Singerocomus atlanticus sp . nov . , and a fi rst record of Singerocomus rubrifl avus ( Boletaceae , Boletales ) for Brazil

Ongoing surveys of macrofungi in the Brazilian Atlantic Forest ecoregion continue to uncover a diverse assemblage of ectomycorrhizal fungi. A new species of Boletaceae, Singerocomus atlanticus sp. nov., is described. Singerocomus rubrifl avus, previously known only from Guyana, is recorded for the fi rst time from the Brazilian Atlantic Forest. Phylogenetic data, macroand microscopic illustrations, and comments are presented for each species.


Introduction
Th e Atlantic Forest ecoregion of Brazil is a globally important biodiversity hotspot, harboring many endemic species across multiple taxonomic groups (Olson & Dinerstein 2002;Paese et al. 2010;Joly et al. 2014).Ectomycorrhizal (ECM) fungi were traditionally poorly known from the Atlantic Forest, although this situation is changing (Meijer 2008;Maia et al. 2015;Barbosa-Silva et al. 2017;Magnago et al. 2017).In particular, macrofungi in ECM basidiomycete lineages of the Agaricales, Boletales, Russulales, and Cantharellales appear to be widespread in the Atlantic Forest region and new species and distribution records are being rapidly described (e.g.Sulzbacher et al. 2013a;b;Roy et al. 2017).
Th e genus Singerocomus was proposed by Henkel et al. (2016) to accommodate a new species from Guyana, Singerocomus rubriflavus, and a new combination for Xerocomus inundabilis, originally described from the Brazilian Amazon (Singer et al. 1983).Diagnostic features for Singerocomus include a pileate-stipitate, epigeous basidioma, pinkish red to red, tomentose pileus, yellow, tubulose, non-cyanescent hymenophore depressed at the stipe, subangular pores, concolorous or lighter stipe with or without squamules, and olivaceous brown basidiospore deposit.The combination of smooth basidiospores, phylloporoid tube trama, trichodermial pileipellis, and lack of clamp connections characterize the genus micromorphologically. Th e new genus was justifi ed by a multi-locus, taxon-dense phylogenetic analysis across the Boletaceae which demonstrated that Singerocomus species had no close relatives at the generic level within the family (Henkel et al. 2016).
As part of our ongoing macrofungal surveys in the Atlantic Forest, Singerocomus atlanticus sp.nov. is described, and S. rubriflavus, previously known only from Guyana, is recorded for the first time for Brazil.

Collections
Collections of Singerocomus T.W. Macroscopic features were described from fresh basidiomata and specimens were dried with a food dehydrator (Total Chef TCFD-05 Deluxe) at about 40 ºC.Color codes (e.g.OAC 640) were based on the Online Auction Color Chart (Kramer 2004).Macrochemical tests were performed according to Singer (1986).Microscopic structures were rehydrated in 3 % KOH and dyed with Congo Red.Melzer´s reagent was used for testing amyloidity.To observe the surface of the basidiospores in scanning electron microscopy (SEM), small fragments of the hymenophore were taken from dried specimens and mounted directly on aluminum stubs using carbon adhesive tabs, coated with 30 nm of gold, and examined with a JEOL JSM-6390LV scanning electron microscope, operating at 10KeV, at the Centro de Microscopia e Microanálise of the Universidade Federal do Rio Grande do Sul.Line drawings of microscopic features were made using digital photographs from the specimens examined.Voucher materials were deposited at ICN and FLOR Herbaria and duplicates will be sent to VIES (Thiers 2017).

DNA extraction, amplification and sequencing
DNA was extracted from dried specimens using the Doyle & Doyle (1987) protocol adapted by Góes-Neto et al. (2005).Full nuclear ribosomal internal transcribed spacer nrITS (ITS1-5.8S-ITS2)and nuclear ribosomal large subunit (nrLSU) were PCR-amplified with primer pairs ITS6-R/ITS8-F, and the nuclear LSU rDNA (28S) were PCRamplified with LR0R/LR7 following Dentinger et al. (2010) and Vilgalys & Hester (1990) respectively.Complementary unidirectional sequence reads were aligned and edited in Geneious 6.1.8(Kearse et al. 2012) and deposited in GenBank (Tab.1).The ITS ribosomal DNA sequence from the new taxon was initially subjected to a BLASTn query against GenBank to explore its putative phylogenetic relationships.A total dataset of 38 sequences (18 nrITS and 20 nrLSU) were used in the analyses, both newly generated in this study or downloaded from Genbank database.Five genera (Butyriboletus, Lanmaoa, Rugiboletus, Rubroboletus, Singerocomus) were included in the phylogenetic analyzes based on Henkel et al. (2016) with Bothia included as the outgroup.

Sequence alignment, and phylogenetic analysis
Each dataset was automatic alignment separately using MAFFT (Katoh & Standley 2013), following the L-INS-i and G-INS-i criteria (for nrITS and nrLSU, respectively).Gblocks (Talavera & Castresana 2007) was used to exclude ambiguous portions of the alignment, producing a final aligned dataset of 775 bp for ITS and 841 bp for 28S.Alignments were deposited in TreeBASE (http://www.treebase.org/treebase/index.html) under accession no.ID 21441.Maximum likelihood (ML) analysis was performed separately on ITS and 28S to determine whether there were any well-supported phylogenetic incongruencies between these two loci.Because no supported incongruence was detected we conducted maximum likelihood and Bayesian inference (BI) analysis based on the concatenated ITS+28S dataset.The dataset was subdivided into four partitions: ITS1, 5.8S, ITS2, nrLSU.Maximum likelihood was carried out with RAxML-HPC v.8 (Stamatakis 2014), available in the CIPRES science gateway (Miller et al. 2010, http:// www.phylo.org/),using GTRGAMMA as the model of evolution (Stamatakis 2006), choosing the rapid bootstrap analysis (command -fa) with a random starting tree and 1000 maximum likelihood bootstrap replications.Bayesian inference was conducted on MrBayes v. 3.2.6 (Ronquist & Huelsenbeck 2003) as implemented on the CIPRES Science Gateway 3.1 (Miller et al. 2010), using four parallel MCMC chains, which were allowed to run for 20 million generations, with sampling every 1000 generations.The best-fitted substitution models were set to each partition calculated by MrModeltest 2.3 (Nylander 2004).The Akaike information criterion was chosen to select the most appropriate model of DNA substitution for each data set or data partition used in the analyses.The convergence diagnostic was calculated every 10 4 generations and its critical value was set in order to automatically stop the analysis when the standard deviation of the split frequencies had reached the value defined by the stopval command (stoprule = yes stopval = 0.01).In all analyses, the first 25 % trees from each run were discarded as burnin.Resulting trees from the two independent runs were then pooled to produce one 50 % majority-rule consensus tree and Bayesian posterior probabilities were generated for the resulting tree.A node was considered well supported if it showed a Bayesian Posterior Probabilities (BPP) ≥ 0.95 and/or Bootstrap (BS) ≥ 80 %, while moderate support was considered BS ≥ 70 %, and non-supported BPP < 0.95.All phylogenetic trees were visualized using FigTree (Morariu et al. 2009).

BLASTn queries and phylogenetic analysis
Ten new sequences of Singerocomus from Brazil were generated (6 nrITS and 4 nrLSU).ITS BLASTn queries of each of the new taxa on GenBank indicated affinities with specimens in the genera Xerocomus and Singerocomus.LSU BLASTn queries indicated affinities with Boletaceae, but were uninformative at the genus level.
All rDNA markers were combined into a single matrix, including 38 sequences from 23 specimens representing 13 putative species, resulting in an aligned matrix of 1116 bp.The respective ML tree showing BS and BPP values on branches are shown in Figure 1.All phylogenetic analysis performed showed that specimens of Singerocomus grouped into one distinct, well supported clade (BS = 0.99, BPP = 1 %) that included the new species, S. inundabilis (Singer) T.W.   Commentary.Singerocomus atlanticus is morphologically similar to S. inundabilis (Tab.2) but differs in its richer red pileus color and its NH4OH macrochemical reactions of instantly blue on the pileus and ferrugineous-orange on the stipe, features lacking in the other known species of the genus (Singer et al. 1983;Henkel et al. 2016).From a molecular standpoint Singerocomus atlanticus is also clearly different at the species level from S. inundabilis (Fig. 1).Singerocomus atlanticus has a broad distribution in the Atlantic Forest region, with conspecific specimens documented from the Espírito Santo type locality and from Santa Catarina ca.1.700 km distant.

S. atlanticus S. inundabilis S. rubriflavus
Pileus 16-37 mm wide, convex to plane convex, velutinous to mattedvelutinous, reddish to pinkish, with olive yellowish pruina all over 25-33(-47)    Habit, habitat, and distribution.Solitary on soil, in the north region of the Atlantic Forest in Bahia.The species was previously known only from Guyana in association with ECM Pakaraimaea dipterocarpacea (Cistaceae).
Only one single-basidiome collection of S. rubriflavus has so far been made in Brazil, approximately 3.000 km from the type locality in Guyana.While the Brazilian specimen agrees well both macro-and micromorphologically with the Guyana type specimen, minor differences include the smaller stipe squamules and slightly shorter basidiospores (8-10 μm vs. 9-11(-12) μm) of the Brazilian specimen.Although the Brazilian specimen and two specimens from Guyana formed a well-supported monophyletic group in our phylogenetic analysis (Fig. 1), the variability in branch lengths among the specimens suggests intraspecific variability.As noted by Henkel et al. (2016) similar genetic variability was found among multiple morphologically identical specimens from Guyana, and more research is needed to determine whether S. rubriflavus may encompass a species complex.

Discussion
Morphological and molecular analyses corroborate the placement of our boletoid specimens in Singerocomus.Given that Singerocomus was first recognized from an Amazonian species described in Xerocomus, renewed taxonomic and phylogenetic scrutiny could be profitably applied to Neotropical Boletaceae.In this respect special attention should be given to the numerous Amazonian boletes described in Xerocomus by Rolf Singer (Singer & Digilo 1957;1960;Singer et al. 1983).Most boletoid specimens deposited at INPA are poorly preserved, and many species have only the holotype, these were observed when INPA Herbarium was visited.. Recollections of these species will help to confirm the occurrence of Xerocomus species in Brazil and phylogenetic analysis may uncover more new genera.
While the Brazilian Atlantic Forest has long been recognized for its unique plants and animals, macrofungal surveys in the region continue to uncover a wealth of undescribed species.In the present study our discoveries of a new bolete species and a large range extension for another highlight the importance of continued mycological exploration in this unique region.

Figure 1 .
Figure 1.Maximum likelihood (ML) tree based on nrITS and nrLSU sequences.Bootstrap values above 70% and Bayesian posterior probability above 0.9 are shown.
Henkel & M.E.Sm. were made between 2011 and 2016 at multiple sites in the Brazilian Atlantic Forest, including Reserva Biológica Augusto Ruschi in Espírito Santo, Parque Estadual da Serra do Conduru in Bahia, and Unidade de Conservação Ambiental do Desterro and Plaza Caldas da Imperatriz in Santa Catarina.

Table 1 .
GenBank accession numbers and voucher numbers of sequences used for the phylogenetic analysis.

Table 2 .
Comparison among taxa of Singerocomus