https://orcid.org/0000-0001-8679-0995
ABSTRACT
Tulasnella (Tulasnellaceae) is a genus of fungus that can form mycorrhizal associations with orchids (Orchidaceae). Here we used molecular phylogenetic analyses and morphological characteristics of pure cultures across four different media to support the description of five new Tulasnella species associated with commonly occurring and endangered Australian orchids. Tulasnella nerrigaensis associates with Calochilus; T. subasymmetrica and T. kiataensis with Thelymitra; and T. korungensis and T. multinucleata with Pyrorchis and Rimacola respectively. The newly described species were primarily delimited by analyses of five loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), C14436 (adenosine triphosphate [ATP] synthase), C4102 (glutamate synthase), C3304 (ATP helicase), and mt large subunit 16S rDNA (mtLSU). Tulasnella subasymmetrica is introduced for some isolates previously identified as T. asymmetrica, and this latter species is characterized from multilocus sequencing of a new isolate that matches ITS sequences from the ex-type culture. Morphological differences between the new species are slight. Tulasnella multinucleata has 6–12 nuclei per hyphal compartment which is the first instance of multinucleate rather than binucleate or trinucleate hyphal compartments in Tulasnella. The formal description of these species of Tulasnella will aid in future evolutionary and ecological studies of orchid-fungal interactions.
INTRODUCTION
Over one hundred Tulasnella J. Schröt. (Cantharellales, Tulasnellaceae Juel) species are described worldwide (www.indexfungorum.org). Previously, asexual forms of Tulasnella were placed in Epulorhiza R.T. Moore (Moore Citation1987); however, based on the concept “one fungus, one name” (Hawksworth Citation2011), Epulorhiza is now treated as a synonym of Tulasnella (Stalpers et al. Citation2021). Species of Tulasnella have been isolated from orchids around the world and have been found to form mycorrhizal associations with over 40 orchid genera (Oberwinkler et al. Citation2017; Rasmussen et al. Citation2015; Yukawa et al. Citation2009). Species of Tulasnella can: form associations with liverworts in the Aneuraceae (Kottke et al. Citation2008; Krause et al. Citation2011; Preußing et al. Citation2010); grow as saprotrophs in decayed wood (Cruz et al. Citation2014; Mack et al. Citation2021; Roberts Citation1992, Citation1993); and play an important role as ectomycorrhizal fungi of forest trees (Bidartondo et al. Citation2004; Solís et al. Citation2017; Tedersoo et al. Citation2010).
Tulasnella appears to have higher molecular evolutionary rates compared with other orchid mycorrhizal fungi in the cantharelloid clade, e.g., Ceratobasidium and Serendipita (Binder et al. Citation2005; Moncalvo et al. Citation2006). This is indicated by the difficulty of aligning the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) across the genus, with four phylogenetic groups identified within Tulasnella by Cruz et al. (Citation2014). Furthermore, morphological features of Tulasnella species are often similar in shape and size (Roberts Citation1993, Citation1994a, Citation1994b). Due to this, and the difficulty of inducing the formation of basidia and basidiospores, multilocus sequence data are necessary to distinguish amongst morphologically cryptic Tulasnella species (Arifin et al. Citation2021).
In recent years, many novel species of Tulasnella have been described, often from orchid hosts. Within Tulasnella group IV, 10 new species have been described from Australian orchids of the subtribes Drakaeinae and Cryptostylidinae, using morphology and multilocus DNA sequencing (Arifin et al. Citation2021; Linde et al. Citation2017). Furthermore, five species were described that fell into group II: T. phuhinrongklaensis C. Rachanarin, N. Suwannarach & S. Lumyong (Rachanarin et al. Citation2018) from Phalaenopsis pulcherrima in Thailand, T. tubericola K. Solís, J. Barriuso, A. Garcés-Claver & V. González (Solís et al. Citation2017) from oak trees in Spain, and three Tulasnella species from Spiranthes sinensis var. amoena in Japan (T. cumulopuntioides S. Fujimori, J.P. Abe, I. Okane & Y. Yamaoka, T. dendritica S. Fujimori, J.P. Abe, I. Okane & Y. Yamaoka, and T. ellipsoidea S. Fujimori, J.P. Abe, I. Okane & Y. Yamaoka) (Fujimori et al. Citation2019). A further four new Tulasnella from endangered orchid species in Brazil (Freitas et al. Citation2020) were described, which were placed in group III based on the classification by Cruz et al. (Citation2014). Recently, Hormomyces Bonord. has been recognized as a synonym of Tulasnella, resulting in the transfer of the type (Hormomyces aurantiacus Bonord.) to Tulasnella, as T. aurantiaca (Bonord.) J. Mack & Seifert, a saprobic species on rotten wood, belonging to group IV (Mack et al. Citation2021).
In recent classifications of the Orchidaceae tribe Diurideae, the subtribe Megastylidinae is composed of seven genera: Burnettia, Leporella, Lyperanthus, Megastylis, Pyrorchis, Rimacola, and Waireia, with each genus consisting of a maximum of two species (except Megastylis where seven species have been described) (Chase et al. Citation2015; Weston et al. Citation2014). The Thelymitrinae consists of two large genera, Calochilus and Thelymitra, with more than 30 and 100 species, respectively, and one monotypic genus, Epiblema (Nargar et al. Citation2018; Nauheimer et al. Citation2018; Weston et al. Citation2014). Many as yet undescribed Tulasnella species were detected in previous studies in some of these orchid genera (Bonnardeaux et al. Citation2007; Reiter et al. Citation2018; Sommer et al. Citation2012).
Over several decades from the 1960s, J. H. Warcup and P. H. B. Talbot made extensive studies on the fungi associated with Australian orchids. According to a compilation of Tulasnella isolates obtained by Warcup and Talbot from Australian orchids (Linde et al. Citation2017: tables 2 and 3), such isolates from orchid genera in the Megastylidinae and Thelymitrinae were assigned to two species described as new from Australian material, T. asymmetrica Warcup & P.H.B. Talbot from Thelymitra and T. cruciata Warcup & P.H.B. Talbot from Acianthus and Thelymitra, and also to T. calospora (Boud.) Juel and T. violea (Quél.) Bourdot & Galzin (Warcup and Talbot Citation1967, Citation1971). Based on morphological features, Roberts (Citation1999) placed T. asymmetrica under T. pinicola Bres., but he did not examine the type collection of the former. Phylogenetic analyses including sequences from ex-type and other cultures indicate that there is diversity among isolates assigned to Tulasnella asymmetrica by Warcup, with Cruz et al. (Citation2014) referring to two distinct clades of T. asymmetrica and “T. asymmetrica.” In addition, phylogenetic analyses indicate that further undescribed species exist in association with orchids in the Megastylidinae and Thelymitrinae (Reiter et al. Citation2018; Sommer et al. Citation2012).
The aim of this study is to investigate the morphology and phylogenetic relationships among Tulasnella from selected Australian orchids in the subtribes Megastylidinae and Thelymitrinae, which we hypothesize will belong to novel species. We formally describe five new Tulasnella species that associate with genera of orchids from both subtribes (Calochilus and Thelymitra from Thelymitrinae and Pyrorchis and Rimacola from Megastylidinae). We clarify the application of the name Tulasnella asymmetrica, placing some isolates formerly assigned to this species under one of the novel species. New species are delimited using multilocus sequence analyses. Details on culture morphology, microscopic characteristics, and nuclear characterization are also provided for each new species.
MATERIALS AND METHODS
Orchid collections and fungal isolation.—
Orchids in Thelymitrinae (Calochilus) and Megastylidinae (Pyrorchis and Rimacola) were collected to explore their Tulasnella associations. Root samples of Rimacola elliptica were collected from one population (two plants) in Morton National Park, New South Wales (Global Positioning System [GPS]: 34°40ʹ30″S, 150°17ʹ31.2″E); Pyrorchis forrestii roots were sampled from Blue Lake Road in Western Australia (three plants; GPS: 34°45ʹ58.5″S, 117°17ʹ0.4″E); P. nigricans roots were obtained from two populations in Western Australia (three plants per population) at Pomeroy Road in Korung National Park (GPS: 32°4ʹ51.6″S, 116°4ʹ51.5″E) and Lake Unicup (GPS: 34°26ʹ11.5″S, 116°37ʹ3.4″E); and Calochilus robertsonii (three plants) was sampled near Oallen Ford Road, Nerriga, in New South Wales (GPS: 35°7ʹ37.2″S, 150°1ʹ26.4″E). During transportation, all samples were kept moist in damp paper towel and fungal isolations were conducted within a maximum of 2 days after sampling. Fungal isolation from root samples onto half-strength (½) fungal isolation medium (½ FIM; glucose was used instead of sucrose in this study) (Clements and Ellyard Citation1979), supplemented with 50 mg/L streptomycin sulfate (Merck, Darmstadt, Germany), followed methods described in Arifin et al. (Citation2021). A small piece (1 cm) of peloton-rich tissue root sampled was lyophilized and preserved for direct sequencing of fungal associates for some plants (subject to root material availability after fungal isolations). Fungi from two undescribed Tulasnella operational taxonomic units (OTUs) (“T. asymmetrica” and OTU1) associated with Thelymitra epipactoides (Diurideae, Thelymitrinae), isolation and collection of which were described in Reiter et al. (Citation2018), were also investigated and described in this study. Voucher specimens of the fungi were deposited as living cultures (stored metabolically inactive at −80 C) in the Royal Botanic Gardens Victoria Living Collection (RBGV-LC) and Victorian Plant Pathology Herbarium (VPRI) or lodged as freeze-dried cultures at the National Herbarium of Victoria (MEL).
DNA extraction and sequencing.—
To obtain fungal mycelium for DNA extraction, Tulasnella-like isolates were inoculated in liquid ½ FIM amended with 50 mg/L streptomycin and incubated for 4 wk at 22 C in the dark. Mycelia were harvested and lyophilized with a freeze dryer for 24 h. Dried mycelium were stored at −18 C prior to DNA extraction using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Nuc rDNA ITS region (ITS1-5.8S-ITS2) was amplified and sequenced using the primers ITS5 and ITS4 (Bruns and Gardes Citation1993; White et al. Citation1990). Two nuclear loci, C4102, which encodes glutamate synthase, and C3304, which encodes adenosine triphosphate (ATP) helicase, were amplified and sequenced using primers C4102F/R and C3304F/R, respectively (Linde et al. Citation2014; Ruibal et al. Citation2013). Furthermore, two mitochondrial loci that encode ATP synthase (C14436) and mitochondrial large subunit 16S rDNA (mtLSU) were amplified and sequenced, following Ruibal et al. (Citation2013) and Bruns et al. (Citation1998). To construct a comprehensive phylogenetic tree of C3304, this locus was also amplified and sequenced in this study for five Tulasnella species previously described by Arifin et al. (Citation2021). Because only ITS sequences of T. asymmetrica were available on GenBank, we sequenced all five loci of T. asymmetrica isolate CLM2287 (isolate kindly provided by John Dearnaley, University of Southern Queensland, isolated from Bulbophyllum bracteatum, Queensland, Australia) and included this specimen in our analyses. Sequencing results were checked and manually edited using Sequencher 5.4.1 (Genes Codes, Ann Arbor, Michigan). All DNA sequences generated in this study were deposited in GenBank ().
Table 1. Tulasnella isolates and GenBank accession numbers of multiple loci used to construct concatenated phylogeny in this study
For direct sequencing from peloton-rich root tissue, genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). The ITS region was amplified using the Tulasnella-specific primer combination of ITS5 and ITS4-Tul (Taylor and McCormick Citation2008) after establishing that the majority of fungi detected in all three orchid species are Tulasnella. Polymerase chain reaction (PCR) products were cloned using a pCR4-TOPO cloning vector (Thermo Fisher Scientific, Waltham, Massachusetts) and transformed using competent Escherichia coli DH10B. Due to difficulty in cultivating some of the fungi in this study, direct sequencing especially for Rimacola and Pyrorchis was employed to obtain more sequences of the Tulasnella symbionts to confirm their host ranges in the studied orchids.
Phylogenetic analyses.—
To confirm isolates represented new species and had not been described elsewhere, all obtained Tulasnella-like ITS sequences were matched against National Center for Biotechnology Information (NCBI) GenBank (http://www.ncbi.nlm.nih.gov/) with BLASTn. All closely related Tulasnella sequences were downloaded from GenBank, along with other sequences representative of the five phylogenetic groups within Tulasnella as identified by Cruz et al. (Citation2014). Due to difficulties in aligning the divergent sequences from different phylogenetic groups in Tulasnella, an initial sequence alignment was undertaken only for the most conserved 5.8S region of the ITS (175 bp), to place sequences into phylogenetic groups. The initial phylogenetic analyses of the 5.8S region revealed that the new Tulasnella in this study belonged to Tulasnella groups III and IV and a new group, subsequently named group V. For each of the three phylogenetic groups represented by our isolates, separate analyses were undertaken of the full-length ITS (approximately 640 bp). Furthermore, across all isolates, each of four other loci (C14436, C4102, C3304, and mtLSU) was analyzed separately and in a concatenated analysis that also included the 5.8S region. In total, 323 sequences from GenBank (172 ITS, 43 C14436, 42 C4102, 10 C3304, and 56 mtLSU) were included in these analyses.
Sequences were aligned using Clustal W (Thompson et al. Citation1994). Phylogenetic trees were constructed using Bayesian inference (BI) analysis using MrBayes 3.2.6 (Ronquist and Huelsenbeck Citation2003). Node support was assessed with Bayesian inference (BI) for 2 500 000 generations, and the tree was sampled every 200 generations with a 25% burn-in. Convergence of runs was confirmed when the average standard deviation was <0.01 with effective sample sizes >200. Additionally, a RAxML maximum likelihood (ML) analysis (Stamatakis et al. Citation2008) phylogenetic tree was constructed using 1000 pseudoreplicates of nonparametric bootstrapping. Both BI and ML analyses were visualized using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). The 5.8S ITS phylogenetic tree was rooted with Serendipita sp. (GenBank accession number DQ983816), whereas phylogenetic trees of the other loci were midpoint rooted. The best-fit substitution model for each locus was determined by ModelFinder (Kalyaanamoorthy et al. Citation2017) in IQ-TREE 2.0 by using the Bayesian information criterion (Nguyen et al. Citation2014). Each gene marker was partitioned by codon positions, and the concatenated alignment was partitioned by each locus and their codon positions. Within each group, pairwise sequence divergences for ITS within and among Tulasnella species were calculated with MEGA X (Kumar et al. Citation2018) from uncorrected p-distance matrixes. The ITS sequences used in comparisons of inter- and intraspecies distance are the sequences in the relevant trees.
Morphological and culture characteristics.—
Morphology of cultures and microcharacters were examined for at least two isolates of each species as identified in the phylogenetic analyses, as well as for the previously described species, T. asymmetrica (isolate CLM2287).
Culture morphology and growth assessment
To assess culture growth and morphological features, stock isolates were initially grown on ½ FIM agar (with streptomycin sulfate 50 mg/L) and incubated for 2 wk at 22 C in the dark. Small agar blocks (5 mm2) were transferred to each of four different media, with three replicates per isolate per medium. The different media used to visualize morphological and culture characteristics were ½ FIM (Clements and Ellyard Citation1979), low carbon-nitrogen Melin-Norkrans medium (Wright et al. Citation2010) + A–Z vitamin (Wyeth Consumer Healthcare, Baulkham Hills, Australia; hereafter 3MN+vitamin) as modified in Ruibal et al. (Citation2017), E-medium (Caldwell et al. Citation1991), and ¼ PDA (potato dextrose agar; Alpha Biosciences, Baltimore, Maryland), all supplemented with streptomycin sulfate 50 mg/L. Culture morphology, including culture characteristics such as shape of colony margin, colony color, presence/absence of aerial hyphae, growth uniformity, any visible concentric zones, hyphal size and types, and other distinctive features, was recorded after 4 wk incubation at 22 C as described in Arifin et al. (Citation2021).
Growth assessment was conducted using four media as above (½ FIM, 3MN+vitamin, E-medium, and ¼ PDA, all supplemented with streptomycin sulfate 50 mg/L) as described in Arifin et al. (Citation2021). Colony diameters were measured perpendicularly using calipers after 4 wk incubation at 22 C in the dark, with three replica plates per isolate per medium. Statistical analysis was performed using a linear-mix model lmertest package in R (R Core Team Citation2019), and a one-way analysis of variance (ANOVA) was used to determine significance differences in colony growth among Tulasnella species within each medium.
Microscopic characteristics and fluorescence staining of fungal nuclei
Isolates were examined for the number of nuclei per hyphal cell based on Hua’an et al. (Citation1991) with modifications by using a Hoechst dye 33342 (Thermo Fisher Scientific) as the staining agent. To prepare the inoculum for staining, glass slides were covered with a thin layer of PDA. PDA-covered glass slide was laid above the damp Whatman filter paper in the sterile Petri dish to keep the humidity during incubation. After the PDA dried up, 1 mm2 of agar block mycelium was inoculated on the middle of the slide. All procedures were prepared aseptically in the airflow cabinet. Petri dishes were sealed and incubated at 22 C for 3 wk in the dark. After incubation, slides were air-dried in a laminar airflow cabinet for approximately 15 min. To make the working solution of Hoechst dye, a buffer pH 7.8 consists of 0.1 M KH2PO4 and 0.1 M NaOH was prepared for best visualization of Rhizoctonia fungi (Fang et al. Citation2013; Hua’an et al. Citation1991). Nuclei examination was conducted using a Leica DM5500 B fluorescence microscope (Leica Microsystems, Wetlzar, Germany) at 1000× magnification. The number of nuclei was counted for at least 20 cells/compartments per isolate. The photographs of cells for each isolate were taken using a Leica DM5500 B automated upright microscope camera.
RESULTS
The 5.8S phylogeny constructed from Tulasnella sequences from new isolates and clones, as well as sequences from GenBank, formed five main clades: four consistent with the previously recognized phylogenetic groups I–IV and one an unassigned group, which is here described as phylogenetic group V (SUPPLEMENTARY FIG. 1). Based on morphological characteristics and multilocus molecular identification of isolates associated with Australian terrestrial orchids in the Megastylidinae and Thelymitrinae, we describe five new Tulasnella species. The five new species (SUPPLEMENTARY TABLE 1) fall into Tulasnella group III—Tulasnella subasymmetrica, sp. nov. (22 isolates), T. kiataensis, sp. nov. (15 isolates), and T. korungensis, sp. nov. (5 isolates); group IV—T. nerrigaensis, sp. nov. (6 isolates); and group V—T. multinucleata, sp. nov. (2 isolates).
In the ITS phylogeny of group III isolates ()
Figure 1. Bayesian inference tree for ITS region of Tulasnella phylogenetic group III. Numbers on the branches are Bayesian posterior probabilities (>0.70)/bootstrap (>70%) support values. Tulasnella described in this study are shown in bold.
In the ITS phylogeny of group IV ()
Figure 2. Bayesian inference tree for ITS region of Tulasnella phylogenetic group IV. Numbers on the branches are Bayesian posterior probabilities (>0.70)/bootstrap (>70%) support values. Tulasnella described in this study are shown in bold.
In the ITS phylogeny of group V, the clade of nine sequences of T. multinucleata formed a well-supported (BPP = 1.00 and bootstrap = 100) clade sister to a single isolate of an un-named Tulasnella from Brazil associated with Cyrtopodium hatschbachii designated as Tulasnella sp. CH01 by De Carvalho et al. (Citation2017) ().
Figure 3. Bayesian inference tree for ITS region of Tulasnella phylogenetic group V. Numbers on the branches are Bayesian posterior probabilities (>0.70)/bootstrap (>70%) support values. Tulasnella described in this study are shown in bold.
Where sequences were available, all five new Tulasnella species could be recovered when either analyzed separately with one of the other four loci (C14436, C4102, C3304, and mtLSU) or in a concatenated alignment (5.8S+C14436+C4102+C3304+mtLSU) (). Unfortunately, some species/isolates failed to amplify for the loci C4102 and C3304 ()
Table 2. Posterior probability/bootstrap support values for clades of five new Tulasnella species across five loci when analyzed separately and concatenated (5.8S+C14436+C4102+C3304+mtLSU)
Figure 4. Bayesian inference tree for Tulasnella using five concatenated loci: 5.8S ITS, C14436, C4102, C3304, and mtLSU region. Numbers on the branches are Bayesian posterior probabilities (>0.70)/bootstrap (>70%) support values. Tulasnella described in this study are shown in bold.
For all new Tulasnella in this study, maximum within-species ITS sequence divergences ranged from 0% to 2.4% (), with T. brigaderoiensis as the closest species of T. kiataensis (pairwise interspecies ITS sequence divergence 7.5–8.4%; ), T. orchidis closely related to T. subasymmetrica and T. korungensis (pairwise interspecies ITS sequence divergence 5.1–10.4% and 9.3–9.9%, respectively; ), and T. violea as the closest relative of T. nerrigaensis (pairwise interspecies ITS sequence divergence 18.1–19.5%; ).
Table 3. Maximum percentage pairwise sequence divergence within species, as well as minimum and maximum percentage pairwise sequence divergences between species, for the ITS region of Tulasnella group III
Table 4. Maximum percentage pairwise sequence divergence within species, as well as minimum and maximum percentage pairwise sequence divergences between species, for the ITS region of Tulasnella group IV
Table 5. Maximum percentage pairwise sequence divergence within species, as well as minimum and maximum percentage pairwise sequences divergence between species, for the ITS region of Tulasnella group V
Among the five new Tulasnella described here, most species had similar growth rates across all media. One exception was that T. multinucleata differed significantly in colony diameter compared with other species on all tested media after 4 wk, except in comparison with T. kiataensis and T. korungensis on 3MN+vitamin (P > 0.1; SUPPLEMENTARY TABLE 2). In general, T. multinucleata had significantly smaller colony diameters among media tested except on 3MN+vitamin, on which T. nerrigaensis was the slowest growing (SUPPLEMENTARY ).
TAXONOMY
Tulasnella subasymmetrica Arifin, Reiter, T.W. May & Linde, sp. nov.
MycoBank MB840177
Typification: AUSTRALIA. VICTORIA: Lake Mundi Flora and Fauna Reserve, isolated from lateral roots of Thelymitra epipactoides, 3 Aug 2010, N.H. Reiter AL.LM5.5.1 (holotype RBGV-LC 211184 culture preserved in a metabolically inactive state, isotype MEL 2431979 specimen, a freeze-dried culture). GenBank: ITS = MH134545.
Etymology: subasymmetrica (Latin), referring to the previous identification of some isolates as T. asymmetrica.
Description: On ¼ PDA, 78–81 mm diam, cream white in color on both sides, with thin, appressed surface mycelium, hyphae mostly submerged, growth moderately dense, radial without dendroid pattern; no concentric zones; minute spirals present; margin even. On ½ FIM, similar to ¼ PDA but off-white in color and minute spirals present. On E-medium and 3MN+vitamin, aerial hyphae more obvious and with dendroid growth in comparison with ½ FIM and ¼ PDA. On 3MN+vitamin, aerial hyphae form scattered pattern of small, irregular denser hyphal aggregations on the colony surface ().
Figure 5. Tulasnella cultures on (from left to right) ¼ PDA, ½ FIM, 3MN+vitamin, and E-medium; all supplemented with streptomycin sulfate 50 mg/L. A. Tulasnella asymmetrica (CLM2287). B. T. subasymmetrica (AL.LM5.5.1). C. T. kiataensis (AL.K14.8). D. T. korungensis (CLM1788). E. T. multinucleata (CLM2222). F. T. nerrigaensis (CLM1870).
Hyphae septate, 4–7 µm diam, length 20–40 µm between septa, cylindrical, thin-walled; terminal elements cylindrical; branched, with branching mostly at right angles, with constriction at base of branch hypha; lacking clamp connections; binucleate ().
Figure 6. Micromorphological features of Tulasnella subasymmetrica showing branches mostly at right angles and often without constrictions (A–B), cylindrical hyphae with cylindrical terminal elements (B, black arrows), and binucleate hyphal compartments (C–D). Bar = 20 µm.
Ecology and distribution: Isolated from and forms a mycorrhizal association with the endangered host Thelymitra epipactoides (Orchidaceae) growing in damp heathland and coastal heathland scrub, on sandy and clay loam soils—Victoria (host also found in South Australia), Australia.
Other specimens examined: AUSTRALIA. VICTORIA: Lake Mundi Flora and Fauna Reserve, isolated from Thelymitra epipactoides, 3 Aug 2010, N.H. Reiter AL.LM5.2 (RBGV-LC 211185); Port Campbell National Park, isolated from T. epipactoides, 3 Aug 2010, N.H. Reiter AL.PC7.2 (RBGV-LC 211186, MEL 2431954); N.H. Reiter AL.PC10.1 (RBGV-LC 211187, MEL 2431961).
Notes: The growth pattern on 3MN+vitamin is distinctive, with scattered denser hyphal aggregations. Tulasnella subasymmetrica has relatively fast growth on all media tested, especially in comparison with T. asymmetrica. This latter species is also well separated on sequence data, when comparison is made against the clade containing the ex-type isolate (). However, included here on the basis of sequence placement within the T. subasymmetrica clade () are isolates Warcup 0302 (MAFF 305808) and Warcup 0591 (MAFF 305809), which were included under the clade name “Tulasnella asymmetrica” in Cruz et al. (Citation2014) and Reiter et al. (Citation2018). Warcup 0302 was originally isolated by Warcup in 1967 from Thelymitra epipactoides (Warcup Citation1973) and identified in that publication to Tulasnella asymmetrica where it was shown to form mycorrhizal associations with Thelymitra aristata and T. longifolia. Isolate Warcup 0591 isolated from T. epipactoides (Warcup Citation1973), also originally identified as Tulasnella asymmetrica, was found to germinate various species of Thelymitra but not members of the genera Diuris, Spiranthes, Orthrocerus, Calochilus, Dendrobium, or Spiranthes (Warcup Citation1981).
Tulasnella kiataensis Arifin, Reiter, T.W. May & Linde, sp. nov.
Figure 7. Micromorphological features of Tulasnella kiataensis showing branches mostly at right angles and often without constrictions (A–B), cylindrical hyphae with cylindrical and/or clavate terminal elements (B), and binucleate hyphal compartments (C–D). Bar = 20 µm.
MycoBank MB840178
Typification: AUSTRALIA. VICTORIA: Kiata Flora and Fauna Reserve, isolated from Thelymitra epipactoides, 5 Aug 2010, N.H. Reiter AL.K.14.8 (holotype RBGV-LC 211188 culture preserved in a metabolically inactive state, isotype MEL 2432006 specimen, a freeze-dried culture). GenBank: ITS = MH134533.
Etymology: Refers to the location where the host orchid was collected, the Kiata Flora and Fauna Reserve.
Description: On ¼ PDA, 79–82 mm diam, off-white above and below, with thin aerial mycelium, hyphae mostly submerged, growth radial without dendroid pattern; no concentric zonation; minute spirals often present; margin even. On E-medium, showing similar features but above and below creamy white and with denser hyphae around the inoculum block. On ½ FIM, also showing similar features compared with ¼ PDA but with sparser mycelium. On 3MN+vitamin, growth dendroid toward edge, with the sparsest mycelium among the four media, and with aerial hyphae forming very scattered, slightly denser irregular hyphal aggregations ().
Hyphae septate, 3–5 µm diam, length 30–40 µm between septa, cylindrical, thin-walled; terminal elements cylindrical or rarely clavate; branched and sometimes tortuous, with branching mostly at right angles, with constriction at base of branch hypha; lacking clamp connections; binucleate ().
Ecology and distribution: Isolated from and forms a mycorrhizal association with the endangered Orchidaceae host Thelymitra epipactoides growing in woodland on shallow sands—Victoria (host also found in South Australia), Australia.
Other specimens examined: AUSTRALIA. VICTORIA: Kiata Flora and Fauna Reserve, isolated from Thelymitra epipactoides, 5 Aug 2010, N.H. Reiter AL.K12.8 (RBGV-LC 211189, MEL 2432002); N.H. Reiter AL.KSE4.6 (RBGV-LC 211190, MEL 2431997); N.H. Reiter AL.K14.1 (RBGV-LC 211191, MEL 2432005).
Notes: Growth of T. kiataensis was markedly slower than T. subasymmetrica on 3MN+vitamin but not on other media (SUPPLEMENTARY FIG. 6). Also, on 3MN+vitamin as diagnostic medium, T. kiataensis showed less obvious scattered aerial hyphae. This species was previously designated as “OTU1” in Reiter et al. (Citation2018).
Tulasnella korungensis Arifin, T.W. May & Linde, sp. nov.
Figure 8. Micromorphological features of Tulasnella korungensis showing swollen hyphal compartments (A–B) and occasionally trinucleate (C, white arrow) but mostly binucleate (D) hyphal compartments, separated by septa (D, white arrows). Bar = 20 µm.
MycoBank MB840179
Typification: AUSTRALIA. WESTERN AUSTRALIA: Korung National Park, Pomeroy Road, isolated from Pyrorchis nigricans, 13 Aug 2016, C. Linde CLM1788 (holotype RBGV-LC 200012 culture preserved in a metabolically inactive state, isotype MEL 2470320 specimen, a freeze-dried culture, isotype VPRI 43719 culture preserved in a metabolically inactive state). GenBank: ITS = MT981409.
Etymology: Refers to the location the host orchid was collected from in Korung National Park.
Description: On ¼ PDA, 72–78 mm diam, above and below off-white, with very thin aerial mycelium, hyphae mostly submerged, growth comparatively dense compared with other media, radial, without dendroid pattern; no concentric zonation; minute spirals absent; margin even. On ½ FIM and E-medium, with similar features to ¼ PDA but growth less dense. On 3MN+vitamin, with obvious aerial mycelium toward the edge of the colony ().
Hyphae septate, 3–5 µm diam, length 35–45 µm between septa, cylindrical, thin-walled; terminal elements clavate or occasionally shorter and subglobose to ellipsoid (10–20 µm long and 6–7 µm diam); branched, with branching usually at right angles, with constriction at the base; lacking clamp connections; mostly binucleate but occasionally trinucleate ().
Ecology and distribution: Isolated from the widespread host Pyrorchis nigricans (Orchidaceae), growing in open areas with jarrah forests, on well-drained gray sandy soil—Western Australia (host also found in South Australia, New South Wales, Victoria, and Tasmania), Australia.
Other specimens examined: AUSTRALIA. WESTERN AUSTRALIA: Korung National Park, isolated from Pyrorchis nigricans, 13 Aug 2016, C. Linde CLM1760 (RBGV-LC 200013, VPRI 43716); C. Linde CLM1761 (RBGV-LC 200014, VPRI 43717); C. Linde CLM1762 (RBGV-LC 200015, VPRI 43718).
Notes: Colony growth of T. korungensis was relatively similar on the four media tested (SUPPLEMENTARY FIG. 6) and lacked diagnostic characteristics on all medium types compared with other Tulasnella described in this study, except that growth tended to be slower than other species, apart for T. asymmetrica, which had particularly slow growth.
Tulasnella multinucleata Arifin, T.W. May & Linde, sp. nov.
Figure 9. Micromorphological features of Tulasnella multinucleata showing branches mostly at right angles (A), swollen cell chains (B), and multinucleate hyphal compartment, cylindrical hyphae, and terminal ends (C, white arrow), with septae (D, white arrows). Bar = 20 µm.
MycoBank MB840180
Typification: AUSTRALIA. NEW SOUTH WALES: Morton National Park, isolated from Rimacola elliptica, 1 Sep 2018, A. Arifin CLM2222 (holotype RBGV-LC 190931 culture preserved in a metabolically inactive state, isotype MEL 2469731 specimen, a freeze-dried culture, isotype VPRI 43509 culture preserved in a metabolically inactive state). GenBank: ITS = MT981410.
Etymology: Refers to the multinucleate hyphal cells of this species.
Description: Cultures on ¼ PDA, 43–62 mm diam, cream above and below inoculum block, otherwise off-white, with cottony to cobwebby, well-developed, off-white aerial mycelium, particularly over the inoculum block and sparse, submerged mycelium extending beyond area of aerial mycelium, growth irregular, radially dendroid; no concentric zonation; minute spirals absent; margin very uneven. On ½ FIM, similar to ¼ PDA, but with less aerial mycelium and submerged mycelium more extensive. On 3MN+vitamin and E-medium, similar to ¼ PDA ().
Hyphae septate, 7–10 µm diam, length 50–100 µm between septa, cylindrical, thin-walled, sometimes shorter and broader, and then clavate or fusoid (to 15 µm diam), these broader cells in short, irregular chains; terminal elements cylindrical or sometimes clavate or subglobose; lacking clamp connections; branched, branches usually at right angles, with constriction at base of branch hypha, but sometimes not constricted and/or with branch not at a right angle, sometimes the direction of chains of swollen cells changes, with one section of the chain arising from the side of the adjacent cell in the chain; multinucleate, with 6–12 nuclei per cell ().
Ecology and distribution: Isolated from the highly localized, evergreen host Rimacola elliptica (Orchidaceae), growing on wet and shaded sandstone cliffs and rock fissures—New South Wales, Australia.
Other specimen examined: AUSTRALIA. NEW SOUTH WALES: Morton National Park, isolated from Rimacola elliptica, 1 Sep 2018, A. Arifin CLM2223 (RBGV-LC 190932, VPRI 43510, MEL 2469732).
Notes: Compared with all Tulasnella species recently described from Australia, including the six species described by Arifin et al. (Citation2021), T. multinucleata in culture has the most irregular growth, with dendroid growth on most media and a very uneven margin, as well as the most well-developed aerial mycelia. Morphologically, T. multinucleata is the only Tulasnella species that is multinucleate and hyphal diameter (7–10 µm) is broader than that of all other species treated here (3–7 µm).
Tulasnella nerrigaensis Arifin, T.W. May & Linde, sp. nov.
Figure 10. Micromorphological features of Tulasnella nerrigaensis showing fusoid to elliptical monilioid cell chains (A–B), branched hyphae usually at right angles without constriction at the base (C), binucleate hyphal compartments (C–D), and rarely observed bridge-like structures linking between cells (D, white arrow). Bar = 20 µm.
MycoBank MB840181
Typification: AUSTRALIA. NEW SOUTH WALES: Nerriga, near Oallen Ford Road, isolated from Calochilus robertsonii, 20 Oct 2017, C. Linde CLM1870 (holotype RBGV-LC 201126 culture preserved in a metabolically inactive state, isotype MEL 2485396 specimen, a freeze-dried culture, isotype VPRI 43964 culture preserved in a metabolically inactive state). GenBank: ITS = MW575585.
Etymology: nerrigaensis (Latin), referring to the locality of the host orchid, Nerriga, New South Wales.
Description: On ¼ PDA, 77–81 mm diam, above and below off-white, with thin aerial mycelium, hyphae mostly submerged, growth radial without dendroid pattern; no concentric zonation, minute spirals present; margin even. On E-medium, with similar features, but with 2–3 concentric zones. On ½ FIM, with similar features compared with ¼ PDA but less aerial mycelium. On 3MN+vitamin, showing the most distinctive features compared with other three media, with underside and upperside creamy white, growth dendroid, slower and with denser hyphae forming a thicker layer of aerial hyphae compared with other media, and with minute spirals absent ().
Hyphae septate, 3–6 µm diam, length 40–60 µm between septa, cylindrical, thin-walled; also with shorter and broader cells that are fusoid to elliptical (15–25 µm long and up to 9 µm diam) and sometimes found in monilioid chains (up to five cell chains in each branch); terminal elements clavate; branched, with branching usually at right angles, without constriction at the base; lacking clamp connections; binucleate ().
Ecology and distribution: Isolated from the widespread host Calochilus robertsonii (Orchidaceae) growing in open forest and woodland, sandy soil—New South Wales (host also found in Queensland, Victoria, and Tasmania), Australia.
Other specimens examined: AUSTRALIA. NEW SOUTH WALES: Nerriga, Oallen Ford Road, isolated from Calochilus robertsonii, 20 Oct 2017, C. Linde CLM1869 (RBGV-LC 201125, VPRI 43963); C. Linde CLM1873 (RBGV-LC 201127, VPRI 43965); C. Linde CLM1875 (RBGV-LC 201129, VPRI 43967).
Notes: T. nerrigaensis appears to have demanding medium requirements, as we could successfully cultivate only five isolates from C. robertsonii. Direct sequencing from C. gracillimus and C. paludosus (one population each) also detected T. nerrigaensis as their mycorrhizal symbiont (data not shown).
Tulasnella asymmetrica Warcup & P.H.B. Talbot, New Phytologist 66:637. Citation1967.
Figure 11. Micromorphological features of Tulasnella asymmetrica CLM2287 showing cylindrical hyphae with branches mostly at right angles (B–C), which are septate (C, white arrow), often with peg-like protuberances (A–B, black arrows; D, white arrow), and binucleate (C–D). Bar = 20 µm.
Typification: AUSTRALIA. SOUTH AUSTRALIA: Mount Lofty Ranges, isolated from Thelymitra luteocilium, 1967, J. Warcup 085 (holotype DAR ADW-16001; ex-type culture MAFF 305806). GenBank: ITS = DQ388046 (Suarez et al. Citation2006), KC152339–KC152344 [clones c001–c006] (Cruz et al. Citation2014).
Other specimen examined: AUSTRALIA. QUEENSLAND: Main Range National Park, isolated from Bulbophyllum bracteatum, 19 Jun 2018, J.D.W. Dearnaley CLM2287 (VPRI 43968).
Description: On ¼ PDA, 27–32 mm diam, above and below off-white, with thin aerial mycelium, hyphae mostly submerged, growth radial without dendroid pattern; no concentric zonation, minute spirals present; margin even. On ½ FIM and 3MN+vitamin, similar to ¼ PDA but less aerial mycelium. On E-medium, showing the most distinctive features compared with the other three media, with slower, dendroid growth, and with denser hyphae forming a thicker layer of aerial hyphae compared with other media ().
Hyphae septate, 2–4 µm diam, length 40–60 µm between septa, cylindrical, thin-walled; terminal elements cylindrical; branched, with branching usually at right angles, without constriction at the base; lacking clamp connections; binucleate ().
Ecology and distribution: Isolated from a lithophytic host Bulbophyllum bracteatum (Orchidaceae) growing on a basalt boulder on the edge of the escarpment in open Eucalyptus woodland—Queensland, Australia.
Notes: The original type citation was “Typus Herb. ADW No. 16,001, J.H.Warcup (085)”, and under the host information Warcup and Talbot (Citation1967) noted the association with Thelymitra luteocilium and listed two isolates, 085 and 091, both from Mount Lofty Ranges. Microfungi collections from ADW (Herbarium, Waite Agricultural Research Institute) were transferred to the New South Wales Plant Pathology and Mycology Herbarium (DAR) in 1999 (Shivas et al. Citation2006). The material in DAR cited above consists of fragmentary and very thin fungal growth on soil in a Petri dish, along with the original ADW packet. The ex-type culture MAFF 305806 derived from Warcup 085 has been sequenced a number of times and is close in sequence information to another isolate identified by Warcup as T. asymmetrica (Warcup 0267, MAFF 305807). A recent isolate from Bulbophyllum (CLM2287) has an ITS sequence that falls within the clade of sequences from these two Warcup isolates, including the ex-type culture (). The intraspecific distance including this isolate is the largest of any of the species examined herein (at 4.1%). Multilocus data from further isolates are required to confirm conspecificity of the Thelymitra and Bulbophyllum isolates placed under T. asymmetrica. Whatever the eventual delimitation of T. asymmetrica, it is clear that it is quite distinct from T. subasymmetrica, with sequence divergence of up to 19% between the two taxa (). In addition, morphological features of T. asymmetrica also confirm it as a distinct species to T. subasymmetrica ( and 11).
Several isolates originally placed under T. asymmetrica are reidentified here as T. subasymmetrica (see descriptions for that species, above). Subsequent to the original description, T. asymmetrica was reported from other orchid hosts in Chiloglottis, Cryptostylis, and Dendrobium in various studies based on morphology by Warcup and Talbot (as compiled by Linde et al. Citation2017), but no specimens or cultures appear to survive to support this identification. Without sequence data, identification of T. asymmetrica in association with these other hosts remains suspect.
Tulasnella cruciata Warcup & P.H.B. Talbot, New Phytologist 70:37. Citation1971.
Notes: The original type citation was “Typus: Herb. ADW No. 16218, J.H. Warcup (0296)”, and Warcup and Talbot (Citation1971) noted the hosts as Acianthus caudatus, from Mount Lofty Ranges, South Australia, Thelymitra fuscolutea, from Pomonal, Victoria, and T. pauciflora, from Mount Lofty Ranges, but it is not clear from which location and host the type isolate was obtained. Roberts (Citation1999) treated T. cruciata as an independent species, noting that it was similar in morphology to T. danica Hauerslev and T. asymmetrica, but that the type collection could not be found. A single specimen labeled T. cruciata has been located in DAR, from South Australia, labeled at DAR as collected by Warcup in 1971, with host indicated merely as “Orchidaceae” (DAR 41994). The original ADW packet is not present, but the DAR label notes that the collection consists of “living and dried cultures … ex ADW 16218.” Later annotation slips in DAR 41994 cite the associations with multiple hosts mentioned in the protolog. The specimen consists of a dried culture prepared in 1982. This cannot be considered to be a type, as that must be either a living culture stored metabolically inactive or a dried down culture prepared at the time (as was the type of T. asymmetrica). Although there are a number of cultures isolated by Warcup held in CBS (Westerdijk Fungal Biodiversity Institute, culture collection of fungi and yeasts) and MAFF (Ministry of Agriculture, Forestry and Fisheries, Japan), none are identified as T. cruciata, nor are there living cultures in DAR. Further isolates from Acianthus, Chiloglottis, and Thelymitra were identified as T. cruciata on the basis of morphology by Warcup and Talbot (as compiled by Linde et al. (Citation2017): ), but it appears that no specimens or cultures survive to support the identification. Given that original material consisted of isolates from multiple orchid genera (Acianthus and Thelymitra) and that no material suitable for lectotypification survives, the name T. cruciata remains of uncertain application.
DISCUSSION
We describe five species of Tulasnella associated with various Australian orchids. The species described here, T. multinucleata, which is the first multinucleate Tulasnella reported, along with T. nerrigaensis, T. korungensis, T. kiataensis, and T. subasymmetrica, were well supported as distinct species in multigene phylogenetic analyses of three nuclear (including ITS) and two mitochondrial loci. Maximum within-species ITS sequence divergences are under 2.5% for all new species (). Consistent with previous studies, all within-species ITS sequence divergences (2.4% for T. subasymmetrica, 1.3% for T. kiataensis, 0% for T. korungensis, 0.3% for T. nerrigaensis, and 1.0% for T. multinucleata) are below the Tulasnella delimitation threshold of 4–6% (Arifin et al. Citation2021; Linde et al. Citation2017) and even show a lower value than the universal Basidiomycota divergence threshold of 3.3% (Nilsson et al. Citation2008). Despite two apparent subclades for T. subasymmetrica for some loci, the subclades are only supported in C3304; thus, we consider T. subasymmetrica to be one species. We also clarify the naming of T. asymmetrica by showing that T. subasymmetrica is the appropriate name for some collections previously assigned as “T. asymmetrica,” such as by Cruz et al. (Citation2014).
Multilocus analysis for fungal delimitation is necessary to avoid misleading classification by a single locus. Ideally, concordant tree topologies are retrieved from different genes (Taylor et al. Citation2000), with greater power coming from inclusion of more genes (Dupuis et al. Citation2012). A genealogical concordance approach has been used successfully for species delimitation across various groups of fungi (e.g., Stefani et al. Citation2014, Linde et al. Citation2014). Because the ITS1 and ITS2 regions of the ITS region are highly diverse and difficult to align, first we analyzed the ITS locus separately for each Tulasnella phylogenetic group () as defined using a 5.8S ITS alignment (SUPPLEMENTARY FIG. 1) in order to place novel species into groups. This was followed by separate analysis of all markers, within each of the main groups of Tulasnella, which gave concordant results, providing strong evidence for species delimitation. In addition, a concatenated analysis across the genus retrieved the same novel species, although it is the occurrence of species clades in each separate marker that provides the strongest support for the novel species. To enable alignment of the variable ITS region across the genus in the multigene concatenated data set, the unalignable less conserved regions of the ITS (ITS1 and ITS2) were trimmed and the conserved 5.8S region was analyzed together with four other loci (C14436+C4102+C3304+mtLSU).
The current classification of Australian terrestrial orchids based on coding exon sequences at 221 loci places genera of interest in relation to Tulasnella symbionts in four subtribes of the tribe Diurideae: i.e., Cryptostylidinae, Drakaeinae, Megastylidinae, and Thelymitrinae (Peakall et al. Citation2021: ). In this classification, several members of the Megastylidinae form a clade at the base of Drakaeinae; Drakaeinae + Megastylidinae is sister to Thelymitrinae, and Cryptostylidinae is sister to the clade of the three other subtribes. In regard to the associated Tulasnella, orchids within the Drakaeinae (Arifin et al. Citation2021; Linde et al. Citation2017, Citation2014) and Cryptostylidinae (Arifin et al. Citation2021; Nguyen et al. Citation2020) all associate with symbionts classified as belonging to group IV. In contrast, orchids within the Megastylidinae associate with Tulasnella belonging to groups II, III, and V, and those in the Thelymitrinae associate with Tulasnella belonging to groups III and IV. Thus, orchid genera in the Cryptostylidinae and Drakaeinae form fungal associations with one Tulasnella group only (group IV), and especially the Drakaeinae orchid genera are mostly fungal genus specialists but species generalists (Linde et al. Citation2017, Citation2014), whereas the genera and species of Megastylidinae and Thelymitrinae seem to be more generalist (associating with multiple Tulasnella taxa and phylogenetic groups). Furthermore, despite Cryptostylidinae and Drakaeinae being not closely related subtribes (Peakall et al. Citation2021; Weston et al. Citation2014), they have in common associations with Tulasnella group IV, even sharing Tulasnella species/OTUs from this phylogenetic group. It is notable that there are no group IV associates of Megastylidinae, despite the occurrence of Tulasnella symbionts from this group in both Drakaeinae and Thelymitrinae (which are part of a single clade that has members of Megastylidinae in a central position).
The groups within Tulasnella are groups of convenience for the purpose of aligning sequences, but they do represent distinct lineages that are significantly divergent. Taking this into account, multiple “gains” in fungal associations appear to have been made by Megastylidinae and Thelymitrinae from the ancestral Cryptostylidinae association with group IV, most of which were apparently lost again in the Drakaeinae. Alternatively, further sampling of orchid hosts may reveal new associations and broaden the range of Tulasnella associated with particular subtribes. Such host range expansion, at the group level, has occurred with Tulasnella nerrigaensis, the only new species described here from group IV. This novel species only associates with Calochilus (subtribe Thelymitrinae), in contrast to the other Australian species in group IV, all recently described, which associate with Drakaeinae and Cryptostylidinae (Arifin et al. Citation2021; Linde et al. Citation2017).
Previously, Pyrorchis (Megastylidinae) was shown to associate with a broad range of Rhizoctonia-like fungi including T. danica (Bonnardeaux et al. Citation2007) and Tulasnellaceae sp. 6 MB-2012 and sp. 7 MB-2012 (Sommer et al. Citation2012), all belonging to Tulasnella phylogenetic group II (SUPPLEMENTARY FIG. 1). In our study, Pyrorchis also associates with the novel species T. korungensis from Tulasnella group III as well as some unassigned Tulasnella from group II (SUPPLEMENTARY TABLE 1, clones only for group II sequences). Therefore, Pyrorchis has the ability to utilize a broad range of fungi (distantly related Tulasnella from groups II and III), which may assist in the orchid’s environmental adaptability to thrive in various habitats (Bonnardeaux et al. Citation2007; De Long et al. Citation2013).
Despite the geographic distance (America vs. Australia) and different habitats (e.g., terrestrial vs. epiphytic and temperate vs. tropical orchids), distantly related orchid hosts associate with closely related Tulasnella symbionts. For example, in Tulasnella group III (), T. brigadeiroensis (epiphytic host Laeliinae from South America; Freitas et al. Citation2020) is the sister species of T. kiataensis (terrestrial host Thelymitrinae from Australia). In addition, Tulasnella sp. OTU3 (host Eulophiinae from North America; Downing et al. Citation2020) is the sister species of T. subasymmetrica (terrestrial host Thelymitrinae from Australia). Furthermore, in Tulasnella group V (), T. multinucleata (lithophytic host Megastylidinae from temperate Australia) is the sister clade of Tulasnella sp. CH01 (terrestrial host Cyrtopodiinae from subtropical South America; De Carvalho et al. Citation2017). Of course, these apparent close relationships may alter with further taxon sampling.
Uni-, bi-, tri-, and multinucleate cell characteristics are common in Rhizoctonia-like fungi such as in Serendipitaceae M. Weiß, Waller, A. Zuccaro & Selosse (Andersen Citation1996; Oktalira et al. Citation2021; Williams and Thilo Citation1989), Sebacinaceae Oberw. & K. Wells (Basiewicz et al. Citation2012), and Ceratobasidiaceae G.W. Martin (Andersen Citation1996; Oberwinkler et al. Citation2013; Veldre et al. Citation2013). However, Tulasnella species described recently, as well as T. nerrigaensis, T. kiataensis, T. subasymmetrica, and T. asymmetrica in this study, all display only binucleate characteristics. Exceptions are T. tubericola (Solís et al. Citation2017) and T. korungensis (this study), which occasionally have trinucleate hyphal compartments. Here we also found that T. multinucleata is the most distinctive species in Tulasnella as far as the number of nuclei is concerned, with 6–12 nuclei in one hyphal compartment. Multiple nuclei per hyphal compartment may provide an adaptive advantage over fungi that are uni- or binucleate. Multinucleate fungi were shown to provide different sets of genes to use in substrate utilization, which led to fungal phenotypic adaptation (Jinks Citation1952) and together with mobile chromosomes contributed to pathogenicity (Ma et al. Citation2010; Rep and Kistler Citation2010). A number of filamentous Ascomycota harbor up to 100 nuclei per hyphal compartment, such as in Neurospora crassa Shear & B.O. Dodge, Aspergillus nidulans (Eidam) G. Winter, Sclerotinia sclerotiorum (Lib.) de Bary, and Fusarium oxysporum Schlecht. (Roper et al. Citation2011). Although little is known about the role of multinucleate hyphae in orchid mycorrhizal fungi, T. multinucleata provides a unique opportunity to explore the mechanisms affected by the multinucleate state. The host of T. multinucleata, R. elliptica, grows in very different habitats compared with other Australian orchids examined here―as a lithophyte growing on sandstone cliffs and is narrowly distributed in eastern Australia (Jones Citation2021). The advantage/disadvantage of being multinucleate should therefore be explored for Tulasnella.
In plant-pathogen interactions, fungal pathogenicity is mediated by small secreted proteins called effectors, determining host infection and colonization (Dodds and Rathjen Citation2010; Stergiopoulos and de Wit Citation2009). The advancement of genome sequencing revealed similar signaling pathways between pathogenic and mycorrhizal fungi (Favre-Godal et al. Citation2020). Effector-like proteins have been expressed in mutualistic plant-fungal interactions (Sezdzielewska-Toro and Delaux Citation2016). Transcriptome analyses of several mycorrhizal fungi (including T. calospora in Serapias orchids) grown symbiotically in plant tissues showed that these fungi have higher expression of effector-like proteins compared with saprotrophic fungi (Plett and Martin Citation2015). Although the molecular basis of orchid mycorrhizal fungi colonization and specificity is less known compared with fungal pathogens, it is clear that effector-like proteins play an important role in developing orchid mycorrhizal host-specific interactions, and further investigation into such effectors will help explain mycorrhizal specificity in orchids.
These newly established species contribute to the number of described Tulasnella taxa in the world, providing names to species based on rigorous species delimitation. The ITS region is an effective barcode for the accurate identification of novel species of Tulasnella and their relatives, which will facilitate further taxonomic and conservation studies of host and fungus. Having names and a rigorous method for identification means that future studies will be able to report on the species involved with confidence, especially those that play a significant role as mycobiont partners for many endangered orchid species. Due to the improvement in molecular-based identifications and ever-increasing sequencing data availability, it is highly likely that our future knowledge of Tulasnella diversity will be further expanded to include more new taxa and phylogenetic groups.
ETHICAL APPROVAL STATEMENT
Permissions were obtained from the state authorities to collect all plant materials in this study: New South Wales National Parks & Wildlife Service (permit number SL100294), Western Australia Department of Parks and Wildlife (permit number SW017980), and Department of Environment, Land, Water & Planning Victoria (permit number 10008918).
Supplemental Material
Download Zip (12.5 MB)Supplemental MaterialACKNOWLEDGMENTS
We thank Ryan Phillips, Rod Peakall, Alyssa Weinstein, Mary Argall, and Mark Clements for assisting with field work and sampling; Tobias Hayashi and Sharyn Wragg for assisting with photography of cultures; Carlos Pavón Vázquez for helping with phylogenetic analyses; Farid Rahimi for microscopy assistance; Leon Smith and Richard Dimon for initial laboratory work with culture collections; Jordan Bailey for information about collections at DAR; and John Dearnaley for providing an isolate of T. asymmetrica. We also thank Brandon Matheny, Alfredo Justo, and two anonymous reviewers for comments on an earlier version of the manuscript.
DISCLOSURE STATEMENT
No potential conflict of interest was reported by the author(s).
SUPPLEMENTARY MATERIAL
Supplemental data for this article can be accessed on the publisher’s Web site.
Additional information
Funding
Related Research Data
LITERATURE CITED
- Andersen TF. 1996. A comparative taxonomic study of Rhizoctonia sensu lato employing morphological, ultrastructural and molecular methods. Mycol Res. 100:1117–1128.
- Arifin AR, May TW, and Linde CC. 2021. New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae. Mycologia. 113:212–230.
- Basiewicz M, Weiss M, Kogel KH, Langen G, Zorn H, and Zuccaro A. 2012. Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov. Fungal Biology. 116:204–213.
- Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, and Read DJ 2004. Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proceedings of the Royal Society B. 271:1799–1806.
- Binder M, Hibbett DS, Larsson K-H, Larsson E, Langer E, and Langer G. 2005. The phylogenetic distribution of resupinate forms across the major clades of mushroom‐forming fungi (Homobasidiomycetes). Systematics and Biodiversity. 3:113–157.
- Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, Sivasithamparam K. 2007. Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions. Mycol Res. 111:51–61.
- Bruns TD, and Gardes M. 1993. Molecular tools for the identification of ectomycorrhizal fungi - taxon-specific oligonucleotide probes for suilloid fungi. Mol Ecol. 2:233–242.
- Bruns TD, Szaro TM, Gardes M, Cullings KW, Pan JJ, Taylor DL, Horton TR, Kretzer A, Garbelotto M, and Li Y. 1998. A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis. Mol Ecol. 7:257–272.
- Caldwell BA, Castellano MA, and Griffiths RP. 1991. Fatty acid esterase production by ectomycorrhizal fungi. Mycologia. 2:233–236.
- Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Van Den Berg C, and Schuiteman A. 2015. An updated classification of Orchidaceae. Botanical Journal of the Linnean Society. 177:151–174.
- Clements MA, and Ellyard RK. 1979. The symbiotic germination of Australian terrestrial orchids. American Orchid Society Bulletin. 48:810–816.
- Cruz D, Suárez JP, Kottke I, and Piepenbring M. 2014. Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella. Mycologia. 106:708–722.
- De Carvalho OC, de Paiva Neto VB, Padilha DRC, Veloso TGR, Bocayuva MF, Soares DCO, and Kasuya MCM. 2017. Cyrtopodium paludicolum germination with two Tulasnella isolates. Acta Botanica Brasilica. 32:107–112.
- De Long JR, Swarts ND, Dixon KW, and Egerton-Warburton LM. 2013. Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media. Ann Bot. 111:409–418.
- Dodds PN, and Rathjen JP. 2010. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet. 11:539–548.
- Downing JL, Liu H, McCormick MK, Arce J, Alonso D, Perez JL. 2020. Generalized mycorrhizal interactions and fungal enemy release drive range expansion of orchids in southern Florida. Ecosphere. 11:e03228.
- Dupuis JR, Roe AD, Sperling FA. 2012. Multi-locus species delimitation in closely related animals and fungi: one marker is not enough. Mol Ecol. 21:4422–4436.
- Fang X, Finnegan PM, Barbetti MJ. 2013. Wide variation in virulence and genetic diversity of binucleate Rhizoctonia isolates associated with root rot of strawberry in Western Australia. PLOS One. 8:e55877.
- Favre-Godal Q, Gourguillon L, Lordel-Madeleine S, Gindro K, Choisy P. 2020. Orchids and their mycorrhizal fungi: an insufficiently explored relationship. Mycorrhiza. 30:5–22.
- Freitas EFS, Da Silva M, Cruz ES, Mangaravite E, Bocayuva MF, Veloso TGR, Selosse MA, Kasuya MCM. 2020. Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species. Sci Rep. 10:7069.
- Fujimori S, Abe JP, Okane I, Yamaoka Y. 2019. Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae). Mycoscience. 60:71–81.
- Hawksworth D. 2011. A new dawn for the naming of fungi: impacts of decisions made in Melbourne in July 2011 on the future publication and regulation of fungal names. MycoKeys. 1:7–20.
- Hua’an Y, Sivasithamparam K, O’Brien PA. 1991. An improved technique for fluorescence staining of fungal nuclei and septa. Australasian Plant Pathology. 20:119–21.
- Jinks JL. 1952. Heterokaryosis: a system of adaptation in wild fungi. The Royal Society Publishing. 140:83–99.
- Jones DL. 2021. A complete guide to native orchids of Australia. Sydney, Australia: Reed New Holland Publishers Pty Ltd.
- Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, and Jermiin LS. 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 14:587–589.
- Kottke I, Haug I, Setaro S, Suárez JP, Weiß M, Preußing M, Nebel M, Oberwinkler F. 2008. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical mountain rain forest. Basic and Applied Ecology. 9:13–23.
- Krause C, Garnica S, Bauer R, and Nebel M. 2011. Aneuraceae (Metzgeriales) and tulasnelloid fungi (Basidiomycota): a model for early steps in fungal symbiosis. Fungal Biol. 115:839–851.
- Kumar S, Stecher G, Li M, Knyaz C, and Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 35:1547–1549.
- Linde CC, May TW, Phillips RD, Ruibal M, Smith LM, Peakall R. 2017. New species of Tulasnella associated with terrestrial orchids in Australia. IMA Fungus. 8:27–47.
- Linde CC, Phillips RD, Crisp MD, Peakall R. 2014. Congruent species delineation of Tulasnella using multiple loci and methods. New Phytologist. 201:6–12.
- Ma LJ, van der Does HC, Borkovich KA, Coleman JJ, Daboussi M-J, Di Pietro A, Dufresne M, Freitag M, Grabherr M, Henrissat B, et al. 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature. 464:367–373.
- Mack J, Assabgui RA, and Seifert KA. 2021. Taxonomy and phylogeny of the basidiomycetous hyphomycete genus Hormomyces. Fungal Systematics and Evolution. 7:177–196.
- Moncalvo JM, Nilsson RH, Koster B, Dunham SM, Bernauer T, Matheny PB, Porter TM, Margaritescu S, Weiß M, Garnica S, et al. 2006. The cantharelloid clade: dealing with incongruent gene trees and phylogenetic reconstruction methods. Mycologia. 98:937–948.
- Moore RT. 1987. The genera of Rhizoctonia-like fungi: Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis, and Rhizoctonia. Mycotaxon. 29:91–99.
- Nargar K, Molina S, Wagner N, Nauheimer L, Micheneau C, and Clements MA. 2018. Australasian orchid diversification in time and space: Molecular phylogenetic insights from the beard orchids (Calochilus, Diurideae). Aust Syst Bot. 31:389–408.
- Nauheimer L, Schley RJ, Clements MA, Micheneau C, and Nargar K. 2018. Australasian orchid biogeography at continental scale: Molecular phylogenetic insights from the Sun Orchids (Thelymitra, Orchidaceae). Mol Phylogenet Evol. 127:304–319.
- Nguyen DQ, Li H, Tran TT, Sivasithamparam K, Jones MGK, Wylie SJ. 2020. Four Tulasnella taxa associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata. Fungal Biol. 124:24–33.
- Nguyen LT, Schmidt HA, Von Haeseler A, and Minh BQ. 2014. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 32:268–274.
- Nilsson RH, Kristianson E, Ryberg M, Hallenberg N, Larsson K-H. 2008. Intraspecific ITS variability in the Kingdom Fungi as expressed in the international sequence databases and Its implications for molecular species identification. Evolutionary Bioinformatics. 4:193–201.
- Oberwinkler F, Cruz D, and Suárez JP. 2017. Biogeography and ecology of Tulasnellaceae. Ecological Studies. 230:237–271.
- Oberwinkler F, Riess K, Bauer R, Kirschner R, and Garnica S. 2013. Taxonomic re-evaluation of the Ceratobasidium-Rhizoctonia complex and Rhizoctonia butinii, a new species attacking spruce. Mycological Progress. 12:763–776.
- Oktalira FT, May TW, Dearnaley JD, and Linde CC. 2021. Seven new Serendipita species associated with Australian terrestrial orchids. Mycologia. 113:968–987.
- Peakall R, Wong DCJ, Phillips RD, Ruibal M, Eyles R, Rodriguez-Delgado C, and Linde CC. 2021. A multitiered sequence capture strategy spanning broad evolutionary scales: Application for phylogenetic and phylogeographic studies of orchids. Mol Ecol Resour. 21:1118–1140.
- Plett JM, Martin F. 2015. Reconsidering mutualistic plant-fungal interactions through the lens of effector biology. Curr Opin Plant Biol. 26:45–50.
- Preußing M, Nebel M, Oberwinkler F, and Weiss M. 2010. Diverging diversity patterns in the Tulasnella (Basidiomycota, Tulasnellales) mycobionts of Aneura pinguis (Marchantiophyta, Metzgeriales) from Europe and Ecuador. Mycorrhiza. 20:147–159.
- R Core Team. 2019. R: a language and environment for statistical computing, Vienna, Austria.
- Rachanarin C, Suwannarach N, Kumla J, Srimuang K, Ehc M, Lumyong S. 2018. A new endophytic fungus, Tulasnella phuhinrongklaensis (Cantharellales, Basidiomycota) isolated from roots of the terrestrial orchid, Phalaenopsis pulcherrima. Phytotaxa. 374:99–109.
- Rasmussen HN, Dixon KW, Jersakova J, Těšitelová T. 2015. Germination and seedling establishment in orchids: a complex of requirements. Ann Bot. 116:391–402.
- Reiter N, Lawrie AC, and Linde CC. 2018. Matching symbiotic associations of an endangered orchid to habitat to improve conservation outcomes. Ann Bot. 122:947–959.
- Rep M, and Kistler HC. 2010. The genomic organization of plant pathogenicity in Fusarium species. Curr Opin Plant Biol. 13:420–426.
- Roberts P. 1992. Spiral-spored Tulasnella species from Devon and the new forest. Mycol Res. 96:233–236.
- Roberts P. 1993. Allantoid-spored Tulasnella species from Devon. Mycol Res. 97:213–220.
- Roberts P. 1994a. Globose and ellipsoid-spored Tulasnella species from Devon and Surrey, with a key to the genus in Europe. Mycol Res. 98:1431–1452.
- Roberts P. 1994b. Long-spored Tulasnella species from Devon, with additional notes on allantoid-spored species. Mycol Res. 98:1235–1244.
- Roberts P. 1999. Rhizoctonia-forming Fungi: a taxonomic guide. Richmond (United Kingdom): Royal Botanic Gardens Kew.
- Ronquist F, and Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 19:1572–1574.
- Roper M, Ellison C, Taylor JW, and Glass NL. 2011. Nuclear and genome dynamics in multinucleate ascomycete fungi. Current Biology. 21:786–793.
- Ruibal MP, Peakall R, Smith LM, Linde CC. 2013. Phylogenetic and microsatellite markers for Tulasnella (Tulasnellaceae) mycorrhizal fungi associated with Australian orchids. Applications in Plant Sciences. 1:1200394.
- Ruibal MP, Triponez Y, Smith LM, Peakall R, Linde CC. 2017. Population structure of an orchid mycorrhizal fungus with genus-wide specificity. Sci Rep. 7:5613.
- Sezdzielewska-Toro K, Delaux P-M. 2016. Mycorrhizal symbioses: today and tomorrow. New Phytologist. 209:917–20.
- Shivas RG, Beasley DR, Pascoe IG, Cunnington JH, Pitkethley RN, and Priest MJ. 2006. Specimen-based databases of Australian plant pathogens: past, present and future. Australasian Plant Pathology. 35:195–198.
- Solís K, Barriuso JJ, Ana G-C, and Gonzalez V. 2017. Tulasnella tubericola (Tulasnellaceae, Cantharellales, Basidiomycota): A new Rhizoctonia-like fungus associated with mycorrhizal evergreen oak plants artificially inoculated with black truffle (Tuber melanosporum) in Spain. Phytotaxa. 317:175–187.
- Sommer J, Pausch J, Brundrett MC, Dixon KW, Bidartondo MI, and Gebauer G. 2012. Limited carbon and mineral nutrient gain from mycorrhizal fungi by adult Australian orchids. Am J Bot. 99:1133–1145.
- Stalpers J, Redhead SA, May TW, Rossman AY, Crouch JA, Cubeta MA, Dai Y-C, Kirschner R, Langer GJ, Larsson K-H, et al. 2021. Competing sexual-asexual generic names in the Agaricomycotina (Basidiomycota), with recommendations for use. IMA Fungus. 12(22):1-32.
- Stamatakis A, Hoover P, and Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol. 57:758–771.
- Stefani FO, Jones RH, and May TW. 2014. Concordance of seven gene genealogies compared to phenotypic data reveals multiple cryptic species in Australian dermocyboid Cortinarius (Agaricales). Mol Phylogenet Evol. 71:249–260.
- Stergiopoulos I, and de Wit PJ. 2009. Fungal effector proteins. Annu Rev Phytopathol. 47:233–263.
- Suarez JP, Weiss M, Abele A, Garnica S, Oberwinkler F, and Kottke I. 2006. Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest. Mycol Res. 110:1257–1270.
- Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology. 31:21–32.
- Taylor DL, and McCormick MK. 2008. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas. New Phytologist. 177:1020–1033.
- Tedersoo L, May TW, and Smith ME. 2010. Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages. Mycorrhiza. 20:217–263.
- Thompson JD, Higgins DG, and Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.
- Veldre V, Abarenkov K, Bahram M, Martos F, Selosse MA, Tamm H, Kõljalg U, and Tedersoo L. 2013. Evolution of nutritional modes of Ceratobasidiaceae (Cantharellales, Basidiomycota) as revealed from publicly available ITS sequences. Fungal Ecol. 6:256–268.
- Warcup JH. 1973. Symbiotic germination of some Australian terrestrial orchids. New Phytologist. 72:387–92.
- Warcup JH. 1981. The mycorrhizal relationships in Australian orchids. New Phytologist. 87:371–381.
- Warcup JH, and Talbot PHB. 1967. Perfect states of Rhizoctonias associated with orchids. New Phytologist. 66:631–641.
- Warcup JH, Talbot PHB. 1971. Perfect states of Rhizoctonias associated with orchids II. New Phytologist. 70:35–40.
- Weston PH, Perkins A, Indsto JO, Clements MA. 2014. Phylogeny of Orchidaceae tribe Diurideae and its implications for the evolution of pollination systems. In: Edens-Meier R, and Bernhardt P, editors. Darwin’s orchids: Then and now. US: The University of Chicago. p. 91–154.
- White TJ, Bruns TD, Lee S, and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: A guide to methods and applications. New York: Academic Press, Inc. p. 315–322.
- Williams PG, and Thilo E. 1989. Ultrastructural evidence for the identity of some multinucleate rhizoctonias. New Phytologist. 112:513–518.
- Wright MM, Cross R, Cousens RD, May TW, and McLean CB. 2010. Taxonomic and functional characterisation of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia. Mycorrhiza. 20:375–390.
- Yukawa T, Ogura-Tsujita Y, Shefferson RP, Yokoyama J. 2009. Mycorrhizal diversity in Apostasia (Orchidaceae) indicates the origin and evolution of orchid mycorrhiza. Am J Bot. 96:1997–2009.