Diversity of polypores in the Dominican Republic: Pseudowrightoporia dominicana sp. nov. (Hericiaceae, Russulales)

Abstract The new species Pseudowrightoporia dominicana is described from the Dominican Republic based on morphological and molecular data (nrITS and nrLSU sequence analyses). It is mainly characterised by pileate basidiomata with a bright pinkish context and a di-trimitic hyphal system. Phylogenetically, it is sister to the African species P. gillesii and to the Asiatic P. japonica.


Introduction
The genus Wrightoporia Pouzar, typified with W. lenta (Overh. & J. Lowe) Pouzar (Pouzar 1966), is traditionally characterised by resupinate to pileate basidiomata, annual to perennial habit, small to medium pores and cottony to hard texture. Hyphal system monomitic to di-trimitic, generative hyphae clamped or rarely with simple septa, skeletal hyphae dextrinoid, partially dextrinoid (only in the tubes) or not dextrinoid. Basidiospores small, cylindrical to globose, smooth to finely asperulate, amyloid (Ryvarden 1982(Ryvarden , 2016David and Raichenberg 1987, Stalpers 1996, Núñez and Ryvarden 2001, Hattori 2008. To date, there are 52 species transferred to or described in the genus (Index Fungorum 2018). This genus belongs to the Hericiaceae, in the Russulales (Larsson andLarsson 2003, Chen et al. 2016). Chen et al. (2016), on the basis of combined nrITS/nrLSU phylogenetic analyses and morphological data, indicated that the genus Wrightoporia, as currently circumscribed, is strongly polyphyletic and recognised six clades in Wrightoporia s.l. Consequently, species previously treated in Wrightoporia were transferred to Amylonotus Ryvarden, Amylosporus Ryvarden and to the three new genera Larssoniporia Y.C. Dai During the species diversity study of wood-inhabiting macromycetes in the Dominican Republic, a pileate Pseudowrightoporia was discovered. The aim of this investigation was to identify and to analyse the Pseudowrightoporia specimens using both morphological and molecular techniques.

Morphology
Photographs of fresh basidiomata were taken in situ by a Nikon Coolpix 8400 digital camera and then dried, while the photos of the microscopical structures were obtained through a Olympus BH-2 light microscope and a Nikon D7100 digital camera. For microscopical analysis, tiny fragments from dried material were mounted in Melzer's anionic reagent for testing amyloid and dextrinoid reactions of spores and other microscopical elements. All microscopic measurements were carried out with a ×1000 oil immersion objective. Basidiospores were measured from hymenophores of mature basidiomes, dimensions are given as: (minimum-) average minus standard deviationaverage -average plus standard deviation (-maximum) of length × (minimum-) average minus standard deviation -average -average plus standard deviation (-maximum) of width; Q = (minimum-) average minus standard deviation -average -average plus standard deviation (-maximum) of the length/width ratio. Spore statistics were produced using R version 3.4.4 (R Core Team 2018). Herbarium acronyms follow Thiers (2018, continuously updated) with the exception of ANGE that refers to the personal herbarium of C. Angelini.

DNA extraction, PCR amplification and DNA sequencing
Genomic DNA was isolated from 10 mg of a dried voucher specimen (JBSD 127410), using the DNeasy Plant Mini Kit (Qiagen, Milan) according to the manufacturer's instructions. Primers LR0R/LR6 (Vilgalys and Hester 1990, Vilgalys lab. http://www. botany.duke.edu/fungi/mycolab) were used for the nrLSU (28S) DNA amplification and universal primers ITS1F/ITS4 for the ITS region amplification (White et al. 1990, Gardes andBruns 1993). Amplification reactions were performed in a PE9700 thermal cycler (Perkin-Elmer, Applied Biosystems, Norwalk) in 25 ml reaction mixtures using the following final concentrations or total amounts: 5 ng DNA, 1 × PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 mM of each primer, 2.5 mM MgCl 2 , 0.25 mM of each dNTP, 0.5 unit of Taq polymerase (Promega, Madison). The PCR programme was as follows: 3 min at 95 °C for 1 cycle; 30 s at 94 °C, 45 s at 50 °C, 2 min at 72 °C for 35 cycles, 10 min at 72 °C for 1 cycle. PCR products were resolved on a 1% agarose gel and visualised by staining with ethidium bromide. The PCR products were purified with the AMPure XP kit (Beckman Coulter, Pasadena) and sequenced by MACROGEN (Seoul). The sequences were submitted to GenBank (http://www.ncbi. nlm.nih.gov/genbank/) and their accession numbers are reported in Figs 1-2.

Sequence alignment, dataset assembly and phylogenetic analysis
Sequences were checked and assembled with Geneious 5.3 (Drummond et al. 2010) and compared to those available in the GenBank database (http://www.ncbi.nlm. nih.gov/Genbank/) using the BLASTN algorithm (Altschul et al. 1990). Based on BLASTN results, sequences were selected according to the recent monographic work on Wrightoporia s.l. by Chen et al. (2016).
Two phylogenetic analyses were performed: the first, based on a combined nrITS and nrLSU sequences dataset, to focus on the phylogenetic position of the new species in the Russulales (Russuloid clade); the second, based only on a nrITS dataset was restricted to the taxa closely related to P. dominicana according with the previous combined data analysis. Alignments were generated for each nrITS and nrLSU dataset using MAFFT (Katoh et al. 2002) with default conditions for gap openings and gap extension penalties. The two alignments were imported into MEGA 6 (Tamura et al. 2013) for manual adjustment. The best-fit substitution model for each single alignment was estimated by both the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) with jModelTest 2 (Darriba et al. 2012). The GTR + G Figure 1. Bayesian phylogram obtained from the combined nrITS-nrLSU sequence alignment of Russulales taxa selected according to Chen et al. (2016). Sistotrema brinkmannii, S. coronilla, S. muscicola and S. sernanderi were used as outgroup taxa. Values for clades that are supported in either the Bayesian (posterior probabilities, BPP) and Maximum likelihood (ML bootstrap percentage, MLB) analyses are indicated. BPP values (in bold) above 0.70 and MLB values above 50% are given above/below branches. The newly sequenced collection is in bold. model was chosen for both the nrITS and nrLSU alignments. The sequences of Sistotrema brinkmannii, S. coronilla, S. muscicola and S. sernanderi were used as outgroup taxa (Larsson andLarsson 2003, Chen et al. 2016) in the combined analysis; Dentipel- lis coniferarum, D. fragilis and Hericium alpestre were selected as outgroup taxa in the nrITS analysis. The ITS dataset was not partitioned into ITS1, 5.8S and ITS2 subsets. Phylogenetic hypotheses were constructed under Bayesian inference (BI) and Maximum likelihood (ML) criteria. The BI was performed with MrBayes 3.2.6 (Ronquist et al. 2012) with one cold and three incrementally heated simultaneous Monte Carlo Markov chains (MCMC) run for 10 million generations, under the selected evolutionary model. Two simultaneous runs were performed independently. Trees were sampled every 1,000 generations, resulting in overall sampling of 10,001 trees per single run; the first 2,500 trees (25%) were discarded as burn-in. For the remaining trees of the two independent runs, a majority rule consensus tree showing all compatible partitions was computed to obtain estimates for Bayesian posterior probabilities (BPP). ML estimation was performed through RAxML 7.3.2 (Stamatakis 2006) with 1,000 bootstrap replicates using the GTRGAMMA algorithm to perform a tree inference and search for a good topology. Support values from bootstrapping runs (MLB) were mapped on the globally best tree using the "-f a" option of RAxML and "-x 12345" as a random seed to invoke the novel rapid bootstrapping algorithm. BI and ML analyses were run on the CIPRES Science Gateway web server (Miller et al. 2010). Only BPP and MLB values over 0.70 and 50%, respectively, are reported in the resulting trees (Figs 1-2). Branch lengths were estimated as mean values over the sampled trees.

Results
The combined nrITS and nrLSU data matrix comprised 118 sequences (including 117 from GenBank) and includes 2132 positions. The nrITS data matrix comprises a total of 25 sequences (including 24 from GenBank) and includes 687 positions. As both Bayesian and Maximum likelihood analyses produced comparable topologies, only the Bayesian trees with both BPP and MLB values are shown (Figs1-2). In the combined two-gene phylogeny of Russulales taxa (Fig. 1), the new species falls, as an independent phylogenetic branch, in the Hericiaceae within the Pseudowrightoporia cluster. Pseudowrightoporia dominicana is sister (BPP = 1.00, MLB = 95) to P. japonica. Pseudowrightoporia is shown to be sister (BPP = 1.00, MLB = 100) to a well-supported clade (BPP = 1.00, MLB = 80) consisting of Wrightoporiopsis and Dentipellicula, as previously highlighted by Chen et al. (2016). The small ITS analysis restricted to species of Pseudowrightoporia and Wrightoporiopsis (Fig. 2) supports P. dominicana as a new species and indicates P. gillesii and P. japonica as its phylogenetically closest species. Fig. 3 Holotype. Dominican Republic. La Vega (Province), Jarabacoa (Municipality), Montaña (Locality), 19°06'39"N, 70°37'57"W, on an unidentified live trunk of a deciduous tree, in a mixed mountain forest with several broadleaved species and pines (Pinus occidentalis), 17 December 2016, Claudio Angelini, (JBSD 127410, isotype ANGE 789).

MycoBank MB824844
Etymology. The epithet refers to the country, The Dominican Republic, where this species was found.
Habit, habitat and distribution. Pileate, gregarious on a live trunk of deciduous tree, so far known only from the type locality.