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Octospora conidiophora (Pyronemataceae) – a new species from South Africa and the first report of anamorph in bryophilous Pezizales
expand article infoZuzana Sochorová§, Peter Döbbeler|, Michal Sochor§, Jacques van Rooy#
‡ Palacký University Olomouc, Olomouc, Czech Republic
§ Crop Research Institute, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic
| Universität München, München, Germany
¶ National Herbarium, South African National Biodiversity Institute, Pretoria, South Africa
# University of the Witwa­tersrand, Johannesburg, South Africa
Open Access

Abstract

Octospora conidiophora is described as a new species, based on collections from South Africa. It is characterised by apothecia with a distinct margin, smooth or finely warted ellipsoid ascospores, stiff, thick-walled hyaline hairs, warted mycelial hyphae and growth on pleurocarpous mosses Trichosteleum perchlorosum and Sematophyllum brachycarpum (Hypnales) on decaying wood in afromontane forests. It is the first species of bryophilous Pezizales in which an anamorph has been observed; it produces long, claviform, curved, hyaline and transversely septate conidia. Three other cryptic species of Octospora were detected using three molecular markers (LSU and SSU nrDNA and EF1α), but these could not be distinguished phenotypically. These are not described formally here and an informal species aggregate O. conidiophora agg. is established for them. The new species and finds of Lamprospora campylopodis growing on Campylopus pyriformis and Neottiella albocincta on Atrichum androgynum represent the first records of bryophilous Pezizales in South Africa.

Keywords

Afromontane forests, bryosymbionts, conidia, cryptic biodiversity, muscicolous parasites, Sematophyllum, Trichosteleum, South Africa

Introduction

The family Pyronemataceae is not only highly diverse in terms of morphology but also ecologically (Perry et al. 2007, Hansen et al. 2013). It includes six related genera that obligately grow on bryophytes – Octospora Hedw., Lamprospora De Not., Neottiella (Cooke) Sacc., Octosporopsis U.Lindem. & M.Vega, Octosporella Döbbeler and Filicupula Y.J.Yao & Spooner. These ascomycetes, known as bryoparasitic, bryophilous or bryosymbiotic Pezizales, form ca. 0.2–15 mm broad apothecia or perithecia-like apothecia (in Octosporella), coloured in shades of orange or red. They infect their hosts by elaborate infection structures consisting of superficial appressoria and intracellular haustoria (Döbbeler 1980). Together with their hosts, they can be found on various substrates like soil, burnt ground, rocks or bark and wood, both in natural and anthropogenic habitats in arctic to tropical regions (e.g. Benkert 1987; Schumacher 1993; Döbbeler 1997; Egertová et al. 2018).

Only rare reports of bryophilous Pezizales from the African continent are known: Lamprospora maireana Seaver, described on the basis of material from Algeria (Seaver 1914); Octospora tetraspora (Fuckel) Korf var. aegyptiaca J.Moravec from Egypt (Moravec 1972), later revised by D. Benkert as O. leucoloma Hedw. var. tetraspora Benkert, as indicated by his revision label; and O. kilimanjarensis J.Moravec, described from Tanzania (Moravec 1997) and later reported from Ethiopia together with a probably undescribed Octospora species (Lindemann 2013).

From southern Africa, thus far, no finds of these fungi have been reported and no vouchers are deposited in the South African National Collection of Fungi (PREM; Riana Jacobs-Venter pers. comm.). Surprisingly, during three weeks of our field excursions in KwaZulu-Natal and Mpumalanga, eastern South Africa, in February and March 2018, 39 populations of bryophilous Pezizales (Octospora, Lamprospora and Neottiella) were recorded. Only three of them could be assigned to described species, based on morphological characters, host association and DNA sequencing: Lamprospora campylopodis W.D.Buckley growing on Campylopus pyriformis (Schultz) Brid. (two collections) and Neottiella albocincta (Berk. & M.A.Curtis) Sacc. on Atrichum androgynum (Müll. Hal.) A.Jaeger (one collection). The remaining specimens were separated into six morphospecies. One of them, an undescribed Octospora species, growing on pleurocarpous mosses from the family Sematophyllaceae (Hypnales), turned out to be very common and remarkable in several aspects after detailed analysis. The aim of this contribution is to provide a description of this species, clarify its phylogenetic relationships and discuss associated taxonomical problems.

Methods

Sample collection and observation

Fungi were collected in February and March 2018 in South African Provinces KwaZulu-Natal and Mpumalanga. The description of Octospora conidiophora is based on 11 collections belonging to the most frequent genotype. Observations of apothecial features were made on vital (marked by *) or rehydrated (†) material mostly in tap water, cresyl blue (CRB), lactophenol cotton blue (LPCB) or lactic acid cotton blue (LACB). Absence of amyloidity of asci was confirmed in Lugol´s solution. Infection structures were observed on rehydrated material. Parts of the host plants (leaves and rhizoids) close to an apothecium were separated, pulled apart, treated with LPCB and studied by light microscopy. The preparations were screened at 100× to 200× magnification for the presence of conidia. Infection structures and conidia usually occurred in the same mounts. Illustrations and measurements of hyphae, appressoria and haustoria, as well as conidia, were done in LPCB. The mosses were identified as hosts, based on the presence of appressoria on leaves or rhizoids. The host species were determined using standard techniques for bryophytes (Magill 1981). Collections are deposited in the Mycological department of the National Museum in Prague (PRM) and the herbarium of the Botanische Staatssammlung München (M).

DNA extraction, PCR amplification and sequencing

DNA was extracted from dried apothecia by the CTAB method as outlined by Doyle and Doyle (1987). Up to three apothecia were homogenised by a pestle and incubated in 300 μl extraction buffer at 65 °C for one hour; the extract was subsequently purified in chloroform-isoamyl alcohol mixture, precipitated by isopropanol and finally dissolved in water and incubated with RNase for 30 min at 37 °C. DNA quality was checked on agarose gel. Molecular sequence data were generated for three loci: the 28S subunit of ribosomal DNA (LSU) was amplified with primers LR0R and LR6 (Vilgalys and Hester 1990), the 18S subunit of rDNA (SSU) with primers NS1 and NS6 (White et al. 1990) and translation elongation factor-1alpha (EF1α) with primers EF1-983F and EF1–1567R (Rehner and Buckley 2005). PCR was performed with Kapa polymerase (Kapa Biosystems, Wilmington, USA) following a standard protocol with 37 cycles and annealing temperature of 54 °C. The PCR products were purified by precipitation with polyethylene glycol (10% PEG 6000 and 1.25 M NaCl in the precipitation mixture) and sequenced from both directions using the same primers by the Sanger method at Macrogen Europe, Amsterdam, The Netherlands.

Phylogenetic analysis

Newly generated sequences were assembled, edited and aligned in Geneious 7.1.7. (Biomatters, New Zealand) using the MAFFT plugin, manually corrected and deposited in NCBI GenBank under accession numbers MK569288MK569376. Datasets were compiled from these and previously published sequences (Table 1), aligned, trimmed in order not to contain too many missing data at the ends and concatenated in Geneious 7.1.7. Bayesian Inference for concatenated data was computed in Mrbayes (ver. 3.2.4; Ronquist et al. 2012) with 2×107 generations, sampling every 1000th tree, in two independent runs, each with 4 chains, the first 50% (107) generations being excluded as burn-in. The most suitable substitution model for each locus was determined in Partitionfinder 2.1.1 (Lanfear et al. 2017) using the AIC corrected for small samples (AICc) and a greedy search. Single-locus phylogenies were computed with similar settings, but with 6×106 MCMC generations and the parameter temp. = 0.01.

Specimens used for the phylogeny inference and their GenBank accession numbers. Newly generated sequences are MK569288MK569376.

Taxon Collection code LSU SSU EF1α
Lamprospora campylopodis W.D.Buckley 48633 MF066054 MK569364 MK569289
Lamprospora dictydiola Boud. ldic MF754056 MK569365 MF754054
Lamprospora miniata De Not. var. parvispora Benkert LMSk MF066065 MK569366 MF754055
Lamprospora sylvatica Egertová & Eckstein UA1 MG947604 MK569367 MK569290
Neottiella rutilans (Fr.) Dennis 46853 MK569313 MK569336 MK569288
Neottiella vivida (Nyl.) Dennis NVZla MF066068 MK569337 MF754051
Octospora affinis Benkert & L.G.Krieglst. OAFZla MF754075 MK569347 MF754045
Octospora conidiophora Sochorová & Döbbeler ZE11/18 MK569315 MK569348 MK569291
Octospora conidiophora ZE23/18 MK569324 MK569349 MK569294
Octospora conidiophora ZE45/18 MK569316 MK569296
Octospora conidiophora ZE46/18 MK569317 MK569350 MK569298
Octospora conidiophora ZE48/18 MK569321 MK569351 MK569297
Octospora conidiophora ZE57/18 MK569318 MK569352 MK569295
Octospora conidiophora ZE62/18 MK569323 MK569354 MK569299
Octospora conidiophora ZE63/18 MK569319 MK569355 MK569292
Octospora conidiophora ZE71/18 MK569322 MK569356 MK569293
Octospora conidiophora ZE75/18 MK569320 MK569357 MK569300
Octospora conidiophora ZE77/18 MK569331 MK569353 MK569301
Octospora conidiophora agg. – lineage B ZE37/18 MK569325 MK569358 MK569302
Octospora conidiophora agg. – lineage B ZE38/18 MK569329 MK569359 MK569303
Octospora conidiophora agg. – lineage B ZE51/18 MK569327 MK569362 MK569306
Octospora conidiophora agg. – lineage B ZE52/18 MK569326 MK569360 MK569304
Octospora conidiophora agg. – lineage B ZE53/18 MK569328 MK569361 MK569307
Octospora conidiophora agg. – lineage B ZE65/18 MK569330 MK569363 MK569305
Octospora conidiophora agg. – lineage C ZE44/18 MK569332 MK569373 MK569308
Octospora conidiophora agg. – lineage C ZE56/18 MK569333 MK569374 MK569309
Octospora conidiophora agg. – lineage D ZE69/18 MK569334 MK569375 MK569310
Octospora erzbergeri Benkert ERZ MF754068 MK569340 MF754042
Octospora excipulata (Clem.) Benkert OExc MF754062 MK569369 MF754047
Octospora fissidentis Benkert & Brouwer Fis MF754073 MK569341 MF754044
Octospora humosa (Fr.) Dennis OHZla MF754074 MK569343 MF754043
Octospora ithacaensis (Rehm) K.B.Khare OLOi MF754071 MK569346 MF754053
Octospora kelabitiana Egertová & Döbbeler Oct-Jat MF754065 MK569372 MF754048
Octospora kelabitiana ZE61/16 MF754064 MK569376 MF754049
Octospora leucoloma Hedw. Oleu MF066067 MK569370
Octospora orthotrichi (Cooke & Ellis) K.B.Khare & V.P.Tewari HR8 MK569314 MK569342 MK569311
Octospora phagospora (Flageolet & Lorton) Dennis & Itzerott PHG44 MF754072 MK569344 MF754046
Octospora pseudoampezzana (Svrček) Caillet & Moyne OP1 MF754069 MK569339 MF754050
Octospora wrightii (Berk. & M.A.Curtis) J.Moravec WRIG MF754070 MK569345
Octosporella perforata (Döbbeler) Döbbeler PERF MF754060 MK569368 MF754052
Octosporopsis erinacea Egertová & Döbbeler DUM20/1 MF754057 MK569338 MF754041
Otidea leporina (Batsch) Fuckel KGOL MK569335 MK569371 MK569312

Divergence times were estimated with Beast 2.5.1 (Bouckaert et al. 2014) using the LSU and SSU data from our sample set (one sample per species or phylogenetic lineage) and six additional species: Caloscypha fulgens (Pers.) Boud., Scutellinia scutellata (L.) Lambotte, Cheilymenia stercorea (Pers.) Boud., Aleuria aurantia (Pers.) Fuckel, Pyronema domesticum (Sowerby) Sacc. and Sarcoscypha coccinea (Gray) Boud. (all sequences obtained from Beimforde et al. 2014; EF1α was not analysed by these authors and, therefore, not included in our molecular dating). Four calibration points were used for the analysis and the divergence times, together with their confidence intervals, were also taken from Beimforde et al. (2014), namely divergence Cheilymenia-Scutellinia, divergence Aleuria-(Cheilymenia+Scutellinia), split-off of Sarcoscypha and split-off of Caloscypha. Monophyly was forced for all of the points except the second one due to an unclear position of the Octospora clade. Analysis was run under GTR+I+G substitution model (as for Mrbayes), with relaxed clock log normal model and 108 MCMC generations, but the first 50% were excluded as burn-in. Priors included the Yule model with uniform birth rate and exponential gamma shape. Convergence and stationarity were analysed using Tracer v1.7.1 (Rambaut et al. 2018) and results were considered when effective sample size (ESS) ≥ 1000. Statistical uncertainty of divergence time estimates was assessed through the calculation of highest probability density (HPD) values.

Results

Phylogenetic and phenotypic analysis

After trimming, the total length of the concatenated alignment was 2702 bp (539 bp from EF1α, 1102 bp from LSU and 1061 bp from SSU, including gaps). Every studied locus provided sufficient polymorphism both amongst and within previously phenotypically delimited groups (Suppl. material 1: Table S1). Four distinct phylogenetic lineages were detected in the concatenated data, as well as in single-locus data within the group of specimens that were hosted by Sematophyllaceae (Fig. 1, Suppl. material 2: Fig. S1). Divergence between them was between 4 and 59 nucleotide differences at every locus (Suppl. material 1: Table S1). The four South African lineages formed a highly supported and distinct clade together with O. kelabitiana (Fig. 1). Molecular dating analysis estimated the basal split of bryophilous Pezizales to be 87–172 Ma old (95% confidence interval; mean = 149 Ma), the basal split of the South African accessions was estimated at 23–73 Ma (mean = 47 Ma; Fig. 2).

Figure 1. 

Bayesian phylogeny inference, based on concatenated alignment of EF1α, LSU and SSU sequences. Bayesian posterior probabilities are shown above branches; Otidea leporina serves as outgroup; trees based on analysis of each locus are shown in Suppl. material 2: Fig. S1.

Figure 2. 

Maximum clade credibility tree with estimated divergence times, based on SSU and LSU data; calibration points are marked by red circles, posterior probabilities shown above branches, bars indicate the 95% highest posterior density (HPD) intervals.

No significant differences in phenotypic traits were detected amongst the South African lineages using standard characters and methods. They shared the structure of excipulum, stiff, thick-walled hyaline hairs, ellipsoid hyaline ascospores which can be either smooth or ornamented with fine warts and which contain 1 or 2 guttules, warted mycelial hyphae, appressoria, haustoria and presence of anamorph. Although differences amongst individual collections were observed, phenotypic characters did not correspond to the molecular markers and many characters exhibited variability both amongst and within the four phylogenetic lineages (Table 2).

Variability of selected characters in O. conidiophora agg.

Voucher Ornament of ascospores (†) Size of ascospores [μm] (†) Mean size of ascospores [μm] (†) Q of ascospores (†) Qm Observation of conidia Host
Lineage A - Octospora conidiophora s. str.
ZE11/18 smooth 14.4–16.0 × 8.0–9.1 15.1 × 8.4 1.65–1.91 1.79 yes S. brachycarpum
ZE23/18 smooth 14.0–16.3 × 7.5–9.0 14.9 × 8.0 1.66–2.03 1.84 yes T. perchlorosum
ZE45/18 smooth 13.6–16.2 × 7.5–8.3 15.2 × 7.9 1.74–2.08 1.92 yes T. perchlorosum
ZE46/18 smooth 15.0–17.0 × 8.0–9.9 15.9 × 8.7 1.63–2.06 1.82 yes T. perchlorosum
ZE48/18 smooth 14.5–17.0 × 8.3–9.9 16.1 × 9.0 1.64–1.99 1.79 yes T. perchlorosum
ZE57/18 smooth 14.9–16.2 × 7.9–9.0 15.5 × 8.2 1.69–1.99 1.87 yes T. perchlorosum
ZE62/18 smooth 14.0–16.7 × 8.0–8.9 15.2 × 8.2 1.72–1.99 1.85 yes T. perchlorosum
ZE63/18 smooth 14.0–17.0 × 8.0–9.2 15.4 × 8.7 1.67–1.89 1.77 yes T. perchlorosum
ZE71/18 warted 13.0–15.0 × 8.0–9.9 14.1 × 8.9 1.39–1.73 1.59 no T. perchlorosum
ZE75/18 smooth 14.0–16.1 × 7.8–9.1 15.1 × 8.4 1.65–1.94 1.79 yes T. perchlorosum
ZE77/18 warted 13.5–15.4 × 8.7–10.5 14.4 × 9.4 1.40–1.67 1.53 yes T. perchlorosum
all specimens 13.0–17.0 × 7.5–10.5 15.2 × 8.5 1.39–2.08 1.79
Lineage B
ZE37/18 warted 13.3–15.5 × 8.3–9.9 14.6 × 9.1 1.47–1.82 1.60 yes S. brachycarpum
ZE38/18 warted 12.5–15.1 × 8.0–9.7 13.8 × 8.8 1.46–1.78 1.57 no T. perchlorosum
ZE51/18 warted 13.5–15.7 × 8.4–10.2 14.5 × 9.4 1.45–1.65 1.54 yes T. perchlorosum
ZE52/18 warted 13.5–16.0 × 8.0–9.5 14.7 × 8.8 1.47–1.83 1.66 yes T. perchlorosum
ZE53/18 warted 14.0–15.3 × 8.0–10.1 14.7 × 9.3 1.47–1.85 1.56 no T. perchlorosum
ZE65/18 warted 13.5–16.2 × 7.7–9.9 14.4 × 8.8 1.47–1.75 1.60 yes T. perchlorosum
all specimens 12.5–16.2 × 7.7–10.2 14.5 × 9.1 1.45–1.85 1.59
Lineage C
ZE44/18 warted 13.5–15.9 × 7.2–8.1 14.6 × 7.8 1.71–2.01 1.87 yes S. brachycarpum
ZE56/18 warted 13.1–15.4 × 7.0–8.2 14.1 × 7.6 1.66–2.11 1.84 yes S. brachycarpum
both specimens 13.1–15.9 × 7.0–8.2 14.3 × 7.7 1.66–2.11 1.86
Lineage D
ZE69/18 smooth or lightly warted 13.5–18.0 × 7.9–9.9 15.5 × 8.6 1.61–2.02 1.80 yes S. brachycarpum

Taxonomy

Octospora conidiophora Sochorová & Döbbeler, sp. nov.

MycoBank No: MB829095
Figs 3, 4, 5, 6, 7, 8, 9

Etymology

Conidiophorus (Gr./Lat.) refers to production of conidia.

Diagnosis

Differs from Octospora kelabitiana by larger apothecia with a distinct margin, infection of pleurocarpous mosses of the family Sematophyllaceae and frequent formation of a Spermospora-like anamorph.

TYPE: SOUTH AFRICA. KwaZulu-Natal Province: Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°7.72'S, 29°25.27'E, 1750 m alt., on Trichosteleum perchlorosum on decaying wood, 2 Mar. 2018, Z. Egertová (Sochorová) and M. Sochor ZE48/18, holotype: PRM 951743, isotype: M; LSU GenBank accession number: MK569321, SSU GenBank accession number: MK569351, EF1α GenBank accession number: MK569297.

Description

Apothecial features: Apothecia in groups on plants of Trichosteleum perchlorosum or Sematophyllum brachycarpum or between them, 0.2–1.5 mm broad, up to 0.65 mm high, first subglobose with a small apical opening, later hemispherical, turbinate to disc-shaped, pinkish-orange, sessile, mostly with a well-developed margin, outer surface of excipulum with adpressed to shortly protruding hairs or hyphae.

Hairs *55–205 × 4–10.5 µm, scattered at flanks, hyaline, scarcely septate, obtuse, thick-walled, wall *0.5–3.5 µm thick. Excipulum at the base *230–330 µm thick, laterally about 50 µm thick, composed of angular to subangular (triangular, trapezoid, rectangular), globose, subglobose or irregularly shaped cells, *6–43 × 5–42 µm, outermost cells thick-walled (neighbouring cells divided by up to *6 µm broad wall). Margin *60–280 µm broad, consisting of globose, subglobose, pyriform or trapezoid cells, *10–38 × 7–30 µm.

Subhymenium *40–75 µm wide, consisting of densely packed cylindrical cells *3–7 µm wide mixed with angular or irregularly shaped cells, *4.5–8 × 4.5–6 µm. Paraphyses filiform, straight or bent, unbranched, septate, uppermost one or two cells containing little very pale droplets (*0.5–2 µm in diameter), *2.1–3.5 µm broad (†1.5–2.3 µm), terminal cell *19–83 × 3–7 µm (†18–57 × 3–5.5 µm). Asci *146–197 × 12–15.5 µm (†135–192 × 9.5–12.5 µm), cylindrical, unitunicate, operculate, inamyloid, arising from croziers, with 8 uniseriate ascospores. Ascospores *13–17.2 × 7–10.5 µm, mean 15.2 × 9 µm, Q = 1.34–1.99, Qm = 1.69 (†13–17 × 7.5–10.5 µm, mean 15.2 × 8.5 µm, Q = 1.39–2.08, Qm = 1.79), ellipsoid to narrowly ellipsoid, hyaline, containing one or two lipid guttules (up to *8 µm in diameter if one, *4–5.5 µm if two), smooth or ornamented with cyanophilous, very small, obtuse warts 0.1–0.3 µm broad; germinating with a single germ tube.

Mycelial (†): Hyphae restricted to the lowermost plant parts, irregularly growing on and between the leaf bases, stems and especially the rhizoids, hyaline, with ramifications and anastomoses, often thick-walled, (2–)3–6(–7) µm in diameter (excluding ornamentation); hyphal surface with minute to large protuberances, in optical section with numerous minute or larger, semi- or subglobose warts or spines, in surface view, these structures sometimes looking like ridges extended perpendicularly to the hyphal axis; largest warts up to 1.5(–2) µm high; hyphae growing within hyphae present; whole hyphal wall slightly cyanophilous, outermost rough part strongly cyanophilous.

Appressoria variable, frequent (even more than 30 per leaf observed) and easy to detect, closely attached to both leaf sides or to rhizoids, colourless, 1-, 2- or 3-celled, from above elliptical, (14–)16–23(–26) µm long, (8–)11–16 µm wide, laterally seen slightly kidney-shaped, (7–)9–13(–16) µm high, with walls up to 2.5(–4) µm thick; surface rough but not warty, cyanophilous; appressorial cytoplasma strongly cyanophilous; appressoria mostly laterally formed on short stalks; stalks often gradually expanding toward the appressorium; perforation of the host cell wall by means of a delicate peg; peg often surrounded by a brown, straight or curved lignituber-like swelling measuring up to 10(–15) × 2–4(–6) µm; rhizoid wall at the perforation point slightly uplifted towards the appressorium; perforation point not always visible from above.

Haustoria within living leaf cells or rhizoidal cells, at first as a thick short filament, later becoming up to 55 µm long, orientated longitudinally in the rhizoid and developing ramifications (in wider rhizoids), rarely filling out the whole host cell; haustorial cytoplasm strongly cyanophilous.

Anamorph (†): Conidia variable in shape and size, claviform, hyaline, transversely septate, ca. (50–)70–115(–154) µm long (including the tail); proximal cell usually distinctly wider than the subproximal cell, rarely cells almost cylindrical, both cells measuring together (30–)35–48(–55) × (6–)7.5–12(–15) µm, subproximal cell continuously attenuating into a tail; tail typically curved to curled, 1- or 2-(3-)celled, (15–)30–60(–100) µm long and (1.5–)2(–2.5) µm in diameter at the distal end; proximal cell of the conidia with a conspicuous, circular, slightly protruding, delicately fringed scar, (3–)4(–4.5) µm in diameter, resulting from detachment from the conidiogenous cell; scar sometimes slightly laterally positioned; walls of conidia cyanophilous; the two proximal cells smooth, the tail sometimes warty (like the hyphae); germ tube one (to three) per conidium, arising from the scar or laterally from different regions of the conidia, including the tail cells.

Conidiogenous cells irregularly shaped, shorter and wider than sterile hyphal cells, rich in cytoplasmic content, usually with 1(–2) scars; shape and size of the scars like those at the conidia, also with a delicately fringed margin.

Figure 3. 

Octospora conidiophora. A–E Apothecia in situ F Habitat A, E ZE63/18 B ZE57/18 C, F ZE11/18 D holotype ZE48/18.

Figure 4. 

Microscopic characters of Octospora conidiophora. A Ascospores inside ascus stained with CRB B Free ascospores in tap water C Paraphyse in tap water D Young ascus in tap water E Margin of apothecium in tap water F Hair and excipular cells in tap water A–F ZE77/18.

Figure 5. 

Microscopic characters of Octospora conidiophora. A Cells of the outermost layer of excipulum from an outside view stained with CRB B Mycelial hypha stained with CRB C Appressoria and hyphae on a leaf of Sematophyllum brachycarpum in tap water D Appressorium stained with LACB E Germinating conidium stained with LPCB F Germinated conidium produced a bifurcate warted hypha (right arrow), appressorium (left arrow) probably not connected to the conidium, in LACB A, B, F ZE77/18 C, E ZE11/18 D holotype ZE48/18.

Figure 6. 

Microscopic characters of Octospora conidiophora. A Hypha with two-celled appresorium closely attached to the cells of the host leaf B Ascospores C Germinating ascospores found on leaves D Variation of appressoria mostly seen from above, infection pegs not always observed, appressoria seen in lateral view with infection pegs (indicated by arrows) A, B, D holotype ZE48/18 C ZE11/18. Scale bar: 30 µm. Illustrated by P.D.

Figure 7. 

Microscopic characters of Octospora conidiophora. A–C, E–H Conidia, distal curved part apparently sometimes broken off, some conidia germinating D Conidogenous cells, on the right with a developing conidium E (on the right) Conidium anastomosing to mycelial hypha with two-celled appressorium F Conidium germinating by a hypha with a warty surface and a two-celled appressorium G Conidium with anastomosis to mycelial hypha H (on the right) Two germinating conidia with an anastomosis between them A, F ZE63/18 B ZE46/18 C ZE77/18 D, E holotype ZE48/18 G ZE57/18 H ZE11/18. Scale bar: 50 µm. Illustrated by P.D.

Hosts

Trichosteleum perchlorosum, Sematophyllum brachycarpum (Sematophyllaceae, Hypnales)

Distribution

South Africa, Mpumalanga and KwaZulu-Natal Provinces (Fig. 8).

Figure 8. 

Geographical distribution of the four lineages of Octospora conidiophora agg. in South Africa. Red circle: lineage A (O. conidiophora s.str.); green triangle: lineage B; light blue square: lineage C, dark blue star: lineage D.

Conservation status

Octospora conidiophora seems to be a common representative of the genus in South Africa, widespread and forming abundant populations. Its hosts are also common and widespread in the region (see below). Although the main habitat (afromontane forest) is naturally fragmented, it is often protected against human activities by nature reserves or national parks. Therefore, O. conidiophora does not fulfil the criteria for categories CR (critically endangered) to NT (near threatened) and we propose its evaluation as LC (least concern) for the present moment.

Additional specimens examined

South Africa. Mpumalanga Province: Ehlanzeni District Municipality, Graskop Gorge, 24°56.74'S, 30°50.8'E, 1355 m alt., on Trichosteleum perchlorosum on decaying wood, 6 Mar. 2018, Z. Egertová and M. Sochor ZE62/18 (PRM 951745); Ehlanzeni District Municipality, Graskop Gorge, 24°56.88'S, 30°50.75'E, 1435 m alt., on Trichosteleum perchlorosum on decaying wood, 6 Mar. 2018, Z. Egertová and M. Sochor ZE63/18 (PRM 951746); Ehlanzeni District Municipality, Buffelskloof Nature Reserve, 25°15.98'S, 30°31.08'E, 1725 m alt., on Trichosteleum perchlorosum on decaying wood, 10 Mar. 2018, Z. Egertová and M. Sochor ZE75/18 (PRM 951748); Ehlanzeni District Municipality, Buffelskloof Nature Reserve, 25°16.37'S, 30°30.62'E, 1605 m alt., on Trichosteleum perchlorosum on decaying wood, 9 Mar. 2018, Z. Egertová and M. Sochor ZE71/18 (PRM 951747); Ehlanzeni District Municipality, Buffelskloof Nature Reserve, 25°16.53'S, 30°30.25'E, 1625 m alt., on Trichosteleum perchlorosum on decaying wood, 10 Mar. 2018, Z. Egertová and M. Sochor ZE77/18 (PRM 951749). KwaZulu-Natal Province: Uthukela District Municipality, Royal Natal National Park, 28°40.88'S, 28°55.73'E, 1760 m alt., on Sematophyllum brachycarpum on decaying stem, 19 Feb. 2018, Z. Egertová and M. Sochor ZE11/18 (PRM 951739); Uthukela District Municipality, Royal Natal National Park, 28°44.05'S, 28°54.85'E, 1800 m alt., on Trichosteleum perchlorosum on decaying stem, 20 Feb. 2018, Z. Egertová and M. Sochor ZE23/18 (PRM 951740). Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°8.95'S, 29°25.35'E, 1665 m alt., on Trichosteleum perchlorosum on decaying wood, 3 Mar. 2018, Z. Egertová and M. Sochor ZE57/18 (PRM 951744); Uthukela District Municipality, uKhahlamba Drakensberg Park, Giants Castle Nature Reserve, 29°16.93'S, 29°30.93'E, 1765 m alt., on Trichosteleum perchlorosum on decaying wood, 1 Mar. 2018, Z. Egertová and M. Sochor ZE46/18 (PRM 951742); Uthukela District Municipality, uKhahlamba Drakensberg Park, Giants Castle Nature Reserve, 29°16.98'S, 29°30.87'E, 1775 m alt., on Trichosteleum perchlorosum on decaying wood, 1 Mar. 2018, Z. Egertová and M. Sochor ZE45/18 (PRM 951741).

Figure 9. 

Microscopic characters of Octospora conidiophora agg. (lineage B). A Conidia, distal curved part apparently sometimes broken off, five conidia germinating by formation of usually a single hypha, conidium on the left connected to a hypha by two anastomoses B Strongly warted hyphae, the left one seen from above, the two others in optical section C Appresssoria infecting rhizoids in lateral view, the right one seen from above, infection pegs surrounded by lignituber-like tubes formed by the host cell wall, intracellular haustoria present apart from the lowermost infection where the peg is completely encapsulated by the host cell wall A, B, C ZE37/18. Scale bars: 50 µm (A); 30 µm (B, C). Illustrated by P.D.

Data to other lineages

Lineage B: Mpumalanga Province: Ehlanzeni District Municipality, 3040 m WSW from the Graskop railway station, 24°56.28'S, 30°48.65'E, 1495 m alt., on Trichosteleum perchlorosum on decaying wood, 7 Mar. 2018, Z. Egertová and M. Sochor ZE65/18 (PRM 951735). KwaZulu-Natal Province: Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°7.62'S, 29°25.33'E, 1725 m alt., on Trichosteleum perchlorosum on decaying wood, 2 Mar. 2018, Z. Egertová and M. Sochor ZE51/18 (PRM 951732); Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°7.57'S, 29°25.38'E, 1715 m alt., on Trichosteleum perchlorosum on decaying wood, 2 Mar. 2018, Z. Egertová and M. Sochor ZE52/18 (PRM 951733); Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°7.98'S, 29°26.25'E, 1500 m alt., on Trichosteleum perchlorosum on decaying wood, 2 Mar. 2018, Z. Egertová and M. Sochor ZE53/18 (PRM 951734); Sisonke District Municipality, Marutswa Forest, 29°48.55'S, 29°47.28'E, 1465 m alt., on Sematophyllum brachycarpum on decaying stem, 24 Feb. 2018, Z. Egertová and M. Sochor ZE37/18 (PRM 951730); Sisonke District Municipality, Marutswa Forest, 29°48.6'S, 29°47.37'E, 1480 m alt., on Trichosteleum perchlorosum on decaying stem, 24 Feb. 2018, Z. Egertová and M. Sochor ZE38/18 (PRM 951731).

Lineage C: KwaZulu-Natal Province: Uthukela District Municipality, uKhahlamba Drakensberg Park, Giants Castle Nature Reserve, 29°17.02'S, 29°30.87'E, 1780 m alt., on Sematophyllum brachycarpum on decaying wood, 28 Feb. 2018, Z. Egertová and M. Sochor ZE44/18 (PRM 951736); Uthukela District Municipality, uKhahlamba Drakensberg Park, Injasuti, 29°8.33'S, 29°25.68'E, 1565 m alt., on Sematophyllum brachycarpum on decaying wood, 3 Mar. 2018, Z. Egertová and M. Sochor ZE56/18 (PRM 951737).

Lineage D: Mpumalanga Province: Ehlanzeni District Municipality, Buffelskloof Nature Reserve, 25°16.93'S, 30°30.45'E, 1470 m alt., on Sematophyllum brachycarpum on decaying wood, 9 Mar. 2018, Z. Egertová and M. Sochor ZE69/18 (PRM 951738).

Taxonomic affinities

The phylogenetically closest and phenotypically most similar species is Octospora kelabitiana described from Borneo, which shares most characters with the African species. It also has apothecia with stiff, thick-walled hyaline hairs, ellipsoid, hyaline ascospores of similar size like O. conidiophora († in H2O (13.5)14.5–17(18) × 7–8(9) μm, in LPCB (12.5)13–16(17) × (6.5)7–8(8.5) μm), filiform, unbranched paraphyses, smooth appressoria of similar size and even the warted mycelial hyphae, which is a character unknown in any other species of bryophilous Pezizales (Egertová et al. 2018). Nevertheless, it can be distinguished easily by growth on a completely different host – thallose liverworts from the genus Riccardia Gray. Furthermore, its apothecia are smaller, often taller than wide and lack a distinct margin. Its appressoria are usually one-celled, less often two-celled, while in O. conidiophora, two-celled appressoria are very common and even three-celled ones were found. Anamorph has not been detected in O. kelabitiana.

Discussion

According to the available literature and data from the main South African public fungarium (PREM), bryophilous Pezizales are completely unknown from southern Africa, despite the fact that this is a large and species-rich region, which hosts a very diverse bryoflora (Van Rooy and Phephu 2016). Our initial work revealed that this group of fungi is relatively common and probably also very diverse in southern Africa, despite the fact that the work was carried out in extraordinarily dry (and thus unsuitable) summer. Amongst others, four phenotypically similar, yet molecularly distinct lineages were discovered on two host species (lineages A and B on Trichosteleum perchlorosum and Sematophyllum brachycarpum, lineages C and D only on S. brachycarpum). This research brings novel insights into evolution and systematics of bryophilous ascomycetes and also raises important questions on taxonomic evaluation of these lineages. Therefore, we briefly discuss the taxonomy of cryptic taxa and suggest a suitable taxonomic solution for our collections. As O. conidiophora is the first species of bryophilous Pezizales with a detected anamorph, we also discuss this finding. Finally, diagnostic characters and data on distribution of the host mosses are provided as they may help expand the known distribution area of O. conidiophora in the future.

Taxonomic approach

The four lineages could not be distinguished phenotypically on the basis of characters that are normally studied in bryophilous Pezizales, although genetic differentiation was very high at all of the three studied loci (Suppl. material 1: Table S1). Such great genetic distances are usually observed amongst different species or even genera. The observed genetic distances, together with molecular dating, imply that the phenotypically more or less homogeneous morphotype actually represents a group of several cryptic species that have already become reproductively isolated in the Tertiary (Fig. 2). Similar cryptic diversity is probably quite common in fungi, including many genera of Pezizales, e.g. Genea Vittad. (Smith et al. 2006, Alvarado et al. 2016), Geopyxis (Pers.) Sacc. (Wang et al. 2016), Helvella L. (Nguyen et al. 2013, Skrede et al. 2017), Terfezia (Tul. & C.Tul.) Tul. & C.Tul. (Ferdman et al. 2009), Trichophaea Boud. (Van Vooren 2016) and Tuber P.Micheli ex F.H.Wigg. (Bonuso et al. 2010). In bryophilous Pezizales, intraspecific sequence variability was observed, e.g. in Octosporopsis nicolai (Maire) U.Lindem., M.Vega & T.Richt. (Lindemann et al. 2014) and Octospora kelabitiana (Egertová et al. 2018). Each of the species comprised two genetic lineages that, nevertheless, were relatively weakly diverged and were therefore not treated taxonomically. Besides the significant genetic distances amongst the South African populations, another fact speaks against the possibility that the four lineages could be treated as a single species; the whole clade includes Octospora kelabitiana (Fig. 1), a distinct species from Borneo infecting liverwort Riccardia. A widely defined species (i.e. including the four lineages but excluding O. kelabitiana) would therefore be paraphyletic.

The current approach of many authors to delimitation of species is based primarily or solely on DNA sequence data and sequence-based diagnoses have become almost a common practice in macromycetes (e.g. Buyck et al. 2016, Leacock et al. 2016, Taşkın et al. 2016, Wang et al. 2016, Korhonen et al. 2018). Some authors even aim to base descriptions of new species on environmental sequence data only (e.g. Hibbett et al. 2011). Although molecular phylogenetics is an excellent tool for evaluation of biodiversity, assignment of scientific binominal to molecularly defined species leads to several practical problems, mainly those related to limited accessibility of the methods for many field mycologists. Especially in developing countries, in which even standard optical microscopy can be barely affordable at the leading institutes, determination of species via DNA sequencing is still a matter for the distant future. This methodological obstacle may soon result (or has already resulted in some groups) in the split of traditional phenotype-based taxonomy and molecular taxonomy. Until recently, molecular taxonomy mostly worked with groups, such as molecular operational taxonomic unit (MOTU; Hibbett et al. 2011), phylogenetic species (O’Donnell et al. 2011), virtual taxon (Öpik et al. 2010) etc. and designated an alphanumeric code to them. Nevertheless, many of the molecular taxa are currently given traditional scientific names, often without studying related, validly described species that cannot be sequenced for various reasons. This process, although justified by the aim of cataloguing of global biodiversity, makes the resulting taxonomy impractical or even unusable for field mycologists (and sometimes also for molecular biologists). Another problem with descriptions of species, based on molecular data, is the fact that the borderline between intraspecific and interspecific molecular variation is often unclear (Thines et al. 2018), dependent on many evolutionary factors (e.g. Leliaert et al. 2014) and may become fuzzy after a more intensive and/or extensive sampling is performed, particularly if only one or few molecular markers are used. Nevertheless, this problem also exists with traditional taxonomy (e.g. Flynn and Miller 1990, Paal et al. 1998, Benkert 2001). One solution to the problems mentioned above is an integrative approach. This takes advantage of both multiple characters (morphology, DNA, ecology etc.) and results in robust, phylogeny-based taxonomy that is accessible to various users (e.g. Araújo et al. 2015, Skrede et al. 2017, Haelewaters et al. 2018).

After thorough consideration of the above-mentioned facts, we decided not to formally describe all of the four discovered cryptic species at the present moment. Instead, we prefer to establish two taxa: O. conidiophora (s.str.), which refers to the most common phylogenetic lineage A and the informal taxon O. conidiophora agg., which applies to all of the four South African cryptic species, but also to the morphologically distinct and host-specific Bornean O. kelabitiana. Although the name O. kelabitiana is older and should therefore be selected for the aggregate, we believe that the name O. conidiophora agg. better suits the pragmatic purposes of this informal taxon. Our approach enables field mycologists to determine their specimens at least on the aggregate level and, at the same time, preserves a monophyletic taxonomical system. Detailed studies may reveal phenotypic differences between the South African lineages of O. conidiophora agg., which can then be formally described as species. Until then, we prefer to leave lineages B, C and D without a Latin binominal.

Anamorph

Conidia have been reported in several genera of Pezizales. The most frequent type of conidia are amerospores which are produced, e.g. in Caloscypha Boud. (Paden et al. 1978), Desmazierella Lib. (Hughes 1951), Iodophanus Korf (Korf 1958, sub Ascophanus Boud.), Pachyphlodes Zobel (Healy et al. 2015), Peziza Fr. (Berthet 1964a, Paden 1967, 1972), Ruhlandiella Henn. (Warcup and Talbot 1989, sub Muciturbo P.H.B.Talbot), Thecotheus Boud. (Conway 1975), Urnula Fr. (Davidson 1950), Cookeina Kuntze, Phillipsia Berk. (Paden 1975), Pithya Fuckel (Paden 1972), Nanoscypha Denison (Pfister 1973), Sarcoscypha (Fr.) Boud. (Harrington 1990), Geopyxis (Paden 1972), Pyropyxis Egger (Egger 1984, Filippova et al. 2016) and Trichophaea (Hennebert 1973). Staurospores can be found in Miladina lecithina (Cooke) Svrček (Descals and Webster 1978). The conidia of O. conidiophora can be classified as scolecospores or phragmospores and are therefore unique amongst Pezizales with known teleomorph. In their shape, they resemble the conidia of the anamorphic genus Spermospora R. Sprague (Ascomycota, Pezizomycotina), a parasite of grasses (Sprague 1948, Seifert et al. 2011).

Detached conidia were regularly found between the rhizoids and leaves in almost all collections of Octospora conidiophora agg. (with the exception of specimens ZE38/18, ZE53/18 and ZE71/18, probably due to limited material). The distal part of the conidia is sometimes short and straight. It is not clear whether this is an artefact caused by breaking off during preparation, although tail fragments have not been found. Germinating conidia are not rare. Longer germination tubes look like normal hyphae with the characteristic warty surface structure (Figs 5F, 7F). Conidia germinating by a two-celled appressorium (Fig. 7F) or connected to a mycelial hypha by an anastomosis (Figs 7E, G, 9A) have been repeatedly observed. Conidiogenous cells (Fig. 7D) are much more difficult to detect than conidia and have only been found in a few collections. The scars formed by detachment of conidia must not be confused with the ends of torn-off hyphae, which inevitably result during preparation. Fully developed conidia still connected to the conidiogeneous cells have not been found. Apparently, mature conidia easily detach from their conidiogenous cells. A developing, still attached conidium was observed once (Fig. 7D).

Octospora conidiophora agg. is the first case amongst bryophilous Pezizales in which an anamorph has been detected. The absence of records of anamorphic states in other species can be caused either by their real rarity or only by their difficulty in detection. The latter can have many reasons. First, bryophilous ascomycetes, in general, stand rather on the periphery of researchers´ interest (see Döbbeler 1997). Second, anamorphs are usually inconspicuous and therefore not easy to encounter. Even if an anamorph is found, it can be difficult to link it with the corresponding teleomorph, because many fungal species commonly occur together. Moreover, anamorphs and teleomorphs are often formed in different environmental conditions (Kendrick 1979) and often at different times. And third, anamorphs are often studied in aseptic cultures and subsequently cultures are used for confirmation of their identity by molecular methods; unfortunately, cultivation of bryophilous Pezizales seems to be problematic (Berthet 1964b) and is not commonly attempted. Although an anamorph has not been confirmed by cultivation methods in O. conidiophora agg., the connection of anamorph and teleomorph is based on the evidence discussed above: conidia were repeatedly found amongst the moss plants near the teleomorph; germinating conidia have hyphae with the same ornamentation as observed in the mycelium bearing apothecia; conidiogenous cells occur on the mycelial hyphae; conidia anastomose with mycelial hyphae; the germlings form appressoria.

Hosts

Sematophyllum brachycarpum (Hampe) Broth.

Syn: Hypnum brachycarpum Hampe

Sematophyllum brachycarpum can be distinguished from other species of Sematophyllum in southern Africa by the complanate, straight leaves with relatively large groups of alar cells (in 3–4 rows) that are not much inflated or coloured (Fig. 10, see also Câmara et al. 2019).

Figure 10. 

Sematophyllum brachycarpum. A Plants B Typical shoot with leaves C Leaf D Leaf base with alar cells.

The species is by far the most common and widespread species of Sematophyllum in South Africa; S. brachycarpum is found in forests and wooded areas of the Limpopo, Mpumalanga, North West, Gauteng, Free State, KwaZulu-Natal, Eastern Cape and Western Cape Provinces (Fig. 11, see also Câmara et al. 2019). It occurs as an epiphyte or occasionally on soil or rocks, from sea level up to 1900 m alt. The species is widely distributed throughout the Afromontane Region, as defined by Van Rooy and Van Wyk (2010) and was found to belong to the Widespread Afromontane Subelement, a subdivision of the Afromontane Forest Element (Van Rooy and Van Wyk 2011). The Widespread Afromontane Subelement is centred in the Midlands of KwaZulu-Natal and the Drakensberg escarpment of Mpumalanga as well as in forests in the south-western Cape. The species has also been recorded from Lesotho, Swaziland, Mozambique, Zimbabwe, Zambia, Uganda and Kenya (O’Shea 2006).

Figure 11. 

Geographical distribution of Sematophyllum brachycarpum in southern Africa based on records in BM, L, MO and PRE.

Trichosteleum perchlorosum Broth. & Bryhn

Trichosteleum perchlorosum is the only southern African species of Sematophyllaceae (sensu stricto) with papillose leaf cells. However, the papillae are sometimes difficult to see or may be absent on some leaves. The falcate leaves with enlarged, inflated and coloured alar cells will also help to identify the species (Fig. 12, see also Câmara et al. 2019).

Figure 12. 

Trichosteleum perchlorosum. A. Plants. B. Leaf. C. Leaf papillae. D. Alar cells.

The species is endemic to the southern part of Africa and occurs as an epiphyte and also on decaying logs or rocks from sea level up to 3090 m high (Drakensberg of KwaZulu-Natal). It is most frequently collected in the KwaZulu-Natal Province of South Africa, but it is also known from Limpopo, Mpumalanga, Eastern Cape and Western Cape Provinces, as well as Swaziland (Fig. 13, see also Câmara et al. 2019). Trichosteleum perchlorosum is widespread throughout the Afromontane Region sensu Van Rooy and Van Wyk (2010), but unknown from Afromontane outliers in the Magaliesberg of Gauteng and the North West, the eastern Free State and the Waterberg of Limpopo. It was therefore included in the Tropical Afromontane Subelement (Van Rooy and Van Wyk 2011), which is centred in the Drakensberg escarpment of Mpumalanga and the Midlands of KwaZulu-Natal. This species was also reported from Zimbabwe (O’Shea 2006).

Figure 13. 

Geographical distribution of Trichosteleum perchlorosum in southern Africa based on records in BM, L, MO and PRE.

Acknowledgements

ZS and MS thank John Manning for financial support of the travel to South Africa. Riana Jacobs-Venter is acknowledged for providing information on the absence of bryophilous Pezizales in the South African National Collection of Fungi and Markéta Šandová and Miriam Brožíková (PRM) for information about Octospora tetraspora var. aegyptiaca. We are grateful to Hester Steyn and Elizma Fouche for editing the maps and the editor Danny Haelewaters and the reviewers, Nicolas Van Vooren and Donald H. Pfister, for comments to the manuscript. The work was partly supported by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO0418. ZS was supported by an internal grant from Palacký University (IGA_PrF_2019_004). Research was conducted under permit no. OP 1264/2018.

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