Porodaedalea cedrina (Basidiomycota ...

Nova Hedwigia 2013 Vol. 96 issue 3–4, 419–426 published online January 22, 2013

Article

Porodaedalea cedrina (Basidiomycota, Agaricomycetes, Hymenochaetaceae) – a new polypore from the Mediterranean area Michal Tomšovský1*and Jiří Kout2 1 Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, CZ-61300, Czech Republic 2 Department of Biology, Faculty of Education, University of West Bohemia, Klatovská 51, Pilsen, CZ-30619, Czech Republic

With 2 figures and 1 table

Abstract: Porodaedalea cedrina is described as a new species. The fungus occurs on Cedrus atlantica and C. libani in North Africa (Algeria, Morocco) and Western Asia (Turkey, Lebanon). The DNA sequences of ITS region of ribosomal RNA genes and translation elongation factor 1-alpha confirmed its separation from other Porodaedalea species in Eurasia. The new species seems to be distributed in localities of Cedrus spp. native distribution in Mediterranean area. Key words: taxonomy, Basidiomycota, Hymenochaetales, Phellinus.

Introduction The genus Porodaedalea Murrill (Basidiomycota, Agaricomycetes, Hymenochaetaceae) is a taxonomically difficult complex of morphologically similar species that inhabit conifers (Picea, Pinus, and Larix). In Europe, three species of genus are currently recognized − P. chrysoloma (Fr.) Fiasson & Niemelä, P. laricis (Jacz. ex Pilát) Niemelä, and P. pini (Brot.) Murrill (Tomšovský et al. 2010). Two more species, P. himalayensis (Y.C.Dai) Y.C.Dai and P. yamanoi (Imazeki) Y.C.Dai, are reported from Asia (Dai 2010). However, the genus can include more undescribed species adapted to less widespread coniferous taxa. The specimens of Porodaedalea (= Phellinus pini group) have been repeatedly reported from Cedrus atlantica (Endl.) Manetti ex Carrière (Atlas Cedar) in North *Corresponding author: [email protected] © 2013 J. Cramer in Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, Germany. DOI: 10.1127/0029-5035/2013/0087

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www.borntraeger-cramer.de 0029-5035/2013/0087 $ 2.00

Africa – Algeria and Morocco (Pilát 1942, Malençon 1955, Telleria 1984). The fungus from Cedar has been identified as Xanthochrous pini var. abietis (Malençon 1955), Phellinus pini (Telleria 1984) or as Phellinus vorax (Černý 1985) – a synonym of Porodaedalea laricis. Tomšovský at al. (2010) incorrectly named a specimen from Morocco (PRM 876397) as "Phellinus chrysoloma var. abietis". In fact, the specimen is labeled "Phellinus chrysoloma = P. abietis". Pilát (1942) intended to create a new form – Phellinus pini var. typica forma cedrina. Nevertheless, the description was not valid due to lack of a Latin diagnosis (for fungi this had been obliged from 1 January 1935 till 31 December 2011; Knapp et al. 2011). The so far molecular studies (Fischer 1996, Tomšovský et al. 2010) resulted in isolated position of specimens from Cedrus atlantica which deserves a detailed molecular taxonomic study. The aim of our work is to assure the taxonomic position of these specimens due to examination of the DNA sequences and morpho-anatomical characters of additional collections from Cedrus spp. from the Mediterranean area. Materials and methods In total, two historical specimens (holotype of Phellinus pini var. cedrina – Pilát 1942 and specimen PRM 876397 sequenced by Tomšovský et al. 2010) and four more recent (not older than 10 years) collections were examined. Macroscopic description of specimens is based on dry basidiomes. Colour abbreviations follow Kornerup & Wanscher (1983), herbarium abbreviations are according to Holmgren & Holmgren (1998). Mycological herbarium of Department of Biology, University of West Bohemia is abbreviated as KBI (not included in Holmgren & Holmgren 1998). Abbreviation JV refers to the Polypores Collection of Dr. Josef Vlasák (Vlasák 2012). Microscopic features are described from dried material mounted in Melzer’s reagent (1.5 g KI, 0.5 g iodine, 20 g chloral hydrate dissolved in distilled H2O) and Congo Red (dissolved in 10% ammonia solution) using an Olympus BX-51 light microscope with a magnification of 1000×. Basidiospores were measured from sections cut from the tubes, in presenting spore size data, 5% of the measurements excluded from extremes in the range are shown in parentheses. The factor Q (quotient of length and width in any one spore), average values of lengths (L) and widths (W) are determined. The pore widths were examined under an Olympus SZX12 stereomicroscope. The specimens selected for DNA extraction and PCR are listed in Table 1. The two loci: ITS region of ribosomal RNA gene (ITS) and partial sequence of translation elongation factor 1-alpha gene (tefa) – were selected for the analysis. The DNA extraction and PCR of ITS was applied according to Tomšovský et al. (2010) and Vasaitis et al. (2009). For the amplification of tefa, the primer pair 983F/2218R was used (Rehner et al. 2005). PCR for the locus was performed using a following touchdown PCR procedure: The amplifications were initiated with a 2 min denaturation at 94°C. The annealing temperature in the first amplification cycle was 60°C, which was subsequently stepwise reduced by 1°C per cycle over the next 9 cycles. An additional 35 amplification cycles were then performed, each consisting of 30 s denaturation at 94°C, a 30 s annealing step at 50°C, and a 1 min extension at 72°C, concluding with a 10 min incubation at 72°C.

Phylogenetic analysis The corresponding sequences of each individual locus were aligned using the MUSCLE algorithm and adjusted manually in BioEdit (Hall 1999). The respective sequences of Onnia leporina were selected as an outgroup. 420 eschweizerbart_xxx

Table 1. Specimens selected for DNA sequencing.

Number PH44 PH114 JV 879*)

Species

Locality

Substrate

Porodaedalea sp., Lebanon, Al Shouf Cedrus libani Cedrus Turkey, FethiyeP. sp., Cedrus Cedrus libani Babadağ Mt. Morocco, Middle Atlas, Cèdre Cedrus P. sp., Cedrus Gouraud Forest by atlantica Azrou Czech Rep., P. chrysoloma Picea abies Moravian Karst

GenBank Herbarium/culture Accession number no. A BRNM 737547 MCF 03/1167 KTI, JK 0908/043 (duplicate JV 0408/42) BRNM 712788

U9*)

P. chrysoloma

Sweden, Uppsala

Picea abies

BRNM 712790

Cui 9320

P. himalayensis

China, Tibet

Picea sp.

Cui 9320 (BJFC)

995*)

P. laricis

France, Pelvoux

Larix decidua

PRM 892094

E7*)

P. laricis

Russia, Primorsk Picea ajanensis territory

TFC 1981-38

PH10*)

P. laricis

Finland, Ilomantsi

H6002109, Niemelä 7399

U1*) TM3 861*) 884*) E3*) Dai 8202B 1107*)

P. pini

Picea abies

Sweden, Uppsala Pinus sylvestris

BRNM 712791

Turkey, Isparta Pinus nigra BRNM 737548 province spp. pallasiana Croatia, Isle of Pinus P. pini BRNM 712792 Korčula halepensis Czech Rep., Podyjí P. pini Pinus sylvestris BRNM 712785 National park Russia, Sakhalin P. yamanoi TFC 1971-24 Picea sp. Island China, Jilin P. yamanoi Dai 8202 (BJFC) Picea sp. province Czech Rep., Jihlava Onnia leporina BRNM 712782 Picea abies town P. pini

JQ772466 JQ772472 JQ772467 JQ772473 JQ772468 JQ772474 FJ775547 FJ775577 FJ775543 FJ775576 JQ772471 JQ772477 FJ775567 FJ775587 FJ775559 FJ775585 FJ775571 FJ775590 FJ775556 FJ775591 JQ772470 JQ772476 FJ775554 FJ775599 FJ775555 FJ775597 FJ775551 FJ775592 JQ772469 JQ772475 FJ775542 FJ775573

*) Sequences published by Tomšovský et al. (2010). A

The first number refers to the ITS sequence, the second one to tefa sequence.

B

The specimen cited by Dai (2010).

To determine whether the datasets of different genetic markers (ITS and tefa) were in significant conflict, two methods were applied. The partition homogeneity test in 421 eschweizerbart_xxx

PAUP* 4.0b10 (Swofford 2003) was used between the markers using 100 replicates and the heuristic general search option. The null hypothesis of congruence was rejected if p < 0.01. A test based on maximum agreement subtrees (de Vienne et al. 2007) was further performed. Phylogenies were generated in MrBayes version 3.2.1 (Ronquist & Huelsenbeck 2003). The GTR model with gamma-distributed rate variation across sites was chosen as a substitution model using the MrModeltest 2.3 (Nylander 2008). Markov chains were initiated from a random tree and were run for 5000000 generations; samples were taken every 100th generation. The number of excluded generations determined as burn-in was used by Tracer 1.5. The Bayesian branch supports were assigned as posterior probabilities (PP) on the consensus trees. In addition, bootstrap branch support values (BS) were estimated in PAUP 4.0b10 using 1000 replicate datasets with the random addition of sequences during each heuristic search. Results Partition homogeneity tests showed no significant conflict between the two genetic markers used (p = 0.23). A test based on maximum agreement subtrees also confirmed the congruence of ITS and tefa trees (Icong = 1.549, p-value = 0.0008). These results allowed us to perform a combined analysis of the generated ITS and tefa sequence data. The complete alignment was 1829 bp long, composed of 1539 constant and 290 variable characters (65 of them were parsimony informative). The Bayesian analysis yielded trees with the likelihood values and the model parameters: ln = −4089.715, alpha: 0.294, tree length: 0.306. Empirical base frequencies were: pi(A):0.218; pi(C): 0.244; pi(G): 0.252; pi(T): 0.286. The specimens from Cedrus form a well supported clade separated from all co-analyzed species (Fig. 1) which supports its independent taxonomic position. The ITS sequences of "Cedrus clade" are almost identical (99%) to previously published ITS sequence no. FJ775550 by Tomšovský et al. (2010). According to the molecular results we decided to describe a new species – Porodaedalea cedrina. Taxonomy Porodaedalea cedrina Pilát ex Tomšovský & Kout, sp. nov. Protologue: Porodaedalea cedrina is morphologically similar to P. pini, but differs in the fixed nucleotide characters in internal transcribed spacers 1 and 2 of ribosomal RNA gene and translation elongation factor 1-alpha. The fungus occurs on Cedrus spp. in localities of host native distribution in North Africa and Western Asia (Morocco, Algeria, Turkey, and Lebanon). The combination of molecular end ecological features is essential for delimitation of the new species. 422 eschweizerbart_xxx

Fig. 1. The phylogram resulted from the Bayesian analysis. Numbers at branches indicate Bayesian posterior probabilities and bootstrap values higher than 50%.

Mycobank no. MB564700 Etymology: cedrina − refers to Cedrus, probably the exclusive host.

Description: Basidiocarps perennial, pileate, solitary to imbricate, hard corky when fresh, heavy and woody hard when dry. Without odour or taste. Pilei usually dimidiate to ungulate, projecting up to 3 cm, 8 cm wide and 8 cm thick at base. Pileal surface greyish brown 6F1-3, 7F3 to anthracite H1, concentrically sulcate with narrow zones, irregularly cracked, becoming encrusted with age, the encrusted zone dominant in old basidiocarps; margin obtuse or acute. Pore surface amber yellow 4B6, yellow ochre or brownish yellow 5C7-8, tan to rust-brown 6E5-8, slightly glancing; sterile margin narrow to almost lacking; pores circular when young to more or less daedaleoid, 1–2(–3) per mm; dissepiments thin, entire. Context tan brown 6E6, woody hard, up to 10 mm thick. Tubes varying from yellow ochre 5C6 of young stage to brown 6E5 of old parts, woody corky, up to 2 cm long, annual layers indistinct, old tubes merging into context. Hyphal system dimitic; generative hyphae simple septate; tissue darkening but otherwise unchanged in KOH. Generative hyphae infrequent in tubes and context, colourless, thin-walled, occasionally branched, frequently simple septate, 2.4–3 μm diam; skeletal hyphae dominant, yellow ochre or brownish yellow 5C7-8, thick-walled with a narrow to wide lumen, rarely branched, occasionally septate (more often in context), loosely interwoven to regularly arranged in dissepiment wall, (2.7–)3–4.5 μm diam. Setae frequent in hymenium, originating from both subhymenial and tramal hyphae, not seen at dissepiment edges, not or rarely embedded in trama, subulate, rusty 423 eschweizerbart_xxx

Fig. 2. Microscopic characters of Porodaedalea cedrina: A. hymenial setae, B. spores, C. basidia, D. tramal skeletal hyphae, E. contextual hyphae. The bar refers to all characters in the figure.

brown 6E7-8, 7E7-8, thick-walled, 30–62 × 6–18 μm; cystidioles infrequent, clavate, colourless, thin-walled; basidia distinctly clavate, with four sterigmata and a simple septum at the base, 14–21 × 5–6.5 μm. Basidiospores broadly ellipsoid, colourless, thin- to slightly thick-walled, smooth, without guttules, moderately cyanophilous, negative in Melzer’s reagent, (4.9–)5.0– 6.0(–6.3) × (3.8–)4.0–5.1(–5.5) μm, L = 5.49 μm, W = 4.60 μm, Q = 1.04–1.38, average Q = 1.20 (n = 104 spores/5 specimens). The fungus causes white pocket rot of Cedrus spp. Holotype: Morocco, Middle Atlas Mts., near the road ca. 8–10 km north from Ifrane, south from Fes, at base of living trunk of Cedrus atlantica. Leg. František Kotlaba, 17.IV.1992, PRM 876397. The DNA sequence of the ITS region of ribosomal RNA gene is deposited in the GenBank (accession no. FJ775550). Other specimens examined: Morocco, Middle Atlas, Cèdre Gouraud Forest by Azrou, Cedrus atlantica, leg. J.Kout, 9.VIII.2004, two specimens in KBI: JK 0908/04-3 (duplicate JV 0408/42) and JK 0908/04-4. − Algeria, Aurès Mts., Chelia, Cedrus atlantica, leg. A.Pfeffer, VI.1932, PRM 889032 (older number PRM 628400). − Turkey, Muğla province, Fethiye-Babadağ Mt., 1300 m a.s.l., Cedrus libani, leg. H.H.Doğan, 3.XI.2003, MCF 03/1167. − Lebanon, Al Shouf Cedar Nature Reserve, Al Barouk Cedar Forest, 33°42'3.2"N, 35°42'9.5"E, ca. 1500 m a.s.l., Cedrus libani, leg. O.Koukol, 31.III.2009, BRNM 737547.

Discussion The results confirmed an independent position of P. cedrina, though the species is hardly distinguishable from related species according to morpho-anatomical characters of basidiomes. Malençon (1955) argued his specimens from Cedrus did not follow 424 eschweizerbart_xxx

description of Phellinus pini f. cedrina (Pilát 1942) due to lack of triquetrous shape of basidiome. According to our observation, the basidiome shape in Porodaedalea is rather variable according to its age or placement on tree and this is unsuitable for delimitation of species. The shape and dimensions of basidiospores and setae are rather uniform within the genus (compare data published by Tomšovský et al. 2010) – only P. chrysoloma has somewhat smaller spores. The pore density of P. cedrina is similar to P. pini but the molecular data do not confirm close relation of the two species. Except DNA sequences, the affinity to host genus seems to be essential for identification of P. cedrina. Nevertheless, the native occurrence of Cedrus may be crucial for colonisation of hosts by the fungus. Similarly, larches are inhabited by P. pini instead of P. laricis in non-native plantations in lower altitudes in the Czech Republic (Tomšovský et al. 2010). Therefore, other Porodaedalea spp. may occur on planted trees of Cedrus in its non-native localities. The distribution of P. cedrina and its particular ecological preferences are poorly known. Our results revealed the ITS sequences of P. cedrina from Morocco differ in two nucleotide positions from Asian specimens. This may be a proof of genetic differentiation of geographically separated disjunct populations growing on different host species (Cedrus atlantica and C. libani A.Rich.). The genetic diversity of Cedrus spp. analysed by Dagher-Kharrat et al. (2007) revealed significant differences between geographically separated Cedrus atlantica (North Africa) and C. libani-C. brevifolia group (Eastern Mediterranean). However, the occurrence of the fungus on other two Cedrus species − C. brevifolia (Hook.f.) A.Henry (Cyprus) and C. deodara (Roxb. ex D.Don) G.Don (Afghanistan, Pakistan, and India) has not been confirmed yet and deserves a further study. Due to fragmentation of natural populations of Cedrus spp., P. cedrina populations may suffer from loss of genetic diversity. Although the fungus is reported as a pathogen of old trees (Malençon 1955), it may be locally endangered and deserves conservation. Overall, Porodaedalea is probably richer in species than it has been expected. Other undescribed species can occur in East Asia (Dai 2010) and North America (Fischer 1996) with affinities to diverse coniferous hosts. Acknowledgements The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, project no. MSM 6215648902. We thank curators of MCF and PRM fungal collections for loans of specimens.

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Manuscript received March 16, 2012; accepted May 31, 2012.
