Introduction

A collection of a Boletopsis species was made in 2009 during the annual foray of the Fungal Network of New Zealand (FUNNZ), at the Orongorongo Valley in the Rimutaka Forest Park east of Wellington, North Island, New Zealand. The material was initially thought to be sterile but subsequent examination showed a few spores with the characteristic thelephoroid morphology of Boletopsis. A subsequent collection in 2010 from South Island and re-collection of material at the North Island site provided more fertile material and is the basis for this description of a new species. This appears to be the first record of the genus in the Southern Hemisphere.

Boletopsis is a genus of ectomycorrhizal, stipitate, poroid fungi phylogenetically related tothe hydnoid (toothed) genera Phellodon, Hydnellum, Bankera and Sarcodon and placed in the Bankeraceae (Kirk et al. 2008). Four species are currently recognised in the subgenus Boletopsis (Stalpers 1993; Watling and Milne 2006); Boletopsis leucomelaena (Pers.) Fayod, the type of the genus, Boletopsis grisea (Peck) Bond. & Sing., Boletopsis smithii K. Harrison and Boletopsis perplexa Watling & J. Milne. Boletopsis leucomelaena and Boletopsis grisea are widely distributed across North America, Europe and Asia, but are nowhere common (Gilbertson 1986). Boletopsis leucomelaena is usually associated with Picea, whereas Boletopsis grisea and Boletopsis perplexa are associatedwith Pinus, but there are records of these species associated with hardwoods. The ectomycorrhizal association of Boletopsis smithii is unknown. Recent molecular data supports the existence of additional taxa in North America (Watling and Milne 2008). Boletopsis grisea has also been reported from oak dominated cloud forest in Costa Rica and this represents the most southerly record of Boletopsis subgenus Boletopsis to date (Mata and Ryvarden 2007). Two other species, B. atrata Ryvarden and Boletopsis subcitrina Corner are pleuropodal, without inflated hyphae, and possess spores with small warts or spines. These species were segregated into Boletopsis subgenus Boletopsina (Stalpers, 1993). Boletopsis subcitrina subsequently formed the basis of a new genus Corneroporus (Hattori, 2001) and it seems likely this group is more distantly related to Boletopsis leucomelaena than the remaining four accepted species.

Methods

DNA was extracted from dried herbarium material of the collection PDD96007 using REDExtract-N-Amp Plant PCR Kits (Sigma, USA). The tissue was ground in extraction buffer with a plastic pestle in the Eppendorf tube, then DNA extraction and PCR were carried out following the manufacturer’s instructions. The extract was sequenced for the rRNA loci ITS1+5.8+ITS2 and LSU following the methods of (Johnston and Park 2005). Primers were ITS1F and ITS4 for the ITS region and LR0R and LR6 for LSU (Gardes and Bruns 1993). The chromatographs were assembled using Geneious (Drummond et al. 2011). Existing sequences were downloaded from GenBank for Boletopsis species, related New Zealand material and selected sequences of members of the Bankeraceae from other parts of the world (Table 1). Few LSU sequences are available for Boletopsis. This study focussed on ITS sequences resulting from an analysis of the relationships between Boletopsis grisea, Boletopsis leucomelaena and Boletopsis perplexa (Watling and Milne 2008).

Data exchange between applications was facilitated using Alter (Glez-Peña et al. 2010). Sequence alignment was carried using MAFFT (Katoh et al. 2002) within Geneious using the G-INS-i algorithm. Gblocks (Castersana 2000) was used to eliminate poorly aligned segments, with the number of contiguous conserved positions set to 8, the minimum block set to 10, and allowed gap positions to half, resulting in an alignment of 493 bases. The alignment was analysed by Jmodeltest (Posada 2008). A best-fit model of nucleotide substitution of GTR+G was proposed by jmodeltest. Phylogenetic analyses were performed MrBayes v3.2 (Huelsenbeck and Ronquist 2001) using the recommended model with two sets of four chains, one cold and three heated, with a chain temperature of 0.2. A sequence of Piptoporus betulinus was selected as an outgroup. All prior probabilities were left on default values. The model was run with a sampling frequency of 500 until the split-deviation frequency had fallen below 0.01, ca 1.2 million iterations. The results were examined to ensure good Metropolis coupling of chains and convergence statistics using Tracer (Rambaut and Drummond 2009). The first 25% of trees were removed in constructing a 50% majority rule consensus phylogram.

Results

Figure 1 shows the results of the phylogenetic analysis. The species concepts Boletopsis grisea and Boletopsis perplexa are supported. Boletopsis sp. (SL23), Boletopsis subsquamosa (SMI350), and Boletopsis sp. (Rec227652) are also referable to Boletopsis perplexa. The latter collections confirm the presence of this taxon in North America, as suggested by (Watling and Milne 2008). A consensus concept of Boletopsis leucomelaena is less well supported by these preliminary data with the collections appearing separately in the analysis. It is possible the current use of the name Boletopsis leucomelaena represents multiple cryptic taxa; it is reported as occurring in widely separate geographic regions and with differing ectomycorrhizal hosts. A similar situation was recently demonstrated in the case of European species of related Hydnellum and Phellodon (Ainsworth et al. 2010). Boletopsis nothofagi is clearly supported as a new taxon differing in 22 sites relative to Boletopsis leucomelaena (AFTOL, DQ484064) and 18 sites relative to Boletopsis leucomelaena (Niemela, DQ408771).

Discussion

The presence of Boletopsis nothofagi in New Zealand beech forests appears to have been overlooked despite a long history of the study of similar fungi in New Zealand (Cunningham 1958) (Maas Geesteranus 1971). This suggests Boletopsis nothofagi is a relatively rare (or rarely fruiting) indigenous member of the New Zealand ectomycorrhizal beech forest mycota. In addition no records of Boletopsis have been traced for any localities of naturally occurring Nothofagus forests in Australia, New Caledonia, New Guinea or South America. An alternative explanation for the presence of Boletopsis nothofagi in New Zealand is as a recent introduction of a species that usually has a different ectomycorrhizal host. Such introductions into New Zealand beech forests have occurred at least once in the case of Amanita muscaria (Johnston et al. 2008).

The absence of previous records of this recognisable species combined with the wide separation of the two currently known sites indicate that Boletopsis nothofagi is most likely a rare indigenous member of the New Zealand beech forest mycota. Its conservation status in New Zealand requires further investigation considering the status of other members of the family elsewhere in the world. For example many hydnoid members of the family are threatened in Europe due to a variety of causes (Arnolds 2010). Many of these species are listed on European national red-data lists of fungi (Dahlberg and Mueller 2011). Boletopsis grisea is currently designated as threatened on five national lists and is subject to a number of management plans (Anon 2011).

Taxonomic treatment