Phylloporia lonicerae (Hymenochaetales, Basidiomycota), a new species on Lonicera japonica from Japan and an identification key to worldwide species of Phylloporia.

Phylloporia, in the Hymenochaetaceae, is a polypore genus with a worldwide distribution. The new taxon Phylloporia lonicerae is introduced, which is the first Phylloporia species to originate from Japan. This species grows exclusively on living Lonicera japonica and is distinguished by annual, sessile basidiocarps that occur in clusters, pileal surface of narrow, concentrically sulcate zones, 6-8 pores per mm, duplex context separated by a black zone, dimitic hyphal system and broadly ellipsoid basidiospores, 3.2-4 × 2.3-3.1 µm. Phylogenetically, P. lonicerae is nested within the Phylloporia clade as a distinct terminal lineage with full statistical supports and sister to the clade of P. minutispora, P. cf. pulla and P. terrestris with weak supports. Besides Phylloporia bibulosa, P. chrysites and P. spathulata, P. lonicerae is the fourth species of Phylloporia recorded from Japan. An identification key to all accepted 48 species of Phylloporia is provided.

Introduction

Murrill, in the Donk, was introduced for an unusual polypore species, Murrill growing on the underside of living leaves in Columbia (Murrill 1904). For nearly 70 years, was forgotten until Ryvarden (1972) transferred five taxa into the genus. Renewed interest in was stimulated by Wagner and Ryvarden’s (2002) phylogenetic and morphological study in which they accepted 12 species. Since then, a number of new species have been described from Africa (Ipulet and Ryvarden 2005, Decock et al. 2015, Yombiyeni et al. 2015, Yombiyeni and Decock 2017), the Americas (Valenzuela et al. 2011, Decock et al. 2013, Ferreira-Lopes et al. 2016) and Asia, especially China (Gafforov et al. 2014, Cui et al. 2010, Zhou and Dai 2012, Zhou 2013, 2015a, 2015b, 2016, Liu et al. 2015, Chen et al. 2017, Ren and Wu 2017).

began as a monophyletic genus based on phylogenic studies of the large subunit of the nuclear ribosomal gene (nLSU) (Wagner and Ryvarden 2002) but is now paraphyletic with the inclusion of (Speg.) Rajchenb. & J.E. Wright in the clade (Valenzuela et al. 2011, Decock et al. 2013). The genus is morphologically quite diverse and includes species with annual or perennial basidiocarps with resupinate, sessile or stipitate habits, homogenous or duplex context, monomitic or dimitic hyphal system and cylindrical to subglobose basidiospores (Wagner and Ryvarden 2002, Cui et al. 2010, Zhou 2015a). Substrate preferences of species are equally diverse. Some species are saprobes that colonise woody debris (Ipulet and Ryvarden 2005, Zhou 2015b, Ferreira-Lopes et al. 2016) and others are parasites usually of specific plant hosts (Zhou 2015a, Ren and Wu 2017, Yombiyeni and Decock 2017).

There are three species of reported from Japan – (Lloyd) Ryvarden, (Berk.) Ryvarden and (Hook.) Ryvarden (Núñez and Ryvarden 2000). In this paper, a new species, , is described from Nara, Japan, growing on living vines of . Morphological and molecular data support the recognition of this new species. In addition, an updated key to the known species of is presented.

Materials and methods

Morphological examination

The studied specimens were deposited at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP) in China. The macroscopic characters were observed from dried specimens with the aid of a stereomicroscope. Specimen sections were mounted in Cotton Blue (CB), Melzer’s reagent (IKI) and 5 % potassium hydroxide (KOH) for observation using a Nikon Eclipse 80i microscope at magnification up to 1000×. Special colour terms follow Petersen (1996). All measurements were taken PageBreakfrom sections mounted in CB. When presenting the size variation of basidiospores, 5% of measurements from each end of the range were put in parentheses. Line drawings of microscopic characters were made with the aid of a drawing tube. The abbreviations used in the description are as follows: L = mean basidiospore length (arithmetic average of all measured basidiospores), W = mean basidiospore width (arithmetic average of all measured basidiospores), Q = variation in the L/W ratios between the specimens studied and n = number of basidiospores measured from a given number of specimens.

Molecular sequencing

The PCR products were directly amplified from the extracts of the basidiocarps with the Phire® Plant Direct PCR Kit (Finnzymes Oy, Finland) according to the manufacturer’s protocol. The PCR protocol was as follows: initial denaturation at 98 °C for 5 min, followed by 39 cycles of denaturation at 98 °C for 5 s, annealing at 48 °C for 5 s and extension at 72 °C for 5 s and a final extension of 72 °C for 10 min. The primers LR0R and LR7 (Vilgalys and Hester 1990) were used for PCR amplification and subsequent sequencing at the Beijing Genomics Institute, China. The newly generated sequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank; Fig. 1).

Figure 1.

Phylogenetic position of inferred from the nLSU dataset. The topology is inferred by maximum likelihood algorithm, while bootstrap values above 50 % and Bayesian posterior probabilities above 0.8 are given at the nodes. Newly sequenced specimens are in boldface.

Phylogenetic analysis

To explore the phylogenetic relationship of , six nLSU sequences were incorporated into previous nLSU datasets of (Zhou 2016, Chen et al. 2017, Ren and Wu 2017, Yombiyeni and Decock 2017). Several species of Murrill and Murrill were included in the dataset and (Bull.) P. Karst. was selected as the outgroup taxon.

The nLSU dataset was aligned with MAFFT 7.110 (Katoh and Standley 2013) with the g-ini-i option (Katoh et al. 2005). The best-fit evolutionary model for the resulting alignment that was deposited in TreeBASE (http://www.treebase.org; accession number S21971), was estimated as GTR + I + G using jModelTest 2.1.4 (Darriba et al. 2012). Following this model, maximum likelihood (ML) and Bayesian Inference (BI) algorithms were used to infer the phylogeny of the alignment. The ML analysis was conducted using raxmlGUI 1.2 (Silvestro and Michalak 2012, Stamatakis 2006) under the auto FC option for bootstrap (BS) replicates (Pattengale et al. 2010). MrBayes 3.2 (Ronquist et al. 2012) was carried out for BI analysis, which employed two independent runs, each including four chains of 10 million generations and starting from random trees. Trees were sampled every 1000th generation. Of the sampled trees, the first 25 % was deleted and the remaining trees were used to construct a 50 % majority consensus tree and calculate Bayesian posterior probabilities (BPPs). Chain convergence was determined using Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/).

Results

Six nLSU sequences of were generated and included in a dataset of 105 sequences and 942 characters. ML analysis was ended after 250 BS replicates. BI analysis converged all chains as indicated by the effective sample sizes of all parameters above 2000 and the potential scale reduction factors close to 1.000. As the ML and BI analyses generated congruent topologies in main lineages, the ML tree is presented in Figure 1. Values of BS above 50 % and BPPs above 0.8 are given at the nodes. The phylogenic tree (Fig. 1) shows that the strongly supported clade (98 % in ML, 1 in BI) consists of 44 terminal lineages and the six samples formed a new lineage with full statistical supports (100 % in ML, 1 in BI). The lineage is sister to the clade that includes Ipulet & Ryvarden, (Mont. & Berk.) Decock & Yombiy and L.W. Zhou with weak supports.

Taxonomy

W.M. Qin, Xue W. Wang, T. Sawahata & L.W. Zhou sp. nov.

MB823715

Figs 2 , 3

Figure 2.

Basidiocarps of in situ. a LWZ 20170703-2 (holotype) b LWZ 20170622-1 (paratype). Scale bars: 2 cm.

Figure 3.

Microscopic structures of (drawn from the holotype, LWZ 20170703-2). a Basidiospores b Basidia and basidioles c Hyphae from trama d Hyphae from lower context e hyphae from upper tomentum. Scale bars: a = 5 µm, b–e = 10 µm.

Holotype.

JAPAN. Nara, Research Forest of Faculty of Agriculture, Kindai University, 3 Jul 2017, on living vine of , LWZ 20170703-2 (IFP 019172).

Etymology.

(Lat.): referring to , the host tree genus.

Basidiocarps annual, sessile, imbricate, rarely solitary, without odour or taste, woody. Pilei semi-circular, flabelliform or fused together, applanate, single pileus projecting up to 1.5 cm long, 3 cm wide and 0.5 cm thick at base. Pileal surface greyish-brown to yellowish-brown, velutinate, concentrically sulcate with narrow zones; margin pale yellow or concolorous, sharp. Pore surface honey-yellow, slightly glancing; sterile margin distinct, curry-yellow, up to 0.5 mm wide; pores circular to angular, 6–8 per mm; dissepiments thin, entire. Context up to 2 mm thick, duplex, with a black zone, lower context olivaceous buff, hard corky, up to 1 mm thick, upper tomentum cinnamon-buff, soft, up to 1 mm thick. Tubes honey-yellow, corky, up to 3 mm long.

Hyphal system dimitic; generative hyphae simple septate; tissue darkening but otherwise unchanged in KOH. Context: in the lower context, generative hyphae hyaline to pale yellowish, slightly thick- to thick-walled with a wide lumen, frequently branched and septate, 2–4 μm in diam; skeletal hyphae golden yellow, thick-walled with a narrow lumen, unbranched, aseptate, interwoven, 2–4.5 μm in diam; in the upper tomentum, generative hyphae infrequent, pale yellowish, slightly thick- to thick-walled with a wide lumen, rarely branched, frequently septate, 2–4 μm in diam; skeletal hyphae golden yellow, thick-walled with a narrow to wide lumen, unbranched, aseptate, loosely interwoven, 2.5–5 μm in diam; in the black zone, hyphae dark brown, thick-walled with a narrow lumen, strongly agglutinated, interwoven. Tubes: generative hyphae hyaline to pale yellowish, thin- to PageBreakslightly thick-walled, occasionally branched, frequently septate, 1.8–4 μm in diam; skeletal hyphae golden yellow, thick-walled with a narrow lumen, unbranched, aseptate, interwoven, 2–4 μm in diam. Setae absent. Cystidia and cystidioles absent. Basidia clavate, with four sterigmata up to 3 μm long and a simple septum at the base, 7–11 × 4–6 μm; basidioles in shape similar to basidia, but slightly smaller. Basidiospores broadly ellipsoid, pale yellowish, thick-walled, smooth, indextrinoid, inamyloid, acyanophilous, (3–)3.2–4 × (2.1–)2.3–3.1(–3.3) μm, L = 3.61 μm, W = 2.77 μm, Q = 1.28–1.33 (n = 90/3).

Additional specimens (paratypes) studied.

(All on living vine of )—JAPAN. Nara, Research Forest of Faculty of Agriculture, Kindai University, 31 Oct 2016, LWZ 20161031-1 (IFP 019173); 27 Feb 2017, LWZ 20170227-1 (IFP 019174); 25 Mar 2017, LWZ 20170325-1 (IFP 019175); 22 Jun 2017, LWZ 20170622-1 (IFP 019176); 3 Jul 2017, LWZ 20170703-1 (IFP 019177).

Discussion

is morphologically distinct from other species in by its annual, sessile basidiocarps that occur in clusters, pileal surface of narrow, concentrically sulcate zones, 6–8 pores per mm, duplex context separated by a black zone, dimitic hyphal system and broadly ellipsoid basidiospores, 3.2–4 × 2.3–3.1 µm. In the field, it is readily identified by fruiting on living vines, >1.5 cm diameter, of . is most similar to Yuan Y. Chen & B.K. Cui and L.W. Zhou by sharing annual, sessile basidiocarps in clusters and a dimitic hyphal system, but easily distinguished from by larger pores (8–9 per mm) and subglobose basidiospores (Chen et al. 2017) and from by larger pores and basidiospores and the specific host (Zhou 2016). An updated key, based on Zhou (2016), to all accepted 48 species of is provided below.

is a well-known and important medicinal plant (Li 1578). Therefore, the potential medicinal applications of fungi growing on this plant are intriguing. Li et al. (2010) studied the medicinal metabolites from basidiocarps of (Schumach.) Ryvarden that were collected on in China. Recent phylogenetic evidence, however, indicates that Chinese specimens of collected on hosts other than are distinct from a specimen collected on in Germany (Zhou and Dai 2012). As was originally described from Denmark (Larsen and Cobb-Poulle 1990), specimens used by Li et al. (2010) in their study are likely or another undescribed species.

Some species of are parasitic and appear to be restricted by host and geographic distribution of its host. For example, L.W. Zhou & Y.C. Dai, which occurs exclusively on living and L.W. Zhou & Y.C. Dai, which colonises living , are widely distributed in China (Zhou and Dai 2012, unpublished data). Similarly, in central African rainforests, Decock & Yombiy is found on living trunks of and whereas Decock & Yombiy occurs only on (Decock et al. 2015). In contrast, has a worldwide distribution and so far is host to a single species of . It will be interesting to determine if is found elsewhere on or if different species of are found on living in other geographic regions.

Since 2010, 21 new species of have been described from China (Cui et al. 2010, Zhou and Dai 2012, Zhou 2013, 2015a, 2015b, 2016, Liu et al. 2015, Chen et al. 2017PageBreak, Ren and Wu 2017). Yet in Japan, only four species, including , are known. It is hoped that this paper will draw attention to this genus in Japan and lead to the discovery of additional species.

1	Basidiocarps resupinate	P. parasitica
–	Basidiocarps sessile or stipitate	2
2	Basidiocarps stipitate and terrestrial (woody debris)	3
–	Basidiocarps sessile and on aerial wood	9
3	Context homogeneous	P. minutispora
–	Context duplex	4
4	Basidiospores > 4 µm long, > 3 µm wide	P. verae-crucis (Berk. ex Sacc.) Ryvarden
–	Basidiospores < 4 µm long, < 3 µm wide	5
5	Cystidia present	6
–	Cystidia absent	7
6	Hyphae in tomentum short and anticlinal	P. elegans Ferreira-Lopes, Robledo & Drechsler-Santos
–	Hyphae in tomentum loosely interwoven	P. nodostipitata Ferreira-Lopes & Drechsler-Santos
7	Pores < 10 per mm	P. spathulata
–	Pores > 10 per mm	8
8	Basidiospores < 3.3 µm long, < 2.3 µm wide	P. terrestris
–	Basidiospores > 3.3 µm long, > 2.3 µm wide	P. afrospathulata Yombiy & Decock
9	Hyphal system dimitic	10
–	Hyphal system monomitic	18
10	Basidiocarps perennial	11
–	Basidiocarps annual	12
11	Pores 6–8 per mm	P. manglietiae Yuan Y. Chen & B.K. Cui
–	Pores 8–11 per mm	P. pectinata (Klotzsch) Ryvarden
12	Basidiocarps solitary	P. nouraguensis Decock & G. Castillo
–	Basidiocarps in cluster	13
13	Pileal surface lighter (greyish-orange to pale cinnamon)	P. fulva Yombiy & Decock
–	Pileal surface darker (yellowish-brown to dark brown)	14
14	Pileus attached by a small vertex and pendant	15
–	Pileus widely attached to the substratum	16
15	Pores 7–9 per mm; basidiospores > 3.5 µm long	P. pendula Yuan Y. Chen & B.K. Cui
–	Pores 11–12 per mm; basidiospores < 3.5 µm long	P. pulla
16	Pores 12–15 per mm; basidiospores < 3 µm long, < 2.5 µm wide	P. minutipora
–	Pores 6–9 per mm; basidiospores > 3 µm long, > 2.5 µm wide	17
17	Pores 6–9 per mm; basidiospores broadly ellipsoid (Q = 1.28–1.33)	P. lonicerae
–	Pores 8–9 per mm; basidiospores subglobose (Q = 1.21–1.23)	P. pseudopectinata Yuan Y. Chen & B.K. Cui
18	Pores 2–4 per mm	19
–	Pores 4–12 per mm	22
19	Basidiospores broadly ellipsoid to subglobose	P. fruticum (Berk. & M.A. Curtis) Ryvarden
–	Basidiospores oblong-ellipsoid, subcylindrical to cylindrical	20
20	Context duplex	P. rzedowskii R. Valenz. & Decock
–	Context homogeneous	21
21	Context < 1 mm thick; on living branch	P. oblongospora Y.C. Dai & H.S. Yuan
–	Context 2–4 mm thick; on living trunk	P. inonotoides Yombiy & Decock
22	Basidiocarps annual to perennial, dense and hard consistency	23
–	Basidiocarps annual, soft corky at least at tomentum layer	29
23	Pores 10–12 per mm; on living Tilia	P. tiliae L.W. Zhou
–	Pores 6–9 per mm; on other angiosperms	24
24	Pileal surface zonate and sulcate	25
–	Pileal surface azonate	P. yuchengii Gafforov, Tomšovský, Langer & L.W. Zhou
25	Pores 6–7 per mm	26
–	Pores 7–9 per mm	27
26	Basidiospores ellipsoid; mostly on Ribes	P. ribis
–	Basidiospores subglobose; mostly on Ephedra, Cotoneaster or Jasminum	P. ephedrae (Woron.) Parmasto
27	Basidiospores > 2.7 µm wide	P. dependens Y.C. Dai
–	Basidiospores < 2.7 µm wide	28
28	Basidiospores ellipsoid to oblong-ellipsoid with a guttule; on Abelia	P. gutta L.W. Zhou & Y.C. Dai
–	Basidiospores broadly ellipsoid without a guttule; on living Crataegus	P. crataegi
29	Basidiospores broadly ellipsoid to subglobose	30
–	Basidiospores ellipsoid, oblong-ellipsoid to cylindrical	40
30	Pores 5–6 per mm	31
–	Pores 6–11 per mm	35
31	Context duplex, separated by a black zone	32
–	Context not separated by a black zone	33
32	Pileal surface azonate, lower context 1–4 µm thick	P. ampelina (Bondartsev & Singer) Bondartseva
–	Pileal surface zonate and sulcate, lower context 1 µm thick	P. weberiana (Bres. & Henn. ex Sacc.) Ryvarden
33	Basidiocarps solitary covered by a thick tomentum layer, pileal surface not radially faintly wrinkled	P. littoralis Decock & Yombiy
–	Basidiocarps in cluster without a distinct tomentum layer, pileal surface radially faintly wrinkled	34
34	Pileus < 1.5 mm thick, margin regular	P. flabelliformis
–	Pileus > 1.5 mm thick, margin irregular	P. gabonensis
35	Basidiocarps > 8 cm wide, > 15 mm thick; contextual hyphae > 5 µm in diam	P. ulloai R. Valenz., Raymundo, Cifuentes & Decock
–	Basidiocarps < 8 cm wide, < 15 mm thick; contextual hyphae < 5 µm in diam	36
36	Contextual hyphae regularly arranged	37
–	Contextual hyphae interwoven	38
37	Pileus distinctly sulcate, not radially striate, margin obtuse, basal context separated by two black zones; hyphae in tomentum > 4 μm in diam; on living angiosperm trunk	P. clausenae L.W. Zhou
–	Pileus faintly sulcate, radially striate, margin sharp, context duplex thoroughly; hyphae in tomentum < 4 μm in diam; on living liana	P. radiata L.W. Zhou
38	Contextual hyphae slightly thick-walled with a wide lumen, frequently septate, large rhomboid crystals absent	39
–	Contextual hyphae thick-walled with a narrow lumen, occasionally septate, large rhomboid crystals present in trama and context	P. chrysites
39	Pores 10–12 per mm; basidiospores < 3 µm long; on living Fontanesia	P. fontanesiae
–	Pores 7–9 per mm; basidiospores > 3 µm long; on other angiosperms	P. oreophila L.W. Zhou & Y.C. Dai
40	Basidiospores mostly > 3 µm wide	41
–	Basidiospores mostly < 3 µm wide	42
41	Pores 4–6 per mm	P. hainaniana Y.C. Dai & B.K. Cui
–	Pores 8–10 per mm	P. capucina (Mont.) Ryvarden
42	Basidiocarp solitary	43
–	Basidiocarp imbricate	46
43	Context homogeneous	P. homocarnica L.W. Zhou
–	Context duplex	44
44	Context not separated by a black zone; on living Flacourtia	P. flacourtiae L.W. Zhou
–	Context separated by a black zone; on other angiosperms	45
45	Pileal surface azonate, pores 6–8 per mm; basidiospores cylindrical	P. cylindrispora L.W. Zhou
–	Pileal surface zonate and sulcate, pores 8–9 per mm; basidiospores ellipsoid	P. lespedezae G.J. Ren & F. Wu
46	Basidiospores mostly < 2.5 µm wide	47
–	Basidiospores mostly > 2.5 µm wide	P. bibulosa
47	Pores 5–6 per mm, context duplex, not separated by a black zone; basidiospores > 3.5 µm long, contextual hyphae interwoven; on living Nandina	P. nandinae L.W. Zhou & Y.C. Dai
–	Pores 7–9 per mm, context duplex, separated by a black zone; basidiospores < 3.5 µm long, contextual hyphae regularly arranged; on living Osmanthus	P. osmanthi L.W. Zhou