Truffle abundance and mycophagy by northern flying squirrels in eastern Washington forests

Although much is known about truffle abundance and rodent mycophagy in mesic Douglas-fir forests in the Pacific Northwest, few data are available for dry interior montane forests dominated by ponderosa pine (Pinus ponderosa), Douglas-fir (Pseudotsuga menziesii), and grand fir (Abies grandis). Our objective was to quantify the relationship between the abundance and diversity of ectomycorrhizal fungal sporocarps in the soil and in the diets of northern flying squirrels (Glaucomys sabrinus) in low-elevation forests of the eastern Washington Cascades. We randomly sampled four stands each of three cover types: dry open ponderosa pine, mesic young mixed-conifer forest, and mesic mature mixed-conifer forest. We sampled the soil for hypogeous sporocarps during the spring of 1999 and 2000. We collected fecal pellets from 318 flying squirrels live-trapped during the fall of 1997–2000. We sampled 2400 m2 of soil surface and found truffles in 40% of 600 plots. Total biomass collected was 609 g. Spring truffle biomass on a kg/ha basis averaged 1.72 in open pine, 3.56 in young, and 4.11 in mature forest. Twenty-two species were collected across all cover types, with all but three species belonging to the Basidiomycotina. Eleven dominant species accounted for 91–94% of truffle biomass in each cover type. Four dominant species accounted for 60–70% of spring truffle biomass: Gautieria monticola, Hysterangium coriaceum, Rhizopogon parksii, and R. vinicolor. Truffle assemblages, richness and total biomass differed among cover types: richness and biomass were highest in young and mature mixed-conifer forest, and lowest in open ponderosa pine forest. Fall squirrel diets were composed of 23 genera or groups of fungi, plus about 22% plant material. Rhizopogon was the most abundant genus in the diet, followed by plant material, then Gautieria, Leucogaster, Alpova, and Hysterangium. Diets in different cover types were similar in the composition, richness, evenness, and the ratio of fungus to plant material. Diet richness varied over the study period. Nineteen truffle genera were detected in fall fecal samples versus 12 in spring soil samples. Management of low-elevation dry forest to maintain or restore stable fire regimes might reduce truffle diversity at stand scales by simplifying stand composition and structure; but, such management might increase long-term beta and landscape truffle diversity and persistence by reducing the occurrence of high-intensity fires and stabilizing inherent fire disturbance regimes

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

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) Gamundı´. 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.Organismic and Evolutionary Biolog

Divergence time estimates (in millions of years) from shared common ancestors for major clades within <i>Tuberaceae</i> as referred to in Figures 3–5.

<p>Nodes D-M represent major genetic groups within <i>Tuber</i>.</p

Bayesian Divergence Time Estimates for Truffles.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052765#pone-0052765-t001" target="_blank">Table 1</a>.</p

Hypothesized evolution of a truffle lineage.

<p>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).</p

Phylogenetic reconstructions of <i>Tuber</i> based on maximum likelihood analysis of four individual loci: internal transcribed spacer region (ITS), 28

<p> <b>s large subunit rDNA (LSU), elongation factor 1-alpha (EF1a), and RNA polymerase II (RPB2).</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052765#pone-0052765-g004" target="_blank">figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052765#pone-0052765-g005" target="_blank">5</a>. Taxa in the <i>Helvellaceae</i> were excluded from the ITS analysis because of the alignment challenges imposed by sequence divergence.</p

Results of statistical dispersal-vicariance (S-DIVA) analyses of the <i>Tuberaceae.</i>

<p>Results of statistical dispersal-vicariance (S-DIVA) analyses of the <i>Tuberaceae.</i></p

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

<p>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 <i>Pinaceae</i>, and gray for ancestors in symbiotic association with angiosperms and <i>Pinaceae</i>. 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 <i>Helvellaceae</i> including species of epigeous <i>Helvella</i> and hypogeous <i>Balsamia</i>. Major lineages of <i>Tuber</i> and <i>Tuberaceae</i> are indicated to the right of the tree. The <i>Tuberaceae</i> 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.</p

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

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