Introduction

Species of the genus Tuber P. Micheli ex F.H. Wigg belong to the family Tuberaceae (Pezizales) and form ectomycorrhizae with many forest tree species (^{Bonito et al., 2011}; ^{Li et al., 2018}). These species are ecologically and economically important (^{Guevara et al., 2013}; ^{Neri-Luna et al., 2012}). Approximately 86 species of Tuber are known worldwide (^{Guevara et al., 2013}; ^{Kirk et al., 2008}). They grow in mycorrhizal symbiotic association with gymnosperm and angiosperm trees. Thirty-eight species of Tuber have been described in North America (^{Guevara et al., 2013}). The diversity of the genus in Mexico is expected to be high because this region is a center of diversification of Quercus Kappelle, Maarten and Pinus Linneo, 2 of the main ectomycorrhizal hosts of Tuber. Knowledge of Tuber in Mexico has recently expanded. For example, new species belonging to the Maculatum clade, such as Tuber castilloi Guevara, Bonito & Trappe, T. guevarai Bonito & Trappe (^{Guevara et al., 2013}), Tuber mexiusanum Guevara, Bonito & Trappe (^{Guevara et al., 2013}), T. mixtecorum J. García, Ayala Vázquez & de la Fuente (^{García-Jiménez et al., 2021}) and T. theleascum M. Leonardi, A. Paz, G. Guevara & Pacioni (^{Leonardi et al., 2019}) associated with Quercus spp., have been described in northeastern Mexico. Tuber incognitum and T. anniae belonging to the Puberulum clade have also been recorded in association with Quercus spp and Pinus montezumae in central Mexico (^{Piña-Páez et al., 2018}). In association with trees of forest interest in Mexican ecosystems, T. guzmanii, T. separans, and T. pseudoseparans have also been found (^{Gómez-Reyes et al., 2018}; ^{Guevara et al., 2015}; ^{Piña-Páez et al., 2018}).

Carya illinoinensis K. Koch (pecan) is an agronomically important ectomycorrhizal nut tree in whose plantations in the southern United States truffle species such as Tuber lyonii have been reported (^{Benucci et al., 2012}; ^{Bonito et al., 2011}; ^{Rodríguez et al., 2018}). Ascomata and ectomycorrhizae of Tuber brennemanii and T. floridanum have also been found in these orchards (^{Grupe et al., 2018}). In Mexico, Tuber diversity has not been explored in pecan plantations, although due to their particular soil and climatic conditions it is to be expected that there are species not yet described. In this paper, Tuber caryophilum sp. nov., a species in the Rufum clade, whose distribution includes Chihuahua and la Comarca Lagunera of Coahuila, Mexico, is described based on morphological, ecological and molecular characters.

Materials and methods

Ascomata were collected from a Carya illinoinensis orchard in the Comarca Lagunera of Coahuila and characterized following the recommendations of ^{Castellano et al. (1989)} and ^{Pegler et al. (1993)}. Duplicates of the specimens were deposited in the José Castillo Tovar herbarium (ITCV) and MEXU. Characters examined included ascoma size and color, asci shape, asci wall thickness, and number of spores per ascus. Sections were cut by hand and then mounted in 5% KOH and Melzer's reagent for light microscopy. Thirty measurements of different structures such as spores and asci were made in 5% KOH. Microscopic structures were measured and photographed on a Velab VE-B3 microscope and a ZEISS Scope A1 optical stereoscope.

DNA was extracted by the CTAB method and amplified by PCR in 25μl reactions according to ^{Sambrook et al. (1989)}. The reactions consisted of 2.5 μl of 10X PCR buffer, 2.0 μl of 2.5 Mm MgCl final concentration, 2.0 μl of dNTPs, 2.0 μl of each primer 10 μM, 1.5 units of Taq polymerase (GoTaq®, Promega, WI), 11.3 μl of MiliQ grade water and 3 μl of DNA. The ribosomal internal transcribed spacer (ITS) region was amplified with the ITS4 and ITS5 oligonucleotides. The PCR program consisted of an initial denaturation at 94 °C for 3 min, followed by 34 cycles of 94 °C, 51 °C and 72 °C for 1 min each and a final extension at 72 °C for 8 min (Taylor et al., 2006). Amplification was carried out on a MiniAmp Plus Thermal Cycler (Applied Biosystems, USA). A section of the large ribosome subunit (LSU) was amplified with the LR0R and LR5 oligonucleotides (^{Vilgalys & Hester, 1990}) and the enzyme Taq & Load (Avantor, PA, USA). The 25 μl reactions consisted of 5 μl of master mix, 0.25 μl of each oligonucleotide 50 μM, water, and 1 μl of DNA. The PCR program began with denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C, 54 °C and 72 °C for 1 min each and final extension at 72 °C for 10 min. PCR products were cleaned with ExoSAP-IT (Thermofisher, USA) with some modifications (Ángeles-Argáiz et al., 2016). DNA and PCR products were reviewed on 0.8% and 1.5% agarose gels with 0.5% TBE buffer. Samples were stained with Gel Red (Bioitium, CA, USA) using a 100 bp molecular weight marker as reference. Gels were photographed on a Multidoc-IT photodocumenter (Analytik Jena Company, CA, USA). ITS PCR products were sequenced in both directions at Macrogen (Rockville, MD, USA), with PCR primers. LSU sequences were obtained at the Biodiversity and Health Sequencing Laboratory of the Institute of Biology, UNAM, using BigDye Terminator 3.1 (Thermofisher), also in both directions.

Nucleotide sequences were edited and aligned in Geneious Prime version 2021 with the MUSCLE algorithm (^{Maddison & Maddison, 2016}). Sequences of T. caryophilum voucher materials were deposited in GenBank under accession numbers MZ092919 and OK642388 for ITS, OK642397 and OK642398 for LSU and OK642406 for the ITS of ectomycorrhizae. The alignment for phylogenetic analyses included the sequences generated in this study, those previously included in analyses of the Rufum clade (^{Eberhart et al., 2020}; ^{Leonardi et al., 2019}) and sequences of high nucleotide similarity obtained from the GenBank database by means of the BLAST algorithm (^{Altschul et al., 1990}) (Table 1). A total of 72 samples from 26 taxa of the Rufum clade and 1 outgroup were aligned (Fig. 1). The concatenated alignment had 1,076 bp where bases 1-556 corresponded with the ITS and bases 557-1,076 with the LSU. The alignments were reviewed manually excluding ambiguous regions.

Figure 1 Phylogenetic placement of Tuber caryophilum sp. nov. in the Rufum clade. The tree is based on an ITS and LSU rDNA concatenated alignment. The consensus tree represents a Bayesian approximation with 1,000 generations and a maximum likelihood analysis with 1,000 bootstrap replicates. Just support values higher than 0.70 and/or 70% are displayed. 

Phylogenetic analyses and evolutionary model selection were performed using IQ-TREE (v2.1.4, ^{Minh et al., 2020}) from the concatenated and partitioned alignment (IQ-TREE execution line: iqtree -s ../data/concat.fasta -p Partition.nex -m MFP --runs 100 --abayes -B 1000 -T AUTO -ntmax 28). The best evolutionary models were selected with ModelFinder (^{Kalyaanamoorthy et al., 2017}). The best evolutionary model for the ITS marker was TIM2+F+I+G4 and for LSU it was TIM3e+I+G4. The resulting tree is the consensus of 100 replicates of 2 phylogenetic analyses; an ultra-fast Maximum Likelihood analysis (^{Hoang et al., 2018}) with 1,000 bootstrap replicates (MVB), complemented with a Bayesian approximation branch support (BPP) analysis (^{Anisimova et al., 2011}). To show the ectomycorrhizal status and distribution of T. caryophilum, the ITS sequence of the holotype was contrasted by means of nucleotide similarity (% ITS NS) against ectomycorrhizal sequences of Carya illinoinensis obtained from an orchard in Chihuahua, Mexico.

Results

The consensus tree of Bayesian approximation and Maximum likelihood shows the Rufum clade as monophyletic and with high support (BPP = 1, MV = 100). Within this clade, T. caryophilum is an independent, monophyletic and well-supported clade (BPP = 1, MVB = 100). This species appears as the sister clade of T. theleascum (BPP = 0.99, MVB = 98). In turn, these 2 species are grouped with the T. lyonii complex in a well-supported clade (BPP = 1, MVB = 100) made up of species from the southern USA and northern Mexico. Moreover, we found that the ITS sequence of the T. caryophilum holotype had a 99.3% NS (4 substitutions/549 bp) with mycorrhizal sequences from a pecan orchard in Chihuahua. This indicates that, like T. lyonii, T. caryophilum is an ectomycorrhizal symbiont of C. illinoinensis. Consequently, Tuber caryophilum is designated as a new species supported by ITS and LSU phylogenetic analyses of rnDNA, morphological characters, and ecology.

Description

Tuber caryophilum J.A. Sánchez, G. Guevara and R. Garibay-Orijel, sp. nov.

Fig. 2a-f

MycoBank 840581

GenBank MZ092919 (ITS), OK642397 (LSU)

Figure 2 Tuber caryophilum (holotype ITCV 1888). a, Ascomata showing peridial surface; b, ascoma in cross section showing glebal surface; c, peridium cross section showing pseudoparenchyma; d, peridium in cross section; e, ascospores within asci showing alveoli; f, ascospore with equinulate surface. a, b (bar = 1 cm); c, d, e, f (bar = 20μm). 

Type. Mexico, Coahuila, Municipality of Viesca, Tierra Blanca Orchard, September 4, 2019, Sánchez st3 (ITCV 1888 "José Castillo Tovar" herbarium).

Diagnosis. Cream peridium with translucent veins towards the epicutis; pseudoparenchymatous epicutis mainly although in some areas it is prosenchymatous, isodiametric hyphae of 3-17 μm; echinulate ascospores of 18-48 x 10-27 μm; it grows in pecan (Carya illinoinensis) orchards.

Ascoma. Subglobose to irregular, 19 × 10 × 18 mm, translucent veins on the light brown to dark brown peridium when dry, with a white to cream furrow and an irregular linear or "V"-shaped margin continuing into the gleba as veins, some areas dark brown with cherry tints and with dark brown to reddish insect caverns; peridium smooth, some areas rough, separable from the gleba, without cystidia. White, cream gleba that is gray to dark when dried, marbled with white to gray, dark brown to reddish brown veins that continue towards the peridium (furrows). Strong, very pleasant and distinctive odor, unrecorded taste.

Peridium. 110-220 μm thick, epicutis 50-75 μm, pseudoparenchymatous in its outermost part, although in some parts it is prosenchymatous with hyphae 3-17 μm wide, versiform to angular or isodiametric, wall 1-4 μm thick, yellow to orange-reddish in KOH; subcutis 70-150 μm wide, pseudoparenchymatous strongly interwoven, hyaline hyphae in KOH, septate 2-4 μm wide. Gleba, intertwined vein hyphae, 2-4 μm at widest part. Asci: 47 105 × 32-50 μm (Q = 1.07-2.63), average 67.1 × 40.6 μm (Q = 1.7) including pedicel, subglobose to broadly ellipsoid, hyaline in KOH, 1-2 μm double wall may have a short to very long pedicel or in some it is absent, 1-5 ascospores per ascus. Ascospores: 18-48 × 10-27 μm (Q = 1-2.40), average 30.1 × 17.2 (Q = 1.76) subglobose to broadly ellipsoid or spindle-shaped excluding ornamentation; echinulate, echinulae mostly free, in some of them a subreticula can be observed, 1-4 μm high. Asci with 1 ascospore 35-48 × 18-27 μm (Q = 1.30-2.33), average 42.1 × 22.4 (Q = 1.91); 2 ascospores 20-38 × 15-21μm (Q = 1-2.4), average 31.3 × 17.7 μm (Q = 1.79); 3 ascospores 18-33 × 14-18 μm (Q = 1.06-2), average 27.1 × 16.6 (Q = 1.63); 4 ascospores 21-33 × 12-18 μm (Q = 1.44-2.13), average 26.4 × 15.1 (Q = 1.76); 5 ascospores 19-33 × 10-17 μm (Q = 1.19-2.36), average 24 × 14.4 (Q = 1.69).

Taxonomic summary

Etymology. Refers to the ectomycorrhizal association between T. caryophilum and Carya illinoinensis.

Distribution and ecology. In northern Mexico in la Comarca Lagunera of Coahuila and Chihuahua, ectomycorrhizal symbiont of Carya illinoinensis. To date it has only been found in pecan orchards, not in natural habitats.

Habitat. Hypogeous, solitary or gregarious under pecan trees (Carya illinoinensis).

Collections examined in Mexico. Coahuila, Municipality of Viesca, Tierra Blanca Orchard, September 4, 2019, Sánchez st3_N2, MEXU 30227; Sánchez st3_N3, ITCV 1890; Sánchez st3_N4, ITCV 1891.

Remarks

Phylogenetic analyses show that Tuber caryophilum belongs to the Rufum clade and, together with T. theleascum (ITS NS = 93.6-93-9%), is related (ITS NS = 90.4-93.1%) to the T. lyonii complex, which is also an ectomycorrhizal species of Carya illinoinensis. Tuber caryophilum differs from T. theleascum because the latter has ascomata without translucent areas on the peridium, a pseudoparenchymatous epicutis with elongated prostrate or intertwined hyphae 4-7 μm wide and smooth, while the former species has translucent areas on the peridium and a pseudoparenchymatous epicutis with isodiametric hyphae 13-17 μm wide. Furthermore, they differ in ascospores size; T. caryophilum has 18-48 × 10-27 μm ascospores, whereas in T. theleascum they are 18-44 × 13-25 μm. Tuber caryophilum is also similar to T. lyonii, an edible truffle species native to the southeastern USA (^{Bonito et al., 2013}; ^{Sharma et al., 2012}). However, they differ macro- and microscopically; in T. lyonii, peridium width is larger (300-500 μm), ascospores are ellipsoid 30-37 × 22-24 μm and epicutis width is 20-40 μm, with hyphae 6-10 μm wide (^{Healy et al., 2016}; ^{Sharma et al., 2012}). In contrast, in T. caryophilum, the peridium width is 110-220 μm, its ascospores are 18-48 × 10-27 μm and epicutis width is 50-75 μm, with 3-17 μm hyphae. These 3 species share important microscopic features such as the pseudoparenchymatous peridium surface and most of their ascospores are subglobose to ellipsoid. Other common features are that all 3 are closely related in the same clade of the Rufum section and that they develop in pecan orchards (^{Grupe et al., 2018}; ^{Sharma et al., 2012}; ^{Trappe et al., 1996}).

Other Tuber species associated with pecan plantations have been described but these do not belong to the Rufum section (Table 2). One of these species is Tuber brennemanii, which belongs to the Maculatum clade and therefore differs morphologically and molecularly from species belonging to the Rufum clade. For example, T. brennemanii presents anamorphic ascospores and a periclinal subperidium (^{Grupe et al., 2018}). Likewise, Tuber floridanum, also on the Maculatum clade, has been found in pecan orchards. This species is distinguished by the presence of dermatocystidia and commonly has 2-4 spores and reticulate ornamentation as present in the Maculatum (^{Grupe et al., 2018}). Regarding T. floridanum, it is known to have been unintentionally introduced into southern Brazil on the roots of pecan tree seedlings.

Given the phylogenetic closeness of T. caryophilum to T. lyonii, its discovery in pecan orchards opens the door to its use in northern Mexico. To develop this, it will be necessary to carry out mycorrhization experiments in nurseries and to know its organoleptic properties. This could promote a system of co-cultivation between T. caryophilum and C. illinoinensis as occurs in orchards in southeastern Florida and Europe where various species of truffles are harvested alongside nut production (^{Bonito et al., 2013}; ^{Lefevre et al., 2012}; ^{Trappe et al., 1996}).