Tuber aztecorum sp. nov., a truffle species from Mexico belonging to the Maculatum clade (Tuberaceae, Pezizales)

Abstract A new species of truffle, T. aztecorum, is described from central Mexico. Tuber aztecorum can be distinguished from other related Tuber species synoptically by a combination of morphological features including ascospore size, pellis cells with irregular thickness, cystidia, ascoma colour and associated host (Abies religiosa an endemic Abies species from central Mexico); sequence variation on the ITS rDNA also distinguishes T. aztecorum from related species. A phylogenetic analysis of the ITS rDNA demonstrates that T. aztecorum belongs to the Maculatum clade and is unique from other similar small, white-cream coloured Tuber species distributed in north-eastern Mexico such as T. castilloi and T. guevarai.


Introduction
Tuber is one of the most important edible truffle genera in the world due to its economic importance and ecological role in forest ecosystems. Tuber spp. are known as 'true truffles' and their fruiting bodies are edible and highly valued. Ecologically, Tuber spp. form symbiotic ectomycorrhizal associations with gymnosperm and angiosperm trees and also orchids (Riousset et al. 2001, Wurzburger et al. 2001, Bidartondo et al. 2004, Walker et al. 2005, Mello et al. 2006, Martin and Bonito 2012, Morcillo et al. 2015. In addition, Tuber spp. are consumed for nutrition by many kinds of invertebrates and vertebrates including primates (McGraw et al. 2002, Hanson et al. 2003, Hochberg et al. 2003, Maser et al. 2008, Beaune et al. 2013. A few Tuber species are now cultivated worldwide, including Tuber melanosporum, T. aestivum and T. borchii (Martin and Bonito 2012).
The Puberulum and Maculatum clades within the genus Tuber are two of the most species diverse and geographically widely dispersed of the eleven recognised clades. More recently, the related Latisporum clade was described from Asia, where the species are endemic (Fan et al. 2016).Tuber species in these three clades are often pale in colour and typically small in size , Bonito et al. 2010a, Guevara et al. 2013b, Payen et al. 2014. Recent molecular analyses of Tuber spp. from northern and central Mexico and USA have shown that Tuber species are genetically unique compared to their European and Asian counterparts (Bonito et al. 2009, 2010a, Lancellotti et al. 2016. Many Tuber species belonging to these clades have been formally named recently. For example, five species within the T. separans complex of the Puberulum clade were described from Mexico. Tuber bonitoi, a large truffle (approx. 5 cm) found recently in Mexico, is morphologically similar to T. borchii. It was found associated with Pinus hartwegi and Abies religiosa. Tuber brunneum, a smaller, brownish truffle from central Mexico, was associated with Quercus magnolifolia as was T. pseudoseparans and T. tequilanum. Tuber guzmanii and T. separans are also found in Mexico and belong to the Puberulum clade (Guevara et al. 2015). Other Tuber species belonging to the Maculatum clade are known from north-eastern and central Mexico including Tuber castilloi, T. gardneri, T. guevarai, T. maculatum, T. mexiusanum and T. miquihuanense (Cázares et al. 1992, Guevara et al. 2008, 2013a,b, 2015. In addition, new findings on asexual anamorphic states have been discovered for some North American Tuber species, however the role of these structures is still unknown (Urban et al. 2004, Ouanphanivanh et al. 2008.
Studies on Tuber species from Mexico are still scarce. In this work, a morphological and molecular analysis was performed on recent Tuber collections. The authors report on a new taxon, which is described here as T. aztecorum. Phylogenetically, T. aztecorum is within the Maculatum clade, a group of small to medium sized, white truffles. It is associated with Abies religiosa, an endemic Abies species from central Mexico. Tuber aztecorum can be differentiated from related taxa by its morphology, ecology, biogeography and nuclear ITS ribosomal DNA. This research contributes to the knowledge of Tuber biodiversity and ecology in North America.

Sampling and morphological characterisation
Tuber fruiting bodies were collected from central México and preserved following recommendations of Harkness (1899) and Castellano et al. (1989). Duplicate splits of sample collections are deposited in the herbaria José Castillo Tovar (ITCV), Oregon State University (OSC), Michigan State University (MSU) and Florida University (FLAS). Previously accessioned herbarium specimens of Tuber, including type collections from OSC and ITCV, were also examined during this study.
Morphological data were obtained by the methods of Castellano et al. (1989), Gilkey (1916Gilkey ( , 1939 and Pegler et al. (1993). Examined characters included ascoma (fruiting body) size, surface texture and colour, peridial structure; spore length and width (excluding ornamentation), length/width ratio (Q), shape, wall thickness, number of reticular meshes, height of the meshes, colour and ascus size, shape, wall thickness and number of spores/ascus. Hand-cut sections were mounted in 5% KOH and Melzer's reagent for light microscopy. Spore measurements of Tuber spp. in KOH compared to those in water showed no KOH effect (J. Trappe, unpublished data). Microscopic structures were measured and photographed under a light microscope and stereo microscope.

DNA sequencing and phylogenetic analyses
Molecular protocols follow those of Guevara et al. (2008). DNA was extracted from truffle fruiting bodies with the chloroform extraction technique using CTAB 2X DNA extraction buffer. The ITS region was amplified with the primer pair ITS1f-ITS4 (Gardes andBruns 1993, White et al. 1990). PCR products were cleaned enzymatically with antarctic phosphatase and endonuclease digestion (New England Biolabs, Ipswich MA). Sanger sequencing was performed by Big Dye chemistry v3.1 (Applied Biosystems, Foster City, CA) with the forward primer ITS1f and reverse primers ITS4. DNA sequences were determined on an ABI 3700 capillary sequencer (Applied Biosystems, Foster City CA). DNA sequences were viewed and manually edited in Sequencher 4.0 (Gene Codes, Ann Arbor, MI). Sequences were aligned with MUSCLE (Edgar 2004). Alignments were manually checked and ambiguous regions were excluded in Mesquite 2.5 (Maddison and Maddison 2009).
Phylogenetic analyses were conducted with maximum likelihood (ML) in PAUP* (Swofford 2002). The best fit nucleotide substitution model (GTR+G+I) was based on the Akaike information criterion and was implemented in PAUP* 4d106 (Swofford 2002). ML bootstrap support based on 1000 replicates was assessed with RAxML (Pattengale et al. 2009, Stamatakis et al. 2008, Stamatakis 2006) and executed on the CIP-RES Science Gateway (Miller et al. 2010). Phylogenetic trees were rooted with species belonging to the Latisporum clade. Sequences produced in this study are deposited in GenBank under accession numbers KY271791 and KY271790, Table 1.

Molecular analyses
A total of 51 taxa including holotypes were analysed (Table 1). As previous studies have shown, the Maculatum clade was distinct from the Puberulum and Latisporum clades in ML and Bayesian Inference analyses (Fig. 1). The designation of Tuber aztecorum as a new species is supported by ITS rDNA analysis, morphological characters and ecology. Tuber aztecorum is a sister species to T. castilloi, but T. castilloi differs by having larger spores, 27-63 × 20-40 µm, is without an irregular thickness to the cell wall on peridial hyphae and is associated mainly with Quercus spp. Also resembles T. guevarai but T. guevarai has narrow spores that are 18-55 × 16-42 µm and creamyellow fruiting bodies and rDNA variation.
Peridium 110-350 µm thick. Outer layer (epicutis) a pseudoparenchyma 62-250 µm thick, of hyphae 5-30 µm diam., versiform, angular or isodiametric, in some areas hyphae arranged perpendicular to the epicutis, hyaline to reddish-brown in mass in KOH, thick-walled (2 µm), without intracellular content. Surface hairs versiform, single hair-like hyphae or cystidia 53-97 µm long × 4-5 µm at the base, tapered to the tip, some with septa, scattered or in clusters, brittle, thin-walled, hyaline in KOH. Other hyphae present are claviform, erect, cylindrical or sinuate with an irregular thickness to the cell wall that resembles knobs or "spines". Some globose or constricted hyphae emerging from isodiametric hyphae, 3-10 µm wide. Inner layer (subcutis) 50-225 µm thick, of prostrate and interwoven, hyphae gradually intermixing into gleba, hyaline in KOH and trypan blue, hyphae Figure 1. Phylogenetic tree inferred under the maximum-likelihood (ML) criterion from the ITS rDNA alignment corresponding to the Tuber dataset. The tree was rooted using midpoint rooting. Numbers on the branches represent support values from 1,000 ML bootstrap replicates. The branches are scaled in terms of the expected number of substitutions per site. The phylogeny is rooted with species belonging to the Latisporum clade. Accession numbers in the sequence labels indicate sequences from Genbank.

Discussion
Molecular data confirm that T. aztecorum belongs to the Maculatum clade, which is distinct from the Puberulum and Latisporum clades. Tuber aztecorum is morphologically and ecologically distinct from other known Tuber species (Fig. 1). Tuber aztecorum is a sister species to T. castilloi, however, T. castilloi differs by having larger spores, 27-63 × 20-40 µm, without irregular thickness to the cell wall and is associated mainly with Quercus spp. (Guevara et al. 2013a, b). Also T. aztecorum resembles T. guevarai but T. guevarai has narrower spores that are 18-55 × 16-42 µm (Guevara et al. 2013b). Although T. aztecorum belongs to the Maculatum clade, it is also morphologically similar to other Tuber species outside this group. It was preliminarily identified as T. gibbosum in the Gibbosum clade due to its association with Abies religiosa and the presence on the peridium of hyphae with irregular swellings in T. aztecorum (Guevara et al. 2013a, Bonito et al. 2010b). However, close morphological analysis and further molecular analysis revealed that it was not closely related to the Gibbosum clade. Tuber aztecorum is also similar to other species that belong to the Puberulum clade. Tuber foetidum is similar in its dark brown to reddish-brown peridium. However, T. foetidum has peridial cells 20-30 µm wide, lacks hairs, ascospores that are 25-44 × 21-32 µm and it grows under Larix, Quercus and Fagus (Jeandroz et al. 2008, Pegler et al. 1993. It is similar to T. puberulum, T. rapaeodorum and T. borchii from Europe. These three Tuber species have a dense and conspicuous fine epicutis. Tuber puberulum is frequently found in association with both Fagus and Larix. Tuber rapaeodorum has a paler ascoma surface with thinner cystidia, ellipsoid spores and is associated with Quercus, Larix, Taxus, Pinus, and Fagus. Tuber borchii has a pale, whitish or yellowish ascoma surface with abundant peridial hairs, 80% of its spores are ellipsoid and it is usually associated with Fagus or Larix (Pegler et al. 1993, Montecchi and Sarasini 2000, Halász et al. 2005, Wang et al. 2007, Jeandroz et al. 2008. Tuber latisporum differs morphologically from T. aztecorum by its conspicuously pubescent peridium, spores that are 24-49 (-51) × 20-40 (-44) µm and its association with Pinus armandii in China (Chen and Liu 2007).
In conclusion, morphological and sequence analysis of ITS rDNA can distinguish T. aztecorum from previously described species with strong bootstrap support and confidence (Halász et al. 2005, Wang et al. 2007, Jeandroz et al. 2008, Bonito et al. 2010a. The number of formally described Tuber species continues to grow (Table 1).