México, Tamaulipas, Ciudad Victoria, Torre de Microondas “Las Mulas”, 23°37'00"N, 99°14'31"W, alt. 1549 m, under Quercus polymorpha Schlecht. & Cham., Quercus sp. and Juglans sp., hypogeous, solitary or in groups of 2, 11 November 2006, col. G. Guevara (holotype: ITCV 896).

Pachyphlodes brunnea is be recognized by the dark brown ascomata and two-layered. Thick (474–570 µm) peridium, white gleba when immature, spores ornamented with capitate columns growing under Quercus and with an odor similar to raw potatoes.

Latin, brunnea in reference to the brown peridium.

Ascomata subglobose to ovoid, 15–17 × 10–15 mm, surface dry, with an irregular basal depression, surface dark brown when fresh (Fig. 2a), with geometric, angular, or pyramidal warts 1 mm wide, with flattened, elevated, or rounded top. Gleba solid (Fig. 2b), marbled with white sterile veins separating brownish, fertile tissue, overall brownish when dried. Odor of corn starch-like or of raw potatoes.

Figure 2. 

Pachyphlodes brunnea (Holotype: ITCV 896) a ascoma dried b gleba in cross-section c peridium in cross-section, showing a wart composed of isodiametric cells d light microscopy of asci and spores e, f SEM microscopy of spores in surface view. Scale bars: 3 mm (a, b), 20 µm (c, d), 5 µm (e, f).

Peridium of two layers. Outer peridium 125–570 µm thick, of textura angularis, with warts up to 300–500 (–800) μm high, outermost cells up to 42 μm broad, some ventricose or irregular, radial arrangement in some areas, walls 2–3 (–5) μm thick, reddish-brown to orange-brown in 5% KOH, innermost cells up to 10 μm broad, walls 1–2 μm thick, hyaline in 3% KOH. Inner peridium 120–500 (–700) μm thick, composed of hyaline, septate, interwoven hyphae (textura intricata), 5–12 µm broad, thin-walled 1–2 μm thick. Asci 8-spored, clavate, subclavate, subfusoid or irregular, 120–238 × 30–45 µm including pedicel, hyaline in 5% KOH, walls 1 µm thick, asci are scattered. Paraphyses not detected. Ascospores irregularly biseriate to uniseriate, hyaline in 5% KOH, globose, including ornamentation 18–22 µm broad, mean = 20 µm; excluding ornamentation 12–18 (–20) µm broad, mean = 15 µm. Ornamentation averaging 1.5 (–2.0) µm high, capitate columns, consisting of columns with a boarder, rounded tip.

Known only from northeastern México (Tamaulipas, Nuevo Leon). Ascomata hypogeous always associated with Quercus polymorpha, and DNA (JN102443) of this species were recovered from sampled roots of oak (JN102443) from Chipinque National Park in Nuevo León. No DNA sequences of this species were found in soil in central or southern México.

México, Tamaulipas, Ciudad Victoria, Torre de Microondas “Las Mulas”, 23°37'00"N, 99°14'31"W, alt. 1549 m, under Quercus polymorpha, Quercus sp. and Juglans sp., hypogeous, solitary or in pairs, November 11, 2006, col. G. Guevara (ITCV 891; No ITS); Carretera Victoria, El Madrono, 23°36'3"N, 99°13'8"W, alt. 1460 m, under Quercus canbyi Trel., Q. polymorpha, and Q. laeta Liebm., hypogeous, August 1, 2008, col. G. Bonito (JT32623; GenBank MT461399). Nuevo León, Municipio de Santiago, El Cercado September 14, 1983, col J. García (UNL 3757; GenBank EU427551).

The ITS sequences of Pachyphlodes brunnea are similar to those of P. marronina (97.79% of identity and 12 nucleotide differences in ITS region), which is why it was originally described as P. marronina. However, the peridium color and geographic location of these two species differ considerably. Spore ornamentation also separates them. The fresh peridium of P. marronina is red with indistinct warts, while that of P. brunnea is dark brown with distinct angular warts. The angular to pyramidal warts in the peridium of P. brunnea aretaller (300–800 µm) than the lower, indistinct warts on P. marronina (160–270 µm). The spines in P. marronina are taller (1.5–3.0 µm) than P. brunnea (1.5–2.0 µm), conferring a different aspect to the spores overall (Fig. 2e, f). Pachyphlodes brunnea superficially resembles P. melanoxantha (Tul. & C. Tul. ex Berk.) Doweld and P. annagardnerae R.A. Healy & M.E. Sm., but the latter two are black to the unaided eye, purple under transmitted light, have acute tipped spiny spores, and P. melanoxantha is said to have a nauseous odor (Berkeley 1844). In contrast, P. brunnea is dark brown to the unaided eye, yellowish-brown under transmitted light, and has a pure white gleba with capitate spore spines and a pleasant odor. Pachyphlodes annagardnerae has no perceptible odor.

México, Michoacán, road Morelia-Atécuaro, Morelia, 19°36'0"N, 101°10'58.8"W, alt. 2280 m, under Quercus deserticola Trel., hypogeous, solitary, 30 September 2012, col. R. Garibay-Orijel (holotype: MEXU 26842).

Pachyphlodes coalescens can be recognized by the brown ascomata and two-layered, thick (600–700 µm) peridium, and a gleba marbled with light yellow, meandering, sterile veins alternating with dark brown fertile veins, spores ornamented with truncated spines, that have material deposited at the tips, which accumulates and coalesces with neighboring tip material to form a broad, meandering, roughened, reticulum that hides the underlying spines, growing under Quercus.

Named for the process that produces the spore ornamentation: material deposited on the spine tips coalesces to form a meandering reticulum, from Latin coalecere, to grow together.

Ascomata irregularly subglobose, slightly compressed, 12 × 14 mm, surface with flat, polygonal warts with 4–6 sides, each wart about 2.5–3.0 mm broad, orange-brown when fresh (Fig. 3a), dark reddish-brown when dried, areole 6 × 4 mm where internal sterile veins emerge. Gleba light yellow with translucent yellowish sterile veins when fresh becoming cream with light brown veins when dried (Fig. 3b).

Figure 3. 

Pachyphlodes coalescens (Holotype: MEXU 26842) a ascoma fresh b gleba in cross-section c peridium in cross-section, showing a wart composed of isodiametric cells d light microscopy of asci and spores e, f SEM microscopy of spores in surface view. Scale bars: 5 mm (a, b), 100 µm (c), 10 µm (d, e), 5 µm (f).

Peridium of two layers. Outer peridium 440–500 μm thick, composed of textura angularis, with warts up to 220 μm high, outermost cells up to 30 μm broad, walls 1 μm broad, orange-brown in 5% KOH, interior cells up to 22 μm broad with notably thinner cell walls <0.5 µm, hyaline (Fig. 3c). Inner peridium about 175–190 μm thick, composed of hyaline, septate, interwoven hyphae 4.5–6.5 µm broad, thin-walled <0.5 μm. Paraphyses filiform, septate, with swollen tips, 200–210 × 8.75 μm, 10–14 μm broad at the apex, pale green with granular contents, thin-walled <0.5 μm. Asci 8-spored, irregularly distributed in fertile brown veins among interwoven hyphae, pyriform to cylindrical with a short pedicel, 180–195 μm long including pedicel, 40–50 μm wide, pedicel 22–26 × 10–12 μm, widening at the base, hyaline in 5% KOH, walls <0.5 µm (Fig. 3d). Spores irregularly biseriate to uniseriate. No reaction of asci in Melzer’s reagent. Ascospores (Fig. 3e, f) globose, hyaline to light yellow, size range including ornaments 20–23 μm, averaging 21.20 μm, spores excluding ornaments 16–18 μm, averaging 17.70 μm. Ornamentation averaging 1.80 μm high, of short capitate spines that accumulate material at the tips that coalesces to produce a nearly solid covering over the spore by maturity.

Ascomata hypogeous, known from Michoacán and Tlaxcala co-occurring with Quercus deserticola Trel, Quercus rugosa Née, and Q. crassifolia Humb. & Bonpl. DNA sequences have also been found in Quercus dry forests or xerophilous pine-oak forests in Libres in Puebla, Tequila volcano in Jalisco, and Cerro del Águila in Michoacán, all in central-southwestern México.

México, Tlaxcala, 1 km east of San Francisco Temezontla, Municipio Panotla, alt. 2600 m, under Quercus rugosa Née, and Q. crassifolia Humb. & Bonpl., September 20, 2007, col. E. Cázares (JT32454; GenBank EU543209).

Pachyphlodes coalescens has a texture and peridial structure of the peridium similar to the other two species of the Marronina clade (P. brunnea and P. marronina) clade, but they vary in other macroscopic or microscopic characteristics. Ascomata of Pachyphlodes brunnea are dark brown to brownish black, whereas P. coalescens ascomata are orange-brown. In addition, they differ in spore size (P. brunnea 18–22 μm vs. P. coalescens 20–23 μm), and the spore ornamentation of P. brunnea is of discreet, capitate columns, whereas in P. coalescens, it is of spines with additional material that is so thickly deposited at the apices as to form a broad, meandering perispore that nearly covers the spore surface. Pachyphlodes coalescens are similar to P. marronina, but the latter has smaller spores (19–22 μm) ornamented with coarse, mostly discreet, truncate to capitate spines, whereas P. coalescens has short spines fully connected at the tips via the material deposited at the apex of each spine (see above). The spore ornamentation of P. coalescens is similar to that of P. nemoralis Hobart, Bóna & A. Paz and P. pfisteri Tocci, M.E. Sm. & Healy, which otherwise differ strongly in color, peridium structure, and phylogenetic placement.