Phytotaxa 327 (1): 047–056 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2017 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.327.1.2

Two new of () from southern China revealed by morphological characters and molecular data

MING ZHANG, TAI-HUI LI & BIN SONG State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China. *Correspondence to: [email protected]

Abstract

Two new species of Chalciporus are described from southern China. Chalciporus citrinoaurantius sp. nov. is characterized by a lemon yellow to grayish orange pileus, light orange to rose pink tubes and elongated basidiospores (9.0)9.5–12.5(13) × 3–4(4.5) μm; C. hainanensis sp. nov. is characterized by small basidiomata with grayish yellow to brownish orange pilei, orange yellow to brownish orange tubes with brownish orange to brownish red pores and elliptic basidiospores 8–10.5(11) × 4–5(5.5) μm. Phylogenetic analysis based on the combined dataset (nrLSU+tef1-α+rpb2) confirmed the affinity of the new species in Chalciporus and indicated their relationships with other species within the .

Key words: Boletes, phylogentic analysis,

Introduction

Chalciporus Bataille (1908:36) is a small and widely distributed genus in the family Boletaceae. It was established in 1908 with C. piperatus (Bull.) Bataille (1908:39) as the type species to accommodate those species with a reddish brown hymenophore, a smooth stipe with yellow to bright yellow basal mycelium, a peppery taste and smooth spores (Bataille 1908). According to recent phylogenetic studies, Chalciporus is a monophyletic group, and together with the genus Pilát (1969:217) forms a basal branch in the Boletaceae (Nuhn et al. 2013; Wu et al. 2014, 2016). The genus Rubinoboletus Pilát & Dermek (1969:81) was proven to be a synonym of Chalciporus based on morphological characteristics (Singer 1973, 1986; Pegler & Young 1981, Klofac 2006) and a classification of subgenus Rubinoboletus was proposed (Klofac 2006). Molecular data has verified the synonymy (Nuhn et al. 2013; Wu et al. 2014, 2016). Thirty-seven names have been recorded in the whole world (http://www.indexfungorum.org), with only two species originally reported from China, viz. C. radiatus Ming Zhang & T.H. Li (2016:21) and C. rubinelloides G. Wu & Zhu L. Yang (2016:74) (Zhang et al. 2015b; Wu et al. 2016). During a recent survey on the bolete diversity in southern China, two new species were found based on morphological observations and phylogenetic analyses. They were formally described and illustrated as follows.

Materials and methods

Sampling, morphological observation and descriptions Photographs of the basidiomata were taken in the type localities when collected. Specimens were dried and deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM). Macroscopic characters were examined from fresh materials. Microscopic observations were carried out on tissue sections stained with 5% KOH and 1% aqueous Congo red, or Melzer’s reagent under a light microscope (Olympus BX51, Tokyo) with magnification up to 1000×. All measurements were made in 5% KOH. The methods of morphological descriptions in the previous study by Zhang et al. (2015a) were followed. The colors were recorded and described in general terms following Kornerup and Wanscher (1978). The notations of basidiospores (n/m/p) indicate that the measurements were made on n basidiospores

Accepted by Genevieve Gates: 26 Jul. 2017; published: 3 Nov. 2017 47 from m basidiomata of p collections. Microstructures were observed from rehydrated materials and line drawings were drawn by free hand.

DNA extraction, PCR amplification and sequencing Genomic DNA of the new species was extracted from material (dried with silica gel) using the Sangon Genomic DNA Extraction kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Primer pairs LR0R/LR5 (Vilgalys & Hester 1990), EF1-B-F1/EF1-B-R and RPB2-B-F1/RPB2-B-R (Wu et al. 2014) were used for the amplification of the large subunit nuclear ribosomal RNA (nrLSU) region, translation elongation factor 1-alpha subunit (tef1-α) and the second largest subunit of RNA polymerase II (rpb2), respectively. PCR was performed in a total volume of 25 ml containing 0.5 ml template DNA, 0.5 ml of each primer,11 ml distilled water, and 12.5 ml PCR mix [DreamTaq™ GreenPCR Master Mix (2×), Fermentas, Vilnius, Lithuania]. Amplifications were performed in a Tprofessional standard thermocycler (Biometra, Göttingen, Germany) under the following conditions: pre-denaturation at 95°C for 4 min, then followed by 35 cycles of denaturation at 95°C for 60 s, annealing at 54°C (nrLSU) /52°C (tef1-α and rpb2) for 60 s, extension at 72°C for 80 s and a final extension at 72°C for 8 min. The PCR product was electrophoresedon 1% agarose gels with known standard DNA marker, and sequenced on an ABI Prism® 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, USA) at Beijing Genomic Institute (BGI) using the same primers as in the PCR amplifications. Newly generated sequences in this work have been submitted to GenBank.

Sequence alignments and phylogenetic analyses DNA sequences were aligned using Clustal X v1.81 (Thompson et al. 1997) and manually modified where necessary in BioEdit v7.0.5.3 (Hall 1999). The ambiguous aligned bases and introns of protein-coding genes were retained in the final analyses. For molecular phylogenetic analyses on the combined dataset (nrLSU+tef1-α+rpb2), Maximum likelihood (ML) and Bayesian inference (BI) were employed by using RAxML v7.2.6 (Stamatakis 2006) and MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003), respectively. Substitution models of partition in the dataset were determined using the Akaike Information Criterion (AIC) implemented in MrModeltest v2.3 (Posada & Crandall 1998; Nylander 2004). The selected substitution models for the partitions are: GTR+I+G for nrLSU, HKY+I+G for tef1-α and rpb2. For ML analysis, all parameters were kept default except for the model choosing as GTRGAMMAI, and the statistic supports were calculated using nonparametric bootstrapping with 1000 replicates. BI analysis using selected models and 4 chains were conducted by setting generations to 1 million and stoprul command with the value of stopval set to 0.01. Trees were sampled every 100 generations. The trees were summarized and statistical values were obtained using the sump and sumt commands with the first 25% generations were discarded as burn-ins. Branches that received bootstrap support for Maximum likehood (ML) and Bayesian posterior probabilities (BPP) greater than or equal to 70 % (ML) and 0.95 (BPP), respectively, were considered as significantly supported. Buchwaldoboletus lignicola (Kallenb.) Pilát (1969:217) was selected as the outgroup based on recent studies (Zhang et al. 2015b, Wu et al. 2016).

Results

Molecular phylogeny The multi-locus (nrLSU+tef1-α+rpb2) dataset included 64 sequences (30 28S, 19 tef1-a, and 15 rpb2) from 30 samples. Among them, 22 sequences were newly generated in the present study (7 28S, 8 tef1-α, and 7 rpb2). The dataset had an aligned length of 2164 bp nucleotide sites. The aligned matrix was submitted to TreeBase (21151). Phylogenetic trees generated from ML and BI analyses share the same topology, and with only minimal differences in statistical support values. Thus, only the ML tree topology is shown (Fig. 1). The phylogenetic tree showed that Chalciporus is a well-supported monophyletic group with strong statistical support (ML=100 %, BPP=1) (Fig. 1). The samples collected from southern China formed two independent lineages, one lineage collected from the subtropical area closely clustered with C. rubinelloides with high statistical support (ML=100 %, BPP=1), and the other one collected from the tropical area formed a sister clade to an unnamed sequence (GDGM43250) produced in this study.

48 • Phytotaxa 327 (1) © 2017 Magnolia Press ZHANG ET AL. FIGURE1. Phylogenetic analysis of Chalciporus species inferred from Maximum Likelihood (ML) analysis of lsu+tef1-α+rpb2 sequences are shown. Bootstrap values (ML ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.95) are shown around branches. The two new species’ sequences are indicated in bold face. Questionable sequences are marked with quotation marks.

Taxonomy

Chalciporus citrinoaurantius Ming Zhang & T.H. Li sp. nov. MycoBank: MB821522 Etymology: citrino- means lemon yellow and aurantius means orange color, therefore, citrinoaurantius refers to the lemon yellow to orange hue of the pileus color. Diagnosis:—This species is similar to C. rubinelloides, but differs in the smaller basidiomata with light yellow to grayish orange pilei, larger pores, and smaller basidospores (9.0)9.5–12.5(13) × 3–4(4.5) μm. The light orange to rose pink tubes unchanging when bruised and the yellowish stipe context changing to light yellow when exposed are also distinguishing features. Type:—CHINA. Hunan Province, Chenzhou City, Yizhang county, Mangshan National Natural Reserve, at 24°57′N, 112°56′E, alt. 1300 m, 14 June 2015, Ming Zhang (GDGM 44776, holotype!).

Description:—Basidiomata small. Pileus 2–5 cm wide, hemispheric at first, then becoming convex to nearly plane with age, occasionally turning upwards at the margin when mature, fleshy, surface dry, viscid when wet, smooth or velvety- tomentose, light yellow, lemon yellow, light orange, grayish yellow to grayish orange (1A5–6A5, 1B5–6B5). Context 2–4 mm thick at stipe, soft, white, yellowish white to pale yellow (1A1–3A1, 1A2–3A2, 1A3–3A3), unchanging when exposed to air. Tubes 3–5 mm deep, depressed to subdecurrent, light orange, pastel red to rose pink (6A5–11A5), often with purplish hue, color unchanging when bruised. Pores 1–1.5 mm in diam, circular to angular, pore surface

Two new species of Chalciporus (Boletaceae) Phytotaxa 327 (1) © 2017 Magnolia Press • 49 concolorous with tubes, unchanging when bruised. Stipe 30–40 × 3–5 mm, central, solid, subcylindrical or clavate, equal or slightly tapered downward, concolorous with pileus or paler, surface smooth or pruinose at apex or upper half, sometimes with indistinct longitudinal striations when mature; stipe context yellowish white to pale yellow (2A2–3A2, 2A3–3A3), changing to light yellow (2A5–3A5) when exposed, then partially becoming concolorous with stipe surface or nearly so; basal mycelium yellowish white, yellow to golden yellow. Odor indistinctive and taste slightly peppery. Basidiospores [80/4/4] (9.0)9.5–12.5(13) × (3)3.5–4(4.5) μm , [Q = (2.5)2.7–3.25(3.4), Qm = 2.96 ± 0.21], fusiform, cylindrical to somewhat oblong, barely inequilateral in profile, smooth, thin-walled, yellowish to yellowish brown in 5% KOH, yellow brown to dark brown in Melzer’s reagent. Basidia 23–32 × 8–10 μm, 4-sterigmate, clavate, yellowish white to hyaline in 5% KOH. Pleuro- and cheilocystidia 30–65 × 8–13 μm, scattered, projecting and conspicuous, narrowly fusiform, thin-walled, smooth, yellowish white to hyaline, and usually containing some yellowish brown oleiferous scattered substance in 5% KOH. Hymenophoral trama subparallel, yellowish white to hyaline in 5% KOH, with hyphae 6–10 μm broad. Pileipellis a trichoderm of erect hyphae 4–7 μm in diam, branched, yellowish white to hyaline in 5% KOH, dark brown to rusty brown in Melzer’s reagent; terminal elements cylindrical, clavate or nearly fusoid. Stipitipellis a tangled layer of repent hyphae 3–7 μm in diam, yellowish white to hyaline in 5% KOH. Clamp connections absent in all tissues. Habit, habitat and distribution:—Solitary or scattered on humus and debris under Pinus massoniana Lamb and mixed with other broadleaf trees, alt. 1000–1500 m; June to September, currently known from Hunan and Zhejiang Province. Additional specimens examined:—CHINA. Hunan Province, Chenzhou City, Yizhang county, Mangshan National Natural Reserve, at 24°57′N, 112°56′E, alt. 1300 m, 14 June 2015, Ming Zhang (GDGM44717); Same locality, 19 May 2015, Ming Zhang (GDGM44480); Same locality and date, Ming Zhang (GDGM44481); Zhejiang Province, Longquan county, Fengyangshan National Natural Reserve, at 27°56′N, 119°12′E, alt. 1000 m, 11 September 2016, Ming Zhang (GDGM46540)

Chalciporus hainanensis Ming Zhang & T.H. Li sp. nov. MycoBank: MB821521 Etymology: hainanensis refers to the type locality in Hainan Island. Diagnosis:—It is characterized by small basidiomata with olive yellow to brownish orange pilei, orange-yellow to brownish orange tubes, white to yellowish white context staining blue at first, then gradually changing to grayish orange to grayish red when exposed, and small basidiospores 8–10.5(11) × 4–5(5.5) μm. Type:—CHINA. Hainan Province, Ledong county, Jianfengling National Natural Reserve, at 18°43′N, 108°51′E, alt. 1000 m, 3 July 2013, Ming Zhang (GDGM44464, holotype!).

Description:—Basidiomata small. Pileus 2–3 cm wide, hemispheric at first, then becoming convex to nearly plane, occasionally turning upwards at margin when mature, fleshy, surface dry, velvety-tomentose to fibrillose, grayish yellow, olive yellow, grayish orange to brownish orange (4B4–7B4, 4C4–7C4). Context 2–4 mm thick, soft, white to yellowish white (3A1–4A1, 3A2–4A2), changing blue at first, then gradually changing to grayish orange to brownish orange or grayish red (4B4–5B4, 4C4–5C4) when bruised. Tubes 3–5 mm deep, slightly depressed to subdecurrent, yellow, orange-yellow to orange (3B8–5B8) at young, brownish yellow to brownish orange (4C8–7C8) when mature, unchanging or slightly changing blue when bruised. Pores 1–1.5 mm in diam, circular to angular, pore surface grayish yellow to brownish yellow (4C8–5C8) initially, becoming brownish orange to brownish red (6C8–9C8) when mature, slightly changing blue when bruised. Stipe 30–40 × 3–5 mm, central, solid, subcylindrical or clavate, equal or slightly enlarged downward, concolorous with pileus, sometimes ribbed by the decurrent lamella lines at apex, usually with a brownish red annulus at apex, gradually darkening to yellowish brown to dark brown (5D8–6D8) downwards to base, often slightly becoming bluish brown where injured, surface dry, smooth to velvety-tomentose, or sometimes longitudinally striate, with yellowish to yellow basal mycelium. Stipe context yellowish brown to brown (5D8–6D8), slightly changing to blue when exposed, and then becoming unevenly reddish brown to dark brown mixed with bluish brown. Odor indistinctive and taste slightly peppery. Basidiospores [80/2/2] 8–10.5(11) × 4–5(5.5) μm, [Q = (1.7)1.9–2.2(2.5), Qm = 2.06 ± 0.15], fusiform, cylindrical to somewhat oblong, barely inequilateral in profile, smooth, thin walled, yellowish to yellowish brown in 5% KOH, yellow brown to dark brown in Melzer’s reagent. Basidia 25–33 × 9–14 μm, clavate, yellowish white to hyaline in 5% KOH, 2, 4-sterigmate, 2–3.5 μm long. Pleurocystidia 43–70 × 7–12 μm, scattered, projecting and conspicuous, narrowly fusiform, thin-walled, smooth, yellowish white to hyaline, and usually containing some yellowish brown scattered oleiferous substance in 5% KOH. Cheilocystidia similar to pleurocystidia, abundant and conspicuous,

50 • Phytotaxa 327 (1) © 2017 Magnolia Press ZHANG ET AL. fusoid to subclavate, smooth, thin-walled, hyaline or, more frequently, with scattered dark yellowish brown content. Hymenophoral trama subparallel, yellowish white to hyaline in 5% KOH, with hyphae 4–13 μm broad. Pileipellis an intricate trichoderm to interwoven subcutis of erect hyphae 3–8 μm in diam, branched, yellowish white to hyaline in 5% KOH, dark brown to rusty brown in Melzer’s reagent; terminal elements cylindrical, clavate or nearly fusoid. Stipitipellis a tangled layer of repent to suberect hyphae 4–13 mm in diam, forming the dotted ornamentation over surface of stipe, yellowish white to hyaline in 5% KOH, end hyphae 6–20 μm diam. Clamp connections absent in all tissues. Habitat and distribution:—Solitary or scattered on humus and debris under Fagaceae mixed with other broadleaf trees, alt. 800–1200 m; currently only known from Hainan Province. Additional specimens examined:―CHINA. Hainan Province, Changjiang county, Bawangling National Nature Reserve, at 19°21′ N, 109°12′ E, alt. 1000 m, 8 July 2013, Chao-Qun Wang (GDGM46161)

FIGURE 2. Basidiomata of Chalciporus. a–d. Chalciporus citrinoaurantius (a–b: from GDGM44776, holotype!; c–d: from GDGM44480); e–f. Chalciporus hainanensis (GDGM44464, holotype!). Bars = 2 cm. Photos by: Ming Zhang.

Two new species of Chalciporus (Boletaceae) Phytotaxa 327 (1) © 2017 Magnolia Press • 51 Discussion

Morphologically, the main characters of the genus Chalciporus are the relatively smaller basidiomata each with a pinkish red to reddish brown hymenophore, smooth stipe with yellow to bright yellow basal mycelium, peppery taste and elongated or short ellipsoid spores. Our two new species fit well with the macro- and microscopic features of the genus as well as the phylogenetic analysis result. Chalciporus citrinoaurantius is closely related to C. rubinelloides originally described from southwest China in both morphological and molecular phylogenic analyses. However, C. rubinelloides differs in having a larger fruitbody with a light orange to brownish orange pileus surface, a decurrent hymenophore with smaller pores, yellowish context unchanging when exposed, and larger basidospores 11.5–15 (16) × 4–5.5 (6) μm (Wu et al. 2016). Two North American species C. pseudorubinellus (A.H. Sm. & Thiers) L.D. Gómez (1997:81), C. rubinellus (Peck) Singer (1973:39) and a European species C. rubinus (W.G. Sm.) Singer (1973:319) also show some similarity to C. citrinoaurantius. However, C. pseudorubinellus differs by its yellowish to cinnamon pileus, bright rose red pore surface gradually fading to orange or yellowish brown when mature and lacks the peppery taste (Smith & Thiers 1971; Gómez 1996; Klofac 2006); Chalciporus rubinellus differs in having a red to reddish pileus, a bright rose red to rose pink hymenophore, bright yellow stipe with context unchanging when bruised and larger basidiospores (12–15 × 3.3– 4.5 μm) (Smith & Thiers 1971; Bessette et al. 2000; Klofac 2006). Chalciporus rubinus can be easily distinguished from C. citrinoaurantius by its smaller ellipsoid basidiospores (5.5–7 × 3.5–4.5 μm) (Watling 1970; Alessio 1985; Klofac 2006).

FIGURE 3. Microscopic characters of Chalciporus citrinoaurantius (GDGM44476, holotype!). a. Basidiospores; b. Basidia; c. Pleurocystidia; d. Pileipellis. Drawings by: Ming Zhang.

52 • Phytotaxa 327 (1) © 2017 Magnolia Press ZHANG ET AL. FIGURE 4. Microscopic characters of Chalciporus hainanensis (GDGM44464, holotype!). a. Basidiospores; b. Pleurocystidia; c. Basidia; d. Pileipellis. Drawings by: Ming Zhang.

Chalciporus hainanensis is similar to C. africanus Degreef & De Kesel (2008:330), C. piperatoides (A.H. Sm. & Thiers) T.J. Baroni & Both (1991:560) and C. piperatus. However, the African species C. africanus differs in its cinnamon to dull red pileus, decurrent and light yellow to oxblood red hymenophore, and narrower basidiospores (7.1) 7.5–9.9(10.2) × 3.4–4.2 μm (Degreef & Kesel 2008); the North American species C. piperatoides differs in having a larger fruitbody, a subviscid and tan to dull orange-cinnamon pileus, a decurrent and dull ochraceous hymenophore, narrower basidiospores 7–9 (10) × 3–3.5 μm and an amyloid reaction of the tube trama (Smith & Thiers 1971; Baroni & Both 1991; Bessette et al. 2000). The worldwide reported species C. piperatus, originally described from Europe, differs in its larger fruitbody with a glabrous and yellow-brown to orange cinnamon pileus, unchanging brownish- reddish pores when bruised and a strong peppery taste (Fries 1821; Saccardo1888; Coker & Beers 1943; Watling 1970; Smith & Thiers 1971; Alessio 1985; Both 1993; Klofac 2006; Bessette et al. 2000). In addition, the pores of C. hainanensis do not develop obvious reddish vinaceous color as is seen in C. piperatus. The blue discoloration of the context is perhaps a good field recognition feature of C. hainanensis. Molecularly, the BLAST search of the GenBank database using nrLSU sequences revealed that sequences labeled as C. amarellus (Quél.) Bataille (1908:39) and C. rubinelloides are closest to those of C. citrinoaurantius, both sharing the identities up to 99%, but having only 95% and 91% common query coverage values and 1528 and 1506 max scores respectively; and sequences named as C. amarellus and C. pseudorubinellus are closest to those of C. hainanensis, both sharing the common query coverage values up to 100%, but having only 98% and 97% identities and 1434 and 1423 max scores respectively. This implied that they should be the members of the same genus. Further phylogenetic analysis showed that C. citrinoaurantius grouped with C. rubinelloides, “C. amarellus” and C. pseudorubinellus from close to distant, and all of them formed a moderately supported lineage (Fig.1). However, C.

Two new species of Chalciporus (Boletaceae) Phytotaxa 327 (1) © 2017 Magnolia Press • 53 pseudorubinellus and C. rubinelloides can easily be separated from C. citrinoaurantius morphologically as mentioned above. Chalciporus amarellus, originally described from Europe, differs in a pale yellowish brown to orange brownish pileus, raspberry red pores and larger basidiospores 9–12.5(14) × 3.5–5(6) μm (Alessio 1985, Klofac 2006). The sequence of C. hainanensis together with the sequences labeled as C. aff. rubinellus and Chalciporus sp. formed an unsupported lineage (Fig. 1). In morphology, the species originally described from North America, C. rubinellus, differs from C. hainanensis in its red to reddish pileus, bright rose red to rose pink hymenophore, bright yellow context unchanging when bruised, and larger basidiospores 12–15 × 3.3– 4.5 μm (Smith & Thiers 1971, Bessette et al. 2000, Klofac 2006); the sequence labeled as Chalciporus sp. (GDGM43250) might represent a close taxon which is sister to C. hainanensis. Its voucher specimen was also collected from a locality close to that of C. hainanensis. However, it can be easily distinguished from C. hainanensis by its decurrent and dull ochraceous hymenophore, larger pores (up to 3 mm wide) and larger basidiospores 8–12 × 4.5–5.5(6) μm (examined in this study). The close relationship of the two taxa implies that they diverged from a recent common ancestor in comparison to the other species. Species in Chalciporus have been widely reported in both northern and southern hemispheres (Klofac 2006). Most species have strict geographic distribution characteristics and relatively narrow distribution ranges and taxa with intercontinental distributions are uncommon. Chalciporus austrorubinellus L.D. Gómez (1997:78) and C. chontae Halling & M. Mata (2004:144) was originally reported from Costa Rica. C. austrorubinellus has a smaller basidimata with alutaceous to brownish colored pileus, an olive yellow hymenophore, pale yellow flesh changing blue when exposed, and smaller basidospores (7.6)9–9.12(10) × 3–5 μm (Gómez 1996; Klofac 2006); C. chontae has a tacky and lemon yellow to pale brown pileus surface, pale brownish pink flesh without discoloration, and smaller thick- walled basidiospores (9–10.5 × 3.5 –4.2 μm) (Halling et al. 2004; Klofac 2006). The Oceanian species C. aurantiacus (McNabb) Pegler & T.W.K. Young (1981:137), originally described from New Zealand, has a golden yellow to reddish orange pileus, a dull red hymenophore, and shorter but broader basidiospores 7–9 × 4.2–6 μm (Pegler & Young 1981; Klofac 2006). The Lesser Antillean species C. caribaeus Pegler (1983:586) has a cream buff to snuff brown colored pileus, an apricot yellow hymenophore with larger (up to 4 mm in diam) and radially arranged pores, cream context unchanging when bruised and moderately thin-walled basidiospores 10.2–12.2 × 3.7–4.7 μm; C. corallinus Pegler (1983:585) has a bright pink to old rose colored pileus, a deep chrome hymenophore with larger (up to 2 mm in diam) and radially arranged pores, pale butter yellow context unchanging when bruised and narrower basidiospores 9–11.2 × 3.2–4 μm; C. persicinus Pegler (1983:586) has an amber yellow to ochraceous buff colored pileus, a pale gray yellow, to pale cream brown hymenophore with larger (2–4 × 1.5–2.5 mm) and radially arranged pores, cream context unchanging when bruised and slightly thicker wall basidiospores 10.5–13.5 × 4–5 μm (Pegler 1983). The Central African species C. griseus (Heinem. & Rammeloo) Klofac & Krisai (2006:51), C. luteopurpureus (Beeli) Klofac & Krisai (2006:50), C. phlebopoides (A.H. Sm. & Thiers) T.J. Baroni & Both (2006:51) and C. virescens (Heinem.) Klofac & Krisai (2006:50) can be easily distinguished by their smaller basidiospores (7–8.4 × 4–5.1 μm, 6–7 × 3.5–4 μm, 6.3–7.1 × 4.1–4.7 μm and 7–8.2 × 4–4.5 μm, respectively) (Beeli 1926; Rammeloo 1983; Heinemann 1964; Klofac 2006). C. phaseolisporus (T.H. Li, R.N. Hilton & Watling) Klofac & Krisai (2006:50) originally described from Australia has a straw colored pileus, a pallid hymenophore, and smaller basidospores 5.5–6.5 × 3–3.5 μm (Li & Watling 1999; Klofac 2006). The Central American species, C. trinitensis (Heinem.) Singer, I.J.A. Aguiar & M.H. Ivory (1983:86), has a reddish brown to brownish yellow pileus, a deep purplish red hymenophore and larger basidiospores 11–13.5 × 5–5.8 μm (Heinemann 1954; Singer et al. 1983; Klofac 2006).

Key to Chalciporus species in China 1. Stipe surface smooth; hymenophore regular, not obviously radially arranged...... 2 1. Stipe surface with brown to reddish brown dots, hymenophore obviously radially arranged, pileus grayish yellow to brownish orange, basidiospores smaller, 7–8 × 3.5–4 μm...... C. radiatus 2. Basidiospores relatively broader, with a Qm < 2.5; staining blue on tubes or context at first when bruised; hymenophore brownish yellow to brownish orange; basidiospores 8–10.5(11) × 4–5(5.5) μm...... C. hainanensis 2. Basidiospores relatively narrower, with a Qm > 2.5; not staining blue anywhere when bruised...... 3 3. Pileus surface olive yellow to orange; hymenophore reddish orange to orange red; pores large 1–1.5 mm in diam; stipe context changing to light yellow when exposed; basidiospores (9.0)9.5–12.5(13) × (3)3.5–4(4.5) μm...... C. citrinoaurantius 3. Pileus surface light orange to brownish orange; hymenophore grayish red to red; pores small 2–3/mm; stipe context yellowish and unchanging when exposed; basidiospores 11.5–15(16) × 4–5.5(6) μm...... C. rubinelloides

54 • Phytotaxa 327 (1) © 2017 Magnolia Press ZHANG ET AL. Acknowledgments

The authors are grateful to the anonymous reviewers for their constructive comments and suggestions. Thanks are given to Dr. Chao-Qun Wang and Mr. Hao Huang (Guangdong Institute of Microbiology) for their kind help in the field work. This study was supported by the Ministry of Science and Technology of the People’s Republic of China (Nos. 2013FY111500, 2014CB460613), the National Natural Science Foundation of China (Nos. 31370072, 31570020), and the GDAS’ Special Project of Science and Technology Development (2017GDAS CX-0825).

References

Alessio, C.L. (1985) Boletus DILL, ex L. (sensu lato). In: Fungi Europaei 2A. Saronno: Biella Gio-vanna, 126 pp + 9 pls. Bataille, F. (1908) Quelques champignons intéressants des environs de Besançon. Bulletin de la Société d’Histoire Naturelle du Doubs 15: 23–61. Baroni, T.J. & Both, E.E. (1991) in North America. Mycologia 83: 559–564. Beeli, M. (1926) Contribution Nouvelle a l’étude de la flore mycologique du Congo. Bulletin de la Société royale de botanique de Belgique 58 (2): 203–216. Bessette, A.E., Roody, W.C. & Bessette, A.R. (2000) North American Boletes, a color guide to the fleshy pored mushrooms. Syracuse: Syracuse University Press, 396 pp. Both, E.E. (1993) The boletes of North America: a compendium. Buffalo Museum of Science, Buffalo, 436 pp. Coker, W.C. & Beers, A.H. (1943) The Boletaceae of North Carolina. University of North Carolina Press, Chapel Hill, 96 pp. Degreef, J. & Kesel, A.D. (2008) Chalciporus africanus, a new bolete species from Africa. Mycotaxon 103: 329–335. Fries, E.M. (1821) Systema Mycologicum 1: 1–520 Gómez, L.D. (1996) Basidiomicet es de Costa Rica: Xerocomus, Chalciporus, , Boletellus, Xanthoconium (Agaricales: Boletaceae). Revista de Biologia Tropical 44 (suppl. 4): 59–89. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98. Halling, R.E. Mata, M. & Mueller, G.M. (2004) Three new boletes for Costa Rica. Memoirs of the New York Botanical Garden 89: 141–147. Heinemann, P.P. (1954) Un bolet de l’ile de la Trinité. Bulletin du Jardin Botanique de l’État à Bruxelles 24 (2): 121–125. Heinemann, P.P. (1964) du Katanga. Bulletin du Jardin botanique de l’Etat à Bruxelles 34 (4): 425–478. Klofac, W. (2006) The genus Chalciporus, a world-wide survey. Österreichische Zeitschrift für Pilzkunde 15: 31–65. Kornerup, A. & Wanscher, J.H. (1978) Methuen handbook of colour. Eyre Methuen, London, 252 pp. Li, T.H. & Watling, R. (1999) New taxa and combinations of Australian boletes. Edinburgh Journal of Botany 56(1): 143–148. Nuhn, M.E., Binder, M., Taylor, A.F., Halling, R.E. & Hibbett, D.S. (2013) Phylogenetic overview of the Boletineae. Fungal Biology 117: 479–511. https://doi.org/10.1016/j.funbio.2013.04.008 Nylander, J.A.A. (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Pegler, D.N. & Young, T.W.K. (1981) A natural arrangement of the with reference to spore morphology. Transactions of the British Mycological Society 76: 103–146. https://doi.org/10.1016/S0007-1536(81)80013-7 Pegler, D.D. (1983) Agaric Flora of the Lesser Antilles. Additional Series IX, Kew Bulletin, London, 668 pp + 27 pls. Pilát, A. (1969) Buchwaldoboletus. Genus novum Boletacearum. Friesia 9: 217–218. Pilát, A. & Dermek, A. (1969) Rubinoboletus and genus novum Boletacearum. Ceská Mykologie 23: 81–82. Posada, D. & Crandall, K.A. (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818. https://doi.org/10.1093/bioinformatics/14.9.817 Rammeloo, J.J. (1983) Combinaisons et taxons nouveaux de Gyrodontaceae (Boletineae). Bulletin du Jardin Botanique National de Belgique 53: 294–297. Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572– 1574. Saccardo, P.A. (1888) Sylloge Hymenomyceteae, Vol. VI. Polyporeae, Hydneae, Thelephoreae, Clavarieae, Tremellineae. Sylloge Fungorum 6: 1–928.

Two new species of Chalciporus (Boletaceae) Phytotaxa 327 (1) © 2017 Magnolia Press • 55 Singer, R. (1973) Notes on bolete taxonomy. Persoonia 7: 313–320. Singer, R. (1986) The Agaricales in modern taxonomy, 4th edn. Koeltz Scientific Books, Koenigstein, 981 pp + 88 pls. Singer, R. Araujo, I. & Ivory, H.M. (1983) The Ectotrophically Mycorrhizal Fungi of the Neotropical Lowlands, Especially Central Amazonia. Beihefte zur Nova Hedwigia 77: 1–352 Smith, A.H. & Thiers, H.D. (1971) The boletes of Michigan. The University of Michigan Press, Ann Arbor, 438 pp. Stamatakis, A. (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690. https://doi.org/10.1093/bioinformatics/btl446 Thomson, J.D., Gibson, T.J., Plewnlak, F., Jianmougin, F. & Higgins, D.G. (1997) The Clustal X windows interfaces: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876–4882. https://doi.org/1 0.1093/nar/25.24.4876 Vilgalys, R. & Hester, M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 Watling, R. (1970) British fungus flora 1: Boletaceae: Gomphidiaceae: Paxillaceae. HMSO, Edinburgh, 124 pp. Wu, G., Feng, B., Zhu, X.T., Li, Y.C., Zeng, N.K., Hosen, M.I. & Yang, Z.L. (2014) Molecular phylogenetic analyses redefine seven major clades and reveal 22 new generic clades in the fungal family Boletaceae. Fungal Diversity 69: 93–115. https://doi.org/10.1007/s13225-01 4-0283-8 Wu, G., Li, Y.C., Zhu, X.T., Zhao, K., Han, L.H., Cui, Y.Y., Li, F., Xu, J. & Yang Z.L. (2016) One hundred noteworthy boletes from China. Fungal Diversity 81: 25–188. https://doi.org/10.1007/s13225-016-0375-8 Zhang, M., Li, T.H., Xu, J. & Song, B. (2015a) A new violet brown Aureoboletus (Boletaceae) from Guangdong of China. Mycoscience 56: 481–485. https://doi.org/10.1016/j.myc.2015.02.002 Zhang, M., Wang, C.Q., Li, T.H. & Song, B. (2015b) A new species of Chalciporus (Boletaceae, Boletales) with strongly radially arranged pores. Mycoscience 57: 20–25. https://doi.org/10.1016/j.myc.2015.07.004

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