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Research Article
Two new species of Verruconis from Hainan, China
expand article infoMin Qiao, Weiguang Tian, Rafael F. Castañeda-Ruiz§, JianPing Xu|, Zefen Yu
‡ Yunnan University, Kunming, China
§ Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro de Humboldt”, Havana, Cuba
| McMaster University, Ontario, Canada
Open Access

Abstract

Two new species of the genus Verruconis, V. hainanensis and V. pseudotricladiata, were described using combined morphological and DNA sequence data. The DNA sequences of respective strains including nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and fragments of three protein-coding genes (ACT1, BT2, TEF1) were sequenced and compared with those from closely-related species to genera Ochroconis and Verruconis (Family Sympoventuriaceae, Order Venturiales). Morphologically, both species showed typical ampulliform conidiophores and conidiogenous cells, features not seen in other species of Verruconis. The conidia of V. hainanensis are fusiform and those of V. pseudotricladiata are Y or T shaped, similar to old members of a closely-related genus Scolecobasidium. The addition of these two new species provides a new perspective on the heterogeneity of Scolecobasidium.

Keywords

Aquatic hyphomycetes, dematiaceous fungi, phylogenetic placement, new taxon

Introduction

The genus Verruconis Samerp. et al. was proposed for the neurotropic opportunist Ochroconis gallopava (W.B. Cooke) de Hoog (Samerpitak et al. 2014). The thermophilic characteristic of this genus is remarkable because all three proposed species of Verruconis can grow at 35–42 °C. In addition to the difference in growth temperature, Verruconis and Ochroconis de Hoog & Arx also differed in conidia colour (Samerpitak et al. 2014). However, a recent molecular phylogenetic analysis placed the mesophilic V. panacis T. Zhang & Y. Zhang into Verruconis, a result suggesting that both genera are more heterogeneous in their morphological and growth requirements than previously thought (Zhang et al. 2018).

Besides V. panacis, other three Verruconis species were transferred from other genera. The type species, V. gallopava (W.B. Cooke) Samerp. & de Hoogs [≡ Dactylaria gallopava (W.B. Cooke) G.C. Bhatt & W.B. Kendr., ≡ Ochroconis gallopava (W.B. Cooke) de Hoog] was transferred from Diplorhinotrichum Höhn.; V. verruculosa (R.Y. Roy et al.) Samerp. & de Hoog (≡ Scolecobasidium verruculosum R.Y. Roy et al.) was transferred from Scolecobasidium and V. calidifluminalis (Yarita et al.) Samerp. & de Hoog (≡ Ochroconis calidifluminalis Yarita et al.) was transferred from Ochroconis. These reclassifications suggested that genera Ochroconis, Verruconis and Scolecobasidium E.V. Abbott are closely related and that both morphological and molecular data are needed in order to derive robust classifications. Ochroconis, typified by O. constricta (E.V. Abbott) de Hoog & Arx, transferred from Scolecobasidium, was set up to comprise species with unbranched, subspherical to cylindrical or clavate conidia. Based on these criteria, many Scolecobasidium species were transferred to Ochroconis, while species in the genus Scolecobasidium were restricted to those with T- or Y-shaped or bi-lobed, two- to many-celled conidia and ampulliform conidiogenous cells, possessing one to three conidium-bearing denticles at the apex of the conidiogenous cells (de Hoog and von Arx 1973). However, there is a significant disagreement amongst mycologists about whether the genus Ochroconis should be established and some researchers still placed species with unbranched conidia under Scolecobasidium (Ellis 1976; Matsushima 1980, 1985, 1987, 1993, 1996; Punithalingam and Spooner 2011; Lu et al. 2013; Ren et al. 2013; Xu et al. 2014).

Samerpitak et al. (2014) revised the genera Ochroconis and Scolecobasidium using DNA sequences of the nuclear ribosomal RNA gene clusters and three protein-coding genes (actin: ACT1, β-tubulin: BT2, translation elongation factor 1-α: TEF1). They found that the type species of Scolecobasidium, S. terreum E.V. Abbott, ex-type strain CBS 203.27, originally described as having the T-shaped conidia, had lost the ability to produce conidia. Interestingly, this strain was phylogenetically distant from other strains with Y-shaped conidia as described for S. terreum in all analyses. Consequently, type strain S. terreum CBS 203.27 is now regarded as a non-representative strain of the species and, indeed, the validity of this species has been questioned and Scolecobasidium is considered to be of doubtful identity.

However, Gams thought that an ex-type culture was not so important to decide if a genus is retained, because there are other cultures of S. terreum available all over the world, which clearly define the identity of this characteristic fungus. He even thought that CBS 510.71, the ex-type of Humicola minima Fassat., a species with characteristic Y-shaped conidia, may replace S. terreum (Gams 2015). However, in Samerpitak’s analysis, many Scolecobasidium species were scattered outside the Family Sympoventuriaceae. Consequently, the genus Scolecobasidium has been questioned (Samerpitak et al. 2014). Since then, several new Ochroconis species have been described under Ochroconis (Giraldo et al. 2014; Samerpitak et al. 2015a; 2015b; 2017; Crous et al. 2016; 2017), while the number of Scolecobasidium species has not increased since 2014 (Index Fungorum 2018). Species with forked conidia, similar to S. terreum, were also added to Ochroconis based on phylogenetic relationships amongst members of Sympoventuriaceae (Giraldo et al. 2014). The strict morphological characters to demarcate Scolecobasidium were abandoned in favour of the molecular phylogenetic approach. Subsequent analyses based on combined molecular sequence information, ecological and physiological traits and morphological differences resulted in the establishment of the genus Verruconis.

Hainan Province, China is a centre of biodiversity for aquatic hyphomycetes. Since 2015, we have reported several new aquatic hyphomycetes from this area (Guo et al. 2015; Qiao et al. 2017, 2018). During further studies of aquatic hyphomycetes on submerged decaying leaves collected from a stream in Hainan Province, we encountered two fungi which resembled species of Scolecobasidium. Based on phylogenetic analyses, we identified that the fungi belonged to Verruconis. In this paper, we describe the two fungi as new species and determined their phylogenetic placement based on the combined sequences of SSU, ITS, LSU, BT2, TEF1 and ACT1.

Materials and methods

Collection of samples, isolation and characterisation

Submerged dicotyledonous leaves were collected from a stream in Hainan. Samples were collected in zip-lock plastic bags and labelled and then transported to the laboratory. The rotten leaves were cut into several 2–4 × 2–4 cm sized fragments in the laboratory and then spread on to the surface of CMA (20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) medium for 10 days; a single conidium was isolated and cultivated on CMA in Petri plates using sterilised needles while viewing with a BX51 microscope. Morphological observations were then made from CMA after incubation at 28 °C for one week. Measurement data were based on 30 random conidia and 10 conidiophores. Pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan) and at the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR and sequencing

Total DNA was extracted from fresh mycelia as described by Turner et al. (1997). Six markers, nuSSU, D1/D2 region of nuLSU, ITS and part of ACT1, BT2 and TEF1 were amplified by PCR using primers as reported earlier (Feng et al. 2013). PCR amplifications were performed using the methods described previously (Wang et al. 2014). The PCR products were then sent to the Beijing Tsingke Biotechnology Co. of China Ltd and sequenced on both strands with the same primers that were used for amplification.

Sequence alignment and phylogenetic analysis

Preliminary BLAST searches with nuSSU and nuLSU gene sequences of the new isolates indicated that they had a close phylogenetic relationship with sequences from the genus Verruconis, Ochroconis and Scolecobasidium. Based on this, we downloaded sequences at the six marker loci from strains belonging to genera Ochroconis and Verruconis, including 42 strains representing 21 species of Ochroconis and four species of Verruconis. The sequences of these representative strains were combined with those from our own cultures (see Table 1 for all GenBank accession numbers). Scolecobasidium excentricum R.F. Castañeda, W. Gams & Saikawa was specified as an outgroup.

Table 1.

Species, strains and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses.

Taxon strain GenBank accession number
ACT BT2 ITS LSU SSU TEF1
Ochroconis anellii (Graniti) de Hoog & Arx CBS 284.64* KF155912 KF156184 FR832477 KF156138 KF156070 KF155995
O. anomala A. Nováková & Mart.-Sánch. CBS 131816* KF155935 KF156194 HE575201 KF156137 KF156065 KF155986
O. constricta (E.V. Abbott) de Hoog & Arx CBS 211.53* KF155941 KF156187 HQ667519 KF156148 KF156073 KF156005
CBS 202.27 KF155942 KF156161 AB161063 KF156147 KF156072 KF156003
CBS 269.61 KF155939 KF156163 KF156024 KF156149 KF156074 KF156004
O. cordanae Samerp., Crous & de Hoog CBS 475.80* HQ916976 KF156197 KF156022 KF156122 KF156058 KF155981
CBS 172.74 KF155906 KF156198 KF156023 KF156121 KF156057 JF440566
CBS 780.83 KF155905 KF156199 HQ667539 KF156120 KF156059 KF155979
O. crassihumicola (Matsush.) de Hoog & Arx CBS 120700 KJ867427 KJ867433 KJ867429 KJ867430 KJ867431 KJ867428
O. gamsii de Hoog CBS 239.78* KF155936 KF156190 KF156019 KF156150 KF156088 KF155982
CBS 101179 KF155937 KF156192 KF156020 KF156151 KF156091
O. globalis Samerp., A.P.M. Duarte, Attili-Angelis & de Hoog CBS 119644* KF956086 KF961065 KF961086 KF961097 KF961108 KF961075
CBS 131956 KF956094 KF961067 KF961088 KF961100 KF961117 KF961081
CBS 135766 KF956087 KF961072 KF961094 KF961106 KF961116 KF961082
O. humicola (G.L. Barron & L.V. Busch) de Hoog & Arx CBS 116655* KF155904 KF156195 HQ667521 KF156124 KF156068 KF155984
O. icarus Samerp., A. Giraldo, Guarro & de Hoog CBS 116645 LM644599 LM644604 HQ667525 LM644565 KF156083
O. lascauxensis A. Nováková & Mart.-Sánch. CBS 131815* KF155911 KF156183 FR832474 KF156136 KF156069 KF155994
O. longiphorum (Matsush.) Samerp. & de Hoog CBS 435.76* KF155908 KF156182 KF156038 KF156135 KF156060 KF155978
O. macrozamiae Crous & R.G. Shivas CBS 102491 KF155938 KF156191 KF156021 KF156152 KF156092 KF155983
O. minima (Fassat.) Samerp. & de Hoog CBS 423.64 KF155943 KF156173 HQ667523 KF156131 KF156085 KF156008
CBS 536.69 KF155944 KF156174 HQ667524 KF156132 KF156084 KF156009
O. mirabilis Samerp. & de Hoog CBS 413.51 KF155957 KF156164 HQ667536 KF156140 KF156076 KF156001
dH 14815 KF155954 KF156170 KF156036 KF156145 KF156079 KF155998
O. musae (G.Y. Sun & Lu Hao) Samerp. & de Hoog CBS 729.95* KF155948 KF156171 KF156029 KF156144 KF156082 KF155999
O. ramosa A. Giraldo, Gené, Deanna A. Sutton & Guarro UTHSC 03-3677 LM644601 LM644606 LM644522 LM644566 LM644549
UTHSC 04-2729 LM644602 LM644607 LM644523 LM644567 LM644550
UTHSC 12-1082 LM644603 LM644608 LM644524 LM644551
O. sexualis Samerp., Van der Linde & de Hoog dH 22953 KF155903 KF156188 KF156017 KF156119 KF156090 KF155977
PPRI 12991* KF155902 KF156189 KF156018 KF156118 KF156089 KF155976
O. tshawytschae (Doty & D.W. Slater) Kiril. & Al-Achmed CBS 130.65 KF155916 KF156178 HQ667566 KF156127 KF156061 KF155989
CBS 228.66 KF155915 KF156179 KF156016 KF156128 KF156064 KF155992
CBS 100438* KF155918 KF156180 HQ667562 KF156126 KF156062 KF155990
O. verrucosa (Zachariah, Sankaran & Leelav.) Samerp. & de Hoog CBS 225.77 KF155909 KF156186 KF156130 KF156066 KF155985
CBS 383.81* KF155910 KF156185 KF156015 KF156129 KF156067
Scolecobasidium excentricum R.F. Castañeda, W. Gams & Saikawa CBS 469.95* KF155934 KF156196 HQ667543 KF156105 KF156096 KF155975
Verruconis calidifluminalis (Yarita, A. Sano, de Hoog & Nishim.) Samerp. & de Hoog CBS 125818* KF155901 KF156202 AB385698 KF156108 KF156046 KF155959
V. gallopava (W.B. Cooke) Samerp. & de Hoog CBS 437.64* HQ916989 KF156203 HQ667553 KF156112 KF156053 KF155968
CBS 118.91 KF156110 KF156047
CBS 863.95 KF156114 KF156052
Verruconis verruculosa (R.Y. Roy, R.S. Dwivedi & R.R. Mishra) Samerp. & de Hoog CBS 119775* KF155919 KF156193 KF156014 KF156106 KF156055 KF155974
Verruconis hainanensis Z.F. Yu & M. Qiao YMF1.04165* MK248271 MK244397 MK248269 MF536879 MF536881
Verruconis panacis T. Zhang & Y. Zhang SYPF8337* MF536883 MF536882 MF536880 MK248267 MK248272
Verruconis pseudotricladiata Z.F. Yu & M. Qiao YMF1.04915* MK253013 MK244396 MK248270 MK248268 MK248273

Six alignment files were generated, one for each gene and converted to NEXUS files with ClustalX 1.83 (Thompson et al. 1997) to identify the phylogenetic positions of two species. The six alignments were then combined with BioEdit 7.1.9.0 (Hall 1999). All characters were weighted equally and gaps were treated as missing characters. Maximum likelihood (ML) analysis was computed by RAxML (Stamatakis 2006) with the PHY files generated with ClustalX 1.83 (Thompson et al. 1997), using the GTR-GAMMA model. Maximum likelihood bootstrap proportions (MLBP) were computed with 1000 replicates. Bayesian inference (BI) analysis was conducted with MrBayes v3.2.2 (Ronquist et al. 2012). The Akaike information criterion (AIC) implemented in jModelTest 2.0 (Posada 2008) was used to select the best fit models after likelihood score calculations were done. The base tree for likelihood calculations was ML-optimised. HKY+I+G was estimated as the best-fit model under the output strategy of AIC, Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 2000000 generations, sampling every 1000th generation. Two independent analyses with four chains each (one cold and three heated) were run until the average standard deviation of the split frequencies dropped below 0.01. The initial 25% of the generations of MCMC sampling were discarded as burn-in. The refinement of the phylogenetic tree was used for estimating Bayesian inference posterior probability (BIPP) values. The Tree was viewed in FigTree v1.4. The values of Maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 0.95 at the nodes are shown along branches.

Results

Phylogenetic analysis

The phylogenetic relationships amongst the known representative taxa are completely congruent with the previous studies (Samerpitak et al. 2014; Giraldo et al. 2014). Ochroconis and Verruconis formed two distinct clades. Within the Ochroconis clade, three species, O. minima (Fassat.) Samerp. & de Hoog, O. ramose A. Giraldo et al., O. icarus Samerp.et al. with T-shaped conidia fell into a highly-supported sub-clade. Both V. hainanensis and V. pseudotricladiata were nested in a well -supported subclade, with V. panacis as the closest sister species. The sub-clade comprising the two new species and V. panacis is closer to the clade composed of V. calidifluminalis and V. gallopava than to V. verruculosa (Figure 1).

Figure 1. 

Phylogenetic tree based on Bayesian analysis of the combined sequences of SSU, ITS, LSU BT2, TEF1 and ACT1. Scolecobasidium excentricum is used as the outgroup. Bayesian posterior probabilities, greater than 0.95, are given above the nodes. Maximum likelihood bootstrap values, greater than 75%, are given below the nodes. The scale bar shows the expected changes per site.

Taxonomy

Verruconis hainanensis Z.F. Yu & M. Qiao, sp. nov.

MycoBank No: MB828550
Figure 2

Etymology

Latin, hainanensis, refers to the collection locality.

Description

Colonies on CMA medium compact, restricted, brown to fuliginous, 13 mm at 20 °C after 20 days, 16 mm at 25 °C, 11 mm at 30 °C, no growth at 35 °C. Aerial hyphae subhyaline to brown, smooth- or somewhat rough-walled. Conidiophores semi-macronematous, mononematous, sometimes slightly moniliform, unbranched or branched at the apex with 2–4 divergent conidiogenous cells, brown basal cell, pale brown branches, smooth, up to 25 μm long. Conidiogenous cells mostly monoblastic, discrete, scattered, brown to fuliginous or pale brown, lageniform to ampulliform, pale brown, 3.4–6.0 × 2.2–3.6 μm, with a fimbriate denticle-like at the conidiogenous locus after rhexolytic conidial secession. Conidia solitary, acrogenous, fusiform, rostrate at the apical cell, 3-septate, dark at the septa, coarsely verrucose, more or less equilateral, slightly constricted at the median septum, bicoloured, with brown middle cells and subhyaline end cells, 23–30.2 × 3.6–5.7 μm, with an inconspicuous basal frill.

Figure 2. 

Culture and anamorph of Verruconis hainanensis (YMF 1.04165). a Culture on CMA at 25 °C after 20 days b conidiophores and monoblastic conidiogenous cells c Conidia; Scale bars: 2 cm (a); 10 μm (b, c).

Type

CHINA. From leaves of an unidentified dicotyledonous plant submerged in a stream, Qixianling, Hainan Province, 18°68'N, 109°69'E, 902 m alt., 16 June 2016, Z.F. Yu (dried slide YMFT 1.04165, holotype; live culture YMF 1.04165 –ex-type culture; CGMCC–3.18974–isotype).

Notes

Verruconis hainanensis shares the fusiform conidial shape with some described Scolecobasidium species, such as: S. cateniphorum Matsush., S. caffrum Matsush., S. houhense D.W. Li & Jing Y. Chen and S. tropicum Matsush., but all these taxa are readily distinguishable from the new Chinese species. Specifically, S. cateniphorum is distinguished by its 1-septate, smooth or inconspicuous echinulate, 10–24 × 2–3.5 μm conidia (Matsushima 1975). S. caffrum and S. tropicum both have 2-septate conidia, but S. caffrum has conidia mostly smooth or inconspicuously rough, 20–35 × 4–7.5 μm, with pale brown central and subhyaline end cells (Matsushima 1996) and S. tropicum has conidia with smooth or inconspicuous verruculose, smaller, 14–20 × 4.5–6 μm, with pale brown central and subhyaline end cells (Matsushima 1983). S. houhense with 3-septate conidia is superficially similar to V. hainanensis, but S. houhense is characterised by minutely verruculose conidia, 26–31 × 4.5–5.5 μm, brown, with central cells darker than end cells and slightly protuberate and with a dark basal scar and its conidiogenous cells and conidiophores are different from those of V. hainanensis (Li et al. 2010). The distinct dark scar, described from S. houhense, has been reported by Matsushima (1975) in Nakataea fusispora (Matsush) Matsush., but it is absent in V. hainanensis.

Verruconis pseudotricladiata Z.F. Yu & M. Qiao, sp. nov.

MycoBank No: MB828551
Figure 3

Etymology

Latin, pseudotricladiata refers to similar conidia shape to Scolecobasidium tricladiatum.

Description

Colonies on CMA medium compact, restricted, brown to fuliginous, surface velvety or floccose, 12 mm at 20 °C after 20 days, 14 mm at 25 °C, 10 mm at 30 °C, no growth at 35 °C. Mycelium subhyaline to pale brown and smooth- or somewhat rough-walled. Conidiophores semi-macronematous, mononematous, straight or flexuous, 1–4 septa, sometimes moniliform (composed of 2–5 globose serial cells), pale brown, smooth, 6.5–27.2 × 2.1–3.5 μm, sometimes reduced to conidiogenous cells that arise from assimilative hyphae. Conidiogenous cells monoblastic, rarely polyblastic after sympodial elongation, globose, ampulliform, lageniform to clavate, 3.0–5.3 × 2.3–3.8 μm, integrated or discrete, mostly determinate, with an inconspicuous or distinct fimbriate denticle-like at the conidiogenous locus after rhexolytic conidial secession. Conidia mostly acrogenous, subhyaline to pale brown, smooth to verruculose, staurosporic, unbranched or branched: i) unbranched conidia (main axis) cylindrical-clavate, 2–4 septate, slightly constricted at the septa, mostly smooth, rarely verruculose, 16–20 × 3.3–4.7 μm, with an inconspicuous basal frill and often with a globose or ellipsoidal, 0–1 septate, 5.6–12.3 × 2.8–4.5 μm primary branch at the apex; ii) branched conidia staurosporic, Y-, or T-shaped, composed of the main axis and two branches (primary and secondary); iia) main axis cylindrical-clavate to clavate, 1–3-septate, mostly 2-septate, smooth or rarely verruculose, very pale brown, 15.6–20.6 × 3.8–5.7 μm; iib) primary branches obclavate, 1–2 septate, verruculose toward the apex, smooth at the basal cell, 17.9–18.2 × 2.9–4.7 μm, at an angle of 45° arising from the apex of main axis; iic) secondary branches ovoid to obclavate, smooth or verruculose towards the apex, 0–2-septate, (–5.6)12.3–17.9 × 2.8–4.5 μm, arising eccentrically from the basal cell of the primary branches.

Figure 3. 

Cultures and anamorph of Verruconis pseudotricladiata (YMF 1.04915). a Cultures on CMA at 25 °C after 20 days b branched Y-shaped conidia c unbranched conidia d T-shaped conidia h Conidiophores and conidiogenous cells. Conidiogenous cells on hyphae (black arrow). Scale bars: 2 cm (a); 10 μm (b–h).

Type

CHINA. From leaves of an unidentified broad-leaf species submerged in a stream, Diaoluo Mountain, Hainan Province, 18°41'N, 109°41'E, 254 m alt., 16 June 2016, Z.F. Yu (dried slide YMFT 1.04915, holotype; live culture YMF 1.04915 ex-type; CGMCC–3.18939–isotype).

Notes

Verruconis pseudotricladiata is similar to S. tricladiatum Matsush. on the general conidial morphology, but in S. tricladiatum, the conidiophores are mostly moniliform, irregularly branched forming profuse fascicules and, on pure culture, lack staurosporic conidia or rarely formed on the conidiogenous cells, the conidia are mostly unbranched, ellipsoidal to fusiform, (1–) 3–4 (–5)-septate, (9.5–)14–22 (–28) × 4–5 (–6) μm, pale olivaceous or pale brown, verruculose conidia (Matsushima 1971).

Discussion

The Index Fungorum currently lists 66 names in Scolecobasidium. However, 22 of these 66 names have been transferred into genera Dactylaria Sacc., Paradendryphiella Woudenb. & Crous, Ochroconis, Trichoconis Clem., Neta Shearer & J.L. Crane and Verruconis (Index Fungorum 2018). Of the remaining 42 species, the majority lacks authentic culture materials and DNA sequence data, making the revision of Scolecobasidium very difficult. However, since 2014, the number of Scolecobasidium species has not increased, while many new species have been reported under Ochroconis, including species with forked conidia (Giraldo et al. 2014). Although Scolecobasidium is still listed as an accepted genus of Ascomycota (Wijayawardene et al. 2017), this genus will likely be phased out. Thus, we have placed our strains into Verruconis based on phylogenetic analysis.

Morphologically, the two new species resemble some members of the genus Scolecobasidium. Conidiophores composed of 2–5 globose serial cells are very typical in old members of Scolecobasidium, such as S. alabamense Matsush., S. amazonense Matsush., S. cateniphorum Matsush. and S. lanceolatum Matsush. However, amongst these species, only the LSU sequence of S. cateniphorum was available. Further, Y- branched conidia of V. pseudotricladiata was previously only described in S. tricladiatum, while T-shaped branched conidia appeared in four species, including the type species S. terreum, O. minima (Fassat.) Samerp. & de Hoog, O. ramosa Samerp. et al. and O. icarus Samerp. et al. In the molecular phylogenetic tree, inferred from the combined sequences of six marker loci, except for the type species, three species with T-shaped branched conidia form a single clade with high support within Ochroconis. In the combined analysis of SSU and LSU, S. tricladiatum strain P051 is closely related to V. pseudotricladiata and S. terreum 043 fell into Ochroconis, nested with other species with T-shaped branched conidia (data not shown). The phylogenetic analysis is partly consistent with the morphological comparison. The article, comprising sequences of S. tricladiatum strain P051 and S. terreum 043, has not been published and we do not know if two species have been identified correctly. Anyhow, molecular data for our strains will help improve the taxonomy and revision of Scolecobasidium.

When the genus Verruconis was established, the thermophilic character was one of the main characteristics distinguishing this genus from Ochroconis. The first three species included in this genus all have a high optimal growing temperature of 35–42 °C and maximum growing temperature of 47–50 °C (Samerpitak et al. 2014). However, both our species and their close relative V. panacis are mesophilic, which blurred a major distinguishing feature between Verruconis and Ochroconis. Morphologically, Verruconis is characterised by poorly differentiated, flexible, mostly cylindrical to acicular, with 0(−1) thin septa conidiophores, sometimes without conidiophores (Samerpitak et al. 2014). However, conidiophores of two new species are distinct, only occasionally reducing to conidiogenous cells. Ampulliform conidiogenous cells also appeared in O. minima and O. icarus, but no species in Verruconis and Ochroconis have similar conidiophores to those of the two new species, which were composed of 2–5 globose serial cells. Based on the phylogenetic relationships amongst the species, the distributions of morphological features indicate that conidiophores and conidiogenous cells are important features for defining these two related genera. Our results suggest that the analyses of more sequences and more cultures in this group of fungi are needed to provide a robust revision of the three genera Verruconis, Ochroconis and Scolecobasidium.

Acknowledgements

This work was financed by the National Natural Science Foundation of PR China (31770026, 31760012). We are grateful to two reviewers for critically reviewing the manuscript and for providing helpful suggestions to improve this paper.

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