Contemporary documentation of the rare eastern North American species Inocybe insignis ( Inocybaceae , Agaricales )

Inocybe insignis, an eastern North American species with greenish blue staining basidiomes, is documented for the first time since its original description circa 75 years ago in the Great Smoky Mountains. Here, we provide a detailed macroscopic and microscopic description and photographs of this rarely collected species, based on material collected in an Illinois oak-hickory forest. Analysis of molecular phylogenetic data strongly supports an evolutionary alliance with Asian and Australasian species of the Inocybe asterospora group. The combination of stellate basidiospores, marginate stipe base, entirely pruinose stipe, rimose pileus, and strong distinctive odor corroborates the molecular results, but the closest relatives of I. insignis lack the greenish blue staining characteristic of I. insignis.


Introduction
Inocybe insignis Smith was described in 1941 on the basis of a collection of a single basidiome, made in Aug. 1938 (AHS 9781, held in MICH) in the Great Smoky Mountains National Park in Tennessee.Smith noted that "[t]he species is known only from the type," and that it "possesses so many unusual characters that it would be difficult to confuse it with any other Inocybe" (p.12).The combination of stellate basidiospores, presence of pleurocystidia, and green to blue bruising is apparently unique; better-known bluing species like I. calamistrata (Fr.: Fr.) Gill.feature smooth spores and lack pleurocystidia.Other bluing species with smooth basidiospores and pleurocystidia include I. corydalina Quél.and allies and I. aeruginascens Babos (Kosentka et al. 2013); these species, however, exhibit their bluing reaction due to presence of hallucinogenic secondary metabolites (Kuyper 1986).Beyond the holotype collection, we find no records of further Inocybe insignis collections in American herbaria, despite Smith's inclusion of the species in a popular identification resource, How to Know the Gilled Mushrooms (Smith et al. 1979).The collection described in the present work was made in 2011 from central Illinois and consists of about a dozen basidiomes.

Methods
Collection of fresh basidiomes followed methods in Kuo and Methven (2014).Colors were recorded and codified following HEX codes displayed on a 2013 iMac with Intel Iris Pro graphics, using GNU Image Manipulation Program (GIMP) 2.8.10.Microscopic features were studied using hand sections of fresh material, and of dried specimens rehydrated in water after immersion in 90% alcohol.Sections were mounted in 2% KOH, Melzer's reagent, and lactophenol and cotton blue, and viewed using a Nikon Alphaphot YS microscope.Specimens are deposited in the New York Botanic Garden (NY) and the University of Tennessee Fungus Herbarium (TENN).Herbaria are cited according to Thiers (continuously updated).
DNA was extracted and sequenced from a recently dried basidiome following protocols outlined in Judge et al. (2010).Three nuclear gene regions were amplified: the internal transcribed spacers (ITS) of the ribosomal RNA operon, the first 1350 bp of the large subunit ribosomal RNA (nLSU), and the second largest subunit of RNA polymerase II (rpb2) between conserved domains six and seven.For the LSU region, primers LR0R and LR7 were used for PCR and sequencing, in addition to use of LR5 as a sequencing primer, as in Baroni and Matheny (2011).DNA sequences of I. insignis have been released on GenBank (KP170913, KM245989).Destructive sampling of the holotype collection at MICH was avoided since the collection is represented by a single specimen.
All sequences were concatenated into a DNA sequence alignment in MacClade (Maddison and Maddison 2005).Based on BLASTn searches of sequences available in GenBank, taxa were sampled with affinities to Inocybe asterospora Quél.Sequences of Inocybe mixtilis (Britzelm.)Sacc.were used for rooting purposes as it is known this species diverged outside the I. asterospora group (Matheny et al. 2009, Ryberg et al. 2012).A GTR model of molecular evolution was used, together with an invariant proportion of sites parameter and gamma distributed rate heterogeneity, using Maximum Likeli-hood (ML) in the program RAxML 7.2.8 (Stamatakis 2006).500 bootstrap replicates were employed to evaluate the statistical significance of groupings within the resulting best ML tree.The concatenated alignment is available at http://www.bio.utk.edu/matheny/Site/Alignments_%26_Data_Sets.html or by request from the authors.

Results
Although an ITS amplicon was produced for I. insignis, heterogeneity in both spacers precluded direct sequencing of this product.Eight coding sites within the rpb2 gene region sequenced were found to be polymorphic, seven of which are silent.One polymorphic site was observed in the nLSU gene region sequenced.
The molecular dataset contains sequences from 19 vouchered specimens (Table 1) and 2874 included sites.28 sites of the ITS region were too ambiguous to align and were thus omitted before phylogenetic analysis.ML analysis supports the autonomous status of Inocybe insignis in the I. asterospora group (Fig. 1).The species is most closely related to a well-supported cluster of Australasian and southeast Asian species (Horak et al. 2015) characterized by a suite of similar characteristics: stellate basidiospores, entirely pruinose stipe, marginate stipe base, thick-walled pleurocystidia, rimose or strongly rimose pileus surface, and generally distinctive odor (spermatic).Pileus 30-60 mm broad; conic to obtusely conic at first, becoming broadly conic to applanate, with a broad, low umbo; surface dry, radially appressed fibrillose, becoming radially rimose, finely squamulose over the disc, dull brown (664139) to medium orangish brown (784333), with a slightly darker center, bruising greenish blue (445253), negative with the application of 15% KOH; context up to 7 mm thick on the disc, whitish, unchanging or slowly turning greenish blue on exposure; odor strongly sweet and fragrant, with a spermatic component; taste not assessed.Lamellae adnate to uncinate; close; with several tiers of lamellulae; at first buff (ded1e2), becoming dull cinnamon brown (904e2e); occasionally staining greenish blue where bruised; edges concolorous, not conspicuously fimbriate.Stipe 50-70 × 3-4 mm; equal above a rimmed, bulbous base; surface dry, densely pruinose the entire length; cortina not observed and presumably absent; dull brown (664139) to pinkish brown (ad8796), bruising greenish blue to blackish blue (33363d); basal bulb 6-8 mm broad, subglobose, rimmed, whitish; basal mycelium whitish; context dull cinnamon brown (735c3a), unchanging or slowly turning greenish blue on exposure, becoming hollow.
Based on a multi-gene phylogenetic analysis (Fig. 1), I. insignis is most closely related, and indeed the sister group to, a small consortium of species from southeast Asia and Australasia.Inocybe pileosulcata E. Horak, Matheny & Desjardin has been recorded from Thailand and Malaysia in association with a wide assortment of ectomycorrhizal plant associates (Dipterocarpus, Shorea, Castanopsis, Pinus) in tropical lowland and montane forests (Horak et al. 2015).We are also aware of this species from dipterocarp forests in tropical regions of India  stellate-shaped basidiospores, distinct marginate basal bulb, and conspicuous odor, typically spermatic.All three Australasian and Asian species discussed above lack the strong sweet smell (but with a spermatic component) and the greenish blue staining of basidiomes that characterize I. insignis.
Collections of Inocybe xanthomelas Boursier & Kühner could possibly be confused with I. insignis due to their discoloration to a fuscous or grayish black color especially after drying.However, I. xanthomelas does not discolor greenish blue or blackish blue, nor is it closely related phylogenetically to I. insignis.Other species from Europe have been documented with nodulose spores, a marginate stipe base, and flesh that changes color, particularly upon drying.The taxonomic status of these species has been addressed by Esteve-Raventós et al. ( 2015), but none of these have the stellate spores that characterize I. insignis, and all are phylogenetically remotely related to the I. asterospora group (Ryberg et al. 2010).
The biogeographical relationship exhibited here (Fig. 1) -a disjunct relationship between eastern North America and southeast Asia and Australasia -is one not often recorded in mushroom-forming fungi.Disjunct relationships between eastern North America and temperate east Asian species have been suggested in various groups of macrofungi (Wu and Mueller 1997;but see Mueller et al. 2001) and between eastern North America and the Caribbean (Baroni et al. 1997).In other groups some cohesion between Asia and western North American macrofungal species has been reported (Petersen and Hughes 2003).A group worthy of investigation that might share a similar southeast Asia/Australasia-eastern North American disjunct is Gloeocantharellus (Corner 1969, Giachini et al. 2010).However, northern South American species have also been ascribed to Gloeocantharellus, and this element is apparently missing from the I. asterospora group.

Conclusion
The combination of outstanding taxonomic features-the greenish blue staining of basidiomes, stellate spores, rimose pileus, stipe with a marginate basal bulb, and strong odor-affirms the identity of this rare agaric as Inocybe insignis.This is the first report of this unusual species since it was described in 1941 from east Tennessee.Molecular phylogenetic analysis confirms close evolutionary relationships to Australasian and Asian species in the Inocybe asterospora group.

Figure 1 .
Figure 1.Maximum likelihood tree for the Inocybe asterospora group.The tree is inferred from combined ITS-nLSU-rpb2 DNA sequence data.Values above branches represent bootstrap proportions (only those >70% are shown).The scale bar represents the number of expected substitutions per site.
. The two provisionally named Australasian species, I. vagata Matheny & Bougher ined.(=I.asterospora sensu E. Horak) and I. nobilissima Matheny & Bougher ined., occur in Australia and/or New Zealand, and have been recorded in forests dominated by sclerophyll vegetation (Eucalyptus, Leptospermum, Allocasuarina, Acacia) and/or mixed with Nothofagus in New Zealand.Despite the wide geographical disjunction in this group, all of these species share the presence of

Figure 2 .
Figure 2. Basidiomes of Inocybe insignis (Kuo 07101101).A-C Kuo 07101101 D Holotype collection (A.H.Smith 9781, held in MICH); The Regents of the University of Michigan, Ann Arbor, MI 48109.Used with permission.Contact the University of Michigan Herbarium for details.

Table 1 .
Taxon sampling and DNA sequences used in this study.