Fungal Genetics and Biology 47 (2010) 672–682 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi A multigene molecular phylogenetic assessment of true morels (Morchella) in Turkey Hatıra Tasßkın a, Saadet Büyükalaca a, Hasan Hüseyin Dog˘an b, Stephen A. Rehner c, Kerry O’Donnell d,* a Faculty of Agriculture, Department of Horticulture, University of Çukurova, 01330 Adana, Turkey b Faculty of Science, Department of Biology, University of Selçuk, 42079 Konya, Turkey c Systematic Mycology and Microbiology Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA d Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL 61604, USA article info abstract Article history: A collection of 247 true morels (Morchella spp.) primarily from the Mediterranean and Aegean Regions of Received 30 September 2009 Southern Turkey, were analyzed for species diversity using partial RNA polymerase I (RPB1) and nuclear Accepted 9 May 2010 ribosomal large subunit (LSU) rDNA gene sequences. Based on the result of this initial screen, 62 collec- Available online 24 May 2010 tions representing the full range of genetic diversity sampled were subjected to multigene phylogenetic species recognition based on genealogical concordance (GCPSR). The 62-taxon dataset consisted of partial Keywords: sequences from three nuclear protein-coding genes, RNA polymerase I (RPB1), RNA polymerase II (RPB2), Conservation translation elongation factor (EF1-a), and partial LSU rDNA gene sequences. Phylogenetic analyses of the EF-1a individual and combined datasets, using maximum parsimony (MP) and maximum likelihood (ML), GCPSR ITS rDNA yielded nearly fully resolved phylogenies that were highly concordant topologically. GCPSR analysis of LSU rDNA the 62-taxon dataset resolved 15 putative phylogenetically distinct species. The early diverging Elata RPB1 (black morels) and Esculenta Clades (yellow morels) were represented, respectively, by 13 and two spe- RPB2 cies. Because a Latin binomial can be applied with confidence to only one of the 15 species (Morchella Species limits semilibera), species were identified by clade (Mel for Elata and Mes for Esculenta) followed by a unique Arabic number for each species within these two clades. Eight of the species within the Elata Clade appear to be novel, including all seven species within the Mel-20-to-31 subclade and its sister designated Mel-25. Results of the present study provide essential data for ensuring the sustainability of morel harvests through the formulation of sound conservation policies. Published by Elsevier Inc. 1. Introduction Hemisphere (Pilz et al., 2007). As a result, it is possible to purchase dried morels in local supermarkets in numerous countries Species of true morels (Morchella spp.) are one of the most throughout the year. In addition, commercial cultivation of morels highly prized and easily identified epigeous macrofungi collected has made it possible to enjoy fresh morels year round as well by mycophiles during the Spring in temperate regions of the (Ower et al., 1986). Northern hemisphere (Weber, 1995; Kuo, 2005). Factors that con- Synapomorphies that united Morchella, together with two other tribute to their economic importance include their extraordinary epigeous genera, Verpa and Discioitis, within the Morchellaceae in- demand among gourmets and foodies alike, coupled with their cluded multinucleate, eguttulate ascospores with a crown of epi- scarcity due to a sporadic and short fruiting season restricted to plasmic polar granules (Berthet, 1964). Molecular phylogenetic a few weeks each spring. With their ever-increasing popularity, analyses using nearly complete SSU rDNA and partial LSU rDNA se- harvest of wild morels has become a commercially successful cot- quences confirmed the monophyly of this family (O’Donnell et al., tage industry in morel-rich regions of countries such as China, In- 1997) and expanded its circumscription to include two hypogeous dia, Mexico, Turkey and the United States in the Northern genera, Fischerula and Leucangium (Hansen and Pfister, 2006). Although these studies provided a robust hypothesis of evolution- ary relationships within the Morchellaceae, and identified the Dis- * Corresponding author. Address: Bacterial Foodborne Pathogens and Mycology cinaceae as its sister, surprisingly few studies have focused on Research Unit, National Center for Agricultural Utilization Research, Agricultural elucidating species limits within Morchella, and none has used Research Service, United States Department of Agriculture, 1815 North University multilocus phylogenetic species recognition based on genealogical Street, Peoria, IL 61604-3999, USA. Fax: +1 (309) 681 6672. E-mail address: [email protected] (K. O’Donnell). concordance (GCPSR; Taylor et al., 2000). To date, species-level 1087-1845/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.fgb.2010.05.004 H. Tasßkın et al. / Fungal Genetics and Biology 47 (2010) 672–682 673 molecular systematic markers employed within this genus have provinces (Fig. 1; Supplemental Table 1) and then air-dried for sub- been limited to isozymes (Gessner et al., 1987; Royse and May, sequent analysis. After the specimens were typed molecularly 1990; Wipf et al., 1996), restriction fragment polymorphisms of using partial RPB1 and LSU rDNA gene sequence data, a subset of the LSU rDNA (Bunyard et al., 1994, 1995) and internal transcribed 62 collections (Table 1), chosen to represent the full range of phy- spacer (ITS) region of the nuclear rDNA (Buscot et al., 1996), and logenetic diversity sampled, were analyzed phylogenetically using phylogenetic analyses of the ITS rDNA sequence data (Wipf et al., a four-locus dataset comprising portions of the RPB1, RPB2, EF-1a 1999; Kellner et al., 2005). However, due to its length variability, genes and domains D1 and D2 of the nuclear large subunit (LSU) ITS rDNA sequences cannot be aligned among members of the rDNA. In addition, sequence of the nuclear ribosomal ITS rDNA re- black (Elata Clade) or yellow (Esculenta Clade) morels unless gion was obtained for members of the Mel-20-to-31 subclade. Phy- extensive regions are excluded from the analyses (Kellner et al., logenetic species identity, geographic origin including GPS 2005). coordinates and dominant vegetation type were recorded for each Although export of fresh morels from Turkey quadrupled over collection (Table 1; Supplemental Table 1). Twenty-seven cultures the past 5 years where in 2008, 238,106 kg averaging $18 US dol- representing 13 of the 15 species sampled in this study were made lars per kilogram were exported primarily to France and Germany, to preserve this novel fungal genetic diversity to insure that it knowledge of Turkish morels is limited to a single morphological would be available to the scientific and biotechnological communi- survey of macrofungi (Solak et al., 2007). Understanding their spe- ties (see Supplemental Table 1; Hawksworth, 2004). Pure cultures cies diversity, however, is critical to elucidating their ecology and obtained from germinated ascospores were typed using a partial systematics, and for formulating sound conservation policies to en- RPB2 gene sequence to insure their authenticity, prior to being sure sustainability of morel harvests (Pilz et al., 2007). Towards stored in the ARS Culture Collection (NRRL) at À175 °C in a cryogen this end, 247 specimens of Morchella, collected in the spring of consisting of 10% skim milk and 1% DMSO. 2007 and 2008 primarily in Mediterranean and Aegean provi- To assess whether ascospore and ascus dimensions could be dences of Southern Turkey, were analyzed phylogenetically using used for morphological species recognition, detailed measure- multilocus DNA sequence data. The primary objectives of the pres- ments were made of ascospore length and width (N = 50) and ascus ent study were threefold: (1) compare the utility of partial LSU length and width (N = 25) from two independent collection of rDNA, RPB1, RPB2 and EF-1a gene sequences for resolving species 11/15 Morchella species detected in Turkey; measurements of limits of Turkish morels using GCPSR (Taylor et al., 2000); (2) as- 4/15 species (Mel-9, Mel-26, Mel-30 and Mes-8) were based on sess the utility of ITS rDNA sequences for species-level phylogenet- single collections (Supplemental Table S2). ics within the Elata Mel-20-to-31 subclade, and (3) use the phylogenetic data to better understand the geographic distribution 2.2. Molecular biology of Morchella spp. within Turkey. Total genomic DNA was extracted following a CTAB (hexadecyl- 2. Methods trimethyl-ammonium bromide (Sigma Chemical Co., St. Louis, MO) protocol (O’Donnell et al., 1997) from approximately 50–100 mg of 2.1. Material studied each dried specimen, which had been pulverized with a pipette tip in a 1.5 ml eppendorf tube. Once the mycelium was resuspended in The 247 Morchella specimens included in the present study 700 ll of the CTAB extraction buffer (100 mM Tris–Cl pH 8.4, 1.4 M were collected in the spring of 2007 and 2008 in 10 Turkish NaCl, 25 mM EDTA pH 8.0, 2% CTAB), it was incubated at 60 °C for Fig. 1. The 10 provinces and five regions (in parentheses) where morels were collected. 674 H. Tasßkın et al. / Fungal Genetics and Biology 47 (2010) 672–682 Table 1 Species subjected to multilocus