Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage

Abstract

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae--the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (~156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.

Figure 1. Hypothesized evolution of a truffle lineage.

In this scenario the habitat of an epigeous species with 8-spored, uniseriate asci becomes more arid (A). Selection for reduced water loss results in an enclosed truffle form that has hymenium-lined chambers and asci that are shorter and more clavate in form (B). The ability to forcibly discharge spores is lost and selection for other means of spore dispersal intensifies, leading to spore dispersal through animal mycophagy. Continued selection results in a truffle species that fruits belowground and has a solid gleba stuffed with spherical asci packed with irregular numbers of spores (C).

Figure 2. Morphological diversity and characters of truffles and their relatives.

A. An “Elfin-saddle” cup-fungus Helvella lacunose Fr. Asci line the outside of the fertile cap, which is borne upon a stipe composed of vegetative tissue; B. the “earth-tooth” fungus Underwoodia singer Gamundí & E. Horak. A layer of fertile tissue lines the outside of the tooth-shaped cap. C. Gymnohydnotrya sp. collected under Nothofagus pumilio (Poepp & Endl.) Krasser in Argentina and similar to sequences from Nothofagus mycorrhizas. Fertile asci line both the inside and the outside the fruiting body. D. Choiromyces alveolatus (Harkn.) Trappe, a Pinaceae associate from western North America. E. A knobby-shaped representative of the/puberulum lineage, a clade of small, whitish truffles. F. Tuber canaliculatum Gilkey has a peridium covered in minute warts and its asci contain one or two reticulate spores. G. Flask-shaped ascus of the spiny-spored Tuber lyonii Butters with a stem at the point of attachment. H. Representative of the/japonicum lineage. I. Swollen beaded hyphae from the outer peridium of species belonging to the/gibbosum lineage. J. Large pyramidal warts cover the outer surface of Tuber aestivum. K. The spores of Choiromyces meandriformis Vittad. are ornamented with unusual pitted tubes. L. Species in the/excavatum lineage have a thick outer peridium and a partially enclosed internal cavity. M. Species in the/maculatum lineage have ellipsoid, alveolate-reticulate ascospores. N. The spores of Tuber sp.13 of the/melanosporum clade are particularly spiny. O. The spores of Tuber spinoreticulatum Uecker & Burds have spines that are irregularly connected by ridges that form a partial reticulation. Scale bars: A, B, C, D, E, F, H, J, L = 1 cm; G, I, K, M, N, O = 10 µm.

Figure 5. Bayesian Divergence Time Estimates for Truffles.

The maximum clade credibility chronogram estimated in BEAST is shown with nodes placed at the median age. Node bars (grey) represent the node age 95% highest posterior density (HPD) for nodes receiving at least 50% Bayesian posterior probability. The median age is provided for labeled nodes (A–P) that are discussed in the text and node age parameters are presented in Table 1.

Figure 3. Phylogenetic reconstructions of Tuber based on maximum likelihood analysis of four individual loci: internal transcribed spacer region (ITS), 28

s large subunit rDNA (LSU), elongation factor 1-alpha (EF1a), and RNA polymerase II (RPB2). Models and likelihood scores for each locus are: ITS = Sym +G+I (–3960.627); LSU = GTR +G+I (−8732.114); EF1a = GTR +G+I (7374.012); RPB2 = K80 (5880.021). Clade names and node labels are consistent with each other and with figures 4 and 5. Taxa in the Helvellaceae were excluded from the ITS analysis because of the alignment challenges imposed by sequence divergence.

Figure 4. Maximum likelihood (ML) phylogenetic reconstruction of the Tuberaceae phylogeny based on ITS, 28S rDNA, EF1α, and RPB2 gene regions.

Thickened branches represent ML bootstrap support >70 and posterior probabilities of 100. ML bootstrap values above nodes are based on 1000 replicates. Posterior probabilities are presented below nodes. Thickened branches without numbers received maximum ML and Bayesian support values. Reconstructed ancestral host plant associations (based on maximum likelihood) are represented at internal nodes by circles; black for ancestors in symbiotic association with angiosperms, white for ancestors in symbiotic association with Pinaceae, and gray for ancestors in symbiotic association with angiosperms and Pinaceae. Nodes supported by transitions in spore ornamentation from alveolate-reticulate to spiny are shown with an asterisk *. Economically important species are denoted by the symbol $ after their name and geographic origin. The phylogeny is rooted with taxa from the Helvellaceae including species of epigeous Helvella and hypogeous Balsamia. Major lineages of Tuber and Tuberaceae are indicated to the right of the tree. The Tuberaceae form a monophyletic group, which is resolved as a sister group to a previously unrecognized Southern hemisphere lineage (/gymnohydnotrya). Type specimens are denoted by the superscripts: ^{h -} holotype, ^{i -}isotype, ^{p -}paratype.

Figure 6. Results of statistical dispersal-vicariance (S-DIVA) analyses of the Tuberaceae.