Boletopsis nothofagi sp . nov . associated with Nothofagus in the Southern Hemisphere

Boletopsis nothofagi sp. nov., an ectomycorrhizal taxon is described from Nothofagus forests in New Zealand. A comparison of available molecular ITS sequences, and morphological data was carried out to confirm the novelty of the taxon. This is the first report of the genus in the Southern Hemisphere.


Boletopsis nothofagi sp. nov. associated with
Nothofagus in the Southern Hemisphere 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 to the 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) (Gilbertson 1986).Boletopsis leucomelaena is usually associated with Picea, whereas B. grisea and B. perplexa are associated with Pinus, but there are records of these species associated with hardwoods.The ectomycorrhizal association of B. 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 B. 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 B. 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 B. grisea, B. leucomelaena and B. 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 fre- quency 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.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 1998(Anon -2011)).
Distribution.North and South Islands of New Zealand Ecology.ectomycorrhizal in southern beech (Nothofagus) forests and so far found only in association with Nothofagus fusca.Etymology.nothofagi for its ectomycorrhizal association with Nothofagus.Conservation status.Although there are no data on the stability of the population size or historical changes in distribution of this species, it is likely to be naturally uncommon according to the New Zealand Threat Classification System (Townsend et al. 2008).

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Discussion.Boletopsis nothofagi differs from described species in the more elongate spores with a narrow central waist, granular extra-cellular material becoming green in KOH and habitat in Nothofagus forests (Niemala and Saarenoska 1989;Harrison 1975;Watling and Milne 2006).

table 1 .
Sequences considered in the analysis.