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Three New Species of from Southwest China

Article in Phytotaxa · October 2016 DOI: 10.11646/phytotaxa.282.2.7

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Three New Species of Rhizopogon from Southwest China

LIN LIa,b,g, YONGCHANG ZHAOa, DEQUN ZHOUb*, FUQIANG YUc, LINYONG ZHENGd, YUN WANGe, XIAOLEI ZHANGa, ZHONGJIAN DUANf, XINGYUN ZHAOf, ZHIHONG HEf & SHUHONG LIa,b* aKey Laboratory for Agricultural Biotechnology of Yunnan, Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650223 Yunnan, China bFaculty of Environmental Sciences and Engineering, Kunming University of Science and Technology, Kunming 650500 Yunnan, China cKunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201 Yunnan, China dSichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China eNew Zealand Institute for Plant and Food Research Limited, Christchurch 8140, fJianchuan County Forestry Bureau, Jianchuan 671300, Yunnan, China gThe College of Agriculture and Biological Science, Dali University, Dali 671000, Yunnan, China *Correspondence author: Shuhong Li Email: [email protected] Dequn Zhou Email: [email protected]

Abstract

Rhizopogon fruiting bodies were collected from Pinus in Yunnan and Sichuan Provinces in southwest China. Based on morphological and molecular phylogenetic analyses, three new species are described as Rhizopogon jiyaozi, Rhizopogon flavidus and Rhizopogon sinoalbidus. Their phylogenetic relationships to each other and previously described species are discussed.

Key words: Hypogeous fungi, ITS, morphology, phylogeny,

Introduction

The Rhizopogon Fr. & Nordholm (, , ) contains approximately 150 species of hypogeous fungi (Kirk et al. 2008). They are associated mainly with and are distributed worldwide in natural and exotic forests (Molina et al. 1999). Despite this cosmopolitan range, most known species are found in (Pinus) and Douglas- [Pseudotsuga menziesii (Mirb.) Franco] forests of the Pacific Northwestern United States, and New Zealand (Smith 1964; Smith and Zeller 1966; Martín 1996; Visnovsky et al. 2010; Walbert et al. 2010; Cooper 2012). Rhizopogon are common ectomycorrhizal fungi in these coniferous forests and play an important role in the ecology of these ecosystems. The first Rhizopogon species was described in Europe by Elias Magnus Fries in 1817. Smith and Zeller published their North American monograph in 1966 and divided the genus into two subgenera, Rhizopogonella and Rhizopogon. Species in the subgenus Rhizopogonella were moved into by Trappe in 1975. Based on macroscopic and microscopic characteristics of the and color changes on the peridium from chemical reactions, the subgenus Rhizopogon was divided into four sections, Amylopogon, Fulviglebae, Rhizopogon and Villosuli (Smith and Zeller 1966). The Morden taxonomy divided the genus Rhizopogon into five subgenera, Versicolores, Rhizopogon, Roseoli, Villosuli and Amylopogon, based on morphological and DNA molecular phylogenetic analyses (Grubisha 2001; Grubisha et al. 2002). To date six Rhizopogon species have been reported from China: (Corda) Th. Fr. Rhizopogon shanxiensis B. Liu, Rhizopogon nigrescens Coker & Couch, Rhizopogon piceus Berk. & M.A. Curtis, Rhizopogon cylindriosporus A.H. Sm., Fr. and Rhizopogon superiorensis A.H. Sm. (Liu B 1985; Tao and Chang 1988; Yu FQ and Liu PG 2005; Dai YC and Yang ZL 2008; Dai YC et al. 2010; Shao DH et al. 2013). In 2012 and 2013, a few Rhizopogon specimens were collected in Yunnan and Sichuan in southwest China (Plate.1). Morphological and molecular phylogenetic analyses determined that the Yunnan and Sichuan collections contained three new species. Here, the new species Rhizopogon jiyaozi belonging to subgen. Roseoli, Rhizopogon flavidus belonging to subgen. Rhizopogon and Rhizopogon sinoalbidus belonging to subgen. Amylopogon are described, and their relationships with other species of Rhizopogon are discussed.

Accepted by Samantha Karunarathna: 2 Oct. 2016; published: 27 Oct. 2016 151 Material & methods

Morphological characteristics examination Twenty-seven specimens were collected in Yunnan and Sichuan and deposited in the YAAS Herbarium, Yunnan Academy of Agricultural Sciences, Kunming, China. Macroscopic characteristics were described from fresh specimens. Sections were cut with a razor blade by hand, mounted in 5% KOH solution or water, and then stained with Melzer’s reagent. The sections were examined under an Olympus BH-2 microscope.

DNA extraction, PCR amplification, sequencing and phylogenetic analyses The total genomic DNA of specimens was extracted from a piece of a fruiting body tissue using the Zomanbio Plant Genomic DNA Kit. The internal transcribed spacer (ITS) rDNA region was amplified with PCR primers ITS4 and ITS5. The PCR reactions were run on a BIO-RAD C1000TM Thermal Cycler with the following settings: initial denaturation for 5 min at 94°C, followed by 32 cycles of 40 s, denaturation at 94°C, annealing at 56°C for 40 s, extension for 1 min at 72°C and final extension at 72°C for 10 min. The purifying and sequencing of PCR products were conducted by the Beijing Genomics Institute. The other sequences data of ITS rDNA used in this study were downloaded from GenBank, as shown in Table 1.

TABLE 1. Database of materials used for molecular analyses. Species name Voucher Origin GenBank No. of References ITS R. abietis A.H.Smith OSC41455 USA KC346843 From GenBank R. abietis A.H.Smith Trappe 7603 USA EU837243 From GenBank R. albidus A.H. Smith AHS 69642 USA AM085519 From GenBank R. alkalivirens A.H. Smith AHS69037 USA AF377154 Bidartondo & Bruns (2002) (holotype) R. arctostaphyli A.H. Smith JPT5705 USA AF377167 Bidartondo & Bruns (2002) (holotype) R. arctostaphyli A.H. Smith - USA EU726304 Hynes et al.(2010) R. atroviolaceus A.H. Smith AHS68263 USA AF377131 Bidartondo & Bruns (2002) R. bacillisporus A.H. Smith OSC63507 USA EU837230 From GenBank R. bacillisporus A.H. Smith SOC1307 USA JN022517 From GenBank R. brunsii Grubisha & Trappe LG1296 USA AY971814 Grubisha et al. (2005) R. brunsii Grubisha & Trappe LG701 USA AY971823 Grubisha et al. (2005) (Holotype) R. burlinghamii A.H.Smith JMT17882 USA AF058303 Grubisha et al. (2002) R. corsicus Demoulin & Moy. V. Demoulin s.n17-IX- Belgium AM085521 From GenBank 1989 R. ellenae A.H.Smith AHS66137 USA AF071445 Grubisha et al. (2002) R. ellenae A.H.Smith JMT17476 USA AF058311 Grubisha et al. (2002) R. ellenae A.H.Smith - USA JF695019 From GenBank R. ellipsosporus Trappe, Castellano OSC138981 USA JX310372 From GenBank & Amar. R. ellipsosporus Trappe, Castellano OSC138996 USA JX310373 From GenBank & Amar. R. evadens A.H.Smith AHS65484 USA AF062927 Grubisha et al. (2002) ...Continued on next page

152 • Phytotaxa 282 (2) © 2016 Magnolia Press LI ET AL. TABLE 1. (Continued) Species name Voucher Origin GenBank No. of References ITS R. evadens A.H.Smith JMT16402 USA AF058312 Grubisha et al. (2002) R. evadens A.H.Smith JMT12321 USA AF062932 Grubisha et al. (2002) R. fallax A.H. Smith AHS66116 USA AF377143 Bidartondo & Bruns (2002) R. fallax A.H. Smith AHS65762 USA AF377144 Bidartondo & Bruns (2002) R. flavidus YAASL2957(holotype) China KP893813 This study R. flavidus YAASL2956 China KP893814 This study R. flavidus YAASL2959 China KP893815 This study R. fuscorubens A.H.Smith JMT17446 USA AF058313 Grubisha et al. (2002) R. fuscorubens A.H.Smith Ash58b - AY880943 Ashkannejhad & Horton (2006) R. graveolens Tul. & C. Tul. PRM619028 Czech AJ810037 Martín & García (2009) R. idahoensis A.H. Smith - - AF224472 Kretzer et al. (2000) R. idahoensis A.H. Smith AHS70715 USA AF377123 Bidartondo & Bruns (2002) R. jiyaozi YAASL2929(holotype) China KP893830 This study R. jiyaozi YAASL2335 China KP893823 This study R. jiyaozi YAASL2336 China KP893824 This study R. jiyaozi YAASL2911 China KP893825 This study R. jiyaozi YAASL2916 China KP893826 This study R. jiyaozi YAASL2918 China KP893827 This study R. jiyaozi YAASL2925 China KP893828 This study R. jiyaozi YAASL2927 China KP893829 This study R. jiyaozi YAASL2930 China KP893831 This study R. jiyaozi YAASL2931 China KP893832 This study R. jiyaozi YAASL2932 China KP893833 This study R. jiyaozi YAASL2950 China KP893834 This study R. jiyaozi YAASL2395 China KP893835 This study R. jiyaozi YAASL2399 China KP893836 This study R. jiyaozi YAASL2409 China KP893837 This study R.luteolus Fr. JMT22516 Sweden AF062936 Grubisha et al. (2002) R.luteolus Fr. DG05-22 Scotland JQ888192 Pickles B.J. et al. (2012) R. milleri A.H. Smith AHS70789 - AF377135 Bidartondo & Bruns (2002) (holotype) R. occidentalis Zeller & Docdge JMT17564 USA AF058305 Grubisha et al. (2002) R. occidentalis Zeller & Docdge LCG211 USA AF062939 Grubisha et al. (2002) R. ochraceorubens A.H.Smith AHS59643 USA AF062928 Grubisha et al. (2002) R. ochraceorubens A.H.Smith JMT19192 USA AF071440 Grubisha et al. (2002) R. odoratus A.H. Smith AHS 71319 USA AM085526 From GenBank ...Continued on next page

New Species of Rhizopogon from Southwest China Phytotaxa 282 (2) © 2016 Magnolia Press • 153 TABLE 1. (Continued) Species name Voucher Origin GenBank No. of References ITS R. pachydermus K.A. Harrison & K.A.Harrison7290 USA AM085527 From GenBank A.H. Smith R. pedicellus A.H. Smith AHS66176 USA AF377126 Bidartondo & Bruns (2002) R. pseudoroseolus A.H.Smith MICH66604 USA AJ810041 Martín & García (2009) R. pseudoroseolus A.H.Smith K98S35 New GQ267484 Walbert et al. (2010) Zealand R. roseolus Corda RrUP175 Sweden DQ179127 From GenBank R. roseolus Corda MA-Fungi 47716 Spain AJ810064 Martín & García (2009) R. roseolus Corda (= R. rubescens isolate T1PK2 Latvia JX907816 Klavina et al. (2013) Tul. & C. Tul. ) R. roseolus Corda (= R. rubescens clone NS182 Lithuania DQ068965 Menkis et al. (2005) Tul. & C. Tul. ) R. salebrosus A.H. Smith R141SCCO USA HQ914327 Dowie et al. (2012) R. salebrosus A.H. Smith AHS69292 USA AF377156 Bidartondo & Bruns (2002) R. sardous Pacioni AQUI27-XI-1981 Italy AM085529 Martín & García (2009) R. semireticulatus A.H.Smith JMT7899 USA AF058307 Grubisha et al. (2002) R. semireticulatus A.H.Smith JMT17562 USA AF062940 Grubisha et al. (2002) R. semireticulatus A.H.Smith AHS71326 USA AF377118 Bidartondo & Bruns (2002) R. semireticulatus A.H.Smith AHS71330 - AF377119 Bidartondo & Bruns (2002) (holotype) R. sinoalbidus YAASL2949(holotype) China KP893820 This study R. sinoalbidus YAASL2944 China KP893816 This study R. sinoalbidus YAASL2946 China KP893817 This study R. sinoalbidus YAASL2947 China KP893818 This study R. sinoalbidus YAASL2948 China KP893819 This study R. sinoalbidus YAASL2953 China KP893821 This study R. sinoalbidus YAASL2954 China KP893822 This study R. subbadius A.H. Smith AHS58998 USA AF377152 Bidartondo & Bruns (2002) (holotype) R. subbadius A.H. Smith AHS68485 USA AF377151 Bidartondo & Bruns (2002) R. subcaerulescens A.H.Smith - - M91613 Grubisha et al. (2002) R. subgelatinosus A.H.Smith JMT7624 USA AF062937 Grubisha et al. (2002) R. subgelatinosus A.H.Smith AHS68558 USA AF377147 Bidartondo & Bruns (2002) R. subpurpurascens A.H.Smith AHS65669 USA AF062929 Grubisha et al. (2002) R. subpurpurascens A.H.Smith JMT19168 USA AF058308 Grubisha et al. (2002) R. subpurpurascens A.H.Smith OSC44691 USA EU837236 From GenBank R. subpurpurascens A.H.Smith - USA JF695022 From GenBank R. subsalmonius A.H.Smith JMT17218 USA AF062938 Grubisha et al. (2002) ...Continued on next page

154 • Phytotaxa 282 (2) © 2016 Magnolia Press LI ET AL. TABLE 1. (Continued) Species name Voucher Origin GenBank No. of References ITS R. subsalmonius A.H.Smith M.P. Martin 1653 Spain AM085530 From GenBank R. succosus A.H.Smith JMT19321 USA AF062933 Grubisha et al. (2002) R. variabilisporus A.H.Smith OSC63496 USA KC346855 From GenBank R. variabilisporus A.H.Smith OSC134688 USA KC346856 From GenBank R. verii Pacioni G. Pacioni s.n.14-XII- Tunisia AM085531 From GenBank 1982 R. vesiculosus A.H.Smith OSC:129170 FS-151 Canada HQ385849 Luoma,D.L. et al. (2011) R. vesiculosus A.H.Smith OSC:129175 FS-225 Canada HQ385854 Luoma,D.L. et al. (2011) R. vulgaris (Vitt.) M. Lange JMT19154 USA AF062934 Grubisha et al. (2002) R. vulgaris (Vitt.) M. Lange JMT17998 USA AF062931 Grubisha et al. (2002)

To establish parsimonious trees of subgen Roesoli, fifteen sequences were made from our samples of YAAS L2335, L2336, L2395, L2399, L2309, L2911, L2916, L2918, L2925, L2927, L2929, L2930, L2931, L2932 and L2409, and analyzed with other 17 retrieved from GenBank. Two sequences of R. ellenae were selected and used as outgroups. For establishing parsimonious trees of subgen Rhizopogon three sequences made from our samples of YAAS L2957, L2956 and L2959 were made, and analyzed with other 26 retrieved from GenBank.Two sequences of R. ellenae were selected and used as outgroups. Seven sequences made from our samples of YAAS L2944, L2946, L2947, L2948, L2949, L2953 and L2954 were made, and analyzed with othre 32 retrieved from GenBank to establish parsimonious trees of subgen. Amylopogon. Two sequences of R. vesiculosus were selected and used as outgroups. Sequences were edited and assembled using SeqMan II (Larsson and Sundberg 2011). Alignment of nucleotide sequences was performed by Mafft-win. Sequences were adjusted manually using BioEdit 7.0.1. Phylogenetic analyses were conducted using PAUP* 4.0 beta. Equally weighted parsimony (MP) was used to search for optimal trees. The specific procedures and analyses followed the methods of Hofstetter et al. (2002) and Larsson and Sundberg (2011).

FIGURE 1. Maps of Yunnan and Sichuan Province, China showing the locations where the Rhizopogon collections were found (in red colour).

Results

Phylogenetic analyses Three most parsimonious trees were produced based on the maximum parsimony analyses (Fig. 2, Fig. 3 and Fig. 4). Fig. 2 shows the strict consensus phylogenetic tree of Rhizopogon subgen. Roseoli, with consistency index (CI)=0.812, retention index (RI)=0.896, homoplasy index (HI)=0.188, and rescaled consistency index (RC)=0.727. In this tree,

New Species of Rhizopogon from Southwest China Phytotaxa 282 (2) © 2016 Magnolia Press • 155 all the samples of R. jiyaozi are grouped together to form Subclade I with 100% bootstrap support, which is clearly separated from other groups of subgen. Roseoli. The phylogenetic analysis shows that R. jiyaozi is distinct from other species of Rhizopogon.

FIGURE 2. Strict consensus phylogenetic tree of Rhizopogon subgen. Roseoli based on ITS sequences. Numbers above branches indicate bootstrap support above 50%.

Fig. 3 shows the strict consensus phylogenetic tree of Rhizopogon subgen. Rhizopogon, with consistency index (CI)=0.734, retention index (RI)=0.895, homoplasy index (HI)=0.266 and rescaled consistency index (RC)=0.657. In this tree, all the samples of R. flavidus form SubClade II with 100% bootstrap support and separated from other groups of subgen. Rhizopogon. The phylogenetic analysis shows that the new species is distinct from other Rhizopogon species, although it is closely related to R. succosus. Fig. 4 shows a strict consensus phylogenetic tree of Rhizopogon subgen. Amylopogon, with consistency index (CI)=0.831, retention index (RI)=0.944, homoplasy index (HI)=0.169, and rescaled consistency index (RC)=0.785. In this tree, all the six samples of R. sinoalbidus form subclade I with 100% bootstrap support and are well separated from other groups of subgen. Amylopogon. The phylogenetic analysis shows that the new species of R. sinoalbidus is distinct from other Rhizopogon species.

156 • Phytotaxa 282 (2) © 2016 Magnolia Press LI ET AL. FIGURE 3. Strict consensus phylogenetic tree of Rhizopogon subgen. Rhizopogon based on ITS sequences. Numbers above branches indicate bootstrap support above 50%.

Taxonomy

Rhizopogon jiyaozi Shu H. Li & L. Li sp. nov. Fig. 5 (a, b and c) and Fig. 6 MycoBank MB811827

Diagonosis:—Basidiocarps globose, slightly soft to rubbery, surface discoloring rose-pink when cut or bruised. hyaline to brown in mass in KOH, non-amyloid in Melzerʼs reagent. Holotype:—CHINA: Yunnan Province, Lijiang, Lashi Lake, in forest of Pinus yunnanensis Franch. 26.87882ºN 100.15820ºE, alt. 2480.6 m, July 31. 2013, S.H. Li, YAAS L2929-Holotype (GenBank KP893830), Herbarium of Yunnan Academy of Agricultural Sciences, Kunming, China. Etymology:—from Latin jiyaozi referring to the local name of the , which points out the shape of the fungus like kidney of chicken. Description:—Basidiocarps (Fig. 5 a) globose to subglobose, 1–5 cm diam., slightly soft to rubbery, white when young, finally becoming yellowish brown, discoloring rose-pink when cut or bruised, attached by rhizomorphs over the surface, depressed, white to yellow. Chemical reactions: KOH on dried peridium, deep yellowish brown becoming black immediately. Odor not distinctive. Peridium (Fig. 6 a and b) thin, easily pulled off, 180–400 μm thick, composed of hyaline interwoven hypha, 5–9 μm in diam., with red-brown pigment, becoming rose-pink when cut. Gleba (Fig. 5 b and c) white when young, becoming dark olive green to brown in maturity, finally gelatinized; chambers small, numerous, labyrinthine. Basidia (Fig. 6 c and e) clavate to cylindrically clavate, thin-walled, commonly six-spored. Cystitia (Fig. 6 f) abundant, subglobose to ovoid or ellipsoid, thin-walled. (Fig. 6 d) obtuse to ellipsoid,

New Species of Rhizopogon from Southwest China Phytotaxa 282 (2) © 2016 Magnolia Press • 157 smooth, (6–)6.5–8.5 (–9) × (2–)2.5–3.5 μm, hyaline to brown in mass in KOH, non-amyloid in Melzerʼs reagent, sometimes with 1–2 guttulate. Clamp connections absent.

FIGURE 4. Strict consensus phylogenetic tree of Rhizopogon subgen. Amylopogon based on ITS sequences. Numbers above branches indicate bootstrap support above 50%.

FIGURE 5. Rhizopogon jiyaozi (a, b and c). a Basidiocarp; b Mature basidiocarps and their cross sections; c Young basidiocarps and their cross sections; Rhizopogon flavidus (d, e and f). d and e Basidiocarp; f Mature, young basidiocarps, and their cross sections. Rhizopogon sinoalbidium (g, h and i). g Basidiocarp; h Mature basidiocarps and their cross sections; i Mature, young basidiocarps, and their cross sections.

158 • Phytotaxa 282 (2) © 2016 Magnolia Press LI ET AL. Additional specimens examined:—CHINA. Sichuan Province, Ganzi, Daocheng, Aden Scenic Area, in P. yunnanensis Mast. forest alt. 3045.3 m, August 22, 2012, S.H. Li, YAAS L2335, L2336; Yunnan Province, Lijiang, in forest of P. yunnanensis alt. 2480.6 m, July 31. 2013, S.H. Li, YAAS L2911, L2916, L2918, L2925, L2927, L2930, L2931 and L2932; Dali, Jianchuan, Mt. Thousands of Lions, in forest of P. yunnanensis Franch. alt. 2339.5 m, August 3. 2013, S.H. Li, YAAS L2950; Shangri-La, Baima mountains, in forest of Pinus densata alt. 3291.2 m, August 26 2013, S.H. Li YAAS L2395, L2399 and L2409. Ecology & Distribution:—Hypogeous under trees of P. yunnanensis, at an elevation of 2,300–3,200 m, fruiting from July to August. Known only from Yunnan and Sichuan, China.

FIGURE 6. Rhizopogon jiyaozi. a Hyphae of the peridium; b Section of peridium in KOH; c and e showing basidia and branchybasidioles; d Basidiospores; f Hymenium showing cystidia.

Rhizopogon flavidus Shu H. Li & L. Li sp. nov. Fig. 5 (d.e and f) and Fig. 7 MycoBank MB811995

Diagonosis:—Basidiocarps globose, surface yellow to flavous, becoming rust-red to deep orange when cut, bruised or drying. Basidiospores hyaline to brown in mass in KOH, nonamyloid in Melzerʼs reagent. Holotype:—CHINA: Yunnan Province, Dali, Jianchuan, Mt. Thousands of Lions, in soil under pure forests of P. yunnanensis Franch. 26.88396ºN 100.17677ºE, alt. 2339.5 m, August 3, 2013, S.H. Li, YAAS L2957-Holotype (GenBank KP893813), Herbarium of Yunnan Academy of Agricultural Sciences, Kunming, China. Etymology:—from Latin flavidus, referring to the color of the peridium. Description:—Basidiocarps (Fig. 5 d and e) 0.6–1.5 cm broad, globose to subglobose, rubbery, yellow to flavous, often discoloring rust-red to deep orange when cut, bruised or drying, with abundant depressed, easily dropped off, branched rhizomorphs, co-colored with the basidiocarp. Chemical reactions: KOH on dried peridium, deep orange becoming olive immediately and soon black. Odor not distinctive. Peridium (Fig. 7 a) 80–120 μm thick, of woven

New Species of Rhizopogon from Southwest China Phytotaxa 282 (2) © 2016 Magnolia Press • 159 hypha, 4–8 μm in diam, often obscured by abundant reddish to orange-brown pigmented debris. Gleba (Fig. 5 f) dirty white when young, becoming olive black to gray black in maturity gelatinized finally, chambers small, numerous, labyrinthine. Trama consisted of interwoven hyaline hyphae, (Fig. 7 c). Basidia (Fig. 7 d) clavate to clavate-cylindrical, (13.0–) 14.5–16.5 (–21.0) × (3–) 4–5 (–6) μm, commonly six-spored. Basidiospore (Fig. 7 b) obtuse to ellipsoid, smooth, 5–7 (–8) × 2.5–3.5 μm, hyaline to brown in mass in KOH, nonamyloid in Melzerʼs reagent, sometimes 1–2 guttulate. Clamp connections absent.

FIGURE 7. Rhizopogon flavidus. a Section of peridium. b Basidiospores; c Trama plates. d Stripped pieces of hymenium.

Additional specimens examined:—CHINA: Yunnan Province, Lijiang, Lashi lake, in forest of P. yunnanensis, alt. 2480.6 m, 2013.7.31. S.H. Li, YAAS L2924. Dali, Jianchuan, Mt. Thousands of Lions, in forest of P. yunnanensis alt. 2339.5 m, August 3, 2013. S.H. Li, YAAS L2956, L2959 and L2961. Ecology & Distribution:—Hypogeous under trees of P. yunnanensis at an elevation of 2300–2500 m, fruiting from July to August. It is only known in Yunnan, China.

Rhizopogon sinoalbidus Shu H. Li & L. Li sp. nov. Fig. 5 (g. h and i) and Fig. 8 MycoBank MB811829

Diagonosis:—Basidiocarps dirty white, finally pale gray, unchanging where cut or bruised, but turning yellow to yellow-brown where pressed against needles or sticks. Peridium composed of interwoven hyaline hyphal strands, with abundant, pink to gray or faintly blue extracellular pigment deposits in KOH solution. Basidiospore no obvious reaction of amyloid in Melzerʼs reagent. Holotype:—CHINA: Yunnan Province, Dali, Jianchuan, Mt. Thousands of Lions, in forests of P. yunnanensis, 26.88396ºN 100.17677ºE, alt. 2339.5 m, August 3. 2013, S.H. Li, YAAS L2949-Holotype (GenBank KP893820), Herbarium of Yunnan Academy of Agricultural Sciences, Kunming, China. Etymology:—from the Latin sinoalbidus, sino-referring to the origin and albidius to the color of the peridium. Description:—Basidiocarps (Fig. 5 g) 0.6–2.5 cm broad, globose to subglobose, dirty white, finally becoming pale gray to pale dingy, becoming brown when dried, unchanging where cut or bruised, but turning yellow to yellow- brown where pressed against needles or sticks; smooth when young, becoming slightly exasperated by mature. Attached by rhizomorphs, loosely covering the base and depressed overall, white to dingy white, unchanging where cut or bruised, easily fall off. Chemical reactions: KOH on dried brown peridium instantly turning pink, soon black. Odor not distinctive. Peridium (Fig. 8 a and b) 500–700 μm thick, composed of interwoven hyaline hyphal strands and hyphae, 5–10 μm in diam., with abundant, pink to gray or faintly blue extracellular pigment deposits in KOH. Gleba (Fig. 5 h and i) dirty white in youth, becoming yellowish-olive to olive and finally olive brown to dark grayish olive, exuding fluid when cut, columella lacking. Trama composed of interwoven hyaline gelatinized hyphae. Basidia (Fig.8 c and d) clavate to clavate-cylindrical, (14–)16–20(–21) × (4–)5–6.5 μm, commonly four to six-spored. Basidiospore

160 • Phytotaxa 282 (2) © 2016 Magnolia Press LI ET AL. (Fig. 8, e and f) obtuse to ellipsoid, smooth, 6.5–7.5 (–8.6) × 2.5–3.0 (–3.5) μm, with 1–2 guttules, hyaline singly and brownish yellow in mass in KOH, no obvious reaction of amyloid in Melzerʼs reagent. Clamp connections absent. Additional specimens examined:—CHINA: Yunnan Province, Dali, Jianchuan, Mt. Thousands of Lions, in forests of P. yunnanensis, alt. 2339.5 m, August 3, 2013. S.H. Li, YAAS L2944, L2946, L2947, L2948, L2953 and L2954.

FIGURE 8. Rhizopogon sinoalbidus. a Section of peridium in KOH; b Hyphae of the peridium; c Hymenium showing basidia and branchybasidioles; d Piece of hymenium; e Basidiocarps in KOH; f Basidiospores in Meltzer’s solution.

Ecology & Distribution:—Hypogeous under trees of P. yunnanensis at an elevation of 2300–2500 m, fruiting from July to August known only in Yunnan, China.

Discussion

Rhizopogon jiyaozi is a delicacy in Yunnnan and Sichuan Provinces, southwestern China. Between august and october each year, locals go to mountains to collect R. jiyaozi. Usually, locals eat some by stir-fry or boiling, and others for sale about 10–20RMB per kg in nearby market. This species was previously misidentified as R. roseolus (=R. rubescens) (Yu and Liu 2005). Morphologically, R. jilaozi resembles R. roseolus (R. rubescens). However, molecular analysis clearly shows that R. jiyaozi is distinct from R. roseolus and is grouped in a different clade (Fig. 2) . Rhizopogon jiyaozi has smaller basidiospores and deeper yellow basidiocarps, which differ from R. roseolus (Smith and Zeller 1966; Pegler et al. 1993; Grubisha et al. 2002). In the strict consensus phylogenetic tree, R. burlinghamii, R. variabilisporus and R. abietis are grouped together in Subclade II and formd Clade I with R. jiyaozi (the Subclade I). Rhizopogon burlinghamii has longer and narrower spores 7–9 × (1.8–)2–2.3μm than R. jiyaozi. The surface of R. abietis basidiocarps quickly

New Species of Rhizopogon from Southwest China Phytotaxa 282 (2) © 2016 Magnolia Press • 161 become tinged lemon-yellow and then ochraceous when touched or injured. In contrast, R. jiyaozi discolors rose-pink when cut or bruised. Rhizopogon variabilisporus has a rough and fibrillose surface whereas the surface of R. jiyaozi is smooth. In addition, R. jiyaozi has smaller basidiospores than R. abietis and R. variabilisporus (Smith and Zeller 1966). Molecular analyses show that R. flavidus is distinct from other species in subgen. Rhizopogon and closely related to R. succosus (Fig. 3). However R. succosus differs from R. flavidus in having larger and thicker (approximately 0.7 μm) walled basidiospores (Smith and Zeller 1966). In addition R. flavidus has more rhizomorphs that are fragile and easily fall off. Basidiospores of R. sinoalbidus have no obvious amyloid reaction with Melzerʼs reagent, which separates it from species in Subgen. Amylopogon. Molecular analyses show that R. sinoabidius is distinct from other species in the subgenus (Fig. 4).

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (No. 31160010 and 31370070) and Key Laboratory for Agricultural Biotechnology of Yunnan (2014006). Our thanks go to the Beijing Genomics Institute for purifying and sequencing the PCR products.

References

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