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Research Article
Outline, phylogenetic and divergence times analyses of the genus Haploporus (Polyporales, Basidiomycota): two new species are proposed
expand article infoHeng Zhao‡, Josef Vlasák§, Yuan Yuan‡
‡ Beijing Forestry University, Beijing, China
§ Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic

Corresponding author: Yuan Yuan ( yuanyuan1018@bjfu.edu.cn )

Academic editor: Zai-Wei Ge
© 2023 Heng Zhao, Josef Vlasák, Yuan Yuan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhao H, Vlasák J, Yuan Y (2023) Outline, phylogenetic and divergence times analyses of the genus Haploporus (Polyporales, Basidiomycota): two new species are proposed. MycoKeys 98: 233-252. https://doi.org/10.3897/mycokeys.98.105684
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Abstract

Haploporus species have a worldwide distribution and 27 species have been accepted. In this study, two new species, Haploporus crystallinus and H. dextrinoideus, are proposed from South America, based on the molecular fragments (ITS, LSU and mtSSU) and morphological evidence. Molecular clock analysis was performed and the result suggests that the ancestor of Polyporales originated between the Late Jurassic and Early Cretaceous period, with a mean stem of 159.8 Mya [95% higher posterior density (HPD) of 142.4–184.1 Mya] and the genus Haploporus occurred at a mean stem of 108.3 Mya (95% HPD of 88.5–128.2 Mya). In addition, most species of the genus are diversified between 60.5 Mya and 1.8 Mya, during the Paleogene to Neogene. A key to the accepted species of the genus Haploporus is provided.

Key words

fungal diversity, new taxa, molecular clock dating, wood-inhabiting fungi

Introduction

Currently, more than 155,755 species of fungi have been described worldwide, based on molecular analyses and morphological features and the numbers of fungal species have been rapidly increasing in the last two decades (https://www.speciesfungorum.org/Names/Names.asp, accessed 21 June 2023). For example, Kirk et al. (2008) recorded 487 species of the family Hymenochaetaceae in the 10th version of Dictionary of the Fungi, but 672 poroid species in 34 genera were recognised in 2022 (Wu et al. 2022a). Still, the fungal diversity is poorly known as only 4.10–7.08% fungal species have been described of an estimated 2,200,000–3,800,000 (Hawksworth and Lücking 2017; Wang et al. 2019).

The genus Haploporus Bondartsev & Singer, proposed by A. S. Bondartsev and R. Singer and typified by Haploporus odorus (Sommerf.) Bondartsev & Singer (Singer 1944), belongs to Polyporaceae, Polyporales, Agaricomycetes in Basidiomycota. The genus is characterised by: 1) basidiomata annual to perennial and resupinate to pileate; 2) hyphal system monomitic, dimitic to trimitic with clamped generative hyphae; 3) cyanophilous skeletal hyphae; 4) cyanophilous and ornamented basidiospores; and 5) causing a white rot (Singer 1944; Dai et al. 2002; Piątek 2003, 2005; Li et al. 2007; Shen et al. 2016; Zhou et al. 2019, 2021; Decock et al. 2021; Man et al. 2023).

A phylogenetic study suggested that the genus Haploporus was sister to the genus Perenniporia, based on the ITS + LSU + mtSSU + rpb2 + TEF1 sequences and 13 species have been accepted (Shen et al. 2016). Later, ITS + LSU and ITS + LSU + mtSSU sequences were used to reconstruct the phylogenetic relationships of the genus Haploporus, respectively (Zhou et al. 2019, 2021; Decock et al. 2021; Man et al. 2023), the results showing that they were compatible.

Until now, 27 species are accepted in the genus Haploporus, widely distributed in Africa, Asia, Europe, North America, Oceania and South America (Wu et al. 2022b; Man et al. 2023). In Africa, four species, H. eichelbaumii, H. grandisporus, H. nanosporus and H. papyraceus, are described (Decock et al. 2021). In Asia, 16 species are reported, accounting for 59.3% (16/27) of total species of the genus and are mainly found in China and Sri Lanka (Dai et al. 2004, 2007, 2021; Li et al. 2007; Dai 2012a, b; Shen et al. 2016; Zhou et al. 2019, 2021; Man et al. 2023). In Europe, three species are accepted, viz. H. odorus, H. subtrameteus and H. tuberculosus (Ryvarden and Gilbertson 1993; Zhou et al. 2019). In North America, four species are reported, including H. alabamae, H. gilbertsonii, H. odorus and H. papyraceus (Zhou et al. 2019). In Oceania, only one species, H. pirongia, is found from Australia and New Zealand (Zhou et al. 2019). In South America, four species are recorded from Brazil and Ecuador (Lira et al. 2018; Zhou et al. 2021; Man et al. 2023). Most species of the genus Haploporus grow on angiosperms, including branches, twigs, fallen trunks, stump, even living trees, while H. latisporus is the sole species growing on gymnosperm wood (Li et al. 2007; Zhou et al. 2019, 2021).

Molecular clock analyses provided new insights into the origin and evolution of fungi, such as reconstructing the divergence time of Basidiomycota and early-diverging fungi, based on multiple gene loci (Cao et al. 2012; Wu et al. 2014; Chen et al. 2015; Cui et al. 2018; He et al. 2019; Varga et al. 2019; Zhao et al. 2022a). The previous study suggested that divergence times of the order Polyporales, the genus Polyporus and its allied genera, originated about 141.81 Mya and 49–63 Mya years ago, based on six DNA fragments (Ji et al. 2022). The divergence times of Haploporus species have not yet been studied.

During the trips in South America, we collected some specimens of the genus Haploporus and carried out detailed studies combining morphology, phylogeny and molecular clock dating. Thus, two new species, Haploporus crystallinus and H. dextrinoideus, are described in this study.

Materials and methods

Morphological studies

In the present study, the newly-studied specimens of the genus Haploporus were collected from South America, deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC, China), the National Museum Prague of Czech Republic (PRM, Czech Republic) and the private herbarium of Josef Vlasák (JV, Czech Republic). The methods of morphological description followed the previous study (Dai 2010; Chen et al. 2016). 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 = x/y = number of spores (x) measured from a given number (y) of specimens (Yuan et al. 2017). The colour terms used by Anonymous and Petersen are followed (Anonymous 1969; Petersen 1996).

DNA extraction, PCR amplification and sequencing

Total genomic DNAs were extracted from dried specimens using a kit (Aidlab Biotechnologies, Beijing, China), following the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to amplify the partial fragments of the internal transcribed spacer (ITS), large subunit ribosomal RNA (LSU) and small subunit mitochondrial rRNA gene (mtSSU) with the fungal-specific primers, according to the previous studies (Zhou et al. 2019, 2021; Man et al. 2023). The amplification of the ITS, LSU and mtSSU partial fragments were carried out under the following conditions: for ITS and mtSSU partial fragments, an initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min; for LSU partial fragments, an initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min and a final extension of 72 °C for 10 min (Zhou et al. 2019, 2021; Man et al. 2023). Sequencing was performed by BGI Tech Solutions (Beijing Liuhe Co., Ltd., Beijing, China) using the ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). All the newly-generated sequences are deposited in GenBank database with the accession numbers listed in Table 1.

Table 1.
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Taxa information and GenBank accession numbers used in this study.

Species Sample no. GenBank Accession no. Country References
ITS LSU mt-SSU
Haploporus alabamae Dollinger 895 KY264038 MK433606 MW463004 USA Zhou et al. (2019)
H. alabamae JV 1704/75 MK429754 MK433607 MW463005 Costa Rica Zhou et al. (2019)
H. angustisporus Dai 10951 KX900634 KX900681 MW463006 China Zhou et al. (2019)
H. bicolor Dai 19951 MW465684 MW462995 – China Zhou et al. (2021)
H. crassus Dai 13580 MW465669 KU941865 – China Zhou et al. (2019)
H. crystallinus JV 2208/36 OQ919235 OQ919238 OQ919241 French Guiana This study
FG-14-870 MT782653 MT777443 – French Guiana Decock et al. (2021)
H. cylindrosporus Dai 15664 KU941854 KU941878 KU941903 China Shen et al. (2016)
H. dextrinoideus JV 2211/1-J OQ919237 OQ919240 OQ919242 Ecuador This study
JV 2106/45-J OQ919236 OQ919239 – Ecuador This study
H. ecuadorensis JV1906/C10-J MW465661 OP948227 OP948226 Ecuador Man et al. (2023)
H. eichelbaumii Congo 1 MT758256 MT758256 – Congo Decock et al. (2021)
KE-17-238 MT758261 MT758261 – Kenya Decock et al. (2021)
H. gilbertsonii JV 1611/5-J MK429756 MK433609 MW463007 USA Zhou et al. (2019)
H. grandisporus KE-16-130 MT758242 MT758242 – Kenya Decock et al. (2021)
KE-17-228 MT758244 MT758244 – Kenya Decock et al. (2021)
H. latisporus Dai 11873 KU941847 KU941871 MW463008 China Shen et al. (2016)
H. longisporus JV 1906/C11-J MW465685 MW462996 – Ecuador Zhou et al. (2021)
H. microsporus Dai 12147 KU941861 KU941885 – China Shen et al. (2016)
H. monomitica Dai 24229 OP725709 OP725712 – China Man et al. (2023)
Dai 24246 OP725710 OP725713 OP725715 China Man et al. (2023)
Dai 24251 OP725711 OP725714 OP725716 China Man et al. (2023)
H. nanosporus MUCL 47447 MT782648 MT777438 – Gabon Shen et al. (2016)
MUCL 47559 MT782650 MT777440 – Gabon Shen et al. (2016)
H. nepalensis Dai 12937 KU941855 KU941879 KU941904 China Shen et al. (2016)
H. odorus Dai 11296 KU941845 KU941869 KU941894 China Shen et al. (2016)
Yuan 2365 KU941846 KU941870 KU941895 China Shen et al. (2016)
H. papyraceus Dai 10778 KU941839 KU941863 KU941888 China Shen et al. (2016)
H. pirongia Dai 18659 MH631017 MH631021 MW463009 Australia Zhou et al. (2019)
H. punctatus Dai19628 MW465687 MW462998 MW463011 Sri Lanka Zhou et al. (2021)
H. septatus Cui 4100 KU941844 KU941868 KU941893 China Shen et al. (2016)
H. srilankensis Dai19523 MW465688 MW462999 MW463012 Sri Lanka Zhou et al. (2021)
H. subpapyraceus Cui 2651 KU941842 KU941866 KU941891 China Shen et al. (2016)
Dai 9324 KU941841 KU941865 KU941890 China Shen et al. (2016)
H. subtrameteus KUC20121102-36 KJ668536 KJ668389 – Korea Shen et al. (2016)
Haploporus sp. 1 LR11231 MT758249 MT758249 – Malawi Decock et al. (2021)
H. thindii Cui 9373 KU941851 KU941875 KU941900 China Shen et al. (2016)
Cui 9682 KU941852 KU941876 KU941901 China Shen et al. (2016)
H. tuberculosus 15559 KU941857 KU941881 KU941906 Sweden Shen et al. (2016)
Perenniporia citrinoalba Dai 13643 NR_171808 NG_075212 KX880705 China Cui et al. (2019)
P. hainaniana Cui 6364 JQ861743 JQ861759 KF051044 China Zhao and Cui (2013)

Note: The sequences generated in this study are in bold. “−” represents sequences unavailable.

Phylogenetic analyses

The ITS, LSU and mtSSU partial sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) and concatenated after excluding the poorly-aligned sites as dataset 1. The aligned dataset 1 was analysed using Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI) phylogenetic analyses with RAxML v.8 (Stamatakis 2014), PAUP v.4.0b10 (Swofford 2002) and MrBayes v.3.2.7a (Ronquist et al. 2012), respectively, followed the previous studies (Zhao et al. 2021; Man et al. 2023). Additionally, the phylogenetic analyses of the aligned ITS, LSU and mtSSU partial sequences were also undertaken using RAxML v.8 (Suppl. materials 1: figs 1–3; Stamatakis 2014), respectively. The optimum model of the aligned dataset 1 was tested using the ModelTest-NG v.0.1.7 (Darriba et al. 2020). For ML and MP analyses, 1,000 bootstrap replications were carried out. For BI analysis, two million generations were conducted with random initial trees and the first 25% were set as burn-in.

Divergence time analysis

Dataset 2, consisting of 58 ITS and LSU partial sequences, was used to infer the divergence times of the species in the genus Haploporus (Table 1, Suppl. material 1: table S1 and file S1). The divergence time was estimated with BEAST v.2.6.5 (Bouckaert et al. 2014), following previous studies (Liu et al. 2022; Zhao et al. 2022b). Four time points were selected for calibration and the offset age with a gamma distributed prior (scale = 20, shape = 1): 1) Archaeomarasmius leggettii Hibbett et al. (1995, 1997) represented the divergence time of Agaricales at 90 Mya; 2) Quatsinoporites cranhamii S.Y. Sm. et al. (Smith et al. 2004; Berbee and Taylor 2010) represented the Hymenochaetaceae at 125 Mya; 3) Paleopyrenomycites devonicus Taylor et al. (Taylor et al. 1999, 2005) represented between Ascomycota and Basidiomycota at 400 Mya; and 4) the estimated mean crown age of Polyporales at 123.74 Mya (Ji et al. 2022). A total of 10,000,000 generations were set, first 20% being burn-in. The results of the log file and trees file were assessed using Tracer v.1.5 and TreeAnnotator v.2.6.5, respectively.

Results

Phylogeny

In this study, dataset 1, including a total of 41 sequences, was used to reconstruct the phylogenetic relationships of the genus Haploporus (Table 1, Fig. 1 and Suppl. material 1: file S1). The aligned dataset 1 has a length of 2,797 characters (ITS, 1–783 characters; LSU, 784–2063 characters; and mtSSU, 2064–2797 characters), of which 1,890 are constant characters, 266 are parsimony-uninformative characters and 641 are parsimony-informative characters. The Maximum Parsimony (MP) analysis yielded a tree of length 779, consistency index 0.4856, homoplasy index 0.5144, retention index 0.7658 and rescaled consistency index of 0.3719. The best model for the aligned dataset 1 was GTR + I + G in the Bayesian analysis and the average standard deviation of split frequencies was 0.004282. The phylograms of three analyses, ML, BI and MP, are similar in topology and the ML tree was selected to represent the phylogenetic relationships (Fig. 1).

Figure 1. 

A Maximum Likelihood phylogenetic tree of Haploporus based on ITS, LSU and mtSSU gene fragments, with two specimens Perenniporia citrinoalba and P. hainaniana used as outgroups. The new species Haploporus crystallinus and H. dextrinoideus are shaded. Maximum Likelihood bootstrap values (≥ 50%)/Maximum Parsimony bootstrap values (≥ 50%)/Bayesian Posterior Probabilities (≥ 0.90) of each clade are indicated along branches. The scale bar left upper indicates the number of substitutions per site.

The phylogenetic analyses indicated that H. crystallinus is clustered with H. nanosporus and H. microsporus and H. dextrinoideus formed an independent lineage in the Haploporus clade, closely related with H. grandisporus, H. ecuadorensis, H. eichelbaumii and H. longisporus (Fig. 1). BLAST results of ITS sequences with the top hit taxa were followed by phylogenetics analyses (Suppl. material 1: table S2).

Divergence times of the Haploporus

In the present study, the molecular clock analysis suggested that the order Polyporales and genus Haploporus emerged at a mean stem of 159.8 Mya [95% higher posterior density (HPD) of 142.4–184.1 Mya] and 108.3 Mya (95% HPD of 88.5–128.2 Mya), respectively (Fig. 2). A total of 27 species of the genus Haploporus was estimated as molecular sequences in H. brasiliensis and H. pileatus were not available (Lira et al. 2018). The results suggested that species of the genus Haploporus may be diversified between 60.5 Mya and 1.8 Mya, during the Paleogene to Neogene in Cenozoic (Fig. 2 and Table 2). Amongst those species, 18 species occurred in the Neogene, accounting for 67% and only nine species originated in the Paleogene.

Table 2.
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Divergence times of estimated taxa in the genus Haploporus.

Species Mean of stem in MCC tree (Mya) 95% HPD
Haploporus alabamae 29.6 14.1–51.2
H. angustisporus 2.9 1.1–5.9
H. bicolor 35.7 21.1–53.0
H. crassus 1.8 0.3–4.5
H. crystallinus 6.7 2.3–14.1
H. cylindrosporus 4.7 1.9–8.8
H. dextrinoideus 10.3 5.4–16.8
H. ecuadorensis 5.9 2.7–10.2
H. eichelbaumii 2.8 0.9–5.6
H. gilbertsonii 34.5 23.0–48.3
H. grandisporus 2.8 0.9–5.6
H. latisporus 59.8 52.7–86.0
H. longisporus 18.5 10.9–28.2
H. microsporus 60.5 39.1–84.1
H. monomitica 48.2 23.0–67.8
H. nanosporus 6.7 2.3–14.1
H. nepalensis 7.7 3.6–13.4
H. odorus 48.2 23.0–67.8
H. papyraceus 13.2 6.0–23.8
H. pirongia 35.2 22.1–50.5
H. punctatus 2.9 1.1–5.9
H. septatus 23.0 12.7–37.5
H. srilankensis 4.5 1.9–8.5
H. subpapyraceus 1.8 0.3–4.5
H. subtrameteus 54.5 44.4–77.1
H. thindii 4.7 1.9–8.8
H. tuberculosus 20.0 10.4–32.4

MCC represented as maximum clade credibility.
Figure 2. 

Estimated divergence of Haploporus generated by molecular clock analyses using the aligned dataset 2 of ITS and LSU sequences. Estimated mean divergence time (Mya) and Posterior Probabilities (PP) > 0.8 are annotated at the internodes. The 95% Highest Posterior Density (HPD) interval of divergence time estimates are marked by horizontal blue bars. The geological timescale is indicated at the bottom.

Taxonomy

Haploporus crystallinus H. Zhao, Vlasák & Yuan Yuan, sp. nov.

MycoBank No: MycoBank No: 849261
Figs 3, 4

Etymology

crystallinus (Lat.): Refers to the species having many crystals amongst the subiculum and tube trama.

Type

French Guiana, Roura, Camp Cayman, rotten log on the road, 27 August 2022, JV 2208/36 (Holotype PRM, isotypes BJFC 039927 and JV 2208/36). GenBank: ITS = OQ919235, LSU = OQ919238, mtSSU = OQ919241.

Basidiomata resupinate, perennial, inseparable from the substrate, more or less corky when dry, up to 10 cm long, 3.5 cm wide and 4 mm thick at centre. Hymenophore pinkish-buff (5A3) to cream buff (4A4) when dry, with indistinct margin; pores angular to round, 5–7 per mm; dissepiments thick, entire. Subiculum darker than tubes, more or less corky. Tubes pinkish-buff (5A3), hard corky.

Figure 3. 

Basidiomata of Haploporus crystallinus (Holotype, JV 2208/36). Scale bar: 1 cm.

Hyphal system dimitic; generative hyphae with clamp connections, hyaline, thin-walled; skeletal hyphae thick-walled, frequently branched, distinctly dextrinoid in Melzer’s reagent, cyanophilous in Cotton Blue; tissues unchanged in 2% potassium hydroxide.

Subicular generative hyphae infrequent, hyaline, thin-walled, sometimes branched, 1.0–1.5 µm in diam.; skeletal hyphae dominant, with a narrow lumen to subsolid, usually branched, flexuous, interwoven, 0.8–2.0 µm in diam. Irregular-shaped and -sized crystals frequently present.

Figure 4. 

Microscopic characteristics of Haploporus crystallinus (Holotype, JV 2208/36) a basidiospores b basidia c basidioles d cystidioles e hyphae from tube trama f hyphae from subiculum. Scale bars: 5 μm (a); 10 μm (b–f).

Tube tramal generative hyphae infrequent, hyaline, thin-walled, sometime branched, 1.0–1.5 µm in diam.; skeletal hyphae dominant, with a narrow lumen to subsolid, usually branched, flexuous, interwoven, 0.8–2.0 µm in diam. Cystidioles fusiform with a sharp tip, thin-walled, hyaline, 12.0–21.0 × 3.0–4.5 µm. Basidia more or less capitate to pyriform, with four sterigmata and a clamp connection at base, 17.5–27.0 × 6.5–9.0 µm; basidioles capitate to pyriform, almost the same size of basidia. Dendrohyphidia absent. Irregular-shaped and -sized crystals frequently present.

Basidiospores ellipsoid, slightly thick-walled, tuberculate, hyaline, some with a guttule, dextrinoid in Melzer’s reagent, cyanophilous in Cotton Blue, (3.8–)4.0–5.5 × (2.1–)2.6–3.5(–3.8) µm, L (arithmetic average length) = 4.60 µm, W (arithmetic average width) = 3.03 µm, Q (L/W ratio) = 1.52 (n = 30/1).

Distribution and ecology

Haploporus crystallinus is distributed in French Guiana and growing on rotten unidentified angiosperm log; causes a white rot.

Haploporus dextrinoideus H. Zhao, Vlasák & Yuan Yuan, sp. nov.

MycoBank No: MycoBank No: 849262
Figs 5, 6

Etymology

dextrinoideus (Lat.): Refers to the species having dextrinoid hyphae.

Type

Ecuador, Papallacta Termas, 3,300 m standing dead tree, 15 June 2021, Josef Vlasák Jr., JV 2106/45-J (Holotype PRM, isotypes BJFC 038566 and JV). GenBank: ITS = OQ919236, LSU = OQ919239.

Basidiomata resupinate, annual, inseparable from the substrate, more or less corky when dry, up to 5.0 cm long, 3.0 cm wide and 0.4 mm thick at centre. Hymenophore cream bubalinus (4A2/3) to pinkish-buff (5A3) when dry, margin indistinct; pores angular to round, 1–3 per mm; dissepiments thick, entire. Subiculum slightly darker than tubes, more or less corky, up to 0.2 mm thick. Tubes pinkish-buff (5A3), hard corky.

Figure 5. 

Basidiomata of Haploporus dextrinoideus (Holotype, JV 2206/45-J). Scale bar: 1 cm.

Hyphal system dimitic; generative hyphae with clamp connections; skeletal hyphae thick-walled, branched, dextrinoid in Melzer’s reagent, cyanophilous in Cotton Blue; tissues unchanged in 2% potassium hydroxide.

Subicular generative hyphae hyaline, thin-walled, sometimes branched, 1.0–3.0 µm in diam.; skeletal hyphae dominant, with a wide lumen, usually branched, flexuous, interwoven, 1.5–3.0 µm in diam.

Tube tramal generative hyphae hyaline, thin-walled, usually branched, 1.0–2.5 µm in diam.; skeletal hyphae dominant, with a wide lumen, usually branched, flexuous, distinctly interwoven, 1.0–3.0 µm in diam. Cystidioles fusiform with a sharp tip, thin-walled, hyaline, 19.0–35.0 × 4.5–6.5 µm. Basidia more or less capitate to pyriform, with four sterigmata and a clamp connection at base, sometimes with a few small guttules, 21.0–34.0 × 7.0–14.0 µm; basidioles capitate to pyriform, almost the same size of basidia. Dissepimental hyphae thick-walled with one or four simple septa. Dendrohyphidia present amongst hymenium, thin-walled, hyaline. Large and irregularly-shaped crystals sometimes present amongst trama.

Figure 6. 

Microscopic characteristics of Haploporus dextrinoideus (Paratype, JV 2211/1-J) a basidiospores b basidia c basidioles and cystidioles d dendrohyphidia e hyphae from tube trama f hyphae from subiculum g dissepiment hyphae. Scale bars: 10 μm (a–g).

Basidiospores oblong to ellipsoid, thick-walled, tuberculate, hyaline, some with a guttule, dextrinoid in Melzer’s reagent, cyanophilous in Cotton Blue, (12.0–)13.2–19.0 × (5.0–)6.0–9.0 µm, L = 15.43 µm, W = 7.78 µm, Q = 1.98–2.16 (n = 60/2).

Additional material studied

Ecuador, Papallacta Termas, 3,300 m, on unidentified angiosperm, November 2022, Josef Vlasák Jr., JV 2211/1-J.

Distribution and ecology

Haploporus dextrinoideus is known from Ecuador high mountains, 3,300 m and growing on dead unidentified angiosperm trees; causes a white rot.

Discussion

Phylogenetic analyses, based on a combined dataset 1 (ITS + LSU + mtSSU sequences), indicates that H. crystallinus forms a sister group to H. nanosporus and H. microsporus with strong support. H. nanosporus differs from H. crystallinus by rarely branched skeletal hyphae, wider generative hyphae (1.5–2.0 µm vs. 1.0–1.5 µm) and indextrinoid basidiospores (David and Rajchenberg 1992). Additionally, H. nanosporus is distributed in Africa (David and Rajchenberg 1992; Decock et al. 2021), while H. crystallinus is found in French Guiana, South America. Haploporus microsporus differs from H. crystallinus by smaller pores (7–9 vs. 5–7 per mm) and annual basidiomata (Zhou et al. 2019). Specimen Haploporus sp. FG-14-870 (GenBank numbers: MT782653 and MT777443), collected by Cony Decock on 7 April 2014 in French Guiana, shows ITS sequence similarity with H. crystallinus JV 2208/36 over 99%, forming together a clade in the phylogenetic tree. Therefore, Haploporus sp. FG-14-870 is another specimen of Haploporus crystallinus.

Phylogenetic analyses also suggest that H. dextrinoideus forms a clade with the H. eichelbaumii, H. grandisporus, H. ecuadorensis and H. longisporus (Fig. 1). Haploporus eichelbaumii differs from H. dextrinoideus by smaller and indextrinoid basidiospores [11.0–14.0 × 5.3–6.5 µm vs. 13.2–19.0 × 6.0–9.0 µm; Decock et al. (2021)]. Haploporus grandisporus differs from H. dextrinoideus by narrower skeletal hyphae (1.3–1.8 µm vs. 1.5–3.0 µm), smaller basidia and basidioles (18.0–20.0 × 8.0–13.0 µm vs. 21.0–34.0 × 7.0–14.0 µm) and indextrinoid basidiospores (Decock et al. 2021). Haploporus ecuadorensis differs from H. dextrinoideus by wider basidioles and basidia (40.0–45.0 µm vs. 19.0–35.0 µm) and hyphae and basidiospores neither amyloid nor dextrinoid (Man et al. 2023). Haploporus longisporus differs from H. dextrinoideus by the indextrinoid skeletal hyphae and basidiospores (Zhou et al. 2021).

In addition, two species, H. brasiliensis and H. pileatus, were described in Brazil from South America without molecular data (Lira et al. 2018). Haploporus brasiliensis differs from H. crystallinus by annual basidiomata, larger pores (1–3 per mm vs. 5–7 per mm), thin dissepiments, wider subicular generative hyphae (2.0–2.5 µm vs. 1.0–1.5 µm) and larger, long-ellipsoid basidiospores [6.0–8.0 × 4.0–5.0 µm vs. 4.0–5.5 × 2.6–3.5 µm; Lira et al. (2018)]. Haploporus pileatus differs from H. crystallinus by pileate basidiomata, larger pores (3–4 per mm vs. 5–7 per mm), thin dissepiments, cylindrical and larger basidiospores [9.0–10.0 × 4.0–5.0 µm vs. 4.0–5.5 × 2.6–3.5 µm; Lira et al. (2018)]. Haploporus brasiliensis differs from H. dextrinoideus by smaller basidiospores (6.0–8.0 × 4.0–5.0 µm vs. 13.2–19.0 × 6.0–9.0 µm) and basidia [12.0–15.0 × 4.0–6.0 µm vs. 21.0–34.0 × 7.0–14.0 µm; Lira et al. (2018)]. Haploporus pileatus differs from H. dextrinoideus by smaller pores (3–4 per mm vs. 1–3 per mm), smaller basidiospores (9.0–10.0 × 4.0–5.0 µm vs. 13.2–19.0 × 6.0–9.0 µm) and basidia [14.0–24.0 × 5.0–7.0 µm vs. 21.0–34.0 × 7.0–14.0 µm; Lira et al. (2018)].

He et al. (2019) estimated the order Polyporales originated at a mean stem of 138 Mya and Ji et al. (2022) suggested that Polyporales originated in the Early Cretaceous, with a mean stem of 141.81 Mya (95% HPD of 102.35–191.91 Mya). Our molecular clock analysis showed that Polyporales originated in a mean stem of 159.8 Mya (95% HPD of 142.4–184.1 Mya), which overlapped previous studies (He et al. 2019; Ji et al. 2022). The genus Haploporus emerged at a mean stem of 108.3 Mya (95% HPD of 88.5–128.2 Mya), earlier than the genus Polyporus and its allied genera (Zhou et al. 2016; Ji et al. 2022). Species of the genus Haploporus probably diversified between 60.5 Mya and 1.8 Mya and 18 species occurred in the Neogene, indicating that species were rapidly diversified in this period. A similar result is evident in the genus Onnia, where most species also appeared in the Neogene (Zhao et al. 2022b).

The study reconstructed the phylogenetic relationships of the genus Haploporus, described two new species based on the molecular fragments and morphological evidence. Molecular clock analysis provided insight into the divergence times of Haploporus species.

Key to species of Haploporus

1 Hyphal system monomitic H. monomitica
1a Hyphal system dimitic to trimitic 2
2 Basidiospores < 8 µm long 3
2a Basidiospores > 8 µm long 7
3 Pores 1–3 per mm H. brasiliensis
3a Pores > 3 per mm 4
4 Cystidioles absent 5
4a Cystidioles present 6
5 Basidiomata annual to perennial, resupinate; pore 7–8 per mm; skeletal hyphae dextrinoid; basidiospores ellipsoid H. nanosporus
5a Basidiomata perennial, effused-reflexed to pileate; pore 3–5 per mm; skeletal hyphae IKI-; basidiospores ovoid H. odorus
6 Basidiomata perennial; pore 5–7 per mm H. crystallinus
6a Basidiomata annual; pore 7–9 per mm H. microsporus
7 Skeletal hyphae dextrinoid 8
7a Skeletal hyphae non-dextrinoid 9
8 Basidiomata annual; pore 1–3 per mm; basidiospores 13.2–19.0 × 6.0–9.0 µm H. dextrinoideus
8a Basidiomata perennial; pore 4–5 per mm; basidiospores 8.5–11 × 4–5.2 μm H. srilankensis
9 Basidiospores cylindrical H. thindii
9a Basidiospores oblong ellipsoid to ellipsoid H. subtrameteus
10 Hyphal system trimitic 11
10a Hyphal system dimitic 13
11 Skeletal hyphae dextrinoid H. tuberculosus
11a Skeletal hyphae non-dextrinoid 12
12 Basidiospores ovoid to ellipsoid H. alabamae
12a Basidiospores oblong-ellipsoid to cylindrical H. pirongia
13 Cystidioles absent 14
13a Cystidioles present 16
14 Basidiomata pileate H. pileatus
14a Basidiomata resupinate 15
15 Pores 4–5 per mm, basidiospores cylindrical, 10–11.5 × 4.5–5 µm H. cylindrosporus
15a Pores 1.5–4 per mm, basidiospores ellipsoid to oblong, 10–15 × 5–6.8 µm H. eichelbaumii
16 Dendrohyphidia present 17
16a Dendrohyphidia absent 21
17 Pores 5–7 per mm H. bicolor
17a Pores < 4 per mm 18
18 Basidiospores cylindrical 19
18a Basidiospores ellipsoid to oblong 20
19 Basidiospores 18.2–22 × 7–9 µm H. longisporus
19a Basidiospores 13–15 × 5–6 µm H. papyraceus
20 Hyphal system trimitic, skeletal hyphae dextrinoid H. grandisporus
20a Hyphal system dimitic, skeletal hyphae non-dextrinoid H. ecuadorensis
21 Pores > 3 per mm 22
21a Pores < 3 per mm 26
22 Pores 5–6 per mm H. septatus
22a Pores 3–5 per mm 23
23 Skeletal hyphae non-dextrinoid H. crassus
23a Skeletal hyphae dextrinoid 24
24 Cystidioles without septum H. angustisporus
24a Cystidioles with a simple septum 25
25 Basidiospores 9–10.8 × 3.8–5 µm H. punctatus
25a Basidiospores 9–12 × 5.5–8 µm H. subpapyraceus
26 Basidiospores 9–10 µm wide H. latisporus
26a Basidiospores < 9 µm wide 27
27 Basidiospores 12–15 × 6–8 µm H. gilbertsonii
27a Basidiospores 8.5–11.5 × 4.5–6.5 µm H. nepalensis

Acknowledgements

We thank Prof. Yu-Cheng Dai (Beijing Forestry University) for revision of the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

The research is supported by the National Natural Science Foundation of China (Nos. 32161143013, 32000010) and Academy of Sciences of the Czech Republic RVO (No. 60077344).

Author contributions

H. Zhao did the drawings, DNA sequencing, data analyses and drafted the paper; J. Vlasák collected and determined specimens and revised the paper; Y. Yuan did the morphological descriptions, revised the paper and provided funding.

Author ORCIDs

Heng Zhao https://orcid.org/0000-0003-2938-5613

Josef Vlasák https://orcid.org/0000-0002-5363-4459

Yuan Yuan https://orcid.org/0000-0001-6674-9848

Data availability

The sequences are deposited in the GenBank database (Table 1).

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Supplementary material

Supplementary material 1 

The supplementary materilas in this study

Heng Zhao, Josef Vlasák, Yuan Yuan

Data type: (docx.file, fasta.file)

Explanation note: figure S1. A Maximum Likelihood phylogenetic tree of Haploporus, based on ITS sequences, with two specimens Perenniporia citrinoalba and P. hainaniana used as outgroups. The new species Haploporus crystallinus and H. dextrinoideus are in bold. Maximum Likelihood bootstrap values (≥ 50%) of each clade is indicated along branches. The scale bar left upper indicates the number of substitutions per site. figure S2. A Maximum Likelihood phylogenetic tree of Haploporus, based on LSU sequences, with two specimens Perenniporia citrinoalba and P. hainaniana used as outgroups. The new species Haploporus crystallinus and H. dextrinoideus are in bold. Maximum Likelihood bootstrap values (≥ 50%) of each clade is indicated along branches. The scale bar left upper indicates the number of substitutions per site. figure S3. A Maximum Likelihood phylogenetic tree of Haploporus, based on mtSSU sequences, with two specimens Perenniporia citrinoalba and P. hainaniana used as outgroups. The new species Haploporus crystallinus and H. dextrinoideus are in bold. Maximum Likelihood bootstrap values (≥ 50%) of each clade is indicated along branches. The scale bar left upper indicates the number of substitutions per site. table S1. Specimens and GenBank accession numbers used in this study. table S2. The top hits of the new species, based on BLAST of ITS sequences from GenBank. file S1. All the aligned sequences.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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