True Morels (Morchella, Pezizales) of Europe and North America: Evolutionary Relationships Inferred from Multilocus Data and a Unified Taxonomy
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Mycologia, 107(2), 2015, pp. 359–382. DOI: 10.3852/14-166 # 2015 by The Mycological Society of America, Lawrence, KS 66044-8897 True morels (Morchella, Pezizales) of Europe and North America: evolutionary relationships inferred from multilocus data and a unified taxonomy Franck Richard1 28S rDNA D1-D2 domains (28S). The 107 newly Jean-Michel Bellanger sequenced collections were from both continents, UMR 5175 CEFE, INSERM, Campus CNRS, 1919 including 48 types, together with previously published Route de Mende, F-34293 Montpellier, France sequences. Names are applied to 30 of the 65 currently Philippe Clowez recognized genealogical species. Results of the present 56 place des Tilleuls, F-60400 Pont-l’Eveˆque, France study revealed that the number of Morchella species in Europe (n 5 21) is nearly identical to that in North Karen Hansen America (n 5 22). Only seven species were found on Swedish Museum of Natural History, Department of both continents, consistent with previous reports of Botany, P.O. Box 50007, SE-104 05 Stockholm, high continental endemism within the genus. Present- Sweden ly it is not possible to tell whether the transoceanic Kerry O’Donnell disjunctions were due to human activities, migration Bacterial Foodborne Pathogens and Mycology Research across a Bering land bridge or long-distance dispersal. Unit, National Center for Agricultural Utilization In an effort to stabilize the taxonomy, due in part to Research, US Department of Agriculture, Agricultural the recent publication of synonyms for 11 of the Research Service, 1815 North University Street, Peoria, Illinois 61604 species, accepted names are presented together with their corresponding later synonyms. A new subclade Alexander Urban that includes holotypes of M. castanea and M. University of Vienna, Faculty of Life Sciences, brunneorosea is identified in sect. Morchella (Esculenta Department of Botany and Biodiversity Research, Division of Systematic and Evolutionary Botany, Clade). Lectotypes for Morchella deliciosa, M. eximia Rennweg 14, A-1030 Wien, Austria and M. tridentina are designated here, as well as epitypes for M. dunalii, M. eximia, M. purpurascens Mathieu Sauve and M. vulgaris. Morchella conica was determined to UMR 5175 CEFE, Universite´ de Montpellier, Campus be illegitimate, and further research is required to CNRS, 1919 Route de Mende, F-34293 Montpellier, France determine the identity of M. elata and M. inamoena. Key words: Ascomycota, Morchellaceae, nomen- Re´gis Courtecuisse clature, Pezizomycetes, taxonomy Pierre-Arthur Moreau De´partement des Sciences ve´ge´tales et fongiques, faculte´ des sciences pharmaceutiques et biologiques, Univ Lille INTRODUCTION Nord de France, F-59000 Lille, France, and EA 4483, UFR Pharmacie, F-59000 Lille, France True morels (Morchella) comprise one of the most intensively collected groups of macrofungi worldwide, but their systematics remains in flux. A series of Abstract: Applying early names, with or without recent multilocus molecular phylogenetic analyses original material, to genealogical species is challeng- (Tas¸kın et al. 2010, 2012; O’Donnell et al. 2011; Du ing. For morels this task is especially difficult because et al. 2012a, b) employing genealogical concordance of high morphological stasis and high plasticity of phylogenetic species recognition (GCPSR, Taylor apothecium color and shape. Here we propose a et al. 2000), showed that morphological species nomenclatural revision of true morels (Morchella, recognition (MSR) within this iconic genus frequently Pezizales) from Europe and North America, based on fails to delimit species, due to widespread cryptic molecular phylogenetic analyses of portions of the speciation. The dearth of phenotypically informative genes for RNA polymerase II largest subunit (RPB1) macro- and micromorphological characters within andsecondlargestsubunit(RPB2), translation this genus greatly reduces the utility of MSR in the elongation factor-1a (TEF1), the nuc rDNA region absence of critical molecular phylogenetic data. As a encompassing the internal transcribed spacers 1 and result, MSR-based taxonomic treatments have pro- 2, along with the 5.8S rDNA (ITS), and partial nuc duced conflicting estimates of species diversity. Some taxonomic treatments have recognized a few, highly Submitted 25 Jun 2014; accepted for publication 10 Nov 2014. variable taxa (e.g. Dennis 1978: three species in 1 Corresponding author. E-mail: [email protected] Britain; Weber 1995: three species in North America; 359 360 MYCOLOGIA Dissing 2000: eight species in the Nordic countries), MATERIALS AND METHODS while others accepted many species, varieties and forms (Krombholz 1834: 11 species; Boudier 1897: 20 Taxon sampling.—Most of the collections analyzed in the present study represent types included in Clowez (2012) species; Jacquetant 1984: 30 species; Clowez 2012: 52 that were deposited in LIP. Also, some additional collec- species). The aforementioned GCPSR studies laid the tions of Pierre Collin and Pierre-Arthur Moreau, as well as foundation for a taxonomic revision of Morchella in recent collections deposited by P. Clowez (LIP), together North America (Kuo et al. 2012). with collections from LUG, O and S (Thiers B [continu- The molecular phylogenetic studies revealed that ously updated]) were included (SUPPLEMENTARY TABLE I). Morchella comprises three clades (O’Donnell et al. Duplicates of all material from LIP used for DNA analyses 2011), including an early diverging basal lineage are kept in the fungal herbarium of the CEFE-CNRS (1919 (section Rufobrunnea sensu Clowez 2012) estimated route de Mende, F – 34293 Montpellier, France). Collec- to have evolved in the late Jurassic. This clade is tions cited in Clowez (2012) for which DNA sequence data represented by two extant species, M. rufobrunnea, could not be generated are not mentioned below. which has been grown commercially (Ower et al. 1986), Nomenclature and typifications.—To support several early and M. anatolica (Is¸ilog˘lu et al. 2010, Tas¸kın et al. lectotypes (i.e. illustrations or material where DNA sequenc- 2012). The origin of the later-diverging sister clades, es could not be obtained), epitypes were selected using the Elata (black morels, section Distantes sensu Clowez following criteria: i. the collection came from the original 2012) and Esculenta (yellow morels, section Morchella continent and biogeographical region as indicated in the sensu Clowez 2012) was dated to the early Cretaceous, protologue; ii. a good photograph and if possible a full approximately 125 Mya. These clades comprise at least description from a freshly collected epitype is available; and iii. DNA sequence data was obtained from at least three loci, 27 and 36 phylogenetically distinct species respectively including the ITS, which has been recently proposed as a (O’Donnell et al. 2011; Du et al. 2012a, b; Voitk et al. universal DNA barcode marker for Fungi (Schoch et al. 2014). Because binomials could be applied to only four 2012). When two names of equal priority (i.e. published species with confidence, phylospecies within these two simultaneously in a same paper) were treated as synonyms clades were informally named using Mes (for Esculenta (McNeill et al. 2012, Art. 11.5), the choice was made in favor Clade) or Mel (for Elata Clade) codes followed by a of the best-documented name (i.e. original material/type in unique Arabic number. Although epithets based on good condition and documented by DNA sequence data European collections are typically used in taxonomic from the most loci). The dates of effective publication were treatments of morels from Asia (Imazeki et al. 1988) 16 Apr 2012 for Clowez (2012) and 29 Aug 2012 for Kuo and North America (Arora 1986, Weber 1995), the et al. (2012). Although a preliminary version of the latter phylogenetic results indicate that these names might was published online on 11 Apr 2012, this does not qualify as effective publication (McNeill et al. 2012, Art. 30.2). be misapplied, given that the majority of morels appear to exhibit high continental endemism and provincial- DNA extraction, amplification and sequencing.—DNA ex- ism in the northern hemisphere, consistent with their traction and PCR amplification were conducted with the proposed evolutionary origin in Laurasia (O’Donnell REDExtract-N-AmpTM Plant PCR Kit (Sigma-Aldrich, St et al. 2011). Louis, Missouri), following the manufacturer’s instructions. Uncertainty over what epithet to accept, especially Efforts were made to PCR amplify portions of five genetic loci for North American taxa, was complicated significantly with the following primer pairs: the nuc rDNA region encompassing the internal transcribed spacer and 5.8S rDNA by the recent publication of independent morpholog- (ITS) with ITS-1F/ITS-4 (Gardes and Bruns 1993), the partial ical (Clowez 2012) and molecular and morphological nuc 28S rDNA D1-D2 domains (28S) with LR0R/LR7 systematic treatments of the genus (Kuo et al. 2012). (Vilgalys and Hester 1990), the translation elongation factor Because the epithets proposed by Clowez (2012) have 1-a gene (TEF1) with EF526F/EF3AR (Rehner and Buckley priority over those applied to conspecifics in Kuo et al. 2005), the RNA polymerase II largest subunit gene (RPB1) (2012), the primary objective of the present study with gRPB1A/aRPB1C (Matheny et al. 2002) and the RNA was to assess what taxa in the latter study represent polymerase II second largest subunit gene (RPB2) with 9F/ nomenclatural synonyms of taxa validly published by 3R (Liu et al. 1999). PCR amplifications were performed in a Clowez (2012). To accomplish this objective, we total volume of 20 mL, including 1 mL genomic DNA, in a attempted to obtain phylogenetically informative master cycler gradient thermos-cycler (Eppendorf AG, DNA sequence data from the holotypes and paratypes Hamburg, Germany). The cycling parameters were as follows: 94 C for 3 min, 35 cycles of 94 C for 30 s, 53 C for available for European species, especially those de- 30 s, 72 C for 1–2 min, followed by 72 C for 7 min. Amplicons scribed by Clowez (2012), to determine what names were purified and sequenced at Genoscope, Evry, France, or should be accepted for the morels sampled.