Persoonia 23, 2009: 86 – 98 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158509X475913 Taxonomy and evolutionary relationships within species of section Rimosae (Inocybe) based on ITS, LSU and mtSSU sequence data E. Larsson1, M. Ryberg1, P.-A. Moreau2, Å. Delcuse Mathiesen1, S. Jacobsson1 Key words Agaricales Basidiomycota molecular systematics phylogeny taxonomy Abstract The present study aimed at elucidating the structure of Inocybe subg. Inosperma sect. Rimosae but included also representatives from subg. Mallocybe and the genus Auritella. Phylogenetic relationships were inferred using ITS, LSU and mtSSU sequence data. The analyses recovered the ingroup as a monophyletic, strongly supported clade. The results indicate that recognizing Auritella on the genus level renders Inocybe paraphyletic. The species traditionally placed in sect. Rimosae were found to be distributed over two strongly supported clades, Maculata and Rimosae s.s. The Maculata clade clusters with sect. Cervicolores and the two represent subg. Inosperma in a strict sense. Rimosae s.s. emerges as an independent, supported clade well separated from Inosperma s.s. Twenty-one terminal groups were correlated with morphologically distinct species. In addition several taxa on single branches and minor less supported clades were recovered. A key to the identified species of the Maculata and Rimosae s.s. clades which occur in Northwest Europe is provided. Article info Received: 7 April 2009; Accepted: 9 July 2009; Published: 21 September 2009. INTRODUCTION Inocybe is a large genus of agaric fungi with an estimated 500 species world wide (Kirk et al. 2008), a number that is likely to increase considerably when tropical and southern temperate areas are more intensively explored. Intrageneric classifications have been based mainly on spore morphology, the form and distribution of cystidia, and stipe morphology. The spores may be ellipsoid, amygdaliform or nodulose/angular. Many species have incrusted thick-walled pleuro- and cheilocystidia (metuloids). Some large groups completely lack the metuloids but then have numerous thin-walled cheilocystidia. The stipe may be of uniform thickness or have a distinctly bulbous base. A number of classifications combining these and other characters in various ways have been proposed (Heim 1931, Kühner & Romagnesi 1953, Kühner 1980, Kuyper 1986, Singer 1986, Stangl 1989, Kobayashi 2002). Several phylogenetic analyses of Inocybe using both ribosomal and protein coding genes have been published (Matheny et al. 2002, Matheny 2005, Matheny & Bougher 2006a, Matheny et al. 2009). These studies confirm that Inocybe is monophyletic. In a multi-gene phylogeny of Agaricomycotina Matheny et al. (2006) showed that Inocybe does not belong in Cortinariaceae, where it has traditionally been placed, but has affinities to Crepidotaceae. Matheny (2005) suggested that Inocybe should be recognized at the family level as Inocybeaceae, a family already proposed and described by Jülich (1982). Matheny (2005) identified five clades within Inocybaceae, which he called Inocybe, Inosperma, Pseudosperma, Mallocybe and Auritella. The Inocybe clade holds the generic type species and 1 2 Department of Plant and Environmental Sciences, University of Gothenburg, P.O. Box 461, SE-405 30 Göteborg, Sweden; corresponding author e-mail: ellen.larsson@dpes.gu.se. Laboratorie de Botanique, Faculté de Sciences Pharmaceutiques et Biologiques, 3 rue du Professeur Laguesse, BP 83, F-59006 Lille Cédex, France. includes all species with incrusted cystidia (metuloids) irrespective of spore shape. Mallocybe, recognized as a separate subgenus (Kuyper 1986), includes species with necropigmented basidia and thin-walled cheilocystidia originating from the subhymenium. Auritella was recently separated from Inocybe as an independent genus (Matheny & Bougher 2006a, b) and seems to represent a unique Paleotropical and Southern hemisphere lineage with species from Australia and Africa. The two clades Inosperma and Pseudosperma basically include the species in sections Rimosae and Cervicolores in the classification by Kuyper (1986). Pseudosperma was introduced as a clade name only and not formally assigned any classification status according to ICBN (McNeill et al. 2006). Section Rimosae is in traditional classifications placed in subg. Inosperma (Kühner 1980, Kuyper 1986, Stangl 1989). The section includes species characterized by radially fibrillose to rimose (squamulose) caps, ellipsoid to phaseoliform spores, and absence of metuloid pleurocystidia but with densely packed, simple, cylindrical, clavate to pyriform hymenial cheilocystidia that make the gill edge look distinctly white in mature specimens. Other characters that may occur are a distinctly bulbous stem base, reddening flesh, yellow to olivaceous tinges on lamellae, and specific odours. Phylogenetic analyses in a recent publication on the biogeography of Inocybaceae (Matheny et al. 2009) included a broader sampling of sect. Rimosae than any previous study, although representatives from Europe were still few. The results indicated that the section is non-monophyletic. Many species in Rimosae are known to occur on more nutrient rich soils, often on calcareous ground, others prefer more nutrient poor acid soils. Several species are found in disturbed places such as along forest paths and roadsides. They form ectomycorrhizal associations with a broad range of host trees of both gymnosperms and angiosperms (Kuyper 1986, Stangl 1989, Jacobsson 2008). Several species occur in arctic and alpine regions and are then associated with shrubs and herbs such as Salix, Dryas, and Polygonum (Favre 1955, Horak © 2009 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 87 E. Larsson et al.: Relationships among species of section Rimosae 1987, Kühner 1988, Bon 1997, Ferrari 2006). A few are found on coastal sand dunes associated with Pinus and Salix (Orton 1960, Bon 1984). The taxonomy in Rimosae has for a long time been confused since many species are described on small differences in macro- and micro-morphology. Authors with a conservative approach recognise 10 – 20 species (Kuyper 1986, Stangl 1989), others include more than 40 (Bon 1997). In the Nordic countries 8–13 species are usually recognized (Stridvall et al. 1989, Jacobsson 2008). Some European species are likely to show a northern circumpolar distribution (Ryberg et al. 2008). However, since a modern comprehensive treatment of Inocybe in North America is lacking the biogeographic knowledge is incomplete (Kauffman 1924, Stuntz 1947, 1954). Inocybe rimosa, taken in a wide sense, shows a considerable morphological variation and also a broad ecological range covering all biomes from nemoral deciduous forests to the arctic-alpine zone. Some authors have advocated a narrow species concept and described a number of species and varieties (Heim 1931, Kühner 1988, Bon 1997). Kuyper (1986) choose the opposite strategy and recognized only one species, listing more than 30 species and varieties as synonyms. The present study had three aims: to examine the phylogenetic structure and position of sect. Rimosae, to identify the number of North European species within Rimosae, and to elucidate the phylogenetic relationship among them. MATERIALS AND METHODS Morphological studies Micro-morphological characters were observed using a Zeiss Axioscope 2, equipped with phase contrast. Spores and cystidia were measured in a 3 % KOH solution at × 400 and 1 000 magnification using microscope photos taken with a Canon G9 digital camera and using software AxioVision (Carl Zeiss AB). Unusually large or small spores were not considered. Collections are deposited in the herbarium of the Department of Plant and Environmental Sciences, University of Gothenburg (GB) if not otherwise indicated. Data on sequenced specimens is provided in Table 1. Taxon sampling Ninety-nine ingroup specimens were sequenced. They represent the majority of species within section Rimosae that occur in North Europe (Jacobsson 2008). In addition specimens from Estonia, France, Great Britain, Slovakia, USA, and Australia were included. Specimens were selected to represent a broad spectrum of morphological characters and ecology. Eight species of sect. Cervicolores and subg. Mallocybe were also sequenced and included in the analyses. Based on results from earlier molecular phylogenetic studies of Agaricales and Inocybe (Matheny 2005, Matheny & Bougher 2006a, Matheny et al. 2006) species of Conocybe, Crepidotus, Naucoria, Pleuroflammula, and Simocybe, were selected as out-group. ITS and LSU sequences for included species of Auritella, Simocybe, and Pleuroflammula, were taken from GenBank (AY380371, AY380395, AY635766, DQ494696, AY745706, AF205707, DQ494685, AF745706). Two GenBank sequences (DQ917657 ITS, EU600863 LSU) representing specimens identified as Inocybe sororia were included in the separately aligned and analysed dataset for the Rimosae s.s. subclade A (see below). DNA extraction, PCR and sequencing Sequences from three regions were generated for the study, the complete ITS region, 1200 base pairs of the 5’ end of the nuclear LSU ribosomal DNA, and the mitochondrial SSU ribosomal DNA. DNA extractions, PCR reactions and sequencing were performed as described in Larsson & Örstadius 2008. Primers used to amplify the complete ITS region and the 5’ end of the LSU region were ITS1F (Gardes & Bruns 1993) and LR21, LR0R and LR7 (Hopple & Vilgalys 1999), the mtSSU MS1 and MS2 (White et al. 1990). Primers used for sequencing were ITS1, ITS3, ITS4, MS1, MS2 (White et al. 1990), Ctb6 (http://plantbio.berkeley.edu/~bruns/), Lr5 and LR3R (Hopple & Vilgalys 1999). Phylogenetic analyses Sequences were edited and assembled using Sequencher 3.1 (Gene Codes, Ann Arbor). Sequences were aligned automatically using the software MAFFT (Katoh et al. 2002) and adjusted manually using the data editor in PAUP* (Swofford 2003). Sequences have been deposited in GenBank and accession numbers are listed in Table 1. Heuristic searches for most parsimonious trees were performed using PAUP*. All transformations were considered unordered and equally weighted. Variable regions with ambiguous alignment were excluded and gaps were treated as missing data. Heuristic searches with 1 000 random-addition sequence replicates and TBR branch swapping were performed. Relative robustness of clades was assessed by the bootstrap method using 1 000 heuristic search replicates with 100 random taxon addition sequence replicate, TBR swapping, saving 100 trees in each replicate. Bayesian analysis of the datasets was performed using MrBayes 3.2 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003). MrModelTest 2.2 (Nylander 2004) was used to estimate separate best-fit models of evolution for ITS (1 and 2 combined), 5.8S, LSU, and mtSSU. The Bayesian inference was set up with model parameters estimated separately for each of the four partitions. Four parallel runs using MetropolisCoupled Markov Chain Monte Carlo (MCMCMC) were implemented instead of the default of two to improve the inference of convergence statistics. To decrease the computational burden two chains, one hot and one cold (temperature difference set to 0.1 to increase the efficiency of metropolis coupling), were used instead of the default of four. Each chain was run for 10 million generations with tree and parameter sampling every 1 000 generations (10 000 trees). Tracer (Drummond & Rambaut 2007) and AWTY (Wilgenbusch et al. 2004) were used to examine when the chains had reached a stationary state and how many generations were appropriate to discard as burn-in. A 50 % majority-rule consensus phylogram was computed from the remaining trees; the proportions of this tree correspond to Bayesian posterior probabilities (BPP). To investigate if there were any conflict between the nuclear and mitochondrial regions, analyses were also made for these partitions separately. It was then checked if there were any conflict between the regions in nodes separating species and with more than 0.7 BPP support (cf. Lutzoni et al. 2004). To improve the resolution and be able to use the complete ITS region in the phylogenetic analysis realignment of sequence data in the Rimosae s.s. subclade A and Rimosae s.s. subclade D were performed as described above. Heuristic searches for most parsimonious trees and bootstrap analysis were performed as above except that no restriction on saving of trees in the replicates was applied. RESULTS For all 99 ingroup specimens the ITS/LSU region was generated. For 54 of these also mtSSU sequences were generated 88 Persoonia – Volume 23, 2009 Table 1 Data of specimens sequenced in this study. Species Original specimen identification Coll. ID. / Origin Ecology, substrate GenBank ITS / LSU Conocybe siliginea Crepidotus mollis C. mollis var. calolepis Inocybe cervicolor I. bongardii I. subhirsuta I. cfr calamistrata I. dulcamara I. terrigena I. fulvipes I. agardhii Naucoria salicis N. bohemica N. submelinoides Inocybe adaequata I. arenicola I. bulbosissima I. cfr cookei I. cfr flavella I. cfr rimosa I. cfr squamata I. cookei I. dulcamaroides I. erubescens I. flavella I. xanthocephala I. hygrophorus I. maculata I. maculata forma fulva I. cfr rimosa I. adaequata I. arenicola I. arenicola I. fastigiata var. alpina I. fastigiata var. alpina I. fastigiata var. alpina I. bulbosissima I. rimosa I. fastigiata var. alpina I. cookei I. rimosa I. majalis I. flavella I. flavella I. flavella I. rimosa I. perlata I. cfr rimosa I. fastigiata I. arenicola I. rimosa I. rimosa I. rimosa I. umbrinella I. rimosa var. umbrinella I. cft obsoleta I. cft squamata I. cft squamata I. squamata I. squamata I. squamata I. squamata I. squamata I. cookei I. cookei I. cookei I. cookei I. cookei I. dulcamaroides I. dulcamaroides I. erubescens I. erubescens I. erubescens I. flavella I. flavella I. flavella I. xanthocephala I. hygrophorus I. maculata I. maculata I. maculata I. maculata I. maculata I. maculata I. maculata I. maculata I. cfr maculata I. cfr rimosa I. maculata forma fulva I. maculata forma fulva I. maculata LÖ93-04 / Swe EL45-04 / Swe EL14-08 / Swe SJ04024 / Swe EL123-04 / Swe EL45-05 / Nor KHL13071 / Costa Rica EL89-06 / Swe EL117-04 / Swe EL37-05 / Nor EL88-04 / Swe EL71a-03 / Swe EL71b-03 / Swe TAA185174 / Est In a pasture deciduous wood deciduous wood Picea forest, calcareous Quercus, calcareous Dryas, Salix, alpine Quercus Salix glauca Picea, calcareous Dryas, Salix, alpine Salix, calcareous Alnus, Betula Alnus, Betula Alnus DQ389731 AM882996 FJ904178 AM882939, AM882941 AM88294 AM882948 FJ904122 AM882864 AM882858 FJ904123 FJ904180 FJ904179 AM882885 PC2008-0014 / GB MR00022 / Swe RC GB99-014 / Fra EL238-06 / Fra EL51-05 / Nor EL66-05 / Nor EL37-06 / Swe EL75-07 / Swe EL88-06 / Swe EL30-06 / Swe EL104-04 / Swe GK080924 / GB PAM05062502 / Fra EL118-05 / Fin BJ920829 / Swe EL90-04 / Swe EL71-04 / Swe JD2008-0241 / GB I116-06 / Australia PAM05061101 / Fra JV26578 / Est EL127-04 / Swe TAA185135 / Est JV22619 / Est PC080925 / GB JV8125 / Fin EL81-06 / Swe I93-04 / Australia I113-05 / Australia SJ92-010 / Swe SM92-013 / Swe SJ92-017 / Swe Stordal18318 / Nor JV2609 / Fin MR00035 / Swe EL191-06 / GB EL70a-03 / Swe EL73-05 / Swe EL109-04 / Swe EL29-08 / USA EL112-06 / Swe TAA185164 / Est KGN980714 / Swe BH910707 / Swe EL56-08 / Swe EL137-05 / Swe LAS89-030 / Swe PAM00100606 / Fra EL97-06 / Swe EL74-05 / Swe MR00020 / Swe EL121-04 / Swe EL58-03 / Swe EL126-04 / Swe EL182-08 / Slov EL78-03 / Swe EL166-08 / Swe EL114-06 / Swe SJ05029 / Swe EL247-06 / Fra PAM01100120 / Fra SJ06007 / Swe Fagus forest Tilia, Corylus Pinus, Salix, sand dune Pinus, sand dune Dryas, Salix, alpine Salix reticulata, alpine Salix polaris, alpine Salix reticulata, alpine Salix lapponum, subalpine Salix polaris, alpine Corylus Quercus, Betula, wet Salix, calcareous soil Salix, Betula, ravine Salix, Betula, hyperit Salix, Betula, calcareous Fagus, calcareous soil Fagus, Corylus deciduous forest Tilia, calcareous Pinus, calcareous Fagus, Quercus, calcareous Pinus, Betula, calcareous Quercus, Corylus, calcareous Pinus, Quercus Picea, Tilia, Populus, rich Salix glauca, subalpine, wet deciduous forest deciduous forest Picea, calcareous Picea, Populus, Betula Pinus, Populus, park Picea mixed forest Picea, Populus, Pinus Corylus, Quercus Corylus, Quercus Fagus, Quercus Betula, Quercus Corylus, Quercus Salix reticulata, alpine Dryas, alpine Quercus, Tilia, calcareous Fagus, Tilia, rich soil Fagus, park Corylus, Salix, Alnus, wet Corylus, Alnus, Quercus wet Alnus, wet Salix Betula, Salix, subalpine meadow Fagus, Quercus Tilia, Corylus, calcareous Fagus, Quercus, calcareous Fagus, rich soil Fagus, Quercus, calcareous Fagus, rich soil Mixed trees, pasture Picea, Corylus, calcareous Dryas, Polygonum, alpine Pinus, Alnus Pinus, Populus Betula Betula FJ904177 AM882706 FJ904134 FJ904133 AM882764 AM882765 FJ904161 FJ904160 FJ904159 FJ904158 AM882952 FJ904129 FJ904128 AM882782 AM882774 AM882773 AM882786 FJ904125 FJ904142 FJ904155 FJ904154 AM882768 AM882766 FJ904157 FJ904153 FJ904152 FJ904135 FJ904141 FJ904140 AM882785 AM882783 AM882784 FJ904139 FJ904138 AM882954 FJ904173 AM882953 AM882955 AM882956 FJ904127 FJ904126 AM882950 AM882951 AM882949 FJ904131 AM882776 AM882775 FJ904130 FJ904137 AM882959 AM882958 AM882957 AM882963 AM882964 FJ904172 AM882962 FJ904171 FJ904170 AM882994 FJ904169 FJ904168 FJ904167 mtSSU FJ904242 FJ904185 FJ904186 FJ904187 FJ904181 FJ904183 FJ904184 FJ904182 FJ904243 FJ904240 FJ904241 FJ904189 FJ904188 FJ904224 FJ904223 FJ904222 FJ904221 FJ904220 FJ904196 FJ904195 FJ904193 FJ904192 FJ904216 FJ904215 FJ904219 FJ904218 FJ904214 FJ904190 FJ904203 FJ904234 FJ904233 FJ904194 FJ904239 FJ904198 FJ904199 FJ904197 FJ904202 FJ904232 FJ904231 FJ904230 89 E. Larsson et al.: Relationships among species of section Rimosae Table 1 (cont.) Species I. melliolens I. cfr microfastigiata I. mimica I. obsoleta I. perlata I. quietiodor I. rhodiola I. rimosa I. sororia I. squamata I. umbrinella Original specimen identification I. umbrinella I. melliolens I. microfastigiata I. mimica I. mimica I. obsoleta I. obsoleta I. perlata I. perlata I. quietiodor I. quietiodor I. quietiodor I. quietiodor I. quietiodor I. quietiodor I. rhodiola I. rhodiola I. rimosa I. rimosa I. rimosa I. rimosa var. umbrinella I. rimosa I. fastigiata var. argentata I. rimosa I. rimosa I. fastigiata var. argentata I. cfr fasigiata I. rimosa coll. I. squamata I. cfr squamata I. cfr squamata I. curreyi I. rimosa var. brunnea I. rimosa coll. I. cfr rimosa I. cfr rimosa I. perlata Coll. ID. / Origin Ecology, substrate PAM05052303 / Fra EL224-06 / Fra EL113-06 / Swe EBJ961997 / Swe TK2004-114 / Swe EL17-04 / Swe BJ890915 / Swe BJ940922 / Swe EL74-04 / Swe RP980718 / Swe LAS97-067 / Swe LAS94-023 / Swe PAM01091310 / Fra EL115-04 / Swe JV20202 / Nor PAM00090117 / Fra EL223-06 / Fra AO2008-0250 / GB EL118-08 / Swe EL102-04 / Swe EL211-06 / Fra TK97-156 / Swe PAM03110904 / Fra EL75-05 / Swe SJ04007 / Swe PAM06112703 / Corsica Kuoljok0512 / Swe JV15200 / Swe SJ08003 / Swe TK96-109 / Swe SJ85048 / Nor PAM05052301 / Fra JV13699 / Fin JV17954 / Est PC081010 / GB PC080816 / GB PAM01102912 / Fra (Table 1). The aligned complete dataset, including sequences downloaded from GenBank, consisted of 119 sequences and 3 461 characters. The majority of the ITS1 and ITS2 regions was found to be too variable to be included in the analyses. After exclusion of ambiguous regions 1 985 characters remained for the analysis. Of these 1 409 were constant, 174 were variable and parsimony uninformative, and 402 were parsimony informative. The maximum parsimony analysis yielded 63 900 equally most parsimonious trees (length = 1 812, CI = 0.4354, RI = 0.8490). Bootstrap analysis recovered Inocybe s.l. (including Auritella) as monophyletic with 91 % support. It forms together with Crepidotus, Simocybe, and Pleuroflammula a clade with 92 % bootstrap support. Four major clades within the ingroup received strong support. They are here called Auritella (100 %), Rimosae s.s. (98 %), Mallocybe (100 %), and Inosperma (98 %). The Inosperma clade was further divided in the Cervicolores clade (98 %) and a moderately supported group here called the Maculata clade (72 %). The Rimosae s.s. clade includes 68 sequences dispersed over 6 strongly supported subclades (Fig. 1A – F) and a number of groups that in most cases seem to correspond to species. Eight of these terminal groups have been identified as Inocybe arenicola, I. mimica, I. dulcamaroides, I. flavella, I. squamata, I. hygrophorus, I. obsoleta, and I. perlata, respectively. One distinct but non-identified clade is reported as Inocybe sp. Specimens identified as I. flavella seem to cover several taxa differing in the shape and size of spores. Small-spored specimens are together lumped as I. cfr flavella but this label seems Tilia, calcareous Salix, Quercus, wet Dryas, alpine Pinus, Picea, calcareous Pinus, Betula, calcareous Picea, Corylus Picea mixed forest Fagus, Betula, meadow Corylus, Betula, calcareous Fagus, Quercus, park Fagus, Quercus, calcareous Fagus, Quercus, calcareous Betula, Salix Quercus, Tilia, park Betula, Alnus, calcareous Salix Salix, wet forest Salix Picea, Betula, calcareous Betula, garden Quercus, Carpinus Corylus, calcareous Quercus Fagus, Quercus, park Tilia Fagus Salix, alpine meadow Salix herbacea, alpine Betula, Pinus Populus, calcareous Populus, calcareous Populus, Picea, park Pinus, Populus, Salix Pinus, calcareous Helianthemum, calcareous Fagus, Quercus, calcareous Quercus ilex GenBank ITS / LSU mtSSU FJ904148 FJ904149 FJ904156 FJ904124 AM882781 AM882769 AM882770 AM882772 AM882771 FJ936169 AM882974 AM882961 FJ936168 AM882960 FJ904174 FJ904176 FJ904175 FJ904147 FJ904146 AM882761 FJ904145 AM882844 FJ904144 AM882762 AM882763 FJ904143 FJ904150 FJ904151 FJ904136 AM882780 AM882778 FJ904132 FJ904165 FJ904166 FJ904164 FJ904163 FJ904162 FJ904211 FJ904217 FJ904191 FJ904204 FJ904205 FJ904238 FJ904237 FJ904236 FJ904235 FJ904210 FJ904209 FJ904208 FJ904207 FJ904206 FJ904212 FJ904213 FJ904201 FJ904200 FJ904228 FJ904229 FJ904227 FJ904226 FJ904225 to cover at least two taxa. Thirty-three sequences cluster to a strongly supported clade that corresponds to I. rimosa s.l. Within such a broadly defined I. rimosa five subclades corresponding to species were recovered. One is the alpine species I. bulbosissima, a second is I. rimosa s.s., a third is I. umbrinella, and the remaining two are tentatively identified as I. cfr sororia and I. melliolens. The specimens originating from deciduous forests in Australia form a strongly supported clade within Rimosae and represent two unidentified species. In addition several taxa on single branches and minor less supported clades were recovered (Fig. 1). These terminals may represent new species, either undescribed or described from other regions but not yet identified. In the Maculata clade six terminal taxa were recovered as strongly supported (Fig. 1). They are identified as Inocybe adaequata, I. rhodiola, I. erubescens, I. quietiodor, I. cookei, and I. maculata, including I. maculata forma fulva. For the separate regions, MrModelTest suggested GTR+I+G (ITS), K80+I (5.8S), GTR+I+G (LSU), and GTR+I+G (mtSSU) as optimal models; this information was employed in MrBayes. The Tracer and AWTY analysis indicated that 5 million generations would be an appropriate burn in time as a stationary state was reached for all chains well before that. This was also supported by the fact that the standard deviation of split frequencies calculated in MrBayes was below 0.01 well before this point. The last 5 000 trees of each run (20 000 trees in total) were therefore summarized into a 50 % majority-rule consensus phylogram (Fig. 1). No conflict was found between the nuclear and mitochondrial regions according to the criteria defined in Materials and Methods. 90 Persoonia – Volume 23, 2009 I. rimosa PAM03110904 I. rimosa EL 102/04 I. rimosa EL 211/06 I. rimosa SJ04/007 I. rimosa I. rimosa EL 75/05 I. rimosa PAM06112703 1/− I. rimosa AO2008/0250 I. rimosa EL 118/08 0.98 / 72 I. rimosa TK97/156 1 1 / 93 I. melliolens PAM05052302 I. melliolens 99 I. melliolens EL224/06 1 0.98 / − I. cfr sororia Kuoljok0512 I. cfr sororia 100 I. cfr sororia JV15200 1 I. cfr rimosa JV8125 81 I. cfr rimosa PC080925 I. bulbosissima EL30/06 I. bulbosissima EL88/06 I. bulbosissima EL37/06 I. bulbosissima 1 0.97 / − I. bulbosissima EL66/05 86 I. bulbosissima EL75/07 I. bulbosissima EL51/05 I. cfr rimosa JV26578 I. cfr rimosa PAM05061101 1/− I. cfr rimosa TAA185135 1 / 100 I. cfr rimosa JV22619 I. cfr microfastigiata EL113/06 0.97 / − I. cfr rimosa EL127/04 I. umbrinella PC081010 1/− I. umbrinella JV17954 1 / 97 0.97 / − I. umbrinella PAM01102912 I. umbrinella I. umbrinella JV13699 I. umbrinella PC080816 1 / 96 I. obsoleta BJ890915 I. obsoleta 1 / 88 I. obsoleta EL17/04 1 / 100 I. perlata EL74/04 I. perlata I. perlata BJ940922 I. cfr rimosa GGI113/05 1 / 99 I. cfr rimosa GGI116/06 I. cfr rimosa GGI93/04 I. cfr flavella EL118/05 1 / 88 I. cfr flavella PAM05062502 I. cfr flavella I. cfr flavella JK240908 I. cfr flavella EL90/04 1 89 I. cfr flavella BJ920829 I. squamata PAM05052301 1 / 98 I. squamata SJ85/048 1/− 1 I. squamata 78 I. squamata SJ08/003 0.99 / − I. squamata TK96/109 0.99 / 84 I. flavella LAS89/030 1 / 72 I. flavella EL137/05 I. flavella 1 I. flavella EL56/08 91 I. xanthocephala PAM00100606 1 / 75 I. hygrophorus EL97/06 I. hygrophorus I. sp. SJ92/010 I. sp. JV2607 1 / 100 0.95 I. sp. Stordal18318 I. sp. I. sp. SJ92/017 I. sp. SM92/013 I. dulcamaroides EL112/06 1 / 100 I. dulcamaroides I. dulcamaroides EL29/08 1 − 1 / 100 I. mimica TK2004/114 I. mimica 0.99 I. mimica EBJ961997 88 1 / 85 I. cfr rimosa EL81/06 I. arenicola EL238/06 1 1 I. arenicola 100 I. arenicola RC GB99/014 79 1 I. cfr rimosa JD2008/0241 − I. cfr rimosa EL71/04 Auritella geoaustralis AY380395 Auritella aureoplumosa AY635766 AURITELLA Auritella dolichocystis AY380371 B C RIMOSAE sensu stricto A D E F 1 / 100 Fig. 1 Bayesian 50 % majority-rule consensus phylogram. Bayesian posterior probabilities and bootstrap values above 70 % from the maximum parsimony analysis are indicated on branches. Recovered major clades are named and marked with a scale bar and minor supported clades discussed in the text have been numbered A–F. Conocybe siliginea was used to root the tree. 91 E. Larsson et al.: Relationships among species of section Rimosae MACULATA I. maculata f. fulva PAM 01100120 I. maculata f. fulva EL 78/03 I. maculata f. fulva EL166/08 1 / 97 I. maculata f. fulva I. maculata f. fulva EL247/06 I. maculata f. fulva SJ05/029 1 / 100 I. maculata f. fulva SJ06/007 I. maculata f. fulva EL 114/06 I. maculata EL121/04 I. maculata MR00020 1 / 83 1 / 91 0.98 / − I. maculata EL74/05 I. maculata I. maculata EL58/03 1 / 100 I. maculata EL126/04 I. maculata EL182/08 I. cookei EL191/06 I. cookei MR00035 0.96 / 73 I. cookei I. cookei EL70A/03 1 / 100 I. cookei EL73/05 1/− I. cookei EL109/04 I. cfr cookei EL104/04 1 / 72 I. quietiodor LAS97/067 I. quietiodor RPI19980718 I. quietiodor PAM01091310 I. quietiodor 1 / 100 I. quietiodor JV20202 I. quietiodor LAS94/023 I. quietiodor EL115/04 1 / 100 I. rhodiola EL223/06 1 / 92 1 / 98 I. rhodiola PAM00090117 I. rhodiola I. adaequata MR00022 1 / 100 I. adaequata 1 / 94 I. adaequata 2008/0014 I. erubescens BH910707 1 / 100 I. erubescens TAA185164 I. erubescens I. erubescens KGN980714 I. subhirsuta EL43/05 1 / 88 0.98 / − I. cfr calamistrata KHL13071 CERVICOLORES 1 / 98 I. cervicolor SJ04/024 I. bongardii EL123/04 I. fulvipes EL37/05 1 / 95 0.98 / − I. agardhii EL88/04 MALLOCYBE 1 / 100 I. terrigena EL117/04 I. dulcamara EL89/06 1 / 100 Crepidotus mollis EL45/04 Crepidotus calolepis EL14/04 1 / 80 1 / 81 Simocybe serrulata DQ494696/AY745706 Simocybe centunculus AF205707 0.99 / − Pleuroflammula flammea DQ494685/AF367962 1 / 99 Naucoria bohemica EL71B/03 Naucoria salicis EL71A/03 Naucoria submelinoides TAA185174 Conocybe siliginea LÖ9304 INOSPERMA Fig. 1 (cont.) 0.1 Also in this analysis Inocybe, including Auritella, is recovered as monophyletic with a BPP value of 1.00. The four major clades recovered in the maximum parsimony analysis, are present also in the Bayesian tree, all of them with BPP values 1.00. The Cervicolores (BPP 1.00) and Maculata clades (BPP 1.00) are also strongly supported. All 21 species clades from the maximum parsimony analysis of sect. Rimosae are also supported in the Bayesian analysis (Fig. 1). The Bayesian tree topology is more or less identical to the MP bootstrap tree. However, some additional clades were recovered with strong support, e.g. Crepidotaceae (BPP 0.99). The realigned dataset of 33 taxa in the Rimosae s.s. subclade A included 2 819 characters. After exclusion of regions with incomplete data, mainly from the mtSSU, 2 362 characters remained for the analysis. Of these 2 126 were constant, 83 were variable and parsimony uninformative, and 153 were parsimony informative. The heuristic searches recovered 4 290 equally most parsimonious trees (length = 340, CI = 0.7853, RI = 0.8933). Fig. 2 illustrates one of these as a mid-point rooted phylogram. The bootstrap analysis recovered the same strongly supported terminal clades as in the complete parsimony analy- sis but also generated moderate support (78 %) for I. rimosa s.s. Support for the remaining species level clades were: I. melliolens (99 %), I. cfr sororia (98 %), I. bulbosissima (98 %), and I. umbrinella (100 %). The North American sequences representing I. sororia clustered with North European sequences with 100 % support. The realigned dataset of 18 taxa in the Rimosae s.s. subclade D included 2 812 characters. After exclusion of regions with incomplete data, mainly from the mtSSU, 2 421 characters remained for the analysis. Of these 2 266 were constant, 57 were variable and parsimony uninformative, and 98 were parsimony informative. The heuristic search recovered 675 most parsimonious trees (length = 190, CI = 0.8526, RI = 0.9111). Fig. 3 illustrates one of these as a mid-point rooted phylogram. The bootstrap analysis recovered five strongly supported (above 90 %) clades of which four have the same topology as in the complete analyses, viz. I. squamata (100 %), Inocybe sp. (100 %), I. flavella (100 %), and I. cfr flavella A (99 %) while I. cfr flavella B is supported only together with I. hygrophorus (100 %). Inocybe flavella + I. xanthocephala (91 % in the large parsimony analysis) is not supported. 92 Persoonia – Volume 23, 2009 found that the terminal clades we have been able to correlate to morphological species, in general show a low within clade sequence divergence. Notable exceptions to this observation are I. rimosa and related species (Fig. 2 subclade A), and I. flavella and related species (Fig. 3 subclade D). DISCUSSION The present study aimed at elucidating the phylogenetic structure of Inocybe subg. Inosperma sect. Rimosae as defined by Kuyper (1986) but included also representatives from the subg. Mallocybe and the recently erected genus Auritella (Matheny & Bougher 2006a, b). The ingroup was recovered as monophyletic and strongly supported. However, our results indicate that recognizing Auritella on the genus level renders Inocybe paraphyletic. The solution is either to sink Auritella as a subgenus within Inocybe, or to split Inocybe into a number of smaller genera. However, our study was not designed to take a decision on that matter. The Maculata clade is here represented by seven species with thin-walled, often clavate to pyriform cheilocystidia and phaseoliform spores. They usually have specific odours that differ from the spermatic smell typical of most species in the Rimosae s.s. clade. Inocybe maculata smells like raw potatoes or Tuber, I. cookei like honey, I. erubescens like perfumed soap, I adaequata and I. rhodiola like beetroot, and I. quietiodor like Lactarius quietus, that is, both sourish and sweetish. Having specific odours is a character they share with the species in sect. Cervicolores. However, odour is a very difficult and subjective character to use and although a spermatic smell characterizes most species in the Rimosae s.s. clade some species have other characteristics, e.g. I. melliolens Kühner (1988), which smells of honey when drying. We found that the species traditionally placed in sect. Rimosae did not form a monophyletic clade. Instead they are distributed over two strongly supported clades: Maculata and Rimosae s.s. (Fig 1). The Maculata group clusters with sect. Cervicolores and the two combined represent subg. Inosperma in a new stricter sense. A more narrowly defined sect. Rimosae emerges as an independent supported clade well separated from Inosperma s.s. There is a trend in the Maculata clade for the stipe base to be distinctly bulbous. This characteristic is present in I. cookie, I. maculata, I. maculata forma fulva, and I. quietiodor, while in I. adaequata, I. erubescens, and I. rhodiola the bulbous base is not as pronounced. Several species are characterized by a reddening of the flesh (I. adaequata, I. erubescens, I. rhodiola) and this trait occurs also among species in Cervicolores. In the study by Matheny (2006) a division of sect. Rimosae was indicated although only one representative of the Rimosae s.s. clade was included. In a recent biogeographic study of Inocybaceae more representatives of the Rimosae s.s. clade were included and the split topology again supported (Matheny et al. 2009). Matheny uses Pseudosperma as the clade name for what is here called Rimosae s.s. Inocybe maculata is known as a variable species with considerable differences in cap colour and presence of velar remnants. These observations correspond to a high divergence among the sequences generated from specimens initially identified as I. maculata in a wide sense. The specimens we have sequenced can be divided in two morphotypes which also seem to correlate with ecological preferences. One type has a chocolate brown cap, often covered with conspicuous, white velar remnants. It grows associated with Carpinus, Corylus, Fagus, Quercus, and Tilia on rich soils, usually on calcareous ground. This type fits with the original description of I. maculata but unfortunately the specimens representing this morphotype do not form a monophyletic clade and the sequence divergence indicates that more than one species are involved (Fig.1, 4a). The other morphotype is more yellow to reddish brown and has less or no Through our morphological investigations we were able to correlate 21 terminal groups within clades Maculata and Rimosae s.s. with morphologically distinct species (Fig. 1). The molecular support is based on data from nLSU, mtSSU, 5.8S and a few conservative regions of ITS, leaving out the variable regions of ITS because of aligning problems. The ITS region is the locus that has been most commonly used for species delimitation of fungi (Kõljalg et al. 2005, Kõljalg & Larsson 1998, Larsson & Örstadius 2008, Nilsson et al. 2008). In general Inocybe species show a high sequence divergence in the ITS region. Closely related species often deviate in several substitutions and insertion/deletion events and are therefore easy to identify using simple sequence comparison (Altschul et al. 1997). We also Fig. 2 One of the equally most parsimonious trees obtained from the maximum parsimony analysis of the Rimosae s.s. subclade A, presented as a mid-point rooted phylogram. Bootstrap values are indicated on branches. 88 5 changes I. rimosa TK97/156 I. rimosa SJ04/007 I. rimosa PAM06112703 I. rimosa EL75/05 I. rimosa PAM03110904 78 I. rimosa EL211/06 I. rimosa AO2008/0250 90 I. rimosa EL118/08 I. rimosa EL 102/04 99 I. melliolens PAM05052303 99 I. melliolens EL 224/06 I. cfr sororia Kuoljok0512 98 100 I. cfr sororia JV15200 I. sororia DQ917657/EU600863 I. cfr rimosa JV8125 98 I. cfr rimosa PC080925 I. cfr rimosa JV26578 I. cfr rimosa PAM05061101 I. bulbosissima EL88/06 I. bulbosissima EL66/05 I. bulbosissima EL51/05 98 I. bulbosissima EL30/06 I. bulbosissima EL37/06 I. bulbosissima EL75/07 I. cfr rimosa EL127/04 I. cfr microfastigiata EL113/06 I. cfr rimosa TAA185135 100 I. cfr rimosa JV22619 I. umbrinella PAM01102912 I. umbrinella PC081010 100 I. umbrinella JV17954 I. umbrinella PC080816 I. umbrinella JV13699 E. Larsson et al.: Relationships among species of section Rimosae 99 100 5 changes 93 I. cfr flavella EL 90/04 I. cfr flavella BJ920829 I. cfr flavella PAM05062502 I. cfr flavella JK240908 I. cfr flavella EL 118/05 I. hygrophorus EL97/06 I. flavella LAS89/030 100 I. flavella EL137/05 81 I. flavella 56/08 I. xanthocephala PAM00100606 I. sp. SJ92/010 I. sp. Stordal18318 100 I. sp. SM92/013 I. sp. SJ92/017 I. sp. JV2607 I. squamata PAM05052301 73 Fig. 3 One of the equally most parsimonious trees obtained from the maximum I. squamata SJ08/003 100 parsimony analysis of the Rimosae s.s. subclade D, presented as a mid-point rooted I. squamata SJ85/048 I. squamata TK96/109 phylogram. Bootstrap values are indicated on branches. velar remnants on the cap. This type seems to be associated with Betula, Picea, Pinus, Populus, and Salix, but also with Dryas and Polygonum in alpine environments. It fits the concept of I. maculata forma fulva Bon (1991, Fig. 4b) described from a coastal dune area with Populus in northern France. Of the seven specimens sequenced two originate from France, one of them from the same region and ecological setting as the type of forma fulva. Sequence data are uniform for this taxon and clade support is strong (97 %, BPP 1.0). The LSU sequences deposited in GenBank as I. sororia originate from a Northwest American conifer stand (Matheny et al. 2009). They are almost identical to our sequences from two specimens from herb-rich locations and dwarf Salix in the alpine zone of Sweden (Fig. 2). Inocybe sororia is described by Kauffman (1924) from North American frondose forests. Since we have only studied the Swedish material and they deviate from the descriptions given by Kauffman (1924) and Stuntz (1947) we feel uncertain about the identity of our collections. The Rimosae s.s. clade includes six strongly supported subclades (Fig. 1A –F) and altogether 15 terminal clades that we could correlate to morphologically distinguishable species. In addition Rimosae s.s. also includes several unidentified minor terminal clades and sequences that occur on single branches (Fig. 1). In general the species in this clade have ellipsoid to indistinctly phaseoliform spores and the cheilocystidia tend to be more cylindrical to clavate than in the Maculata clade. However, the shape of the cheilocystidia is very variable even within the same species. In this clade we also more often find that the apex of the stipe is distinctly white pruinose to flocculose. The spermatic odour and the presence of yellow to olive-yellow pigments in the lamellae are characteristic for many species in Rimosae s.s. The occurrence of oily refracting contents in hyphae is notable and may be connected to the spermatic odour. Kuyper (1986) found a correlation between the intensity of the olive-yellow tone of the lamellae and the strength of the spermatic odour. Inocybe bulbosissima includes specimens associated with dwarf Salix and Dryas in the alpine zone. The species is usually regarded as a variety of I. rimosa and then named I. fastigiata var. alpina. Another alpine species with similar characteristics is I. microfastigiata which is said to differ by smaller spores and a darker brown cap (Kühner 1988). A specimen with such morphology was included in this study and did not cluster with I. bulbosissima (Fig. 2, 4d). More specimens and sequences are required before the circumscription of I. microfastigiata can be clarified. Rimosae subclade A. This clade corresponds to I. rimosa in a wide sense and includes 33 specimens (Fig. 2) originating from England, Estonia, France, and Scandinavia. They represent most of the large variation in macro-morphological characters and ecology demonstrated by the many varieties described (Heim 1931, Favre 1955, Kühner 1988, Bon 1997). Five terminal clades with moderately strong to strong support were recovered and are further discussed below. Still other sequences form unsupported groups or occur on single branches (Fig. 1, 2). Inocybe rimosa s.s. includes nine specimens that show a great variation in cap colours from pale to ochraceous yellow brown to dark brown. In micro-morphology they are all very similar. The specimens originate from different habitats ranging from nemoral deciduous forests to boreal Picea forest. There is a nine base pair insertion in the beginning of the ITS region in five of the specimens but this difference could not be correlated to differences in morphology or ecology. The specimens named I. melliolens originate from France. In morphology this species looks like a typical I. rimosa s.s. but has a strong smell of honey. We have not seen any specimens with this character from Northern Europe and the species may have a more southern distribution, even if it is described from Dryas vegetation in the French Alps (Bon 1997). Inocybe umbrinella includes specimens with warm yellowish to reddish brown caps with a dark centre and contrasting strongly rimose and lighter periphery. The micro-morphology is almost identical to I. rimosa. In Bresadola´s description (Bresadola 1905) it is said to grow in gravely places with Populus nigra. Three of our specimens were collected in rather dry, sandy environments with Helianthemum, Pinus, and Quercus ilex. In our opinion these specimens fit the descriptions by Bresadola (1905) and Enderle & Stangl (1981). Some other specimens with a dark brown cap clustering with or close to I. rimosa were first determined as I. umbrinella (Table 1). These specimens have fruit-bodies that in general are somewhat smaller than typical I. umbrinella. Inocybe umbrinella may also be confused with I. perlata but I. perlata usually has a dull dark brown cap and a flattened, less pronounced umbo. Rimosae subclade B. Inocybe obsoleta and I. perlata were recovered as independent species. Both have rather large and robust fruit-bodies and occur in mixed deciduous forests and parks, often on somewhat calcareous and nutrient rich soils. In micro-morphology they are hard to separate from I. rimosa. The best characters for identification are the robust fruit-bodies and the dark greyish brown colour without any yellow flush on the cap in I. perlata and the pale clay yellowish brown cap with distinct, white velum remnants in young specimens of I. obsoleta. Our interpretation of the name I. obsoleta is here based on specimens from North Europe only. Rimosae subclade C includes three specimens that originate from Australia. We were not able to correlate them to known species and they are provisionally named I. cfr squamata and I. cfr rimosa. They represent two species closely related to I. rimosa but may not occur in North Europe. 94 Rimosae subclade D includes species that are often encountered in mixed forests with Salix and Populus. We identified I. flavella s.l., I. hygrophorus, I. squamata, and one undescribed species (Inocybe sp.). Inocybe hygrophorus is represented by only one specimen. It was collected in a subalpine meadow in forest with Salix and Betula and fits the original morphological description. Sequence data confirm that it is distinct from I. flavella. It seems to be rare or maybe overlooked as it may be mistaken for I. flavella or I. rimosa. The two species I. squamata and Inocybe sp. are very similar in macro-morphology, with scattered appressed scales on the cap. The spores are distinctly phaseoliform and narrow in Inocybe sp. while in typical I. squamata they are broadly ellipsoid and only occasionally slightly phaseoliform. We observed that the lamellae were more yellow and the fruit-bodies on average larger in Inocybe sp. This possibly undescribed species also seems to have a boreal distribution judging from the known records from Sweden, Finland, and Norway. Inocybe squamata is, in the Nordic countries, only known from nemoral and hemiboreal regions. The specimens determined as I. flavella split into three supported subclades. We have included specimens that originate from Sweden, Finland, England, and France. We identified two morpho-types, here named I. flavella and I. cfr flavella. The specimens are all rather similar in macro-morphology, but show a variation in the presence of yellow pigments on lamellae and stipe and in the colour and structure of the cap. No special odour was detected. In micro-morphology a variation in the length and shape of the spores can be observed. In I. flavella s.s. the spores are 10–12 × 5–6 µm and usually not phaseoliform. In the clade named I. cfr flavella the spores are shorter 9–10.5 × 5–6 µm and often more or less phaseoliform. The analysis including the ITS region confirm the high sequence divergence within I. flavella s.l. (Fig. 3). There are several species and forms described as close to I. flavella (Heim 1931, Orton 1960, Kuyper 1986, Kühner 1988, Bon 1997) and additional sequence data is needed to disentangle the entities involved within this clade. Rimosae subclade E. Inocybe dulcamaroides is an arcticalpine species associated with Dryas and Salix reticulata. The two sequences representing this species are 100 % identical throughout the ITS region despite originating from Sweden and USA (Montana), respectively. It is reminiscent of I. dulcamara in that it has a short stipe in comparison to its cap diameter. This makes it a morphologically characteristic species but it is little collected and seemingly rare or overlooked (Fig. 4c). Rimosae subclade F includes the two species, I. arenicola and I. mimica, which are characterized by rather large spores. They cluster together with sequences of three specimens which could not be matched with any species descriptions. The only uniting factor we found in this clade was a preference for calcareous soil conditions. Other morphological and ecological traits show a large variation. Most of the species discussed in this paper are typified with material that turned out to be less suitable for DNA extraction. Some collections are simply old, others, e.g. those of Marcel Bon, are apparently dried under conditions that did not preserve DNA well. Still others are very scanty, e.g. most of Kühner’s type specimens. This situation is not at all unusual within fungal taxonomy. If we shall be able to take full advantage of the higher precision of species definitions made possible by molecular data, we must make extensive use of the epitypification tool offered by ICBN. Like all typification measures, also the selection of an epitype must be done with utmost care in order to preserve the intentions of the original author. Our preferred method is to first seek a profound understanding of the regional species Persoonia – Volume 23, 2009 diversity through intense field work, then match our collections with existing names, compare them to authoritative material, and finally, if necessary, select epitypes from rich, molecularly characterized collections. This study had a focus on the species that occur in European arctic and alpine environments and in temperate regions of North Europe. Many of the species belonging in sect. Rimosae which are described from North America and from the Mediterranean have yet to be sampled before a more complete understanding of the phylogenetic diversity of the Maculata and Rimosae s.s. clades can be achieved. Only then will it be appropriate to fix names through epitypification. TAXONOMY Species of the Maculata clade identified in Northwest Europe Inocybe adaequata (Britzelm.) Sacc., Syll. Fung. (Abellini) 5: 767. 1887 Specimens examined. GREAT BRITAIN, England, Bucks, Kings Wood Tylers Green, 19 Aug. 2008, P. Cullington 2008/0014. – SWEDEN, Bohuslän, Valla, Sundsby, 1 Sept. 1979, SJ79154; Bohuslän, Tanum, Lammö, 29 Sept. 2004, MR00022; Västergötland, Kinnekulle, Medelplana, Råbäcks munkängar, 6 Aug. 1977, SJ77120: Inocybe cookei Bres., Fungi Trident. 2, 8–10: 17. 1892 Specimens examined. GREAT BRITAIN, Scotland, Ledmore oakwood, 14 Sept. 2006, EL191-06. – NORWAY, Buskerud, Hönefoss, Grunntjern, 28 Aug. 2003, J. Vauras 20202 (TUR-A). – SWEDEN, Västergötland, Alingsås, Nolhagaparken, 30 Aug. 2003, EL70A-03; Alingsås, Nolhagaparken, 21 Aug. 2005, EL73-05; Alingsås, Nolhagaparken, 2 Sept. 2006, EL150-06; Östad, Österäng, 26 Sept. 2004, EL109-04; Östad, Ekedalen, 26 Sept. 2004, EL104-04; Östad, Östad säteri, Djurgården, 4 Sept. 2008, EL67-08; Göteborg, Botaniska Trädgården, 19 Sept. 1975, SJ386; V. Tunhem, Hunneberg, 4 Oct. 2004, MR00035; Töreboda, Älgarås, Velen, 6 Sept. 2003, EL50-03; Grimmered, Björräsakulle, 17 Aug. 1989, SJ89002. Inocybe erubescens A. Blytt, Videnskabs-Selskabets Skrifter. I Math.-Naturv. Kl., 6: 54. 1905 (‘1904’) Specimens examined. ESTONIA, Saaremaa, Tagamösa, 29 July 2004, TAA185164. – Sweden, Närke, Askersund, Stjärnsund, 14 July 1998, K-G Nilsson; Skåne, Genarp, Häckeberga, 7 July 1991, Bernt Hägg. Inocybe maculata Boud., Bull. Soc. Bot. France 32: 283. 1885 Specimens examined. DENMARK, Falster, Nykøbing, Fuglsang Storskov, 3 Oct. 2007, EL136-07. – SLOVAKIA, Rimavská Sobota, Drña, 3 Oct. 2008, EL182-08. – SWEDEN, Bohuslän, Tanum, Lammö, 29 Sept. 2004, MR00020; Halland, Fjärås, Tjolöholm, 20 Sept. 1975, SJ389; Västergötland, Karlsborg, Undenäs, Bölet, 5 Sept. 2003, EL45-03; Västergötland, Berg, Högsböla ängar, Melldalaskogen, 7 Sept. 2003, EL58-03; Västergötland, Medelplana, Råbäcks Munkängar, 5 Sept. 2003, EL41-03; Västergötland, Kinnekulle, Medelplana, Råbäcks Munkängar, 27 Sept. 2004, EL121-04; Västergötland, Kinnekulle, Medelplana, Råbäcks Munkängar, 27 Sept. 2004, EL126-04; Västergötland, Alingsås, Nolhagaparken, 30 Aug. 2003, EL68-03; Västergötland, Alingsås, Nolhagaparken, 21 Aug. 2005, EL74-05. Inocybe maculata forma fulva Bon, Doc, Mycol. 21 (no. 81): 47. 1991 Specimens examined. FRANCE, Merlimont, Pas de Calais, 3 Nov. 2006, EL247-06; Isére, Chireas, 1 Oct. 2001, PAM01100120 (LIP). – SWEDEN, Härjedalen, Hamra, Hamrafjället, 18 Aug. 2006, EL114-08; Dalarna, Rättvik, Ö. om Gärdsjöns sydände, 23 Aug. 1982, R. Morander 4320; Dalarna, Rättvik, Rättviksheden, 29 Aug. 2005, SJ05-029; Närke, Lerbäck, Udden, 13 Aug. 1997, K-G Nilsson; Närke, Lekhyttan, Lunnasjön, 13 Sept. 2008, EL166-08; Närke, Lerbäck, Runsala ravin, 11 Sept. 2008, EL134-08; Närke, Kvistbro, Sixtorp, Gammelhyttan, 9 Sept. 2008, EL82-08; Skåne, Hässleholm, Igna- 95 E. Larsson et al.: Relationships among species of section Rimosae a b c d Fig. 4 a. Inocybe maculata (EL182/08); b. Inocybe maculata forma fulva (EL82/08); c. Inocybe dulcamaroides (EL112/06); d. Inocybe bulbosissima (EL75/07). berga, 15 Sept. 2003, EL78-03; Värmland, Övre Ullerud, Torsberget SSO, 17 Aug. 1991, Bo Jansson; Lule Lappmark, Jokkmokk, SSO Messaure, 23 Aug. 2003, S. Kuoljok 0337. Inocybe quietiodor Bon, Doc. Mycol. 6 (no. 24): 46. 1976 Specimens examined. DENMARK, Lolland, Flintinge byskov, 4 Oct. 2007, EL142-07. – FRANCE, Isére, Claix, Le Penil, 13 Sept. 2001, PAM01091310 (LIP); Namps-au-Val, Fremoutiers, 14 Sept. 1994, RC/F94064 (LIP). – SWEDEN, Västergötland, Alingsås, 18 July 1998, RP98/048; Västergötland, Götene, Medelplana, 28 Sept. 1997, LAS97/067; Västergötland, Kinnekulle, Medelplana, Råbäck, 10 Sept. 1994, LAS 94/023; Västergötland, Kinnekulle, Österplana, 27 Sept. 2004, EL115-04; Västergötland, Kinnekulle, Medelplana, Råbäcks Munkängar, 8 Sept. 2008, EL73-08. Inocybe rhodiola Bres., Fungi Trident. 1: 80. 1884 Specimens examined. FRANCE, Saint-Amand, Dréve des Prés Charniers, 31 Oct. 2006, EL223-06; Isére, Saint Laurent du Pont, 1 Sept. 2000, PAM00090117 (LIP). – ITALY, Cuneo, Ceva, Alessio, 12 Sept. 1980, Bon80091207 (LIP). Species of the Rimosae s.s. clade identified in Northwest Europe Inocybe arenicola (R. Heim) Bon, Doc. Mycol. 12 (no. 48): 44. 1983 (‘1982’) Specimens examined. ESTONIA, Saaremaa: Kaarma, Mändjala, 19 Sept. 2008, J. Vauras 26578 (TUR-A). – FRANCE, Merlimont, Pas de Calais, 2 Nov. 2006, EL238-06; Quend-les-Pines, 18 May 1983, Bon83047 (LIP). – GREAT BRITAIN, Sandscale, Haws-Nears, Barrow on Furness, 31 Aug. 1999, RC/ GB99.014. Inocybe bulbosissima (Kühner) Bon, Bull. Mycol. Bot. Dauphiné-Savoie 32 (no. 126): 19. 1992 Specimens examined. FRANCE, Les Arcs 2000 (73), Lac Marloup, 24 Aug. 2000, Bon (LIP). – NORWAY, Hordaland, Ulvik, Finse, Sandalsnut, 12 Aug. 2005, EL51-05; Hordaland, Ulvik, Finse, Sandalsnut, 12 Aug. 2005, EL4705; Hordaland, Ulvik, Finse, Sandalsnut, 14 Aug. 2005, EL66-05. – SWEDEN, Härjedalen, Tännäs, Hamra, Hamrafjället, 15 Aug. 2006, EL88-06; Torne Lappmark, Jukkasjärvi, Latnjajaure, 3 Aug. 2006, EL30-06; Torne Lappmark, Jukkasjärvi, Latnjajaure, 4 Aug. 2006, EL37-06; Torne Lappmark, Jukkasjärvi, Latnjajaure, 11 Aug. 2007, EL75-07. Inocybe dulcamaroides Kühner, Doc. Mycol. 19 (no. 74): 18. 1988 Specimens examined. SWEDEN, Härjedalen, Tännäs, Hamra, Hamrafjället, 18 Aug. 2006, EL112-06. – USA, Montana, Carbon County, Quad Creek, 8 Aug. 2008, EL29-08. Inocybe flavella P. Karst., Meddel. Soc. Fauna Fl. Fenn. 16: 100. 1890 Specimens examined. FINLAND, Etelä-Hämä, Juupajoki, Korkeakoski, 9 Sept. 2005, EL118-05. – FRANCE, Abscon, Carriere des Peupliers, 25 Apr. 2005, PAM05042502 (LIP); Isère, Saint Laurent du Pont, 6 Oct. 2000, PAM00100606 (LIP). – GREAT BRITAIN, England, Haycop, Shropshire, 24 Sept. 2008, G. Kibby. – SWEDEN, Bohuslän, Torslanda, Sillvik, 3 July 2004, SJ04-005; Bohuslän, Tanum, Kalvö, 16 Sept. 2004, EL90-04; Bohuslän, Munkedal, Foss, 9 Sept. 1989, LAS89/030; Skåne, Fågeltofta, Kronovalls sumpskog, 21 Aug. 2005, EL137-05; Skåne, Fågeltofta, Kulladal, 21 Sept. 2005, EL137-05; Västergötland, Östad, Djurgården, 3 Sept. 2008, EL56-08; Värmland, Övre Ullerud, Torberget, 30 Aug. 1990, B. Jansson; Värmland, Fryksände, Fensbol, 29 Aug. 1992, B. Jansson. 96 Inocybe hygrophorus Kühner, Bull. Trimestriel Soc. Mycol. France 71: 169. 1956 (‘1955’) Specimens examined. FRANCE, Beaufort, Savoie, Col des Pris, 28 Aug. 2008, PAM08082801 (LIP).– SWEDEN, Härjedalen, Tännäs, Sandåsvallen, 16 Aug. 2006, EL97-06. Inocybe mimica Massee, Ann. Bot., Lond. 18: 492. 1904 Specimens examined. SWEDEN, Öland, Penåsa, 8 Sept. 2004, TK2004114; Gotland, Tjaukle, 7 Oct. 1996, Elsa Bohus-Jensen. Inocybe obsoleta Romagn., Bull. Trimestriel Soc. Mycol. France 74: 145. 1958 Specimens examined. ESTONIA, Saaremaa, Torgu, Viieristi Nature Reserve, 20 Sept. 2008, J. Vauras 26619 (TUR-A). – SWEDEN, Bohuslän, Valla, Sundsby, 12 Sept. 1982, SJ82068; Bohuslän, Torslanda, Röds skalgrusbank, 28 Aug. 2008, SJ08-006; Bohuslän, Resteröd, Ulvesund, 25 July 2004, EL17-04; Bohuslän, Resteröd, Ulvesund, 18 Sept. 2004, EL10004; Västergötland, Trollhättan, Åkerströms naturreservat, 10 Aug. 1986, LAS86/024; Västergötland, Fors, nära Slumpån, 5 Aug. 1977, Leif Stridvall; Värmland, Karlstad, Trangård, 15 Sept. 1989, Bo Jansson. Inocybe perlata (Cooke) Sacc., Syll. Fung. (Abellini) 5: 774. 1887 Specimens examined. FINLAND, Varsinais-Soumi, Lohja rural commune, Vitkkala, 7 Aug. 1988, J. Vauras 3091. – SWEDEN, Bohuslän, Uddevalla, Kuröds skalbankar, 15 Sept. 2004, EL74-04; Värmland, Visnum, Värmlands Säby, 22 Sept. 1994, Bo Jansson. Inocybe rimosa (Bull.: Fr.) P. Kumm., Führer Pilzk. (Zwickau): 78. 1871 Specimens examined. ESTONIA, Hiiumaa, Käina, Kassari, Sääre, 17 Sept. 2001, J. Vauras 17954 (TUR-A). – FINLAND, Varsinais-Soumi, Lohja, Virkkala Kyrkstad, 16 July 1998, J. Vauras 13699 (TUR-A); Varsinais-Soumi, Lohja, Virkkala, Pähkinäniemi, 10 Aug. 1993, J. Vauras 8125 (TUR-A). – FRANCE, Lille, Faculté du Pharmacie, 23 May 2005, PAM05052303 (LIP); Lille (Nord), 11 June 2005, PAM05061101 (LIP); Pyrénées Atlantiques, Odres, 9 Nov. 2003, PAM03110906 (LIP); Corsica, Bonifacio, La Tonnare, 27 Nov. 2006, PAM06112703 (LIP). – GREAT BRITAIN, England, Cambs, Fowlmere RSPB reserve, 2008, A. Outen 2008/0250; England, Nesscliffe, Shropshire, 25 Sept. 2008, P. Cullington 08.09.25; England, Essex, Hales Wood, 25 Sept. 2008, J. Darby 2008 / 0241. – SWEDEN, Bohuslän, Tanum, Kville Hjärterön, 14 Sept. 2004, EL54-04; Bohuslän, Sotenäs, Hogsäms bokskog, 15 Sept. 2004, EL71-04; Bohuslän, Tanum, Lur, Galtö, 15 Sept. 2004, EL80-04; Torne Lappmark, Jukkasjärvi, Abisko, Björkliden, 17 Aug. 1999, J. Vauras 15200; Västergötland, Göteborg, Sahlgrenska, 13 July 2004, SJ04-007; Västergötland, Alingsås, Kullingsberg, 19 Sept. 2004, EL102-04; Västergötland, Alingsås, Nolhagaparken, 21 Aug. 2005, EL75-05; Västergötland, Meldelplana, Råbäcks Munkängar, 27 Sept. 2004, EL127-04; Öland, Torslunda, S. om Tvetabäcken, 12 Oct. 1997, TK97-156. Inocybe squamata J.E. Lange, Dansk Bot. Ark. 2 (no. 7): 39. 1917 Specimens examined. FINLAND, Varsinais-Soumi, Turku, Ilpoinen, 14 July 1987, J. Vauras 2607 (TUR-A). – FRANCE, Monbéqui, Tarn et Garonne, 11 Nov. 2003, PAM03111204 (LIP); Lille, Faculté du Pharmacie, 23 May 2005, PAM05052301 (LIP). – NORWAY, Oppland, Östra Toten, 5 July 1977, J.Stordal 18318 (O); Oppland, Lunner, 30 July 2004, T.E. Brandrud 102-04 (O); Oslo, Hovedöya, 19 Aug. 1985, SJ85048. – SWEDEN, Bohuslän, Torslanda, Röds skalgrusbank, 10 Aug. 2008, SJ08-003; Bohuslän, Torslanda, Röds skalgrusbank, 28 Aug. 2008, SJ08-007; Jämtland, Lit, Niklasbodarna, 10 Aug. 1992, SJ 92010; Jämtland, Östersund, Lövbergaparken, 11 Aug. 1992, SJ92-017; Medelpad, Tuna, Uvberget, 19 Aug. 1992, S.Muskos 92-013; Öland, Gräsgård, Löt, SSV Solberga, 27 Aug. 1997, TK96-109. Persoonia – Volume 23, 2009 Porquerolles, 29 Oct. 2001, PAM01102912 (LIP). – GREAT BRITAIN, England, Oxon, Watlington Hill, 10 Oct. 2008, P. Cullington 10.10.08; England, Bucks, Kings Wood, 16 Aug. 2008, P. Cullington 16.08.08. – ITALY, Alto Adige, Trento, Desert, 3 June 1899, Bresadola (holotype S). KEY TO THE SpECIES OF THE MACulATA AND RIMosAE S.S. CLADES OCCURRINg IN NORTHwEST EUROpE 1. Basidiocarp ± reddening with age or handling . . . . . . . 2 1. Basidiocarp not reddening . . . . . . . . . . . . . . . . . . . . . . 4 2. Pileus robust, whitish, slowly turning brick red with age or from damage . . . . . . . . . . . . . . . . . . . . . . . I. erubescens 2. Frb red brown with a ± vinaceous tinge . . . . . . . . . . . . 3 3. Pileus robust, fibrillose, sometimes breaking up into scales, stipe slowly staining somewhat vinaceous. Smell rather strong, reminding of beetroot . . . . . . . . . . . I. adaequata 3. Pileus more slender, fibrillose–squamose. Stipe becoming distinctly vinaceous red towards the base. Smell weak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. rhodiola 4. With evident, distinct smell of various compounds but not spermatic. Spores generally phaseoliform. Cheilocystidia broadly clavate to pyriform . . . . . . . . . . . . . . . . . . . . . . 5 4. Smell, if present, spermatic. Spores variable, mostly ellipsoid. Cheilocystidia generally slenderly clavate or cylindrical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5. Pileus predominantly yellow. Stipe with a distinct bulb . 6 5. Pileus brown or brownish. Stipe equal or subbulbous . 7 6. Smell of honey. Spores 7–9 × 4–5 µm, distinctly phaseoliform. Cheilocystidia pyriform . . . . . . . . . . . . . . I. cookei 6. Smell recalling Lactarius quietus, spores 8–11 × 5.5 –6.5 µm, less distinctly phaseoliform . . . . . . . . . I. quietiodor 7. Pileus hazel brown to dark brown, often with white, conspicuous velar patches at centre. Growing in nutrient-rich Fagus or Quercus forests . . . . . . . . . . . . . . . I. maculata 7. Pileus brownish, generally with a ± fulvous tinge, white velar patches often less evident. Growing with various broad-leaved or coniferous trees I. maculata forma fulva 8. Pileus with scales (may sometimes disappear) . . . . . . 9 8. Pileus fibrillose-rimose, without scales . . . . . . . . . . . . 11 9. Spores 12 –15 × 6 – 8 µm, ellipsoid. Pileus > 65 mm, reminding of I. rimosa, gills with a faint olivaceous tinge. Stipe initially whitish, then reddish brown. Under deciduous trees on calcareous soils. Very rare and poorly known . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. mimica 9. Spores smaller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 10. Spores 8–10 × 5.5–6.5 µm, broadly ellipsoid (Q = 1.4–1.6). Pileus 20–50 mm, yellowish brown; lamellae initially without or with only weak yellow tinge. With deciduous trees on calcareous soils. In temperate or hemiboreal areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. squamata 10. Spores 8.5 –11 × 4.5 –6 µm, often somewhat phaseoliform (Q = 1.6 – 2.0). Pileus 30 – 90 mm, yellowish to reddish brown, outwards more yellow. Lamellae initially pale yellow. With deciduous and coniferous trees, boreal Inocybe sp. Inocybe umbrinella Bres., Ann. Mycol. 3 (2): 161. 1905 11. Growing with Salix repens or Pinus in dune sand along coasts in western Europe. Pileus 25 –70 mm, initially whitish due to thick velipellis, beneath this straw yellow or ochraceous; gills initially white. Stipe solid, often deeply buried in sand. Spores 12 –16 × 6–8.5 µm . . I. arenicola 11. In other habitats. Pileus generally yellow to brown . . . 12 Specimens examined. ESTONIA, Hiiumaa district, Käina commune, Kassari, 17 Sept. 2001, J. Vauras 17954 (TUR). – FINLAND, Varsinais-Suomi, Lohja, Virkala, 16 July 1998, J. Vauras 13699 (TUR-A). – FRANCE, Ile de 12. Pileus flocculose from a thick universal veil, ochraceous brown, 10 – 25 mm (reminding of I. dulcamara). Cheilocystidia with internal drops of brown pigments, broadly E. Larsson et al.: Relationships among species of section Rimosae clavate to utriform. Spores 11–14 × 7–8.5 µm. With dwarf Salix in arctic/alpine zones . . . . . . . . . . I. dulcamaroides 12. Pileus fibrillose-rimose. Cheilocystidia without drops . 13 13. Arctic/alpine species with a distinct, white bulb at the base and large spores, 12–15 × 6–8 µm. Pileus 10–35 mm, pale yellow, then ochraceous to reddish brown. Smell spermatic . . . . . . . . . . . . . . . . . . . . . . . . I. bulbosissima 13. Not so . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 14. Pileus only finely fibrillose (almost smooth in centre), pale brownish with a yellow tinge, lamellae with a yellowish olive tinge. Stipe almost equal, 50–80 × 8–12 mm. Spores 9–11 × 4.5–5.5 µm, phaseoliform. In mountainous areas, mixed forest with Salix . . . . . . . . . . . . . . . . . . . . I. hygrophorus 14. Pileus distinctly fibrillose/rimose . . . . . . . . . . . . . . . . . 15 15. Pileus brown without yellow tinges . . . . . . . . . . . . . . . 16 15. Pileus not brown, with ± yellow pigments . . . . . . . . . . 17 16. Large species with acute umbo, reminding of I. rimosa. Pileus 35–100 mm. Stipe 80–120 × 8–13 mm, becoming brownish with age. Spores 10–13 × 6–8 µm. Under deciduous trees in forests and parks . . . . . . . . I. perlata 16. Smaller species. Pileus 20–45 mm, hazel to cinnamon brown, usually with blunt umbo. Spores 10–13 × 5.5 –6.5 µm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. umbrinella 17. Pileus with a distinct white velipellis. Lamellae without olivaceous tinge. Odour absent. Microscopically as I. rimosa. Under deciduous trees on calcareous ground I. obsoleta 17. Pileus without distinct white velipellis . . . . . . . . . . . . . 18 18. Pileus typically distinctly umbonate and strongly rimose. Lamellae with an olivaceous yellow tinge. Smell spermatic. Spores 9.5 –12.5 × 6–7 µm, generally ellipsoid and only exceptionally somewhat phaseoliform . . . . . . . I. rimosa 18. Pileus fibrillose-rimulose, not strongly rimose. Lamellae initially clay-coloured lacking or with only a weak yellow tinge. Smell absent. Spores 9–12 × 5–6 µm, often phaseoliform. Cheilocystidia narrow. Pileus more yellow and less rimose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 19. Spores 10–12 × 5–6.5 µm, not phaseoliform. Pileus 20–60 mm, fibrillose, yellowish brown, predominantly yellow towards the margin . . . . . . . . . . . . . . . . . . . . . . . I. flavella 19. Spores 9–10.5 × 5–6 µm, ± phaseoliform . I. cfr flavella Acknowledgements We are grateful to Penny Cullington, Alan Outen, Leif and Anita Stridvall, Tommy Knutsson, Elsa Bohus-Jensen, Sonja Kuoljok, Genevieve Gates, and Jukka Vauras for sharing interesting collections. We also thank the herbaria C, GB, LIP, O, S, TAA, TUR, TUR-A, UPS for sending loans and Abisko Scientific Research Station and the Royal Swedish Academy of Sciences for providing lodging and working space at Latnjajaure Field Station. 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