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Podoserpula ailaoshanensis sp. nov. (Amylocorticiales, Basidiomycota) from China based on morphological and sequence analyses
m y c o s c i e n c e 5 7 ( 2 0 1 6 ) 2 9 5 e3 0 1
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Podoserpula ailaoshanensis sp. nov. (Amylocorticiales, Basidiomycota) from China based on morphological and sequence analyses Jun-Liang Zhou, Hong Chen, Bao-Kai Cui* Institute of Microbiology, Beijing Forestry University, Beijing 100083, China
article info
abstract
Article history:
Podoserpula is described from Australia and mainly collected from the southern hemi-
Received 21 February 2016
sphere. It is characterized by the brightly colored multi-storied basidiocarps, non-amyloid
Received in revised form
and thick-walled ellipsoid to subglobose basidiospores, distributed in subtropical to trop-
22 April 2016
ical regions. In this study, P. ailaoshanensis is described and illustrated as a new species
Accepted 23 April 2016
based on morphological and molecular evidence. This species is characterized by its multi-
Available online 2 June 2016
storied basidiocarps, zonate and fuzzy pileus, light ivory to yellow orange pileal surface, hollow main axis, and globose to subglobose, thick-walled basidiospores (4.1e5 3.9
Keywords:
e4.8 mm). Phylogenetically, on the basis of combined dataset of internal transcribed spacer
Amylocorticiales
(ITS), nuclear large subunit rDNA (nLSU), translation elongation factor 1-a gene (EF1-a) and
Phylogeny
small subunit nuclear ribosomal RNA gene (nSSU), P. ailaoshanensis is strongly supported as
Taxonomy
a new species of Podoserpula. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Podoserpula D.A. Reid is usually known as “pagoda fungus” for its multi-storied basidiocarps. It was erected by Reid (1963) based on the type species P. pusio (Berk.) D.A. Reid. Features distinguishing Podoserpula from others are as follows: stipitate basidiocarps with a simple or very sparingly branched axis; pilei often brightly colored, normally vary in shape, joined to the main axis by stipe-like base or the main axis appearing to pierce the centre of each individual pileus; hyphal structure monomitic, generative hyphae with clamps; cystidia and gloeocystidia absent; basidiospores small, thick-walled, inamyloid, ellipsoid to subglobose; growing on the ground or in
well rotted stump-wood (Reid 1963). On the basis of morphological features and geographic distributions, four varieties, including P. pusio var. austro-americana, P. pusio var. ellipsospora, P. pusio var. pusio and P. pusio var. tristis, were described by Reid (1963). Podoserpula miranda B. Buyck, B. Duhem, G. Eyssartier & M. Ducousso, which is limited to New Caledonia, is the second Podoserpula species (Buyck et al. 2012). Besides Australia, New Zealand, Venezuela and New Caledonia, Podoserpula has been collected from several other places, such as Brazil (Pegler 1988), Chile and Guiana (Hjortstam and Ryvarden 2007), Falklands Islands (Watling 2012) and Madagascar (Buyck 2008). Although it has been collected from many places, there is no record from Asia.
* Corresponding author. Tel./fax: þ86 10 62336309. E-mail address: [email protected] (B.-K. Cui). http://dx.doi.org/10.1016/j.myc.2016.04.003 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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During field collections in Ailaoshan of Yunnan Province, southwestern China, a beautifully unknown fungus growing on the ground was found. It was identified as a new species of Podoserpula according to morphological and phylogenetic analyses.
2.
Materials and methods
2.1.
Morphology
each specimen. The following abbreviations are used: IKIe ¼ neither amyloid nor dextrinoid, L ¼ mean basidiospore length (arithmetic average of all basidiospores), W ¼ mean basidiospore width (arithmetic average of all basidiospores), Q ¼ variation in the L/W ratios between the specimens studied, Qm ¼ mean Q, n (a/b) ¼ number of basidiospores (a) measured from given number (b) of specimens.
2.2.
Specimens examined in this study are deposited in the herbaria of Institute of Microbiology, Beijing Forestry University (BJFC) and Kunming Institute of Botany, Chinese Academy of Sciences (HKAS). Macro-morphological descriptions were based on field notes and photographs. Micro-morphological data were obtained from dried specimens, and observed under a light microscope following methods in Li et al. (2014). The free-hand sections mounted in 5% KOH solution after staining with 1% Congo Red, while the Melzer solution was used to check for the presence of amyloid or not. Microscopic features, such as basidiospores, basidia, hyphae and cystidioles, were observed and photographed at a magnification of up to 1000 by Nikon Digital Sight DS-Fi1 microscope (Nikon, Tokyo, Japan), and quantified by Image Pro Plus 6.0 Software (Media Cybernetics, Silver Spring, USA). The size of basidiospores were measured based on 60 basidiospores of
Molecular phylogeny
Total genomic DNA were extracted from dried specimens using FH Plant DNA Kit II (Demeter Biotech Co. Ltd., Beijing, China) according to the manufacturer's procedure. The internal transcribed spacer (ITS) regions were amplified with the primer pair ITS4 and ITS5 (White et al. 1990) while the nuclear large subunit rDNA (nLSU) with LR0R and LR7 (Vilgalys and Hester 1990), the translation elongation factor 1-a gene (EF1a) with EF1-983F and EF1-1567R (Rehner and Buckley 2005), the small subunit nuclear ribosomal RNA gene (nSSU) with PNS1 and NS41 (Hibbett 1996). The final PCR volume is 50 mL each tuber which contains 1.5 mL of primer (10 pM), 2 mL DNA extract, 20 mL ddH2O and 25 mL 2 EasyTaq PCR SuperMix (TransGen Biotech Co. Ltd., Beijing, China). PCR was performed on S1000™ Thermal Cycler (Bio-Rad Laboratories, California, USA). PCR procedures for ITS, EF1-a, nLSU and nSSU were as follows: (1) initial denaturation 95 C for 2 min, (2)
Table 1 e Species, specimens and GenBank accession number of sequences used in this study. Species
Amylocorticiales Amyloathelia crassiuscula Amylocorticium cebennense Amylocorticium subincarnatum Amylocorticium subsulphureum Anomoporia bombycina Anomoloma albolutescens Anomoloma flavissimum Anomoloma luteoalba Anomoloma myceliosum Anomoloma submyceliosum Anomoporia kamtschatica Athelia rolfsii Ceraceomyces americanus Ceraceomyces borealis Ceraceomyces serpens Hypochniciellum subillaqueatum Irpicodon pendulus Leptosporomyces septentrionalis Plicaturopsis crispa Plicaturopsis crispa Podoserpula ailaoshanensis Podoserpula ailaoshanensis Podoserpula pusio Podoserpula pusio Jaapiales (Outgroup) Jaapia argillacea Jaapia ochroleuca
Specimen no.
Country
GenBank accession no. ITS
nLSU
EF1-a
nSSU
GB/K169-796 CFMR:HHB-2808 AS-95 CFMR:HHB-13817 CFMR:L-6240 CFMR:L-6088 Cui12303 Cui 2687 CFMR:MJL-4413 Dai 7402 KHL11072 AFTOL-664 FP-102188 CFMR:L-8014 HHB-15692-Sp KHL 8493 GB/B.Norden JS16122 FP-101310-SP AFTOL-ID 1924 ZJL2015015 Liu170 PERTH:E6761 H. Lepp 329 ACT
Sweden USA Unknown USA USA USA China China Canada China Unknown Unknown USA USA USA Unknown Unknown Norway USA USA China China Australia Australia
DQ144610 GU187505 AY463377 GU187506 GU187508 GU187507 KT954955 KT954961 GU187500 KT954963 AY463379 DQ484060 KP135409 GU187512 KP135031 AY463431 DQ144619 GU187497 DQ534576 DQ494686 KU324484a KU324485a DQ494688 GU187555
DQ144610 GU187561 AY586628 GU187562 GU187564 GU187563 KT954969 KT954975 GU187559 KT954977 AY586630 AY635773 KP135277 GU187570 KP135200 AY586679 DQ144619 GU187664 AY293203 DQ470820 KU324487a KU324488a DQ470821 EF535271
e GU187675 e GU187680 GU187674 GU187671 e e GU187677 e e e e GU187686 e e e e e e KU324494a e e e
e GU187612 e GU187617 GU187611 GU187618 e e GU187614 e e AY665774 e GU187624 e e e e AY293148 e KU324491a KU324492a e e
CBS 252.74 KHL 8433
The Netherlands Sweden
GU187524 EU118637
GU187581 EU118637
GU187711 e
e e
‘e’ indicates that sequences are unavailable. a Sequences newly generated in this study.
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denaturation at 94 C for 45 s, (3) annealing at 50 C (for nLSU)/ 53 C (for ITS and nSSU)/54 C (for EF1-a) for 1 min 10 s, (4) extension at 72 C for 2 min, (5) repeat for 36 cycles of last three steps, (6) final extension at 72 C for 10 min. All PCR products were purified and sequenced in the Beijing Genomics Institute (BGI), China, with the same primers. All newly generated sequences were deposited at GenBank and listed in Table 1. Jaapia argillacea Bres. and J. ochroleuca (Bres.) Nannf. & J. Erikss of Jaapiales were selected as outgroups. Sequences were aligned with additional sequences downloaded from GenBank (Table 1) using BioEdit 7.2.5 (Hall 1999). Alignment was manually adjusted to allow maximum alignment and to minimize gaps. Sequence alignments were deposited at TreeBase (submission ID 19154; www.treebase. org). One thousand partition homogeneity test (PHT) replicates of the combined dataset of ITS, nLSU, EF1-a and nSSU sequences were tested by PAUP* version 4.0b10 (Swofford 2002) to determine whether the partitions were homogeneous. The PHT result indicated all the four different DNA sequences display a congruent phylogenetic signal (P value ¼ 0.145, much greater than 0.01). Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian inference (BI) analyses were conducted for the combined datasets of ITS, nLSU, EF1-a and nSSU sequences. Phylogenetic analysis was done as Han et al. (2016).
297
The best-fit evolutionary model was selected by hierarchical likelihood ratio tests (hLRT) and Akaike information criterion (AIC) in MrModeltest 2.2 (Nylander 2004) after scoring 24 models of evolution by PAUP* version 4.0b10 (Swofford 2002). The ML searches were conducted with RAxmlGUI 1.31 (Michalak 2012) involved 1000 ML searches under the GTRGAMMA model and only the best tree from all searches was kept. The ML bootstrap values (ML-BS) obtained from 1000 replicates were performed using RAxmlGUI 1.31 (Michalak 2012) with the GTRCAT model to assess the reliability of the nodes. The MP topology and bootstrap values (MP-BS) obtained from 1000 replicates were performed using PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. One thousand max-trees were set, branches of zero length were collapsed and all parsimonious trees were saved. Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree (MPT) generated. Bayesian phylogenetic inference and Bayesian posterior probabilities (BPP) were performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were
Fig. 1 e Phylogeny of Podoserpula and related species based on combined ITSþnLSUþEF1-aþnSSU sequences. Topology is from ML analysis with maximum likelihood bootstrap support value (≥50, former), parsimony bootstrap support value (≥50, middle) and Bayesian posterior probability value (≥0.95, latter).
298
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run for 2,000,000 generations until the split deviation frequency value <0.01, and sampled every 100 generation resulting in 20,000 trees. The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian posterior probabilities (BPP) of the clades. Trees were viewed in FigTree 1.4.0 (http://tree.bio.ed.ac.uk/ software/figtree/). Branches that received bootstrap support for ML, MP and BI greater than or equal to 50% (ML-BS and MPBS) and 0.95 (BPP) were considered as significantly supported, respectively.
3.
Results
The concatenated dataset resulted in an alignment with 5079 total characters, of which 3640 characters were constant, 412 variable characters were parsimony-uninformative and 1027 parsimony-informative characters. In the MP analysis, 13,285,213 rearrangements were tried and one most parsimonious tree scored 3462 (TL ¼ 3462, CI ¼ 0.634, RI ¼ 0.629, RC ¼ 0.398, HI ¼ 0.366) was retained. The ML analysis had an analogical topology with the Bayesian trees, and topology from ML analysis was presented along with ML-BS (above 50), MP-BS (above 50) and BPPs (above 0.95) values (Fig. 1). Phylogenetic analyses revealed that two newly sampled specimens formed a distinct lineage with high supports (100/ 100/1.00) and strongly clustered (100/100/1.00) with P. pusio in Amylocorticiales clade.
4.
Taxonomy
Podoserpula ailaoshanensis J.L. Zhou & B.K. Cui, sp. nov. Figs. 2, 3 MycoBank no.: MB 815485 This species differs morphologically from P. pusio and P. miranda in its zonate and villus covered pilei, hollow main axis covered with white powder or tomentum, globose to subglobose basidiospores (4.1e5 3.9e4.8 mm) and geographical distribution limited to Ailaoshan, southwestern China. Type: CHINA, Yunnan Province, Jingdong County, Ailaoshan Nature Reserve, elevation 2450 m, on angiosperm forest ground, 10 Sep 2015, Jun-Liang Zhou (ZJL2015015, holotype in BJFC). Gene sequences ex-holotype: KU324484 (ITS), KU324487 (nLSU), KU324494 (EF1-a), KU324491 (nSSU). Etymology: the word “ailaoshanensis” (Lat.) refers to the place, Ailaoshan, where the holotype was collected. Fruiting body: Basidiomata about 6.6e10.5 cm high, multistoried with an erect and branched main axis. Pilei light ivory to yellowish orange when fresh, becoming yellowish brown to pale brown after drying; margin white when fresh; normally varying in shape from reniform, spatulate to infundibuliform, with slightly involute margin; upper surface zonate and covered with villus; soft and flexible when fresh and fragile upon drying; generally the main axis appearing to pierce the pilei centrally or eccentrically, besides few pilei
Fig. 2 e Basidiocarps of Podoserpula ailaoshanensis. A: Liu170 (photographed by Xiao-Bin Liu), B: ZJL2015015. Bar: 1 cm.
joining to the main axis by a lateral stipe-like base; pilei usually tend to decrease in size toward the top of the main axis, 0.6e4 cm wide and less than 1.5 mm thick when fresh. Hymenophore ivory to beige; densely wrinkled over the entire surface, Cantharellus-like, radially arranged and always decurrent on the main axis. Main axis subcylindrical and hollow; upper partial surface covered with white powder or tomentum and slightly pearl dark grey at the basal part; changing to brown beige after touching or injuring; up to 9.8 cm long, 0.7e1.1 cm in diam. Context soft, cream and less than 1.5 mm thick when fresh. Hyphal structure: Hyphal system monomitic; generative hyphae bearing clamp connections, IKIe; tissue unchanged in KOH. Context: Generative hyphae colorless and smooth, thinwalled, occasionally ramified, regularly arranged, 3.1e10.9 mm in diam. Hyphae between context and subhymenium up to 50 mm wide, loosely interwoven. Hyphae in cuticle thin-walled with clamp connections, incrustations absent, loosely interwoven, 2.7e7.3 mm in diam.
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Fig. 3 e Microscopic structures of Podoserpula ailaoshanensis (Holotype). A: basidiospores. B: basidia and basidioles. C: vertical section of the cap, 1: hymenium and subhymenium, 2: hyphae of context. D: hyphae in cuticle. E: hyphae from context. F: hyphae of the hymenophoral context. G: hyphae from the main axis. Bars: A, B, DeG 10 mm; C 100 mm. Hymenophore: Hymenium and subhymenium about 100e175 mm wide, densely; generative hyphae colorless and smooth, thin-walled, 2.5e10.5 mm in diam. Cystidia or gloeocystidia absent; basidia slenderly clavate, with a basal clamp and four sterigmata, 22.6e44.6 4.1e7.5 mm; basidioles in shape similar to basidia but slightly smaller. Main axis: Generative hyphae colorless and smooth, thinwalled, occasionally ramified, formed of axially oriented, interwoven, 3.2e10.1 mm in diam. Basidiospores: globose to subglobose, rarely broadly ellipsoid; thick-walled, colorless, smooth, often bearing a large and hyaline guttule, IKIe, (3.8e)4.1e5(e5.1) (3.7e)3.9e4.8(e4.9) mm, L ¼ 4.5 mm, W ¼ 4.24 mm, Q ¼ 1e1.23, Qm ¼ 1.06 (n ¼ 120/2). Habit: scattered or gregarious on angiosperm forest ground. Additional specimen examined: CHINA, Yunnan Province, Jingdong County, Ailaoshan Nature Reserve, elevation 2491 m, on angiosperm forest ground, 22 Jul 2013, Xiao-Bin Liu (Liu170, paratype in HKAS).
5.
Discussion
Morphologically, Podoserpula was considered as a member of Coniophoraceae for its folded or blistered hymenial surface, a monomitic hyphal construction and hyphae with clamp connections at the septa (Reid 1963; Donk 1964). Later, phylogenetic results revealed that P. pusio clustered with Plicaturopsis crispa (Pers.) D.A. Reid and Athelia rolfsii (Curzi) C.C. Tu & Kimbr. (¼ Sclerotium rolfsii Sacc.) in the Plicaturopsidoid clade (Matheny et al. 2006). Binder et al. (2010) put Podoserpula into Amylocorticiales according to a six-locus phylogenetic analysis. A recent phylogenetic analysis also indicated that Podoserpula belongs to Amylocorticiales (Song et al. 2016). Our current phylogenetic result also proved Podoserpula as a member of Amylocorticiales and was closely related to Hypochniciellum subillaqueatum (Litsch.) Hjortstam, which is characterized by whitish to cream colored or yellowish basidiocarps, presence of chlamydospores, and small ellipsoid
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n et al. basidiospores with a greyish Melzer's reaction (Gorjo 2011). Phylogenetic analyses based on combined ITS, nLSU, EF1-a and nSSU sequences revealed that P. ailaoshanensis grouped together with P. pusio with high supports (Fig. 1). Both of them have superimposed basidiocarps, yellowish orange pilei which vary in shape from reniform to spatulate, and similar hyphal structure. But the pileus of P. pusio normally joined to the main axis by a short, flattened, stipe-like base (Reid 1963), while the main axis of P. ailaoshanensis usually appeared to pierce the pileus centrally or eccentrically. In addition, P. pusio usually has pink hymenial surface and ellipsoid to subglobose basidiospores (2.8e6 2e3.5 mm; Reid 1963), while P. ailaoshanensis has ivory to beige hymenial surface and much wider basidiospores (4.1e5 3.9e4.8 mm). Moreover, the hollow main axis covered with white powder or tomentum, the zonate and fuzzy pileus, and the geographical distribution of P. ailaoshanensis are also different from P. pusio. Previously, four varieties of P. pusio were reported: P. pusio var. austro-americana, P. pusio var. ellipsospora, P. pusio var. pusio and P. pusio var. tristis (Reid 1963), among them, P. pusio var. tristis different from the other three varieties by its pale fawn pileus, main axis appears to pierce the pileus centrally or eccentrically, and geographical distribution limited to New Zealand. Podoserpula pusio var. ellipsospora has large and distinct ellipsoid basidiospores (4e5 2.8e3.5 mm), and it is known only from Australia. Podoserpula pusio var. austroamericana has broadly ellipsoid to subglobose basidiospores (3.5e4.2 2.5e3.2 mm), typically large fruiting bodies which reaching 18 cm, and known only from Venezuela. Podoserpula pusio var. pusio has broadly ellipsoid to subglobose (2.8e4 2e3.5 mm), small fruiting bodies which seldom exceeding 7.5 cm, and known only from Australasia (Reid 1963). These four varieties may represent different taxa at species level. Podoserpula pusio var. pusio shares orange or yellow pilei which vary in shape from reniform to spatulate with P. ailaoshanensis, but its pilei joined to the main axis by a lateral stipe-like base; moreover, P. pusio var. pusio has smaller basidiocarps (Reid 1963), pink hymenial surface (Willis 1954; Reid 1963), and smaller basidiospores (2.8e4 2e3.5 mm; Reid 1963). Podoserpula pusio var. austro-americana has cream, cream ochraceous to yellowish orange fruiting body (Buyck et al. 2012), which may be confused with P. ailaoshanensis, but it can be easily separated from P. ailaoshanensis by its distinctly pink hymenophores, narrow stipe-like base of pilei and smaller basidiospores (3.5e4.2 2.5e3.2 mm; Reid 1963); in addition, the main axis of P. pusio var. austro-americana often folks and arises a number of broad superimposed pilei (Reid 1963). Podoserpula pusio var. ellipsospora differs from P. ailaoshanensis in its stipe-like base of pilei and distinctly ellipsoid basidiospores (4e5 2.8e3.5 mm; Reid 1963). Podoserpula pusio var. tristis differs from P. ailaoshanensis by its pale fawn pileus when fresh and distinctly smaller basidiospores (3e3.5 2e2.8 mm; Reid 1963). Unfortunately, no DNA sequences data are available for P. miranda, so this species is not included in the phylogenetic analysis. But, morphologically P. miranda has pinkish lilac basidiocarps, upward and outward growing pilei, solid main axis and somewhat smaller basidiospores (3.5e5 3.3e4.6 mm), these characters are different from P.
ailaoshanensis; in addition, P. miranda geographical distribution limited to New Caledonia (Buyck et al. 2012).
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of China.
Acknowledgments The authors would like to thank Prof. Zhu-Liang Yang and Dr. Xin-Yu Wang (Kunming Institute of Botany, Chinese Academy of Sciences, China) for loan of specimens. Mr. Xiao-Bin Liu (Kunming Institute of Botany, Chinese Academy of Sciences, China) is grateful for collecting specimen and offering photograph. The research was financed by the Fundamental Research Funds for the Central Universities (Project No. 2016ZCQ04) and the National Natural Science Foundation of China (Project No. 31422001).
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journal homepage: www.elsevier.com/locate/myc
Full paper
Podoserpula ailaoshanensis sp. nov. (Amylocorticiales, Basidiomycota) from China based on morphological and sequence analyses Jun-Liang Zhou, Hong Chen, Bao-Kai Cui* Institute of Microbiology, Beijing Forestry University, Beijing 100083, China
article info
abstract
Article history:
Podoserpula is described from Australia and mainly collected from the southern hemi-
Received 21 February 2016
sphere. It is characterized by the brightly colored multi-storied basidiocarps, non-amyloid
Received in revised form
and thick-walled ellipsoid to subglobose basidiospores, distributed in subtropical to trop-
22 April 2016
ical regions. In this study, P. ailaoshanensis is described and illustrated as a new species
Accepted 23 April 2016
based on morphological and molecular evidence. This species is characterized by its multi-
Available online 2 June 2016
storied basidiocarps, zonate and fuzzy pileus, light ivory to yellow orange pileal surface, hollow main axis, and globose to subglobose, thick-walled basidiospores (4.1e5 3.9
Keywords:
e4.8 mm). Phylogenetically, on the basis of combined dataset of internal transcribed spacer
Amylocorticiales
(ITS), nuclear large subunit rDNA (nLSU), translation elongation factor 1-a gene (EF1-a) and
Phylogeny
small subunit nuclear ribosomal RNA gene (nSSU), P. ailaoshanensis is strongly supported as
Taxonomy
a new species of Podoserpula. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Podoserpula D.A. Reid is usually known as “pagoda fungus” for its multi-storied basidiocarps. It was erected by Reid (1963) based on the type species P. pusio (Berk.) D.A. Reid. Features distinguishing Podoserpula from others are as follows: stipitate basidiocarps with a simple or very sparingly branched axis; pilei often brightly colored, normally vary in shape, joined to the main axis by stipe-like base or the main axis appearing to pierce the centre of each individual pileus; hyphal structure monomitic, generative hyphae with clamps; cystidia and gloeocystidia absent; basidiospores small, thick-walled, inamyloid, ellipsoid to subglobose; growing on the ground or in
well rotted stump-wood (Reid 1963). On the basis of morphological features and geographic distributions, four varieties, including P. pusio var. austro-americana, P. pusio var. ellipsospora, P. pusio var. pusio and P. pusio var. tristis, were described by Reid (1963). Podoserpula miranda B. Buyck, B. Duhem, G. Eyssartier & M. Ducousso, which is limited to New Caledonia, is the second Podoserpula species (Buyck et al. 2012). Besides Australia, New Zealand, Venezuela and New Caledonia, Podoserpula has been collected from several other places, such as Brazil (Pegler 1988), Chile and Guiana (Hjortstam and Ryvarden 2007), Falklands Islands (Watling 2012) and Madagascar (Buyck 2008). Although it has been collected from many places, there is no record from Asia.
* Corresponding author. Tel./fax: þ86 10 62336309. E-mail address: [email protected] (B.-K. Cui). http://dx.doi.org/10.1016/j.myc.2016.04.003 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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During field collections in Ailaoshan of Yunnan Province, southwestern China, a beautifully unknown fungus growing on the ground was found. It was identified as a new species of Podoserpula according to morphological and phylogenetic analyses.
2.
Materials and methods
2.1.
Morphology
each specimen. The following abbreviations are used: IKIe ¼ neither amyloid nor dextrinoid, L ¼ mean basidiospore length (arithmetic average of all basidiospores), W ¼ mean basidiospore width (arithmetic average of all basidiospores), Q ¼ variation in the L/W ratios between the specimens studied, Qm ¼ mean Q, n (a/b) ¼ number of basidiospores (a) measured from given number (b) of specimens.
2.2.
Specimens examined in this study are deposited in the herbaria of Institute of Microbiology, Beijing Forestry University (BJFC) and Kunming Institute of Botany, Chinese Academy of Sciences (HKAS). Macro-morphological descriptions were based on field notes and photographs. Micro-morphological data were obtained from dried specimens, and observed under a light microscope following methods in Li et al. (2014). The free-hand sections mounted in 5% KOH solution after staining with 1% Congo Red, while the Melzer solution was used to check for the presence of amyloid or not. Microscopic features, such as basidiospores, basidia, hyphae and cystidioles, were observed and photographed at a magnification of up to 1000 by Nikon Digital Sight DS-Fi1 microscope (Nikon, Tokyo, Japan), and quantified by Image Pro Plus 6.0 Software (Media Cybernetics, Silver Spring, USA). The size of basidiospores were measured based on 60 basidiospores of
Molecular phylogeny
Total genomic DNA were extracted from dried specimens using FH Plant DNA Kit II (Demeter Biotech Co. Ltd., Beijing, China) according to the manufacturer's procedure. The internal transcribed spacer (ITS) regions were amplified with the primer pair ITS4 and ITS5 (White et al. 1990) while the nuclear large subunit rDNA (nLSU) with LR0R and LR7 (Vilgalys and Hester 1990), the translation elongation factor 1-a gene (EF1a) with EF1-983F and EF1-1567R (Rehner and Buckley 2005), the small subunit nuclear ribosomal RNA gene (nSSU) with PNS1 and NS41 (Hibbett 1996). The final PCR volume is 50 mL each tuber which contains 1.5 mL of primer (10 pM), 2 mL DNA extract, 20 mL ddH2O and 25 mL 2 EasyTaq PCR SuperMix (TransGen Biotech Co. Ltd., Beijing, China). PCR was performed on S1000™ Thermal Cycler (Bio-Rad Laboratories, California, USA). PCR procedures for ITS, EF1-a, nLSU and nSSU were as follows: (1) initial denaturation 95 C for 2 min, (2)
Table 1 e Species, specimens and GenBank accession number of sequences used in this study. Species
Amylocorticiales Amyloathelia crassiuscula Amylocorticium cebennense Amylocorticium subincarnatum Amylocorticium subsulphureum Anomoporia bombycina Anomoloma albolutescens Anomoloma flavissimum Anomoloma luteoalba Anomoloma myceliosum Anomoloma submyceliosum Anomoporia kamtschatica Athelia rolfsii Ceraceomyces americanus Ceraceomyces borealis Ceraceomyces serpens Hypochniciellum subillaqueatum Irpicodon pendulus Leptosporomyces septentrionalis Plicaturopsis crispa Plicaturopsis crispa Podoserpula ailaoshanensis Podoserpula ailaoshanensis Podoserpula pusio Podoserpula pusio Jaapiales (Outgroup) Jaapia argillacea Jaapia ochroleuca
Specimen no.
Country
GenBank accession no. ITS
nLSU
EF1-a
nSSU
GB/K169-796 CFMR:HHB-2808 AS-95 CFMR:HHB-13817 CFMR:L-6240 CFMR:L-6088 Cui12303 Cui 2687 CFMR:MJL-4413 Dai 7402 KHL11072 AFTOL-664 FP-102188 CFMR:L-8014 HHB-15692-Sp KHL 8493 GB/B.Norden JS16122 FP-101310-SP AFTOL-ID 1924 ZJL2015015 Liu170 PERTH:E6761 H. Lepp 329 ACT
Sweden USA Unknown USA USA USA China China Canada China Unknown Unknown USA USA USA Unknown Unknown Norway USA USA China China Australia Australia
DQ144610 GU187505 AY463377 GU187506 GU187508 GU187507 KT954955 KT954961 GU187500 KT954963 AY463379 DQ484060 KP135409 GU187512 KP135031 AY463431 DQ144619 GU187497 DQ534576 DQ494686 KU324484a KU324485a DQ494688 GU187555
DQ144610 GU187561 AY586628 GU187562 GU187564 GU187563 KT954969 KT954975 GU187559 KT954977 AY586630 AY635773 KP135277 GU187570 KP135200 AY586679 DQ144619 GU187664 AY293203 DQ470820 KU324487a KU324488a DQ470821 EF535271
e GU187675 e GU187680 GU187674 GU187671 e e GU187677 e e e e GU187686 e e e e e e KU324494a e e e
e GU187612 e GU187617 GU187611 GU187618 e e GU187614 e e AY665774 e GU187624 e e e e AY293148 e KU324491a KU324492a e e
CBS 252.74 KHL 8433
The Netherlands Sweden
GU187524 EU118637
GU187581 EU118637
GU187711 e
e e
‘e’ indicates that sequences are unavailable. a Sequences newly generated in this study.
m y c o s c i e n c e 5 7 ( 2 0 1 6 ) 2 9 5 e3 0 1
denaturation at 94 C for 45 s, (3) annealing at 50 C (for nLSU)/ 53 C (for ITS and nSSU)/54 C (for EF1-a) for 1 min 10 s, (4) extension at 72 C for 2 min, (5) repeat for 36 cycles of last three steps, (6) final extension at 72 C for 10 min. All PCR products were purified and sequenced in the Beijing Genomics Institute (BGI), China, with the same primers. All newly generated sequences were deposited at GenBank and listed in Table 1. Jaapia argillacea Bres. and J. ochroleuca (Bres.) Nannf. & J. Erikss of Jaapiales were selected as outgroups. Sequences were aligned with additional sequences downloaded from GenBank (Table 1) using BioEdit 7.2.5 (Hall 1999). Alignment was manually adjusted to allow maximum alignment and to minimize gaps. Sequence alignments were deposited at TreeBase (submission ID 19154; www.treebase. org). One thousand partition homogeneity test (PHT) replicates of the combined dataset of ITS, nLSU, EF1-a and nSSU sequences were tested by PAUP* version 4.0b10 (Swofford 2002) to determine whether the partitions were homogeneous. The PHT result indicated all the four different DNA sequences display a congruent phylogenetic signal (P value ¼ 0.145, much greater than 0.01). Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian inference (BI) analyses were conducted for the combined datasets of ITS, nLSU, EF1-a and nSSU sequences. Phylogenetic analysis was done as Han et al. (2016).
297
The best-fit evolutionary model was selected by hierarchical likelihood ratio tests (hLRT) and Akaike information criterion (AIC) in MrModeltest 2.2 (Nylander 2004) after scoring 24 models of evolution by PAUP* version 4.0b10 (Swofford 2002). The ML searches were conducted with RAxmlGUI 1.31 (Michalak 2012) involved 1000 ML searches under the GTRGAMMA model and only the best tree from all searches was kept. The ML bootstrap values (ML-BS) obtained from 1000 replicates were performed using RAxmlGUI 1.31 (Michalak 2012) with the GTRCAT model to assess the reliability of the nodes. The MP topology and bootstrap values (MP-BS) obtained from 1000 replicates were performed using PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. One thousand max-trees were set, branches of zero length were collapsed and all parsimonious trees were saved. Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree (MPT) generated. Bayesian phylogenetic inference and Bayesian posterior probabilities (BPP) were performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were
Fig. 1 e Phylogeny of Podoserpula and related species based on combined ITSþnLSUþEF1-aþnSSU sequences. Topology is from ML analysis with maximum likelihood bootstrap support value (≥50, former), parsimony bootstrap support value (≥50, middle) and Bayesian posterior probability value (≥0.95, latter).
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run for 2,000,000 generations until the split deviation frequency value <0.01, and sampled every 100 generation resulting in 20,000 trees. The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian posterior probabilities (BPP) of the clades. Trees were viewed in FigTree 1.4.0 (http://tree.bio.ed.ac.uk/ software/figtree/). Branches that received bootstrap support for ML, MP and BI greater than or equal to 50% (ML-BS and MPBS) and 0.95 (BPP) were considered as significantly supported, respectively.
3.
Results
The concatenated dataset resulted in an alignment with 5079 total characters, of which 3640 characters were constant, 412 variable characters were parsimony-uninformative and 1027 parsimony-informative characters. In the MP analysis, 13,285,213 rearrangements were tried and one most parsimonious tree scored 3462 (TL ¼ 3462, CI ¼ 0.634, RI ¼ 0.629, RC ¼ 0.398, HI ¼ 0.366) was retained. The ML analysis had an analogical topology with the Bayesian trees, and topology from ML analysis was presented along with ML-BS (above 50), MP-BS (above 50) and BPPs (above 0.95) values (Fig. 1). Phylogenetic analyses revealed that two newly sampled specimens formed a distinct lineage with high supports (100/ 100/1.00) and strongly clustered (100/100/1.00) with P. pusio in Amylocorticiales clade.
4.
Taxonomy
Podoserpula ailaoshanensis J.L. Zhou & B.K. Cui, sp. nov. Figs. 2, 3 MycoBank no.: MB 815485 This species differs morphologically from P. pusio and P. miranda in its zonate and villus covered pilei, hollow main axis covered with white powder or tomentum, globose to subglobose basidiospores (4.1e5 3.9e4.8 mm) and geographical distribution limited to Ailaoshan, southwestern China. Type: CHINA, Yunnan Province, Jingdong County, Ailaoshan Nature Reserve, elevation 2450 m, on angiosperm forest ground, 10 Sep 2015, Jun-Liang Zhou (ZJL2015015, holotype in BJFC). Gene sequences ex-holotype: KU324484 (ITS), KU324487 (nLSU), KU324494 (EF1-a), KU324491 (nSSU). Etymology: the word “ailaoshanensis” (Lat.) refers to the place, Ailaoshan, where the holotype was collected. Fruiting body: Basidiomata about 6.6e10.5 cm high, multistoried with an erect and branched main axis. Pilei light ivory to yellowish orange when fresh, becoming yellowish brown to pale brown after drying; margin white when fresh; normally varying in shape from reniform, spatulate to infundibuliform, with slightly involute margin; upper surface zonate and covered with villus; soft and flexible when fresh and fragile upon drying; generally the main axis appearing to pierce the pilei centrally or eccentrically, besides few pilei
Fig. 2 e Basidiocarps of Podoserpula ailaoshanensis. A: Liu170 (photographed by Xiao-Bin Liu), B: ZJL2015015. Bar: 1 cm.
joining to the main axis by a lateral stipe-like base; pilei usually tend to decrease in size toward the top of the main axis, 0.6e4 cm wide and less than 1.5 mm thick when fresh. Hymenophore ivory to beige; densely wrinkled over the entire surface, Cantharellus-like, radially arranged and always decurrent on the main axis. Main axis subcylindrical and hollow; upper partial surface covered with white powder or tomentum and slightly pearl dark grey at the basal part; changing to brown beige after touching or injuring; up to 9.8 cm long, 0.7e1.1 cm in diam. Context soft, cream and less than 1.5 mm thick when fresh. Hyphal structure: Hyphal system monomitic; generative hyphae bearing clamp connections, IKIe; tissue unchanged in KOH. Context: Generative hyphae colorless and smooth, thinwalled, occasionally ramified, regularly arranged, 3.1e10.9 mm in diam. Hyphae between context and subhymenium up to 50 mm wide, loosely interwoven. Hyphae in cuticle thin-walled with clamp connections, incrustations absent, loosely interwoven, 2.7e7.3 mm in diam.
m y c o s c i e n c e 5 7 ( 2 0 1 6 ) 2 9 5 e3 0 1
299
Fig. 3 e Microscopic structures of Podoserpula ailaoshanensis (Holotype). A: basidiospores. B: basidia and basidioles. C: vertical section of the cap, 1: hymenium and subhymenium, 2: hyphae of context. D: hyphae in cuticle. E: hyphae from context. F: hyphae of the hymenophoral context. G: hyphae from the main axis. Bars: A, B, DeG 10 mm; C 100 mm. Hymenophore: Hymenium and subhymenium about 100e175 mm wide, densely; generative hyphae colorless and smooth, thin-walled, 2.5e10.5 mm in diam. Cystidia or gloeocystidia absent; basidia slenderly clavate, with a basal clamp and four sterigmata, 22.6e44.6 4.1e7.5 mm; basidioles in shape similar to basidia but slightly smaller. Main axis: Generative hyphae colorless and smooth, thinwalled, occasionally ramified, formed of axially oriented, interwoven, 3.2e10.1 mm in diam. Basidiospores: globose to subglobose, rarely broadly ellipsoid; thick-walled, colorless, smooth, often bearing a large and hyaline guttule, IKIe, (3.8e)4.1e5(e5.1) (3.7e)3.9e4.8(e4.9) mm, L ¼ 4.5 mm, W ¼ 4.24 mm, Q ¼ 1e1.23, Qm ¼ 1.06 (n ¼ 120/2). Habit: scattered or gregarious on angiosperm forest ground. Additional specimen examined: CHINA, Yunnan Province, Jingdong County, Ailaoshan Nature Reserve, elevation 2491 m, on angiosperm forest ground, 22 Jul 2013, Xiao-Bin Liu (Liu170, paratype in HKAS).
5.
Discussion
Morphologically, Podoserpula was considered as a member of Coniophoraceae for its folded or blistered hymenial surface, a monomitic hyphal construction and hyphae with clamp connections at the septa (Reid 1963; Donk 1964). Later, phylogenetic results revealed that P. pusio clustered with Plicaturopsis crispa (Pers.) D.A. Reid and Athelia rolfsii (Curzi) C.C. Tu & Kimbr. (¼ Sclerotium rolfsii Sacc.) in the Plicaturopsidoid clade (Matheny et al. 2006). Binder et al. (2010) put Podoserpula into Amylocorticiales according to a six-locus phylogenetic analysis. A recent phylogenetic analysis also indicated that Podoserpula belongs to Amylocorticiales (Song et al. 2016). Our current phylogenetic result also proved Podoserpula as a member of Amylocorticiales and was closely related to Hypochniciellum subillaqueatum (Litsch.) Hjortstam, which is characterized by whitish to cream colored or yellowish basidiocarps, presence of chlamydospores, and small ellipsoid
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n et al. basidiospores with a greyish Melzer's reaction (Gorjo 2011). Phylogenetic analyses based on combined ITS, nLSU, EF1-a and nSSU sequences revealed that P. ailaoshanensis grouped together with P. pusio with high supports (Fig. 1). Both of them have superimposed basidiocarps, yellowish orange pilei which vary in shape from reniform to spatulate, and similar hyphal structure. But the pileus of P. pusio normally joined to the main axis by a short, flattened, stipe-like base (Reid 1963), while the main axis of P. ailaoshanensis usually appeared to pierce the pileus centrally or eccentrically. In addition, P. pusio usually has pink hymenial surface and ellipsoid to subglobose basidiospores (2.8e6 2e3.5 mm; Reid 1963), while P. ailaoshanensis has ivory to beige hymenial surface and much wider basidiospores (4.1e5 3.9e4.8 mm). Moreover, the hollow main axis covered with white powder or tomentum, the zonate and fuzzy pileus, and the geographical distribution of P. ailaoshanensis are also different from P. pusio. Previously, four varieties of P. pusio were reported: P. pusio var. austro-americana, P. pusio var. ellipsospora, P. pusio var. pusio and P. pusio var. tristis (Reid 1963), among them, P. pusio var. tristis different from the other three varieties by its pale fawn pileus, main axis appears to pierce the pileus centrally or eccentrically, and geographical distribution limited to New Zealand. Podoserpula pusio var. ellipsospora has large and distinct ellipsoid basidiospores (4e5 2.8e3.5 mm), and it is known only from Australia. Podoserpula pusio var. austroamericana has broadly ellipsoid to subglobose basidiospores (3.5e4.2 2.5e3.2 mm), typically large fruiting bodies which reaching 18 cm, and known only from Venezuela. Podoserpula pusio var. pusio has broadly ellipsoid to subglobose (2.8e4 2e3.5 mm), small fruiting bodies which seldom exceeding 7.5 cm, and known only from Australasia (Reid 1963). These four varieties may represent different taxa at species level. Podoserpula pusio var. pusio shares orange or yellow pilei which vary in shape from reniform to spatulate with P. ailaoshanensis, but its pilei joined to the main axis by a lateral stipe-like base; moreover, P. pusio var. pusio has smaller basidiocarps (Reid 1963), pink hymenial surface (Willis 1954; Reid 1963), and smaller basidiospores (2.8e4 2e3.5 mm; Reid 1963). Podoserpula pusio var. austro-americana has cream, cream ochraceous to yellowish orange fruiting body (Buyck et al. 2012), which may be confused with P. ailaoshanensis, but it can be easily separated from P. ailaoshanensis by its distinctly pink hymenophores, narrow stipe-like base of pilei and smaller basidiospores (3.5e4.2 2.5e3.2 mm; Reid 1963); in addition, the main axis of P. pusio var. austro-americana often folks and arises a number of broad superimposed pilei (Reid 1963). Podoserpula pusio var. ellipsospora differs from P. ailaoshanensis in its stipe-like base of pilei and distinctly ellipsoid basidiospores (4e5 2.8e3.5 mm; Reid 1963). Podoserpula pusio var. tristis differs from P. ailaoshanensis by its pale fawn pileus when fresh and distinctly smaller basidiospores (3e3.5 2e2.8 mm; Reid 1963). Unfortunately, no DNA sequences data are available for P. miranda, so this species is not included in the phylogenetic analysis. But, morphologically P. miranda has pinkish lilac basidiocarps, upward and outward growing pilei, solid main axis and somewhat smaller basidiospores (3.5e5 3.3e4.6 mm), these characters are different from P.
ailaoshanensis; in addition, P. miranda geographical distribution limited to New Caledonia (Buyck et al. 2012).
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of China.
Acknowledgments The authors would like to thank Prof. Zhu-Liang Yang and Dr. Xin-Yu Wang (Kunming Institute of Botany, Chinese Academy of Sciences, China) for loan of specimens. Mr. Xiao-Bin Liu (Kunming Institute of Botany, Chinese Academy of Sciences, China) is grateful for collecting specimen and offering photograph. The research was financed by the Fundamental Research Funds for the Central Universities (Project No. 2016ZCQ04) and the National Natural Science Foundation of China (Project No. 31422001).
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