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 Quercus oleoides 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.

ectomycorrhizal fungi, Neotropical fungi, oak forest

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 and Halling 2010; Neves et al. 2010, 2012; Zeng 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 (1973, 1978) and Singer and Gómez (1984), to more recent contributions by Ortiz et al. (2007), Neves and Halling (2010) 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. 1987; Montoya and Bandala 1991, Ortiz-Santana et al. 2007, Neves 2007, Neves and Halling 2010).

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 (Montoya and 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, 1978; Singer 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 2006; Cavender-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 km², 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 micro-morphological 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.

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.

Figure 1. 

Concatenated three-locus (nrLSU, tef-1α and ITS) phylogenetic analysis by maximum likelihood of Phylloporus species. Bootstrap values (BS> 75) / Posterior probabilities (PP > 0.85) are indicated above branches. New species are indicated in bold letters.

Description of the new species

Phylloporus rimosus Bandala, Montoya & Garay, sp. nov.

MycoBank No: 829439

Figs 2a, b, 3, 4

Holotype

MEXICO. Veracruz: Municipality of Coatepec, Vaquería, gregarious in soil, under Quercus oleoides Schltdl. & Cham., 27 June 2012, Montoya 4834 (XAL).

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. Ortiz & M.A. Neves.

Gene sequences ex-holotype

MK226546 (ITS), MK226554 (LSU), MK314102 (tef-1α).

Etymology

Referring to the rimose pileus surface.

Description

Pileus 27–80 mm diam, convex to plane-convex, at times faintly depressed at center or even infundibuliform; surface velvety, uniform but frequently rimose-areolate, or fracturing and forming rivulose patches, cracked when seen under lens, vinaceous to grayish-vinaceous (7D4–D5, 7C4; 5YR 3/4, 4/3, 4/4–25Y 6/6), darker in some areas especially towards the margin, or yellowish, reddish-yellow, reddish-brown or even yellowish-beige (10YR 5/4, 6/6) in other parts especially towards the center, some specimens even reddish-vinaceous (7E8–E7) with brownish tinges (7D6–6E8), mature specimens fading to brownish when exposed to the sun; margin slightly incurved, edge entire, at times undulate. Lamellae subdecurrent to decurrent, 9–15 mm broad, close, bright yellow (3A7, 5A6–A7; 5Y8/8; 4A16), mustard yellow with age (4A6–A7; 4B7–B8), staining blue or greenish-blue when handled, stains becoming reddish or brownish-vinaceous after several minutes, old specimens or specimens long exposed to the sun developing reddish spots at lamellae sides or even dark brownish red or brown at edge; somewhat sinuous when the hymenophore is seen frontally, veined or anastomosed mostly in the area below the pileus and intervenose or even somewhat labyrinthiform, especially when young; lamellullae of different sizes, edge entire. Stipe 27–80 × 7–12 mm, cylindrical, curved, somewhat sinuous, compact, apex with ribbed appearance by decurrent lines of the lamellae, surface pruinose, scabrous or even with tiny rigid scales, cracked after long exposure to the sun, beige (10YR 6/6–8) or pale yellow (4A/2), or whitish at the bottom of the surface and covered with a reddish or oxide-red (25YR 4/6) pruina, at the middle area reddish-beige (8D16), at times caespitose. Basal mycelium whitish-cream with some yellow spots or even mustard yellow (5Y8/6). Context yellow, staining pinkish or pinkish-brown. KOH 3% reddish (10YR 3/6 to 2.5YR 3/4) on pileus, stipe surface and context; NH₄OH 10% greenish-blue (5Y 2.5/1) on pileus surface, the center of the stain becoming reddish (2.5YR 3/6), brownish at the hymenium, negative in the context and faintly green or negative on stipe surface. Odor mild to slightly citric. Taste mild.

Basidiospores (9–) 9.5–14 (–15) × 3.5–5 µm, X‒ = 11–12.3 × 4.3–4.6 µm, Q‒ = 2.5 2.8 µm, subfusiform, with suprahilar depression, somewhat ventricose, apex attenuated, yellow to amber yellow in KOH, wall slightly thickened (up to 0.5 µm thick). Basidia 29–50 (–55) × 7–10 (–11) µm, clavate, tetrasporic, rarely trisporic, hyaline, thin walled, unclamped. Pleurocystidia 42–105 (–120) × 9–27 µm, narrowly to broadly utriform, at times cylindrical or subclavate, rarely sphaeropedunculate (52–58 × 20–23 μm), thin-walled, at times thickened in some areas, some with incrustations, hyaline, abundant, unclamped. Cheilocystidia (33–) 34–70 (–75) × 8–17 (–19) µm, narrowly utriform, hyaline, thin-walled, at times thickened towards the apex, unclamped. Pileipellis a trichodermis, with anticlinally oriented hyphae, tightly interwoven, frequently disposed in mounds, hyphae 8–16 µm broad, wall slightly thickened (up to 1 µm), hyaline yellowish-brown; terminal elements 23–64 × 8–14 µm, cylindrical, slightly inflated, other or clavate, pale yellowish-brown. Pileus trama hyphae 5–16 µm broad, in a lax interwoven arrangement, hyaline, thin walled. Hymenophoral trama arranged in a more or less regular central strand and somewhat divergent on both sides of the strand, with cylindrical hyphae 7–19 µm broad; some slightly inflated, hyaline, thin-walled, unclamped.

Habitat

In soil, solitary or gregarious, in tropical oak forest, under Quercus oleoides and Q. sapotifolia.

Additional studied material

MEXICO. Veracruz: Alto Lucero Co., NE Mesa de Venticuatro, 4 Oct 2016, Garrido14; 19 Sep 2017, Gutiérrez 37. Zentla Co. Road Puentecilla-La Piña, 2 July 2009, Ramos 195. Around town of Zentla, 15 June 2016, Montoya 5232a; Montoya 5238; 23 June 2016, Gutiérrez 5, Hervert 84; 30 June 2016, Cesar 61, Hervert 93; 6 July 2016, Caro 69; 30 Aug 2016, Garrido 3; 24 Aug 2017, Garay 368; 7 Sep 2017, César 84 (all at XAL).

Figure 2. 

Basidiomes of Phylloporus species. a, b P. rimosus (a Garrido 3, b Montoya 5232a) c P. quercophilus (LM5239 holotype). Scale bars: 10 mm.

Figure 3. 

Phylloporus rimosus (Montoya 4834, holotype). a Basidiospores b hymenophoral trama c longitudinal section of pileipellis. Scale bars: 10 μm (a), 25 μm (b), 100 μm (c).

Figure 4. 

Phylloporus rimosus (Montoya 4834, holotype). a Basidiospores b basidia c cheilocystidia d pleurocystidia. Scale bars: 5 μm (a), 10 μm (b–d).

Phylloporus quercophilus Montoya, Bandala & Garay, sp. nov.

MycoBank No: 829440

Figs 2c, 5, 6

Holotype

MEXICO. Veracruz: Municipality of Zentla, around town of Zentla, 850 m a.s.l., in soil, in small groups, at tropical oak forest, under Quercus oleoides 15 June, 2016, Montoya 5239 (XAL).

Diagnosis

Its reddish pileus tinges together with, context staining reddish, basidiospores 9–13 × 3–4 µm and narrowly utriform or subcylindrical cystidia and its habitat distinguish it from close related species, such as P. caballeroi Singer.

Gene sequences ex-holotype

MK226550 (ITS), MK226558 (LSU), MK314106 (tef-1α).

Etymology

In reference to the habitat.

Description

Pileus 15–65 mm diam., hemispheric at first, then becoming convex to plane-convex,; surface velvety, reddish-vinaceous (8D7, 8E7–8), dark reddish-brown (9E6–7), brown (7C5) with pinkish tinges to pinkish-vinaceous (7C6) with paler zones and dark vinaceous tinges (7D6–D7); margin straight to slightly decurved to incurved, undulate. Lamellae 5–8 mm width, adnate to subdecurrent, close to slightly subdistant, yellow (3A5, 3B7), mustard-yellow (4B7–B8), staining pale brown or blue-greenish when handled, veined or anastomosed mostly below pileus surface and with interparietal veins, margin finely fimbriate, lamellullae of different sizes, with reddish spots. Stipe 25–55 × 3–13 mm, central, attenuated towards the base, sinuous, compact, reddish-vinaceous (9E7), middle and basal part yellowish to pale brown, bright yellow (3A2, 4A6), with olive to pinkish-vinaceous tinges when young, frequently with a reddish pruina and fine appressed scales over the apex, surface smooth, with peeling fibers especially in mature specimens. Basal mycelium whitish to yellowish. Context dirty whitish, staining reddish especially towards the pileus area where it is hygrophanous; stipe at times fistulose but mostly compact, especially at apical area. KOH 3% blackish on pileus, greenish to brown in lamellae, negative in context; NH₄OH 10% bluish on pileus, or bluish-greenish at the beginning, later blackish in pileus and stipe, dark grayish-blue in context and lamellae. Odor fruity. Taste mild.

Basidiospores 9–13 × 3–4 µm, X‒ = 10–10.7 × 3.6–3.7 µm, Q‒ =2.7–2.9 µm, subcylindrical, with a faint suprahilar depression, attenuated towards apical area and with rounded apex, frontal view subcylindrical, hyaline, with very pale greenish tinges, wall slightly thickened (up to 0.5 µm) 10 to 30% in a field of view dextrinoid. Basidia 28–42 (–46) × 6–10 µm, clavate, tetrasporic, hyaline, unclamped. Pleurocystidia 50–102 × 8–16 µm, narrowly utriform, subutriform or irregularly subcylindric, hyaline, pale yellowish, not incrusted, thin walled, at times the wall slightly thickened up to 1 µm, unclamped. Cheilocystidia 42–90 × 8–14 µm, hyaline, narrowly fusiform to subcylindrical, thin-walled, at times incrusted, unclamped. Pileipellis a trichodermis composed of more or less erect and tightly interwoven hyphae, at times disposed in mounds, hyphae 7–14 µm broad, thin walled, unclamped; terminal elements 20–48 × 7–14 µm, hyaline, other cells with pale yellow contents, this layer yellowish-brown in KOH at lower magnifications, thin walled, unclamped. Pileus trama hyphae 6–13 µm broad, in a compact interwoven arrangement, cylindrical to subcylindrical, hyaline, thin walled, at times incrusted in a faintly circumferential striate pattern, unclamped. Hymenophoral trama divergent; hyphae 6–12 µm broad, thin-walled (< 1 µm thick), at times with resinous like incrustations, some hyphae with a faintly striate appearance, hyaline, unclamped.

Habitat

In soil, in small groups or solitary, in tropical oak forest, under Quercus oleoides Schltdl. & Cham.

Additional studied material

MEXICO. Veracruz: Zentla Co., around town of Zentla, 850 m a.s.l., 12 July 2017, Gutiérrez 29; 24 Aug 2017, Garay 366; 7 Sep 2017, Garay 373a (all at XAL).

Figure 5. 

Phylloporus quercophilus (Montoya 5239, holotype). a Basidiospores b hymenophoral trama c longitudinal section of pileipellis. Scale bars: 10 μm (a), 100 μm (b), 25 μm (c).

Figure 6. 

Phylloporus quercophilus (Montoya 5239, holotype). a Basidiospores b basidia c pleurocystidia d cheilocystidia. Scale bars: 5 μm (a), 10 μm (b–d).

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.

Phylloporus quercophilus appeared as a sister species (Fig. 1) to specimens identified by Neves et al. (2012) and Zeng et al. (2013) as P. caballeroi Singer and P. leucomycelinus Singer. Phylloporus caballeroi described by Singer (1973) from Argentina, differs from P. quercophilus by the pileus with olivaceous tinges, lamellae in a closer arrangement, context not staining reddish, and association with Alnus. Neves and Halling (2010) offered a broader concept of P. caballeroi, and congruent with the original diagnosis, they cite similar basidiospores [4–5 (–6) μm diam. (Q= 2.21)] and ampullaceous cystidia. Phylloporus leucomycelinus differs from P. quercophilus by the smalller basidiomes (pileus 28–34 mm diam; stipe 27–45 × 3–5 mm), with deep red-brown pileus, lamellae brownish yellow to yellow-brown with olive tinge, and shorter [50–71 × (6–) 11–12] ampullaceous cystidia (Singer 1978).

Considering some morphological and color resemblance, P. rimosus and P. quercophilus should be compared with P. scabripes B. Ortiz and M.A. Neves from Belize, P. bellus (Massee) Corner and P. rufescens Corner from Singapore (Corner 1970; Singer and Gómez 1984; Ortiz-Santana et al. 2007). However, they are genetically distinct according to the phylogeny inferred here (Fig. 1) [that include sequences produced by Neves et al. (2012) and Zeng et al. (2013)]. Phylloporus scabripes is similar to P. rimosus because of its distinctly scabrous stipe surface, an unusual feature for a species of Phylloporus. The former species differs however, from P. rimosus by shorter basidiospores [9.8–12.8 × 3.2–4.8 µm vs. (9–) 9.5–14 (–15) × 3.5–5 µm] and shorter and broader pleurocystidia [43.2–80 × 13.6–15.2 µm vs. 42–105 (–120) × 9–27 µm)]. The pleurocystidia size also distinguishes P. scabripes from P. quercophilus (50–102 × 8–16 µm) as also the pileus color of P. scabripes [“...pale reddish brown (6D4), paler to tan with age (near 5C4...”] is paler and brownish in range, not as vinaceous, as in both of the Mexican taxa, and the context of P. scabripes does not stain reddish. Moreover, P. scabripes apparently lacks cheilocystidia.

We concur with Zeng et al. (2013) that the name P. bellus has been too widely applied. So we refer here to the original description (Corner 1970) which defines this species with shorter basidiomes than those of P. rimosus [stipe (30–40 × 4–10 mm)], with narrower lamellae (4–7 mm width) and shorter basidiospores [8.5–10 × 4.5–5.5 (–6) μm]. The basidiospores of P. bellus are even shorter than in P. quercophilus. The Asian species P. bellus differs also from both Mexican taxa by the context not staining reddish. On the contrary, P. rufescens Corner shares with both Mexican taxa the reddening of the context but it finally turns black on exposure (Corner 1970), which does not occur either in P. rimosus or in P. quercophilus. Other differences among P. rufescens and the Mexican species are the shorter size of basidiospores (8–9 × 4–5 μm) and more robust basidiomes (pileus 50–140 mm diam and stipe 25–120 × 6–25 mm).

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.

We recognize the support of CONACYT (CB 252431) to study the EcM fungi associated with tropical species of Quercus in Veracruz, Mexico. E. Garay is grateful for the postdoctoral scholarship grant from Fondo SEP-CONACYT for the project CB-252431 and from the INECOL. We acknowledge the assistance in field and laboratory of Biol. D. Ramos (Instituto de Ecología, A.C.). We appreciate the support given by CONACYT (225382) to the Laboratorio de Presecuenciación, Red Biodiversidad y Sistemática, INECOL. IBT Bertha Pérez assisted us in molecular procedures. We appreciate the English revision by Dr. Andrew Vovides, INECOL.