Greetings from belowground: two new species of truffles in the genus Pachyphlodes (Pezizaceae, Pezizales) from México

Abstract Pachyphlodes is a lineage of ectomycorrhizal, hypogeous, sequestrate ascomycete fungi native to temperate and subtropical forests in the Northern Hemisphere. Pachyphlodes species form ectomycorrhizae mainly with Fagales hosts. Here we describe two new species of Pachyphlodes, P.brunnea, and P.coalescens, based on morphological and phylogenetic analysis. Pachyphlodesbrunnea is distributed in the states of Tamaulipas and Nuevo León in northern México, occurring with Quercus and Juglans species. It is characterized by its dark brown peridium, white gleba, and spores with capitate columns. Pachyphlodescoalescens is distributed in the states of Michoacán and Tlaxcala in central and southwestern México co-occurring with Quercus and is distinguished by its reddish-brown peridium, light yellow gleba, and spore ornamentation. Both species, along with P.marronina, constitute the Marronina clade. This clade contains North American species characterized by a brown peridium and spores ornamented with capitate spines to coalesced spine tips that form a partial perispore.


Introduction
Pachyphlodes Zobel, 1854 (Pezizaceae, Pezizales) is characterized by truffle-like ascomata with a thick peridium of large isodiametric cells and globose spores ornamented with spines or columns. The spores are either naked or covered with a perispore (Tulasne and Tulasne 1844;Healy et al. 2018). There are currently 16 recognized Pachyphlodes species and two varieties in the genus (Kirk 2016;Ting et al. 2019). Pachyphlodes was known as Pachyphloeus, but this name was declared illegitimate (Healy et al. 2018), so its species were transferred to the oldest legitimate name Pachyphlodes (Doweld 2013a, b). Pachyphlodes species are distributed across the Northern Hemisphere; in North America and Europe; they form ectomycorrhizae with hosts in the Betulaceae, Fagaceae, and Juglandaceae in temperate and subtropical regions (Smith et al. 2007;Lindner and Banik 2009;Stefani et al. 2009;Tedersoo et al. 2009Tedersoo et al. , 2010Bonito et al. 2011;García-Guzmán et al. 2017). With the use of molecular techniques, the number of Pachyphlodes species has nearly doubled from eight species and two varieties in 2000 to 16 species and two varieties in 2020. Four species have been described from México; Cázares et al. (1992) reported P. citrina (Berk. and Broome) Doweld (unverified by molecular methods) from Nuevo León and P. virescens (Gilkey) Doweld (unverified by molecular methods) from Nuevo León and Tamaulipas; Healy et al. (2009) reported Pachyphlodes cf. carnea from Nuevo León, and described a new species P. marronina Healy, Bonito & Guevara from Nuevo León, Tamaulipas and Tlaxcala. Healy et al. (2009) remarked on morphological differences between the P. marronina collections from the upper Midwestern USA and the P. marronina collections from México and proposed they may be part of a species complex in need of further analysis. With the aim to solve this species complex, here we report new collections along with results from further analyses that support the description of the Mexican collections as two new species of Pachyphlodes in the Marronina clade.

Morphological observations
Ascomata of P. brunnea were collected from the state of Tamaulipas, while P. coalescens collections were found across the states of Michoacán and Tlaxcala. All the specimens are deposited in the following herbaria: Oregon State University (OSC), Instituto Tecnológico de Ciudad Victoria (ITCV) and Herbario Nacional de México (MEXU). Macroscopic characters were described from fresh specimens under natural light, and colors of fresh ascomata are described in general terms by the authors. Microscopic characters were described from razor-blade sections of fresh specimens mounted in 5% KOH and Melzer's reagent. Fifty measurements were taken per structure; measurements of structures are length by width (this is the order of appearance in the descriptions). For scanning electron microscopy (SEM), ascospores were scraped from the dried gleba onto double-sided tape, which was mounted directly on an SEM stub, coated with platinum-palladium, and examined and photographed with a HITACHI TM 3000 scanning electron microscope, or they were prepared and imaged as outlined in Healy et al. (2018).

DNA sequencing and phylogenetic analyses
A tissue sample from collection MEXU 26842 was sent to the Canadian Center of Barcoding (CCDB) for extraction, amplification, and sequencing of the Internal Transcribed Spacer (ITS). DNA was extracted from JT32454, JT32623, and ITCV-GGG-896 at the University of Minnesota with a modified CTAB method (Healy et al. 2009). The ITS1-5.8s-ITS2 (ITS) region was amplified with ITS1 and ITS4 (White et al. 1990) and ITS1f (Gardes and Bruns 1993). DNA sequences were deposited in GenBank (Table 1). Sequences were edited in Geneious 7.1 (Kearse et al. 2012) or Sequencher 4.0 (Gene Codes, Ann Arbor, MI). As done in Piña Páez et al. (2018), the distribution of species was complemented with soil DNA data from central and south México through a BLASTn search against the Mexican Soil Fungi Database in Geneious 10.1. This database includes ITS2 sequences of soil fungi from México and has been partially published in Argüelles-Moyao and Garibay-Orijel (2018).
Phylogenetic analyses of ITS rDNA have been implemented to describe and resolve species delimitation in Pachyphlodes (Healy et al. 2015;Li et al. 2019;Liu et al. 2020). Phylogenetic analyses utilizing the 28S rDNA, ß-Tubulin, and RPB2 markers showed that Pachyphlodes is a member of the Pezizaceae, that Plicariella (Sacc.) Rehm (as Scabropezia Dissing and Pfister) is sister to Pachyphlodes, and that the sister lineage to Pachyphlodes and Plicariella is Amylascus Trappe (Hansen et al. 2005). Healy et al. (2018) showed that Plicariella is within or sister to the Melanoxanthus clade of Pachyphlodes. Our phylogenetic analysis consisted of 42 sequences from 16 described species, including nine sequences from type specimens of Pachyphlodes and from Amylascus Trappe. Amylascus was selected as an outgroup based on previous phylogenetic analyses. DNA sequences were aligned with MAFFT v 6.822 (Katoh and Toh 2010) and manually improved in SE-AL v2.0a11 (Rambaut 2007) for a final alignment with 754 positions. Phylogenetic inferences were estimated with maximum likelihood in RAxML 7.2.8 (Stamatakis 2006) with a GTR + G model of nucleotide substitution. For Bayesian posterior probability, priors were selected with jModeltest 2.1.4 (Darriba et al. 2012), under the Aikake information criterion, and posterior probability was estimated in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) with 20,000,000 generations with trees sampled every 1000 generations. The first 25% of samples were discarded as burn-in, and stationarity was checked in Tracer (Rambaut and Drummond 2007). RAxML and MrBayes were both runs on the Cipres Portal (Miller et al. 2010). Trees were visualized and optimized in FigTree 1.4.4 (http://tree.bio.ed.ac.uk/ software/figtree/), and font and color were added in Adobe Illustrator vCS4 (Adobe Systems, Inc., San Jose, CA). Alignment is available in OSF (Open Space Framework, to be uploaded prior to journal submission).

Results
The nucleotide substitution model selected by jModeltest was TPM1uf+I+G. The final optimization likelihood was -lnL 4774.259669, and the most likely tree is shown in Fig. 1. Both the Maximum Likelihood and Bayesian analyses (Fig. 1) show that P. brunnea forms a new strongly supported clade (100/1), which includes sequences from  voucher collections and ectomycorrhizae. This clade is placed as a sister taxon of P. marronina, which also forms a strongly supported clade (99/1). The sister taxon (100/1) of these two species is P. coalescens, which is also a new strongly supported clade (99/1). Etymology. Latin, brunnea in reference to the brown peridium. Description. 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.
Distribution and ecology. 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.
Taxonomic comments. 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. Diagnosis. 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.

Pachyphlodes coalescens
Etymology. 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.

Discussion
The Pachyphlodes marronina original description included collections from Iowa, U.S.A., Nuevo León and Tlaxcala, México. Cryptic diversity within this species was addressed by Healy et al. (2009) concerning molecular differences between the P. marronina collection from the U.S. and the Mexican collections. We now have additional molecular, geographical, and morphological evidence that the P. marronina complex includes three distinct species across North America: P. brunnea, P. coalescens, and P. marronina. Our evidence indicates that P. brunnea is associated with Quercus on the basis of molecular information from an ectomycorrhizal sequence (JN102443). However, no direct evidence exists that Quercus is the ectomycorrhizal host for the other two species. Their habitat descriptions suggest these two species associate with Quercus, but we need more environmental data to corroborate the association.
The three members of the Marronina clade ( Fig. 1) have an ornamented peridium with flat warts, which are more conspicuous in P. brunnea and P. coalescens. The struc-ture and composition of the peridium are also similar; all three have a two-layered peridium composed of an outermost layer of textura angularis and an inner layer of texture intricata. The biseriate to uniseriate arrangement of the spores in the asci is similar across the three species. Pachyphlodes brunnea resembles P. marronina in spore ornamentation; both species have spores with spines columns that are joined by the accumulation of material at the apex of the spines. However, the shorter spines in P. brunnea confer a clumpier appearance overall. The spore ornamentation in P. coalescens is different from the other two Marronina clade members but is simply the result of the coalescence of spine tip material, which occurs only occasionally in P. marronina. The late-stage spore ornamentation of the coalescence of spore tip material is also seen in P. nemoralis and P. pfisteri (Healy et al. 2015), but these species otherwise differ in color, peridium structure, and phylogenetic placement (Fig. 1).
The sister species of the Marronina clade is P. oleifera (Fig. 1), a European taxon with very distinct morphological characters, peridium with coarse warts, an unusual gray blueish gleba, and finely verrucose spores (Cabero and Pérez-Pérez 2012). Another characteristic of P. oleifera that separates it from the rest of the known species of Pachyphlodes is the oily content in all the microscopic structures, particularly hymenial cells.