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

Abstract 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
Phylloporia Murrill, in the Hymenochaetaceae Donk, was introduced for an unusual polypore species, P. parasitica Murrill growing on the underside of living leaves in Columbia (Murrill 1904). For nearly 70 years, Phylloporia was forgotten until Ryvarden (1972) transferred five taxa into the genus. Renewed interest in Phylloporia 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, 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.
Phylloporia 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 Coltricia cf. stuckertiana (Speg.) Rajchenb. & J.E. Wright in the Phylloporia 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 Phylloporia 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 Phylloporia reported from Japan -P. bibulosa (Lloyd) Ryvarden, P. chrysites (Berk.) Ryvarden and P. spathulata (Hook.) Ryvarden (Núñez and Ryvarden 2000). In this paper, a new species, Phylloporia lonicerae, is described from Nara, Japan, growing on living vines of Lonicera japonica. Morphological and molecular data support the recognition of this new species. In addition, an updated key to the known species of Phylloporia is presented.

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 from 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).

Phylogenetic analysis
To explore the phylogenetic relationship of P. lonicerae, six nLSU sequences were incorporated into previous nLSU datasets of Phylloporia (Zhou 2016, Chen et al. 2017, Ren and Wu 2017, Yombiyeni and Decock 2017. Several species of Fomitiporella Murrill and Fulvifomes Murrill were included in the dataset and Inonotus hispidus (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 andMichalak 2012, Stamatakis 2006) under the auto FC option for bootstrap (BS) replicates (Pattengale et al. 2010). Mr-Bayes 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 P. lonicerae 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 Phylloporia clade (98 % in ML, 1 in BI) consists of 44 terminal lineages and the six P. lonicerae samples formed a new lineage with full statistical supports (100 % in ML, 1 in BI). The Phylloporia lonicerae lineage is sister to the clade that includes P. minutispora Ipulet & Ryvarden, P. cf Etymology. Lonicerae (Lat.): referring to Lonicera, the host tree genus. Description. 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.

Discussion
Phylloporia lonicerae is morphologically distinct from other species in Phylloporia 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 Lonicera japonica. Phylloporia lonicerae is most similar to P. pseudopectinata Yuan Y. Chen & B.K. Cui and P. minutipora L.W. Zhou by sharing annual, sessile basidiocarps in clusters and a dimitic hyphal system, but easily distinguished from P. pseudopectinata by larger pores (8-9 per mm) and subglobose basidiospores (Chen et al. 2017) and from P. minutipora by larger pores and basidiospores and the specific host (Zhou 2016). An updated key, based on Zhou (2016), to all accepted 48 species of Phylloporia is provided below.
Lonicera japonica 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 Phylloporia ribis (Schumach.) Ryvarden that were collected on Lonicera japonica in China. Recent phylogenetic evidence, however, indicates that Chinese specimens of P. ribis collected on hosts other than Ribes are distinct from a P. ribis specimen collected on Ribes in Germany (Zhou and Dai 2012). As P. ribis was originally described from Denmark (Larsen and Cobb-Poulle 1990), P. ribis specimens used by Li et al. (2010) in their study are likely P. lonicerae or another undescribed species.
Some species of Phylloporia are parasitic and appear to be restricted by host and geographic distribution of its host. For example, Phylloporia crataegi L.W. Zhou   . In contrast, Lonicera japonica has a worldwide distribution and so far is host to a single species of Phylloporia. It will be interesting to determine if P. lonicerae is found elsewhere on Lonicera japonica or if different species of Phylloporia are found on living Lonicera japonica in other geographic regions.
Since 2010, 21 new species of Phylloporia 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. 2017, Ren and Wu 2017). Yet in Japan, only four Phylloporia species, including P. lonicerae, are known. It is hoped that this paper will draw attention to this genus in Japan and lead to the discovery of additional species.
Key to worldwide species of Phylloporia Pores 6