Description and distribution of Tuberincognitum sp. nov. and Tuberanniae in the Transmexican Volcanic Belt

Abstract The genus Tuber is a lineage of diverse ectomycorrhizal, hypogeous, sequestrate ascomycete fungi that are native to temperate forests in the Northern Hemisphere. Recently, many new species of Tuber have been described in North America and Asia, based on morphological characteristics and molecular data. Here we describe and illustrate a new species, Tuberincognitum, based upon phylogenetic analysis and morphological description. We also present a new record for Tuberanniae in México. These two Tuber species are distributed in the Transmexican Volcanic Belt in the states of México, Michoacán, Guanajuato, Querétaro and Tlaxcala at altitudes between 2,000 and 3,200 meters. These species are associated with Pinus (T.anniae) and Quercus forests (T.incognitum).


Introduction
Fungal species, within the genus Tuber, produce hypogeous, sequestrate ascomata, that are more commonly known as truffles.These fungi are ectomycorrhizal (EcM) symbionts of angiosperm or gymnosperm hosts, including many species of trees as well as orchids.Plant hosts provide their EcM symbionts with carbohydrates in exchange for greater access to water and nutrients (Wurzburger et al. 2001;Bidartondo et al. 2004;Walker et al. 2005;Shefferson et al. 2008).The genus Tuber has been studied intensively over the past century, largely due to its economic importance as an edible fungus.Most of these efforts have been directed towards European species with economic value (e.g.Tuber melanosporum, Tuber magnatum, Tuber aestivum), which reside in a few clades, neglecting most of the diversity in this genus.Reference and environmental sequences data were recently used to infer a minimum of 180-230 species of Tuber worldwide (Bonito et al. 2010) and substantiate that most Tuber diversity resides within less studied and non-economically important lineages delimitated as the Rufum, Puberulum and Maculatum clades.In México, eighteen Tuber species are known and have been formally described.The majority of the collections of these described species are from northeast and central México.In this study, we propose the new species Tuber incognitum and provide the first report of T. anniae in México based on morphological characteristics and phylogenetic analyses.

Morphological observation
Ascomata were collected from the states of Guanajuato, México, Michoacán, Querétaro, Tlaxcala and were deposited in herbaria at Oregon State University (OSC), Herbario Nacional de México (MEXU) and Herbario José Castillo Tovar (ITCV).Macroscopic characters were recorded from fresh specimens and microscopic characters were described from both sections of fresh specimens and dried specimens mounted in 5% potassium hydroxide (KOH) following protocols from Castellano et al. (1989).

DNA sequencing and phylogenetic analyses
DNA was extracted from ascomata of collections OSC157842 and OSC150066 using a CTAB chloroform extract protocol and ITS rDNA was amplified and sequenced as previously described (Bonito et al. 2010).Tissue samples from collections MEXU 26504, MEXU 26541, MEXU 26218 and MEXU25995 were sent to the Canadian Center of Barcoding (CCDB) for extraction, amplification, sequencing and barcoding of the Internal Transcribed Spacers (ITS).The ITS region was amplified with ITS1f and ITS4 primers (White et al. 1990).The sequences were edited in Geneious 7.1 (http://www.geneious.com,Kearse et al. 2012).The distribution and ecology of these species was complemented with soil DNA data from central and south México through a BLASTn search against the Mexican Soil Fungi Database in Geneious 7.1.This database includes ITS2 sequences of soil fungi (total DNA soil extractions) from central and southern México as part of an ongoing project, which has been partially published by Argüelles-Moyao and Garibay-Orijel (2018).
DNA sequences were manually trimmed and edited with Sequencher 4.0 (Gene Codes Corp., Ann Arbor, Michigan).ITS sequences were queried against the NCBI public database GenBank by use of the BLASTn algorithm to retrieve similar sequences (Altschul et al. 1990).Collated DNA sequences were aligned with MUSCLE in Mesquite 3.04 (Maddison and Maddison 2015;Edgar 2004).Ambiguously aligned regions were excluded from the alignment.Phylogenetic analyses were conducted on ITS rDNA alignments through the CIPRES portal (www.phylo.org,Miller et al. 2010).Maximum Likelihood (ML) searches were conducted with RAxML v.7.2.8 using rapid bootstrapping of 1,000 pseudoreplicates (Stamatakis 2014).Bayesian Inference (BI) was carried out with MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003).To estimate posterior probabilities, 20,000,000 Markov chain Monte Carlo (MCMC) simulation generations were run in two parallel searches on four chains, with trees sampled every 1,000 th generation, with the first 5,000 trees discarded as burn-in (convergence of parallel runs).Bootstrap support, based on 1,000 iterations, was considered informative where it was ≥ 70% and posterior probability was considered significant where it was ≥ 99%.Sequences, generated in this study, are available on GenBank under the accession numbers GQ221447, KC152267, KC152256, KJ595013, KJ595014 and MH174661 (Table 1) and in the BOLD systems database (www.barcodinglife.org).
Etymology.Incognitum is Latin for unknown.The name incognitum is not derived from its morphology, rather from the fact that it was overlooked for so long.The holotype was collected in 1996 and not described until now.
Description.Ascomata 10-15 mm broad, subglobose to slightly irregular, white with light brown areas when dry, glabrous, with canals that continue with the veins into the gleba.Gleba pinkish to purplish pale-brown in youth, dark brown at maturity, marbled with white veins.Odour fruity, pleasant.
Distribution and ecology.Only known from central and southwest México (Querétaro, Michoacán, State of México, Guanajuato and Hidalgo).Ascocarps always associated with Quercus species (Q.crassifolia, Q. polymorpha).An EcM association with Quercus has been verified (MH174661) and its DNA has been recovered only from soil in Quercus forest in Hidalgo, México.
Distribution and ecology.Wang et al. (2013) reported from Europe; Colgan and Trappe 1997;Bonito et al. (2010) reported in North America.Here, we extended the distribution to central México (State of México and Tlaxcala).In Finland, T. anniae has been confirmed to establish association with P. sylvestris L. (Wang et al. 2013).In Washington, this species has been confirmed to establish association with Pseudotsuga menziesii (Mirbel) Franco (Bonito et al. 2010).In México, sporocarps always collected co-occurring with Pinus leiophylla Schiede and Deppe and Abies religiosa (Kuntch) Schldl.and Cham.In México, the only environmental DNA of this species has been recovered from soil in conifer forests in Tlaxcala associated with Pinus montezumae and in State of México associated with A. religiosa (Argüelles-Moyao and Garibay-Orijel 2018).
Taxonomic comments.Tuber anniae is similar to Tuber pacificum Trappe, Castellano and Bushnell, however, the latter species has narrower, ellipsoid spores (23-15 × 16-35 µm) and a thicker peridium (250-400 µm) than the former.T. pacificum has also been found co-occurring with Pseudotsuga menziesii and Tsuga heterophylla (Raf.)Sarg.along costal Oregon, while T. anniae has been found co-occurring with P. leiophylla and A. religiosa.Tuber anniae was first described by Colgan and Trappe (1997).The holotype (from Washington) and the other collections reported were from the Pacific Northwest in the US and reported co-occurring with P. menziesii.The T. anniae complex of species has been proposed based on phylogenetic analysis using ITS region (Wang et al. 2013).The collection from México is very similar to the holotype collection, however, the latter has a brown to dark olive-brown peridium and its spores have thicker (up to 5 µm) spore walls than the former.Additionally, T. anniae, as described by Colgan and Trappe (1997), has mostly globose spores with 10-16 alveolae across the spores.The Finnish collections exhibit subtle morphological differences in comparison with the collections from North America.The Finnish specimens have a smooth peridial surface, except along the grooves and around the pits (Wang et al. 2013), while the holotype specimen was reported to be smooth and lack dermatocystidia (Colgan and Trappe 1997).It seems that the presence of dermatocystida only along the grooves and/or at the bottom of pits in Tuber collections is likely the result of handling of the ascoma during processing (Dr.D. Luoma, personal communication).Additionally, the spores from the Finnish specimens have larger dimensions (27-60 × 27-56 µm; Q = (1.00) 1. 05-1.20 (1.33)) than the specimens described for T. anniae by Colgan and Trappe from Washington (1997).

Discussion
Species in Puberulum clade can be found in North America, Europe and Asia and some regions of North Africa and South America (Bonito et al. 2010(Bonito et al. , 2013;;Jeandroz et al. 2008;Payen et al. 2014).There are some records of species in the Puberulum clade (e.g.Tuber rapaeodorum) that have been introduced into Australia and New Zealand (Bonito et al. 2010).Species in this clade show a wider range of host associations than other species within the Rufum, Excavatum, Aestivum, Maculatum and Gennadii clades (Payen et al. 2014).Species within the Puberulum clade commonly form associations with angiosperms and conifers (Bidartondo et al. 2004;Bonito et al. 2010).In México, twenty species of Tuber have been reported, including the two species from this study.Eight of the twenty belong to the Puberulum clade.Both the Maximum Likelihood and Bayesian analyses (Figure 3) show that T. incognitum forms a strongly supported clade (Maximum Likelihood bootstrap= 97), which includes sequences from both voucher collections and EcM root tip collections.This clade is placed as a sister taxon of the undescribed species Tuber sp. 3 (KC152267) also from México.This study combines sporocarp anatomy, molecular analyses and phylogenetic analyses to support the erection of T. incognitum as a unique species within the Puberulum clade.
The T. anniae species complex is recovered as a strongly supported clade.There is an internal structure in this clade, with different branch lengths and nested subclades, but additional markers are needed to resolve relationships within this species complex.The Mexican specimens' group with those from Alaska (JX094351), form a nested clade that is closely related to a collection from Canada (EU554720).The members in the T. anniae species complex are closely related to T. pacificum from Oregon, USA.Given the relatively high ITS similarity, phylogenetic position and similar morphology to the T. anniae holotype collection, we have identified the Mexican collection as T. anniae, extending its known range and southernmost distribution of this species in North America.

Figure 1 .
Figure 1.Tuber incognitum (Holotype, OSC 150066).a Ascoma, surface and cross-section view b Peridium in cross-section c Light microscopy of spores in cross-sectional view, highlighting the spines and ornamentation d Light microscopy of spores in surface view, highlighting the surface and reticulum.Scale bars: 5 mm (a), 20 µm (b), 15 µm (c, d).

Figure 2 .
Figure 2. Tuber anniae (OSC 157842).a Ascoma, surface and cross-section view b Peridium in crosssection c Light microscopy of spores in cross-sectional view, highlighting the spines and ornamentation d Light microscopy of spores in surface view, highlighting the surface and reticulum.Scale bars: 5 mm (a), 15 µm (b, c, d).

Figure 3 .
Figure 3.Most likely tree based on maximum likelihood phylogenetic inference showing the placement of Tuber incognitum within the Puberulum clade.Bootstrap values ≥ 70% are labelled above nodes.Nodes with posterior probabilities ≥ 99% are blacked.Holotype collections are labelled.The phylogeny is rooted with species belonging to the Maculatum clade.Scale bar corresponds to the mean number of nucleotides substitutions per site.

Table 1 .
Accession and voucher numbers of sequences generated in this paper.