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Research Article
Brahmaculus gen. nov. (Leotiomycetes, Chlorociboriaceae)
expand article infoPeter R. Johnston, Duckchul Park, Matthew E. Smith§, Alija B. Mujic§, Tom W. May|
‡ Manaaki Whenua–Landcare Research, Auckland, New Zealand
§ University of Florida, Gainesville, United States of America
| Royal Botanic Gardens Victoria, Melbourne, Australia
Open Access

Abstract

A second genus in Chlorociboriaceae is described here as Brahmaculus gen. nov. Macroscopically distinctive, all species have bright yellow apothecia with several apothecial cups held on short branches at the tip of a long stipe. The genus is widely distributed across the Southern Hemisphere; the four new species described here include two from Chile (B. magellanicus sp. nov., B. osornoensis sp. nov.) and one each from New Zealand (B. moonlighticus sp. nov.) and Australia (B. packhamiae sp. nov.). They differ from species referred to Chlorociboria, the only other genus in Chlorociboriaceae, in their terrestrial habitat and ascomata that are noticeably more hairy than the known Chlorociboria species, most of which have apothecia with short, macroscopically indistinct hair-like elements. Based on our analyses, Chlorociboria as accepted here is paraphyletic. Additional study is needed to clarify where alternative, monophyletic generic limits should be drawn and how these genera may be recognised morphologically. Also described here are three new Chlorociboria spp. from New Zealand (C. metrosideri sp. nov., C. solandri sp. nov., C. subtilis sp. nov.), distinctive in developing on dead leaves rather than wood and in two of them not forming the green pigmentation characteristic of most Chlorociboria species. New Zealand specimens previously incorrectly identified as Chlorociboria argentinensis are provided with a new name, C. novae-zelandiae sp. nov.

Keywords

Chlorociboria, Cyttariaceae, fungi, molecular phylogeny, systematics, 9 new taxa

Introduction

The modern-day distribution of Nothofagaceae forests of the Southern Hemisphere and their associated fungi are often explained in terms of vicariance in relation to the breakup of Gondwana (e.g. Horak 1983). This explanation has been challenged in recent years (May 2017), with their distribution now thought to be due to a complex mix of ancient vicariant and geologically more recent long distance dispersal events, with evidence from Nothofagaceae phylogeny (e.g. Knapp et al. 2005) along with the phylogeny of some of their specialised fungal associates (e.g. Peterson et al. 2010b). The importance of these forests to the vegetation of southern South America and New Zealand has meant they have been amongst the most intensively studied mycologically in these regions (McKenzie et al. 2000; Johnston et al. 2012, Gamundí et al. 2017; Romano et al. 2017a). Despite this, much of the fungal diversity in these forests remains undiscovered (e.g. Johnston et al. 2012; Romano et al. 2017b).

An example of this undiscovered diversity comes from recent collections of a beautiful, small terrestrial fungus from Nothofagaceae forests in South America, New Zealand and Australia that could not be matched to any known genus. Microscopically they had a clear affinity to Leotiomycetes. The unique apothecia are morphologically complex with a branched stipe and each branch ending in one or more cups, the hymenial surface in these cups forming a complex pattern comprising separate regions with asci and paraphyses, and with hair-like elements. Preliminary sequencing of ribosomal genes of both Australasian and South American specimens showed that these fungi are phylogenetically closely related and that they are also related to the Leotiomycetes genus Chlorociboria.

Here we describe four species in the newly erected genus Brahmaculus based on a combination of unique morphological and molecular characters. We incorporate Brahmaculus DNA sequences into a broad multigene Leotiomycetes phylogeny to show that these fungi represent a second genus in Chlorociboriaceae. Including Brahmaculus in the phylogeny makes Chlorociboria paraphyletic but the morphological and ecological differences between Chlorociboria and Brahmaculus species means that it is not sensible to treat them as a single genus. More intensive genetic sampling of additional Chlorociboria species will be needed to better resolve phylogenetic relationships within Chlorociboriaceae and to clearly define the phylogenetic and morphological limits of the genus Chlorociboria.

It is surprising that specimens of the morphologically spectacular Brahmaculus have not been collected more often in the Nothofagaceae forests of the Southern Hemisphere. Although clearly widespread geographically, these fungi presumably fruit rarely.

Methods

Samples

Specimens were collected during surveys of fungal diversity in Southern Hemisphere forests. Brief notes on macroscopic appearance were prepared and then the specimens dried and stored in the New Zealand Fungarium (PDD), National Herbarium of Victoria (MEL), Museo Nacional de Historia Natural (SGO) and the Florida Museum of Natural History (FLAS).

Morphology and culturing

Microscopic examinations were made from dried material routinely rehydrated and in 3% KOH and mounted in Melzer’s Reagent, or where indicated, rehydrated and mounted in water. Vertical sections about 10 µm thick were prepared from apothecia rehydrated in 3% KOH using a freezing microtome and mounted in lactic acid. Where available, living cultures were grown from germinated ascospores and are stored in the ICMP culture collection, Manaaki Whenua–Landcare Research, Auckland.

DNA extraction and PCR amplification

DNA was extracted from apothecia that had been placed in buffer when fresh, from dried apothecia, or from mycelium from living cultures, using a QIAamp DNA mini kit (QIAGEN, US) on the QIAcube nucleic acid extraction robot (QIAGEN, US). Amplification primers used for each of the genes were: SSU – NS1 and NS4 (White et al. 1990); ITS – ITS-1F and ITS4 (White et al. 1990; Gardes and Bruns 1993); LSU – LROR and LR5 (Bunyard et al. 1994; Vilgalys and Hester 1990); MCM7 – mcm7-709for and mcm7-1348rev (Schmitt et al. 2009); RPB1 – RPB1-Af and RPB1-Cr (Stiller and Hall 1997; Matheny et al. 2002); and RPB2 – RPB2-5f2 and fRPB2-7cR (Liu et al. 1999; Sung et al. 2007).

Phylogenetic analyses

Two phylogenetic analyses were carried out. In the first, LSU, ITS, MCM7, RPB1 and RPB2 sequences from Brahmaculus specimens from South America and New Zealand, together with a set of Chlorociboria and Cyttaria specimens with multi-gene data available (Table 1), were incorporated into the alignments from Johnston et al. (2019 – data available from https://doi.org/10.7931/T5YV-BE95). Cyttaria was added because the analysis presented by Peterson and Pfister (2010a) suggested a relationship to Chlorociboriaceae and additional genes had recently become available for Cyttaria nigra. The expanded dataset was reanalysed using the same methods as Johnston et al. (2019). Briefly, genes were aligned using MAFFT (Katoh and Standley 2013), a maximum likelihood (ML) analysis of the concatenated alignments was run using IQ-TREE (Nguyen et al. 2015; Chernomor et al. 2016), using models selected by ModelFinder (Kalyaanamoorthy et al. 2017) for each partitioned gene, and ultrafast bootstrap (BS) analysis with 1000 replicates estimated branch support in the ML tree (Hoang et al. 2018). Xylaria hypoxylon and Neurospora crassa were used as outgroups.

Table 1.

GenBank accession numbers for DNA sequences of Brahmaculus, Chlorociboria and Cyttaria specimens used for phylogeny in Fig. 1, and for newly generated sequences used in phylogeny in Fig. 2. Sequences generated as part of this project in bold. Data for other taxa included in the Fig. 1 phylogeny from Johnston et al. (2019), see https://doi.org/10.7931/T5YV-BE95.

Species Voucher (T = type specimen) SSU ITS LSU MCM7 RPB1 RPB2 TEF mtSSU β–tubulin
Brahmaculus magellanicus PDD 116650 (T) MW364563 MW364557 MW364560 MW350087 MW350085
B. moonlighticus PDD 112225 (T) MK248054 MK248036 MK248011 MK241483 MK241482 MK241484
B. osornoensis FLAS-F-65492 (T) MW575608
B. packhamiae PDD 117311 (T) MW364556
Chlorociboria aeuruginascens TNS-F-36241 LC434588 LC425045 LC429376 LC431689 LC431723
C. aeuruginascens DSM 107184 (isolate IHIA39, genome) PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475 PRJNA382475
C. aeuruginascens ssp. australis ICMP 15642 (T) JN939873 NR_119520 JN939932 JN993274 JN985222 JN985532
C. aeruginosa AFTOL-ID 151 AY544713 DQ491501 AY544669 DQ471125 DQ470886
C. aeruginosa TNS-F-13596 LC434578 LC425047 LC429383 LC431687
C. aeruginella TAAM 198514 KX090875 MH752067 KX090769 KX090722
C. argentinensis ICMP 16995 JN939876 EF520123 JN939930 JN993275 JN985197 JN985515
C. novae-zelandiae ICMP 18766 (T) JN939875 JN943456 JN939940 JN993286 JN985223 JN985514
C. awakinoana ICMP 15631 JN939870 JN943461 JN939921 JN993273 JN985219 JN985504
C. clavula ICMP 15634 JN939866 JN943465 JN939924 JN993299 JN985215 JN985519
C. duriligna ICMP 18763 (T) JN939863 JN943468 JN939934 JN993279 JN985212 JN985500
C. glauca TAAM 198458 KX090872 KX090821 KX090766
C. halonata ICMP 18764 JN939860 JN943471 JN939935 JN993296 JN985209 JN985502
C. metrosideri ICMP 23410 (T) MW364558
C. poutoensis ICMP 15618 AY755352 MH700576 MH682247
C. solandri ICMP 23686 (T) MW364559
C. spathulata ICMP 18760 JN939868 JN943463 JN939923 JN993272 JN985217 JN985530
C. subtilis PDD 112247 MH921854
Cyttaria darwinii FH (Peterson and Pfister 2010, isolate 40, 45) EU107180 EU107209 EU107250 EU107236
Cyttaria hariotii FH (Peterson and Pfister 2010, isolate 44) EU107194 EU107217 EU107251 EU107245
Cyttaria nigra PDD 117571 MW364564 MW364561 MW350086 MW363493 MW350084 MW350088 MW364562 MW350089

The second analysis used ITS sequences only, treating all four Brahmaculus species, together with all Chlorociboria species with ITS sequences available, using Cenangiaceae as the outgroup. The methods were the same as those used for the multi-gene analysis, except with the TIM2+F+I+G4 model, selected using ModelFinder.

Alignments and partitions for each of the analyses are provided through the Landcare Research – Manaaki Whenua Datastore, https://doi.org/10.7931/2xet-fc88.

Results

Phylogenetic analyses

Helotiales form a strongly supported monophyletic clade, and most families accepted within this order also form strongly supported clades (Fig. 1). The family-level clades of those families clustered in collapsed clades in Fig. 1 have 100% bootstrap support. Chlorociboriaceae and Cyttariaceae are strongly supported as monophyletic but their relationship to each other, and to other basal family-level clades within the Helotiales, is poorly resolved. A fully expanded version of Fig. 1 is available as a nexus file from the Landcare Research – Manaaki Whenua Datastore, https://doi.org/10.7931/2xet-fc88.

Figure 1. 

ML tree based on a multi-gene alignment, placing Brahmaculus within Chlorociboriaceae and both Chlorociboriaceae and Cyttariaceae in Helotiales. Taxa newly named in this paper in bold. Bootstrap values where >90%. See Methods and Table 1.

In both the multi-gene and ITS analyses, Brahmaculus forms a monophyletic clade within Chlorociboriaceae, but Chlorociboria as accepted here is paraphyletic (Figs 1, 2). The Brahmaculus species form a well-supported clade sister to a well-supported clade comprised of Chlorociboria aeruginella and C. halonata (from Northern Europe and New Zealand, respectively). The clade comprised of Brahmaculus plus these two species of Chlorociboria is sister to a clade containing the bulk of sequenced species of Chlorociboria, including the type species of the genus, C. aeruginosa.

Figure 2. 

ML tree based on an ITS alignment, treating all Chlorociboria and Brahmaculus species with ITS sequences available. Taxa newly named in this paper in bold. Bootstrap values where >90%.

At the species level, the ITS analysis supports the molecular phylogenetic distinctiveness of the novel species of Brahmaculus and Chlorociboria accepted here (Fig. 2).

Taxonomy

Brahmaculus P.R.Johnst., gen. nov.

MycoBank No: 838724

Type species

Brahmaculus moonlighticus P.R.Johnst.

Etymology

From Hindu mythology, named after Brahma, the four-headed creator god, reflecting the multiple heads of the apothecia, and the masculine diminutive -culus.

Diagnosis

Phylogenetically Chlorociboriaceae, distinguished from Chlorociboria by its terrestrial habitat, and apothecium with stipe branched near apex, each branch with an apothecial cup.

Description

Apothecia stipitate, yellow rhizomorphs at base of stipe, the stipe branched apically several times, each branch holding an apothecial cup. Receptacle and stipe densely covered with short hairs. Hairs more or less straight, cylindric, thin walled, with a few septa, pale brown intracellular pigment, externally densely encrusted with yellowish material, encrusting material dissolving in KOH + Melzer’s reagent. The hymenium within each apothecial cup is typically divided into smaller segments, with areas comprising asci and paraphyses separated by clumps of hair-like elements. Excipulum comprises cylindric cells arranged more or less parallel to the surface, cells mostly long-cylindric, but sometimes with outermost 1–2 layers of cells short and broad-cylindric, cell walls slightly thickened, hyaline, cells near base of hairs with pale brown vacuolar pigment. Asci with wall thickened at apex, amyloid pore extending through the wall, flaring near the inside and especially toward outside of the wall, 8-spored, with croziers. Paraphyses simple or tapering to apex, of similar length as asci. Ascospores oblong-elliptic, 0-septate, hyaline.

Notes

The four species described below are phylogenetically distinct but remarkably similar morphologically. There appear to be small differences in size and colour of the apothecia and shape of the paraphyses and hairs but having only a single specimen available for each species makes the significance of these differences difficult to assess. The rhizomorphs at the base of the stipe appear to be associated with tree roots. Based on the collecting sites, in South America and New Zealand the roots are likely to be Nothofagaceae, in Australia they may also be Nothofagaceae but Eucalyptus species were also growing in the vicinity. Observations from the South American specimens showed a loose weft of mycelium around the Nothofagaceae roots but there was no clear evidence of a mantle or ectomycorrhizal association. It is possible that these fungi are root endophytes, or perhaps parasites of Nothofagaceae-associated ectomycorrhizal fungi.

Brahmaculus magellanicus M.E.Sm. & P.R.Johnst., sp. nov.

MycoBank No: 838730
Figure 3

Typification

Chile – Magallanes • Puente San Pedro, south of Punta Arenas, stream near the end of the road, Nothofagus betuloides forest; -53.6993, 70.9695; Alija Mujic (MES2454) leg.; 5 Apr 2017; SGOHolotype; FLAS-F-65086 – isotype; PDD 116650 – isotype.

Etymology

Refers to the Magellanic forests of the type locality.

Diagnosis

Phylogenetically distinct from other known Brahmaculus spp., apothecia 3–8 × 1–2.5 mm, paraphyses undifferentiated to rounded apex, ascospores 5.5–9 × 1.5–2 µm (average 7.3 × 1.7 µm).

Description

Apothecia 3–8 mm high, stipe 0.4–0.6 mm wide, cap 1–2.5 mm wide, the more or less globose cap comprising several closely packed apothecial cups, these arising from short, branches at the top of the stipe, hymenium pale yellow, hymenial areas broken into smaller segments by groups of bright yellow, hair-like elements amongst the fertile parts of the hymenium. Receptacle densely covered with stiff, bright yellow hairs, stipe with shorter hairs. Hairs 45–70 × 2.5–4.5 µm, straight, cylindric, tapering gradually in apical half toward small, rounded apex, thin-walled, sparsely septate, pale brown vacuolar pigment, densely encrusted with coarse, bright yellow crystals that dissolve in KOH + Melzers. Ectal excipulum comprising narrow-cylindric cells 8–20 × 2.5–3 µm oriented at low angle to receptacle surface, wall slightly thickened, mostly hyaline except cells at the base of hairs have pale brown vacuolar pigment. Medullary excipulum similar in structure but cells wider, 4.5–8 µm diam. Paraphyses 1.5–2.5 µm diam., undifferentiated at rounded apex, about same length as asci. Asci 40–55 × 4–5 µm, cylindric, apex rounded, wall thickened, amyloid pore extends through the wall, diffuse and flaring slightly towards the outside of the wall, crozier at base, 8–spored. Ascospores 5.5–9 × 1.5–2 µm (average 7.3 × 1.7 µm, n = 12), oblong elliptic, ends rounded, flattened on one side, straight to slightly curved, 0-septate, hyaline.

Figure 3. 

Brahmaculus magellanicus (PDD 116650) A, B fresh apothecia C dried apothecia D ascoma in vertical section, showing multiple apothecial cups on short branches E ascoma in vertical section showing excipular tissue and hairs, and a clump of hairs within the hymenium F squash mount showing hairs in KOH G asci H paraphyses I ascospores. Scale bars: 1 mm (A–C); 100 µm (D); 10 µm (E–I).

Notes

The two Chilean species differ macroscopically, B. magellanicus having noticeably thinner stipes than B. osornoensis. The only known collection of B. magellanicus is from Magellanic subpolar forest in Patagonia that is dominated by Nothofagus betuloides. It is possible that this Brahmaculus species is restricted to these sub Antarctic cold southern forests but more specimens are needed to determine the range of the species.

Brahmaculus moonlighticus P.R.Johnst., sp. nov.

MycoBank No: 838733
Figure 4

Typification

New Zealand – Buller • Moonlight Creek; -42.2713, 171.4587; on soil under Nothofagaceae; A. Chinn leg.; 10 May 2018; PDD 112225 – holotype.

Etymology

From the type locality. Historically important as a gold mining area (where T.H. Chinn, the great-great grandfather of the collector of the type specimen, prospected for gold in the 1880’s), the name also reflects the deep golden colour of the apothecia of this fungus.

Diagnosis

Phylogenetically distinct from other known Brahmaculus spp., apothecia 1.5–3 × 1–1.8 mm, paraphyses taper slightly to rounded apex, ascospores 6.5–8.5 × 1.5–2(–3) µm (average 7.7 × 1.9 µm).

Description

Apothecia 1.5–3 mm high, stipe 0.25–0.5 mm wide, cap 1–1.8 mm wide, bright golden-yellow when fresh, consistently with four short branches arising from top of stipe, each branch with its own apothecial cup, hymenium pale yellow, divided into a complex pattern with hymenial areas separated by narrow groups of golden yellow hair-like elements. Receptacle densely covered with stiff, bright yellow hairs. Hairs 40–60 × 3–4 µm, straight, cylindric, tapering slightly towards rounded apex, pale brown vacuolar pigment, wall smooth, encrusted with coarse yellow-brown crystals that dissolve in KOH + Melzer’s, few-septate. Ectal excipulum comprising long-cylindric cells 15–25 × 3–5 µm, but with the outermost 1–2 layers of cells short and broad-cylindric, 6–8 µm diam., cell walls slightly thickened, hyaline, cells near base of hairs with pale brown vacuolar pigment. Medullary excipulum comprising partly tangled hyphae 3–4 µm diam. with walls thin, hyaline. Paraphyses 2–3 µm diam., tapering slightly towards rounded apex, about same length as asci. Asci 45–55 × 5.5–6 µm, cylindric, tapering slightly to broad subtruncate apex, wall uniformly thickened across apex, amyloid pore extending through wall, flaring slightly towards both inside and outside of wall. Ascospores 6.5–8.5 × 1.5–2 (–3) µm (average 7.7 × 1.9 µm, n = 50), oblong-elliptic to subfusoid, sometimes tapering suddenly to a narrower lower half, ends rounded, flat one side in side view, sometimes slightly curved or sigmoid, 0-septate, hyaline.

Figure 4. 

Brahmaculus moonlighticus (PDD 112225) A fresh apothecia (dried apothecium inset) B detail, fresh apothecia C ascoma in vertical section, showing multiple apothecial cups D ascoma in vertical section showing excipular tissue and hairs E hairs in squash mount in KOH F asci and ascospores G asci and paraphyses H, I ascospores. Scale bars: 1 mm (A, B); 100 µm (C); 10 µm (D–I).

Notes

Brahmaculus moonlighticus has a stipe that consistently has 4 distinct branches near the apex. The other species have several separate hymenial cups, but these are held on very short branches arising from across the apex of the stipe, the margins of these cups superficially forming a more or less continuous layer.

Brahmaculus osornoensis M.E.Sm. & P.R.Johnst., sp. nov.

MycoBank No: 838734
Figure 5

Typification

Chile • Parque Nacional Vicente Perez Rosales, Volcan Osorno, on the road to the ski area just above Mirador el Bosque, Nothofagus dombeyi forest; -41.1382, 72.5370; Matthew Smith and Alija Mujic (MES2942) leg.; 17 April 2017; SGOholotype; FLAS-F-65492 – isotype; PDD 116649 – isotype.

Etymology

Refers to the type locality, Volcan Osorno.

Diagnosis

Phylogenetically distinct from other known Brahmaculus spp., apothecia 3–6 × 1–2.5 mm, paraphyses taper slightly to rounded apex, ascospores 6.5–10(–11) × 1.5–2 µm (average 8.3 × 2 µm).

Description

Apothecia 3–6 mm high, stipe 0.5–1 mm wide, cap 1–2.5 mm wide, the more or less globose cap comprising several closely packed apothecial cups, these arising from short, branches at the top of the stipe, hymenium pale yellow, hymenial areas broken into smaller segments by groups of bright yellow, hair-like elements amongst the fertile parts of the hymenium. Receptacle densely covered with stiff, bright yellow hairs, stipe with shorter hairs. Hairs 50–85 × 2.5–4 µm, straight, with a broad basal cell then cylindric, apically tapering suddenly to the narrow-rounded apex, thin-walled, sparsely septate, densely encrusted with coarse, bright yellow crystals that dissolve in KOH + Melzers. In squash mount, excipular cells broad-cylindric, about 15–30 × 8–12 µm, wall slightly thickened, hyaline. Paraphyses 2–2.5 µm, tapering slightly to rounded apex, about same length as asci. Asci 40–50 × 4–4.5 µm, cylindric, apex rounded, wall thickened, amyloid pore extends through the wall, flaring slightly towards the outside. Ascospores 6.5–10(–11) × 1.5–2.5 µm (average 8.3 × 2.0 µm, n = 50), oblong elliptic, ends rounded, one side flat in side view, sometimes slightly curved, 0-septate, hyaline.

Figure 5. 

Brahmaculus osornoensis (PDD 116649) A fresh apothecia (dried apothecia inset) B squash mount, excipulum and hairs in 3% KOH + Melzer’s reagent C squash mount, hairs in water showing encrusting crystals D paraphyses and asci E ascospores. Scale bars: 1 mm (A); 10 µm (B–E).

Notes

The two Chilean species differ macroscopically, Brahmaculus osornoensis having noticeably broader stipes than B. magellanicus and slightly longer ascospores. B. osornoensis is known only from Nothofagus dombeyi forest in northern Patagonia on Volcan Osorno in the Vicente Perez Rosales National Park. It is possible that this Brahmaculus species is restricted to the wetter and warmer forests in northern Patagonia, but more specimens are needed to determine the range of the species.

Brahmaculus packhamiae T.W.May & P.R.Johnst., sp. nov.

MycoBank No: 838729
Figure 6

Typification

Australia – Tasmania • Geeveston District, Hermons Rd; -43.2652, 146.8613; J.M. Packham (6/R6/26) leg.; 5 June 1995; MEL 2363173 – holotype; PDD 117311 – isotype.

Etymology

Named after the late Jillian (“Jill”) Mary Packham whose assiduous collecting activities detected the type collection.

Diagnosis

Phylogenetically distinct from other known Brahmaculus spp., apothecia up to 11 × 2.5 mm, paraphyses undifferentiated to rounded apex, ascospores 5.5–8.5 × 1.5–2.5 µm (average 7.2 × 1.8 µm).

Description

Apothecia up to 11 mm high, stipe up to 0.8 mm wide, cap up to 2.5 mm wide, the cap comprising several closely packed apothecial cups, these arising from short branches at the top of the stipe, hymenium white when fresh. Receptacle densely covered with stiff, bright yellow hairs, stipe with shorter hairs, yellow rhizomorphs at base. Hairs 40–60 × 2.5–3.5 µm, straight, narrow flask-shaped, broad near base then tapering suddenly to narrow-cylindric apical part, apex rounded, thin-walled, 1–2 septate near the base, densely encrusted with coarse, bright yellow crystals, that dissolve in KOH + Melzers. Ectal excipulum comprising cylindric cells 8–15 × 3–5 µm, oriented at a low angle to the receptacle surface, walls slightly thickened, hyaline. Medullary excipulum comprises partly tangled hyphae 3–5 µm diam. with walls thin, hyaline. Paraphyses 2–2.5 µm diam., undifferentiated at the rounded apex, about the same length as the asci. Asci 35–45 × 4.5–5.5 µm, cylindric, tapering slightly to broad, subtruncate apex, wall thickened across apex, amyloid pore extending through wall, flaring toward outside of wall. Ascospores 5.5–8.5 × 1.5–2.5 µm (average 7.2 × 1.8 µm, n = 20), oblong-elliptic, tapering slightly to rounded ends, one side flat in side view, sometimes slightly curved, 0-septate, hyaline.

Figure 6. 

Brahmaculus packhamiae (PDD 117311) A, B dried apothecia C detail of head of dried apothecium D hymenial surface of rehydrated apothecium, showing multiple separate apothecial cups E squash mount showing excipular cells and hairs in KOH + Melzer’s reagent F ascoma in vertical section G paraphyses, asci, and ascospores H ascospores. Scale bars: 1 mm (A–C); 0.1 mm (D); 10 µm (E–H).

Notes

Brahmaculus packhamiae is macroscopically and microscopically similar to the Chilean B. magellanicus, both species having relatively long and narrow stipes. Notes with the specimen, indicate that when fresh the ascomata “seem to be attached to roots”.

Chlorociboria metrosideri P.R.Johnst., sp. nov.

MycoBank No: 838735
Figure 7

Typification

New Zealand – Bay of Plenty • vic. Rotorua, Tarawera Falls (-38.1573, 176.5193); on fallen leaves Metrosideros excelsa; P.R. Johnston (D2565) leg.; 16 May 2019; PDD 116740 – holotype; ICMP 23410 – ex type culture.

Etymology

Refers to the host substrate of the known specimens.

Diagnosis

Phylogenetically a Chlorociboria, differs in developing on dead leaves rather than wood and in the asci being 4-spored when mature.

Description

Apothecia developing on partly decomposed fallen leaves, not associated with pigmentation of substrate. Apothecia less than 1 mm diam., sessile, with short, matted hairs around the margin, hymenium yellow. Hairs 20–45 × 4 µm, cylindric, walls thin, roughened. Apothecium in vertical section with ectal excipulum 30–40 µm wide, comprising short, broad-cylindric cells 5–7.5 µm diam., with walls hyaline, slightly thickened, rows of cells arranged at a high angle to the receptacle surface near the base of the cup, more parallel to the surface near edge of cup; medullary excipulum of narrow-cylindric cells with thin walls. Paraphyses 2–3 µm diam., taper slightly and gradually to rounded apex, extending 5–10 µm beyond asci. Asci 40–55 × 5.5–7 µm, cylindric, tapering slightly to broad, subtruncate apex, wall thickened at apex with amyloid pore extending as two narrow, parallel bands extending through the wall, initially with 8 spores, 4 spores aborting and 4–spored at maturity, crozier present. Ascospores 7.5–9.5 × 2.5–3.5 µm (average 8.3 × 3.1 µm, n = 20), oblong-elliptic, tapering to rounded ends, one side flat in side view, widest point towards one end, 0–septate, hyaline.

Figure 7. 

Chlorociboria metrosideri (PDD 116740) A fresh apothecia B margin of receptacle in vertical section C surface of receptacle in squash mount showing rough-walled hairs D apex of asci and paraphyses E immature ascus with 8 spores, and mature asci with 4 spores. Scale bars: 0.1 mm (A); 20 µm (B, C); 10 µm (D, E).

Additional specimen examined

New Zealand – Auckland • Rangitoto Island, Kidney Fern Glen; -36.805544, 174.860064; on fallen, partly rotten Metrosideros excelsa leaves; P.R. Johnston (D2329) leg.; 23 Apr 2012; PDD 102723.

Notes

The substrate in both specimens was partly rotted leaves. It is possible that this fungus has a broader host range as most host-specialised, leaf-inhabiting Leotiomycetes are found on recently fallen leaves of their preferred host. Cultures are slow growing (on PDA, 9 mm after 8 weeks) with sparse mycelium and pale brownish pigmentation, remaining sterile.

Chlorociboria novae-zelandiae P.R.Johnst., sp. nov.

MycoBank No: 838736

Typification

New Zealand – Fiordland • Kepler Track, control gates; -45.4396, 167.6822; on Nothofagaceae sp. dead wood; P.R. Johnston (D1484), R.E. Beever, S.R. Pennycook, R. Leschen, T. Lebel leg.; 10 May 2000; PDD 77447 – holotype; ICMP 18766 – ex type culture.

Etymology

Refers to the country of origin, in contrast to Argentina and the morphologically similar C. argentinensis, with which C. novae-zelandiae was previously confused.

Diagnosis

Similar to Chlorociboria argentinensis in having small, allantoid ascospores and lacking tomentum hyphae, but phylogenetically distinct and with smaller ascospores and narrower asci.

Additional specimens examined

C. novae-zelandiae : New Zealand – Fiordland. • Borland Lodge nature trail; on Nothofagaceae sp. dead wood; P.R. Johnston (D1471.2) leg.; 9 May 2000; PDD 77446 – North Canterbury. • Mt Thomas Forest, Richardson Track; on Nothofagaceae sp. dead wood; P.R. Johnston (D679) leg.; 15 Mar 1991; PDD 58574, ICMP 15616 – Nelson. • Arthur Range, Graham Valley Rd, track from Flora car park to Mt Arthur Hut; P.R. Johnston (D993) leg.; 6 May 1994; PDD 77444.

C. argentinensis : Argentina – Tierra del Fuego • Lago Fagnano, vic. Kosobo, road to hot springs; on Nothofagus pumilio fallen wood; P.R. Johnston (SA86), L. Lorenzo leg.; 22 Mar 1996; PDD 92026; ICMP 16994 – Patagonia. • Rio Negro, Nahuel Huapai National Park, path from Puerto Blest to Los Cantaros; on Nothofagus dombeyi fallen wood; P.R. Johnston (SA 188), I. Gamundí, C. Brion leg.; 2 Apr 1996; PDD 92027; ICMP 16995.

Notes

Johnston and Park (2005: 690–693) provided a description of C. novae-zelandiae, from New Zealand specimens reported under the name C. argentinensis. Subsequent DNA sequencing of specimens from Argentina identified as C. argentinensis, showed that the New Zealand species is phylogenetically distinct. Morphologically, the two species are similar, both with an excipulum comprising highly gelatinous textura intricata, the apothecia lacking hair-like tomentum hyphae, and with small, allantoid ascospores. The New Zealand species has somewhat smaller ascospores (average 7.0 × 1.5 µm versus 9.9 × 1.9 µm) and narrower asci (3.5–4.5 µm versus 4–5.5 µm) compared with the Argentinian specimens recognised here as C. argentinensis. The Argentinian specimens match closely the description of Dixon (1975).

Chlorociboria solandri P.R.Johnst., sp. nov.

MycoBank No: 838737
Figure 8

Typification

New Zealand – Fiordland • Fiordland National park, Kepler Track, Rainbow Reach; -45.4429, 167.6802; on Fuscopora solandri fallen leaves; P.R. Johnston (D686) leg.; 17 Mar 1991; PDD 58580 – holotype; ICMP 23686 – ex-type culture.

Etymology

Refers to the host substrate of the holotype.

Diagnosis

Phylogenetically a Chlorociboria, developing on fallen leaves rather than wood, differs from Chlorociboria metrosideri in having flexuous, coiled hairs and lanceolate paraphyses.

Description

Apothecia developing on fallen leaves, not associated with any pigmentation of substrate. Apothecia less than 1 mm diam., short-stipitate, receptacle densely covered with short, white hairs, hymenium pale yellow. Hairs 30–40 × 3–4 µm, short-cylindric, undifferentiated to apex, septate, thin-walled, roughened all over, flexuous, coiled and tangled. Apothecia in vertical section with ectal excipulum 45 µm wide, comprising short-cylindric to subglobose cells 5–8 µm diam. oriented at high angle to receptacle surface, with walls hyaline, thickened, agglutinated, amyloid in some specimens. Medullary excipulum non-gelatinous, comprising narrow-cylindric hyphae with thin walls. Tissue at base of stipe of gelatinous textura intricata. Paraphyses up to 5 µm diam., lanceolate, tapering to narrow rounded apex, extending 20–30 µm beyond asci, wall distinctively thickened at base, amyloid in some specimens. Asci 40–55 × 4.5–5.5 µm, cylindric, tapering gradually to small, subtruncate apex, wall thickened at apex, amyloid pore extending through wall, flaring towards outside of wall, crozier present, 8–spored. Ascospores 8–11.5 × 1.5–2 µm (average 10.0 × 1.7 µm, n = 20), oblong-elliptic to subfusoid, widest point slightly towards the upper end, taper to narrow-rounded ends, 0–septate, hyaline.

Figure 8. 

Chlorociboria solandri A dried apothecium B fresh apothecium C margin of receptacle in vertical section D ascospores E paraphyses F amyloid paraphysis G base of paraphysis with thick wall H details of coiling, rough-walled excipular hairs. Images: PDD 58580 (A, C, D, F–H); PDD 61833 (B, E). Scale bars: 0.1 mm (A, B); 20 µm (C); 10 µm (D–H).

Additional specimens examined

New Zealand – Taupo • Kaimanawa Forest Park, Tree Trunk Gorge; on Fuscopora solandri fallen leaves; P.R. Johnston (D877), I. Gamundí leg.; 1 Feb 1993; PDD 61833 – Mid Canterbury • Craigieburn, Cave Stream; on Fuscopora solandi fallen leaves; E. Horak leg.; 31 Mar 1983; PDD 92925.

Notes

Chlorociboria solandri is micromorphologically distinctive in having scattered, large, lanceolate paraphyses, short-cylindric to more or less globose, thick-walled excipular cells, excipular tissue reacting either blue or red to Melzer’s reagent, and coiling, rough-walled hairs. Known from two specimens from Fuscopora solandri leaves. A third specimen in poor condition, PDD 92925, could be the same species; it is morphologically similar but has longer hairs than the other two specimens. Cultures on agar are very slow growing (10 cm after 4 weeks), have little aerial mycelium and pale yellow brown pigments, remaining sterile.

Chlorociboria subtilis P.R.Johnst., sp. nov.

MycoBank No: 838738
Figure 9

Typification

New Zealand – Westland • Haast Pass Summit, Lookout Track; -44.1063, 169.3519; on fallen leaves Dracophyllum sp.; P.R. Johnston (D2515), M. Padamsee leg.; 16 May 2018; PDD 112247 – holotype.

Etymology

From subtilis (delicate) referring to the stature of the apothecia.

Diagnosis

Blue-green apothecia on blue-green stained fallen, partly decomposed leaves, hairs on receptacle rough-walled, somewhat flexuous, ascospores filiform, 45–55 × 1 µm.

Description

Apothecia erumpent from blue-green stained leaf tissue. Apothecia less than 1 mm diam., cupulate with short, broad stipe, receptacle pale blue-green with tangled, white hairs, especially near the edge of the cup. Hairs 55–75 × 3–4 µm, somewhat flexuous, wall roughened. Apothecia in vertical section with ectal excipulum up to 30 µm wide, cells 6–10 µm diam., short-cylindric to square, walls thick, cells arranged in rows with a high angle to the receptacle surface; medullary excipulum poorly developed, two or three rows of narrow-cylindric cells, walls encrusted with blue-green material; stipe with thick-walled textura intricata. Paraphyses 1.5–2 µm diam., slightly wider towards the apex, often branched in the upper 20–30 µm, extending 15 µm beyond asci. Asci 85–105 µm × 5.5–6.5 µm cylindric, tapering gradually to small, truncate apex, wall thickened at apex, amyloid pore in inner half of wall, reaction most intense on inner edge of wall, pore appears more or less U-shaped, sloping outwards slightly through the wall, 8-spored, spores confined to the upper 60–100 µm of ascus, crozier present. Ascospores 45–55 × 1 µm, filiform, straight, 0–septate, hyaline.

Figure 9. 

Chlorociboria subtilus (PDD 112247) A dried apothecium B apothecium in vertical section C detail of margin of receptacle in vertical section D asci, ascospores and paraphyses E detail of apex of paraphyses and asci F excipular hairs (squash mount). Scale bars: 0.1 mm (A); 100 µm (B); 10 µm (C–F).

Additional specimens examined

New Zealand – Nelson • Arthur Range, Graham River Valley Rd, track from Flora car park to Mt Arthur Hut; on Dracophyllum pyrimidale fallen leaves; P.R. Johnston (D990) leg.; 6 May 1994; PDD 105292 – Central Otago •vic. Dunedin, Great Moss Swamp; on Dracophyllum uniflorum fallen leaves; P.R. Johnston (D82) leg.; 12 May 1984; PDD 105293 – Mid Canterbury • Craigieburn, Cave Stream; on Dracophyllum uniflorum fallen leaves; P.R. Johnston (D248) leg.; 23 Feb 1988; PDD 105294 – Taupo • Tongariro National Park, Ohakune Mountain Road, Blyth Track; on Fuscopora cliffortioides fallen leaves; P.R. Johnston (D353) leg.; 20 May 1989; PDD 55523 • Rangitoto Station, Ranginui Summit; on Dracophyllum pyrimidale fallen leaves; P.R. Johnston (D1622), S.R. Whitton leg.; 6 May 2001; PDD 117584.

Notes

Most specimens are on fallen leaves of Dracophyllum spp., but the host range may be more extensive. A specimen on Fuscopora cliffortioides (PDD 55523) is morphologically similar, but perhaps with longer ascospores.

Discussion

The Brahmaculus species described here are so morphologically and ecologically divergent from Chlorociboria that they must be placed in their own genus. All four new species are members of a well-supported monophyletic lineage within Chlorociboriaceae (Fig. 2). However, in both the multigene and ITS analyses (Figs 1, 2) the Brahmaculus clade makes Chlorociboria, as currently understood in a morphological sense, paraphyletic. If alternative generic limits were to be drawn to recognise only monophyletic genera within Chlorociboriaceae, it is unclear how these genera could be distinguished morphologically. The type of Chlorociboria (C. aeruginosa) sits within the main Chlorociboria clade, and hence the name Chlorociboria will remain attached to the bulk of the species so far described in the genus. However, further sampling of Chlorociboria, including of species lacking green pigments (see below) is required before redrawing generic limits, especially in regard to the distinguishing morphological characters of the main Chlorociboria clade in relation to the phylogenetically differentiated species C. halonata and C. aeruginella.

The multi-gene phylogeny places Chlorociboriaceae in an isolated position near the base of Helotiales. Earlier analyses had suggested a relationship between Chlorociboriaceae and Cyttariaceae (Peterson and Pfister (2010a). The multiple genes newly available from a Cyttaria nigra specimen (PDD 117571) allowed Cyttariaceae to be treated in the multi-gene analysis. This showed that although Cyttariaceae was similar to Chlorociboriaceae in having an isolated position near the base of Helotiales, no particular phylogenetic relationship was found between the families. Cyttariales is treated here as a synonym of Helotiales.

Direct observations of the mycelium at the base of the stipes of several of the Brahmaculus spp. suggests a biotrophic relationship with either the roots of Nothofagaceae (possibly as root endophytes), or the mycorrhizal fungi associated with those roots (possibly as parasites). Johnston and Park (2005) noted a possible ecological relationship between wood rotting basidiomycetes and some of the wood-inhabiting Chlorociboria spp.

Not all of the specimens accepted here as Chlorociboria develop green pigment on their substrate. These include C. glauca and two of the newly described species from New Zealand (C. metrosideri and C. solandri). Both of these newly described species develop on fallen leaves, they have whitish rather than green apothecia, form no green pigment on their substrate, but have an excipular structure and the short, rough-walled, hair-like elements typical of several of the New Zealand representatives of the genus. The third newly named species from New Zealand, Chlorociboria subtilis, also develops on fallen leaves, but both the apothecia and the adjacent parts of the leaf have a blue-green pigment. The apothecial hairs of this species are better developed than those of most Chlorociboria species. Fungi morphologically similar to C. subtilis occur on fallen leaves in both eastern Australia (e.g. PDD 117581) and southern South America (unpubl. data) but they are not named here as only small specimens, and no DNA sequences, are available for these fungi.

Most known Chlorociboria species develop on green-stained, fallen wood. It is likely that there are other unrecognised Chlorociboria species, placed in other genera because they lack green pigment and have substrates apart from wood, the visually obvious features historically regarded as characteristic of Chlorociboria. Their true phylogenetic relationship may be revealed only when DNA sequence data becomes available for them, unless an alternative set of morphological features is discovered that is found to be characteristic of the Chlorociboriaceae clade. Huhtinen et al. (2010) discuss other seemingly ecologically or morphologically atypical Chlorociboria spp. from Europe. If these are shown to be Chlorociboria phylogenetically, they may be key to discovering phylogenetically informative morphological characters for the genus and family.

Conclusions

The phylogenetic breadth of Chlorociboriaceae is becoming better understood with the identification of Brahmaculus as a distinct lineage. For Chlorociboria, recognising that not all species form apothecia on green-stained wood is an important step in characterising the genus and family both morphologically and phylogenetically, and in resolving more accurately its geographic distribution globally.

Acknowledgements

Anna Chinn is thanked for recognising the importance of a tiny fungus she had never seen before, her collection initiating the preparation of this paper, and became the type material of Brahmaculus moonlighticus. Permits giving permission to collect the specimens reported here were issued by the New Zealand Department of Conservation to Manaaki Whenua–Landcare Research and to the Fungal Network of New Zealand for the 14th and 32nd New Zealand Fungal Forays, and by the New Zealand Forests Restoration Trust. Permission to collect fungi in the Chilean National System of Protected Wild Areas was provided by Corporación Nacional Forestal under permit No. 014/2014. We thank Giuliana Furci and Daniela Torres of Fundacion Fungi and Pablo Sandoval-Leiva for facilitating fieldwork in Chile. We thank Rosanne Healy from the FLAS herbarium and the staff at SGO for help with accessioning specimens specimens, and Marcos Caiafa for help with molecular biology lab work. The Argentinian collections of C. argentinensis were collected with the support of the Flora Criptogámica Tierra del Fuego project with the particular assistance of Irma Gamundí and Laura Lorenzo. Collecting activities in Australia were supported by the Tasmanian Forest Research Council.

Johnston and Park were supported through the Manaaki Whenua Biota Portfolio with funding from the Science and Innovation Group of the New Zealand Ministry of Business, Innovation and Employment. Smith was supported by the US National Science Foundation grant DEB 1354802 and the Institute for Food and Agricultural Sciences at the University of Florida (NIFA-USDA award FLA-PLP-005289).

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