# An Updated Global Species Diversity and Phylogeny in the Forest Pathogenic Genus Heterobasidion (Basidiomycota, Russulales)

Yuan Yuan, Jia-Jia Chen, Kari Korhonen, Francis Martin, Yu-Cheng Dai

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Yuan Yuan, Jia-Jia Chen, Kari Korhonen, Francis Martin, Yu-Cheng Dai. An Updated Global Species Diversity and Phylogeny in the Forest Pathogenic Genus Heterobasidion (Basidiomycota, Russulales). Frontiers in Microbiology, 2021, 11, pp.596393. 10.3389/fmicb.2020.596393. hal-03994857

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# An Updated Global Species Diversity and Phylogeny in the Forest Pathogenic Genus *Heterobasidion* (Basidiomycota, Russulales)

Yuan Yuan<fnref n="1" />,²†, Jia-Jia Chen³†, Kari Korhonen⁴, Francis Martin²,⁵ and Yu-Cheng Dai²*

OPEN ACCESS

**Edited by:**
Peter Edward Mortimer,
Kunming Institute of Botany, Chinese
Academy of Sciences, China

**Reviewed by:**
Jan Stenlid,
Swedish University of Agricultural
Sciences, Sweden
Samantha Chandranath
Karunarathna,
Kunming Institute of Botany, China

*Correspondence:
Yu-Cheng Dai
yuchengd@yahoo.com

**Specialty section:**
This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology

Received: 19 August 2020

Accepted: 30 November 2020

Published: 07 January 2021

**Citation:**
Yuan Y, Chen J-J, Korhonen K, Martin F and Dai Y-C (2021) An Updated Global Species Diversity and Phylogeny in the Forest Pathogenic Genus *Heterobasidion* (Basidiomycota, Russulales). *Front. Microbiol.* 11:596393.
doi: 10.3389/fmicb.2020.596393

*Heterobasidion* species are amongst the most intensively studied polypores because several species are aggressive white rot pathogens of managed coniferous forests mainly in Europe and North America. In the present study, both morphological and multilocus phylogenetic analyses were carried out on *Heterobasidion* samples from Asia, Oceania, Europe and North America. Three new taxa were found, i.e., *H. armandii*, *H. subinsulare*, and *H. subparviporum* are from Asia and are described as new species. *H. ecrustosum* is treated as a synonym of *H. insulare*. So far, six taxa in the *H. annosum* species complex are recognized. *Heterobasidion abietinum*, *H. annosum*, and *H. parviporum* occur in Europe, *H. irregulare*, and *H. occidentale* in North America, and *H. subparviporum* in East Asia. The North American *H. irregulare* was introduced to Italy during the Second World War. Species in the *H. annosum* complex are pathogens of coniferous trees, except *H. subparviporum* that seems to be a saprotroph. Ten species are found in the *H. insulare* species complex, all of them are saprotrophs. The pathogenic species are distributed in Europe and North America; the Asian countries should consider the European and North American species as entry plant quarantine fungi. Parallelly, European countries should consider the American *H. occidentale* and *H. irregulare* as entry plant quarantine fungi although the latter species is already in Italy, while North America should treat *H. abietinum*, *H. annosum* s.s., and *H. parviporum* as entry plant quarantine fungi. Eight *Heterobasidion* species found in the Himalayas suggest that the ancestral *Heterobasidion* species may have occurred in Asia.

**Keywords:** taxonomy, phylogeny, new taxa, *Bondarzewiaceae*, pathogenic fungi

<footnote n="1">¹ Institute of Microbiology, School of Ecology and Nature Conservation, Beijing Forestry University, Beijing, China, ² Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Beijing, China, ³ Permanent Research Base of National Forestry and Grassland Administration, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang, China, ⁴ Natural Resources Institute Finland (Luke), Helsinki, Finland, ⁵ University of Lorraine, INRAE, Tree-Microbes Interaction Department, Champenoux, France</footnote>

<footnote>† These authors have contributed equally to this work and share first authorship</footnote>
# INTRODUCTION

The polypore genus *Heterobasidion* Bref., which belongs to the family *Bondarzewiaceae*, is one of the most intensively studied basidiomycetous genera because some species of *Heterobasidion* are aggressive pathogens of managed coniferous forests in Europe and North America (Woodward et al., 1998). Two morphological taxa, *H. annosum* (Fr.) Bref. and *H. insulare* (Murrill) Ryvarden, had generally been accepted in *Heterobasidion* (Murrill, 1908; Gilbertson and Ryvarden, 1986; Ryvarden and Gilbertson, 1993; Núñez and Ryvarden, 2001). However, mating studies have revealed that both *H. annosum* and *H. insulare* are in fact species complexes (Korhonen, 1978; Dai and Korhonen, 1999; Dai et al., 2002, 2003).

Three species, *Heterobasidion abietinum* Niemelä and Korhonen (Eur F-group), *H. annosum* (Fr.) Bref. sensu stricto (Eur P-group) and *H. parviporum* Niemelä and Korhonen (Eur S-group), have been recognized in Europe (Niemelä and Korhonen, 1998), and two species, *H. irregulare* Garbel. and Otrosina (NAm P-group) and *H. occidentale* Otrosina and Garbel. (NAm S-group), were described from North America (Otrosina and Garbelotto, 2010). Based on mating studies, the East Asian taxon in the *H. annosum* species complex was considered as *H. parviporum* (Dai and Korhonen, 1999, 2003; Dai et al., 2006; Dai, 2012; Chen et al., 2015). Similarly, investigations based on mating tests, morphological characteristics and molecular analyses revealed several species also within the Asian *H. insulare* complex: *H. linzhiense* Y. C. Dai and Korhonen (Dai et al., 2007), *H. australe* Y. C. Dai and Korhonen (2009), *H. ecrustosum* Tokuda, T. Hatt. and Y. C. Dai, *H. orientale* Tokuda, T. Hatt. and Y. C. Dai (Tokuda et al., 2009), *H. amyloideum* Y. C. Dai, Jia J. Chen and Korhonen, *H. tibeticum* Y. C. Dai, Jia J. Chen and Korhonen (Chen et al., 2014) and *H. amyloideopsis* Saba, C. L. Zhao, Khalid and Pfister (Zhao et al., 2017). In addition, *H. araucariae* P. K. Buchanan from Australia and adjacent regions (Buchanan, 1988) was confirmed to be a member of the *H. insulare* species complex (Chen et al., 2015).

Earlier phylogenetic analyses on the *H. annosum* complex used sequences of the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear genes, and manganese peroxidase genes, and laccase genes (Maijala et al., 2003; Asiegbu et al., 2004). Later, several attempts were made to resolve the taxonomy of the *H. annosum* complex or *H. insulare* complex using multilocus phylogenetic approaches (Johannesson and Stenlid, 2003; Ota et al., 2006; Linzer et al., 2008; Chen et al., 2014). Recently, five species in the *H. annosum* species complex and eight species in *H. insulare* species complex were also recognized and confirmed by multilocus phylogenetic approaches, and divided into three groups based on five nuclear genes and two mitochondrial genes, i.e., ITS, the large nuclear ribosomal RNA subunit (nrLSU), the largest subunit of RNA polymerase II (RPB1), the second subunit of RNA polymerase II (RPB2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), mitochondrial ATP synthase subunit 6 (ATP6), and mitochondrial small subunit rDNA (mtSSU) (Chen et al., 2015).

Several hypotheses on the evolutionary scenarios of the *Heterobasidion* have been put forward (Otrosina et al., 1993; Ota et al., 2006; Linzer et al., 2008). Dalman et al. (2010) proposed that the *H. annosum* complex originated in Laurasia, *H. annosum* s.s./*H. irregulare* arose in Eurasia, and *H. parviporum*/*H. abietinum*/*H. occidentale*, which occurred in eastern Asia or western North America, emerged between 45 and 60 Ma in the Palaearctic; this conclusion was based on non-coding regions of elongation factor 1-α (EFA), glutathione-S-transferase (GST1), GAPDH, and transcription factor (TF). Recently, based on more species and samples of *Heterobasidion* and the fossil record, molecular dating suggested that ancestral *Heterobasidion* species originated in Eurasia occurred mainly during the Early Miocene (Chen et al., 2015; Zhao et al., 2017).

Based on a larger set of *Heterobasidion* samples from Asia, Oceania, Europe and North America, and using combined RPB1 and RPB2 sequence dataset, a further phylogenetic investigation on the genus is carried out. Four new taxa are detected, and three of them are described and illustrated in the present paper. Moreover, most relevant morphological characteristics of different species of *Heterobasidion* are compared.

# MATERIALS AND METHODS

## Morphological Studies

The studied specimens and cultures (Table 1) are deposited at the herbaria of Institute of Microbiology of the Beijing Forestry University (BJFC, Beijing, China), Natural Resources Institute Finland (Luke, Helsinki, Finland), U.S. Forest Service, Northern Research Station (CFMR, Madison, WI, United States), private herbarium of J. Vlasák (JV, České Budějovice, Czechia), and Landcare Research, New Zealand (PDD, Lincoln, New Zealand). Ecology and some macromorphological characters were based on field notes. Anatomy was studied, and measurements and drawings were made from slide preparations stained with Cotton Blue. Drawings were made with the aid of a drawing tube. In presenting the variation in the size of the spores, the 5% of the measurements at each end of the range are shown in parentheses. Basidiospore spine lengths are not included in the measurements. The following abbreviations are used: IKI = Melzer's reagent, IKI- = both non-amyloid and non-dextrinoid, IKI+ = amyloid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n = number of spores measured from given number of specimens. Color terms are from Petersen (1996).

## DNA Extraction, PCR Amplification and Sequencing

The Rapid Plant Genome kit based on acetyl trimethylammonium bromide extraction (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to
<table><caption>TABLE 1 | Information on samples of *Heterobasidion* used in this study.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Sample no.</th><th rowspan="2">Geographic origin</th><th rowspan="2">Host</th><th colspan="5">GenBank accessions</th></tr><tr><th>ITS</th><th>nrLSU</th><th>RPB1</th><th>RPB2</th><th>GAPDH</th></tr></thead><tbody><tr><td>Bondarzewia occidentalis</td><td>HHB 14803</td><td>Washington, United States</td><td>Picea sitchensis</td><td>DQ200923</td><td>DQ234539</td><td>DQ256049</td><td>AY218474</td><td>-</td></tr><tr><td>B. submesenterica</td><td>Cui 10724</td><td>Sichuan, China</td><td>Abies ernestii</td><td>KJ583205</td><td>KJ583219</td><td>KJ651627</td><td>KJ651720</td><td>KJ651752</td></tr><tr><td>Heterobasidion abietinum</td><td>00053/1</td><td>Trentino, Italy</td><td>Picea abies</td><td>KJ651451</td><td>KJ651509</td><td>KJ651630</td><td>KJ651723</td><td>KJ651754</td></tr><tr><td>H. abietinum</td><td>00055/6</td><td>Trentino, Italy</td><td>Picea abies</td><td>KJ651452</td><td>KJ651510</td><td>KJ651631</td><td>KJ651724</td><td>KJ651755</td></tr><tr><td>H. abietinum</td><td>00057/2</td><td>Trentino, Italy</td><td>Abies alba</td><td>KJ651453</td><td>KJ651511</td><td>KJ651632</td><td>KJ651725</td><td>KJ651756</td></tr><tr><td>H. amyloideum</td><td>Cui 12656</td><td>Tibet, China</td><td>Pinus</td><td>MT146480</td><td>MT446029</td><td>MT157738</td><td>MT157761</td><td>MT157721</td></tr><tr><td>H. amyloideum</td><td>Cui 12274</td><td>Tibet, China</td><td>Abies</td><td>MT146481</td><td>MT446030</td><td>MT157739</td><td>MT157762</td><td>MT157722</td></tr><tr><td>H. annosum</td><td>06071/1</td><td>Lazio, Italy</td><td>Pinus pinea</td><td>KJ651458</td><td>KJ651516</td><td>KF453497</td><td>KF453491</td><td>KJ651761</td></tr><tr><td>H. annosum</td><td>06125/2</td><td>Krasnoyarsk, Russia</td><td>Pinus sylvestris</td><td>KJ651459</td><td>KJ651517</td><td>KF453498</td><td>KF453492</td><td>KJ651762</td></tr><tr><td>H. annosum</td><td>06129/6</td><td>Krasnoyarsk, Russia</td><td>Pinus sylvestris</td><td>KJ583211</td><td>KJ583225</td><td>KF006499</td><td>KF033133</td><td>KJ651763</td></tr><tr><td>H. araucariae</td><td>65008</td><td>Queensland, Australia</td><td>Araucaria cunninghamii</td><td>KJ651462</td><td>KJ651520</td><td>KJ651636</td><td>KJ651729</td><td>KJ651766</td></tr><tr><td>H. araucariae</td><td>82001</td><td>Queensland, Australia</td><td>Araucaria cunninghamii</td><td>KJ651463</td><td>KJ651521</td><td>KJ651637</td><td>KJ651730</td><td>KJ651767</td></tr><tr><td>H. armandii</td><td>Dai 17605</td><td>Yunnan, China</td><td>Pinus armandii</td><td>MT146482</td><td>MT446031</td><td>MT157740</td><td>MT157763</td><td>-</td></tr><tr><td>H. armandii</td><td>Dai 17606</td><td>Yunnan, China</td><td>Pinus armandii</td><td>MT146483</td><td>MT446032</td><td>MT157741</td><td>MT157764</td><td>-</td></tr><tr><td>H. armandii</td><td>Dai 17607</td><td>Yunnan, China</td><td>Pinus armandii</td><td>MT146484</td><td>MT446033</td><td>MT157742</td><td>MT157765</td><td>-</td></tr><tr><td>H. australe</td><td>Cui 12602</td><td>Yunnan, China</td><td>Pinus sp.</td><td>MT146485</td><td>MT446034</td><td>MT157743</td><td>MT157766</td><td>MT157723</td></tr><tr><td>H. australe</td><td>Dai 13507</td><td>Yunnan, China</td><td>Pinus sp.</td><td>MT146486</td><td>MT446035</td><td>MT157744</td><td>MT157767</td><td>MT157724</td></tr><tr><td>H. australe</td><td>Dai 13863</td><td>Yunnan, China</td><td>Pinus sp.</td><td>MT146487</td><td>MT446036</td><td>MT157745</td><td>MT157768</td><td>MT157725</td></tr><tr><td>H. insulare</td><td>FPRI 429</td><td>Philippines</td><td>Pinus sp.</td><td>MT146488</td><td>MT446037</td><td>MT157746</td><td>MT157769</td><td>MT157726</td></tr><tr><td>H. insulare</td><td>Dai 13933</td><td>Chongqing, China</td><td>Pinus massoniana</td><td>MT146489</td><td>MT446038</td><td>MT157747</td><td>MT157770</td><td>MT157727</td></tr><tr><td>H. insulare</td><td>Dai 15095</td><td>Jiangxi, China</td><td>Pinus massoniana</td><td>MT146490</td><td>MT446039</td><td>MT157748</td><td>MT157771</td><td>MT157728</td></tr><tr><td>H. irregulare</td><td>57001/TI</td><td>North Carolina, United States</td><td>Pinus strobus</td><td>KJ651473</td><td>KJ651531</td><td>KJ651638</td><td>KJ651731</td><td>KJ651777</td></tr><tr><td>H. irregulare</td><td>88010/1</td><td>Vermont, United States</td><td>Pinus sp.</td><td>KJ651475</td><td>KJ651533</td><td>KJ651640</td><td>KJ651733</td><td>KJ651779</td></tr><tr><td>H. irregulare</td><td>01062</td><td>Ontario, Canada</td><td>Pinus resinosa</td><td>KJ651477</td><td>KJ651535</td><td>KJ651642</td><td>KJ651735</td><td>KJ651781</td></tr><tr><td>H. linzhiense</td><td>Cui 7216</td><td>Sichuan, China</td><td>Abies sp.</td><td>KJ651480</td><td>KJ651538</td><td>KF006524</td><td>KF033148</td><td>KJ651784</td></tr><tr><td>H. linzhiense</td><td>Cui 9645</td><td>Tibet, China</td><td>Picea sp.</td><td>KJ651481</td><td>KJ651539</td><td>KF033147</td><td>KF006523</td><td>KJ651785</td></tr><tr><td>H. linzhiense</td><td>Dai 5408</td><td>Tibet, China</td><td>Picea sp.</td><td>KJ651484</td><td>KJ651542</td><td>KF033154</td><td>KF006533</td><td>KJ651788</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 1 | Continued</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Sample no.</th><th rowspan="2">Geographic origin</th><th rowspan="2">Host</th><th colspan="5">GenBank accessions</th></tr><tr><th>ITS</th><th>nrLSU</th><th>RPB1</th><th>RPB2</th><th>GAPDH</th></tr></thead><tbody><tr><td>H. occidentale</td><td>79034/TI</td><td>Alaska, United States</td><td>Picea sp.</td><td>KJ651485</td><td>KJ651543</td><td>KJ651645</td><td>KJ651738</td><td>KJ651789</td></tr><tr><td>H. occidentale</td><td>98004/TI</td><td>Oregon, United States</td><td>Picea engelmannii</td><td>KJ651488</td><td>KJ651546</td><td>KJ651648</td><td>KJ651741</td><td>KJ651792</td></tr><tr><td>H. occidentale</td><td>98005/TI</td><td>Oregon, United States</td><td>Abies magnifica var. shastensis</td><td>KJ651489</td><td>KJ651547</td><td>KJ651649</td><td>KJ651742</td><td>KJ651793</td></tr><tr><td>H. orientale</td><td>Cui 11637</td><td>Heilongjiang, China</td><td>Unknown</td><td>MT146491</td><td>MT446040</td><td>MT157749</td><td>MT157772</td><td>MT157729</td></tr><tr><td>H. orientale</td><td>Cui 11815</td><td>Heilongjiang, China</td><td>Pinus sp.</td><td>MT146492</td><td>MT446041</td><td>MT157750</td><td>MT157773</td><td>MT157730</td></tr><tr><td>H. orientale</td><td>Cui 12026</td><td>Heilongjiang, China</td><td>Picea sp.</td><td>MT146493</td><td>MT446042</td><td>MT157751</td><td>MT157774</td><td>MT157731</td></tr><tr><td>H. parviporum</td><td>04121/3</td><td>Artjärvi, Finland</td><td>Picea abies</td><td>KJ583212</td><td>KJ583226</td><td>KF453493</td><td>KF453499</td><td>KJ651800</td></tr><tr><td>H. parviporum</td><td>08021/7</td><td>Krasnoyarsk, Russia</td><td>Picea abies</td><td>KJ651498</td><td>KJ651556</td><td>KF453494</td><td>KF453500</td><td>KJ651803</td></tr><tr><td>H. parviporum</td><td>08123/TI</td><td>Irkutsk, Russia</td><td>Picea abies</td><td>KJ651500</td><td>KJ651558</td><td>KF453495</td><td>KF453501</td><td>KJ651805</td></tr><tr><td>H. sp.</td><td>Korhonen 05030</td><td>California, United States</td><td>Pinus ponderosa</td><td>MT146494</td><td>MT446043</td><td>MT157752</td><td>MT157775</td><td>–</td></tr><tr><td>H. sp.</td><td>Korhonen 05038</td><td>California, United States</td><td>Pinus ponderosa</td><td>MT146495</td><td>MT446044</td><td>MT157753</td><td>MT157776</td><td>–</td></tr><tr><td>H. sp.</td><td>Korhonen 05039</td><td>California, United States</td><td>Pinus ponderosa</td><td>MT146496</td><td>MT446045</td><td>MT157754</td><td>MT157777</td><td>–</td></tr><tr><td>H. subinsulare</td><td>Dai 13842</td><td>Yunnan, China</td><td>Pinus sp.</td><td>MT146497</td><td>MT446046</td><td>MT157755</td><td>MT157778</td><td>MT157732</td></tr><tr><td>H. subinsulare</td><td>Li 140804-30</td><td>Yunnan, China</td><td>Pinus sp.</td><td>MT146498</td><td>MT446047</td><td>MT157756</td><td>MT157779</td><td>MT157733</td></tr><tr><td>H. subparviporum</td><td>Cui 6961</td><td>Hubei, China</td><td>Abies fargesii</td><td>KJ651504</td><td>KJ651562</td><td>KJ651658</td><td>KJ651751</td><td>KJ651809</td></tr><tr><td>H. subparviporum</td><td>Cui 9267</td><td>Tibet, China</td><td>Picea sp.</td><td>MT146499</td><td>MT446048</td><td>MT157757</td><td>MT157780</td><td>MT157734</td></tr><tr><td>H. subparviporum</td><td>Dai 14803</td><td>Jilin, China</td><td>Picea sp.</td><td>MT146500</td><td>MT446049</td><td>MT157758</td><td>MT157781</td><td>MT157735</td></tr><tr><td>H. tibeticum</td><td>Cui 12257</td><td>Tibet, China</td><td>Pinus sp.</td><td>MT146501</td><td>MT446050</td><td>MT157759</td><td>MT157782</td><td>MT157736</td></tr><tr><td>H. tibeticum</td><td>Cui 12335</td><td>Tibet, China</td><td>Pinus sp.</td><td>MT146502</td><td>MT446051</td><td>MT157760</td><td>MT157783</td><td>MT157737</td></tr></tbody></table>

New sequences are shown in bold.

extract genomic DNA from dried fungal specimens and cultures, according to the manufacturer's instructions with some modifications (Chen et al., 2014). The PCR primers for all genes are listed in Table 2. The PCR procedure for nrLSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min, 72°C for 1.5 min, and a final extension at 72°C for 10 min. The following PCR protocol for GAPDH, and ITS was used: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, (50°C for GAPDH, 54°C for ITS), 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR procedure for RPB1 and RPB2 followed Justo and Hibbett (2011) with slight modifications: initial denaturation at 94°C for 2 min,

followed by 10 cycles at 94°C for 40 s, 60°C for 40 s, 72°C for 2 min, then followed by 37 cycles at 94°C for 45 s, 55°C for 1.5 min and 72°C for 2 min, and a final extension at 72°C for 10 min. PCR products were purified with a Gel Extraction and PCR Purification Combo Kit (Spin-column) in Beijing Genomics Institute, Beijing, China. The purified products were then sequenced on an ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, CA, United States) using the same primers as in the original PCR amplifications. All newly generated sequences were deposited at GenBank<fnref n="1" /> and listed in Table 1.

<footnote n="1">¹http://www.ncbi.nlm.nih.gov/</footnote>
<table><caption>**TABLE 2** | PCR primers used in this study.</caption><thead><tr><th>Gene</th><th>Primer</th><th>Primer sequences (5'-3')<sup>a</sup></th><th>References</th></tr></thead><tbody><tr><td rowspan="2">GAPDH</td><td>GAPDH-F</td><td>YGG TGT CTT CAC CAC CAC YGA SSA</td><td>Johannesson et al., 2000</td></tr><tr><td>GAPDH-R</td><td>RTA NCC CCA YTC RTT RTC RTA CCA</td><td>Johannesson et al., 2000</td></tr><tr><td rowspan="2">ITS</td><td>ITS5</td><td>GGA AGT AAA AGT CGT AAC AAG G</td><td>White et al., 1990</td></tr><tr><td>ITS4</td><td>TCC TCC GCT TAT TGA TAT GC</td><td>White et al., 1990</td></tr><tr><td rowspan="2">nrLSU</td><td>LROR</td><td>ACC CGC TGA ACT TAA GC</td><td>Vilgalys and Hester, 1990</td></tr><tr><td>LR7</td><td>TAC TAC CAC CAA GAT CT</td><td>Vilgalys and Hester, 1990</td></tr><tr><td rowspan="2">RPB1</td><td>RPB1-Af</td><td>GAR TGY CCD GGD CAY TTY GG</td><td>Matheny et al., 2002</td></tr><tr><td>RPB1-Cf</td><td>CCN GCD ATN TCR TTR TCC ATR TA</td><td>Matheny et al., 2002</td></tr><tr><td rowspan="2">RPB2</td><td>fRPB2-5F</td><td>GAY GAY MGW GAT CAY TTY GG</td><td>Liu et al., 1999; Matheny, 2005</td></tr><tr><td>fRPB2-7cR</td><td>CCC ATR GCT TGY TTR CCC AT</td><td>Liu et al., 1999; Matheny, 2005</td></tr></tbody></table>

## Phylogenetic Analysis

*Bondarzewia occidentalis* Jia J. Chen, B. K. Cui and Y. C. Dai and *B. submesenterica* Jia J. Chen, B. K. Cui and Y. C. Dai were used as outgroups (Chen et al., 2015). Sequences were aligned with BioEdit (Hall, 1999) and ClustalX (Thompson et al., 1997). Sequence alignments were deposited at TreeBase<fnref n="2" /> (submission ID 25908).

Maximum parsimony (MP) analysis was applied to single-locus genealogies for ITS, nrLSU, RPB1, PPB2, and GAPDH, and combination datasets that contained the RPB1-RPB2 sequences. The tree construction procedure was performed in PAUP* version 4.0b10 (Swofford, 2002). All characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1000 replicates (Felsenstein, 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RCI), and homoplasy index (HI), were calculated for each maximum parsimonious tree generated. Phylogenetic trees were visualized using Treeview (Page, 1996).

MrMODELTEST2.3 (Nylander, 2004) was used to determine the best-fit evolution model for the combined dataset for Bayesian inference (BI). The BI was calculated with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) with a general time reversible model of DNA substitution and an invgamma distribution rate variation across sites. Eight Markov chains were run from random starting tree for 1 M generations of RPB1 and RPB2 dataset, and sampled every 100 generations. The burn-in was set to discard the first 25% of the trees. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap values for MP and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP) and 0.95 (BPP) were considered as significantly supported.

To determine if the datasets were significantly conflicted, the partition homogeneity test option in PAUP 4.0b was used between the loci in all possible pairwise combinations using

1000 replicates and the heuristic general search option. This test randomly shuffles phylogenetically informative sites between two paired loci: if the datasets are compatible, shuffling sites between the loci should not produce summed tree lengths that are significantly greater than those produced by the observed data (Farris et al., 1994; Huelsenbeck et al., 1996).

## RESULTS

### Molecular Phylogeny

All targeted DNA loci were successfully amplified and sequenced from our *Heterobasidion* samples and the outgroup species. Partition homogeneity test showed no conflicts for the RPB1 and RPB2 combined loci (*P* = 0.019, *P* ≥ 0.01). Therefore, the amino acid sequences from RPB1 and RPB2 were combined into a single sequence set. The combined dataset included sequences from 46 specimens representing 18 species. The dataset had an aligned length of 2505 characters, of which 1796 characters were constant, 671 were variable and parsimony-uninformative, and 38 were parsimony-informative. The maximum parsimony analysis yielded four equally parsimonious tree (TL = 1033, CI = 0.789, HI = 0.925, RI = 0.730, RC = 0.211). The best model for the combined RPB1 + RPB2 estimated and applied in the Bayesian analysis: GTR + I + G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). The Bayesian analysis resulted in a topology similar to the MP analysis, with an average standard deviation of split frequencies = 0.006737, and only the MP tree was provided. Both bootstrap values (> 50%) and BPPs (≥0.90) were shown at the nodes (Figure 1).

Three new species, *Heterobasidion armandii*, *H. subinsulare*, and *H. subparviporum* formed a well-supported phylogenetic lineages, respectively (100% MP, and 1 BPPs), and phylogenetically distinct from other known species of *Heterobasidion*.

According to the present phylogenetic analyses, *Heterobasidion* spp. consists of three lineages: (1) the lineage associated to pines, firs and spruces (*H. amyloideopsis*, *H. amyloideum*, *H. araucariae*, *H. armandii*, *H. australe*, *H. insulare*, *H. linzhiense*, *H. orientale*, *H. subinsulare*, and *H. tibeticum*); (2) lineage mainly associated to pines (*H. annosum*

<footnote><sup>a</sup>Degenerac codes: S = G or C, W = A or T, R = A or G, Y = C or T, N = A or T or C or G, D = G or A or T, M = A or C.</footnote>

<footnote n="2">²https://treebase.org/treebase-web/home.html</footnote>
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** | Phylogeny of *Heterobasidion* and related species generated by Maximum Parsimony based on combined RPB1 + RPB2 sequences. Parsimony bootstrap values (before the slash markers) higher than 50% and Bayesian posterior probabilities (after the slash markers) more than 0.95 were indicated along branches.</figcaption></figure>

s.s., *H. sp.* and *H. irregulare*); and (3) the lineage associated to firs and spruces (*H. abietinum*, *H. occidentale*, *H. parviporum*, and *H. subparviporum*).

## Taxonomy

*Heterobasidion armandii* Y. C. Dai, Jia J. Chen and Yuan Yuan, sp. nov. Figures 2, 3
MycoBank MB 834572.

### Type

China, Yunnan Province, Xiping County, Mopanshan Forest Park, alt.135 m, on stump of *Pinus armandii*, June 15, 2017, YC Dai 17605 (BJFC025137, holotype).

### Diagnosis

Differs from other *Heterobasidion* species by its contextual skeletal hyphae are positive in Melzer's reagent, absence of
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** | *Basidiocarps of Heterobasidion armandii* (holotype, Dai 17605). Bar = 2 cm.</figcaption></figure>

cystidia, presence of cystidioles, and subglobose to broadly ellipsoid basidiospores measuring 4.9–5.9 × 3.9–4.5 µm.

## Etymology

*Armandii* (Lat.): referring to the species growing on *P. armandii*.

## Description

*Basidiocarps* annual, pileate, usually imbricate, leathery and without odor or taste when fresh, corky when dry. *Pilei* semicircular to fan-shaped, projecting up to 3 cm, 7 cm wide, and 8 mm thick at base. *Pileal surface* white to cream when juvenile, becoming olivaceous buff with age, at least reddish brown to dark reddish brown at base, crustose, distinctly zonate; margin cream, blunt. *Pore surface* white when fresh, cream when dry, not glancing; pores mostly round to angular, (3–4)–5 per mm; dissepiments thin, entire to slightly lacerate. *Context* cream, woody hard when dry, azonate, up to 5 mm thick, with a thin black line under crust except for the margin. *Tubes* cream to buff, hard corky, up to 3 mm long. *Hyphal system* dimitic; generative hyphae without clamp connections; tramal skeletal hyphae dextrinoid, CB+, contextual skeletal hyphae weakly IKI+, CB+; hyphae unchanged in KOH (not dissolved). Contextual generative hyphae frequently present, colorless, thin- to slightly thick-walled, frequently simple septate, occasionally branched, 3–4 mm diam; contextual skeletal hyphae dominant, colorless, thick-walled with a wide to narrow lumen, rarely branched, flexuous, interwoven, 4–5.5 mm diam. Tramal generative hyphae infrequent, hyaline, thin-walled, frequently simple septate, occasionally branched, 2–3 mm diam; tramal skeletal hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, rarely branched, flexuous, strongly interwoven without orientation, 3–4 mm diam. *Cystidia* absent. *Cystidioles* present, fusiform, occasionally with an apical simple septum. *Basidia* clavate to ampullaceal, with a simple basal septum and four stermigata, 14–24 × 6–8 mm. *Basidioles* in shape similar to basidia, but distinctly shorter. *Basidiospores* subglobose to broadly ellipsoid, hyaline, fairly thick-walled,

asperulate, mostly bearing a small guttule, IKI–, CB+, (4.8–)4.9–5.9(–6) × (3.8–)3.9–4.5(–4.8) mm, *L* = 5.12 mm, *W* = 4.11 mm, *Q* = 1.24–1.25 (*n* = 60/2).

## Additional materials (paratypes) examined

China, Yunnan Province, Xiping County, Mopanshan National Park, alt.1450 m, on stump of *P. armandii*, June 15, 2017, Dai 17606 (BJFC025138), Dai 17607 (BJFC025139); August 16, 2019, Dai 20410 (BJFC032078). Luquan County, Jiaozishan Forest Park, alt.2650 m, on stump of *P. armandii*, November 4, 2018, Dai 19258 (BJFC027726), Dai 19259 (BJFC027727), Dai 19260 (BJFC027728) and Dai 19261 (BJFC027729).

## *Heterobasidion subinsulare* Y. C. Dai, Jia J. Chen and Yuan Yuan, sp. nov. Figures 4, 5

MycoBank MB 834573.

## Type

China, Yunnan Province, Tengchong County, Shuanghe Village, on stump of *Pinus* sp., August 5, 2014, YC Dai 13842 (BJFC017572, holotype).

## Diagnosis

Differs from *Heterobasidion* species by big pores (1–3 per mm), a non-glancing pore surface, its contextual skeletal hyphae are negative in Melzer's reagent, presence of cystidia and cystidioles, and subglobose to broadly ellipsoid basidiospores measuring 5–5.7 × 3.8–5 µm.

## Etymology

*Subinsulare* (Lat.): referring to the similarity to *H. insulare*.

## Description

*Basidiocarps* annual, effused-reflexed to pileate, usually imbricate, leathery and without odor or taste when fresh, woody hard when dry. *Pilei* semicircular to fan-shaped, projecting up to 4 cm, 10 cm wide, and 4 cm thick at base. *Pileal surface* cream when dry, becoming buff to buff-yellow, crustose, azonate; margin buff-yellow to honey-yellow, blunt. *Pore surface* white when fresh, buff to clay-buff when dry, not glancing; pores angular to elongated, 1–3 per mm; dissepiments thin, entire to slightly lacerate. *Context* cream to buff-yellow, corky when dry, azonate, up to 0.5 cm thick. *Tubes* white to buff, hard corky, up to 3.5 cm long. *Hyphal system* dimitic; generative hyphae without clamp connections; skeletal hyphae CB+, dextrinoid near to the tube mouths, IKI– in other parts; hyphae unchanged in KOH (not dissolved). Contextual generative hyphae frequent, hyaline, thin- to slightly thick-walled, frequently simple septate and branched, 2–4 µm diam.; contextual skeletal hyphae dominant, hyaline, thick-walled with a wide lumen, rarely branched, flexuous, interwoven, 2.5–6 µm diam. Tramal generative hyphae frequent, hyaline, thin- to slightly thick-walled, occasionally simple septate, frequently branched, 2–3.5 µm diam; tramal skeletal hyphae dominant, hyaline, thick-walled with a wide lumen, rarely branched, flexuous, strongly interwoven without orientation, 2–5 µm diam. *Cystidia* present, thin-walled, clavate, moniliform or ventricose, 27–40 × 4–8 mm. *Cystidioles* present, thin-walled, fusiform, mostly with an apical simple septum, 22–25 × 4–8 µm. *Basidia* clavate to uniform, with a
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** | Microscopic structures of *Heterobasidion armandii* (drawn from the holotype). (A) Basidiospores; (B) Basidia and basidioles; (C) Cystidioles; (D) Hyphae from trama; (E) Hyphae from context.</figcaption></figure>
simple basal septum and four sterigmata, 18–28 × 4–6 µm. *Basidioles* in shape similar to basidia, but distinctly shorter. *Basidiospores* subglobose to broadly ellipsoid, hyaline, fairly thick-walled, asperulate, mostly bearing a small guttule, IKI–, CB+, (4.5–)5–5.7(–6) × (3.6–)3.8–5(–5.5) µm, *L* = 5.17 µm, *W* = 4.22 µm, *Q* = 1.22 (*n* = 30/1).

**Additional material (paratype) examined**

China, Yunnan Province, Tengchong County, on stump of *Pinus* sp., August 4, 2014, Li 140804-30 (BJFC018422).

**Heterobasidion subparviporum** Y. C. Dai, Jia J. Chen and Yuan Yuan, sp. nov. **Figures 6, 7**

MycoBank MB 834574.

**Type**

China, Hebei Province, Xinglong County, Wulingshan Nature Reserve, on fallen trunk of *Larix* sp., July 30, 2009, BK Cui 6961 (BJFC005448, holotype).

**Diagnosis**

Differs from *Heterobasidion* species by mostly round pores (3–5 per mm), amyloid contextual skeletal hyphae, absence of cystidia, presence of cystidioles, and subglobose to broadly ellipsoid basidiospores measuring 5–6.5 × 4–5.2 µm.

**Etymology**

*Subparviporum* (Lat.): referring to the similarity to *H. parviporum*.

**Description**

*Basidiocarps* perennial, pileate, usually imbricate, leathery and without odor or taste when fresh, hard corky when dry. *Pilei* semicircular to fan-shaped, projecting up to 6 cm, 9 cm wide, and 2.2 cm thick at base. *Pileal surface* buff to grayish brown or grayish dark, at least dark brown at base, crustose, distinctly zonate; margin cream to buff, dull, up to 2 mm. *Pore surface* white when fresh, cream to buff when dry, glancing; pores mostly round, occasionally irregular, 3–5 per mm; dissepiments thin, entire. *Context* buff to brown, corky when dry, azonate, up to 2 mm thick, with a thin black line under crust except for the margin. *Tubes* cream, hard corky, up to 20 mm long. *Hyphal system* dimitic; generative hyphae mostly simple septate; tramal skeletal hyphae dextrinoid, CB+; contextual skeletal hyphae IKI+, CB+, hyphae unchanged in KOH (not dissolved). Contextual generative hyphae infrequent, hyaline, thin-walled to slightly thick-walled, frequently simple septate and branched, 2–4 µm diam; contextual skeletal hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, rarely branched, flexuous, interwoven, 2–4.5 µm diam. Tramal generative hyphae frequent, hyaline, thin-walled to slight thick-walled, frequently simple septate and branched, 1.7–3 µm diam; tramal skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen, rarely branched, flexuous, strongly interwoven without orientation 1.5–3.5 µm diam. *Cystidia* absent. *Cystidioles* present, thin-walled, subulate, and ventricose, 13–26 × 4–6 µm, sometimes with a septum at the top. *Basidia* clavate to barrel-shaped, with a simple basal septum and four sterigmata, 18–24 × 4.5–8 µm. *Basidioles* in shape similar to

basidia, but slightly smaller. *Basidiospores* subglobose to broadly ellipsoid, hyaline, fairly thick-walled, asperulate, IKI–, CB+, 5–6.5(–7) × (3.8–)4–5.2 µm, *L* = 5.65 µm, *W* = 4.35 µm, *Q* = 1.30–1.32 (*n* = 60/2).

**Additional materials (paratypes) examined**

China, Jilin Province, Antu County, Changbaishan Nature Reserve, on fallen trunk of *Picea* sp., September 13, 2014, Dai 14803 (BJFC017915); on living tree of *Abies* sp., September 21, 2019, Dai 20873 (BJFC032542). Xizang Autonomous Region (Tibet), Linzhi County, Lulang, on stump of *Picea* sp., September 16, 2010, Cui 9267 (BJFC008206).

**Other materials examined**

—*Heterobasidion abietinum*. Italy, on *Abies* sp., April 28, 2005, Dai 6557 (BJFC000943).

—*Heterobasidion amyloideum*. China, Xizang Auto. Reg. (Tibet), Linzhi County, Lulang, Sejila Mt., on fallen trunk of *Abies* sp., September 23, 2014, Cui 12274 (BJFC017155); Motuo County, on dead tree of *Abies* sp., September 21, 2014, Cui 12240 (BJFC017154); Milin County, Naligou, on fallen gymnosperm trunk, August 18, 2012, Li 1675 (isotype BJFC16026).

—*Heterobasidion annosum*. Belgium, on *Betula* sp., December 3, 2005, Dai 7445 (BJFC000949).

<figure><img src="image_6.png" /><figcaption>**FIGURE 4** | *Basidiocarps of Heterobasidion subinsulare* (holotype, Dai 13842). Bars (A) = 5 cm, (B) = 1 cm.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**FIGURE 5** | Microscopic structures of *Heterobasidion subinsulare* (drawn from the holotype). (A) Basidiospores; (B) Basidia and basidioles; (C) Cystidioles; (D) Cystidia; (E) Hyphae from trama; (F) Hyphae from context.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**FIGURE 6** | Basidiocarps of *Heterobasidion subparviporum* (paratype, Dai 14803). Bar = 4 cm.</figcaption></figure>

—*Heterobasidion araucariae*. New Zealand, March 20, 1985, PDD 49003 (PDD).

—*Heterobasidion australe*. China, Zhejiang Province, Jinan County, Tianmushan Nat. Res., stump of *Pinus* sp., October 15, 2004, Dai 6330 (paratype BJFC000979); Yunnan Province, Nanhua County, Dazhongshan Nature Reserve, on fallen trunk of *Pinus* sp., September 11, 2015, Cui 12602 (BJFC028381); Huaning County, September 6, 2013, Dai 13507 (BJFC014968); Kunming, Wild duck Lake, July 28, 2014, Dai 13863 (BJFC017593).

—*Heterobasidion insulare*. Philippines, Luzon, Mountain Province, on *Pinus insularis*, 1962, FPRI 429 (CFMR). China, Chongqing, Geleshan Forest Park, stump of *Pinus* sp., July 23, 2014, Dai 13933 (BJFC017663); Jiangxi Province, Anyuan County, Sanbaishan Forest Park, root of *Pinus* sp., December 19, 2014, Dai 15095 (BJFC018207).

—*Heterobasidion irregulare*. United States, on *Pinus* sp., 2004, JV0405/3-J (JV).

—*Heterobasidion linzhiense*. China, Xizang Auto. Reg. (Tibet), Linzhi County, Lulang, fallen trunk of *Picea* sp., September 24, 2010, Cui 9645 (holotype, BJFC008582).

—*Heterobasidion occidentale*. United States, on *Abies* sp., August 2001, JV0108/87 (JV).

—*Heterobasidion orientale*. China, Heilongjiang Province, Yichun, Liangshui Nature Reserve, on fallen gymnosperm trunk, August 26, 2014, Cui 11637 (BJFC016831); on fallen trunk of *Pinus* sp., August 28, 2014, Cui 11815 (BJFC016890); on dead tree of *Picea*, August 31, 2014, Cui 12026 (BJFC016964).

—*Heterobasidion parviporum*. Estonia, on *Picea* sp., May 13, 1995, Dai 1930 (BJFC001016).

—*Heterobasidion* sp. United States, California, Lassen National Forest, on *Pinus ponderosa*, March 2005, Korhonen 05030, Korhonen 05038 and Korhonen 05039 (LUKE).

—*Heterobasidion tibeticum*. China, Xizang Auto. Reg. (Tibet), Bomi County, Tongmai, on fallen trunk of *Pinus* sp., September 22, 2014, Cui 12257 (BJFC017171); Linzhi County, September 24, 2014, Cui 12335 (BJFC017249); July 31, 2004, Dai 5468 (paratype, BJFC000958).

DISCUSSION

The current phylogeny considers that *Heterobasidion* species belong to three species complexes: the *H. annosum* F complex (previously treated as the *H. annosum* S group, Woodward et al., 1998), the *H. annosum* P complex and the *H. insulare* complex. The F complex of *H. annosum* includes four species which are mainly associated to true fir species (*Abies* Mill., *Picea abies* (L.) Karst. and *Tsuga* (Endl.) Carrière; Linzer et al., 2008; Dalman et al., 2010). *H. subparviporum* is mostly found on *Picea* in Asia, while *H. parviporum* is mostly associated to *Picea* in Europe and *H. abietinum* to *Abies* in Europe. *H. occidentale* is colonizing mostly *Tsuga* and *Abies* in western North America. The *H. annosum* P complex includes two taxa which mostly grow on pines: *H. annosum* s.s. in Eurasia, *H. irregulare* in North America (Linzer et al., 2008; Dalman et al., 2010). The *H. insulare* complex includes ten species which are associated to many species of Pinophyta (*Abies*, *Araucaria* Juss., *Keteleeria* Carr., *Larix* Mill., *Picea*, *Pinus* L., *Pseudolarix* Gordon, *Pseudotsuga* Carrière and *Tsuga*). *H. araucariae*, a species from Southern Hemisphere, is clustered into *H. insulare* complex, and is closely related to the species *H. insulare* and *H. subinsulare* (Figure 1).

*Heterobasidion insulare* (=*Trametes insularis* Murrill) was originally described from Philippines (Murrill, 1908) and its type specimen was collected from fallen log of *P. insularis* in the Benguet Province, Luzon, Philippines in 1905. In 1962, Mendoza obtained the isolate FPRI-429 from *P. insularis* in the Mountain Province, Luzon. The Benguet Province and Mountain Province are both located in the Cordillera Administrative Region of Luzon Island (Figure 8). FPRI-429 can thus be considered as the type locality of *H. insulare*. The present results confirmed that FPRI-429 and representatives of *H. ecrustosum* are nested in the same lineage; the latter taxon was described from central Japan to Okinawa, and from southern China (Tokuda et al., 2009). We did not find any distinct morphological difference between the *H. insulare* type and samples of *H. ecrustosum*. Hence, according to the current phylogeny and morphological studies, *H. ecrustosum* is treated as a synonym of *H. insulare*.

*Heterobasidion armandii* is closely related to *H. australe* (Figure 1) and the geographical distributions of the two species are overlapped in China. However, *H. australe* is characterized by a glancing pore surface, lacks cystidioles, and its contextual skeletal hyphae are negative in Melzer's reagent. Morphologically, *H. armandii* resembles *H. amyloideum* and *H. tibeticum* by similar pores (4–6 per mm in *H. amyloideum* and 3–6 per mm in *H. tibeticum*), basidiospores (4.9–5.8 × 3.9–4.5 µm in *H. amyloideum* and 4.5–6 × 3.6–5.3 µm in *H. tibeticum*) and amyloid contextual skeletal hyphae, but *H. amyloideum* and *H. tibeticum* differ from *H. armandii* by the presence of cystidia (Dai and Korhonen, 2009; Chen et al., 2014).

*Heterobasidion subinsulare* is closely related to *H. insulare* (Figure 1), but the latter lacks cystidia. *H. subinsulare* resembles *H. amyloideum* and *H. tibeticum* by having cystidia, but the latter two species have smaller pores (3–6 per mm) and amyloid contextual skeletal hyphae (Chen et al., 2014). *H. subinsulare*, *H. araucariae*, and *H. orientale* share similar pores, but *H. araucariae* can be distinguished from *H. subinsulare* by longer
<figure><img src="image_9.png" /><figcaption>**FIGURE 7** | Microscopic structures of *Heterobasidion subparviporum* (drawn from the holotype). (A) Basidiospores; (B) Basidia and basidioles; (C) Cystidioles; (D) Hyphae from trama; (E) Hyphae from context.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**FIGURE 8** | Type locality of *Heterobasidion insulare* in Philippines.</figcaption></figure>

basidiospores (5.8–6.5 µm vs. 5–5.7 µm) and lacking of cystidia, and *H. orientale* differs from *H. subinsulare* by the sharp pileal margin, dark reddish pileal surface and lacking of cystidia. In addition, *H. subinsulare* is distantly related to *H. amyloideum*, *H. tibeticum*, *H. araucariae*, and *H. orientale* in our current phylogeny (**Figure 1**).

*Heterobasidion subparviporum* is closely related to *H. parviporum* (**Figure 1**), and the latter was considered as same as the former according to the mating tests (Dai and Korhonen, 1999, 2003; Dai et al., 2006; Dai, 2012). Although both taxa are compatible in laboratory, they form two distinct lineages in our phylogeny (**Figure 1**). Morphologically, *H. subparviporum* differs from *H. parviporum* by longer cystidia (18–24 µm vs. 13–17 µm) and bigger basidiospores (5–6.5 × 4–5.2 µm vs. 4.2–5 × 3.8–4.2 µm). In addition, *H. parviporum* is a pathogen on *P. abies* in Europe, while *H. subparviporum* seems to be a saprophytic species according to our investigations. Based on the above data, we suggest that this Asian taxon is a new species *H. subparviporum*. The situation is similar with the European

taxa *H. parviporum* and *H. abietinum*. These two taxa are partly sexually compatible (Capretti et al., 1990; Stenlid and Karlsson, 1991; Woodward et al., 1998), but they do not produce hybrids in nature. So they have been accepted at the species level (Niemelä and Korhonen, 1998; Otrosina and Garbelotto, 2010; Ryvarden and Melo, 2017).

*Heterobasidion irregulare* was proposed by Otrosina and Garbelotto (2010), and it was originally described as *Polyporus irregularis* Underwood on pine log from Auburn, Alabama, eastern United States (Underwood, 1897), although *P. irregularis* is an illegitimate name because there was earlier a fungus named *P. irregularis* Pers. (Persoon, 1825). The lectotype (NY730756) of *H. irregulare* was selected from the type material of *P. irregularis* Underwood, and the epitype (UC1935442) was selected from stump of *P. ponderosa* in the Modoc National Forest, California, western United States. However, three isolates Korhonen 05030, Korhonen 05038, and Korhonen 05039 associated to *P. ponderosa* from Lassen National Forest in California formed another lineage which is closely related to *H. irregulare* (**Figure 1**). Hence it is possible that another taxon exists in western North America. We did not have the basidiocarps of isolates Korhonen 05030, Korhonen 05038, and Korhonen 05039, and no information on their ecology. For the time being we treat this possible taxon as *H. sp*.

*Heterobasidion amyloideopsis* was described from Pakistan mostly based on phylogenetic analysis (Zhao et al., 2017). We studied its ITS (KT598384, KT598385), nrLSU (KT598386, KT598387), RPB1 (KT598390, KT598391), and RPB2 (KT598388, KT598389) sequences and found some of the sequences are uncorrect, and some of these sequences were deleted by NCBI. So the status of *H. amyloideopsis* is ambiguous.

A comparison of these three new species and their morphological and/or phylogenetically related species is also provided in **Supplementary Appendix 1**. The phylogenetic analyses on single loci (ITS, nrLSU, RPB1, RPB2, and GAPDH) were shown in **Supplementary Figures 1–5**.

## CONCLUSION

To date, 15 species are recorded in the genus *Heterobasidion*, including three new species described in the present study.

Five species, *H. abietinum*, *H. annosum* s.s., *H. irregulare*, *H. occidentale*, and *H. parviporum*, distributed in Europe and North America are forest pathogens. Ten Asian taxa are all saprotrophs, and the Asian countries ought to consider these five European and North American species as entry plant quarantine fungi. Parallelly, European countries should consider the American *H. occidentale* and *H. irregulare* as entry plant quarantine fungi (although the latter species is already in Italy), while North America should treat *H. abietinum*, *H. annosum* s.s. and *H. parviporum* as entry plant quarantine fungi. Eight *Heterobasidion* species found in the Himalayas suggest that the ancestral *Heterobasidion* species may have occurred in Asia, as was proposed also in the previous divergence and biogeographic studies on the genus.
**DATA AVAILABILITY STATEMENT**

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ **Supplementary Material**.

**AUTHOR CONTRIBUTIONS**

YY, J-JC, and Y-CD designed the research and contributed to data analysis and interpretation. YY and J-JC performed the research. Y-CD and KK collected the materials. All authors wrote and revised the manuscript, contributed to the article, and approved the submitted version.

**FUNDING**

The research is supported by the Second Tibetan Plateau Scientific Expedition and Research Program (STEP, No. 2019QZKK0503).

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**ACKNOWLEDGMENTS**

We thank Prof. Bao-Kai Cui (China) for allowing us to study his collections. We express our gratitude to Dr. Daniel L. Lindner (CFMR, United States) for loan of stock, to Dr. De-Wei Li (United States) for providing some literatures and improving this manuscript, and to Chang-Lin Zhao (China) for providing sequences.

**SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.596393/full#supplementary-material

**Supplementary Figure 1** | Phylogeny of ITS.

**Supplementary Figure 2** | Phylogeny of nrLSU.

**Supplementary Figure 3** | Phylogeny of RPB1.

**Supplementary Figure 4** | Phylogeny of RPB2.

**Supplementary Figure 5** | Phylogeny of GAPDH.

**Supplementary Appendix 1** | A comparison of taxa in the *Heterobasidion*.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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