Two new species of Phylloporus (Fungi, Boletales) from tropical Quercus forests in eastern Mexico

Abstract We present a proposal of two new species of Phylloporus discovered in tropical oak forests from central Veracruz, Mexico. Both species were distinguished based on macro and micro-morphologic features and supported with a molecular phylogenetic analysis, based on sequences of nuc rDNA ITS, D1, D2 and D3 domains of nuc 28S rDNA (LSU), and transcription elongation factor 1-alpha (tef-1α). In the phylogenetic reconstruction inferred, the new species clustered in two different clades related to species from USA, Costa Rica and Panama. The recollection of fructifications in monodominant stands of either Quercusoleoides or Q.sapotifolia, allowed recognizing the distribution of one of the Phylloporus species under both Quercus species, and the other under Q.oleoides only. Detailed macro and microscopic descriptions accompanied by illustrations, photos and a taxonomic discussion are provided.


Introduction
The genus Phylloporus is widely distributed worldwide with approximately 100 species occurring among conifers and broad-leaf trees as potential hosts (Neves 2007;Ortiz-Santana et al. 2007;Neves et al. , 2012Zeng et al. 2013;Ye et al. 2014). Recent research on Phylloporus systematics revealed that some species placed under this genus in the past are related to other groups. Such is the case of P. boletinoides, that was found to be genetically distant, representing an independent genus, described recently as Phylloporopsis (Farid et al. 2018). Erythrophylloporus Ming Zhang & T.H. Li recently described, is a lamellate lineage in the Boletaceae, having morphological resemblance to Phylloporus (Zhang and Li 2018). Additionally, a high species diversity is being detected in the genus; for example in China, Zeng et al. (2013) recognized at least 21 phylogenetic species of Phylloporus, 17 of which represented newly discovered taxa. Most Phylloporus species have a tropical and subtropical range of occurrence, although some species, e.g. P. imbricatus and P. pachycystidiatus, are known to occur in alpine ecosystems (Zeng et al. 2013;Ye et al. 2014). In the Neotropics, an important diversity of Phylloporus has been documented since the early works by Singer (1973Singer ( , 1978 and Singer and Gómez (1984), to more recent contributions by Ortiz et al. (2007),  and Neves et al. (2012). In the Neotropics, Quercus, Pinus, Abies, Alnus, Dicymbe, and Neea, represent some potential ectomycorrhizal hosts of Phylloporus spp. mentioned in the literature (Singer 1978, Montoya et al. 1987Montoya and Bandala 1991, Ortiz-Santana et al. 2007, Neves 2007. In Mexico, Phylloporus has been collected mainly in temperate and mesophytic forests. Phylloporus guzmanii Montoya & Bandala, and P. fagicola Montoya & Bandala were described as new species, the former found in Pinus and Pinus-Quercus forests, while the latter in mesophyll forest under Fagus grandifolia var. mexicana Bandala 1991, 2011). Other records in Mexico correspond to P. bellus (Massee) Corner, P. rhodoxanthus (Schw.) Bres. (inhabiting Quercus and mixed Pinus-Quercus, Pinus-Abies forests), P. centroamericanus Singer & Gómez and P. foliiporus (Murr.) Singer (in Quercus and mesophyll forests), P. phaeoxanthus var. simplex Singer & Gómez and P. leucomycelinus (Singer) Singer (in Quercus forest) (Singer 1957(Singer , 1978Singer and Gómez 1984;Montoya et al. 1987;Montoya and Bandala 1991;García 1999).
Mexico harbors the greatest center of Quercus species diversity with about 160-165 species of the 500-600 known worldwide (Valencia 2004, Nixon 2006Cavender-Bares 2016). Some species of Quercus dominate the canopy of lowland tropical forest relicts in the country (Challenger and Soberón 2008). In the state of Veracruz (eastern Mexico) such forest ecosystems currently cover around 905 km 2 , and are listed by CONABIO as priority terrestrial regions considered Pleistocene relicts (Arriaga et al. 2000). Such tropical Quercus forests are seriously fragmented but still shelter populations of diverse biological groups, including endemic species of flora and ectomycorrhizal fungi associated with native Quercus trees. Many species of this trophic group of fungi in their tropical range are poorly known in Mexico.
As part of a weekly monitoring of macrofungi in two lowland relicts of tropical Quercus forests in eastern Mexico, we have detected, among other ectomycorrhizal fungi, the common presence of Phylloporus fructifications. After a macro-and micromorphological study of the collections, that included molecular phylogenetic analyses based on ITS, LSU and tef-1α sequences, we concluded that the specimens represent two new species inhabiting the tropical Quercus forests from eastern Mexico.

Sampling and morphological study
A weekly monitoring developed during June-October 2016-2017 in two tropical Quercus forests from Central Veracruz (eastern Mexico) were the basis of the present study, including some collections made in 2009 and 2012. The two forests are within private properties, one located at Zentla Co. (850 m alt.) and the other one at Alto Lucero Co. (400-500 m alt.); both forests present monodominant stands of Q. oleoides Schltdl. & Cham. and Q. sapotifolia Liebm. where the Phylloporus samples were gathered.
Macromorphological and color studies of specimens were conducted on different growth stages of fresh material. In the description of each species, alphanumeric nomenclature of colors is based on Kornerup and Wanscher (1967) (e.g. 3A4-5) and Munsell color chart (1994) (e.g. 2.5YR 4/4). Basidiomes were dried in a hot air dehydrator (45 °C) for a week. Measurements and colors of micromorphological structures were recorded in 3% KOH and Melzer´s solution. Thirty five basidiospores per collection were measured in lateral view. Basidiospore sizes are accompanied by the symbols: X -, representing the range of X (where X is the average of basidiospores length and width in each collection) and Q refers to the range of Q (where Q is the average of the ratio of basidiospore length/basidiospore width in each collection). Line drawings were made under a compound microscope (Nikon Eclipse E400) using an attached drawing tube. Line drawings were made under a compound microscope, using an attached drawing tube. Specimen vouchers are kept at XAL herbarium (Thiers B., continuously updated, Index Herbariorum: http://sweetgum.nybg.org/science/ih/).

Phylogenetic analysis
ITS, LSU and tef-1α sequences of Phylloporus generated in this study and sequences of closely related species downloaded after a BLAST search from GenBank database (http://www.ncbi.nlm.nih.gov/), were incorporated in independent datasets (one for each molecular marker) in the PhyDE program v.0.9971 (Müller et al. 2010). Each dataset (TreeBASE accession 23913) was independently aligned on the online Mafft service (Katoh et al. 2017) and inconsistencies were adjusted manually. The best evolutionary model for every dataset was calculated with MEGA 6.06 (Tamura et al. 2013). A concatenated dataset with previously aligned sequences of ITS + LSU + tef-1α was integrated. Maximum Likelihood (ML) analysis for every dataset and concatenated multilocus dataset were performed for phylogenetic inference, with 1000 bootstrap replicates in the same program. Phylogenetic analyses were also performed with Mr-Bayes v 3.2.6 (Ronquist et al. 2012) for 1,000,000 of replicates. The phylogenies from ML and BI analyses were displayed using Mega 6.06 and FigTree v1.4.3 (Rambaut 2016) respectively.

Results
Eighteen fresh collections of Phylloporus were gathered in the tropical Quercus forests studied. Twenty four ITS, LSU and tef-1α sequences (indicated in bold in Table 1) were obtained from eight specimens, and together with 146 sequences of worldwide Phylloporus species worldwide were included in the phylogenetic analyses developed (Fig. 1). The best resolution in the phylogenetic analyses was obtained in the combined dataset (nrLSU, tef-1α and ITS). In the individual datasets, both species here described were recognized as independent clades with good BS values. We present here the concatenated three-locus phylogenetic tree ( Fig. 1), where the sequences of the Mexican specimens clustered in two strongly supported isolated clades, suggesting that they can be recognized as two different species. Sequences supporting three collections grouped in one clade (BS= 100%, PP= 1.0) sister to sequences of specimens from USA and Panama, identified by Neves et al. (2012) as P. leucomycelinus and P. caballeroi. Another group of five sequences from Mexican specimens also cluster in a well-supported clade (BS= 89%, PP= 1.0) sister to a sequence identified by those authors as P. purpurellus from Costa Rica. Within this latter Mexican clade, sequences recorded as NC 7285-1 and as NC 7286-1, of an unidentified Phylloporus species from USA, appear nested in the phylogeny, suggesting that they belong to the same taxon ( Fig. 1). Considering the distinctive set of morphological features that the Mexican Phylloporus specimens possess (see descriptions below) and with the results of the phylogenetic analysis, we concluded that they represent two new Phylloporus species in tropical Quercus forests from eastern Mexico and both are proposed here.  Diagnosis. Recognized by the combination of pileus vinaceous to grayish-vinaceous, surface becoming rimose-areolate with development, the stipe apex with ribbed appearance and scabrous or even with tiny rigid scales and gills staining blue. Its stature (pileus 27-80 mm diam., stipe 27-80 × 7-12 mm), basidiospores and pleurocystidia size and shape, prevents confusion with P. purpurellus Singer or with P. scabripes B.
Habitat. In soil, in small groups or solitary, in tropical oak forest, under Quercus oleoides Schltdl. & Cham.

Discussion
The multilocus phylogeny inferred demonstrated that Phylloporus rimosus and P. quercophilus are genetically distant, clustered in separate well-supported clades, and apart from other Phylloporus species. Both were found co-habiting in the Quercus forests studied. Although they are somewhat similar in their general habit, when comparing the pileus surface, the velvety texture in P. rimosus becomes rimose-areolate with development, while in P. quercophilus the surface remains uniform. Phylloporus rimosus has more robust basidiomes, with a thicker, scabrous and more rigid stipe. The basidiospore sizes, shape and color are different, being larger in P. rimosus [(9-) 9.5-14 (-15) × 3.5-5 μm, X -= 11-12.3 × 4.3-4.6 μm vs. 9-12.5 × 3-4 μm, X -= 10 -10.7 × 3.6-3.7 μm] more ventricose and attenuated towards the apex, and more pigmented, in contrast to P. quercophilus. The cystidia appear wider (8-27 μm vs. 8-16 μm) and more versiform (including sphaeropedunculate pleurocystidia) in P. rimosus. Another difference is that the latter has a hymenophoral trama with the hyphae arranged in a regular central strand and somewhat divergent on both sides, while in P. quercophilus that trama is distinctly divergent.
In the phylogenetic analysis (Fig 1) P. rimosus grouped close to a Costa Rican specimen identified as P. purpurellus Singer by Neves et al. (2012). According to Singer (1973) the basidiomes of P. purpurellus in comparison with the Mexican species, present a tiny habit, with pileus only up to 26 mm diam. and stipe 30 × 4-4.5 mm; shorter basidiospores (7.5-11.3 × 3.3-4 μm) and with cystidia 48-65 × 8.5-12 μm shorter and narrower. In the analysis, the P. rimosus clade includes two ITS sequences (NC 7285-1, NC 7286-1) obtained from basidiomes growing in a Loblolly pine (Pinus taeda) plantation from North Carolina, USA (after Edwards et al. (2004). Both sequences are inferred to be conspecific with the P. rimosus Mexican collections. Loblolly pine is widely distributed in the SE United States (USDA, https://www.fs.fed.us/database/ feis/plants/tree/pintae/all.html). Currently, the provenance of our specimens and those of Edwards et al. (2004), reveal that P. rimosus displays a range at the eastern portions of both USA and Mexico.
In Costa Rica, Singer and Gomez (1984) concluded that Phylloporus species are present in tropical montane zones forming ectomycorrhiza with Quercus spp. and Alnus jorullensis. They observed however, that this group of fungi did not occur in lower mountains of the country, and suggested that, it is possibly extremely rare there or perhaps, it is not adapted to Q. oleoides or that unknown edaphic or climatic limitations prevent its distribution. Current records of Phylloporus in tropical monodominant stands of Q. oleoides here described suggest the potential ectomycorrhizal association of Phylloporus with this tree species. Additionally, P. rimosus represents a first report of Phylloporus growing in association with Q. sapotifolia trees and even with Pinus taeda.