Cortinarius bovarius (Agaricales), a new species from western North America

Cortinarius bovarius sp. nov., a conifer associated taxon growing on calcareous ground, is described from western North America. Phylogenetic relationships and species limits were investigated using rDNA ITS and nuclear rpb2 sequences, morphological and ecological data. The species belongs to section Bovini and its closest relative is European C. bovinus.


Introduction
Cortinarius is the most species rich genus of the Agaricales with a worldwide distribution.Cortinarius species are important ectomycorrhizal fungi associated with different trees and shrubs belonging e.g. to the order Fagales and families Pinaceae and Salicaceae.Lately it has also been suggested that they have a key role in the carbon cycling of boreal forests (Bödeker et al. 2011).
In recent years there have been a number of publications on the taxonomy, evolution and biogeography of species found in North America (Seidl 2000, Moser and Peintner 2002, Matheny and Ammirati 2006, Garnica et al. 2009, 2011, Bojantchev 2011a, b, Bojantchev and Davis 2011, Harrower et al. 2011, Ammirati et al. 2012, 2013, Niskanen et al. 2012, 2013a, in press a).These studies show several patterns of species distributions.There are species common to North America and Europe, especially those species from more northern and montane conifer forests, i.e.Corti-narius aureofulvus M. M. Moser and C. napus Fr.There are also presumably endemic species in both Western North America, eastern North America and Europe, i.e. ).The species are characterized by brown to dark brown basidiomes without bluish colors and exsiccatae with a dark brown to blackish brown pileus.The universal veil is white, brownish white or grayish white, in some species becoming grayish brown with age, and the odor is indistinct or slightly raphanoid.To date, species are only known from Europe, except C. oulankaënsis which also occurs in Canada in British Columbia.By studying more material from western North America, we wanted to determine if C. bovinus found from Alaska, U.S.A. and Alberta, Canada is conspecific with European samples or does it represent an autonomous species.

Methods
Material gathered by the authors from North America was studied morphologically, ecologically and sequenced to infer phylogentic relationships with other species in Bovini.DNA was extracted from dried material (a piece of lamella) with the NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany).Primers ITS 1F and ITS 4 (White et al. 1990, Gardes andBruns 1993) were used to amplify ITS regions, and specific primers cort6F and b7.1R (Frøslev et al. 2005) for the rpb2 region.The same primer pairs were used in direct sequencing.PCR amplification and sequencing followed Niskanen et al. (2009).Sequences were assembled and edited with Sequencher 4.1 (Gene Codes, Ann Arbor, Mich., USA).Using a BLAST query of the public databases (GenBank: http://www.ncbi.nlm.nih.gov/ and UNITE: http://unite.ut.ee/), we checked if identical or similar sequences for our species exist in public databases.For the phylogenetic analysis ITS and rpb2 sequences of the species belonging to the well-supported ingroup of section Bovini, C. bovinus, C. bovinaster, C. bovinatus, and C. oulankaënsis, were included.Cortinarius anisatus, C. neofurvolaesus, and C. sordidemaculatus were chosen as outgroup species.
The combined ITS and rpb2 alignment of 11 specimens was produced with the program MUSCLE (Edgar 2004) under default settings.The alignment comprised 1286 nucleotides (including gaps).The alignment is available at TreeBASE under S14159 (http://www.treebase.org/treebase-web/home.html).
Bayesian inference (BI) was performed with MrBayes 3.1.2(Ronquist and Huelsenbeck 2003).The best substitution model for the alignment was estimated by both the Akaike information criterion and the Bayesian information criterion with jModelTest version 0.1.1 (Posada 2008).A GTR model, including a gamma shape parameter, was chosen for both DNA regions.Two independent runs with four chains in each were performed for 1 000 000 generations sampling every 100th generation.All trees sampled before stationarity were discarded with a 25% safety margin (burn-in of 2 500 trees [250 000 generations]).Sampled trees from both runs were combined in a 50% majority rule consensus phylogram and posterior probabilities (PP) were calculated.The analysis was run with computer clusters of the CSC, IT Center for Science, Espoo, Finland.
Morphological descriptions are based on material collected by the authors including specimens in all stages of development.Color notations in the description follow Munsell (2009) soil color charts.Microscopic characteristics were observed from dried material mounted in Melzer's reagent (MLZ).Measurements were made in MLZ with an ocular micrometer using 100× oil-immersion lens.Basidiospores were measured from the veil or top of the stipe, 20 spores from one basidiocarp.The length and width were measured for each spore, and their length/width ratios (Q value) were calculated.The lamellar trama and basidia also were examined, and the pileipellis structure was studied from scalp sections taken from the pileus center.
Ecology and distribution.In mesic coniferous forests with Picea, on rich, calcareous soil.Known from U.S.A, Alaska and Canada, Alberta.Fruiting from late August to September.
Etymology.bovarius for its affinity to C. bovinus.Discussion.Cortinarius bovarius is a typical member of section Bovini, a brown species with at first a white universal veil that later becomes brownish, indistinct or slightly raphanoid smell, and occurrence on calcareous ground.It differs from its European sister species, C. bovinus, by on average narrower, less dextrinoid and less verrucose spores (those of C. bovinus on average 6.1-6.4 μm wide, fairly strongly to strongly verrucose at the apex, and fairly strongly dextrinoid).The other known species of section Bovini s. str.from western North America, C. oulankaënsis, has a more grayish brown pileus, more distant lamellae, and relatively narrower spores (Qav.= 1.61-1.65).Cortinarius bovarius is a well-defined species based on morphology and molecular data, and therefore, is here describe as new to science.

Figure 1 .
Figure 1.The Bayesian 50% majority-rule consensus tree inferred from combined ITS and rpb2 regions.PP > 0.50 are indicated above branches.