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AN INTEGRATED TAXONOMIC APPROACH TO SURVEY IN IRAN 1 Saeideh Jafarpour1, Khalil-B erdi F otouhifar1, M ohanimad Javan-Nikhhah , M ohhamad Reza Ase/, 3 4 4 5 Anthony S Davis , Amy L Ross-Davis , John W Hanna , Simona Margaritescu , Jean-Marc 6 4 Moncalvo , and Ned B Klopfenstein

INTRODUCTION Armillaria distribution is needed for disease Iran's most valuable forests are located on the coast management and comparisons with other regions. of the Caspian Sea and cover 1.85 million ha in the In recent years, the utility of DNA sequence-based northern region of the Alborz mountain range, identification has been demonstrated for Armillaria which is the highest mountain range in the Middle spp, and translation elongation factor 1a (tef-1 a) East. Dense forests cover two major provinces, gene sequences have been especially useful for Gilan and Mazandaran; however, less than 10% of phylogenetic analysis to differentiate closely Iran is forested. These forests comprise temperate, related Armillaria spp (e.g., Maphosa et al. 2006; deciduous, broad-leaved tree species. are Hasegawa et al. 2010; Ross-Davis et al. 2012). usually absent in Iranian forests, with only a few Previously, no DNA sequence data were available relics of coniferous species remaining. for validating Armillaria spp in Iran. The objective of this study is to identify Armillaria spp from Iran Armillaria root disease can cause significant using integrated taxonomic methods based on damage in Iranian forests and it is widely morphology, interfertility, and DNA distributed throughout these forests. Armillaria sequences. mellea is a well-known cause of root disease of Caucasian or Persian oak (Quercus macranthera) MATERIALS AND METHODS within the Hatam Baigh forest in northwestern Iran (Davari et al. 2005). Based on the biological We are basing species identification of Armillaria species concept (Korhonen 1978), a previous study in Iran on basidiocarp morphology, interfertility of Armillaria spp in Iran showed the existence of (biological species), and phylogenetic analyses of four intersterility groups, which represented A. DNA sequences from the tef-la gene. Fresh and mellea, A . cepistipes, A. gallica and A. borealis dried were used to determine classical (Asef et al. 2003) . However, the distribution of morphological characteristics DNA sequences Armillaria spp m Iran remams largely (e.g., tef-la) from a set of European biological undocumented a better understanding of tester strain cultures of annulate Armillaria spp, obtained from Kari Korhonen (Finland) and Nenad In: Ramsey , A. & P. Palacios (Comps). Proceedings of the 63rd Annual Western International Forest Disease Work Keca (Serbia), were also included in the analyses. Conference; 2015 Sept. 21-15; Newport, OR. 1Department of Additional tef-la sequences of North American Plant Protection, Faculty of Agricultural Science and Armillaria spp were obtained from GenBank. Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. 2Department of Botany, Sequences were aligned using the MAFFT Research Institute of Plant Protection, Tehran, Iran . software (Katoh & Standley 2013) and a 3 Department of Forest, Rangeland, and Fire Sciences, College phylogenetic tree was constructed using of Natural Resources, University of Idaho, Moscow, Idaho. 4USDA Forest Service, Rocky Mountain Research Station, maximum-likelihood in Garli 2.2 (Bazinet et al Moscow, Idaho. 5Department of Natural History, Royal 2014). Interfertility tests from ­ 6 Ontario Museum, Toronto, Canada. Department of Natural derived cultures between Iranian collections and History, Royal Ontario Museum and Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, European biological species testers are ongoing. Canada.

125 Figure IA. Sampling Armillaria in Iranian forests.

Figure lB. Morphological study of Armillaria.

126 A. ostoyae Europe

A. borealis clade 1 Europe

A. gemina NA A. solidipes NA A. barealis clade 2 Europe

A. cepistipes Europe

A. sinapina NA

A. me/lea Iran & Europe

.: .... ____.. A. me/lea Iran '-----"'--~ ·~::._~ - I A. me/lea NA A. cepistipes NA A. nabsnona NA A. calvescens NA A. gallica NA A. altimantana NA

A. gallica clade 2 Europe

A. gallica clade 1 Europe

new Armillaria species from Iran A. tabescens NA

Figure 2. Preliminary phylogenetic tree of global Armillaria spp constructed in Garli 2.2. (NA =North America).

RESULTS AND DISCUSSION within the A. mellea complex. Although the A. mellea complex is widely distributed around the In this study, approximately 100 Armillaria northern hemisphere, phylogenetic analysis shows basidiocarps were collected from 11 different that North American A. mellea and Iranian A. forest sites in northern Iran (Figure lA and lB). mellea reside in distinct clades. Further studies are Based on morphology and preliminary needed to resolve the of the polyphyletic phylogenetic analyses, most samples are associated A. mellea and A. gallica complexes. with A. mellea and A. gallica complexes (Figure 2) . In Iran, A. mellea is associated with wide-spread A better understanding of the taxonomy and the tree mortality and windthrow, which suggests that global distribution of Armillaria spp will provide A. mellea is a primary pathogen in Iranian forests . information on the potential invasiveness and Interestingly, three A. mellea isolates generated tef- potential impacts of climate change on Armillaria 1 a sequences containing 15 different variable sites root disease. that separated these isolates into a distinct subclade

127 REFERENCES Katoh, K. and Standley, D. M. 2013. MAFFT multiple sequence alignment software version 7: Asef, M.R., Goltapeh, E.M., and Alizadeh, A. improvements in performance and usability. 2003. Identification of Armillaria biological Molecular Biology and Evolution 30(4):772-780. species in Iran. Fungal Diversity 14:51-60. Korhonen, K. 1978. Infertility and clonal size in Bazinet, A.L., Zwickl, D.J., and Cummings, M.P. the Armillariella mellea complex. Karstenia 2014. A gateway for phylogenetic analysis 18(2):31-42. powered by grid computing featuring GARLI 2.0. Systematic Biology 63(5):812-818 . Maphosa, L., Wingfield, B.D., Coetzee, M.P.A., Mwenje, E., and Wingfield, M.J. 2006. Davari, M. and Askari, B. 2004. Armillaria mellea Phylogenetic rel~tionships among Armillaria as a cause of oak decline in Hatam-baigh forest of species inferred from partial elongation factor 1- Iran. Communications in Agricultural and Applied alpha DNA sequence data. Australasian Plant Biological Sciences 70(3):295-304. Pathology 35(5):513-520.

Hasegawa, E., Ota, Y., Hattori, T., and Kikuchi, Ross-Davis, A.L., Hanna, J.W., Kim, M.S., and T. 2010. Sequence-based identification of Klopfenstein, N.B. 2012. Advances toward DNA­ Japanese Armillaria species using the elongation based identification and phylogeny of North factor-I alpha gene. Mycologia 102(4):898-910. American Armillaria species using elongation factor-I alpha gene. Mycoscience 53(2):161-165.

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