Amalfi, Mario

Decock, Cony

Douanla Meli, C.

IMC 9

Yombiyeni, Prudence

DIAL UCLouvain

Phellinus gabonensis and related species evidenced by morphological and molecular studiesP. Yombiyeni, C. Douanla‐Meli, M. Amalfi, C. DecockMycothèque de l’Université catholique de Louvain (BCCM/MUCL), Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgiume-mail : mario.amalfi@uclouvain.be, decock@mbla.ucl.ac.be, prudence_yom@yahoo.frINTRODUCTIONThe taxonomic knowledge of Phellinus sensu lato, and more globally of the poroid Hymenochaetaceae in tropical area or evergreen humid equatorial forest phytogeographicregions is still very fragmentary. A fortiori, we know even less about the phylogenetic relationships of other species occurring in these areas, either with allopatric populations or other related allopatric or sympatric species. The poroid Hymenochaetaceae is characterized by many species complexes, for which morphology poorly discriminate taxa. During extensive fieldwork in tropical and equatorial areas of Africa, South America and  Asia, numerous collections have been made among which several collections all characterized by resupinate basidiomes, ventricose, apically curved to distinctly hamate hymenial setae, and ellipsoid, slightly thick-walled, and pale yellowish basidiospores. Morphologically, these collections could be hardly distinguished, or by some subtle characteristics, which taxonomic pertinence remain uncertain.Hooked setae are known in several Hymenochaetaceae but, above all, the combined characteristics of these specimens certainly call to mind the pattern found in Phellinus caribaeo-quercicolus (Decock et al. 2006), Phellinus setulosus (Lloyd) Imazeki (Corner 1991).The taxonomic status and phylogenetic relationships between these collections are discussed below.2. Phylogenetic relationships within the “Hooked setae clade”RESULTS1. Phylogenetic relationship of Phellinus within the Hymenochaetaceae10Phellinus sp MUCL51476Phellinus sp MUCL51483Phellinus sp MUCL51478Phellinus caribaeo quercicolus MUCL46003Phellinus caribaeo quercicolus MUCL46004Phellinus gabonensis MUCL47562Phellinus gabonensis MUCL52023Phellinus gabonensis MUCL52025Phellinus gabonensis MUCL51275Phellinus sp MUCL47867Phellinus sp MUCL45929Phellinus ellipsoideus MUCL47820Phellinus ellipsoideus MUCL47822Phellinus sp MUCL52001Phellinus sp MUCL52000Phellinus bicuspidatus AY059022Phellinus chaquensis MUCL46742Phellinus populicola AF311038Phellinus ignarius AF311033Phellinus lundellii AF311035Phellinus alni AF311025Phellinus cinereus AF311027Phellinus tuberculosus AF311043Phellinus arctostaphyli AY059026Phellinus tremulae AF311042Phellinus laevigatus AF311034Phellinus orienticus AY059017Phellinus spiculosus AY059055Phellinus glaucescens sensu Hattori MUCL52270Phellinus glaucescens sensu Hattori MUCL52272Phellinus extensus MUCL43928Phellinus linteus AY059018Phellinus vaninii AY059056Phellinus baumii AY058058Inonotus pruinosus AY059045Inonotus tropicalis AY059037Phellinus undulatus MUCL44139Phellinus glaucescens sensu Hattori MUCL52271Phellinus glaucescens sensu Hattori MUCL52273Inonotus hispidus AF311014Inonotus cuticularis AF311010Inonotus obliquus AF311017Phellinus pachyphloeus AY059020Inonotus sp MUCL44666Inocutis dryophilus AF311012Inocutis tamaricis AF311021Inocutis ludovicianus AY059044Inocutis jamaicensis AY059048Fomitiporia polymorpha MUCL46166Fomitiporia aethiopica MUCL44777Fomitiporia hartigii AF3110051Fomitiporia punctata MUCL34101Fomitiporia robusta AF311008Fomitiporia hippophaeicola MUCL31746Phellinus uncisetus MUCL46231Porodaedalea pini AF311037Porodaedalea niemelaei AY059054Onnia triquetra AF311024Pseudoinonotus dryadeus AF311011Fuscoporia torulosa MUCL45967Fuscoporia senex MUCL46182Fuscoporia ferruginosa MUCL45983Fuscoporia contigua AF311029Fuscoporia ferrea MUCL45984Fomes fomentarius AFPhellinus sensu strictoFomitiporiaInonotus sensu Wagner & Fischer  (2002)InocutisPorodaedaleaPseudoinonotusOmniaFuscoporiaHoocked setae clade10Phellinus gabonensis MUCL52007Phellinus gabonensis MUCL51277Phellinus gabonensis MUCL51275Phellinus gabonensis MUCL52014Phellinus gabonensis MUCL52012Phellinus gabonensis MUCL52023Phellinus gabonensis MUCL47562Phellinus gabonensis MUCL52025Phellinus caribaeo quercicolus MUCL46003Phellinus caribaeo quercicolus MUCL46004Phellinus caribaeo quercicolus MUCL46005Phellinus sp MUCL51476Phellinus sp MUCL51483Phellinus sp MUC51478Phellinus sp MUCL47867Phellinus sp MUCL45929Phellinus sp MUCL47866Phellinus ellipsoideus MUCL47820Phellinus ellipsoideus MUCL47822Phellinus sp MUCL52001Phellinus sp MUCL52000GabonEcuadorCaribbean/south-east USA Thailand /China (Hainan)China (Fujian) PRELIMINARY CONCLUSIONS1) Multiloci (partial LSU, ITS-5.8S, and tef 1-α) -based phylogenetic inferences confirm the close proximity of all collections characterized by resupinate basidiomata, hooked (hamate) setae, and broadly ellipsoid, pale yellowish basidiospores. 2) These collections are distributed into several, distinct clades, according to their geographic origin. The collection from Ecuador are closely related to Ph. caribaeo-quercicolus, both forming sister clades. Morphologically, they are slightly different but their main difference could be related to their ecology especially and geographic distribution. in the NEOTROPICAL CLADE, two subclades are evidenced: Ph. caribeao-quercicolus grow on living oak in Cuba and southeastern USA, while the Ecuadorian collections were found so far only on dead fallen trunk, in very humid Amazonian forest.in the ASIAN CLADE, two subclades are evidenced. The Thailandese/Chinese collections originating from the humid, tropical forest in northern Thailand/southeast china form a sister clade to collections originating from broadleaf forest of eastern china (Fujian province).in the AFRICAN CLADE , Phellinus gabonensis is alone, and known from the western edge of the Guineo-Congolian rainforest.The African subclade – known so far from the western edge of the Guineo-Congolian forest, is more closely related to the Neotropical species than to the east Asian species. Multiloci based phylogenetic inferences shows that the South American subclade shares a common ancestor with the African subclade in accordance with previous hypotheses of strong cryptogamic floristic affinities and close biogeographic relationship between South America and the western edge of Africa3) The setal and basidiospores morphology of this clade could be related to Ph. setulosus (Lloyd) Imazeki group (Corner 1991, Robledo et al 2003). However no sequence of Ph. setulosus is available at the moment thus impeding any phylogenetic (DNA based) inference of its relationships with these other taxa.4) Hooked to hamate hymenial setae are widespread over the poroid Hymenochaetales and their presence does not indicate any kind of phylogenetic relationships apart from those with closely related species in the species complex. Hooked to hamate hymenial setae are found for instance in Inonotus sensu Wagner and Fischer, Inonotus P. Karst. s.s. (Ryvarden and Gilbertson 1994), Mensularia Laz. (Gilbertson and Ryvarden 1987), Phellinus Quél. s.s., Fuscoporia Murrill and, in all probability, Fomitiporia Murrill. This morphological feature has arisen independently on several occasions.Materials and methods1. DNA was extracted from freshly collected mycelium grown on Petri dishes on OA, following a protocol of Lee et al. (1988) and purified with Geneclean® III kit (Q-Biogene), following the recommendations of the  manufacturer. 2. DNA extraction, amplification and sequencing of the nuclear ribosomal 5ｴ end of the LSU and ITS regions (including 5.8S) are described in Decock et al (2007). Fortef1-α 1200 bp fragment located between exons 4 and 8 was amplified using the primer pair 983F and 2218R. In this case, a touchdown PCR was used with an initial annealing temperature of 60 C following Rehner and Buckley (2005). Successful PCR reactions resulted in a single band observed on a 0.8% agarosegel, corresponding to approximately 1200 bp. Every PCR-products were cleaned using the QIAquick® PCR purification kit (250) (QUIAGEN Inc.), following the recommendations of the manufacturer. Sequencing reactions were performed using CEQ DTCS Quick Start Kit® (Beckman Coulter), according to the manufacturer’s recommendations, with the primers LROR, LR3, LR3R, LR5 for the LSU, ITS1, ITS2, ITS3 and ITS4 for the ITS (http://biology.duke.edu/fungi/mycolab/primers.htm), and 2212R, 1953R, 983F and 2218R for the tef1-α. Sequencing reactions were performed using the primers.3. Nucleotide sequences were automatically aligned with Clustal X (version 2.0.11 ), then manually adjusted as necessary with the text editor in PAUP* (version 4.0b10). Phylogenetic analyzes were performed separately for each gene region and concatenated using maximum parsimony (MP) as implemented in PAUP* version 4.0b10 and Bayesian inference (BI) as implemented in MrBayes v3.1.2. In MP analysis, gaps were treated as fifth base. Models of evolution for Bayesian inference were estimated using the AIC (Akaike Information Criterion) as implemented in Modeltest 3.7 . For MP analyses the most parsimonious trees (MPT) for each data set were identified using heuristic searches with 1000 random addition sequences, further evaluated by bootstrap analysis, retaining clades compatible with the 50% majority-rule in the bootstrap consensus tree. Analysis conditions were: tree bisection addition branch swapping (tbr), starting tree obtained via stepwise addition, steepest descent not in effect, MulTrees effective. A bootstrap support value (BS) above 70% was considered significant.Bayesian analyzes were implemented with two independent runs, each with four simultaneous independent chains for twelve million generations, starting from random trees, and keeping one tree every 1000th generation. All trees sampled after convergence (ave. standard deviation of split frequencies <0.01 and confirmed using Tracer v1.4 ) were used to reconstruct a 50% majority-rule consensus tree (BC) and to estimate posterior probabilities. The posterior probability (BPP) of each node was estimated based on the frequency at which the node was resolved among the sampled trees with the consensus option of 50% majority-rule  BPP above 0,95 was considered a significant value.Acknowledgments. Prudence Yombiyeni gratefully acknowledges the financial support received from the OIBT short-term grant program (OIBT: 036/08A) and from the ACP-FORENET project funded by the EU (project 9ACP RPR91#1). Mario Amalfi gratefully acknowledges the financial support received from the Belgian State – Belgian Federal Science Policy through an Interuniversity Attraction Poles Program (phase VI, contract P6/06) and the logistic support received from MUCL. Cony Decock gratefully acknowledges the financial support received from the Belgian State – Belgian Federal Science Policy (contract BCCM C3/10/003) and the Fonds de la Recherche Fondamentale Collective (FRFC, contract # 2.4515.06). 

Phellinus gabonensis and related species evidenced by morphological and molecular studies.

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Phellinus gabonensis and related species evidenced by morphological and molecular studies.

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