomes. Habitat and distribution : Xerocomus porosporus is widespread but rather rare in Europe with a preference for climatically favoured regions. It is found from May to November under deciduous trees (Fagus, Carpinus betulus, Quercus cerris, Q. petraea, Q. robur, Q. suber, Betula, Crataegus, Rubus) and in mixed forests, exceptionally also under Pinus and Picea. Collections examined: Spain : Montseny, park of the Castel Montseny, with Fraxinus, Cedrus, Sorbus aucuparia, 18 Oct. 1988, R. Po¨der (IB1988304). – Italy: South Tyrol : Naturns, with Quercus pubescens, Juniperus communis and Fraxinus ornus, 28 Aug. 1999, H. Ladurner (IB1999957) ; Emilia Romagna, Villaminozzo, Pian Vallese, alt. 1350 m, with Fagus sylvatica, 18 Jul. 1999, G. Simonini (GS2106). For additional material see Ladurner (2001a).

Xerocomus pruinatus (Fr.) Que´l. 1888 (Figs 1, 11, 25–26) Basionym : Boletus pruinatus Fr., in Fries & Ho¨k, Boleti : 9 (1835). Pileus 30–100 (–150) mm broad, young convex, pulvinate, later applanate to flattened or depressed, fleshy, initially usually blackish brown, deep purple-brown, deep brown, later more dark brown to purple, exceptionally purple to blood-red. In old basidiomes the brown colours may change into dull olive tinges. Pileus margin lighter coloured, whitish, yellowish, apricot to rusty. Pileus surface dry, when young alveolate-rugose and fugaceous pruinose, later mat and sometimes almost smooth. Pileipellis rarely cracking and if so, then the cracking usually begins at the pileus margin as fine fissures ; subsequently the whole pileipellis may also break into coarse clods. Tubes long, even longer than the diam. of the pileus context, reaching more than 10 mm, young lemon-yellow, bright yellow, chrome-yellow, then

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex greenish-yellow, adnexed with a tooth, usually weakly bluing when bruised. Pores concolorous with the tubes, small to medium sized, only exceptionally wider than 1 mm, roundish to isodiametrically angular, ageing without green tinges, turning to rust-brown when injured. Stipe 30–80(–120)r10–30(–40) mm, cylindrical to ventricose-fusiform, solid, sturdy, weakly fibrillose, young bright yellow, minutely floccose, the initially yellow scales turning red in older basidiomes or after collecting, so that the stipe, with exception of the yellow apex, appears ¡entirely reddish. The stipe base often turns brownish when bruised. Context firm, ¡purely yellow, in older specimen sometimes more ochraceous, exceptionally almost white, then often also reddish tinges are observed in the stipe context ; inconstantly weakly bluing. Smell and taste banal, somewhat acidulous. Fresh spore deposit mustard-yellow, dry with olive tinges. Macrochemical reactions the context of fresh material turns olive-green with Melzer. Basidiospores (9–)14¡1(–17)r(4–)5¡0.5(–6.5) mm, Q=(2.0) 2.7¡0.2 (3.6), V=(68) 195¡38 (340) mm3 (n=806) in KOH 3 %, subfusiform, with well-pronounced supra-apicular depression, with slightly thickened walls (wall to 0.5 mm thick), intensely honey coloured and with one to two guttules when mature, spore surface finely longitudinally striate, inamyloid, not dextrinoid. Basidia 30–45r9.5–15 (n=15), clavate, with hyaline to yellowish content in KOH 3 %, mainly 4-spored. Pleurocystidia scattered, 50–95r10–16 mm, ventricose-fusiform, with hyaline to yellowish content in KOH 3 % (n=15). Cheilocystidia scarce, similar to pleurocystidia. Pileipellis a palisadoderm formed of rather variable, versiform elements, the shape of the terminal elements ranging from small cylindrical to spherical. The slender and short cylindrical terminal elements often show an apical widening, reminiscent of a drumstick (this apical widening of the pileipellis elements is typical of X. pruinatus), the subterminal element usually wider than the terminal one ; sometimes a slender, cylindrical terminal element is followed by a spherical subterminal element of multiple diameter downwards or even chains of one to three spherical elements are observed, their diameter increases downwards. The size of the terminal pileipellis elements is also highly variability : (8–)25.5¡9(–67)r(3.5–)11¡5 (–47.5) mm, Q=(0.5–)2.5¡1 (7) mm in Congo red (n=806). Depending on the developmental stage of the basidiomes and the collection, two principal pileipellis types can roughly be distinguished : pileipellis type 1 : terminal elements rather slender, ¡cylindrical to drumstick-shaped, smooth to weakly incrusted, the smooth ones usually with an intracellular brown pigment, the subterminal cell often widened and as short or even shorter than the terminal element and normally without intracellular pigmentation, the whole pileipellis with weakly to moderately developed incrustations.

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Pileipellis type 2 terminal elements broad, often bulletshaped to subspherical, only exceptionally slender and cylindrical, in the latter case often followed by a roundish penultimate element, often with chains of two to three roundish elements, but the terminal element also in this case narrower than the subterminal cell, intracellular pigment only in the appendage-like narrow terminal cells, the subspherical and bullet-shaped elements normally moderately to heavily incrusted, the whole pileipellis in type 2 with much stronger developed incrustations than in type 1. Transitional stages between these two extremes are frequently found. Stipitipellis consisting of slender, loosely interwoven, mainly hyaline, 3–5 mm broad hyphae ; well-developed caulohymenium on the entire length of the stipe with a nearly closed layer of caulobasidia and caulocystidia. Stipe texture formed of parallel running, ¡hyaline, 2–4 mm broad hyphae, which in the lower half of the stipe differentiate in irregular intervals into the ‘pruinatus-hyphae’: inconspicuous, thin-walled and moderately broad hyphae extend more or less abruptly to strikingly thick-walled, to 30 mm broad, irregularly formed hyphae of variable length, which, depending on their individual developmental stage, show a weak to strong amyloid reaction. The inner wall of these hyphae often shows a particular, uneven surface of crater-like elements (Fig. 1). In all textures thromboplere hyphae occur in thrombomorph or meromorph state. Apart from the amyloid reacting hyphae in the stipe context no particular microchemical reactions were observed. In a few collections, spores reacted with a slight greyish hue when mounted in Melzer’s and the basidia reacted dextrinoid. No metachromatic reaction was observed in spores or hyphae. Habitat and distribution : Xerocomus pruinatus is widespread in Europe, it was collected in Austria, the former Czechoslovakia, Denmark, England, France, Germany, Hungary, Italy, Poland, Sweden, Spain, and Switzerland ; it is probably often misinterpreted as X. chrysenteron. The main fruiting period of X. pruinatus is autumn (September to November), being only exceptionally collected during summer. Basidiomes fruit under conifers (Abies, Picea, Pinus) as well as under deciduous trees (Acer, Carpinus, Castanea, Fagus, Fraxinus, Quercus, etc.), in open grassy sides, on trail borders or in forests. No preferences for particular soil types have been observed. Collections examined: Austria : Carintia : Knappenberg, with Fagus and Picea, 8 Oct. 1995, M. Kirchmair & R. Po¨der (IB19950970). Ka¨rnten : Knappenberg, with Picea between mosses, 8 Oct. 1998, R. Po¨der (IB19980366). – Italy: Parma : Stabielle, with Quercus and Castanea, 2 Oct. 1996, R. Po¨der & H. Ladurner: (IB19961955). Liguria : Savona, Bardineto, alt. 750 m, with Fagus sylvatica and Castanea sativa, 10 Oct. 1998, G. Simonini (GS2017). – Ukraine : Kiev, Angarskii, Prereval, road from Alutscha to Simferopol, 6 Oct. 1992, M. M. Moser (IB19920066). – Spain : Cantabria : malataja, Quercus pyrenaica, 24 Oct. 1999, A. Munoz & R. Luis (IB19991024). For additional material see Ladurner (2001a).

U. Peintner, H. Ladurner and G. Simonini Xerocomus ripariellus Redeuilh, Docums. Mycol. 26(104) : 30 (1997) (Figs 12, 24) Boletellus catalaunicus Po¨der, G. Moreno, Rocabruna & Tabares, Mycotaxon 62 : 232–234 (1997). Pileus 30–70 mm broad, young convex, then applanate, initially ¡red, blood-red, cherry-red, sometimes vinaceous, but also more brownish-red, fading to clay, greyish-brown or dust-coloured from the centre. Pileus margin brighter, whitish, yellowish, long time involute. Pileus surface uneven, gibbose, ¡velvety, pruinose. Tubes young nearly white, then lemon yellow, turning greenish yellow, ¡bluing when bruised. Pores concolorous, irregular, noticeable wide when old, ¡bluing when bruised. Stipe 30–60(–90)r10–20(–25) mm, ¡smooth, subcylindrical to slightly fusiform, slightly tapering at the base, usually rather robust, the apex concolorous with the pores, downwards ¡reddish floccose as in X. chrysenteron, sometimes longitudinally striate, in older basidiomes with haematoma-like spots of various number, the stipe base turning dull brown on pressure. Context initially almost whitish to light lemonyellow, sometimes also bright yellow, more brownish beneath the pileipellis and the stipitipellis, ¡intensely ochraceous in the stipe base, in the lower half of the stipe often vinaceous to violet, elsewhere whitish to yellow, often strongly bluing in the middle part of the stipe. Taste and smell banal. Basal mycelium whitish to yellowish. Colour of dried specimens not significant. Spore deposit brownish with some olive tinges. Macrochemical reactions all collections but one (IB 19940617) showed a distinct ‘fleeting-amyloid ’ reaction in stipe context and hymenophoral trama ; this exception might be explained by the bad preservation of this collection. No other macrochemical reactions observed. Basidiospores (10.5–)13.5¡1(–17)r(4–)4.5¡0.5 (–5.5) mm, Q=(2.2) 2.9¡0.2 (3.6), V=(85) 152¡25 (228) mm3 in KOH 3 % (n=713), slender, subfusiform, with weakly developed but distinct supra-apicular depression, with slightly thickened walls (wall to 0.5 mm thick), intensely honey-coloured and with one to two guttules when mature, the surface finely longitudinally striate (striation more pronounced than in X. pruinatus), inamyloid and not dextrinoid. Basidia 30–45r 9.5–14 mm (n=15), clavate, with hyaline to yellowish to intensely yellow content in KOH 3% ; mainly 4-spored. Pleurocystidia scarce, 50–90r8–16 mm (n=15), ventricose-fusiform, with hyaline to yellowish content in KOH 3 %. Cheilocystidia scattered, similar to the pleurocystidia. Pileipellis a physalo-palisadoderm of extremely variably shaped elements, usually a palisade of versiform elements is observed but in some basidiomes the pileipellis appears nearly epitheloid, the terminal elements are slender cylindrical to broadly clavate to pear shaped, ovoid, spherical, with or without elongated terminal outgrow. Single spherical elements or chains of two to three widened, ¡spherical

675 terminal elements usually turn abruptly into slender cylindrical hyphae. In some basidiomes ‘brushes ’ of widened terminal elements sit on a common, reverse pear-shaped subterminal cell, forming ‘floriform ’ structures ; apart from the shape the dimensions of the terminal pileipellis elements also vary strongly within a single specimen as well as between different basidiomes or different collections : (7–)29.5¡10(–63.5)r(4.5–) 16.5¡5.5(–46) mm, Q=(0.6) 2¡0.9 (7.4) mm in Congo red (n=1023), terminal and subterminal elements finely to heavily incrusted ; in some basidiomes the terminal elements are nearly smooth and hyaline in KOH 3%, in other basidiomes (often of the same collection) heavy incrustations peel off the terminal and subterminal elements forming coarse scabs in KOH 3%. Spherical and subspherical terminal elements of larger diameter usually show much heavier incrustations than slender cylindrical ones. In some basidiomes large, refractive, not congophile plaques are observed (KOH 3 %). Stipitipellis consisting of loosely interwoven, 3–5 mm broad hyphae with hyaline to yellowish content, the hyphal ends mainly rounded, sometimes slightly widened. In the upper half of stipe a well-developed caulohymenium is observed. Stipe context formed of parallel, densely packed, 4–8 mm broad, thin-walled hyphae with hyaline to yellowish, sometimes granular content in KOH 3%. No clamp connections observed. No particular microchemical reactions observed. Habitat and distribution : Xerocomus ripariellus is mostly found in humid habitats, at lake shores or at river banks, in dunes, but also in (humid) gardens, parks, etc., associated with deciduous trees (especially with Populus, Alnus and Salix). Collections examined: France : Yvelines, 78, Etang d ’Or, beside the water, alt. 50 m, with Salix, 29 Sept. 1995, Redeuilh, (GR22541P – holotype p. p.); Paris, Foret d’Orient, 20 Sept. 1993, Mahieu (GR93092); Paris, Nouvelle station, 17 Sept. 1995, Mahieu (GR22465) ; sine loc., 5 Oct. 1994, Anonymous (GR21189). – Spain: Catalonia : Riells de Montseny, among litter on sandy soil in mixed hardwoods, 16 Sept. 1998, A. Rocabruna (IB19980360, as Boletellus catalaunicus). For additional material see Ladurner (2001a).

Xerocomus rubellus Que´l., Ass. Fr. Avanc. Sci. 5 : 620 (1895). (Figs 13, 27–28) Pileus 40–100(–150) mm broad, young nearly hemispherical, then applanate, also slightly depressed at the centre, sometimes irregularly bent, of extremely variable colours, blood-red, cherry-red, orange-red, rose-red, pink, ochraceous-pink to ochraceous-red, incarnadine, ochraceous, yellowish-brown, brown, grey-brown, olive-grey, grey, dark brown, black-brown, typically fading with age. Pileus margin somewhat paler, initially involute, then plane or also revolute, acute. Pileus surface dry, ¡finely tomentose, often cracking especially at the centre, showing the yellow or also red context in the fissures and then resembling X. chrysenteron.

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex Tubes to 12 mm long, adnate to slightly decurrent, initially lemon-yellow, later greenish-yellow, slightly bluing when injured. Pores large, wider than 1 mm, roundish to angular, concolorous with the tubes, in mature basidiomes with orange-rusty tinges, only weakly bluing when bruised. Stipe 25–70(–120)r4–35 mm, solid, versiform, often slender, cylindrical, also stout, bulbose to ventricosesubfusiform, always tapering towards the base and slightly rooting, ornamented with darker brownish or reddish scales or fibrils on ochraceous ground, colour of the stipe surface similar to the pileus colour. Context soft, pale yellow, in base of the stipe with a typical flame-red pigment in form of small dots or a thin red line. Context only weakly bluing and exceptionally also reddening in the cap, the reddish discoloration fading on drying. Taste and smell inconspicuous. Basal mycelium whitish-yellow. Colour of dried specimens not significant. Spore deposit brown with olive tinges. Macrochemical reactions weak ‘fleeting-amyloid ’ reaction in stipe context and exceptionally also in the hymenophoral trama of exsiccata, but most collections show a strong dextrinoid reaction in all textures ; no further macrochemical reactions observed. Basidiospores (9–)12¡1(–15)r(3.5–)5¡0.5 (–6.5) mm, Q=(1.8–)2.3¡0.2(–3.3), V=(76) 169¡35 (–355) mm3 in KOH 3 % (n=1767), broadly elliptical to broadly fusiform, with weak to distinct supra-apicular depression, honey-coloured and with one to two guttules when mature, smooth, inamyloid and not dextrinoid. Basidia 30–45r9–13 mm (n=15), inconspicuous, clavate, hyaline to yellowish in KOH 3 % mainly 4-spored. Pleurocystidia scattered, 40–60r 8–13 mm (n=15), slender, ventricose-fusiform, often with elongated neck, most hyaline, rarely with yellowish content in KOH 3 %. Cheilocystidia similar, numerous, often with yellowish content in KOH 3%. Pileipellis a physalo-palisadoderm consisting of moderately long, cylindrical, septate hyphae. Terminal cells versiform, acorn-shaped, bullet-shaped, subspherical, broadly cylindrical with rounded apex to cystidioid, sometimes also with elongated neck, but also slender, cylindrical, slightly tapering towards the tip (7.5–) 32¡11(–93.5)r(5.5–)12¡4(–41) mm, Q=(0.8) 2.8¡ 1.0 (9.4) in Congo red (n=1798) ; subterminal elements sometimes slightly widened. Pileipellis elements smooth to distinctly incrusted by a fine granular, yellowish pigment, exceptionally also heavily incrusted elements are observed (KOH 3%). Stipitipellis consisting of slender, loosely interwoven, mainly hyaline, 3–5 mm broad hyphae. In the upper third of the stipe bundles of caulohymenium consist of clavate, hyaline to yellow coloured basidia, basidioles and broadly ventricosefusiform cystidia with an often extremely elongated neck (to 30 mm long). Stipe texture not significant, formed of parallel running, ¡hyaline, 2–4 mm broad hyphae; scattered to scarce flame-red crystals (hyphal excretions) dissolving in KOH 3% are found in the

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lower stipe context. Without clamp connections. No particular microchemical reactions observed in any part of the basidiomes. Habitat and distribution: Xerocomus rubellus is widespread in Europe. Brown forms are more frequently found in central, northern and western Europe than the typical red forms. X. rubellus fruits between July and November under deciduous trees and shrubs (e.g. Quercus spp., Tilia sp., Corylus avellana), often in urban areas, on lawn, on disturbed road sides, also in open grass land, but only exceptionally in open, undisturbed woods. This species is reported from Austria, Croatia, England, Finland, France, Germany, the Netherlands, Spain, but occurs probably in all European countries with moderate to warm climate. Collections examined: Croatia : Veli Loscinji, with Quercus ilex and Olea europea, 8 Oct. 1999, H. Ladurner (IB19990917). – Italy : Reggio Emilia: Viale Ramezzini, with Quercus cerris and Tilia, 5 Sept. 1993, G. Simonini (GS961) ; Grosseto, Orbetello, ‘ Le Piane ’, with Quercus suber, 30 Sept. 1993, G. Simonini (GS1044) ; Emilia Romagna, Viano, Pulpiano, alt. 520 m, with Quercus cerris, 7 Jun. 1998, G. Simonini (GS1894). For additional material see Ladurner (2001a).

DISCUSSION Nomenclatural notes Some modern authors (e.g. Hansen & Knudsen 1992) have rejected the name Xerocomus chrysenteron due to considerable nomenclatural confusion and ambiguous interpretations. Bulliard’s (1791) original description of Boletus chrysenteron seems to mix characters of both X. chrysenteron and the ‘brown aspect ’ of X. rubellus (i.e. X. communis (Bull.) Bon 1985). Moreover, Bulliard himself synonymised B. chrysenteron with B. cupreus Schaeff. 1774. However, we regard the name X. chrysenteron as a name in current use, which should be conserved (Redeuilh & Simonini 1994). The old name Boletus subtomentosus var. pascuus Pers. 1825 (syn. Xerocomus pascuus (Pers.) Gilb. 1931) is a nomen dubium, which should not be further applied. The brief macroscopical description given by Persoon (1825) (‘ pileo subtomentoso, poris flesuosis crassis, carne immutabili, stipite brevi squamuloso toto coccineo ’) fits with many Xerocomus species, making it impossible to attribute Persoon’s description to any taxon as it is currently circumscribed. Krombholz’s (1831–46) description and pictures of B. pascuus seem to agree well with the modern concept of X. chrysenteron (dark brown cap, yellow stem, purple red minutely squamulose at the base), but the cap cuticle was not reported as cracking. Corda’s (1837–54) description and picture of B. pascuus (dark brown, velvety, cracked at margin cap, purple red stem basis internally and outwardly) agrees perfectly with the modern concept of X. chrysenteron. Also the habitat on pastures supports the interpretation of B. pascuus as X. chrysenteron.

U. Peintner, H. Ladurner and G. Simonini Klofac & Krisai-Greilhuber (1992) and Lannoy & Estades (2001) used the name X. pruinatus var. pascuus (Pers.) Klofac & Krisai-Greilhuber for a taxon with a smooth cap (not wrinkle-folded as is typical for X. pruinatus), and orange colours on cap and stipe. We interpret X. pruinatus var. pascuus in the sense of Klofac & Krisai-Greilhuber (1992) as a synonym of X. pruinatus : the range of X. pruinatus end cells also circumscribes taxa attributed to the nomen dubium X. pruinatus var. pascuus (Ladurner 2001a, b). In addition, the name X. rubellus is embedded in a considerable amount of nomenclatural confusion. The lack of type material and the varying interpretations (Krombholz 1836, Que´let 1895, Singer 1945, Snell & Dick 1958, Binyamini & Avizohar-Hershenzon 1973, Klofac & Krisai-Greilhuber 1992, Simonini 1998a) make this taxon in the original sense uninterpretable. However, we consider this species epithet as a name in current use and so we will continue to apply it to taxa as delimited above. Epiypification of X. rubellus might contribute to a badly needed unification of the X. rubellus species concept. Phylogeny of the species complex Our morphology-based species concepts were confirmed by the results of the molecular phylogenetic analyses based on the same material. Thus, sequence data of the rDNA-LSU are useful for the delimitation of groups in the Boletales, as already shown in earlier studies (Fischer et al. 1997, Binder 1999, Jarosch & Besl 2001, Binder & Bresinsky 2002). Basal relationships are not fully resolved, but several studies using rDNA sequences have shown that basal branches are often difficult to resolve based on LSU phylogenies only (Binder 1999, Moncalvo et al. 2000a, b, 2002, Wagner & Fischer 2001, Thomas et al. 2002). Our phylogenetic analyses prove that the X. chrysenteron group is a distinct evolutionary lineage, which can clearly be separated from other boletes by sequence data and by morphological characters. Binder (1999) proposed the provisional generic name ‘Paraxerocomus ’ for this species complex based on a few sequences as well as morphological and chemotaxonomical characters. However, as long as relationships of Boletales at the genus level are not satisfactorily understood, especially between Boletus, Xerocomus, Boletellus and Pseudoboletus, we do not recommend nomenclatural changes at generic level. One striking result is the isolated, basal position of X. rubellus within the X. chrysenteron complex. Like most other species of this group, X. rubellus is a macroscopically very variable species. Brown forms are easily confused with X. chrysenteron (from which they differ by the lower spore quotient) and red forms are often confused with X. dryophilus (which differs by broader spores). Red forms of X. pruinatus, X. ripariellus and X. fennicus differ by their striate spores, and the lack of a flame red pigment in the base of the stipe.

677 The well-supported, basal position of X. rubellus indicates that smooth spores are a synapomorphic character: this implies that both, spore striation and spores with a truncate apex (sometimes reminding a germ pore), are secondarily derived within the X. chrysenteron complex. Although basal relationships are not fully resolved, tree topologies indicate that taxa with striate spores do not form a monophyletic group. Thus, the section Striatulisporae does probably not represent natural relationships. The slight spore striation, which is often hard to discern with a light microscope, appears to be a derived character. It can be speculated, that spore striation evolved independently in several lineages of this species complex. In X. fennicus the spore striation is most obvious. Therefore, this species was originally placed in Boletellus. Our phylogenetic analyses demonstrate that X. fennicus belongs to the X. chrysenteron species complex, where it is closely related to X. ripariellus. Therefore we proposed a recombination of this species epithet into Xerocomus. Truncate spores as found in X. fennicus and X. porosporus are secondarily derived characters with two independent origins. Also ‘pruinatus-hyphae’ have multiple origins as they are found in X. pruinatus, X. cisalpinus and sporadically also in X. fennicus. All species within the X. chrysenteron complex can clearly be distinguished based on LSU sequences. However, sequences retrieved from GenBank often do not match with the respective clade. Our analyses show that ‘ X. rubellus ’ AF050649 is a misidentified X. ripariellus, and that ‘ X. chrysenteron ’ AF050647 might be a misinterpreted X. pruinatus. The American ‘X. chrysenteron ’ AF071537 could be conspecific with X. cisalpinus indicating that this new taxon might also occur in California. ‘ X. pruinatus ’ AF402140 could not be assigned to any clade, but it could be within the morphological and molecular range of X. chrysenteron. In summary, sequence data are a valuable and promising tool for Boletales systematics. But the utilisation of material that is macro- and micro-morphologically well characterised is an indispensable prerequisite for reliable results in molecular systematics. Delimiting micromorphological characters Micromorphological characters as well as the ecology are often neglected in species concepts of boletes. The determination and delimitation of taxa was, up to nowadays, based mainly on macroscopical characters, which are often very variable and therefore misleading. All attempts in molecular systematics to address questions on species level are doomed to fail, when the material used is not reliably identified. Bolete species concepts must be placed on a solid base and include concrete, micromorphological characters such as spore size and shape and characters of the pileipellis structure. As we have shown, features of the spores and of the pileipellis are particularly reliable characters for

Xerocomus cisalpinus sp. nov. and the X. chrysenteron complex species differentiation in the Xerocomus chrysenteron complex. But spore dimensions are often very variable, as far as minima and maxima are concerned. Therefore, a statistically significant number of spores must be measured for a reliable identification. Truncate spores and spore striation are good delimiting characters, but spore striation is often difficult to detect with a light microscope. The degree of striation also depends on the maturity of the spores : young spores are more or less smooth, while mature spores are faintly striate. The ornamentation is faintest in X. pruinatus and X. cisalpinus, and increasingly pronounced in X. ripariellus and X. fennicus. A reliable identification of species in the X. chrysenteron complex is also possible based on the structure of the pileipellis and the dimensions of its end cells. In particular, the length of the pileipellis end cells is a good delimiting character, although infraspecific variability is always present to a certain degree. ‘Pruinatushyphae’ are currently known only from three species, and therefore are a good character for their identification. Thus, an accurate identification of species within the X. chrysenteron group can easily be achieved with the combination of the three morphological characters spores, pileipellis and ‘ pruinatus-hyphae ’. In such way reliably identified and characterised material is the key to the often puzzling phylogeny of boletes. The combination of classical and molecular methods can then form a solid base for a more natural system of mushrooms that is urgently needed. ACKNOWLEDGEMENTS We are grateful to our friends Daniele and Massimo Antonini, Antonella Bartocchi, Marco Contu, Roberto Fontenla, Franca Franceschetti, Carmine Lavorato, Mauro Manavella, Eugenio, Giulio e Lanfranco Mariotti, Guy Redeuilh, Alfredo Riva, Giancarlo Sassi, Pietro Signorello for having supported our study with a large amount of collections from all over the Mediterranean area. We thank Guy Redeuilh and Jukka Vauras for permission to publish their photographs of Xerocomus ripariellus and X. fennicus, respectively. Special thanks go to Giovanni Consiglio for the Latin diagnosis, to Esteri Ohenoja for loans of material and translation of Finnish specimen labels, and to Bart Buyck for loaning us the type of Boletellus episcopalis.

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Corresponding Editor: S. A. Redhead