Three new species of Hydnophlebia ( Polyporales , Basidiomycota ) from the Macaronesian Islands

The genus Hydnophlebia includes two species of wood-inhabiting fungi, Hydnophlebia chrysorhizon and Hydnophlebia omnivora. Both are characterized by cream to reddish-orange, resupinate basidiome, with hydnoid hymenophore, margin with strands, a monomitic hyphal system, tubular to ventricose cystidia and elliptical spores. In this paper, a taxonomic study of Hydnophlebia, using morphology and molecular analyses of large subunit nuclear ribosomal DNA (LSU) and the internal transcribed spacer nrDNA operon (ITS), is reported. Three new species, Hydnophlebia canariensis, H. gorgonea and H. meloi, from the Macaronesia bioregion (Canary Islands and Cape Verde Archipelago), are described.

During our survey of corticioid fungi from Macaronesia (Canary Islands and Cape Verde Archipelago), nine hydnoid specimens were initially identified as belonging to the genus Phanerochaete.BLAST search of the large subunit of the nrDNA (LSU) sequences showed high similarity with a sequence published in Wu et al. (2010) and identified as Phanerochaete chrysorhizon (Torr.)Budington & Gilb. (AF139967).In the analysis by Wu et al. (2010) this sequence was recovered within a clade (clade V) containing i.a.Phlebia sensu stricto and a number of taxa with typically odontoid or hydnoid hymenophore, quite far from the Phanerochaete core group.BLAST search of the internal transcribed spacers of the nuclear ribosomal gene (ITS) sequences, which gave high similarity to sequences labelled as Phanerochaete chrysorhizon (AY219359) and Phanerochaete omnivora (Shear) Burdsall & Nakasone (AY219360) published in de Koker et al. (2003).Like later Wu et al. (2010) also de Koker et al. (2003) found that these taxa were not related to the Phanerochaete core group.
The aim of this study was to characterize and classify our specimens from Macaronesia, using morphological data and molecular analyses of LSU and ITS regions.

Sampling, morphological studies and line drawings
Specimens were collected in the Canary Islands and Cape Verde Archipelago (Table 1), and are deposited in the mycological collection (MA-Fungi) of the Real Jardín Botánico herbarium in Madrid, Spain; the initials MD correspond to M. Dueñas, and Tell. to M.T. Telleria.The type specimens of Hydnum chrysorhizon (NY!) and Hydnum omnivorum Shear (BPI!) were included in the morphological analyses.Colours of dried basidiomata are given according to ISCC-NBS Centroid Color Charts (Kelly and Judd 1976).Dried specimens were also used for light microscope studies and drawings.Measurements and drawings were made from microscopic sections mounted in 3% aqueous KOH and/or Congo Red solution and examined at magnifications up to 1250× using an Olympus BX51 microscope.The length and width of 30 spores and 10 basidia were measured from each sample.Line drawings were made with a Leica DM2500 microscope with the aid of a drawing tube.

DNA isolation and sequencing
Genomic DNA was extracted from eight collections (Table 1) using DNeasy® Plant Mini Kit (QIAGEN, Valencia, CA), following the manufacturer's instructions.Basidiomes were disrupted using Tissue-Lyser II (QIAGEN, Germany) and glass beads.Lysis buffer incubation was overnight at 55 °C.
Total DNA was used for PCR amplification of the D1−D2 region of the large subunit (LSU) and the internal transcribed spacer region (ITS) of the nuclear ribosomal gene.The primers LR0R (Rehner and Samuels 1994) and LR7 (Vilgalys and Hester 1990) were used to amplify the region of the LSU nrDNA; the primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) were used to obtain amplifications of both ITS regions, including the 5.8S of the ribosomal RNA gene cluster and flanking parts of the small subunit (SSU) and large subunit (LSU) nuclear ribosomal genes.Individual reactions to a final volume of 25 μL were carried out using illustra TM PuReTaq TM Ready-To-Go TM PCR Beads (GE Healthcare, Buckingham) with a 10 pmol/μL primer concentration, following the thermal cycling conditions used in Martín and Winka (2000).
Negative controls lacking fungal DNA were run for each experiment to check for contamination.The reactions were run with the following parameters for the LSU nrDNA: initial denaturation at 94 °C for 5 min, then 36 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1.5 min, with a final extension at 72 °C for 10 min, and 4 °C soak; for the ITS nrDNA: initial denaturation at 95 °C for 5 min, then 5 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min, followed by 33 cycles of denaturation at 72 °C for 1 min, annealing at 48 °C for 30 s, and extension at 72 °C, with a final extension at 72 °C for 10 min and 4 °C soak.
PCR products were checked on 2% agarose D1 low EEO (CONDA, Pronadisa TM ) gels and subsequently purified using the QIAquick Gel PCR Purification (QIAGEN) kit according to the manufacturer's instructions.The purified PCR products were sequenced using the same amplification primers at Macrogen Korea (Seoul, Korea).
Sequencher v. 4.2 (Gene Codes Corporation, Ann Arbor, MI) was used to edit the resulting electropherograms and to assemble contiguous sequences (Table 1 in bold).BLAST searches (Altschul et al. 1997), using the MEGABLAST option were done to compare the sequences obtained against the sequences in the EMBL/GenBank/DDBJ databases (Cochrane et al. 2011(Cochrane et al. , 2016)).

Sequence alignment and phylogenetic analyses
The LSU and ITS sequences obtained were aligned separately using Se-Al v. 2.0a11 Carbon (Rambaut 2002) for multiple sequences.
Where ambiguities in the alignment occurred, the alignment generating the fewest potentially informative characters was chosen (Baum et al. 1994).Alignment gaps were marked "-", unresolved nucleotides and unknown sequences were indicated with "N".
A maximum parsimony analysis (MP) was carried out; minimum length Fitch trees were constructed using heuristic searches with tree-bisection-reconnection (TBR) branch swapping, collapsing branches if maximum length was zero and with the MulTrees option on in PAUP*4.0b10(Swofford 2003), with a default setting to stop the analysis at 100 trees.Gaps were treated as missing data.Nonparametric bootstrap (MP-BS) support (Felsenstein 1985) for each clade, based on 10,000 replicates using the fast-step option, was tested.The consistency index, CI (Kluge and Farris 1969), retention index, RI, and rescaled consistency index, RC (Farris 1989) were obtained.The maximum likelihood (ML) analysis was done in PAUP*Version 4.0b10, with the GTR+I+G model selected by this programme; for assessing branch support, 1000 non-parametric bootstrap replicates (ML-BS) were performed with the fast-step option.A third analysis was done by a Bayesian approach (Larget andSimon 1999, Huelsenbeck et al. 2001) using MrBayes 3.2 (Ronquist et al. 2012) and assuming the general time reversible model (Rodríguez et al. 1990), including estimation of invariant sites and a discrete gamma distribution with six categories (GTR+I+G), as selected by PAUP*Version 4.0b10.Two independent and simultaneous analyses starting from different random trees were run for two million generations with 12 parallel chains, and trees and model scores saved every 100 th generation.The initial 1000 trees were discarded as burn-in before calculating the 50% majority-rule consensus tree and the posterior probability (PP) of the nodes, as described in Telleria et al. (2010).A combination of bootstrap proportions and posterior probabilities was used to assess the level of confidence for a specific node (Lutzoni et al. 2004;Wilson et al. 2012).The phylogenetic trees were viewed with FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/ figtree/) and edited with Adobe Illustrator CS3 v. 11.0.2(Adobe Systems).
Based on our previous phylogenetic trees obtained from LSU, two sequences of Phlebia radiata Fr. were selected as outgroup (AY219366, EF491867).Alignment gaps were marked "-", unresolved nucleotides and non-sequenced nucleotide positions within the data matrix were indicated with "N".A maximum parsimony analysis (MP) was carried out under heuristic search, following the same criteria as mentioned above for LSU; maximum likelihood (ML) and Bayesian approaches were also performed, using the GTR+I+G as selected by PAUP*Version 4.0b10 and MrModeltest 2.3.The ML and Bayesian analyses were done with the same programs, and followed the same criteria as mentioned above for LSU.

Results
Sixteen new sequences from the Macaronesian specimens were generated in this study (Table 1).The LSU sequence contains the domain D1-D2, and the ITS sequence the regions ITS1, 5.8S nrDNA and ITS2.

LSU analyses
The LSU dataset contains 57 sequences and 908 aligned positions, of which 682 were constant, 82 parsimony uninformative, and 144 parsimony-informative.Maximum parsimony analysis yielded 100 most parsimonious trees (613 steps long, CI = 0.4731, HI = 0.6164, RI = 0.7399) under a heuristic search.Almost identical tree topologies were generated after parsimony and Bayesian analyses.The 50% majority-rule consensus tree from the Bayesian analysis is shown in Fig. 1, with percentage of bootstrap (MP-BS and ML-BS) and posterior probabilities indicated on the branches.The circumscription of clade V from Wu et al. (2010) is indicated in this figure.

ITS analyses
The ITS nrDNA dataset contains 26 sequences and 851 aligned positions, of which 575 were constant, 103 parsimony uninformative, and 173 parsimony-informative.After heuristic search, the 100 trees had 447 steps, CI = 0.7136, HI = 0.3798 and RI = 0.7831.Almost identical tree topologies were generated after parsimony (data not shown), maximum likelihood (data not shown) and Bayesian analyses.The 50% majority-rule consensus tree from the Bayesian analysis is shown in Fig. 2, with percentage of bootstrap (MP-BS and ML-BS), and posterior probabilities indicated on the branches.Similar to the LSU analyses, the sequences from Macaronesian specimens form a clade (MP-BS = 76%, ML-BS = 75%, PP = 1.0), together with downloaded sequences of Hydnophlebia from the USA (Arizona, Florida, Illinois, New York and Puerto Rico) and Canada, identified in Floudas and Hibbett (2015) as H. chrysorhizon, H. cf.chrysorhizon, H. omnivora 1 and H. omnivora 2; as well as sequences of H. chrysorhizon from Illinois and H. omnivora from Arizona published by de Koker et al. ( 2003) that we consider represent the modern interpretation of these two species.Sequence UDB016816 from Madagascar, in Fig. 2, labelled as Hydnophlebia sp. 2, (under Phlebia sp. in UNITE database) also clusters in this clade.
The five sequences from Canada and the USA identified as H. chrysorhizon grouped in a highly supported clade (MP-BS > 80%, ML-BS > 82%, PP = 1.0).The sequences The new sequences generated for this work are distributed over three clades.These clades are here described in the order they occur from top to bottom in Fig. 2.
Etymology.Named after the Canary Islands where the holotype and paratypes were collected.
Other Remarks.This species has very long and well-developed strands and, microscopically, it is the only species in the genus with spores narrowly ellipsoid to cylindrical (L/W = 1.9) and scattered clamps in the aculei hyphae.
Etymology.Named after Gorgades, an ancient name for the Cape Verde Islands, Atlantic Ocean.
Ecology and distribution.This species is known from only two localities of São Vicente Island, Cape Verde Archipelago, on decayed wood of Phoenix atlantica and Prosopis juliflora in arid habitats.
Distribution.Rocky steep slopes, on Sarcostemma daltonii, endemic climbing herb of Cape Verde Archipelago.Only known from the type locality in Fogo Island.
Ecology and distribution.Described from Texas (Shear 1925).According to Burdsall (1985) this species is distributed in the arid regions of southwestern United States, and probably into southern California and northern Mexico.Also reported from Florida (Ginns and Lefebvre 1993) and Uruguay (Martínez and Nakasone 2005).

Discussion
In this study a taxonomic analysis of Hydnophlebia, based on morphological and molecular data, is provided.Hydnophlebia has been confused with Phanerochaete and the two species included, Hydnophlebia chrysorhizon and Hydnophlebia omnivora, were assigned to the latter genus (Burdsall 1985).
For a long time, Hydnophlebia was considered a monospecific genus; however, based on the molecular analyses, both LSU and ITS sequences, as well as a point-by-point comparison of the morphological characters, five species can be discriminated, two already described by other authors (H.chrysorhizon and H. omnivora), and the three new species from Macaronesia described here (H. canariensis, H. gorgonea, and H. meloi).

Figure 1 .
Figure 1.The 50% majority rule Bayesian tree inferred from D1-D2 LSU nrDNA assuming the GTR + I + G model of corticioid fungi included in Table 1.Parsimony bootstrap values (> 50%) maximum likelihood bootstrap values (> 50%) and Bayesian posterior probabilities (> 0.95) are indicated on the branches.Clade V from Wu et al. (2010) is indicated.Taxon name between parentheses indicate specimens with uncertain generic placement.Sequences of the new species described in this paper, H. canariensis, H. gorgonea and H. meloi are in bold.The asterisk (*) after the taxon names denotes type species of the genus.

Figure 2 .
Figure 2. The 50% majority rule Bayesian tree inferred from ITS nrDNA assuming the GTR + I + G model of corticioid fungi included in Table 1.Parsimony bootstrap values (> 50%), maximum likelihood bootstrap values (> 50%) and Bayesian posterior probabilities (> 0.95) are indicated on the branches.Taxon name between parentheses indicate specimens with uncertain generic placement.Sequences of the new species described in this paper, H. canariensis, H. gorgonea and H. meloi are in bold.The asterisk (*) after the taxon names denotes type species of the genus.

Table 1 .
Specimens of Hydnophlebia species described as new, and EMBL/GenBank/DDBJ and UNITE accessions included in the LSU and ITS nrDNA analyses.The asterisk (*) after the taxon names denotes type species of the genus.The specimens with uncertain generic placement are listed at the end of the table; in Fig.1and 2, the uncertainty is indicated by brackets around the name.Isolates and/or voucher specimens are indicated as they appear in GenBank and UNITE accessions.