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Mycological Progress

, Volume 18, Issue 1–2, pp 91–105 | Cite as

Stilbocrea walteri sp. nov., an unusual species of Bionectriaceae

  • Hermann VoglmayrEmail author
  • Walter M. Jaklitsch
Open Access
Original Article
  • 588 Downloads

Abstract

The new species Stilbocrea walteri is described and illustrated from Quercus ilex collected in Portugal. Phylogenetic analyses of LSU rDNA, rpb1, rpb2 and tef1 sequence matrices place S. walteri in the Bionectriaceae, Hypocreales, within a clade of specimens morphologically identified as Stilbocrea macrostoma, the generic type of Stilbocrea. Stilbocrea walteri differs from S. macrostoma in dark olive green to blackish ascomata basally immersed in a stroma, KOH+ and LA+ ascomata and the lack of a stilbella-like asexual morph on natural substrate and pure culture. A simple phialidic asexual morph is formed in pure culture. To enable a morphological comparison, Stilbocrea macrostoma is illustrated.

Keywords

Ascomycota Hypocreales Nectria Phylogenetic analysis Sordariomycetes Taxonomy 

Introduction

During a collecting trip to Portugal, a black stromatic pyrenomycete was collected on dead corticated branches of Quercus ilex. Microscopic analyses revealed a nectriaceous fungus, which could not be identified to genus or species, and also the familial affiliation remained unclear. The partial immersion of ascomata in a well-developed stroma and reddening of the ascomatal walls in KOH pointed towards Nectriaceae, but molecular phylogenetic analysis based on LSU rDNA, rpb1, rpb2 and tef1 sequences revealed a placement within Bionectriaceae. Based on this evidence, a new species of Stilbocrea is described.

Materials and methods

Culture preparation, isolates and specimens

Cultures were prepared from ascospores and maintained as described previously (Jaklitsch 2009). Germinating ascospores were placed on CMD (CMA: Sigma, St Louis, Missouri; supplemented with 2% (w/v) D(+)-glucose-monohydrate) or 2% malt extract agar (MEA; 2% w/v malt extract, 2% w/v agar-agar; Merck, Darmstadt, Germany). The plates were sealed with laboratory film and incubated at room temperature. Cultures used for the study of the asexual morph were grown on 2% MEA or CMD at room temperature (22 ± 3 °C) under alternating 12 h daylight and 12 h darkness. The ex-type culture was deposited at the Westerdijk Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands, and specimens in the Fungarium of the Institute of Botany, University of Vienna (WU). The following specimens of Stilbocrea macrostoma were sequenced for the phylogenetic analyses and/or used for morphological illustration and comparison but are not described in detail here: Panama, Parque Nacional Altos de Campana, on dead branch of an unidentified tree, 5 May 2012, E. Esquivel (WU 32032); culture SM, prepared and maintained on PDA (Merck). Sri Lanka, Nuwara Eliya, Hakgala Sanctuary Botanical Gardens, 12 Feb. 1984, I. Krisai-Greilhuber IK 2346 (WU 26101).

Morphological observations

Microscopic preparations were mounted in water, 3% potassium hydroxide (KOH) or lactic acid (LA). Stereomicroscopy illustrations and measurements were done with a Keyence VHX-6000 system. Light microscopy was performed with Nomarski differential interference contrast (DIC) using the Zeiss Axio Imager.A1 compound microscope, and images and data were gathered using the Zeiss Axiocam 506 colour digital camera and measured by using the Zeiss ZEN Blue Edition software. Measurements are reported as maxima and minima in parentheses and the mean plus and minus the standard deviation of a number of measurements given in parentheses.

DNA extraction, PCR and sequencing

Growth of liquid culture and extraction of genomic DNA was done according to Voglmayr and Jaklitsch (2011), using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany). To confirm the identity of the culture, DNA was also extracted from stromata following the protocol of Voglmayr and Jaklitsch (2011) for herbarium specimens, but using the DNeasy Plant Mini Kit. The complete ITS region and D1 and D2 domains of 28S rDNA region (ITS-LSU) were amplified with primers V9G (de Hoog and Gerrits van den Ende 1998) and LR5 (Vilgalys and Hester 1990), a ca. 750 bp fragment of the RNA polymerase II subunit 1 (rpb1) gene with primers RPB1-Ac (Schoch et al. 2012) and RPB1Cr (Sung et al. 2007b), a ca. 1.1 kb fragment of the RNA polymerase II subunit 2 (rpb2) gene with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999) or dRPB2-5f and dRPB2-7r (Voglmayr et al. 2016) and a ca. 1.4 kb fragment of the translation elongation factor 1-α (tef1) gene with primers EF1-728F (Carbone and Kohn 1999) and EF1-2218R (Rehner and Buckley 2005). From stromatal DNA, only the ITS-LSU was amplified and sequenced. PCR was performed with a Taq polymerase, with annealing temperatures of 55 °C for ITS-LSU, tef1 and rpb2 (primer pair fRPB2-5F, fRPB2-7cR) and 51 °C for rpb1 and rpb2 (primer pair dRPB2-5f, dRPB2-7r). PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr and Jaklitsch (2008). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington) and the PCR primers; in addition, primers ITS4 (White et al. 1990), LR3 (Vilgalys and Hester 1990) and LR2R-A (Voglmayr et al. 2012) were used for the ITS-LSU region. Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyser, Applied Biosystems).

Phylogenetic analyses

As the LSU rDNA is the most representative marker available for many genera of Bionectriaceae, an extended LSU matrix was produced for phylogenetic analyses. For this, the sequence matrix of Jaklitsch and Voglmayr (2011a) was supplemented with selected sequences from Summerbell et al. (2011) and a few additional GenBank sequences. Only few rpb1, rpb2 and tef1 sequences of Bionectriaceae were available from GenBank to phylogenetically place Stilbocrea. For the same reason, ITS rDNA was not phylogenetically analysed. The GenBank accession numbers of sequences downloaded for phylogenetic analyses are given in Table 1 and in the phylogenetic trees (Figs. 1 and 2), following the taxon names. Generic classification of the Nectriaceae follows Lombard et al. (2015), of Stachybotryaceae Lombard et al. (2016) and of Bionectriaceae the taxonomy implemented in NCBI GenBank, with a few additions of recently published new genera.
Table 1

List of taxa and GenBank accessions used in the current phylogenetic study. The references are according to the NCBI Nucleotide database. Sequences in bold were generated during the present study

Taxon

LSU

rpb1

rpb2

tef1

References

Acremoniumacutatum

NG_056976

   

Summerbell et al. (2011)

Acremoniumalternatum

NG_056977

   

Summerbell et al. (2011)

Acremoniumfusidioides

NG_056984

   

Summerbell et al. (2011)

Acremoniumhennebertii

NG_056987

   

Summerbell et al. (2011)

Acremoniumsclerotigenum

NG_057139

 

KC998999

KC998988

Hijikawa et al. (2017), Grum-Grzhimaylo et al. (2013b)

Acremoniumzeylanicum

HQ232154

   

Summerbell et al. (2011)

Bryocentria brongniartii

EU940125

   

Stenroos et al. (2010)

Bryocentria metzgeriae

EU940106

   

Stenroos et al. (2010)

Bulbithecium hyalosporum

AF096187

   

Suh and Blackwell (1999)

Bullanockia australis

KY173506

   

Crous et al. (2016a)

Calonectria cylindrospora

U17409

   

Rehner and Samuels (1995)

Chaetopsina fulva

DQ119554

   

Zhang and Zhuang (unpubl.)

Clonostachys buxi

  

KM232416

 

Lombard et al. (2015)

Clonostachys byssicola

 

GQ506040

 

LT220768

Hirooka et al. (2010), Sharma and Marques (unpubl.)

Clonostachys compactiuscula

 

GQ506036

  

Hirooka et al. (2010)

Clonostachys epichloe

DQ363259

   

Kirschner (unpubl.)

Clonostachys grammicospora

AF193238

   

Rossman et al. (2001)

Clonostachys pityrodes

AY489728

AY489658

  

Castlebury et al. (2004)

Clonostachys rosea

AY283558

GQ506038

DQ522415

AY489611

Seifert et al. (2003), Hirooka et al. (2010), Spatafora et al. (2007), Castlebury et al. (2004)

Clonostachys setosa

AF210670

   

Schroers (2001)

Cosmospora coccinea

AY489734

   

Castlebury et al. (2004)

Cyanonectria cyanostoma

FJ474081

   

Samuels et al. (unpubl.)

Cylindrocladiella microcylindrica

AY793432

   

Crous et al. (2005)

Dialonectria episphaeria

AY015625

   

Zhang and Blackwell (2002)

Emericellopsis alkalina

  

KC999029

KC998993

Grum-Grzhimaylo et al. (2013)

Emericellopsis glabra

GQ505993

GQ506023

  

Hirooka et al. (2010)

Emericellopsis maritima

FJ176861

 

KC999033

KC998997

Grum-Grzhimaylo et al. (2013b)

Emericellopsis minima

  

KC999031

KC998996

Grum-Grzhimaylo et al. (2013b)

Emericellopsis pallida

  

KC999034

 

Grum-Grzhimaylo et al. (2013b)

Emericellopsis terricola

U57082

   

Glenn and Bacon (unpubl.)

Eucasphaeria capensis

EF110619

   

Crous et al. (2007)

Eucasphaeria rustici

KY173501

   

Crous et al. (2016a)

Flammocladiella decora

NG_058175

   

Crous et al. (2015a)

Geonectria subalpina

MH155487

   

Lechat et al. (2018)

Geosmithia brunnea

   

KY872747

Huang et al. (unpubl.)

Geosmithia langdonii

  

HG799928

HG799879

Kolarik et al. (unpubl.)

Geosmithia lavendula

KT155289

   

Stielow et al. (unpubl.)

Geosmithia microcorthyli

  

FM986794

 

Kolarik and Kirkendall (2010)

Geosmithia pallida

  

HG799930

HG799871

Kolarik et al. (unpubl.)

Geosmithia proliferans

   

KY872749

Huang et al. (unpubl.)

Geosmithia putterillii

KT155185

 

HG799907

HG799853

Stielow et al. (unpubl.), Kolarik et al. (unpubl.)

Gliomastix masseei

HQ232060

   

Summerbell et al. (2011)

Gliomastix murorum

  

FJ238363

 

Schoch et al. (unpubl.)

Gliomastix roseogrisea

HQ232122

   

Summerbell et al. (2011)

Heleococcum aurantiacum

JX158463

 

JX158463

JX158397

Grum-Grzhimaylo et al.(2013a)

Heleococcum japonense

JX158442

 

JX158464

JX158398

Grum-Grzhimaylo et al.(2013a)

Heleococcum japonicum

U17429

   

Rehner and Samuels (1995)

Hydropisphaera erubescens

 

DQ518182

AY545731

DQ522344

James et al. (unpubl.), AFTOL (unpubl.), Spatafora et al. (2007)

Hydropisphaera fungicola

 

GQ506025

  

Hirooka et al. (2010)

Hydropisphaera peziza

AY489730

AY489661

DQ522444

AY489625

Castlebury et al. (2004), Spatafora et al. (2007)

Hydropisphaera suffulta

KU237207

   

Lechat (unpubl.)

Hypocreales sp.

GU017530

   

Sakayaroj et al. (2010)

Ijuhya chilensis

KY607553

KY607579

  

Ashrafi et al. (2017)

Ijuhya corynospora

 

KY607580

  

Ashrafi et al. (2017)

Ijuhya faveliana

 

KY607582

  

Ashrafi et al. (2017)

Ijuhya fournieri

KP899118

   

Lechat et al. (2015)

Ijuhya paraparilis

 

GQ506041

  

Hirooka et al. (2010)

Ijuhya parilis

 

KY607584

  

Ashrafi et al. (2017)

Ijuhya peristomialis

KY607559

KY607585

  

Ashrafi et al. (2017)

Ijuhya vitellina

 

KY607577

  

Ashrafi et al. (2017)

Kallichroma glabrum

AF193233

   

Rossman et al. (2001)

Kallichroma tethys

AF193234

   

Rossman et al. (2001)

Lasionectria mantuana

 

GQ506024

  

Rossman et al. (2001)

Lasionectriella rubioi

KU593581

   

Lechat and Fournier (2016)

Leuconectria clusiae

U17412

   

Rehner and Samuels (1995)

Leucosphaerina arxii

NG_057892

   

Summerbell et al. (2011)

Mycoarachis inversa

NG_059437

GQ506021

 

HM484840

Hirooka et al. (2010), Chaverri et al. (2011)

Myrothecium inundatum

KU846474

   

Lombard et al. (2016)

Nectria aurantiaca

HM534892

   

Jaklitsch and Voglmayr (2011b)

Nectria cinnabarina

HM534894

HM484577

JQ014125

AF543785

Jaklitsch and Voglmayr (2011b), Hirooka et al. (2011), Schoch et al. (2012), Currie et al. (2003)

Nectria pseudotrichia

HM534899

   

Jaklitsch and Voglmayr (2011b)

Nectriopsis epimycota

 

GQ506037

  

Hirooka et al. (2010)

Nectriopsis exigua

 

GQ506014

 

HM484852

Hirooka et al. (2010), Chaverri et al. (2011)

Nectriopsis violacea

AF193242

AY489646

  

Rossman et al. (2001), Castlebury et al. (2004)

Neocosmospora haematococca

DQ119558

  

AY489624

Zhang and Zhuang (unpubl.), Castlebury et al. (2004)

Neocosmospora vasinfecta

U17406

   

Rehner and Samuels (1995)

Neonectria coccinea

AY677327

   

Halleen et al. (2004)

Neonectria ditissima

AY677330

   

Halleen et al. (2004)

Neonectria punicea

HM534901

   

Jaklitsch and Voglmayr (2011b)

Niesslia exilis

AY489720

   

Castlebury et al. (2004)

Nigrosabulum globosum

AF096195

   

Suh and Blackwell (1999)

Ochronectria calami

AF193243

AY489644

EF692515

AY489612

Rossman et al. (2001), Castlebury et al. (2004), Sung et al. (2008)

Ovicillium attenuatum

KU382232

   

Zare and Gams (2016)

Paracylindrocarpon aloicola

KX228328

   

Crous et al. (2016b)

Peethambara spirostriata

AY489724

   

Castlebury et al. (2004)

Peethambara sundara

AF193245

   

Rossman et al. (2001)

Penicillifer diparietispora

AY489735

   

Castlebury et al. (2004)

Persiciospora africana

AY015631

   

Zhang and Blackwell (2002)

Protocreopsis korfii

KT852955

   

Lechat and Fournier (2015)

Protocreopsis pertusa

GQ506002

   

Hirooka et al. (2010)

Pseudocosmospora vilior

AY015626

   

Zhang and Blackwell (2002)

Rosasphaeria moravica

JF440985

   

Jaklitsch and Voglmayr (2012)

Roumegueriella rufula

EF469082

GQ506029

EF469116

EF469070

Sung et al. (2007a), Hirooka et al. (2010)

Sarcopodium macalpinei

DQ119566

   

Zhang and Zhuang (unpubl.)

Selinia pulchra

AF193246

GQ506022

 

HM484841

Rossman et al. (2001), Hirooka et al. (2010), Chaverri et al. (2011)

Stachybotrys chartarum

KU846792

   

Lombard et al. (2016)

Stephanonectria keithii

AY489727

  

AY489622

Castlebury et al. (2004)

Stilbocrea macrostoma

AY489725, GQ506004, MH562716

GQ506033, AY489655, MH562716

EF692520, MH577043

AY489620

Hirooka et al. (2010), Castlebury et al. (2004), Sung et al. (2008), this study

Stilbocrea sp.

JQ733407

   

Supaphon et al. (2017)

Stilbocrea” sp.

KX578037

   

Lechat (unpubl.)

Stilbocrea walteri

MH562717

MH562715

MH577042

MH562714

this study

Stromatonectria caraganae

HQ112287

 

HQ112290

HQ112286

Jaklitsch and Voglmayr (2011a)

Synnemellisia aurantia

KX866396

   

Lisboa et al. (unpubl.)

Thyronectria aquifolii

HM534891

   

Jaklitsch and Voglmayr (2011b)

Thyronectria berolinensis

HM534893

   

Jaklitsch and Voglmayr (2011b)

Thyronectria coryli

HM534895

   

Jaklitsch and Voglmayr (2011b)

Thyronectria lamyi

HM534898

   

Jaklitsch and Voglmayr (2011b)

Thyronectria rhodochlora

 

KJ570728

KJ570751

 

Jaklitsch and Voglmayr (2014)

Thyronectria sinopica

HM534900

   

Jaklitsch and Voglmayr (2011b)

Verrucostoma freycinetiae

GQ506013

GQ506018

  

Hirooka et al. (2010)

Verrucostoma martiniciensis

KP192672

   

Crous et al. (2015b)

Volutella buxi

U17416

   

Rehner and Samuels (1995)

Xanthonectria pseudopeziza

KU946964

   

Lechat et al. (2016)

Fig. 1

Phylogram obtained by PAUP from an analysis of the LSU matrix of selected Hypocreales, showing one of 24 most parsimonious trees 1202 steps long. Stilbocrea walteri is revealed as a species of the Bionectriaceae. GenBank accession numbers of sequences are given following the taxon names. The country of origin is provided for accessions within the Stilbocrea clade. Isolates in bold were sequenced during the present study; thickened internal branches are present in the strict consensus of all 24 MP trees. MP and ML bootstrap support above 50% are given at first and second position, respectively, above or below the branches

Fig. 2

Phylograms revealed by PAUP from MP analyses of the rpb1 (a), rpb2 (b) and tef1 (c) matrices, showing the phylogenetic position of Stilbocrea walteri within Bionectriaceae. a One of two MP trees 2320 steps long; asterisk (*) denoting node that collapsed in the strict consensus of the two MP trees. b Single MP tree 2597 steps long. c Single MP tree 957 steps long. GenBank accession numbers of sequences are given following the taxon names; isolates in bold were sequenced during the present study. MP and ML bootstrap support above 50% are given at first and second position, respectively, above or below the branches

The downloaded GenBank sequences were aligned with the sequences generated in our study with the server version of MAFFT (www.ebi.ac.uk/Tools/mafft) using the default settings and checked and refined with BioEdit v. 7.0.9.0 (Hall 1999). The four matrices were analysed separately. The final matrices used for phylogenetic analyses contained 863, 750, 1072 and 951 alignment characters for the LSU, rpb1, rpb2 and tef1, respectively.

Maximum parsimony (MP) analyses were performed with PAUP v. 4.0a161 (Swofford 2002), using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MAXBRLEN. Bootstrap analysis with 1000 replicates was performed in the same way, but using 5 rounds of random sequence addition and subsequent TBR branch swapping during each bootstrap replicate, with the COLLAPSE command set to MINBRLEN; in addition, each replicate was limited to 1 million rearrangements in the LSU analyses. All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to minbrlen.

Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012) using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates.

Bootstrap support below 70% was considered low, between 70 and 90% moderate and above 90% high.

Results

Sequencing and molecular phylogeny

The ITS-LSU sequences obtained from the culture and the stromata of the newly described fungus were identical. Sequence similarity of the ITS of the newly described fungus and the newly sequenced Stilbocrea macrostoma accession from Panama (SM) was 83.5% (71 nucleotide substitutions and 14 gaps).

Of the 866 nucleotide characters included in the LSU analyses, 163 were parsimony informative. Maximum parsimony analyses revealed 24 MP trees 1202 steps long, one of which is shown as Fig. 1. The MP trees differed mainly in the deeper nodes of Nectriaceae (Fig. 1); in some of the MP trees, Stachybotryaceae were embedded within the Nectriaceae (not shown). In the phylogenetic analyses, the Stachybotryaceae were moderately supported, while the clade comprising Bionectriaceae plus Flammocladiellaceae received high support. The Flammocladiellaceae were revealed as sister group to Bionectriaceae in the MP analyses; however, the latter did not receive significant bootstrap support (Fig. 1). Within Bionectriaceae, backbone support of deeper nodes was mostly low or absent. The GenBank accessions of Stilbocrea included in our LSU analyses did not form a monophylum as the unpublished accession KX578037 from Spain labelled Stilbocrea sp. was placed outside the Stilbocrea clade. The three accessions of Stilbocrea macrostoma, the fungus from Portugal and two GenBank accessions of endophyte isolates from tropical marine seagrasses (JQ733407; GU017530) formed a monophylum with low support (Fig. 1). However, the various accessions of Stilbocrea macrostoma did not form a monophylum, as the newly sequenced S. macrostoma specimen from Panama was in a basal position to a highly supported subclade containing the new Stilbocrea species from Portugal, the GenBank accessions of S. macrostoma from New Zealand and Sri Lanka and the two endophyte isolates.

Of the 750 nucleotide characters included in the rpb1 analyses, 367 were parsimony informative. Maximum parsimony analyses revealed two MP trees 2320 steps long, one of which is shown as Fig. 2a. The two MP trees were identical except for an interchanged position of Ijuhya peristomialis and Ijuhya parilis (not shown). Of the 1072 nucleotide characters included in the rpb2 analyses, 533 were parsimony informative. Maximum parsimony analyses revealed a single MP tree 2597 steps long which is shown as Fig. 2b. Of the 951 nucleotide characters included in the tef1 analyses, 231 were parsimony informative. Maximum parsimony analyses revealed a single MP tree 957 steps long which is shown as Fig. 2c.

In the analyses of the protein-coding genes (rpb1, rpb2, tef1), many of the deeper nodes within Bionectriaceae received no or low support (Fig. 2a–c), and only limited comparisons are possible between these trees due to a different taxon selection. However, the new fungus from Portugal and the GenBank accessions of Stilbocrea macrostoma from New Zealand (all three markers available) and Sri Lanka (only rpb1 available) consistently formed a clade with maximum support (Fig. 2a–c), while the newly sequenced Panamese accession of Stilbocrea macrostoma was not contained in this clade (Fig. 2a, b). Remarkably, in the rpb1 tree (Fig. 2a), the fungus from Portugal was placed in a sister group position to the GenBank accessions of Stilbocrea macrostoma from New Zealand and Sri Lanka with medium (84%; MP) to high (95%; ML) support.

Considering morphological and molecular data, the specimen from Portugal is described as a new species.

Taxonomy

Stilbocrea walteri Voglmayr & Jaklitsch, sp. nov. Figs. 3 and 4.
Fig. 3

Stilbocrea walteri, sexual morph (WU 39972). af Stromata/ascomata. gi Stromata in vertical section. j, k Ostiolar region in vertical section. l Ostiole in face view. m Periphyses. n, o Peridium in vertical section. p Peridium in face view. q Stroma tissue in vertical section (f, i, j, l, m, p in 3% KOH; g, h, n, q in water; k, o in LA). Scale barsa 1 mm; b–f 200 μm; g 100 μm; h, i 50 μm; j 20 μm; kq 10 μm

Fig. 4

Stilbocrea walteri, sexual morph (WU 39972), cultures and asexual morph (NQI = CBS 144627). a–d Asci with ascospores (b–d in 3% KOH). e–n, p–v Ascospores (e–j vital, k–n in LA; p–v in 3% KOH; note verruculose and smooth ascospore walls in water/LA and KOH, respectively). o Detail of verruculose ascospore wall (in LA). w, x Cultures (w MEA, 31 d; x CMD, 20 d). y–g1 Conidiophores, pegs and phialides (y, z, d1 CMD, 4 days; a1–g1 CMD, 20 days). h1 Conidia (CMD, 4 days); i1 Blastoconidia (CMD, 20 days). (all in water except where noted). Scale barsa–d, y–c1 10 μm; e–n, p–v, d1–i1 5 μm; o 1 μm

MycoBank MB 826919.

Etymology: in honour of Walter Gams.

Stromata when dry (460–)680–1100(–1600) μm diam (n = 50), (260–)300–430(–520) μm high (n = 30), scattered, less commonly in groups of 2–3, erumpent from bark, pulvinate; round, elliptical or irregular in outline. Stromata at the base compact, white in section. Perithecia (2–)5–15(–20) per stroma, basally immersed in the uppermost layer of the stroma, dark olive green to black when dry, black in water; in 3% KOH with a reddish tinge, reversible after addition of LA, no pigment dissolved. Ostiolar dots (24–)31–42(–47) μm diam (n = 33), umbilicate, black.

Subperithecial and basal tissue of the stroma mostly consisting of a t. angularis of thin-walled, hyaline cells (6–)7.5–15(–18.5) × (3.5–)5–8.5(–11) μm (n = 30), becoming hyphal adjacent to the host tissue; stroma tissue without colour change in KOH or LA. Perithecia (205–)216–271(−277) μm high, (153–)171–234(–250) μm wide (n = 12), globose to subglobose, partially immersed in stroma, apical parts exposed. Ostioles periphysate, periphyses 12–34 μm long, 1.2–2 μm wide (n = 10). Peridium 35–90 μm thick, consisting of three layers: a 6–24-μm thick inner layer of hyaline to subhyaline, thick-walled (outermost) to thin-walled (innermost) elongate cells (6–)8–15(–19) × (1–)2–4(–5) μm (n = 50); a 13–24-μm-thick medium layer of dark olive green, thick-walled, elongate cells (6–)8–15(–18.5) × (4–)5–8(–11) μm (n = 30) turning red brown in KOH and olivaceous to umber brown in LA; and a 16–49-μm-thick outer layer of subhyaline to light olive green, thick-walled, elongate to isodiametric cells (5.5–)6.5–10.5(–12.5) × (3–)3.5–5.5(–7) μm (n = 30); surface sometimes covered by a thin outer layer of collapsed cells and amorphous material. Asci oblong, narrowly clavate or fusoid, lacking a differentiated apical apparatus, upper part with eight uniseriate ascospores (45–)53–66(–72) × (8–)9–10.5(–11) μm (n = 27), lower part stipe-like, ca. 8–20 μm long. Ascospores (8.5–)9.5–11(–12.5) × (4.0–)4.5–5(–5.5) μm, l/w (1.6–)1.9–2.4(–2.9) (n = 130), ellipsoid, oblong or fusoid, hyaline, with a median or slightly eccentric septum, straight, symmetric or slightly curved, slightly constricted at the septum, with broadly rounded ends, distinctly verruculose in water and LA, smooth in 3% KOH, with one large guttule per cell. Asexual morph on natural substrate not seen.

Cultures and asexual morph: colonies slow-growing, reaching 29 mm diam in 10 days on CMD; on MEA compact, flat, with white surface and yellowish reverse, after 1 month irregularly lobate, greyish brown in the centre, ochraceous with whitish patches at the margin; on CMD cottony, white, with abundant surface mycelium of hyphae commonly aggregated to hyphal strands, reverse yellowish. Conidiophores consisting of intercalary phialides with short lateral conidiiferous pegs (0.7–)0.8–3.0(–4.3) × (0.9–)1.1–1.6(–1.8) μm (n = 22), and terminally and laterally formed phialides. Phialides abundant on aerial mycelium, lageniform to cylindrical, (3–)7–15.5(–22) × (1.2–)1.6–2.3(–2.5) μm (n = 40). Conidia (3.5–)4.5–5.5(–6.5) × (1.3–)1.5–2.0(–2.5) μm, l/w (2.0–)2.6–3.3(–3.7) (n = 100), unicellular, allantoid, hyaline, smooth, commonly with a guttule at or towards one or both ends; after few days swelling to irregular shapes and up to ca. 9 × 3.5 μm. Blastoconidia formed on CMD in masses in the colony centre a few days after inoculation, hyaline, first ellipsoid to subglobose, globose and thick-walled with age, (2.5–)3–4.5(–5.5) μm diam (n = 120).

Distribution: Only known from a single collection in Portugal

Host: On dead corticated branches of Quercus ilex; probably saprobic

Holotype: Portugal, Parque Natural de Sintra-Cascais, S Monserrate, on Quercus ilex, 18 Feb. 2017, H. Voglmayr (WU 39972); ex-holotype culture NQI = CBS 144627; ex-holotype sequences MH562717 (ITS-LSU rDNA), MH562715 (rpb1), MH577042 (rpb2), MH562714 (tef1)

Discussion

In the phylogenetic analyses (Figs. 1 and 2), the fungus described here was unexpectedly placed in Bionectriaceae. Dark stromata and/or ascomata are not typically seen in Hypocreales, although they are formed by numerous nectriaceous species such as Nectria eustromatica (Jaklitsch and Voglmayr 2011b) or Thyronectria obscura (Jaklitsch and Voglmayr 2014). The species also shows a KOH-positive reaction of the ascomatal wall which is commonly seen in Nectriaceae (Rossman et al. 1999), but phylogenetic analyses of LSU sequences clearly placed the new fungus within Bionectriaceae, in a clade containing three accessions identified as Stilbocrea macrostoma (Fig. 1). Based on morphological distinctness, we consider the specimen from Portugal to represent a new species, described here as S. walteri. It differs substantially from S. macrostoma, and all putative synonyms listed in Seifert (1985) and Rossman et al. (1999), in its dark olive green to black perithecia, KOH and LA-positive reactions, compact stromata and a lack of a stilbella-like asexual morph. Stilbocrea walteri also contains much fewer perithecia which are apically free and only basally immersed in the stroma, whereas S. macrostoma contains numerous, up to several hundred ascomata almost entirely immersed in the stroma, resulting in a hypocrea-like appearance (Seifert 1985). Also, the stroma texture differs between the two species (a textura angularis-globulosa of thick-walled cells in S. walteri; a hyphal textura intricata with a surface layer of irregularly branched hyphae (cf. Figs. 2q and 4f–i; Seifert 1985, Rossman et al. 1999) in S. macrostoma). In addition, S. macrostoma is primarily a tropical to subtropical species, which to our knowledge has not been recorded from Europe. Notably, there are also a few characters of Stilbocrea walteri shared with S. macrostoma, like ascospores of similar size with a verruculose ornamentation disappearing in KOH (see Figs. 4e–v and 5j–q). Due to these marked discrepancies which could cast doubts on the reliability of the DNA sequences, DNA extraction was repeated directly from stromata, which revealed identical ITS-LSU sequences from stromata and culture, confirming that the sequences originate from the target fungus.
Fig. 5

Stilbocrea macrostoma (ad, f, hj, n, o WU 32032; e, g, km, p, q WU 26101). ad Stromata (bd showing stilbella-like asexual morph). e Peridium in vertical section. fh Stroma tissue in vertical section (f below perithecia; g stroma basis; h stroma surface). i Irregularly branched hyphae from stroma surface. j–p Ascospores (j–m in water; note verruculose and smooth ascospore walls in water and KOH, respectively). q ascus with ascospores (in water). (e–q in 3% KOH except where noted). Scale barsa 1 mm; b 500 μm; c, d 200 μm; e 20 μm; fh, q 10 μm; ip 5 μm

Our analyses (Figs. 1 and 2) may suggest that morphology of the sexual morph is not a good character for classification within Bionectriaceae and Nectriaceae. Asexual morphs, however, are not superior in this regard, as e.g. synnematous, stilbella-like asexual morphs also occur in the Nectriaceae, e.g. in Nectria pseudotrichia (Hirooka et al. 2012), and acremonium-like forms also in several other unrelated families of the Sordariomycetes (see, e.g. Summerbell et al. 2011). Also, the simple phialidic asexual morph of S. walteri observed in pure culture does not provide much phylogenetic information, as similar asexual morphs occur in various hypocrealean lineages.

Except for the commonly sequenced LSU, very few additional sequence data are available for most genera of Bionectriaceae. Apart from the well-studied genera Geosmithia and Clonostachys, even the ITS rDNA is lacking for many taxa. From the four species currently accepted in Stilbocrea (Rossman et al. 1999, de Beer et al. 2013), sequence data are available only for the generic type, Stilbocrea macrostoma. However, all three LSU sequences labelled as S. macrostoma differ substantially, and the two accessions from Sri Lanka and New Zealand form a highly supported subclade with the morphologically deviating S. walteri (Fig. 1), which is also seen in the analyses of the protein-coding genes (Fig. 2). Remarkably, this clade also contains two LSU sequences of endophyte isolates from the tropical marine seagrasses Enhalus acoroides (Sakayaroj et al. 2010) and Thalassia hemprichii (Supaphon et al. 2017), but unfortunately, no morphological data are available for them. In the LSU tree, the newly sequenced Panamese accession of S. macrostoma occupies a basal position in the poorly supported Stilbocrea clade (Fig. 1), but it is placed outside the Stilbocrea clade in the rpb1 and rpb2 trees (Fig. 2a, b), indicating that these accessions represent distinct species which may even not be congeneric. These results, together with the poor backbone support in the phylogenetic analyses (Figs. 1 and 2), suggest that a single gene alone is insufficient to provide a sound basis for defining phylogenetic generic concepts within the Bionectriaceae. A wide pantropical to warm-temperate distribution of S. macrostoma has been derived in the premolecular era (Seifert 1985), but if all sequences are correct in terms of generation from morphologically identical fungal material, then S. macrostoma will most probably be split into several species in future. Several taxa described from the Old and New World and synonymised with S. macrostoma based on morphology (Seifert 1985, Rossman et al. 1999) will then need to be re-considered and re-examined in detail. Remarkably, in their description of S. macrostoma, Seifert (1985) and Rossman et al. (1999) mentioned ascomata occasionally becoming red-brown to dark olive green with age; however, we have not seen any dark green colour in our material investigated. Much more sampling and generation of molecular data including protein-coding phylogenetic markers of Bionectriaceae are necessary to reveal a clearer picture of phylogenetic relationships within the family and to achieve a robust species classification and delimitation.

Notes

Acknowledgments

We thank Eduardo Esquivel for providing fresh material of Stilbocrea macrostoma from Panama.

Funding Information

Open access funding provided by Austrian Science Fund (FWF).

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Authors and Affiliations

  1. 1.Division of Systematic and Evolutionary Botany, Department of Botany and Biodiversity ResearchUniversity of ViennaWienAustria
  2. 2.Institute of Forest Entomology, Forest Pathology and Forest Protection, Department of Forest and Soil SciencesBOKU-University of Natural Resources and Life SciencesWienAustria

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