Three new species of foetid Gymnopus in New Zealand

We describe three new species, Gymnopus imbricatus, G. ceraceicola and G. hakaroa, from New Zealand that are similar to G. foetidus (= Micromphale foetidum), growing on wood, with an insititious stipe and foetid odour. The position of these species within the /gymnopoid clade is confirmed by ITS sequence analysis.


Introduction
The new species we describe are members of the family Omphalotaceae Matheney et al. (2006) and have the morphological characteristics of Gymnopus section Vestipedes subsection Impudicae (Antonín & Noordeloos 2010) which contains Micromphale foetidum (Sowerby) Singer, the type species of the formerly recognised genus Micromphale (e.g. in the sense of Singer 1986).Fruitbodies of the group often have a foetid odour when crushed, described as like rotting cabbage or garlic.The only existing records of this group in New Zealand were found to be misapplications of names applied to northern hemisphere species.Moncalvo et al. (2002) investigated nLSU rDNA sequence data for a large number of agarics and recognised a /micromphale clade containing M. foetidum (AF261328).The clade also contained Gymnopus pro parte, Caripia, Setulipes and Micromphale.
Their /micromphale clade was nested within a broader /lentinuloid clade including Rhodocollybia, Marasmiellus ramealis (Bull.)Singer, Marasmius scorodonius (Fr.)Fr. and Lentinula.Mata et al. (2004), based on an LSU analysis, also identified a clade containing sequences of M. foetidum and material named as Setulipes androsaceus (L.) Antonín and Gymnopus fusipes (Bull.)Gray, the type species of those respective genera.They adopted a broad concept of Gymnopus incorporating these genera together with Marasmiellus.Wilson and Desjardin (2005) used LSU to examine the group and identified a /gymnopus clade containing G. fusipes, M. foetidum, S. androsaceus at its core with Micromphale perforans (Hoffm.)Gray lying on its boundary.These results were broadly supported by Mata et al. (2006) in their analysis using ITS1-5.8-ITS2but they demonstrate clustering of G. fusipes, S. androsaceus and Micromphale on the periphery of a concentration of Gymnopus-labelled samples.On the basis of these results the currently generally accepted concept of Gymnopus is broad (e.g.Noordeloos 2012), and incorporates a number of previously recognised genera.Hughes et al. (2010) erected the genus Connopus to accommodate the Gymnopus acervatus group within the gymnopoid clade and presented LSU and ITS data indicating its placement close to Rhodocollybia.Their LSU analysis supports a core gymnopoid clade containing G. fusipes, S. androsaceus, which once again places Micromphale foetidum and M. perforans on a boundary with a sister group containing Rhodocollybia, Marasmiellus juniperinus Murrill and various Gymnopus species.The /gymnopus, clade as interpreted by Hughes et al., contains significant substructure.A multi-gene analysis including more representatives may indicate the recognition of further segregates at genus-level.In this paper we accept our newly described species within the current broad concept of Gymnopus whilst recognising their close alliance to the historical concept of the genus Micromphale.
For this study we analysed ITS1-5.8-ITS2data for related New Zealand collections together with representative sequences from Genbank, many from the studies cited above.The structure of our ITS tree is consistent with these previous analyses, and once again identifies a /micromphale clade closely linked to core Gymnopus species.ITS data generated for a number of representative collections of our newly described taxa support species concepts based on morphology.

Morphological protocols
Spore dimensions are stated as the mean ± 1.5 SD of 20 measurements, thus covering 86% of measurements under an assumed normal distribution model.Fresh or dried material was examined mounted in 10% KOH or Melzer's reagent.Material was handsectioned.Some micrographs were obtained under DIC conditions.Measurements were always taken without DIC optics and an extended objective iris in order to maximise boundary contrast.

Phylogenetic protocols
DNA extraction and sequencing followed the protocols outlined in Cooper and Leonard (2012).We downloaded from Genbank selected sequences used in cited publications, together with close BLAST matches, Table 1.General sequence management was carried out using Geneious (Drummond et al. 2011).Data exchange between applications was facilitated using Alter (Glez-Peña et al. 2010).Sequence alignment was carried out using MAFFT within Geneious (Katoh et al. 2002).A maximum likelihood analysis was executed using RAxML (Stamatakis 2006), with 100 bootstrap runs, launched from Topali 2.5 (Milne et al. 2004).The substitution model of GTR+G was recommended by Topali 2.5.We selected a sequence of Anthracophyllum archeri (Berk.)Pegler as the outgroup.

Results
Our analysis places the New Zealand taxa in a monophyletic clade close to G. foetidum and G. brassicolens historically recognised in the genus Micromphale (Fig. 1).The combination of sequence data and morphological analysis of many collections indicate two major groups which we equate with the newly described species G. imbricatus and G. ceraceicola.In addition we recognise a further species, G. hakaroa, which is poorly distinguished from G. imbricatus on the basis of ITS sequences but which is morphologically consistently different.Minor sequence variation in the G. ceraceicola group does not correlate with morphology and we choose to recognise these specimens as a single species.More information and images of collections may be found on the Landcare Research website (Systematics Collections Data).Etymology.Ceraceicola, indicating association with a basal waxy layer, although this feature is common to the three species described here.

Gymnopus ceraceicola
Notes.Sequence data indicate variability in the taxon but the morphological details are constant and we choose to recognise a single species.New Zealand records of Gymnopus (Micromphale) foetidum and Gymnopus (Micromphale) brassicolens are attributable to G. ceraceicola.Authentic New Zealand material of these two species has not been identified.Gymnopus brassicolens has paler pileus colours, non-gelatinized pileipellis, cheilocystidia and pileipellis elements with lateral projections, and larger basidiospores.Gymnopus foetidus is macroscopically similar but does not possess the agglutinate fascicles of caulocystidia of G. ceraceicola.Macromorphology.Pileus 3-20 mm in diameter convex, cream to fawn, minutely felty, radially furrowed and striate towards the margin, margin fimbriate.Lamellae cream to creamy yellow, adnate.Lamellae present, in series of two: short/long.Stipe mostly eccentric, cartilaginous, to 3 × 0.5 mm, equal, umber to black, sometimes paler towards base, always entirely smooth.Stipe base insititious and usually associated with a thin waxy to chalky cream layer of partially gelatinised hyphae covering the substrate, usually green with algal cells.Fruitbodies with garlic/rotten cabbage smell, especially when crushed.
Habitat.Forming imbricate colonies of dozens to hundreds of fruitbodies on bark and decorticate wood of dead branches and twigs, especially Kunzea and Leptospermum but occurs with other trees.Also occurs at the stem base of live trees.
Distribution.Broadly distributed and common in both North and South Islands of New Zealand.
Etymology.Imbricatus, pertaining to the often tiered and overlapping eccentrically stemmed caps.
Specimens examined.New Zealand, North Island: PDD 80766, on bark of Beilschmedia tawa, Erua Forest, Taupo, collector J.A. Macromorphology.Pileus 3-10 mm diam.convex, rusty tawny to umber, minutely felty, weakly radially furrowed and striate towards the margin.Lamella cream to yellow, waxy.Lamellae present, in series of three: intercalated short/long/short.Stipe central, cartilaginous, to 5 × 0.6 mm, equal, umber to black, paler towards base, smooth to minutely pruinose.Stipe base insititious and always associated with an obvious waxy to chalky cream layer of partially gelatinised hyphae covering the substrate, usually green with algal cells.Fruitbodies with garlic/rotten cabbage smell, especially when crushed.

Dicussion
Gymnopus imbricatus, as its name suggests forms dense populations of small imbricate fruitbodies.It is most commonly associated with tea-tree (Kunzea ericoides and Leptospermum scoparium) and often found on the bark at the base of living trees.Gymnopus hakaroa is larger, with a dark minutely pruinose cap and again forms dense populations  on the bark of dead logs.These two species have smooth stems.Gymnopus ceraceicola is distinguished by larger fruitbodies, a pruinose stipe, and is more commonly associated with southern-beech forests on dead fallen logs.The species of Gymnopus described here belong in the /micromphale clade of Moncalvo et al. (2002) and share the diagnostic feature of this clade of a foetid odour likely due to the presence of mercaptan-like compounds.In New Zealand this feature is shared with Mycetinis curraniae (G.Stev.)J.A. Cooper & P. Leonard, a marasmioid fungus distinguished by its ornamented hymeniderm pileipellis.Another very distinctive character common to all three Gymnopus species, and visible in the accompanying photographs (Figs 2 and 6), is the presence of a waxy layer of partially gelatinised hyphae on the substrate from which the fruitbodies emerge.This layer is usually green from the presence of embedded algal cells.Interestingly, some published images of G. foetidus in the northern hemisphere also show a similar layer, e.g.Antonín and Noordeloos (2010).Detailed examination of our material does show algal cells deeply embedded within the context of the waxy layer and the basal portion of the stipe (Figs 9 and 10), but it would seem unlikely that algal cells are present in sufficient numbers to confer any significant nutritional benefit to the fungus.The morphologically similar Marasmiellus affixus (Berk.)Singer, described from Australia and commonly known as the 'little stinker', is also associated with a waxy algae-infected layer.The association of M. affixus with alga was noted by Singer (1973) and has been speculated to be a basidio-lichen, although this has not proven (Lepp 2011).A partial, poor quality ITS1 sequence for M. affixus obtained during this work (not deposited) suggests it has affinity with Marasmiellus ramealis (Bull.)Singer rather than the taxa treated here.

Table 1 .
ITS Sequences used in the analysis.New sequences generated for this analysis are in bold.