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Available from: Gang wu Retrieved on: 15 October 2015 Phytotaxa 191 (1): 081–098 ISSN 1179-3155 (print edition) www.mapress.com/phytotaxa/ PHYTOTAXA Copyright © 2014 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.191.1.5

The genus Imleria (Boletaceae) in East Asia

Xue-Tai Zhu1,2, Yan-Chun Li1, Gang Wu1,2, Bang Feng1, Kuan Zhao1,2, Matteo Gelardi3, Gerhard W. Kost4 & Zhu L. Yang1* 1Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kun- ming 650201, Yunnan, China 2University of Chinese Academy of Sciences, Beijing 100049, China 3Via Traversa della Selciatella 2B, I-00062 Bracciano, RM, Italy 4Systematic Botany & , FB17, Philipps-University Marburg, 35043 Marburg, Germany *e-mail: [email protected]

Abstract

The genus Imleria can be easily distinguished from other genera of Boletaceae by the combination of the following charac- ters: a chestnut brown pileus and , a cream to pale yellow hymenophore, a viscid pileal surface when moist, a cyanescent context and hymenophore on handling or exposure, an ixotrichodermal and smooth . The monophyly of Imleria and the detection of four independent were highly supported by phylogenetic analyses using 5 gene markers. The four species, including the previously recognized Im. badia, Im. obscurebrunnea, plus the newly discovered Im. parva and Im. subalpina, are fully documented with taxonomic descriptions and illustrations, and a key to the taxa is provided. Geographically, Im. badia is widely distributed in Europe, North America and probably in Far East Asia, yet there is some intraspecific divergence between specimens from Europe and North America caused probably by geographic isolation. Imleria subalpina and Im. obscurebrunnea might diverge from each other relatively recently with the uplifts of the eastern Himalayas and Hengduan Mountains. An epitype collected from Sweden was designated for Im. badia.

Keywords: boletes, multiple gene analysis, molecular phylogeny, , biogeography

Introduction

Boletus badius Fr. (1828: 126), commonly known as the Bay Bolete, badius (Fr. 1818: 247) Gilbert (1931: 92), is an edible originally described from Europe (Fries 1818). This species can easily be recognized by its chestnut brown pileus and stipe, cream to pale yellow hymenophore and viscid pileipellis when moist (Alessio 1985; Singer 1986; Bas et al. 1988; Breitenbach & Kränzlin 1991; Ladurner & Simonini 2003; Zang 2006; Hills 2008). Its systematic position in the Boletaceae, however, has been unsettled for a very long time. Due to its viscid pileus, it was placed in Rostkovites P. Karst. (1881: 16), Viscipellis (Fr. 1838: 419) Quél. (1886: 155), Ixocomus Quél. (1888: 411) and Gray (1821: 646) by Karsten (1881), Quélet (1886), Quélet (1888), and Kuntze (1898) respectively. However, all these four settlements were not broadly accepted, and the former three generic names are all in fact nomenclatural synonyms of the latter name Suillus. Since B. badius shares with other species of Xerocomus (1887: 477) the subtomentose pileus when dry, Gilbert (1931) transferred it into the genus Xerocomus, and this placement was then accepted by some mycologists (Singer 1945, 1986; Alessio 1985; Breitenbach & Kränzlin 1991; Horak 2005; Hills 2008). Others, such as Smith & Thiers (1971), Both (1993), and Kirk et al. (2008), however, maintained the name B. badius, as the generic rank of Xerocomus was considered doubtful in their opinion. Šutara (2008) also retained the species in the genus L. (1753: 1176), as the sticky pileal surface and the gelatinous ixotrichodermal pileipellis distinguished it from other xerocomoid taxa. Nevertheless, he pointed out that this solution seemed not to be fully satisfactory and the generic position of B. badius required further research. Multi-gene phylogenetic analysis has been applied to the phylogenetic studies of boletes in the last fifteen years and several new genera and many new species have been uncovered in , supported with morphological and molecular evidence (Binder & Bresinsky 2002; Halling et al. 2007; Desjardin et al. 2008, 2009; Li et al. 2009, 2011; Zeng et al. 2012, 2013; Feng et al. 2012; Neves et al. 2012; Halling et al. 2012; Hosen et al. 2013; Zhao et al. 2014).

Accepted by Genevieve Gates: 8 Oct. 2014; published: 30 Dec. 2014 81 Meanwhile, Boletus and Xerocomus have been revealed to not be monophyletic (Binder & Besl 2000; Peintner 2003; Šutara 2008; Desjardin et al. 2009; Dentinger et al. 2010; Feng et al. 2012; Nuhn et al. 2013; Wu et al. 2014). Nuhn et al. (2013) recognized the ‘badius’ , including B. badius and B. pallidus Frost (1874: 105), Wu et al. (2014), however, indicated that B. badius appeared in Clade 22 and B. pallidus in Clade 19 and these two species are probably not monophyletic. Vizzini (2014) erected genus Imleria based on Boletus castaneus β badius Fr. over the last few years, we have collected some samples of Im. badia from Europe, and related specimens from various parts of China. Heterogenetic habitats, variable macro- and micro-morphological features suggested that the samples from China might be different from those gathered in Europe. Here we test the hypothesis that the Chinese collections represent different taxa from those in Europe using a multi-gene phylogenetic approach.

Materials and methods

Morphological studies Macroscopic descriptions are based on detailed field notes and photos of fresh basidiomata. Microscopic structures were observed based on dried material through light microscopes. Tissue from dried material was rehydrated in 5% hydroxide (KOH); the color of microstructures was examined in water. Sections of the pileipellis were cut radially/vertically at a position halfway to the center of the pileus. Sections of the squamules on the stipe were taken from the middle part along the longitudinal axis of the stipe. Color codes follow Kornerup & Wanscher (1981). Basidiospores were examined with a Hitachi S-4800 scanning electron microscope (SEM) at 10.0 kV. Specimens examined are deposited in Herbarium of Cryptogams, Kunming Institute of Botany of the Chinese Academy of Science (HKAS), Swedish Museum of Natural History (S), Herbarium of the Institute of Microbiology of the Chinese Academy of Science (HMAS), Royal Botanic Garden, Kew (K) and Mycological Herbarium of the National Museum of Nature and Science (TNS). The following abbreviations are used: B. = Boletus, Im. = Imleria and X. = Xerocomus. The notations ‘(n/m/p)’ indicate that the measurements were made on ‘n’ basidiospores from ‘m’ basidiomata of ‘p’ collections. The expressions ‘(a) b-c (d)’ stand for the dimensions of the basidiospores, in which ‘b-c’ contains a minimum of 90% of the measured values, and ‘a’ and ‘d’ within parentheses stand for the extreme values. Q is the length/width of the basidiospores and Qm is the average Q ± standard deviation.

Molecular procedures and phylogenetic analyses DNA isolation, PCR amplification and DNA sequencing Total DNA was extracted from the pileus context of dried herbarium material using the CTAB procedure of Doyle & Doyle (1987). Five DNA fragments were amplified in this study, including two non-protein-coding fragments, the ribosomal internal transcribed spacer (ITS) and the large nuclear ribosomal RNA subunit (nrLSU), and three protein- coding fragments, the translation elongation factor 1-α (tef1-α), the largest subunit of RNA polymerase II (rpb1) and the second largest subunit of RNA polymerase II (rpb2). The primer pairs used in this study are as follows, ITS1 and ITS4 for ITS/5.8S (White et al. 1990), LR0R and LR5 for nrLSU (Vilgalys & Hester 1990), EF1-α-F and EF1-α-R for tef1-α (Mikheyev et al. 2006), RPB1-Af and RPB1-Cr for rpb1, bRPB2-6F and bRPB2-7.1R for rpb2 (Matheny 2005). PCR amplifications were conducted on an ABI 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), containing 1μl DNA template, 2.5μl PCR reaction buffer, 0.5μl dNTP mix (0.2mM), 1μl per primer (5μM), 1U

Taq polymerase. The final volume was adjusted to 25μl with distilled H2O. The PCR conditions were set as follows: pre-denaturation at 95oC for 4min, 35 cycles of denaturation 1min at 94oC, annealing 40s (for ITS) and 80s (for nrLSU, tef1-α, rpb1 and rpb2 ) at 50oC (for ITS and nrLSU) and 53oC (for tef1-α, rpb1 and rpb2), elongation 80s at 72oC, and a final elongation of 8min at 72oC was included after the cycles. The PCR products were purified using the Bioteke Purification Kit (Bioteke Corporation, Beijing, China), and then the purified products were sequenced on an ABI-3730- XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA), using the same primer pairs as in the original PCR amplifications. DNA sequences were edited with SeqMan (DNASTAR in Lasergene 9) and Bioedit v7.0.9 (Hall 1999), then aligned with MUSCLE v3.6 (Edgar 2004). The aligned sequences were manually adjusted where necessary.

Dataset assembly The ITS and the 5-loci (5.8S, nrLSU, tef1-α, rpb1 and rpb2) combined datasets, were compiled for different analytical purposes. For species delimitation, we used multi-locus DNA data and phylogenetic species recognition based on genealogical concordance (Taylor et al. 2000) in addition to morphological features. The 5.8S, nrLSU, tef1-α, rpb1

82 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. and rpb2 generated in this study and sequences retrieved from GenBank (Binder et al. 2010; Li et al. 2011; Feng et al. 2012; Hosen et al. 2013) were used in phylogenetic analysis to further determine the phylogenetic position of this genus in Boletaceae, to recognize the species in the genus and to evaluate the relationships among them.

Table 1. Species used in the five-gene combined dataset, their vouchers and GenBank accession numbers. GenBank Accessions Species Name voucher Locality 5.8S nrLSU tef1-α rpb1 rpb2 luteolus 00-436 Africa - KF030238 KF030397 KF030392 - thibetanus AFTOL-450 China DQ200917 AY700189 DQ029199 DQ435800 DQ366279 projectellus AFTOL-713 - AY789082 AY684158 AY879116 AY788850 AY787218 Boletellus shichianus AFTOL-532 China DQ200921 AY647211 DQ408145 - DQ366280 Boletinellus merulioides AFTOL-575 - DQ200922 AY684153 DQ056287 DQ435803 DQ366281 BD380 USA EU231984 HQ161848 - HQ161847 - Boletus edulis Be953(5.8S)/ Germany AY680988 AF456816 GU187682 GU187444 GU187774 B2(nrLSU) Be3(tef1-α,rpb1 and rpb2) 179/97 Germany DQ534564 AF457409 KF030424 KF030396 - Calostoma cinnabarinum AFTOL-439 - AY854064 AY645054 AY879117 AY857979 AY780939 aff. piperatus HKAS 50214 China JQ928610 JQ928621 - JQ928594 - Chalciporus piperatus MB 04-001 USA - DQ534648 GU187690 GU187453 - Gomphidius roseus AFTOL-1780 Germany DQ534570 DQ534669 GU187702 GU187459 GU187818 lividus REG G11 Germany DQ534568 AF098378 GU187461 GU187786 GU187701 HKAS 53502 Germany KC215204 KC215213 KC215247 KC215226 KC215235 Imleria badia S-F119691 Sweden KJ806970 KJ806971 KJ806969 KJ806972 KJ806973 Imleria badia HKAS 74714 Germany - KC215212 KC215242 KC215224 - Imleria obscurebrunnea HKAS 52557 China KC215207 KC215220 KC215243 KC215225 KC215234 Imleria obscurebrunnea HKAS 50477 China KC215206 - KC215245 KC215233 KC215241 Imleria parva HKAS 59437 China KC215203 KC215215 KC215250 KC215228 KC215237 Imleria parva HKAS 55341 China KC215202 KC215216 KC215252 KC215229 KC215238 Imleria subalpina HKAS 56375 China KC215209 KC215217 KC215244 KC215231 KC215240 Imleria subalpina HKAS 74712 China KC215208 KC215218 KC215246 KC215230 KC215239 sp. HKAS 50221 China JQ928612 JQ928624 JQ928583 JQ928593 - aff. aurantiacum HKAS 57390 China JQ928611 JQ928625 JQ928581 JQ928591 JQ928602 sphaerosporus MB06-066 USA GU187540 GU187593 GU187737 - GU187803 vernalis AFTOL-715 China DQ647827 AY645059 DQ457629 - - Phlebopus portentosus REG Php1 - DQ534569 AF336260 GU187735 GU187476 GU187801 pelletieri KM 12805 England JQ967258 JQ967215 - - - arrhizus REG 588 USA GU187538 AF336262 - GU187473 GU187798 porphyroporus AFTOL-1779 Germany DQ534563 DQ534643 GU187734 GU187475 GU187800 parasiticus Xpa1 Germany - AF050646 KF030443 KF030394 - aff. griseus HKAS 59460 China JQ928613 JQ928626 JQ928580 JQ928590 JQ928601 Retiboletus aff. nigerrimus HKAS 59699 China - JQ928627 JQ928582 JQ928592 JQ928603 Rhizopogon nigrescens MB 06-070 USA - GU187594 GU187744 GU187478 GU187806 squarepantsii LHFB14 Malaysia HQ724511 HQ724509 - - - Spongiforma thailandica DED7873 Thailand EU685113 EU685108 - - - floccopus AFTOL-ID 716 - AY854068 AY684155 AY883428 AY858963 AY786065 Suillus spraguei AFTOL-717 - AY854069 AY684154 AY883429 AY858965 AY786066 felleus AT 2001011 Sweden - JQ326993 JQ327015 - - Tylopilus ferrugineus MB 06-053 USA - JQ326994 JQ327016 - - stramineus 3518 USA - KF030353 KF030428 KF030386 - chrysenteron HKAS 56495 Germany KC215210 KC215211 KC215251 KC215227 KC215236 KM 167686 England KC215201 KC215222 KC215248 - - Xerocomus subtomentosus KM 168813 England KC215200 KC215223 KC215249 - - roseola HKAS 52661 China JQ928614 JQ928623 JQ928584 JQ928595 JQ928604 Accessions numbers in boldface indicate sequences obtained in this study.

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 83 Figure 1. Phylogenetic tree generated from combined 5.8S, nrLSU, tef1-α, rpb1 and rpb2 dataset using RAxML (by X.T. Zhu). RAxML likelihood bootstrap support values (>50 %) and Bayesian posterior probabilities (PP >0.95) are indicated above or beneath the branches as RAxML BS/PP. Herbarium voucher or isolate number is provided behind the species name. Imleria is highlighted in boldface.

Because sequences of ITS are much more abundant than those of any other loci in GenBank and United Database, they were primarily used to elucidate the geographic distribution patterns of the species within Imleria. The ITS dataset contains sequences generated in this study and the related ITS sequences from GenBank and United Database. The outgroups were selected according to the topology of the combined dataset analyses. All datasets have been deposited in TreeBASE (S14538).

84 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. Figure 2. Phylogram resulting from the ITS dataset using RAxML (by X.T. Zhu). RAxML likelihood bootstrap support values (>50 %) and Bayesian posterior probabilities (PP >0.95) are indicated above or below the branches as RAxML BS/PP. GenBank and United Database accession numbers and address of collection are provided behind the species name. SW = southwestern, NE = northeastern.

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 85 Phylogenetic analyses Bayesian Inference (BI) and Maximum Likelihood (ML) methods were used to analyze the two compiled datasets. Substitution models of each partition in two datasets were determined using Akaike Information Criterion (AIC) implemented in MrModeltest v2.3 (Nylander 2004). BI and ML were performed using MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003) and RAxML v7.2.6 (Stamatakis 2006) respectively. The partitioned mixed model was used to estimate the model parameters for each gene separately. The selected substitution models for these six partitions are as follow: GTR+I+G for nrLSU, tef1-α and rpb1, SYM+G for 5.8S, K80+I for ITS, HKY+I+G for rpb2. All parameters in the ML analysis were kept default, and statistical support values were obtained using nonparametric bootstrapping with 1000 replications. BI analyses using selected models and 4 chains were conducted by setting generations to 3 million and stoprul command with the value of stopval set to 0.01 (0.001 for ITS dataset). Trees were sampled every 100 generations. The first 25% generations were discarded as burn-in, and Bayesian Posterior Probabilities (PP) were then calculated from the posterior distribution of the retained Bayesian trees. In order to interpret the genetic divergence between specimens of Im. badia from Europe and North America, the intra- and inter-specific variation of the four species were calculated in MEGA 5 using the Maximum Composite Likelihood model (Tamura et al. 2004, 2011).

Results

Molecular data A total of 55 sequences were newly generated in this study, of which 13 were for ITS, 13 for nrLSU, 11 for tef1-α, 10 for rpb1 and 8 for rpb2 (Table 1). The multi-locus combined dataset consisted of 45 taxa and 4802 nucleotide sites (including gaps) for each taxon, of which 156 characters were from 5.8S, 900 from nrLSU, 1146 from tef1-α, 1324 from rpb1 and 1276 from rpb2, 193 sites (1468–1507, 1844–1892, 2727–2799, 3134–3168) were excluded due to alignment ambiguities. In the analyzed 4802 nucleotides, 2596 were constant, 2206 were variable, of which 459 were parsimony uninformative and 1747 were parsimony informative. The monophyly of Imleria was strongly supported by the results from the combined dataset (RAxML BS = 100, PP = 1) and four phylogenetic species of Imleria were segregated (Fig.1). The ITS dataset contained 26 sequences and 645 nucleotide sites (including gaps) for each taxon, 414 bases were constant, while 231 were variable, of which 134 were parsimony uninformative and 97 were parsimony informative. ML and BI analyses produced very similar tree topologies. The four species segregation by multi-locus sequence was also supported by ITS data (Fig. 2). ITS phylogenetic analysis indicated that Im. badia is widely distributed in Europe and North America, yet there is some intraspecific divergence between specimens from the two continents. This species probably occurs also in Far East Asia and South Africa (Fig. 2).

Morphological data A total of thirty-five specimens were examined, including twenty two recent collections of Imleria from China, Japan and Europe, four Chinese collections (HKAS 7764, HKAS 8693, HKAS 8705 and HKAS 31380) regarded as B. badius (Zang 2006), and nine samples of Xerocomus and Boletus. Imleria can be easily distinguished from Xerocomus and Boletus by its bay brown pileus and stipe, viscid pileus with an ixotrichodermal pileipellis, cream to pale yellow hymenophore, bluing context and hymenophore when bruised, and smooth spores. The phylogenetic species can be substantiated by morphology and ecology. Four species, Im. badia, Im. obscurebrunnea, Im. parva and Im. subalpina, are described in the taxonomy section below.

Taxonomy

Imleria Vizzini, Index Fungorum 147: 1, 2014

Basidiomata stipitate-pileate with tubular hymenophore. Pileus hemispherical to applanate, bay, chestnut brown to dark brick brown; margin shortly appendiculate; surface of pileus subtomentose or fairly floccose when dry, viscid when wet. Hymenophore cream to pale lemon yellow, becoming dull yellow with age, bluing when injured; pores compound, angular, concolorous with tube. Stipe central, concolorous with pileus or slightly paler, densely covered with minute yellow-brown to purplish brown squamules. Contexts of pileus and stipe white to yellow, becoming blue or bluish particularly around the tubes and at the stipe apex when bruised. Basal

86 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. whitish to cream. Taste and odor mild. Basidiospores boletoid, smooth under both light microscopy and scanning electron microscopy (Fig. 4), slightly thick-walled. Pleuro- and cheilocystidia lanceolate to subfusoid-mucronate, thin-walled. Pileipellis an ixotrichoderm, consisting of yellowish interwoven hyphae, which are slightly incrusted with yellow-brown and embedded in gelatinous substance. Clamp connections absent.

Imleria badia (Fr.) Vizzini, Index Fungorum 147: 1, 2014 (Figs. 3a, 4a and 5)

Basionym: Boletus castaneus β badius Fr., Obs. mycol. (Havniae) 2: 247, 1818 Boletus castaneus var. badius (Fr.) Fr., Syst. Mycol. 1: 392, 1821 Boletus badius (Fr.) Fr., Elench. Fung. 1: 126, 1828 Xerocomus badius (Fr.) Gilbert, Les Livres du Mycologue Tom. III: Les Bolets:92,1931

Basidioma medium to large. Pileus 30–180 (250)mm in diameter, nearly hemispherical when young, then becoming pulvinate-convex, and finally applanate, golden brown (5D7), light brown (6D8) to rust brown (6E8) or chestnut brown (7E7–7E8); margin short appendiculate, involute when young; pileal surface minutely subtomentose or fairly floccose when dry, becoming viscid when wet. Hymenophore adnate to sinuate, sometimes depressed around stipe apex, initially cream (2A2) to pale lemon yellow (1A5), becoming dull yellow (3B7) with age, bluing (21E4) when injured. Pores compound, angular, 0.5–1.5mm wide, concolorous with tubes, bluing when bruised, fading to dull brown after a few hours. Stipe 40–140 × 10–50mm, solid, cylindrical when young, becoming somewhat clavate with age, concolorous with pileus or slightly paler (3A3–3A6) in the upper part, covered with minute brown (5E8–5F8) squamules. Context of pileus and stipe cream to lemon yellow, brownish beneath the pileipellis, becoming blue particularly above the tubes and at the stipe apex. Basal mycelium whitish to cream (2A1–2A2). Taste and odor mild.

Figure 3. Basidiomata of Imleria species. a. Im. badia (HKAS 74713, by M. Gelardi). b. Im. obscurebrunnea (HKAS 50477, photo by Z.L. Yang). c. Im. parva (HKAS 55341, holotype, photo by B. Feng). d. Im. subalpina (HKAS 74712, photo by B. Feng).

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 87 Figure 4. Basidiospores of Imleria species from herbarium materials under SEM. a. Im. badia (HKAS 74713). b. Im. obscurebrunnea (HKAS 50477). c. Im. parva (HKAS 55341, holotype). d. Im. subalpina (HKAS 74712). Note basidiospores with smooth surface.

Basidia 25–48 × 8–14μm, clavate, 4-spored. Basidiospores [160/8/5], 11–15(17.5) × (3.5)4–5μm, Q = (2.3) 2.5–

3.6(4.2), Qm = 3.0 ± 0.33, boletoid, subfusiform in side view, ellipsoid to somewhat oblong in face view, smooth, slightly thick-walled (0.5μm), nearly hyaline to pale olivaceous brown (2B6) in 5% KOH, with one to two guttulae when mature. Pleuro- and cheilocystidia scattered, 35–65 × 6–10μm (n = 15), lanceolate to subfusoid-mucronate or ventricose, thin-walled, hyaline when young, sometimes with yellowish brown (4B8) plasmatic pigment when mature (in 5% KOH). Caulocystidia 29–65 × 5–9μm, clavate, ventricose, caespitose, forming squamules over the stipe surface. Pileipellis an ixotrichoderm in youth, becoming an ixo-intricate-trichoderm when mature, consisting of yellowish interwoven hyphae, which are slightly incrusted by yellow-brown (4B8) pigment and embedded in gelatinous substance; terminal cells cylindrical with rounded to tapering apex, 30–60 × 4–7μm. Clamp connections absent in all tissues. Habitat: Under (e.g. Abies, Picea, Pinus, Pseudotsuga, Thuja, ) and deciduous trees (e.g. Alnus, Castanea, Carpinus, Fagus, Quercus). Known distribution: A common species in Europe and North America, found from the lowlands up to the timberline (Alessio 1985; Singer 1986; Ladurner & Simonini 2003). Our ITS phylogenetic analysis indicated that this species also occurs in East Asia and South Africa (also see discussion below). Materials examined: SWEDEN, Angermanland, Nordingra, 28 August 1993, A. Strid 19707 (S-F119691, epitype, here designated; GenBank acc. n. KJ806970, KJ806971, KJ806969, KJ806972 and KJ806973 for ITS, nrLSU, tef1- α, rpb1 and rpb2 respectively). GERMANY, Marburg, 4 Dec 2006, Z.L. Yang M3 (HKAS 74714); same location, 24 November 2006, L. Wang 4 (HKAS 53502). ITALY, Lazio, Caldara di Manziana (Rome), elev. 265m, 25 November 2010, M. Gelardi, MG396a (HKAS 74713); Lombardy, Cevo (Brescia), elev. 1750m, 20 August 2012, M. Gelardi, MG463a (HKAS 75797).

88 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. Figure 5. Microscopic features of Imleria badia (drawings by X.T. Zhu). a. Basidia with basidioles. b. Basidiospores. c. Pleuro- and cheilocystidia. d. Pileipellis from a young basidioma. e.Pileipellis from a mature basidioma. (a, b, c and e from HKAS 74713, d from HKAS 53502). (bar = 10μm)

Comments: Imleria badia is characterized by its (usually) large basidioma, with a light brown to bay brown pileus which is slightly viscid when wet, a stout stipe concolorous with the pileal surface or paler, a pale colored

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 89 hymenophore and pileal context staining blue on bruising or exposure. Up to now there are no formal designated types for Im. badia. S-F119691 collected from Sweden is designated here as the epitype of this taxon with DNA nucleotide sequences of five loci in order to help to interpret the concept of the species consistently.

Imleria obscurebrunnea (Hongo) Xue T. Zhu & Zhu L. Yang, comb. nov. (Figs. 3b, 4b and 6) MycoBank: MB 803970

Basionym: Xerocomus obscurebrunneus Hongo, J. Jap. Bot. 54: 301 (1979) Boletus obscurebrunneus (Hongo) Har. Takah., MSJ News. 19: 38 (1992)

Figure 6. Microscopic features of Imleria obscurebrunnea (HKAS 50477). (drawings by X.T. Zhu) a. Basidia with basidioles. b. Basidiospores. c. Pleuro- and cheilocystidia. d. Pileipellis. (a and d from HKAS 50477; b and c from HKAS 50477 and TNS-F-237653, holotype, respectively). (bar = 10μm)

90 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. Basidioma small to medium. Pileus 20–60mm broad, nearly hemispherical when young, then convex and finally applanate, rust brown (6E8), chestnut brown (7E7–7E8) or gray-brown (6E3), margin slightly appendiculate, pileal surface minutely subtomentose or fairly floccose when dry, becoming viscid when wet. Hymenophore sinuate, depressed around the stipe apex, initially cream or pale yellow (2A2–2A3), olivaceous yellow (2B6) or dull yellow (3B7) when mature, becoming greyish to bluish slowly when injured. Pores compound, angular, 0.5–1mm in diameter, concolorous with tubes, bluing slightly when bruised, fading to dull brown after a few hours. Stipe slender, 40–80 × 3– 8mm, slightly enlarged towards the base, concolorous with pileus, densely covered with dark brown (7E8) squamules, apical part yellowish (3A3), basal part whitish to cream. Context of pileus cream to yellowish, bluing slowly when bruised, context of stipe dirty white to brownish. Basal mycelium dirty white (1A1). Taste and odor mild. Basidia 26–35 × 9–11μm, clavate, hyaline, 4-spored. Basidiospores (270/13/10), (9.0)9.5–12 × (3.5)4–

4.5(5.0)μm, Q = 2.2–2.8(3), Qm = 2.55 ± 0.16, subfusiform in side view, ellipsoid in face view, smooth, slightly thick- walled (up to 1μm), pale olivaceous brown (2B6) in 5% KOH, with one to two guttulae when mature. Pleuro- and cheilocystidia scattered, 26–48 × 7–10μm (n = 15), lanceolate to subfusoid-mucronate, thin-walled, hyaline when young, sometimes with yellowish brown (4B8) plasmatic pigment in 5% KOH when mature. Caulocystidia 20–32 × 7–9μm, fusoid-ventricose, clavate, lanceolate, distributed in clusters, forming squamules over the stipe surface. Pileipellis an ixotrichoderm consisting of yellowish interwoven hyphae, which are slightly incrusted by yellow-brown (4B8) pigment and embedded in a gelatinous substance; terminal cells cylindrical with rounded to tapered apex, 40–80 × 5–8μm. Clamp connections absent. Habitat: In forest of Fagus, Quercus, Castanopsis and Lithocarpus. Known distribution: Currently known from southwestern China and Japan. Materials examined: JAPAN, Niigata-Pref., Myokokogen, Sasagamine, 7 October 1977, Tsuguo Hongo 5684 (TNS-F-237653, holotype); Funagata-yama, Taiwa-cho, Kurokawa-gun Miyagi, 29 July 2006, Yoichi Hoshino (TNS- F-12319); Tottori-Pref., Saihaku-gun, Houki-Daisen Mountain, elev. 100m, 26 September 1986, S. Murata (TNS-F- 28438); Ishikawa-ken Hakusan Nature Reserve, 1 August 1980, Yoshiyuki Ikeda (TNS-F-22050); same location, 24 July 1999, Kyouiti Akeyama (TNS-F-22052); same location, 23 September 1994, Yamashita Yoshiaki (TNS-F-22051); CHINA, Yunnan Province: Jingdong County, Ailao Mountain, elev. 2500m, 20 July 2006, Z.L. Yang 4680 (HKAS 50477); same location, elev. 2500m, 15 July 2008, Y.C. Li 1229 (HKAS 56083); same location, elev. 2450m, 20 July 2006, Y.C. Li 592 (HKAS 50346); same location, elev. 1450 m, 17 July 2007, Y.C. Li 872 (HKAS 52557). Comments: Imleria obscurebrunnea is well characterized by its small to medium basidioma with a slender stipe which is rust brown or chestnut brown with a yellowish apical part, and a whitish to cream basal part, an ixotrichodermal pileipellis and its association with Fagus, Quercus, Castanopsis and Lithocarpus. The color and size of the basidiomata of Im. obscurebrunnea are similar to those of X. prebadius (Corner) E. Horak (2011: 120) (Corner 1972; Horak 2011). However, the latter species has a reddish context at the stipe base, and can be found only in tropical lowland dipterocarp forests.

Imleria parva Xue T. Zhu & Zhu L. Yang, sp. nov. (Figs. 3c, 4c and 7) MycoBank: MB 802392

Etymology: parva (small), referring to the small basidioma.

Basidioma small. Pileus 25–35mm broad, hemispherical to broadly convex, chestnut brown (7D8) to dark brown (8E7), margin slightly appendiculate, pileal surface fairly floccose when dry, becoming viscid when wet. Hymenophore sinuate, depressed around the stipe apex, initially cream or pale yellow (2A2–2A3), to pale olivaceous yellow (2B6) when mature, becoming bluish when injured. Pores compound, angular, 0.5–1mm wide, concolorous with tubes, becoming bluish when bruised, finally dull brown. Stipe 40–70 × 3–7mm, slender, cylindrical or enlarged towards the base, concolorous with pileus or slightly paler, densely covered with dark brown squamules (7E8). Context of pileus cream to pale yellow, bluing slowly when bruised, context of stipe dirty white to brownish. Basal mycelium white (1A1).Taste and odor mild. Basidia 26–32 × 9–11μm, clavate, hyaline in 5% KOH, 4-spored. Basidiospores (90/3/3), (8.5)9–11(12) × (3)3.5–

4.5μm, Q = (2.3)2.5–2.7(3.0), Qm = 2.61 ± 0.14, subfusiform in side view, ellipsoid in face view, smooth, slightly thick walled (0.5–1μm), pale olivaceous brown (2B6) in 5% KOH, with one to two guttulae when mature. Pleuro- and cheilocystidia scattered, 30–65 × 7–11μm (n = 15), lanceolate to subfusoid, thin-walled, hyaline when young, sometimes with yellow-brown (4B8) plasmatic pigment with age in 5% KOH. Caulocystidia fusoid-ventricose, clavate, 25–40 × 7–9μm, forming squamules upon the stipe surface. Pileipellis an ixotrichoderm consisting of yellowish interwoven hyphae, which are slightly incrusted by yellow-brown pigment (4B8) and embedded in a gelatinous substance, the

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 91 pigment and gel slowly disappear in 5% KOH; terminal cells cylindrical with rounded to tapered apex, 50–95 × 5–9μm. Clamp connections absent.

Figure 7. Microscopic features of Imleria parva (HKAS 55341, holotype). (drawings by X.T. Zhu) a. Basidia with basidioles. b. Basidiospores. c. Pleuro- and cheilocystidia. d. Pileipellis. (bar = 10μm)

Habitat: In mixed forests of Castanopsis and Pinus. Known distribution: Currently known only from southern and southwestern Yunnan. Materials examined: CHINA, Yunnan Province: Simao City, Caiyang River Natural Reserve, elev. 1500m, 28

92 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. July 2008, B. Feng 231 (HKAS 55341, holotype); Dehong City, Yingjiang County, elev.1940m, 18 July 2009, Y.C. Li 1690 (HKAS 59437); Nujiang City, Lushui County, elev. 2000m, 7 August 2010, Y.J. Hao 190 (HKAS 68162). Material of Xerocomus subdaedaleus examined: China, Province: Minya Konka, H.A. Wen 1169 (HMAS 47904, holotype).

Figure 8. Microscopic features of Imleria subalpina (HKAS 56375 holotype). (drawings by X.T. Zhu) a. Basidia with basidioles. b. Basidiospores. c. Pleuro- and cheilocystidia. d. Pileipellis. (bar = 10μm)

Comments: Imleria parva has small basidiomata (up to 35mm) with a cream to pale olivaceous yellow hymenophore, and the whole stipe is densely covered with dark brown squamules. According to the characters cited above, this species can easily be distinguished from other species of Imleria. Imleria parva is similar to Xerocomus subdaedaleus

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 93 J.Z. Ying (1986: 313) in the small size of basidioma and the chestnut brown pileus and stipe, but the latter has a dry, never viscid, subtomentose pileus, a subdaedaloid hymenophore with irregularly angular pores 1–2mm in diameter and a trichodermal pileipellis (Ying 1986; observations of X.T. Zhu).

Imleria subalpina Xue T. Zhu & Zhu L. Yang, sp. nov. (Figs. 3d, 4d and 8) MycoBank: MB 802394

Etymology: subalpina, indicates that this species is distributed only in subalpine areas.

Basidioma small to medium. Pileus 40–80mm broad, hemispherical to broadly convex, margin slightly appendiculate, red-brown (8E8) to dark brown (8E7), pileal surface minutely tomentose when dry, becoming viscid when wet. Hymenophore sinuate, depressed around the stipe apex, initially yellowish (2A3) or pale lemon yellow (1A5), becoming olivaceous yellow (2B6) when mature, bluing slowly when injured, pores compound, angular, 0.5–1mm in diameter, concolorous with tubes, bluing slowly when bruised, fading to dull brown after a few hours. Stipe 50–70 × 8–17mm, clavate, slightly bulbous at the base, concolorous with pileus, covered with brownish to dark brown (7E8) squamules, apical part of stipe yellowish (3A3). Context of the pileus cream to yellow, bluing slowly when bruised, context of stipe dirty white to brownish. Basal mycelium dirty white (1A1). Taste and odor mild. Basidia 24–35 × 10–13μm, clavate, hyaline in 5% KOH, 4-spored. Basidiospores (93/5/3), (10.5)11–15(17) ×

(4)4.5–6.0(6.5)μm, Q = (2.0)2.4–2.6(3.3), Qm = 2.5 ± 0.23, subfusiform in side view, ellipsoid to somewhat oblong in face view, smooth, slightly thick-walled (0.5–1μm), pale olivaceous brown (2B6) in 5% KOH, with one to two guttulae when mature. Pleuro- and cheilocystidia scattered, 35–60 × 7–10μm (n = 15), lanceolate to subfusoid, thin-walled, hyaline when young, some with yellowish brown (4B8) plasmatic pigment in 5% KOH when mature. Caulocystidia 25–40 × 7–9μm, clavate, forming squamules over stipe surface. Pileipellis an ixotrichoderm composed of yellowish interwoven hyphae, which are slightly incrusted by yellow-brown pigment (4B8) and embedded in gelatinous substance, the pigment and gel disappear slowly in 5% KOH; terminal cells cylindrical with rounded to tapered apex, 20–60 × 5–8μm (n = 30). Clamp connections absent. Habitat: In subalpine mountains (elev. 3300–3900m), under mixed forests dominated by Picea and Abies. Known distribution: Currently known only from northwestern Yunnan. Materials examined: CHINA, Yunnan Province, Shangri-La County, Daxueshan Mountain, elev. 3919m, 21 August 2008, Y.C. Li 1535 (HKAS 56375, holotype); Lijiang City, Yulong County Laojunshan Mountain, elev. 3384m, 3 September 2009, B. Feng 773 (HKAS 74712); Deqing County, Baima Snow Mountain, elev. 3700m, 11 July 1981, L.S.Wang 122 (HKAS 8693). Comments: Imleria subalpina is characterized by its small to medium basidioma with a red-brown to dark brown pileus which is slightly viscid when wet, a yellowish apex to the stipe, a cream to pale olivaceous yellow hymenophore, a context slowly bruising blue. It is similar to Im. obscurebrunnea, however, the basidiospores of Im. subalpina (11–15 × 4.5–6.0μm) are larger than those of Im. obscurebrunnea (9.5–12 × 4–4.5μm), and Im. subalpina is putatively associated with Picea and Abies in subalpina areas, whereas Im. obscurebrunnea is associated with Fagus, Quercus, Castanopsis and Lithocarpus. The color and size of pileus of Im. subalpina is similar to that of Xerocomus rectus (Corner) E. Horak (2011: 127) (Corner 1972; Horak 2011), however X. rectus has a whitish to cream stipe and narrower hyphae (2–3μm diam) in the pileipellis.

Discussion

The systematic position and species diversity of Imleria Molecular techniques have revolutionized our knowledge of basidiomycete evolution (Yang 2011). The results of the phylogenetic analysis from the combined dataset indicate that Imleria is nested within the family Boletaceae (RAxML BS = 90, PP = 0.99) and clusters as a monophyletic group (RAxML BS = 100, PP = 1), clearly separated from Xerocomus s. str., Xerocomellus Šutara (2008: 44), Pseudoboletus Šutara (2008: 55) and Boletus s. str. by genetic distance (Fig.1). The genus Imleria corresponds to the clade 22 of Wu et al. (2014). Interestingly, two badione A and norbadione A, which are responsible for the intense brown colors of the pileus of Im. badia, are unique in the Boletaceae (Steffan & Steglich 1984; Aumann et al. 1989; Velíšek & Cejpek 2011). In addition, these pigments are only found in the gleba of Pisolithus tinctorius (Mont.) E. Fisch. (1900: 338)

94 • Phytotaxa 191 (1) © 2014 Magnolia Press Zhu ET AL. and Scleroderma citrinum Pers. (1801: 153), gasteroid members of Boletales (Winner et al. 2004). It would be a good chemotaxonomical character of Imleria if these pigments were found in the pileus of the rest of the species of this genus in the future. Before this work, all Chinese specimens sharing similar macro-morphological features with Im. badia were regarded as Boletus badius. Our study on samples collected from China showed that three species can be recognized, all of which are distinct from Im. badia. The combination of macro- and micro-morphology, as well as distribution and range can be used as informative characters to distinguish these species from each other. In summary, Im. parva was found in tropical Yunnan and has small basidiomata, Im. obscurebrunnea is known from subtropical regions, and has a medium-sized basidioma with a slender stipe, while Im. subalpina, putatively associated with Picea and Abies, is found only in subalpine areas in southwestern China so far. It should be noted that Boletus pallidus was regarded as a member of the ‘badius’ clade (Nuhn et al. 2013), which was, however, not supported by Wu et al. (2014). In our present work, B. pallidus formed a clade with Imleria (Fig.1), but in view of its pallid to glaucous pileus and pale stipe (Smith & Thiers 1971), and the genetic distance between it and the species of Imleria, B. pallidus is excluded from Imleria. Further studies on Boletaceae with extensive sampling from wider ranges over the world would help us to understand the relationship between B. pallidus and Imleria to a higher degree.

Key to the species in Imleria

For the identification of the four species in the genus, a dichotomous key is provided as follows.

1. Basidiomata tiny to medium (pileus ≤ 80mm in diameter), stipe slender when mature (≤ 15mm in diameter), in East Asia...... 2 1. Basidiomata usually large (pileus ≥ (30) 90mm in diameter), stipe stout (≥ 15mm in diameter usually when mature), in the north temperate zone...... Im. badia 2. Basidiomata usually medium-sized (pileus 50–80mm in diameter), apical part of stipe always white to pale yellow...... 3 2. Basidiomata tiny to small (pileus ≤ 40mm in diameter), apical part of stipe chestnut brown to dark brown...... Im. parva 3. Basidiospores 11–15 × 4.5–6.0μm, in subalpine areas in forests dominated by Abies and Picea, in subalpine mountains of south- western China...... Im. subalpina 3. Basidiospores smaller (9.5–12 × 4–4.5μm), in subtropical areas in forest of Fagus, Quercus Castanopsis and Lithocarpus, in Japan and China...... Im. obscurebrunnea

Intraspecific divergence of Imleria badia due to geographical isolation Imleria badia is widely distributed in north temperate regions according to the result of phylogenetic analysis obtained in this study based on ITS dataset. The occurrence in Southern Hemisphere was probably due to introduction of species, which can be a host plant for Im. badia, into South Africa from Europe and North America. A similar phenomenon has also been reported in the study of the porcini (Feng et al. 2012). Some genetic divergence was found between specimens from Europe and North America in the phylogenetic analysis of the ITS dataset (Fig.2). It can perhaps be interpreted as intraspecific divergence of Im. badia due to geographic isolation, as there is no morphological difference that could be found between them and the intraspecific variation (0.0026) in the species Im. badia is obviously smaller than the lowest interspecific variation (0.0176) in the genus Imleria.

Possible evolutionary history of the closely related Im. obscurebrunnea and Im. subalpine Imleria subalpina and Im. obscurebrunnea formed a sister relationship based on the multi-locus combined dataset, Im. obscurebrunnea is currently known in China and Japan, at 100–2600m altitude, associated with Castanopsis, Lithocapus, Fagus and Quercus while Im. subalpina only fruits at 3300–3900m altitude in northwestern Yunnan and is possibly associated with subalpine trees (Picea and Abies). The geographical distribution and phylogenetic relationships of the two species suggest they may have recently diverged from each other, probably as a result of the uplifts of the Himalaya-Hengduan Mountains (Yang 2005). Tulloss et al. (2005) and Kauserud et al. (2011) suggested that larger spores may contain more water, as well as more , which are essential resources during the germination process and the phase of initial growth of the primary mycelia. Interestingly, we found that the spores of Im. subalpina are significantly larger than those of Im. obscurebrunnea (df = 120.93, t = 17.24, P<0.001). The larger spores of Im. subalpina could be an evolutionary adaption to the environmental pressure (less precipitations, lower temperature, etc.) in subalpine areas.

Imleria (Boletaceae) Phytotaxa 191 (1) © 2014 Magnolia Press • 95 Acknowledgements

The authors are very grateful to Dr. B.M. Spooner and Dr. B. Aguirre-Hudson (Kew), Dr. K. Hosaka (TNS), Pro. Y.J. Yao (HMAS), Dr. K. Hansen (S), Dr. X. H. Wang (Kunming Institute of Botany, Chinese Academy of Sciences, KIB) for providing specimens. Dr. Z.W. Ge, Dr. Md. Iqbal Hosen, Miss J. Qin and Y. J. Hao (KIB) are thanked for providing valuable literature and helpful suggestions. Dr. Z.X Ren (KIB) is acknowledged for helping us in using the SEM facility. This study was supported by the Funds for International Cooperation and Exchange of the National Natural Science Foundation of China (No. 31210103919), and the National Basic Research Program of China (No. 2014CB138305), and the Fundamental Research Program of the Ministry of Science and Technology, China (2013FY110400).

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