Phylogenetic and Morphological Evidence Reveal Five New Species of Boletes from Southern China

Fungi of the order Boletales are extremely important in both ecology and economy, since most of them are ectomycorrhizal fungi, which play vital roles in maintaining forest ecosystems, water and soil protection, vegetation restoration and so on. Although previous studies have shown that this order has a very high species diversity in China, there are few reports on the species diversity of boletes in Jiangxi Province, China. Based on morphological (macroscopic and microscopic morphological characteristics) and phylogenetic analyses (ITS, LSU, and TEF1-α sequences), in this study, the wild boletes in Jiangxi Province were investigated, and five new species are described: Austroboletus albus, Xanthoconium violaceipes, Xanthoconium violaceofuscum, Xerocomus rutilans and Xerocomus subsplendidus. Descriptions and hand drawings of the new species are presented.


Introduction
The order Boletales is one of the largest groups of Basidiomycota and most of them are not only ectomycorrhizal fungi but also edible fungi. They are economically and ecologically important [1]. They could form mycorrhizal relationships with more than ten families of plants, including Betulaceae, Dipterocarpaceae, Fagaceae, Pinaceae and Salicaceae, which play vital roles in maintaining forest ecosystems, water and soil protection, vegetation restoration, and contributing to the diversification of both fungi and their host plants [2].
Jiangxi has the second highest forest coverage in China, which is dominated by evergreen broad-leaved forests with a subtropical warm and humid monsoon climate and abundant rainfall. The climatic conditions and vegetation types are very suitable for the growth Table 1. Names, voucher numbers, localities and corresponding GenBank accession numbers of the sequences used in phylogenetic analyses in this study. Sequences in bold were generated in this study.

Species
Voucher Locality GenBank Accession No. "/" means the data is not applied in this study, "-" means the data is missing.

ITS
Sequence datasets were aligned on the online website MAFFT version 7 (http:// mafft.cbrc.jp/alignment/server/, (accessed on 6 October 2022)) [61], the concatenation of the sequences of the two or three genes was completed in PhyloSuite [62]. The gene fragments of some taxa that could be found or sequenced were regarded as missing data. The intron regions of protein-coding genes were retained in the final analyses [27]. These datasets were then analyzed using RAxML version 8 [63] and MrBayes v3.2 [64] for maximum likelihood (ML) and Bayesian inference (BI), respectively. For ML analyses, under GTRGAMMAI model [65], nonparametric bootstrap analysis with 1000 repetitions [66] was used to determine the statistical support of phylogeney. For BI analyses, substitution models of partition in the datasets were determined using the Bayesian information criterion (BIC) implemented in PartitionFinder 2 [67]. Two or four MCMC runs and trees were sampled every 1000 generations. At the end of the runs, the average deviation of split frequencies was below 0.01. Other parameters were kept at their default settings. Trees were summarized, and posterior probabilities (PPs) were calculated after discarding the first 25% of generations as burn-in. Figtree v1.4.4 was used for visualization of phylogenetic analysis results. Branches that received bootstrap support for maximum likelihood (ML) and Bayesian posterior probabilities (BPP) greater than or equal to 50% (BS) and 0.95 (PP) are shown above.

Molecular Phylogenetic Results
For Austroboletus, six new sequences (two of ITS, two of nrLSU, two of TEF1-α) from two collections were generated. For the combined dataset, HKY + I + G, TIM + I + G and TRNEF + G were evaluated as best-fit substitution models for the ITS, nrLSU and TEF1-α partitions, respectively. The ITS dataset consisted of 43 taxa and 1097 characters. The nrLSU dataset included 60 taxa and 926 characters. The TEF1-α dataset comprised 23 accessions and 623 characters. The combined nuclear dataset (ITS + nrLSU + TEF1-α) contains 126 sequences with 2646 nucleotide sites, and the alignment was deposited in TreeBASE (S30554). The maximum likelihood and Bayesian phylograms have no conflict in topology, the ML trees with both BS and PP values are shown in Figure 1, the green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup. For Xanthoconium, ten new sequences (five of nrLSU, five of TEF1-α) were generated. For the combined datasets, TRNEF + I + G and TRNEF + G were the best-fit substitution models for the nrLSU and TEF1-α partitions, respectively. The nrLSU dataset included 23 taxa and 883 characters. The TEF1-α dataset comprised 20 accessions and 633 characters, and the alignment was deposited in TreeBASE (S30556). The Maximum likelihood and Bayesian phylograms have no conflict in topology, the ML trees with both BS and PP val- Figure 1. Maximum likelihood phylogenetic tree of Austroboletus inferred from the combined nuclear dataset (ITS + nrLSU + TEF1-α). Bootstrap frequencies ≥ 50% and posterior probabilities ≥ 0.95 are shown above supported branches. New sequences are shown in bold. The different colors are for decoration. The green background represents the sequence of new species discovered in this study, the red background represents the outer group, and the blue background represents other known species of this genus used in this study.
For Xanthoconium, ten new sequences (five of nrLSU, five of TEF1-α) were generated. For the combined datasets, TRNEF + I + G and TRNEF + G were the best-fit substitution models for the nrLSU and TEF1-α partitions, respectively. The nrLSU dataset included 23 taxa and 883 characters. The TEF1-α dataset comprised 20 accessions and 633 characters, and the alignment was deposited in TreeBASE (S30556). The Maximum likelihood and Bayesian phylograms have no conflict in topology, the ML trees with both BS and PP values are shown in Figure 2, the green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup. For Xerocomus, ten new sequences (five of nrLSU, five of TEF1-α) were generated. For the combined dataset, TRN + I + G and TRNEF + I + G were the best-fit substitution models for the nrLSU and TEF1-α partitions, respectively. The nrLSU dataset included 45 taxa and 897 characters. The TEF1-α dataset comprised 36 accessions and 619 characters, and the alignment was deposited in TreeBASE (S30557). The Maximum likelihood and Bayesian phylograms have no conflict in topology, the ML trees with both BS and PP values are shown in Figure 3, the green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup. For Xerocomus, ten new sequences (five of nrLSU, five of TEF1-α) were generated. For the combined dataset, TRN + I + G and TRNEF + I + G were the best-fit substitution models for the nrLSU and TEF1-α partitions, respectively. The nrLSU dataset included 45 taxa and 897 characters. The TEF1-α dataset comprised 36 accessions and 619 characters, and the alignment was deposited in TreeBASE (S30557). The Maximum likelihood and Bayesian phylograms have no conflict in topology, the ML trees with both BS and PP values are shown in Figure 3, the green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup. Maximum likelihood phylogenetic tree of Xerocomus inferred from the combined nuclear dataset (nrLSU + TEF1-α). Bootstrap frequencies ≥ 50% and posterior probabilities ≥ 0.95 are shown above supported branches. New sequences are shown in bold. Different colors are used to highlight different contents. The green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup.

Taxonomy
Austroboletus albus F. Zhou, H.Y. Song and D.M. Hu, sp. nov. (Figures 4-6). Mycobank: MB844812. Etymology-Latin "albus" refers to the color of pileus, which is white. Diagnosis: Austroboletus albus is characterized by a gray-green reticulate texture pileus, emerald green when young, white to pale brown when mature and an uneven stipe, similar to a gully, covered with obvious white to yellowish white reticulation, subfusiform to amygdaliform basidiospores measuring 13-17 × 6.5-7.5 µm, surface with intricate reticulum or irregular pits, becoming shorter to nearly smooth toward both poles, yellow to golden yellow in KOH.
Description: Basidiomata are small to medium-sized. Pileus 2-5 cm diam, hemispherical when young, becoming convex to subhemispherical with maturity, surface dry, emerald green when young, and white (5A1) to pale brown (4A2) when mature, with gray-green (26B3) reticulate texture, margin decurved, marginal veil white (5A1), serrated to irregular. Context is 0.5-1.6 cm in thickness in the center of the pileus, white (5A1), no . Maximum likelihood phylogenetic tree of Xerocomus inferred from the combined nuclear dataset (nrLSU + TEF1-α). Bootstrap frequencies ≥ 50% and posterior probabilities ≥ 0.95 are shown above supported branches. New sequences are shown in bold. Different colors are used to highlight different contents. The green background represents the new species identified in this study, the blue represents other known species of the genus, and the red represents the outgroup.

Taxonomy
Austroboletus albus F. Zhou, H.Y. Song and D.M. Hu, sp. nov. (Figures 4-6). Mycobank: MB844812. Etymology-Latin "albus" refers to the color of pileus, which is white. Diagnosis: Austroboletus albus is characterized by a gray-green reticulate texture pileus, emerald green when young, white to pale brown when mature and an uneven stipe, similar to a gully, covered with obvious white to yellowish white reticulation, subfusiform to amygdaliform basidiospores measuring 13-17 × 6.5-7.5 µm, surface with intricate reticulum or irregular pits, becoming shorter to nearly smooth toward both poles, yellow to golden yellow in KOH. Holotype Description: Basidiomata are small to medium-sized. Pileus 2-5 cm diam, hemispherical when young, becoming convex to subhemispherical with maturity, surface dry, emerald green when young, and white (5A1) to pale brown (4A2) when mature, with gray-green (26B3) reticulate texture, margin decurved, marginal veil white (5A1), serrated to irregular.
the center of the pileus, white (1A1) and does not change when injured. Hymenophore adnate around the stipe are quite crowded; pores are circular to angular, 2-3 per mm, white (1A1) when young and yellow (1A5) to yellow-brown (1B5) when old; tubes up to 1 cm long are white (2A1) to pale brown (2B2) and do not change in color when injured. Stipe 2-10.5 × 0.6-1.5 cm, central, cylindrical, solid; surface dry, with obvious dark purple (16D3) stripes, slightly interwoven near the pileus; context is white (16A1) to light pink (16A2), no reaction when bruised; basal mycelium white. Odor indistinct.    (Figures 9 and  10).
Mycobank: MB847089. Etymology-Latin "violaceofuscum" refers to the color of pileus, which is purple- Mycobank: MB847090. Etymology-Latin "violaceipes" refers to the color of stipe, which is purple. Diagnosis: Xanthoconium violaceipes have a hemispherical to convex to applanate pileus, purple-black to purple when young, brownish green when mature, a obvious dark purple stripes, slightly interwoven near the pileus. Basidiospores measure approximately 12.5-16.5 × 4-5.5 µm, ellipsoid to subfusiform to cylindrical, pileipellis is 60-156 µm thick and stipitipellis is 55-110 µm thick. Description: Basidiomata are medium-sized to large. Pileus 1-10 cm in diameter, initially hemispherical, becomes convex to applanate with maturity, surface is dry, purpleblack (11F1) to purple (11B3) when young, brownish green (30B6-30E6) when mature, margin is lighter and deeper in the center. Context is 0.3-0.7 cm in thickness in the center of the pileus, white (1A1) and does not change when injured. Hymenophore adnate around the stipe are quite crowded; pores are circular to angular, 2-3 per mm, white (1A1) when young and yellow (1A5) to yellow-brown (1B5) when old; tubes up to 1 cm long are white (2A1) to pale brown (2B2) and do not change in color when injured. Stipe 2-10.5 × 0.6-1.5 cm, central, cylindrical, solid; surface dry, with obvious dark purple (16D3) stripes, slightly interwoven near the pileus; context is white (16A1) to light pink (16A2), no reaction when bruised; basal mycelium white. Odor indistinct.
Holotype Description: Basidiomata are medium-sized to large. Pileus is 2.9-9 cm in diameter, initially hemispherical, becomes convex to applanate with maturity and the surface is dry, purple (11A2) to ochre (9B4) when young, brownish yellow (5B6) when mature. Context is up to 1 cm in thickness in the center of the pileus, white (5A1) and does not change when injured. Hymenophore adnate to slightly depressed around the stipe, quite crowded; pores iiragular, 2-3 per mm, yellow (2A6) when young and yellowish brown (5C6) when old, slightly higher around the stipe; tubes are up to 1 cm long, golden (2A7) to yellowish brown (5C6) and do not change in color when injured. Stipe is 4-9 × 1-3 cm,     (Figures 13 and 14). Mycobank: MB847092. Etymology-Latin "subsplendidus" refers to the color of pileus, which is yellowish brown.
Diagnosis: Xerocomus subsplendidus has a subhemispherical to applanate to infundibulate pileus, and the surface is always covered with yellowish brown tomentose when young, cracking into brown to dark brown scales with age, always has a crooked stipe, covered with pale brown to reddish brown tomentose in the upper part, and pores and tubes change to blue when injured, has ellipsoid to subfusiform to cylindrical; basidiospores are 9-15.5 × 4-5.5 µm, pale yellow to yellow-brown in KOH.
Description: Basidiomata are small to medium-sized. Pileus is 2.2-5.6 cm in diameter, subhemispherical to applanate to infundibulate, initially earth-yellow (6B5) to yellowish brown (6B7), and brown (6C7) to dark brown (6E5) later and have lighter margins; surface dry, always covered with yellowish brown (6C5) tomentose when young, cracking into brown (6A4) to dark brown (6E5) scales with age. Context is white (6A1), up to 0.8 cm in thickness in the center of the pileus, and does not change when injured. Hymenophore is slightly depressed and decurrent around the apex of the stipe, quite scattered; pores are irregular, 1 per mm or less, yellow (3A6), changes to blue (22D5) when bruised; tubes are up to 1 cm long, golden (2A6) to yellowish brown (2D7), changes to blue (22D5) when Mycobank: MB847091. Etymology-Latin "rutilans" refers to the color of pileus, which is ochre. Diagnosis: The pileus of Xerocomus rutilans is purple to ochre when young, brownish yellow when mature, the pores are yellow when young and yellowish brown when old and the stipes are covered with white tomentose scales, ellipsoidal to elongated to fusiform basidiospores measuring 8.5-11.5 × 4-5.5 µm, pale yellow to yellow brown in KOH.
Holotype Description: Basidiomata are medium-sized to large. Pileus is 2.9-9 cm in diameter, initially hemispherical, becomes convex to applanate with maturity and the surface is dry, purple (11A2) to ochre (9B4) when young, brownish yellow (5B6) when mature. Context is up to 1 cm in thickness in the center of the pileus, white (5A1) and does not change when injured. Hymenophore adnate to slightly depressed around the stipe, quite crowded; pores iiragular, 2-3 per mm, yellow (2A6) when young and yellowish brown (5C6) when old, slightly higher around the stipe; tubes are up to 1 cm long, golden (2A7) to yellowish brown (5C6) and do not change in color when injured. Stipe is 4-9 × 1-3 cm, central, cylindrical, solid, dry and covered with white (2A1) tomentose scales; context is white (2A1) and has no reaction when bruised; basal mycelium is white (1A1). Odor indistinct.
Diagnosis: Xerocomus subsplendidus has a subhemispherical to applanate to infundibulate pileus, and the surface is always covered with yellowish brown tomentose when young, cracking into brown to dark brown scales with age, always has a crooked stipe, covered with pale brown to reddish brown tomentose in the upper part, and pores and tubes change to blue when injured, has ellipsoid to subfusiform to cylindrical; basidiospores are 9-15.5 × 4-5.5 µm, pale yellow to yellow-brown in KOH.
Phylogenetically, the specimens we collected formed an independent clade with strong support (BS = 100%, PP = 1), and were closed to A. albovirescens (HKAS:59624 and HKAS74743) [1,2,31] with high statistical support (BS = 100%, PP = 1). However, A. albovirescens was described by Li and Yang [2], which is characterized by the matte green to gray-green pileus, the ornamentation of basidiospores with unequal pits, and the ixocutis pileipellis. Meanwhile, they formed a large clade with A. mutabilis (BS = 100%, PP Description: Basidiomata are small to medium-sized. Pileus is 2.2-5.6 cm in diameter, subhemispherical to applanate to infundibulate, initially earth-yellow (6B5) to yellowish brown (6B7), and brown (6C7) to dark brown (6E5) later and have lighter margins; surface dry, always covered with yellowish brown (6C5) tomentose when young, cracking into brown (6A4) to dark brown (6E5) scales with age. Context is white (6A1), up to 0.8 cm in thickness in the center of the pileus, and does not change when injured. Hymenophore is slightly depressed and decurrent around the apex of the stipe, quite scattered; pores are irregular, 1 per mm or less, yellow (3A6), changes to blue (22D5) when bruised; tubes are up to 1 cm long, golden (2A6) to yellowish brown (2D7), changes to blue (22D5) when injured. Stipe is 2.5-5 × 0.4-0.7 cm, central, cylindrical or crooked, solid; covered with pale brown (2B3) to reddish brown (9B4) tomentose in the upper part; context is white (3A1) to pale brown (3A2), no reaction when bruised; basal mycelium white. Odor indistinct.
Morphologically, Xa. violaceipes is similar to Boletus violaceofuscus and Xa. separans. Based on the phylogenetic analyses, the collections of Xa. violaceipes are clustered on one branch within Xanthoconium with 100% bootstrap support, and sister to Xa. separans with 82% statistical support, but they have a long genetic distance.
In phylogenetic analyses, the collections of Xa. violaceofuscum are clustered on an independent branch within Xanthoconium with 100% bootstrap support.
Morphologically, Xerocomus subsplendidus is similar to X. fraternus, X. subparvus and X. yunnanensis [1]. However, X. fraternus can be distinguished by the changes in pileus and stipe context when injured, pileus context changing from cream to yellowish and staining bluish slowly when injured, stipe context cream on upper part, staining pale blue slowly when injured, and lower part pale red-brown near stipe base; X. fraternus has shorter basidiospores [(8.5)9.5-12 (13) µm], and the shape of pileipellis terminal cells is subcylindrical. X. subparvus has cream to yellowish pileus context and a red tinge near pileipellis, staining bluish slowly or indistinctly when injured, context of the stipe is pale yellow at the upper part, staining bluish slowly when injured and pale brown to pale-red brown at the lower part; X. subparvus has smaller basidiospores [(8.5)9-10.5(11.5) × (3)3.5-4(4.5) µm], and the shape of pileipellis terminal cells is subcylindrical. X. yunnanensis is characterized by the tubes staining red-brown slowly when young and becoming bluish when mature on injury, the fresh yellow context of pileus near the pileipellis, and the white to yellowish context of stipe, bluing indistinctly when cut [1]; X. yunnanensis has smaller basidiospores [(9)10-11.5(13) × 4-4.5(5) µm], and the terminal cells of pileipellis are tapered or bullet-shaped.
According to our phylogeny, the sequences of Xerocomus subsplendidus form an independent branch within Xerocomus with 100% bootstrap support, and are sistered to Xerocomus sp. (voucher: HKAS90207, HKAS 74927 and HKAS 75076) with 58% statistical support, but they have a long genetic distance. Institutional Review Board Statement: Not applicable.