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Research Article
Six new species of Sporothrix from hardwood trees in Poland
expand article infoAgnieszka Ostafińska‡§, Robert Jankowiak§, Piotr Bilański§, Halvor Solheim|, Michael J. Wingfield
‡ State Forets, Forest District Dynów, Dynów, Poland
§ University of Agriculture, Krakow, Poland
| Norwegian Institute of Bioeconomy Research, Ås, Norway
¶ University of Pretoria, Pretoria, South Africa

Corresponding author: Robert Jankowiak ( rljankow@cyf-kr.edu.pl )

Academic editor: Pedro Crous
© 2021 Agnieszka Ostafińska, Robert Jankowiak, Piotr Bilański, Halvor Solheim, Michael J. Wingfield.
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: Ostafińska A, Jankowiak R, Bilański P, Solheim H, Wingfield MJ (2021) Six new species of Sporothrix from hardwood trees in Poland. MycoKeys 82: 1-32. https://doi.org/10.3897/mycokeys.82.66603
Open Access

Abstract

Sporothrix (Sordariales, Ascomycota) is a well-supported monophyletic lineage within the Ophiostomatales, species of which occur in a diverse range of habitats including on forest trees, in the soil, associated with bark beetles and mites as well as on the fruiting bodies of some Basidiomycota. Several species have also been reported as important human and animal pathogens. During surveys of insect- and wound-associated Ophiostomatales from hardwood trees in Poland, many isolates with affinity to Sporothrix were recovered. In the present study, six undescribed Sporothrix spp. collected during these surveys are characterized based on their morphological characteristics and multi-locus phylogenenetic inference. They are described as Sporothrix cavum, Sporothrix cracoviensis, S. cryptarchum, S. fraxini, S. resoviensis, and S. undulata. Two of the Sporothrix spp. reside in the S. gossypina-complex, while one forms part of the S. stenoceras-complex. One Sporothrix sp. is a member of lineage F, and two other species grouped outside any of the currently defined species complexes. All the newly described species were recovered from hardwood habitats in association with sub-cortical insects, wounds or woodpecker cavities. These species were morphologically similar, with predominantly asexual states having hyaline or lightly pigmented conidia, which produce holoblastically on denticulate conidiogenous cells. Five of the new taxa produce ascomata with necks terminating in long ostiolar hyphae and allantoid ascospores without sheaths. The results suggest that Sporothrix species are common members of the Ophiostomatales in hardwood ecosystems of Poland.

Keywords

6 new species, bark beetle-associated fungi, Ophiostomatales, phylogeny, tree wounds

Introduction

Sporothrix was established by Hektoen and Perkins (1900) based on the morphological description of the human pathogen, Sporothrix schenckii. Species of Sporothrix (Ascomycota, Ophiostomatales, Ophiostomataceae) were first accommodated in Sporotrichum (De Beurmann and Gougerot 1911). Until the latter half of the 20th century, these fungi were also treated in various other genera, including Cephalosporium, Cladosporium (Hedgcock 1906; Münch 1907; Lagerberg et al. 1927; Melin and Nannfeldt 1934; Siemaszko 1939; Davidson 1942; Bakshi 1950; Mathiesen-Käärik 1953; Hunt 1956), Cylindrocephalum, Hormodendron (Robak 1932), Hyalodendron (Goidànich 1935; Georgescu et al. 1948), and Rhinotrichum (Georgescu et al. 1948; Sczerbin-Parfenenko 1953), in order to accommodate the asexual morphs of Ophiostoma. de Hoog (1974) published a monograph of the Sporothrix species and proposed the placement of S. schenckii as the asexual morph of O. stenoceras. That monograph expanded the concept of Sporothrix and included new Sporothrix species causing human infections as well as those associated with wood and bark beetles.

de Hoog et al. (1985) recognized that Sporothrix is not a homogenous group. As DNA sequencing technology was applied to resolve taxonomic relationships for fungi, evidence emerged that S. schenckii is phylogenetically related to species of Ophiostoma (Berbee and Taylor 1992; Hausner et al. 1993, 2000). In these studies, species producing only sporothrix-like asexual states were treated as members of the S. schenckii–O. stenoceras complex in Ophiostoma sensu lato (De Beer et al. 2003; Villarreal et al. 2005; Roets et al. 2006; Zipfel et al. 2006; De Meyer et al. 2008; Linnakoski et al. 2010; Kamgan Nkuekam et al. 2012). The genus Sporothrix was recently redefined and emended based on the analysis of partial 18S and 28S rDNA sequences for species in the Ophiostomatales (De Beer et al. 2016). Sporothrix was consequently separated from species of Ophiostoma and various complexes were defined within Sporothrix. Sporothrix is now defined as one of nine relatively clearly defined genera in the Ophiostomataceae (De Beer and Wingfield 2013; De Beer et al. 2013a, 2013b, 2016).

As currently recognized, Sporothrix includes 56 species (De Beer et al. 2016; Ngubane et al. 2018; Wang et al. 2019; Musvuugwa et al. 2020), which are characterized by their dark brown to black, globose ascomata with elongated necks up to 1600 μm, occasionally terminating in an ostiole, often surrounded by ostiolar hyphae. Ascospores are usually curved and lunate to reniform, without a sheath (De Beer and Wingfield 2013). The asexual states have conidiophores that proliferate sympodially and produce hyaline or occasionally pigmented conidia on denticulate conidiogenous cells (De Beer and Wingfield 2013).

Sporothrix includes a large assemblage of species that are widely distributed across various climatic zones of the world (De Beer and Wingfield 2013; De Beer et al. 2016). Species also occupy a wide range of habitats. The greatest numbers of species are found on bark, in the infructescences of Protea spp. and on the wood of different forest trees (e.g., Roets et al. 2008, 2009, 2013; De Errasti et al. 2016). Other species have been described from soil, bark beetles, ambrosia beetles, mites, and from the fruiting bodies of basidiomycetes (e.g., Constantinescu and Ryman 1989; Marmolejo and Butin 1990; De Meyer et al. 2008; Roets et al. 2008; De Errasti et al. 2016). Several species are also well-known as human and animal pathogens (Travassos and Lloyd 1980; Summerbell et al. 1993; Barros et al. 2004; Lòpez-Romero et al. 2011; Zhang et al. 2015).

Jankowiak et al. (2019a) conducted the first extensive survey of fungal associates of hardwood-infesting bark and ambrosia beetles in Poland. In the same year, Ophiostomatales associated with wounds on hardwood trees were also studied in Poland (Jankowiak et al. 2019b). These studies reported several Sporothrix species, which were apparently new to science, but names were not provided for them. In addition, one unknown Sporothrix species was isolated from cavities of woodpeckers in Poland (Jankowiak et al. 2019c). In this study, morphological characters and DNA sequence data for the ITS region (ITS1–5.8S–ITS2) and three protein coding genes (β-tubulin, calmodulin, translation elongation factor 1-α) were analyzed to characterize six new species of Sporothrix. These were compared with closely related known species and formal descriptions have been provided for them.

Materials and methods

Fungal isolates

The collection details for the isolates included in the present study (Table 1) are provided in previous studies (Jankowiak et al. 2019a, 2019b, 2019c). The cultures are maintained in the culture collection of the Department of Forest Ecosystems Protection, University of Agriculture in Krakow, Poland, and in the culture collection of the Natural Resources Institute Finland (Luke), Helsinki, Finland. The ex-type isolates and representative isolates of the new species described were deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. Dried cultures were deposited as holotype specimens in the Mycological Herbarium (O), Natural History Museum, University of Oslo, Norway.

Table 1.
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Isolates used in the present study.

Fungal species Previous identificationA Isolate no. Source Site GenBank accessionsE
CBS B O-FC KFL=NRFID ITS1-5.8S-ITS2 βT TEF 1-α CAL
Sporothrix cracoviensis sp. nov Sporothrix sp. 7 CBS 147940 KFL17FRJTD Adult of Trypodendron domesticum on Fagus sylvatica Krzeszowice MH283148 MH283365 MH283500 MH283526
CBS 147939 KFL2114bRJTD Adult of Trypodendron domesticum on Fagus sylvatica Krzeszowice MH283149 MH283366 MH283501 MH283527
CBS 147941 O-F-258629 KFL2514aRJTDF Adult of Trypodendron domesticum on Fagus sylvatica Krzeszowice MW768963 MH283367 MH283502 MH283528
CBS 147942F,T O-F-258628 KFL2514bRJTD Adult of Trypodendron domesticum on Fagus sylvatica Krzeszowice MW768964 MH283368 MH283503 MH283529
Sporothrix fraxini sp. nov Sporothrix sp. 8 CBS 147936F,T O-F-258630 KFL21BS16bRJHV Gallery of Hylesinus varius on Fraxinus excelsior Zbylitowska Góra MH283150 MH283370 MH283504 MH283530
CBS 147938F O-F-258631 KFL21BS16dRJHV Gallery of Hylesinus varius on Fraxinus excelsior Zbylitowska Góra MW768968 MH283371 MW768973 MH283531
CBS 147937 KFL21BS16cRJHV Gallery of Hylesinus varius on Fraxinus excelsior Zbylitowska Góra MH283151 MH283372 MH283505 MH283532
Sporothrix resoviensis sp. nov Sporothrix sp. 10 CBS 147927F,T O-F-258632 KFL204ABRZN16AO Wound on Betula pendula Borownica MH740962 MH741100 MH741189 MH741228
Sporothrix cryptarchum sp. nov. Sporothrix sp. 11 KFL1097NOL16RJ Wound on Alnus incana Wierzchosławice MH740963 MH741101 MH741190 MH741229
KFL1146NDB16RJ Wound on Quercus robur Ispina MH740964 MH741102 MH741191 MH741230
CBS 147935 KFL48716NDBRJ Wound on Quercus robur Wierzchosławice MW768967 MH741103 MH741192 MW768977
CBS 147934F,T O-F-258633 KFL410DB16bRJCU Adult of Cryptarcha undata Wierzchosławice MW768966 MH741104 MH741193 MH741231
CBS 147933E O-F-258634 KFL404DB16aRJCU Adult of Cryptarcha undata Wierzchosławice MW768965 MH741105 MH741194 MH741232
Sporothrix undulata sp. nov. Sporothrix sp. 12 CBS 147931E O-F-258636 KFL13NDB15bRJ Wound on Quercus robur Wierzchosławice MH740965 MH741106 MW768974 MW768978
CBS 147930 KFL12NDBCZ15RJ Wound on Quercus rubra Wierzchosławice MH740967 MH741108 MH741196 MW768979
CBS 147928 KFL221NBK16RJ Wound on Fagus sylvatica Czajowice MH740970 MH741112 MH741199 MH741235
CBS 147932 KFL430NDB16RJ Wound on Quercus robur Ispina MH740971 MH741113 MH741200 MH741236
KFL1099NOLCZ16RJ Wound on Alnus incana Wierzchosławice MH740973 MH741115 MH741202 MH741237
KFL1140NDB16bRJ Wound on Quercus robur Ispina MH740975 MH741117 MH741203 MH741238
KFL6117NWB17RJ Wound on Salix fragilis Babimost MW768970 MH741119 MH741204 MW768980
CBS 147929F,T O-F-258635 KFL398DB16RJEG Adult of Epuraea guttata Wierzchosławice MH740976 MH741121 MH741205 MH741239
KFL404DB16bRJCU Adult of Cryptarcha undata Wierzchosławice MW768969 MH741124 MH741208 MH741242
Sporothrix cavum sp. nov Sporothrix sp. 18 CBS 147943F,T O-F-258637 KFL42215aDRJ Cavity of Dendrocopos major on Salix fragilis Kraków MF782813 MF782850 MW768972 MW768976
O-F-258638 KFL35614DRJF Cavity of Dendrocopos medius on Malus domestica Książ Wielki MF782814 MF782851 MW768971 MW768975

A Isolates collected and identified during previous surveys in Poland (Jankowiak et al. 2019a, 2019b, 2019c). Sporothrix sp. 18 in the study of Jankowiak et al. (2019c) was labelled as Sporothrix sp. B CBS Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. C Herbarium of the Natural History Museum, University of Oslo, Norway, D KFL Culture collection of the Department of Forest Ecosystems Protection, University of Agriculture in Krakow, Poland; NRIF The Natural Resources Institute Finland (Luke), Helsinki, Finland. E ITS1-5.8S-ITS2-ITS2 = the internal transcribed spacer 1 and 2 regions of the nuclear ribosomal DNA gene, 5.8S rRNA gene; βT= Beta-tubulin; TEF1-α = Translation elongation factor 1-alpha; CAL = Calmodulin. F Isolates used in growth and morphological studies; Ttype strain Sequences obtained during the survey in this study are indicated in bold.

Microscopy and growth studies

Morphological characters were examined for selected isolates as well as for the herbarium specimens selected as types. Cultures were grown on 2% Malt Extrat Agar (MEA) made up of 20 g Bacto malt extract, 20 g agar Bacto agar powder (Becton Dickinson and Company, Franklin Lakes, USA) in 1 l deionized water. In attempts to induce the formation of ascomata, autoclaved twigs of host trees including the bark were placed at the centres of agar plates containing MEA. Fungal cultures were derived from single spores. To promote the production of ascomata, single conidial isolates were crossed in all possible combinations, following the technique described by Grobbelaar et al. (2009). These cultures were incubated at 25 °C and monitored regularly for the appearance of fruiting structures.

Morphological features were examined by mounting fungal tissue in 80% lactic acid on glass slides, and fruiting structures were observed using a Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) with an Invenio 5S digital camera (DeltaPix, Maalov, Denmark) to capture photographic images. Microscopy followed the technique described by Kamgan Nkuekam et al. (2011). Colour designations were based on the colour charts of Kornerup and Wanscher (1978).

For each taxonomically relevant structure, fifty measurements were made, when possible, using the Coolview 1.6.0 software (Precoptic, Warsaw, Poland). Averages, ranges and standard deviations were calculated for the measurements, and these are presented in the format ‘(min–)(mean–SD)–(mean+SD)(–max)’.

Growth characteristics for the novel species were determined by analysing the radial growth for 12 isolates (two for each species) (Table 1). Agar disks (5 mm diam.) were cut from the actively growing margins of fungal colonies and these disks were placed at the centres of plates containing 2% MEA. Four replicate plates for each of the six putative new species were incubated at temperatures between 5, and 35 °C at 5 °C intervals. The radial growth (two measurements perpendicular to each other per plate) was determined 14 d after inoculation, and growth rates were calculated as mm/d.

PCR, sequencing and phylogenetic analyses

DNA extractions were performed as described by Jankowiak et al. (2019d). For sequencing and phylogenetic analyses, four loci were amplified: the internal transcribed spacer region (ITS, consisting of ITS1, 5.8S, and ITS2), beta tubulin (βT), calmodulin (CAL), and the translation elongation factor 1-alpha (TEF1-α). The primers used for PCR and sequencing of the various gene regions were as follows: ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for ITS; T10 (O’Donnell and Cigelnik 1997) or Bt2a together with Bt2b (Glass and Donaldson 1995) for βT; F-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998) were used for TEF1-α; CL1 and CL2a (O’Donnell et al. 2000) or CL3F and CL3R (De Beer et al. 2016) were used for CAL. PCR and sequencing protocols were as described by Jankowiak et al. (2019d), other than the annealing temperature being optimised for some individual reactions. All analyses were run independently for each gene partition (Figs 1–4). Resulting trees were visually compared for topological incongruence. Gene partitions showing no topological incongruence (βT, CAL) were combined and presented as a concatenated construct (Fig. 5).

For phylogenetic analyses, sequence alignments were performed using the online version of MAFFT v7 (Katoh and Standley 2013). The ITS, βT, CAL, and TEF1-α datasets were aligned using the E-INS-i strategy with a 200PAM/κ=2 scoring matrix, a gap opening penalty of 1.53 and an offset value of 0.00. The alignments were checked manually with BioEdit v.2.7.5 (Hall 1999). The resulting alignments and trees were deposited into TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S27966).

Phylogenetic trees were inferred for each of the datasets using three different methods: Maximum likelihood (ML), Maximum Parsimony (MP) and Bayesian inference (BI). For ML and BI analyses, the best-fit substitution models for each aligned dataset were established using the corrected Akaike Information Criterion (AICc) in jModelTest 2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012). ML analyses were carried out with PhyML 3.0 (Guindon et al. 2010), utilizing the Montpelier online server (http://www.atgc-montpellier.fr/phyml/). The ML analysis included bootstrap analysis (1000 bootstrap pseudoreplicates) in order to assess node support values and the overall reliability of the tree topology. The best evolutionary substitution model was GTR+I+G for ITS (-lnL = 4497.47), GTR+G for CAL (-lnL = 4112.25) and TEF1-α (-lnL = 4218.36), HKY+G for βT (-lnL = 2641.05) and HKY+I+G for combined βT-CAL (-lnL 6798.48).

MP analyses were performed using PAUP* 4.0b10 (Swofford 2003). Gaps were treated as fifth state. Bootstrap analysis (1000 bootstrap replicates) was conducted to determine the levels of confidence for the nodes within the inferred tree topologies. Tree bisection and reconnection (TBR) was selected as the branch swapping option. The tree length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI) and Rescaled Consistency Index (RC) were recorded for each analysed dataset after the trees were generated.

BI analyses using Markov Chain Monte Carlo (MCMC) methods were carried out with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Four MCMC chains were run for 10 million generations applying the best-fit model for each dataset. Trees were sampled every 100 generations, resulting in 100,000 trees. Tracer v1.4.1 (Rambaut and Drummond 2007) was utilized to determine the burn-in value for each dataset. The remaining trees were utilised to generate a 50% majority rule consensus tree, which allowed for calculating posterior probability values for the nodes.

Results

Phylogenetic Analyses

Alignments for the ITS dataset contained 575 characters; for the βT 303 characters; for CAL 543 characters; and for TEF1-α 812 characters; for the concatenated combined dataset 826 (including gaps), of which respectively 202, 123, 271, 439, 390 were parsimony-informative. The exon/intron arrangement of the βT data included exons 5 and 6, interrupted by intron 5. The exon/intron arrangement of the CAL data included exons 4 and 5, interrupted by intron 4. The aligned TEF1-α gene region consisted of intron 3 and exons 4 and 5, but lacked intron 4.

DNA sequence data were generated for 24 isolates considered in this study (Table 1). Blast analyses of the ribosomal DNA sequences placed all the isolates in Sporothrix. Based on phylogenetic analyses of the ITS (Fig. 1), the isolates emerged as six undescribed taxa. Phylogenetic analysis of the ITS indicated that the unknown species resided in two previously defined Sporothrix species complexes, including the S. gossypina- and S. stenoceras- species complexes, and lineage “F”. Additionally, isolates representing two new species grouped outside any of the currently defined species complexes (Fig. 1). Based on the availability of sequence data for these complexes, different datasets were assembled and analysed separately for each species complex.

Figure 1. 

Phylogram obtained from Maximum Likelihood (ML) analyses of the ITS1-5.8S-ITS2 data for the Sporothrix spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Graphilbum fragrans represent the outgroup.

Seven isolates from hardwood-infesting bark beetles identified as Sporothrix 7 and Sporothrix 8 by Jankowiak et al. (2019a) resided in the S. gossypina-complex (Fig. 1). All three gene regions (ITS, βt, CAL) separated Sporothrix sp. 8 from the other known species with strong statistical support (Figs 2–4). The ITS and βt gene regions grouped isolates of this species together with the ex- type isolate of S. variecibatus, while CAL gene region placed it with S. aurorae (Figs 1–3). Isolates representing Sporothrix sp. 7 had ITS sequences that were almost identical to the ITS sequences for S. fusiformis, S. lunata and S. prolifera (Fig. 1). In the βt and CAL trees (Figs 2, 3), Sporothrix sp. 7 formed lineages that clearly separated this species from the known species in the S. gossypina complex, and although there were differences in the βt sequence compared to other species, the node lacked statistical support (Fig. 2). The combined analyses of the βt and CAL datasets clearly distinguish Sporothrix sp. 7 and Sporothrix sp. 8 into separate lineages within the S. gossypina-complex (Fig. 5).

Figure 2. 

Phylogram obtained from Maximum Likelihood (ML) analyses of βT data for the Sporothrix spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Graphilbum fragrans represent the outgroup.

Figure 3. 

Phylogram obtained from Maximum Likelihood (ML) analyses of CAL data for the Sporothrix spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Graphilbum fragrans represent the outgroup.

The single isolate from a wound on Betula pendula identified as Sporothrix sp. 10 by Jankowiak et al. (2019b), resided in S. stenoceras-complex and grouped closely with S. stenoceras sensu stricto based on analysis of ITS, βt, CAL, and TEF1-α gene regions (Figs 1–4). All three gene regions separated Sporothrix sp. 10 from S. stenoceras, although this separation was not statistically supported by the ITS gene region (Figs 1–4). The combined analyses of the βt and CAL datasets clearly distinguish Sporothrix sp. 10 into separate lineages within the S. stenoceras-complex (Fig. 5).

Two isolates from woodpecker cavities identified as Sporothrix sp. 18 by Jankowiak et al. (2019c), belonged to the lineage F defined by De Beer et al. (2016) based on the ITS tree. All the three gene regions (ITS, βt, CAL) separated Sporothrix sp. 18 from the other known species in lineage F with strong statistical support (Figs 1–4). The combined analyses of the βt and CAL datasets clearly distinguish Sporothrix sp. 18 into separate lineages within the Sporothrix spp. (Fig. 5).

Figure 4. 

Phylogram obtained from Maximum Likelihood (ML) analyses of TEF1-α data for the Sporothrix spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Graphilbum fragrans represent the outgroup.

Fourteen isolates from wounds on different species of hardwood trees and nitidulid beetles identified as Sporothrix sp. 11 and Sporothrix sp. 12 by Jankowiak et al. (2019b) did not group in any of the defined Sprothrix species complexes based on analysis of ITS gene region and formed a monophyletic lineage within Sporothrix (Fig. 1). Isolates of Sporothrix sp. 11 had ITS sequences that were identical with ITS sequences noted in Sporothrix sp. 12. In the βt, CAL, and TEF1-α trees (Figs 2–4), Sporothrix sp. 11 and Sporothrix sp. 12 formed well-supported lineages that clearly separated these two putative new species from each other. The combined analyses of the βt and CAL datasets also separated Sporothrix sp. 11 and Sporothrix sp. 12 from the other known species in Sporothrix spp. and also from each other (Fig. 5).

Figure 5. 

Phylogram obtained from Maximum Likelihood (ML) analyses of the combined βT and CAL sequences of the Sporothrix spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Graphilbum fragrans represent the outgroup.

Morphological characteristics

The six new taxa in Sporothrix emerging from the phylogenetic studies showed differences in colony colour. The cultures of Sporothrix spp. 7, 8, 10 and 11 were white. The cultures of Sporothrix sp. 12 were white or pigmented (white grey) whereas cultures of Sporothrix sp. 18 were greyish green. With the exception of Sporothrix sp. 7 cultures that had an optimum growth at 25 °C followed by 20 °C, all of the undescribed taxa displayed optimum growth at 25 °C followed by 30 °C.

All the new taxa emerging from this study produced micronematous conidiophores and hyaline or pigmented conidia formed holoblastically on denticulate conidiogenous cells. Sporothrix sp. 11 and Sporothrix sp. 12 were characterized by the formation of hyaline and pigmented conidia. Other than Sporothrix sp. 18, which remained asexual, a sexual morph was induced in all five of the other emerging taxa. Ascomata were black and globose with straight necks and up to 700 μm long. Ostiolar hyphae were well-developed and up to 74 μm long. Ascospores were allantoid (Sporothrix sp. 7, 8) or kidney-shaped (Sporothrix spp. 10–12), and they lacked sheaths.

Taxonomy

Sporothrix cracoviensis R. Jankowiak, sp. nov.

MycoBank No: 840460
Fig. 6

Etymology

From Latin, referring to the capital of Małopolskie Voivodeship and the former capital of Poland (Cracovia in Latin, Kraków in Polish); the region where this fungus was collected.

Type

Poland, Małopolskie Province, Krzeszowice, from adult Tryopodendron domesticum beetle on Fagus sylvaticum, January 2014, R. Jankowiak (O-F-258628 holotype, culture ex-type CBS 147942).

Description

Sexual and asexual structures produced on sterilised beech twigs on surface of malt agar in Petri dishes. Ascomata abundant, superficially or partly embedded in the agar, single or in groups; ascomatal bases black, globose, (66–)89–153(–245) μm diam., with brown hyphal hairs, 12 to 165 μm long and 1 to 1.8 μm wide at the base; ascomatal necks black, straight or curved, (187–)272–462(–611) μm long, diameter (9–)10.4–16.7(22.5) μm at the apex and (26.8–)29.9–50.5(–63.9) μm at the base. Ostiolar hyphae present, pale brown, septate, straight or slightly waved, tapering towards the apex or sporadically dichotomous branching at the tip, (7–)8–16(–22) in number (17.8–)29.6–48.4(–64.5) μm long, (0.3–)0.5–1(–1.5) μm at the apex and (1.2–)1.6–2.3–(3) μm at the base. Asci evanescent. Ascospores one-celled, allantoid in side view (2.8–)3.1–3.8(–5.1) × (1–)1.1–1.4(–1.6) μm, elliptical in front view (2.8–)3.1–4.2(–4.8) × (1–)1.2–1.5(–1.8) μm, sometimes with residual sheath up to 1 μm thick, accumulated in creamy-colored mass at the tip of the neck. Conidiophores hyaline, micronematous, simple or branched, straight, simple or branched, bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastics, cylindrical, terminal, lateral or intercalary, straight or curved, tapering towards the apex, swollen apical part forming conidia by sympodial proliferation on visible denticles, (4.2–)17.5–43.1(–72.2) μm long, (0.8–)1.1–1.7(–2.1) μm wide at the base. Apical part with denticles (0.8–)1.3–3.7(–7.3) μm long and (1.2–)1.7–3.7(–7.3) μm wide. Conidia hyaline, unicellular, smooth, obovoid to clavate, sometimes slightly curved, with slightly pointed bases, (2.8–)3.2–6.4(–8.7) × (1.1–)1.4–2.1(–2.7) μm, formed directly on denticles. Culture characteristics: Cultures showing optimal growth at 25 °C (1 mm/d) with somewhat slower growth by at 20 °C (0.8 mm/d), white, flat, floccose, growing in a circular pattern with smooth margins.

Figure 6. 

Sporothrix cracoviensis sp. nov. (CBS 147942) a ascoma b ascomatal base c ostiolar hyphae d ascospores e, f conidiogenous cell with an inflated cluster of denticles at the apex g conidiogenous cells arising directly from hyphae h conidia i fourteen-day-old culture on MEA. Scale bars: 50 μm (a, b), 25 μm (c), 10 μm (d–h).

Host tree

Fagus sylvatica.

Insect vector

Trypodendron domesticum, T. signatum.

Distribution

Poland

Additional specimen examined

Poland, Małopolskie Province, Krzeszowice, from adult Tryopodendron domesticum beetle on Fagus sylvaticum, January 2014, R. Jankowiak (O-F-258629, cultures CBS 147941).

Notes

Sporothrix cracoviensis is phylogenetically distinct from the other Sporothrix species based on the βT, CAL and TEF1-α sequences. This species is closely related to S. fusiformis, S. lunata and S. prolifera. Sporothrix cracoviensis has smaller ascomatal necks (187–611 μm) compared to S. fusiformis (301–1168) μm (Aghayeva et al. 2004). Their conidial dimensions and shapes showed also differences. Sporothrix fusiforme has fusiforme conidia (Aghayeva et al. 2004), whereas S. cracoviensis has obovoid to clavate conidia. Sporothix lunata has also different shape of conidia (crescent) (Aghayeva et al. 2004) compared to S. cracoviensis (obovoid to clavate). In addition, S. lunata has smaller conidia (2.3–6.2 × 0.8–1.6 μm) (Aghayeva et al. 2004) compared to S. cracoviensis (2.8–8.7 μm × 1.1–2.7 μm). Sporothrix prolifera could be distinguished from S. cracoviensis by its smaller ascomatal base (S. prolifera: 65–90 μm (Kowalski and Butin 1989); S. cracoviensis: 66–245 μm) and smaller ascomatal necks (S. prolifera: 75–160 μm (Kowalski and Butin 1989); S. cracoviensis: 187–611 μm). In addition, S. prolifera has shorter ostiolar hyphae (S. prolifera: 15–30 μm (Kowalski and Butin 1989); S. cracoviensis: 26.8–63.9 μm) and shorter and wider ascospores (S. prolifera: 3.2–3.8 × 1.8–2 μm (Kowalski and Butin 1989); S. cracoviensis: 2.8–5.1 × 1–1.6 μm). The conidia of S. prolifera are also smaller (S. prolifera: 4–5.8 × 1.6–2.2 μm (Kowalski and Butin 1989) compared to S. cracoviensis (2.8–8.7 × 1.1–2.7 μm).

Sporothrix cracoviensis was represented by four isolates collected from adult Trypodendron domesticum beetles on Fagus sylvatica. It corresponds to Sporothrix sp. 7 in the study of Jankowiak et al. (2019a).

Sporothrix fraxini R. Jankowiak, sp. nov.

MycoBank No: 840463
Fig. 7

Etymology

From Latin, referring to the genus name of the host (Fraxinus excelsior).

Type

Poland, Małopolskie Province, Zbylitowska Góra, from the gallery of Hylesinus varius on Fraxinus excelsior, May 2016, R. Jankowiak (O-F-258630 holotype, culture ex-type CBS 147936).

Description

Sexual and asexual structures produced on sterilized ash twigs and on surface of malt agar in Petri dishes. Ascomata abundant, superficially or partly embedded in the agar, single or in groups; ascomatal base black, globose, (89–)110–161(–216) μm diam., with brown hyphal hairs, 14 to 65 μm long and 1.1 to 2.1 μm wide at the base; ascomatal necks black, straight or curved, (222–)332–461(–526) μm long, diameter (10.1–)11.3–16(–20.4) μm at the apex and (26.2–)29.1–41.4(–53) μm at the base. Ostiolar hyphae present, pale brown, septate, straight or rather tortuous, tapering towards the apex or sporadically dichotomous branching at the tip, (8–)10–20(–24) in number (21.4–)31.1–52.1(–73.6) μm long, (0.4–)0.7–1.1(–1.4) μm at the apex and (1.4–)1.8–2.4–(3.1) μm at the base. Asci evanescent. Ascospores one-celled, allantoid in side view (2.7–)2.9–3.5(–4.4) × (0.9–)1–1.4(–1.8) μm, elliptical in front view (2.2–)2.9–3.8(–4.7) × (0.8–)1.2–1.6(–1.8) μm, sometimes with residual sheath up to 1 μm thick, accumulated in white-color mass at the tip of the neck. Conidiophores hyaline, micronematous, simple or branched, straight, simple or branched, bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastic, cylindrical terminal or intercalary, straight or curved, tapering towards the apex, swollen apical part forming conidia by sympodial proliferation on hardly visible denticles, (13.6–)14.6–47.7(–99.6) μm long, (0.9–)1.2–1.6(–1.9) μm wide at the base. Apical part (0.8–)1.7–5.1(–10.6) μm long and (0.8–)1.1–2(–3) μm wide. Conidia hyaline, unicellular, smooth, obovoid to ellipsoidal, ends slightly rounded or truncate, (2.6–)3.4–5(–6.8) × (0.8–)1.1–1.6(–2) μm, formed directly on denticles. Culture characteristics: Cultures showing optimum growth at 25 °C (1 mm/d) followed by at 30 °C (0.9 mm/d), white, flat, growing in a circular pattern with smooth margins, with sparse aerial mycelium, often fading around the edges.

Figure 7. 

Sporothrix fraxini sp. nov. (CBS 147936) a ascoma b ascomatal base c ostiolar hyphae d ascospores e conidiogenous cell with an inflated cluster of denticles at the apex f conidia g fourteen-day-old culture on MEA. Scale bars: 100 μm (a), 50 μm (b), 25 μm (c), 10 μm (d–f).

Host tree

Fraxinus excelsior.

Insect vector

Hylesinus crenatus, H. varius.

Distribution

Poland

Additional specimen examined

Poland, Małopolskie Province, Zbylitowska Góra, from the gallery of Hylesinus varius on Fraxinus excelsior, May 2016, R. Jankowiak (O-F-258631, cultures CBS 147938).

Notes

This species is phylogenetically distinct from the other Sporothrix species based on the ITS, βT, CAL and TEF1-α sequences. Sporothrix fraxini is closely related to S. variecibatus. However, S. variecibatus does not produce a sexual morph, and has narrower conidia (2–3 μm) (Roets et al. 2008) compared to S. fraxini (0.8–2 μm). In addition, the conidia of S. variecibatus are clavate while S. fraxini has obovoid to ellipsoidal conidia.

Sporothrix fraxini was represented by three isolates collected from the galleries of Hylesinus varius on Fraxinus excelsior. It corresponds to Sporothrix sp. 8 in the previous study of Jankowiak et al. (2019a).

Sporothrix resoviensis R. Jankowiak & A. Ostafińska, sp. nov.

MycoBank No: 840475
Fig. 8

Etymology

From Latin, referring to the capital of Podkarpackie Voivodeship (Resovia in Latin, Rzeszów in Polish), the region from which this fungus was collected.

Type

Poland, Podkarpackie Province, Borownica, from the wound on Betula pendula, June 2016, A. Ostafińska, (O-F-258632 holotype, culture ex-type CBS 147927).

Description

Sexual and asexual structures produced on sterilised birch twigs and on surface of malt agar in Petri dishes. Ascomata abundant, superficially or partly embedded in the agar, single or in groups; ascomatal bases black, globose, (87–)113–184(–232) μm diam., with brown hyphal hairs, 14 to 44 μm long and 0.9 to 2.2 μm wide at the base; ascomatal necks black, straight or curved, often extended at the base, (228–)378–624(–700) μm long, diameter (10–)11.2–17(–20.2) μm at the apex and (26.2–)34–47.7(–56) μm at the base. Ostiolar hyphae present, pale brown, septate, straight or curved, tapering towards the apex and often swollen at the tip, (7–)9–15(–18) in number, (15.7–)26.1–47.7(–67.6) μm long, (0.3–)0.7–1.5(–2.5) μm at the apex and (1.3–)2–3–(3.4) μm at the base. Asci evanescent. Ascospores one-celled, kidney-shaped to almost triangular in side view (2.7–)3.2–3.9(–4.4) × (1.4–)1.7–2.1(–2.3) μm, oblong-elliptical in front view (2.6–)3–3.8(–4.9) × (1.4–)1.7–2.2(–2.6) μm, without residual sheath accumulated in white-colored mass at the tip of the neck. Conidiophores hyaline, micronematous, straight, simple and bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastic, cylindrical, terminal, lateral or intercalary, straight or curved, swollen apical part forming conidia by sympodial proliferation on easily visible denticles, (3.1–)9.3–57(–120.1) μm long, (1–)1.1–1.6(–2.2) μm wide at the base. Apical part (1.3–)1.9–3.5(–4.4) μm long and (1.4–)2.4–3.9(–4.5) μm wide. Conidia hyaline, unicellular, smooth, obovate to ellipsoidal, pointed at the base, (3.9–)4.3–6.7(–8.5) × (2.1–)2.4–3.4(–4) μm, formed singly on denticles or on the side of vegetative hyphae. Culture characteristics: Cultures showing optimum growth at 25 °C (1.8 mm/d) followed by at 30 °C (1.7 mm/d), white, growing in a circular pattern with smooth margins, funiculose and woolly.

Figure 8. 

Sporothrix resoviensis sp. nov. (CBS 147927) a ascoma b, c ostiolar hyphae d ascospores e–g conidiogenous cell with an inflated cluster of denticles at the apex h conidia i fourteen-day-old culture on MEA. Scale bars: 250 μm (a), 25 μm (b, c), 10 μm (d), 25 μm (e), 10 μm (f–h).

Host trees

Betula pendula.

Insect vector

unknown.

Distribution

Poland.

Note

Sporothrix resoviensis is phylogenetically distinct from the other Sporothrix species based on the ITS, βT, CAL and TEF1-α sequences. This species grouped most closely with S. stenoceras but can be distinguished by its larger ascospores (S. resoviensis: 2.7–4.4 × 1.4–3.3 μm; S. stenoceras: 2.0–2.9 × 1.3–1.4 μm (Robak 1932). Perithecia developing on the agar medium and twigs have significantly shorter necks (S. resoviensis: 228–700 μm; S. stenoceras: 450–1500 μm (Robak 1932). Sporothrix resoviensis has larger conidia (3.9–8.5 × 2.1–4 μm) compared to S. stenoceras (3.4–6.9 × 2–3.4 μm). This new species also differs from S. stenoceras based on culture morphology, where S. resoviensis produces wooly cultures, different to the sparse and flat mycelium of S. stenoceras (Robak 1932).

Sporothrix resoviensis was represented by one isolate collected from a wound on Betula pendula. It corresponds to Sporothrix sp. 10 in the study of Jankowiak et al. (2019b).

Sporothrix cryptarchum R. Jankowiak & A. Ostafińska, sp. nov.

MycoBank No: 840477
Fig. 9

Etymology

Referring to the genus name of the beetle, Cryptarcha sp. (Coleoptera: Nitidulidae), with which this fungus is associated.

Type

Poland, Małopolskie Province, Wierzchosławice, from Cryptarcha undata on Quercus robur, June 2016, R. Jankowiak, (O-F-258633 holotype, culture ex-type CBS 147934).

Description

Sexual and asexual structures produced on the sterilised oak twigs and on the surface of malt agar in Petri dishes. Ascomata abundant, superficially or partly embedded in the agar, single or in groups; ascomatal bases black, globose, (55–)115–172(–210) μm diam., with brown hyphal hairs, 15 to 141 μm long and 0.9 to 3.8 μm wide at the base; ascomatal necks black, straight or curved, (126–)198–412(–544) μm long, diameter (10.9–)13–19(–23.8) μm at the apex and (17.6–)29.3–47.6(–59.6) μm at the base. Ostiolar hyphae present, pale brown, with small granules, septate, straight or curved, simple or dichotomous branching, tips tapering or sometimes thickened, (9–)13–24(–31) in number, (15.8–)30.5–51.8(–60.9) μm long, (0.2–)0.3–0.5(–0.7) μm at the apex and (0.9–)1.6–2.4–(3) μm at the base. Asci subglobose to ovoid, (5.5–)6.7–9(–11) × (4–)4.9–6.2(–7.2) μm. Ascospores one-celled, kidney-shaped to almost triangular in side view in side view (3.2–)3.8–4.7(–5.8) × (0.8–)1–1.3(–1.5) μm, elliptical in front view (3.1–)3.6–4.4(–5) × (1–)1.2–1.6(–1.8) μm, sometimes with residual sheath up to 0.6 μm thick, accumulated in white-colored mass at the tip of the neck. Conidiophores hyaline, micronematous, simple or occasionally branched and bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastic, cylindrical, terminal, lateral or intercalary, straight or curved, tapering towards the apex, swollen apical part forming conidia by sympodial proliferation on narrow denticles, (2.2–)13.9–51.2(–102.8) μm long, (0.7–)1.2–1.8(–2.2) μm wide at the base. Apical part (0.6–)1.4–3.1(–5.3) μm long and (1–)1.7–3(–3.8) μm wide, single denticles often below. Conidia of two types: 1) abundant in cultures, often produced, hyaline, unicellular, smooth, obovate to ellipsoid, pointed at the base, (3.3–)4.6–8.1(–10.3) × (1–)1.3–1.9(–2.2) μm, formed directly on denticles; 2) sparse in cultures, subhyaline to lightly pigmented, unicellular, smooth, subglobose to globose, (2.3–)3.1–4.1(–4.5) μm diam, formed singly, either directly on the side of vegetative hyphae or on short lateral branches. Culture characteristics: Cultures showing optimum growth at 25 °C (1.3 mm/d) followed by at 30 °C (1.1 mm/d), mostly pigmented or white or pig, flat, growing in a circular pattern with smooth margins.

Figure 9. 

Sporothrix cryptarchum sp. nov. (CBS 147934) a ascoma b ascomatal base c, d ostiolar hyphae e ascospores f asci g conidiogenous cell with an inflated cluster of denticles at the apex h conidia i globose conidia arising on long conidiophore j globose conidia arising directly from hyphae k fourteen-day-old culture on MEA. Scale bars: 100 μm (a), 25 μm (b–d), 10 μm (e), 25 μm (f, g), 10 μm (h, i), 5 μm (j).

Host tree

Alnus glutinosa, Quercus robur.

Insect vector

Cryptarcha undata, C. strigata.

Distribution

Poland.

Additional specimen examined

Poland, Małopolskie Province, Wierzchosławice, from Cryptarcha undata on Quercus robur, June 2016, R. Jankowiak, (O-F-258634, cultures CBS 147933).

Notes

This species is phylogenetically distinct from the other Sporothrix species based on the ITS, βT, CAL and TEF1-α sequences. Sporothrix cryptarchum is phylogenetically closely related to S. undulata (Sporothrix sp. 12) described in the present study. This species also shares morphological similarities such as kidney-shaped ascospores and two morphological forms of conidia with S. undulata. However, S. cryptarchum has narrow ascospores (0.8–1.5 µm) compared to S. undulata (1.1–2 µm). It also has distinct ostiolar hyphae, with those in S. cryptarchum often dichotomously branching while in S. undulata these hyphae occur only sporadically and do not have dichotomous branching. Both species produce hyaline and pigmented conidia. However, S. cryptarchum cultures are predominantly hyaline whereas those in pure cultures of S. undulata are mostly pigmented. Their conidial shapes in these two species are similar but their dimensions are distinct. Sporothrix cryptarchum has conidia that are smaller than those of S. undulata. In addition, cultures of S. cryptarchum are white and grow in a circular pattern with smooth margins while those of S. undulata grow in a circular pattern with undulate margins and some have grey pigmentation.

Sporothrix cryptarchum was represented by four isolates collected from Poland. It corresponds to Sporothrix sp. 11 in the study of Jankowiak et al. (2019b). Sporothrix cryptarchum was isolated from wounds on hardwood trees and nitidulid beetles (Coleoptera: Nitidulidae), which visited fresh wounds on Quercus robur.

Sporothrix undulata R. Jankowiak & A. Ostafińska, sp. nov.

MycoBank No: 840478
Fig. 10

Etymology

Referring to the aerial mycelium growing in undulating concentric zones on MEA.

Type

Poland, Małopolskie Province, Wierzchosławice, from Epuraea guttata on Quercus robur, June 2016, R. Jankowiak, (O-F-258635 holotype, culture ex-type CBS 147929).

Description

Sexual and asexual structures produced on sterilised oak twigs and on surface of malt agar in Petri dishes. Ascomata abundant, superficially or partly embedded in the agar, single or in groups; ascomatal base black, globose, (65–)95–186(–223) μm diam., with brown hyphal hairs, 8 to 134 μm long and 1.2 to 3.1 μm wide at the base; ascomatal necks black, straight or curved, (114–)174–482(–697) μm long, diameter (9.1–)12.3–18.7(–24.2) μm at the apex and (14.7–)22–40.3(–58.7) μm at the base. Ostiolar hyphae present, pale brown, with small granules, septate, straight or slightly waved, tapering towards the apex or sporadically dichotomously branched at the tip, (9–)16–28(–31) in number, (29.4–)39.9–59.5(–72) μm long, (0.4–)0.6–1(–1.1) μm at the apex and (1.5–)1.8–2.7–(3.3) μm at the base. Asci subglobose to ovoid, (5.7–)6.7–8.5(–9.4) × (3.4–)4.4–5.8(–6.4) μm. Ascospores one-celled, kidney-shaped to almost triangular in side view (3.4–)3.8–4.6(–4.9) × (1.1–)1.4–1.7(–2) μm, elliptical in front view (3.2–)3.5–4.5(–5.6) × (0.9–)1.5–2.1(–2.8) μm, sometimes with residual sheath up to 0.6 μm thick, accumulated in white-colored mass at the tip of the neck. Conidiophores hyaline, micronematous or semimacronematous, simple or occasionally branched and bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastic, cylindrical, terminal, lateral or intercalary, straight or curved, slightly tapering towards the apex, swollen apical part forming conidia by sympodial proliferation on small or hardly visible denticles, (5.2–)11.3–50.4(–112.2) μm long, (0.9–)1.3–1.8(–2.1) μm wide at the base. Apical part (1.1–)1.6–3.4(–5.9) μm long and (1.1–)1.7–3.5(–5.4) μm wide. Conidia of two types: 1) sparsely in cultures, hyaline, unicellular, smooth, ellipsoid, pointed at the base, (3.2–)4.2–7.8(–11.7) × (1.4–)1.7–2.4(–3.5) μm, formed directly on denticles; 2) abundant in cultures, subhyaline to lightly pigmented, unicellular, smooth, subglobose to globose, sometimes pointed at the base, (2.1–)2.9–4.2(–5.5) μm diam, formed singly or in chains, either directly on the side of vegetative hyphae, on short lateral branches or denticles. Culture characteristics: Cultures showing optimum growth at 25 °C (1.2 mm/d) with growth somewhat slower at 20 °C and 30 °C (0.9 mm/d), white or white grey, flat, growing in a circular pattern with undulate margins.

Figure 10. 

Sporothrix undulata sp. nov. (CBS 147929) a ascoma b ascomatal base c ostiolar hyphae d asci e ascospores f–h globose conidia arising on long conidiophore or directly from hyphae i globose conidia j conidiogenous cell with an inflated cluster of denticles at the apex k conidia l–m fourteen-day-old culture on MEA (left- pigmented CBS 147929, right – white KFL404DB16bRJCU). Scale bars: 100 μm (a), 25 μm (b–d), 10 μm (e), 25 μm (f), 10 μm (g, h), 5 μm (i), 10 μm (j), 5 μm (k).

Host tree

Alnus glutinosa, Carpinus betulus, Fagus sylvatica, Quercus robur, Quercus rubra, Salix fragilis.

Insect vector

Cryptarcha undata, Epuraea guttata.

Distribution

Poland.

Additional specimen examined

Poland, Małopolskie Province, Wierzchosławice, from wound on Quercus robur, October 2015, R. Jankowiak (O-F-258636, cultures CBS 147931).

Notes

This species is phylogenetically distinct from the other Sporothrix species based on the ITS, βT, CAL and TEF1-α sequences. Sporothrix undulata is phylogenetically closely related to S. cryptarchum described in this study. The morphological differences between S. undulata and S. cryptarchum are described in the section above treating S. cryptarchum.

Sporothrix undulata was represented by nine isolates collected from Poland. It corresponds to Sporothrix sp. 12 in the study of Jankowiak et al. (2019b). In this study S. undulata was isolated from wounds on hardwood trees and from adults of nitidulid beetles (Coleoptera: Nitidulidae), which visited wounds on Quercus robur.

Sporothrix cavum R. Jankowiak, sp. nov.

MycoBank No: 840479
Fig. 11

Etymology

From Latin, referring to the hollow cavities produced by woodpeckers and from which this fungus was collected.

Type

Poland, Małopolskie Province, Kraków, from the cavity of Dendrocopos major on Salix fragilis, December 2015, R. Jankowiak, (O-F-258637 holotype, culture ex-type CBS 147943).

Description

Sexual morph not observed. Asexual structures produced on sterilized beech twigs placed on the surface of malt agar in Petri dishes. Conidiophores hyaline, micronematous, simple, straight, simple or branched, bearing several conidiogenous cells, either borne on vegetative hyphae or on upright hyphae. Conidiogenous cells blastic, cylindrical, terminal, lateral or intercalary, straight or curved, slightly tapering toward the apex, swollen apical part forming conidia by sympodial proliferation on well-developed denticles, (2.8–)11.5–32.8(–54.4) μm long, (0.7–)1.1–1.7(–2.4) μm wide at the base. Apical part with denticles (1.2–)1.5–2.8(–4.4) μm long and (1.4–)1.8–2.6(–3.1) μm wide, individual denticles often formed below aplical part. Conidia hyaline, unicellular, smooth, obovoid, with pointed bases, (3.1–)3.6–5.5(–7.8) × (1.7–)2–2.7(–3.2) μm, formed on terminal or lateral denticles, either directly on the side of vegetative hyphae. Culture characteristics: Cultures having optimum growth at 25 °C (1.7 mm/d) followed by at 30 °C (1.5 mm/d), growing well at 35 °C (0.6 mm/d), greyish green, with a darker centre, flat, growing in a circular pattern with smooth margins and abundant aerial mycelium.

Figure 11. 

Sporothrix cavum sp. nov. (CBS 147943) a–c conidiogenous cell with an inflated cluster of denticles at the apex and below apex d conidiogenous cells arising directly from hyphae e conidia f fourteen-day-old culture on MEA. Scale bars: 10 μm (a), 25 μm (b), 10 μm (c–e).

Host tree

Malus domestica, Salix fragilis

Insect vector

unknown

Distribution

Poland

Additional specimen examined

Poland, Małopolskie Province, Książ Wielki, from the cavity of Dendrocopos medius on Malus domestica, (O-F-258638, cultures ex-paratype KFL=NRFI 35614DR).

Notes

This species is phylogenetically distinct from the other Sporothrix species based sequences for the ITS, βT, CAL and TEF1-α regions. Sporothrix cavum is related to S. polyporicola based on analyses of the ITS sequences. However, S. cavum in contrast to S. polyporicola, does not produce a sexual morph (Constantinescu and Ryman 1989). In addition, S. cavum has obovoid and short conidia (3.1–7.8 μm), whereas S. polyporicola has clavate and longer conidia (6–14 μm) (Constantinescu and Ryman 1989).

Sporothrix cavum was represented by two isolates collected from the cavities produced by the woodpeckers Dendrocopos major on Salix fragilis and Dendrocopos medius on Malus domestica. It corresponds to Sporothrix sp. 18 in the study of Jankowiak et al. (2019c).

Discussion

Our work (Jankowiak et al. 2019a, 2019b, 2019c; this study) has led to the discovery of six novel Sporothrix species associated with hardwood trees in Poland. Description of these new species brings the total number of species in this genus to 62, of which 16 occur in Poland. These include the six species described here as well as S. aurorae (Jankowiak et al. 2019b), S. cantabriensis (Jankowiak et al. 2017), S. dentifunda (Aghayeva et al. 2005, Jankowiak et al. 2019b), S. eucastaneae (Jankowiak et al. 2019a, 2019b, 2021), S. fusiformis (Jankowiak et al. 2019a, 2019b), S. inflata (Jankowiak et al. 2012; Jankowiak and Bilański 2013a, 2013b), S. inflata ‘2’ (Jankowiak et al. 2019a, 2019b), S. prolifera (Kowalski and Butin 1989; Jankowiak et al. 2019a, 2019b), S. stenoceras, (Kowalski and Butin 1989; Jankowiak and Bilański 2013b, Jankowiak et al. 2019b) and S. variecibatus (Jankowiak and Bilański 2013b).

All of the species described in this study are morphologically similar, having asexual states with hyaline or lightly pigmented conidia produced holoblastically on denticulate conidiogenous cells or directly from the hyphae. Where ascomata were present, these tended to have globose bases with elongated necks terminating in long ostiolar hyphae and allantoid or kidney-shaped ascospores not surrounded by hyaline sheaths. All of the newly described species grew optimally at 25 °C and all also grew well at 30 °C on MEA. Sporothrix undulata and S. cavum differed from the other four species in having pigmented as opposed to white cultures on MEA. All of the newly described species were recovered from hardwood ecosystems in Poland in association with bark and ambrosia beetles, nitidulid beetles, naturally occurring tree wounds or woodpecker cavities.

The six species described in this study can easily be distinguished from each other and from the other species of Sporothrix based on the DNA sequence comparisons. Analyses of the ITS sequence data were insufficient to distinguish between S. cryptarchum and S. undulata or between S. cracoviensis and S. fusiformis. However, analyses of sequence data for the protein-coding genes, including the βT, CAL and TEF1-α showed that S. cracoviensis, S. cryptarchum, and S. undulata represent distinct taxa. Furthermore, the two closely related species, S. cryptarchum and S. undulata formed a new and well-supported lineage in Sporothrix including species infecting wounds on a variety of hardwood trees. The species in this lineage are characterised by having both hyaline as well as pigmented conidia and kidney-shaped ascospores.

The asexual morphs of the Sporothrix species described in this study had variable morphology. All species had hyaline conidia produced holoblastically on denticulate conidiogenous cells that proliferate sympodially or arise directly from hyphae. Sporothrix cryptarchum and S. undulata also had pigmented globose conidia formed singly or in chains, either directly on the sides of the vegetative hyphae or on short lateral branches. The presence of two different conidial types has previously been found in other Sporothrix species, including Sporothrix dimorphospora and S. brunneoviolacea (Madrid et al. 2010) as well as S. brasiliensis, S. globose, and S. mexicana (Marimon et al. 2007).

Recently, Jankowiak et al. (2019b) provided evidence that fresh wounds on hardwood trees in Europe are preferred habitats for some Sporothrix species. These authors isolated 15 Sporothrix species from trees belonging to 12 species of angiosperms. Likewise, nine Sporothrix species have been described from fresh wounds on non-native Eucalyptus spp. and various genera of native trees in South Africa (Kamgan Nkuekam et al. 2012; Musvuugwa et al. 2016, 2020; Osorio et al. 2016).

Three species of wound-associated Sporothrix spp. collected during a survey reported in the study of Jankowiak et al. (2019b) were included in the present study. The greatest number of isolates (194) obtained during that survey were those of S. undulata. This species was found as a common associate of bleeding wounds on Quercus robur and Salix fragilis, suggesting that they might have some level of pathogenicity. The other species inhabiting wounds on hardwood trees that was collected during the survey of Jankowiak et al. (2019a) was S. cryptarchum (34 isolates). Transfer of this species to the sampled tree wounds was most likely by nitidulid (Coleoptera, Nitidulidae) beetles as previously noted by Jankowiak et al. (2019b) who suggested that these insects commonly transmit Ophiostomatales, including Sporothrix species to tree wounds in Poland. Likewise, Kamgan Nkuekam et al. (2012) have demonstrated that the nitidulid beetles Brachypeplus depressus and Carpophilus spp. vector S. candida and S. fumea in the Eucalyptus plantations of South Africa. This association is also consistent with other studies providing compelling evidence that nitidulid beetles act as vectors of the well-known pathogens, such as Bretziella fagacearum (De Beer et al. 2017; Jagemann et al. 2018) and Ceratocystis albifundus (Heath et al. 2009).

The second largest number of isolates (81 in total) included in this study represented two species in the S. gossypina-complex, bringing the total number of species in that complex to 15 (De Beer et al. 2016; Wang et al. 2019). Sporothrix cracoviensis was represented by 45 isolates from the ambrosia beetles Trypodendron domesticum and T. signatum collected on Fagus sylvatica (Jankowiak et al. 2019a). This is not unusual given that an association between ambrosia beetles has recently been recorded by De Errasti et al.(2016) in a study on Nothofagus pumelo in Patagonia. The other species residing in this complex collected during the survey of Jankowiak et al. (2019a) is S. fraxini (36 isolates). This fungus was found on Fraxinus excelsior in association with the bark beetles Hylesinus crenatus and H. varius (Jankowiak et. al. 2019a).

The Polish study by Jankowiak et al. (2019a) revealed that, apart from S. cracoviensis and S. fraxini, five other Sporothrix species (S. fusiformis, S. prolifera, S. eucastanea, Sporothrix sp. 4, Sporothrix sp. 9) were associated with bark beetles. These findings confirm that most species in the S. gossypina complex are associated with galleries of conifer-infesting bark beetles worldwide (De Beer et al. 2016). The other species in the S. gossypina-complex were isolated from the stained oak wood (Kowalski and Butin 1989; Aghayeva et al. 2004), cankers caused by Cryphonectria parasitica on chestnut (Davidson 1978), a hardwood tree native to South Africa (Musvuugwa et al. 2016), and from mites infesting the infructescences (flower heads) of Protea in South Africa (Roets et al. 2008).

Sporothrix cavum, the remaining taxon collected from hardwood trees during the surveys that formed the basis of the present study, resided in lineage F defined by De Beer et al. (2016). This lineage includes three species, namely S. polyporicola, S. dimorphospora, and S. inflata ‘2’. Two of these species (S. dimorphospora, and S. inflata ‘2’) are known from soil and S. polyporicola was isolated from basidiocarps of the polypores Fomitopsis pinicola and Amaropostia stiptica (Constantinescu and Ryman 1989; Madrid et al. 2010). The results of the present study show that species in this complex also accommodate wood-inhabiting Sporothrix species. Other than the fact that S. cavum was isolated from cavities on Salix fragilis and Malus domestica made by woodpeckers (Jankowiak et al. 2019c), nothing is known regarding the ecology or distribution of the fungus. It could, for example, be introduced into these cavities by arthropods or have some relationship with the woodpeckers themselves.

The results of this study have substantially expanded our knowledge of Sporothrix and the ecology of species in this genus. Broadly, the results suggest that Sporothrix species are common members of the Ophiostomatales in hardwood ecosystems in Poland. Furthermore, interesting questions have arisen that should shape future investigations regarding these fungi.

Acknowledgements

This work was supported by the National Science Centre, Poland (contract No. UMO-2014/15/NZ9/00560) and the Ministry of Science and Higher Education of the Republic of Poland.

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