Using mating-type loci to improve taxonomy of the <i>Tuber indicum</i> complex, and discovery of a new species, <i>T</i>. <i>longispinosum</i>

<div><p>Black truffles that morphologically resemble <i>Tuber indicum</i> have been known to occur in Japan since 1979. Our previous studies showed that there are two phylotypes of these truffles, both of which fell into the <i>T</i>. <i>indicum</i> complex (hereinafter “<i>Tuber</i> sp. 6” and “<i>Tuber</i> sp. 7”). However, their taxonomic treatment is still unclear. In this study, we conducted morphological and phylogenetic analyses for a total of 52 specimens from Japan (16 <i>Tuber</i> sp. 6 and 13 <i>Tuber</i> sp. 7), China (10 <i>T</i>. <i>himalayense</i> and 8 <i>T</i>. <i>indicum</i>), and Taiwan (5 <i>T</i>. <i>formosanum</i>). We compared ascospore ornamentation, size, distribution of asci with average number of spores per ascus, spine size and shape of the Japanese specimens with their allied taxa. For phylogenetic analysis, we sequenced two mating loci (MAT1-1-1 and MAT1-2-1) and three commonly used loci (ITS, β-tubulin, and TEF1-α). Three distinct lineages were recognized by phylogenetic analyses based on the sequences of the two mating-related loci and three independent loci. The <i>Tuber</i> sp. 6 sequences clustered with those of <i>T</i>. <i>himalayense</i> and <i>T</i>. <i>formosanum</i>, and there was no clear difference in morphology among them. <i>Tuber</i> sp. 7 formed a distinct lineage in each phylogram. The specimens tended to have five-spored asci more frequently than other allied species and could be characterized as having ascospore ornamentation with longer spines and narrower spine bases. We therefore described <i>Tuber</i> sp. 7 as a new species (<i>T</i>. <i>longispinosum</i>), and treat <i>Tuber</i> sp. 6 and <i>T</i>. <i>formosanum</i> as synonyms of <i>T</i>. <i>himalayense</i>.</p></div

<i>Tuber himalayense</i> photographs (TFM: S17015).

<p>A. Ascomata (bar = 1 cm). B. Peridial warts, (bar = 3 mm). C. Asci and ascospores (bar = 30 μm). D. Peridium and glebal tissue in cross section (bar = 50 μm). E. Ascospore (bar = 10 μm).</p

Phylogenetic relationships among Asian black truffles based on three combined datasets (ITS, β-tublin, and TEF1-α).

<p>The phylogram was obtained by maximum likelihood inference under the TN93+I model. SH-aLRT values and Bayesian posterior probabilities are shown as ML/BPP.</p

Using mating-type loci to improve taxonomy of the <i>Tuber indicum</i> complex, and discovery of a new species, <i>T</i>. <i>longispinosum</i> - Fig 2

<p><b>Ascospore spine height of four-spored asci (A) and width of spine bases (B), for the studied taxa.</b> Different letters above boxes indicate significant differences between mean according to Tukey–Kramer honestly significant difference test (<i>P</i> < 0.01).</p

Relative frequencies of asci with one to six ascospores per ascus for the studied taxa.

<p>Relative frequencies of asci with one to six ascospores per ascus for the studied taxa.</p

Phylogenetic relationships among Asian black truffles based on MAT1-1-1 and MAT1-2-1 sequences.

<p>The phylogram was obtained by maximum likelihood inference under the TN93 model. SH-aLRT values and Bayesian posterior probabilities are shown as ML/BPP.</p

Chronogram and estimated divergence times of porcini s.l. generated from molecular clock analysis using the RPB1-nrLSU data.

<p>Chronogram obtained using the Ascomycota – Basidiomycota divergence time of 582 Mya as the calibration point is shown in panel A. The calibration point and objects of this study are marked in the chronogram. The geological time scale is in millions of years ago (Mya). Estimated divergence times of main nodes are summarized in panel B, with divergence times of lineages in the porcini s.l. highlighted in blue color.</p

Average evolutionary divergence over ITS sequence pairs within and between groups (provincially adopted phylogenetic species) calculated by MEGA 5 using the Maximum Composite Likelihood model.

<p>Average evolutionary divergence over ITS sequence pairs within and between groups (provincially adopted phylogenetic species) calculated by MEGA 5 using the Maximum Composite Likelihood model.</p

Phylogenetic tree inferred from the Maximum Likelihood (ML) analysis based on the RPB1-nrLSU data.

<p>Bootstrap values (ML)/posterior possibilities (from Bayesian Inference) are shown above or beneath individual branches. The positions of five lineages are marked with bold lines, where 1, 2, 3 represent our newly identified lineage (<i>Boletus</i> sp. HKAS71346), “<i>Inferiboletus</i>”, and “<i>Obtextiporus</i>”, respectively. All names in the porcini s.s. lineage were the same as those from ITS analysis (see Fig. 2).</p

Divergence time estimation and ancestral area reconstruction of porcini s.s. using the ITS data.

<p>The Chronogram is obtained from molecular clock analysis using BEAST. Pie chart in each node indicates the possible ancestral distributions inferred from Baysian Binary MCMC analysis (BBM) implemented in RASP, while characters above and beneath each clade identify the possible ancestral distribution estimated by maximum likelihood-based program LAGRANGE. Red circles around pie charts indicate possible dispersal and vicariance events as suggested by BBM analysis, while green circles show only dispersal events. Red branches identify dispersal events inferred by LAGRANGE. Characters beyond species names show current distribution area of each species.</p