gabonensis and related species evidenced by morphological and moView metadata,citationandsimilarpapersatcore.ac.uk lecular studies

P. Yombiyeni, C. Douanla‐Meli, M. Amalfi, C. Decock

Mycothèque de l’Université catholique de Louvain (BCCM/MUCL), Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium e-mail : [email protected], [email protected], [email protected]

INTRODUCTION

The taxonomic knowledge of Phellinus sensu lato, and more globally of the poroid in tropical area or evergreen humid equatorial forest phytogeographic regions 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.

Materials and methods RESULTS 2. Phylogenetic relationships within the “Hooked setae clade” 1. Phylogenetic relationship of 1. DNA was extracted from freshly collected mycelium grown on Petri dishes on Phellinus gabonensis MUCL52007 OA, following a protocol of Lee et al. (1988) and purified with Geneclean® III kit (Q- Phellinus within the Hymenochaetaceae Biogene), following the recommendations of the manufacturer. Phellinus gabonensis MUCL51277 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). For

Phellinus gabonensis MUCL51275 tef1-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 Phellinus sp MUCL51476 Phellinus sp MUCL51483 initial annealing temperature of 60 C following Rehner and Buckley (2005). Phellinus sp MUCL51478 Phellinus gabonensis MUCL52014 Phellinus caribaeo quercicolus MUCL46003 Successful PCR reactions resulted in a single band observed on a 0.8% agarose Phellinus caribaeo quercicolus MUCL46004 Gabon Phellinus gabonensis MUCL47562 Phellinus gabonensis MUCL52012 gel, corresponding to approximately 1200 bp. Every PCR-products were cleaned Phellinus gabonensis MUCL52023 Hoocked setae clade Phellinus gabonensis MUCL52025 using the QIAquick® PCR purification kit (250) (QUIAGEN Inc.), following the Phellinus gabonensis MUCL51275 Phellinus sp MUCL47867 Phellinus gabonensis MUCL52023 recommendations of the manufacturer. Sequencing reactions were performed using Phellinus sp MUCL45929 Phellinus ellipsoideus MUCL47820 CEQ DTCS Quick Start Kit® (Beckman Coulter), according to the manufacturer’s Phellinus ellipsoideus MUCL47822 recommendations, with the primers LROR, LR3, LR3R, LR5 for the LSU, ITS1, Phellinus sp MUCL52001 Phellinus sensu stricto Phellinus gabonensis MUCL47562 Phellinus sp MUCL52000 ITS2, ITS3 and ITS4 for the ITS Phellinus bicuspidatus AY059022 Phellinus chaquensis MUCL46742 (http://biology.duke.edu/fungi/mycolab/primers.htm), and 2212R, 1953R, 983F and Phellinus populicola AF311038 Phellinus gabonensis MUCL52025 Phellinus ignarius AF311033 2218R for the tef1-α. Sequencing reactions were performed using the primers. AF311035 Phellinus alni AF311025 Phellinus caribaeo quercicolus MUCL46003 3. Nucleotide sequences were automatically aligned with Clustal X (version 2.0.11 ), Phellinus cinereus AF311027 Phellinus tuberculosus AF311043 then manually adjusted as necessary with the text editor in PAUP* (version 4.0b10). Phellinus arctostaphyli AY059026 AF311042 Phellinus caribaeo quercicolus MUCL46004 Phylogenetic analyzes were performed separately for each gene region and Phellinus laevigatus AF311034 Caribbean/south-east USA Phellinus orienticus AY059017 concatenated using maximum parsimony (MP) as implemented in PAUP* version Phellinus spiculosus AY059055 Phellinus glaucescens sensu Hattori MUCL52270 Phellinus caribaeo quercicolus MUCL46005 4.0b10 and Bayesian inference (BI) as implemented in MrBayes v3.1.2. In MP Phellinus glaucescens sensu Hattori MUCL52272 analysis, gaps were treated as fifth base. Models of evolution for Bayesian Phellinus extensus MUCL43928 AY059018 inference were estimated using the AIC (Akaike Information Criterion) as Phellinus vaninii AY059056 Phellinus sp MUCL51476 Phellinus baumii AY058058 implemented in Modeltest 3.7 . pruinosus AY059045 Inonotus tropicalis AY059037 Phellinus sp MUCL51483 For MP analyses the most parsimonious trees (MPT) for each data set were Phellinus undulatus MUCL44139 Inonotus sensu Wagner Ecuador Phellinus glaucescens sensu Hattori MUCL52271 identified using heuristic searches with 1000 random addition sequences, further Phellinus glaucescens sensu Hattori MUCL52273 & Fischer (2002) AF311014 Phellinus sp MUC51478 evaluated by bootstrap analysis, retaining clades compatible with the 50% majority- AF311010 AF311017 rule in the bootstrap consensus tree. Analysis conditions were: tree bisection Phellinus pachyphloeus AY059020 Inonotus sp MUCL44666 Phellinus sp MUCL47867 addition branch swapping (tbr), starting tree obtained via stepwise addition, dryophilus AF311012 steepest descent not in effect, MulTrees effective. A bootstrap support value (BS) Inocutis tamaricis AF311021 Inocutis Inocutis ludovicianus AY059044 Phellinus sp MUCL45929 above 70% was considered significant. Inocutis jamaicensis AY059048 Thailand /China (Hainan) polymorpha MUCL46166 Bayesian analyzes were implemented with two independent runs, each with four Fomitiporia aethiopica MUCL44777 Fomitiporia hartigii AF3110051 Phellinus sp MUCL47866 simultaneous independent chains for twelve million generations, starting from Fomitiporia punctata MUCL34101 Fomitiporia Fomitiporia robusta AF311008 random trees, and keeping one tree every 1000th generation. All trees sampled Fomitiporia hippophaeicola MUCL31746 Phellinus uncisetus MUCL46231 Phellinus ellipsoideus MUCL47820 after convergence (ave. standard deviation of split frequencies <0.01 and confirmed Porodaedalea pini AF311037 Porodaedalea niemelaei AY059054 China (Fujian) using Tracer v1.4 ) were used to reconstruct a 50% majority-rule consensus tree triquetra AF311024 Omnia (BC) and to estimate posterior probabilities. The posterior probability (BPP) of each Pseudoinonotus dryadeus AF311011 Pseudoinonotus Phellinus ellipsoideus MUCL47822 torulosa MUCL45967 node was estimated based on the frequency at which the node was resolved Fuscoporia senex MUCL46182 Fuscoporia ferruginosa MUCL45983 Phellinus sp MUCL52001 among the sampled trees with the consensus option of 50% majority-rule BPP Fuscoporia contigua AF311029 Fuscoporia Fuscoporia ferrea MUCL45984 above 0,95 was considered a significant value. Fomes fomentarius AF

10 Phellinus sp MUCL52000

10 PRELIMINARY CONCLUSIONS

1) 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 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). brought toyouby in the AFRICAN CLADE , Phellinus gabonensis is alone, and known from the western edge of the Guineo-Congolian rainforest. provided byDIALUCLouvain 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 Africa 3) 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 and their presence does not indicate any kind of phylogenetic relationships apartCORE 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.

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).