Fungal Genetics and Biology 49 (2012) 455–469 Contents lists available at SciVerse ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Multigene molecular phylogenetics reveals true morels (Morchella) are especially species-rich in China ⇑ Xi-Hui Du a,c, Qi Zhao a, Kerry O’Donnell b, Alejandro P. Rooney b, Zhu L. Yang a, a Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road, No. 132, Kunming, 650201 Yunnan Province, PR China b Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, US Department of Agriculture, Agricultural Research Service, 1815 North University Street, Peoria, IL 61604, United States c Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China article info abstract Article history: The phylogenetic diversity of true morels (Morchella) in China was estimated by initially analyzing Received 22 December 2011 nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences from 361 specimens collected in Accepted 23 March 2012 21 provinces during the 2003–2011 growing seasons, together with six collections obtained on loan from Available online 6 April 2012 three Chinese herbaria. Based on the results of this preliminary screen, 40 Esculenta Clade (yellow mor- els) and 30 Elata Clade (black morels) were chosen to represent the full range of phylogenetic diversity Keywords: sampled. To investigate their species limits, we generated DNA sequences from portions of three protein- Ascomycota coding genes (RPB1, RPB2 and EF-1a) and domains D1 and D2 of the nuclear large subunit (LSU) rDNA for Biodiversity all 70 collections. To fully assess evolutionary relationships, previously published multilocus DNA Conservation biology DNA sequence sequence data representing all known Morchella species was included in this study. Phylogenetic analyses Fungi employing maximum parsimony and maximum likelihood frameworks resolved 30 species in China com- Range evolution pared with 22 in Europe and 19 within North America. Eleven novel phylogenetically distinct species were discovered in China, including two species within the Elata Clade and nine within the Esculenta Clade. Of the 30 species in China, 20 appear to be endemic, nine were also represented in Europe, and four putatively fire-adapted species have disjunct distributions in China, Europe and western North America. Although the diversification time estimates place the Esculenta Clade in China as early as the late Creta- ceous and the Elata Clade by the early Oligocene, 27 of the 30 species evolved between the middle Mio- cene 12 Mya and present. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Although Morchella spp. are easily distinguished from other macrofungi by their sponge-like pileus, morphological species rec- True morels (Morchella spp., phylum Ascomycota) are largely ognition (MSR) within the genus is problematic due to their pheno- restricted to temperate regions of the Northern Hemisphere where typic plasticity, dearth of taxonomically useful characters, and they typically fruit for only a few weeks each spring. Due to their body plan which appears to have remained remarkably static over highly desirable flavor and short fruiting season, morels are among the past 100 million years (O’Donnell et al., 2011). Molecular sys- the world’s most prized edible fungi collected by mycophiles and tematic studies, based on analyses of nuclear ribosomal DNA se- gourmets. To meet the demand created by their growing popular- quence data (Hansen and Pfister, 2006; O’Donnell et al., 1997), ity, wild morels are harvested commercially and exported exten- have confirmed Morchellaceae monophyly as defined by synapo- sively from China, India, Turkey, Mexico, and the United States morphic eguttulate, multinucleate ascospores with a cluster of epi- (Pilz et al., 2007). In China, the annual export of dried morels in- plasmic granules at each pole. Although a number of molecular creased fivefold over the past 5 years to 900,000 kg, averaging systematic studies have been published on Morchella, employing $160 US dollars per kilogram. In addition to the export of commer- diverse molecular markers (reviewed in Pagliaccia et al. (2011)), cially harvested wild morels, efforts to meet the growing demand species limits have only recently been investigated (O’Donnell have included cultivation in a specialized indoor facility using pat- et al., 2011; Tasßkın et al., 2010, 2012) using multilocus DNA se- ented technology within the US (Ower et al., 1986). In addition, quence data and phylogenetic species recognition based on genea- preliminary efforts have been made at growing morels outdoors logical concordance and non-discordance (i.e., GCPSR; Dettman in Yunnan Province, China (Zhao et al., 2009). et al., 2003; Taylor et al., 2000). Collectively, the three published GCPSR studies resolved the Esculenta Clade (yellow morels) and ⇑ Corresponding author. Fax: +86 871 5150227. Elata Clade (black morels) as reciprocally monophyletic sisters, E-mail address: [email protected] (Z.L. Yang). comprising 18 and 31 species, respectively, and a basal monotypic 1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.03.006 456 X.-H. Du et al. / Fungal Genetics and Biology 49 (2012) 455–469 sister clade represented by Morchella rufobrunnea, the only species To rigorously investigate species diversity represented by the that has been cultivated commercially indoors (Ower et al., 1986). 73 collections, we sequenced portions of the nuclear large subunit Results of the molecular phylogenetic studies also revealed that (LSU) rDNA, RNA polymerase largest (RPB1) and second largest most Morchella species appear to exhibit continental endemism subunit (RPB2), and translation elongation factor 1-alpha (EF-1a) and provincialism, which has greatly facilitated reconstructing genes. These sequences were added to published multilocus DNA their historical biogeography (O’Donnell et al., 2011). sequence datasets previously published for Morchella (O’Donnell Due to the limitations of MSR, field guides typically recognize et al., 2011; Tasßkın et al., 2010, 2012), and analyzed phylogeneti- fewer than five species and use European names for collections cally employing the criteria of genealogical exclusivity and non- made in Asia (Huang, 1998; Imazeki et al., 1988; Mao, 2000; Ying discordance (Dettman et al., 2003) under GCPSR. Phylogenetic spe- and Zang, 1994; Zang, 1996) and North America (Arora, 1979; We- cies were recognized as genealogically exclusive under GCPSR, if ber, 1988). To date, only four Morchella species and one subspecific they were supported by maximum parsimony (MP) and maximum taxon have been described from China, and these were based likelihood (ML) bootstrapping of one or more partition, and/or the exclusively on MSR (Chen and Liu, 2005; Li et al., 2006; Mou, combined dataset and no partition contradicted their monophyly 1987; Zang, 1987). Given the high level of cryptic speciation and (i.e., non-discordance; Dettman et al., 2003). In the absence of provincialism discovered within Morchella (O’Donnell et al., the ability to test the monophyly of two lineages within the Escu- 2011; Tasßkın et al., 2010, 2012), and the rich floristic diversity lenta Clade (Fig. 2) and five within the Elata Clade (Fig. 3) repre- within China (Liu, 1988; Ying, 2001), we hypothesized that sented by single collections, these seven lineages were GCPSR-based studies of geographically diverse collections of Mor- interpreted as putatively phylogenetically distinct because they chella might result in the discovery of multiple novel species lin- were significantly divergent from and not sympatric with their eages in heretofore unexplored regions of China, especially given putative sisters. that relatively few collections from eastern Asia were included in O’Donnell et al. (2011). Knowledge gained from such a survey is 2.2. DNA isolation essential for developing scientifically informed conservation prac- tices to enhance sustainability of morel harvests (Pilz et al., 2007) Prior to extracting total genomic DNA, all of the Chinese collec- and to advance our understanding of their genetic diversity, evolu- tions were dried over several exchanges of silica gel in tightly tionary relationships and geographic distribution. sealed plastic bags for one to several days. Approximately 10 mg Towards this end, we generated ITS rDNA sequences from 361 of dried pileus tissue was ground to a fine powder in a 1.5 ml collections of Morchella we collected from 21 provinces together microcentrifuge tube using a Kontes pellet pestle (Kaimu, China). with six collections obtained from three Chinese herbaria to ob- Once pulverized, the samples were suspended in 700 ll of CTAB tain an initial estimate of Morchella species diversity in China. extraction buffer (100 mM Tris–Cl pH 8.4, 1.4 M NaCl, 25 mM Based on our molecular phylogenetic analyses of the aligned ITS EDTA, 2% CTAB), and incubated for 1.5–2.0 h at 65 °C, during which rDNA sequences, 70 collections were chosen to represent the ge- time they were gently inverted 3–5 times. After the samples were netic diversity sampled for which portions of four additional nu- cooled to room temperature, 700 ll of chloroform-isoamyl alcohol clear genes were obtained. Phylogenetic analyses were conducted (24:1) was added to each tube. The mixture was vortexed briefly, on the individual