Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
Paraeutypella guizhouensis gen. et sp. nov. and Diatrypella longiasca sp. nov. (Diatrypaceae) from China
expand article infoLakmali S. Dissanayake, Nalin N. Wijayawardene§,|, Monika C. Dayarathne, Milan C. Samarakoon#, Dong-Qin Dai§, Kevin D. Hyde#, Ji-Chuan Kang
‡ Engineering Research Centre of the Utilization for Characteristic Bio-Pharmaceutical Resources in Southwest, Ministry of Education, Guizhou University, Guiyang, Guizhou Province 550025, China
§ Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China
| State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China
¶ Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, Guizhou Province, 550025, China
# Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand
Open Access

Abstract

Background

In this study, we introduce a novel genus, Paraeutypella, of the family Diatrypaceae comprising three species viz. Paraeutypella guizhouensis sp. nov. and P. citricola (basionym: Eutypella citricola) and P. vitis (basionym: Sphaeria vitis). Diatrypella longiasca sp. nov. is also introduced, which forms a distinct clade in Diatrypella sensu stricto. The discovery of this new genus will contribute to expanding the knowledge and taxonomic framework of Diatrypaceae (Xylariales).

New information

Generic delimitations in Diatrypaceae are unsettled because the phylogeny has yet to be resolved using extensive taxon sampling and sequencing of ex-type cultures. During an investigation of xylarialean fungi, we collected eutypella-like fungi which is distinct from Eutypella sensu stricto in our phylogenetic analyses (ITS and β-tubulin), thus, introduced as Paraeutypella guizhouensis gen. et sp. nov.. Paraeutypella is characterised by having 4–25 perithecia in a stroma each with 3–6 sulcate, long ostiolar necks. Paraeutypella citricola comb. nov. (basionym: Eutypella citricola) is introduced on Acer sp. from China. Diatrypella longiasca sp. nov. is introduced as a new species in Diatrypella sensu stricto. which has 2–5 ascomata per stroma and long ascospores, unusual when compared to other Diatrypella species and distinct phylogenetically.

Keywords

Acer, morphology, novel taxa, phylogeny, Xylariales

Introduction

Diatrypaceae Nitschke (Ascomycota, Xylariales) comprises 21 genera and more than 1,500 species (Senwanna 2017, Mehrabi et al. 2019, Dayarathne et al. 2020b, Wijayawardene et al. 2020). Species of this family are characterised by erumpent to immersed, rarely superficial, black or dark brown, eustromatic or pseudostromatic stromata and 8-spored or polysporous asci with hyaline to light brown, allantoid ascospores (Konta et al. 2020) in their sexual morph. Several asexual morph genera have been linked to the family Diatrypaceae, including Cytosporina Sacc. and Libertella Desm. (Glawe and Rogers 1984). Cytosporina Sacc. includes species with pycnidial and filiform conidia; Libertella Desm. includes species with acervula and filiform conidia (Glawe and Rogers 1984).

Members of Diatrypaceae are saprobes, pathogens or endophytes, associated with a wide range of hosts in terrestrial and aquatic environments (Mehrabi et al. 2019, Dayarathne et al. 2020a, Dayarathne et al. 2020b Konta et al. 2020). Dayarathne et al. (2020a), Dayarathne et al. (2020b) introduced two novel genera, Halocryptosphaeria Dayar. et al. and Halocryptovalsa Dayar. & K.D. Hyde from marine environments. Species of Anthostoma Nitschke, Cryptosphaeria Ces. & De Not., Cryptovalsa Ces. & De Not. ex Fuckel, Diatrype Fr., Diatrypella (Ces. & De Not.) De Not. and Eutypella (Nitschke) Sacc. have been reported as causal agents of canker diseases on a wide range of host plants worldwide (Hyde et al. 2020). The taxonomy and phylogeny of Diatrypaceae need to be resolved, as many genera are polyphyletic. Hence, fresh collections and sequences are required to define genera and establish their phylogenetic placement within the family.

Diatrypella was introduced by Cesati and De Notaris (1863) with D. verruciformis (Ehrh.) Nitschke as the type. The genus is characterised by conical to truncate, cushion-like or discoid stromata usually delimited by a black zone in host tissues, umbilicate or sulcate ostiolar necks, cylindrical, polysporous, long-stalked asci and allantoid, hyaline or yellowish ascospores in their sexual morph and a libertella-like coelomycetes asexual morph (Kirk et al. 2008, Hyde et al. 2020). Both Cryptovalsa and Diatrypella have polysporous asci and cannot easily be distinguished, based only on morphological comparisons (Acero et al. 2004, Vasilyeva and Stephenson 2005). Therefore, molecular data are essential for defining genera in Diatrypaceae (Mehrabi et al. 2015). There are 65 names of Diatrypella in Species Fungorum (2020) (http://www.indexfungorum.org/names/names.asp), but only 15 have molecular data in GenBank (Hyde et al. 2020).

In this study, we introduce a new genus, Paraeutypella, which shows eutypella-like morphology, but is distinct phylogenetically. Paraeutypella comprises three species viz. Paraeutypella guizhouensis sp. nov. and P. citricola (basionym: Eutypella citricola) and P. vitis (basionym: Sphaeria vitis). Diatrypella longiasca sp. nov. is also introduced, which forms a distinct clade in Diatrypella sensu stricto. Species novelties are confirmed by morphological comparisons along with micro-photographs and the phylogeny of combined ITS and β-tubulin sequence data.

Materials and methods

Sample collection and morphological observations

Dead twigs of Acer palmatum and undetermined plants were collected from China (Guiyang, Guizhou Province) during September to October 2019. Samples were observed with a stereomicroscope (SZX16, Olympus). Hand sections of the ascomata were mounted in distilled water and the following characters were measured: diameter and height of ascomata, width of the peridium, diameter and height of ostiolar necks, length and width of asci and ascospores. Melzer’s Reagent was used for testing the ascal apical ring reaction. Images were captured with a Canon EOS70D digital camera fitted to a compound microscope. Measurements were made with the Tarosoft (R) Image Frame Work programme and images used for figures processed with Adobe Photoshop CS6 software (Adobe Systems, USA). Single spore isolation was performed according to Chomnunti et al. (2014) and germinating spores were transferred to potato dextrose agar (PDA- Shanghai Bio-way Technology Co. Ltd.). The pure cultures were incubated at 18–20ºC for four weeks. The type specimens were deposited in the Cryptogamic Herbarium, Kunming Institute of Botany, Academia Sinica (HKAS), Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium (HMAS), Beijing, China. Ex-type cultures were deposited in the Kunming Institute of Botany Culture Collection (KUMCC). Facesoffungi and Index Fungorum numbers are provided as mentioned in Jayasiri et al. (2015) and Index Fungorum (http://www.indexfungorum.org) respectively.

DNA extraction, PCR amplifications and sequencing

Fungal isolates were grown on PDA for 3–4 weeks at 25°C and total genomic DNA was extracted from 50 to 100 mg of axenic mycelium scraped from the edges of the growing cultures (Wu et al. 2001). EZgneTM fungal gDNA extraction kit (BIOMIGA, Hangzhou City, Zhejiang Province, China) was used to extract DNA by following the manufacturer’s protocol. DNA extracts were stored at – 4°C for use in regular work and duplicated at –20°C for long term storage.

DNA sequence data were obtained from the internal transcribed spacer (ITS) and partial β-tubulin gene. ITS and β-tubulin were amplified by using the primers ITS5/ITS4 (White et al. 1990) and T1/T22 (O'Donnell and Cigelnik 1997), respectively. Polymerase chain reaction (PCR) was carried out in a volume of 25 μl, which contained 9.5 μl of ddH2O, 12.5 μl of 2× PCR Master Mix (2× Bench TopTM Taq Master Mix, BIOMIGA, China), 1 μl of DNA template and 1 μl of forward and reverse primers (10 μM each) in each reaction. The PCR thermal cycle programme for all gene amplifications was as follows: initialisation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 50s, elongation at 72°C for 90s and final extension at 72°C for 10 min. Purification and sequencing of PCR products were done by Sangon Biotech, Shanghai, China.

Molecular phylogenetic analyses

Sequence alignment

The sequence data generated in this study were analysed with closely-related taxa retrieved from GenBank (Table 1), based on BLASTn searches (https://www.ncbi.nlm.nih.gov) and recently published data (Mehrabi et al. 2019, Dayarathne et al. 2020b, Konta et al. 2020). ITS and β-tubulin were used for the analyses according to the previous studies listed above. Sequences (ITS and β-tubulin) were aligned using MAFFT v. 6.864b (Katoh et al. 2019) and manually improved when necessary in BioEdit v. 7.0 (Hall 1999). The single gene alignments were used to perform model test in MrModeltest 2.3 to estimate the best-fit evolutionary model under the Akaike Information Criterion (AIC) (Nylander 2004) and resulted in a GTR+I+G substitution model for each. Ambiguously aligned areas of each gene region were excluded and gaps were treated as missing data. Missing characters were assessed to be unordered and equally weighted.

Table 1.

Taxa used in the phylogenetic analysis and their corresponding GenBank accession numbers.

Species

Strain no.

GenBank Accession no.

Reference

ITS

β-tubulin

Allocryptovalsa cryptovalsoidea T

HVFIG02

HQ692573

HQ692524

Trouillas et al. (2011)

A. elaeidis

MFLUCC 15-0707

MN308410

MN340296

Konta et al. (2020)

Allodiatrype arengae T

MFLUCC 15-0713

MN308411

MN340297

Konta et al. (2020)

A. elaeidicola T

MFLUCC 15-0737

MN308415

MN340299

Konta et al. (2020)

A. elaeidis

MFLUCC 15-0708b

MN308413

NA

Konta et al. (2020)

Anthostoma decipiens

IPV-FW349

AM399021

AM920693

Nitschke (1867)

A. decipiens

JL567

JN975370

JN975407

Luque et al. (2012)

Cryptosphaeria eunomia

CBS 216.87

AJ302417

NA

Acero et al. (2004)

C. var. eunomia

CBS 223.87

AJ302421

NA

Acero et al. (2004)

Cryptovalsa ampelina

A001

GQ293901

GQ293972

Trouillas et al. (2015)

C. ampelina

KHJ20

KJ767718

KY352426

Mehrabi et al. (2015)

Diatrypasimilis australiensis T

ATCC MYA 3540

FJ430590

NA

Chalkley et al. (2010)

Diatrype bullata

UCDDCh400

DQ006946

DQ007002

Rolshausen et al. (2006)

D. disciformis T

MFLUCC 15-0538

KR092795

NA

Senanayake et al. (2015)

D. lijiangensis T

MFLU 19-0717

MK852582

MK852583

Thiyagaraja et al. (2019)

D. stigma

DCASH200

GQ293947

GQ294003

Trouillas et al. (2015)

Diatrypella atlantica T

HUEFS 194228

KM396615

KR363998

de Almeida et al. (2016)

D. atlantica

HUEFS 192148

KM396633

KT175563

de Almeida et al. (2016)

D. delonicis T

MFLUCC 15-1014

MH812994

MH847790

Hyde et al. (2019)

D. delonicis

MFLU 16-1032

MH812995

MH847791

Hyde et al. (2019)

D. elaeidis T

MFLUCC 15-0279

MN308417

MN340300

Konta et al. (2020)

D. favacea

ANM 96

KU320616

NA

de Almeida et al. (2016)

D. frostii

UFMGCB 1917

HQ377280

NA

Vieira et al. (2012)

D. heveae T

MFLUCC 17-0368

MF959501

MG334557

Senwanna (2017)

D. heveae

MFLUCC 15-0274

MN308418

MN340301

Konta et al. (2020)

D. iranensis T

IRAN 2280C

KM245033

KY352429

Mehrabi et al. (2015)

D. longiasca T

KUMCC 20-0021

MW039349

MW239658

This study

D. longiasca

KUMCC 20-0022

MW036141

MW239659

This study

D. macrospora T

IRAN 2344C

KR605648

KY352430

Mehrabi et al. (2016)

D. major

ANM 1947

KU320613

NA

de Almeida et al. (2016)

D. pulvinata

H048

FR715523

FR715495

Pazoutova et al. (2012)

D. verruciformis

UCROK1467

JX144793

JX174093

Lynch et al. (2013), Luque et al. (2012)

D. verruciformis

UCROK754

JX144783

JX174083

Lynch et al. (2013)

D. vulgaris

HVFRA02

HQ692591

HQ692503

Trouillas et al. (2015)

D. vulgaris T

HVGRF03

HQ692590

HQ692502

Trouillas et al. (2015)

Eutypa laevata

CBS 291.87

AJ302449

NA

Acero et al. (2004)

E. lata

ATCC 28120

DQ006948

DQ006975

Rolshausen et al. (2006)

E. lata

EP18

HQ692611

HQ692501

Trouillas et al. (2011)

E. lata

RGA01

HQ692614

HQ692497

Trouillas et al. (2011)

E. lata var. aceri

CBS 290.87

HM164736

HM164770

Trouillas et al. (2010)

Eutypella caricae

EL51C

AJ302460

NA

Acero et al. (2004)

E. cerviculata

EL59C

AJ302468

NA

Acero et al. (2004)

E. cerviculata

M68

JF340269

NA

Arhipova et al. (2012)

E. leprosa

EL54C

AJ302463

NA

Acero et al. (2004)

E. leprosa

ANM 85

KU320622

NA

de Almeida et al. (2016)

E. microtheca

ADEL200

HQ692559

HQ692527

Trouillas et al. (2011)

E. microtheca

BCMX01

KC405563

KC405560

Paolinelli-Alfonso et al. (2015)

E. parasitica

CBS 210.39

MH855984

NA

Vu et al. (2019)

E. persica T

IRAN 2540C

KX828144

KY352451

Mehrabi et al. (2019)

E. quercina T

IRAN 2543C

KX828139

KY352449

Mehrabi et al. (2019)

E. semicircularis T

MP4669

JQ517314

NA

Chacón et al. (2013)

E. tamaricis

MFLUCC 14-0445

NA

KX453302

Thambugala et al. (2016)

E. virescens

CBS 205.36

MH855778

MH867286

Vu et al. (2019)

Halocryptovalsa salicorniae

MFLUCC 15-0185

MH304410

MH370274

Dayarathne et al. (2020b)

Halodiatrype avicenniae

MFLUCC 15-0948

MH304414

MH370278

Dayarathne et al. (2020b)

H. salinicola T

MFLUCC 15-1277

KX573915

KX573932

Dayarathne et al. (2016)

H. salinicola

MFLUCC17-2468

MN047113

NA

Dayarathne et al. (2016)

Kretzschmaria deusta T

CBS 826.72

KU683767

KU684190

U’Ren et al. 2016

Monosporascus cannonballus T

ATCC 26931

FJ430598

NA

Unpublished

M. cannonballus

CMM3646

JX971617

NA

Sales et al. (2010)

Neoeutypella baoshanensis

MFLUCC 16-1002

MT310662

NA

Phukhamsakda et al. (2020)

N. baoshanensis T

LC 12111

MH822887

MH822888

Hyde et al. (2019)

Paraeutypella citricolca

HVGRF01

HQ692579

HQ692512

Trouillas et al. (2011)

P. citricola

HVVIT07

HQ692589

HQ692521

Trouillas et al. (2011)

P. citricola

IRAN 2340C

KR605647

KY352439

Mehrabi et al. (2016)

P. citricola

KUMCC 20-0023

MW040050

MW239663

This study

P. citricola

KUMCC 20-0024

MW040049

MW239662

This study

P. guizhouensis T

KUMCC 20-0016

MW036142

MW239660

This study

P. guizhouensis

KUMCC 20-0017

MW039348

MW239661

This study

P. vitis

UCD2291AR

HQ288224

HQ288303

Úrbez-torres et al. (2012)

P. vitis

UCD2428TX

FJ790851

GU294726

Úrbez-Torres and Gubler (2009)

Pedumispora rhizophorae

BCC44877

KJ888853

NA

Klaysuban et al. (2014)

P. rhizophorae

BCC44878

KJ888854

NA

Klaysuban et al. (2014)

Peroneutypa curvispora

HUEFS 136877

KM396641

NA

de Almeida et al. (2016)

P. rubiformis T

MFLUCC 17-2142

MG873477

NA

Shang et al. (2018)

P. scoparia T

MFLUCC 11-0478

KU940151

NA

Dai et al. (2016)

Quaternaria quaternata

CBS 278.87

AJ302469

NA

Acero et al. (2004)

Q. quaternata

GNF13

KR605645

KY352464

Mehrabi et al. (2016)

Xylaria hypoxylon T CBS-122620 KY610407 KX271279 Peršoh et al. (2009)

T: Types strains, newly-generated sequences are indicated in bold, NA: No sequence available in GenBank, ATCC: American Type Culture Collection, Manassas, USA, BCC: BIOTEC Culture Collection, Bangkok, Thailand, CBS: Centra albureau voor Schimmel cultures, Utrecht, The Netherlands, MFLU: Mae Fah Luang University, Chiang Rai, Thailand, CCMB: Bahia Culture Collection of Microorganisms, CMM: Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes,” Federal Rural University of Pernambuco, Brazil, MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand, HKAS: The Herbarium Mycologium, Institute of Microbiology Chinese Academy of Sciences, Beijing, China, HUEFS: Herbarium of the State University of Feira de Santana, IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran, KUMCC: Kunming Institute of Botany Culture Collection, Kunming, China.

Phylogenetic Analyses

Maximum Likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using the GTR+I+G model of evolution. Bootstrap supports were obtained by running 1,000 pseudo-replicates. Bayesian analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate Bayesian posterior probabilities (BYPP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). GTR+I+G was used as the substitution model. Six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 200th generation. The distribution of log-likelihood scores was examined to determine the stationary phase for each search and to decide if extra runs were required to achieve convergence, using the programme Tracer 1.5. The first 10% of generated trees were discarded and remaining 90% of trees were used to calculate posterior probabilities of the majority rule consensus tree. All trees were visualised in FigTree v.1.4.0 (Rambaut 2012) and the final layout (Fig. 1) was done with Microsoft PowerPoint (2013). The final alignment and tree were registered in TreeBASE under the submission ID: 27435 (http://purl.org/phylo/treebase/phylows/study/TB2:S27435?x-access-code=3101b93c442e7aa253174d89df7a500c&format=html).

Figure 1.

Phylogram generated from Maximum Likelihood (RAxML) analysis, based on ITS- β-tubulin matrix. ML bootstrap supports (≥ 70%) and Bayesian posterior probability (≥ 0.95) are indicated as ML/BYPP. The tree is rooted to Kretzschmaria deusta (CBS 826.72) and Xylaria hypoxylon (CBS 122620). Newly-generated strains are in red and type strains are in bold. The asterisks represent unstable species.

Taxon treatments

Diatrypella  (Ces. & De Not.) De Not. 1863

Type species

Diatrypella verruciformis  (Ehrh.) Nitschke(Fr.)

Description

Notes – Diatrypella was introduced by Cesati & De Notaris (1863) and is typified as Diatrypella verruciformis (Ehrh.) Nitschke. There are 146 epithets listed in Index Fungorum (2020). This genus was established to accommodate members of stromatic Sphaeriales which were characterised by ovoid and numerous ascospores and we introduce a new species viz. Diatrypella longiasca, based on new collections from China.

Diatrypella longiasca L.S. Dissan., J.C. Kang & K.D. Hyde, sp. nov.

Materials    Download as CSV 
Holotype:
  1. kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Sordariomycetes
    ; order:
    Xylariales
    ; family:
    Diatrypaceae
    ; taxonRank:
    species
    ; genus:
    Diatrypella
    ; specificEpithet:
    longiasca
    ; scientificNameAuthorship:
    L.S. Dissan., J.C. Kang & K.D. Hyde, sp. nov.
    ; country:
    China
    ; stateProvince:
    Guizhou Province
    ; county:
    Guiyang
    ; locality:
    Guizhou University Garden (South)
    ; identifiedBy:
    L.S. Dissanayake
    ; institutionID:
    HMAS 290656
    ; collectionID:
    HMAS 290658
    ; institutionCode:
    Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium
    ; collectionCode:
    Kunming Institute of Botany Culture Collection
    ; datasetName:
    CLD 42
Other material:
  1. type:
    isotype
    ; institutionID:
    HMAS 290658
    ; collectionID:
    KUMCC 20-0022
    ; institutionCode:
    Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium
    ; collectionCode:
    Kunming Institute of Botany Culture Collection

Description

Saprobic on dead twigs (Fig. 2). Sexual morph: Stromata 0.5–0.7 mm in diam., well-developed, solitary to gregarious, erumpent, black, immersed, globose to subglobose. Ascomata 525–540 μm high, 470–510 μm diam. (x̄ = 532 × 490 μm, n = 15), perithecial, surrounded by white entostroma, immersed in stromata, 2–5 perithecia arranged in a valsoid configuration, subglobose, individual ostiole with a long neck. Neck 180–190 μm long (x̄ = 185 μm, n = 15), cylindrical, with periphyses. Peridium 36–45 μm wide (x̄ = 40.5 μm, n = 20), composed of two layers: outer layer of black, thick-walled cells; inner layer; hyaline, thick-walled cells forming textura angularis. Hamathecium 259–287 μm wide (x̄ = 273 μm, n = 20), composed of cells 3–5 μm wide (x̄ = 4 μm, n = 20), paraphyses arising from base of perithecia, hyaline, long, narrow, unbranched, septate, guttulate, narrowing and tapering towards apex. Asci 105–155 × 10–16 μm (x̄ = 130 × 14 μm, n = 30), polysporous, unitunicate, clavate, apically pointed, with a J-apical ring, long pedicellate (40–50 μm). Ascospores 4–8 × 1–2 μm (x̄ = 6 × 1.5 μm, n = 30), overlapping, hyaline, yellowish in mass, allantoid, aseptate, guttulate, smooth-walled. Asexual morph: Undetermined.

Figure 2.  

Diatrypella longiasca (HMAS 290656, holotype) a. stromata on substrate; b. cross section of a stroma; c, d. vertical section through stroma showing ostiole and perithecia; e. ostiolar canal; f. paraphyses; g–i. asci; j–l. ascospores; m, n. culture on PDA from m above, n below after 6 weeks. Scale bars: 500 µm (a, b), 100 µm (d), 50 µm (e, g–i), 5 µm (f, j–l).

Culture characteristics – Colonies on PDA reaching 21 mm diam. after 2 weeks at 20–25oC, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish white at margin, yellow to brown at centre; mycelium yellowish.

Etymology

The specific epithet longiasca refers to the long asci.

Notes

Diatrypella longiasca shares similar characters with D. vulgaris in having erumpent stromata through the bark often surrounded by remaining adherent epidermis or woody fragments and asci with many ascospores. However, D. vulgaris is different from D. longiasca in having longer ascospores (8–10 × 2–2.5 μm vs. 4–8 × 1–2 μm) (Trouillas et al. 2011). Diatrypella vulgaris has 4–8 ascomata per stromata, while D. longiasca comprises 2–5 ascomata per stromata. Comparison of the ITS 12% (73/570) and β-tubulin 13% (56/432) nucleotide differences, phylogenetic analyses and significant morphological differences indicate that D. longiasca and D. vulgaris are distinct taxa. Thus, D. longiasca is introduced as a new species in Diatrypella, based on its morphology coupled with high support values from the phylogenetic analysis (96% ML, 0.99 BYPP, Fig. 1). A key to species related to Diatrypella longiasca is given below.

Paraeutypella  L.S. Dissan., J.C. Kang, Wijayaw. & K.D. Hyde, gen. nov.

Type species

Paraeutypella guizhouensis L.S. Dissan., J.C. Kang & K.D. Hyde, sp. nov.

Description

Saprobic on dead twigs. Sexual morph: Stromata immersed in bark of dead branches, erumpent, solitary or aggregated. Ascomata with groups of 4–25 perithecia arranged in a valsoid configuration, surrounded by white, powdery entostroma, perithecial, black or brown, subglobose, clustered, immersed in stromata. Necks papillate, with an elongated ostiolar neck, central ostiolar canal filled with periphyses, 3–6 sulcate. Peridium composed of two layers of cells of textura angularis; inner layers cells hyaline or light brown, outer layers cells dark brown to black. Hamathecium composed of paraphyses arising from the base of perithecia, elongate, filiform, narrow, unbranched, septate, guttulate, narrowing and tapering towards apex. Asci 8-spored, unitunicate, thin-walled, clavate to cylindrical clavate or spindle-shaped, long pedicellate, apical rings J-. Ascospores overlapping biseriate, allantoid, slightly to moderately curved, allantoid, several oil droplets in each end, hyaline to light brown, sometimes yellow in mass, aseptate. Asexual morph: Coelomycetous. Conidiomata black, subconic, multiloculate, largely prosenchymatous, producing yellowish conidial masses. Conidiophores not recorded. Conidiogenous cells cylindrical, tapering, arising from pseudoparenchyma or interwoven hyphae, proliferating percurrently or sympodially, rarely both ways. Conidia hyaline, single-celled, slightly to moderately curved, with flattened bases, becoming guttulate (description of asexual morph adapted from Glawe and Jacobs 1987).

Etymology

With reference to the morphological resemblance to Eutypella.

Notes

Paraeutypella is introduced to accommodate three species viz. P. guizhouensis sp. nov., as well as P. citricola and P. vitis, two species previously placed in Eutypella sensu lato. Paraeutypella is typified by P. guizhouensis, which was collected from undetermined dead twigs. Paraeutypella can be distinguished from Eutypella species by stromata with perithecia in groups of 4–25 arranged in a valsoid configuration, 3–6 sulcate, long ostiolar necks, while stromata of Eutypella comprise groups of 20–70 perithecia having comparatively shorter ostiolar necks with sulcate or smooth ostiolar necks. Strains of both genera appear in distinct clades in a phylogeny based on ITS and Beta tubulin data (Fig. 1), thereby justifying the erection of the new genus Paraeutypella. However, sequence data are not available for the type of P. citricola and P. vitis. A co-elomycetous asexual morph has been recorded for P. vitis as Eutypella vitis in culture (Glawe and Jacobs 1987).

Paraeutypella guizhouensis L.S. Dissan., J.C. Kang & K.D. Hyde, sp. nov.

Materials    Download as CSV 
Holotype:
  1. kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Sordariomycetes
    ; order:
    Xylariales
    ; family:
    Diatrypaceae
    ; genus:
    Paraeutypella
    ; specificEpithet:
    guizhouensis
    ; country:
    China
    ; stateProvince:
    Guizhou Province
    ; county:
    Guiyang
    ; locality:
    Guizhou University Garden (North)
    ; habitat:
    Saprobic on dead twigs.
    ; fieldNumber:
    CLD018
    ; identifiedBy:
    L.S.Dissanayake
    ; type:
    Holotype
    ; institutionID:
    HMAS 290654
    ; collectionID:
    KUMCC 20–0016
    ; institutionCode:
    Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium
    ; collectionCode:
    Kunming Institute of Botany Culture Collection
    ; datasetName:
    CLD018
Other material:
  1. type:
    isotype
    ; institutionID:
    HKAS 290655
    ; collectionID:
    KUMCC 20-0017
    ; institutionCode:
    Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium
    ; collectionCode:
    Kunming Institute of Botany Culture Collection

Description

Saprobic on dead twigs (Fig. 3). Sexual morph: Stromata immersed in bark of dead branches, erumpent, aggregated, circular to irregular, superficial, carbonaceous. Ascomata 590–600 × 470–480 μm (x̅ = 595 × 475 µm, n = 10), perithecial, with groups of 6–12 perithecia arranged in a valsoid configuration, subglobose, clustered, immersed in stromata, ostiolate. Neck 400–418 μm long (x̅ = 409 µm, n = 10), papillate, central ostiolar canal filled with periphyses, 3–4 sulcate. Peridium 22–35 μm wide, composed of two layers of textura angularis; inner layer cells light brown to hyaline, outer layers cells dark brown to black. Hamathecium hyaline. Paraphyses 1–2 μm wide (x̅ = 1.5 µm, n = 10), arising from base of perithecia, long, narrow, unbranched, septate, guttulate, narrowing and tapering towards apex. Asci 55–80 × 5–9 μm (x̅ = 67.5 × 7 μm, n = 20), 8-spored, unitunicate, thin-walled, clavate to cylindrical clavate, long pedicellate (25–30 μm), with a J- apical ring. Ascospores 7–11 × 1–3 μm (x̅ = 9 × 2 μm, n = 30), overlapping biseriate, allantoid, hyaline to light brown, smooth, aseptate, usually with 2–3 guttules. Asexual morph: Undetermined.

Figure 3.  

Paraeutypella guizhouensis (HMAS 290654, holotype) a–c. stromata on substrate; d. cross section of a stromata; e. vertical section through an ascostroma showing ostioles and perithecia; f. ostiolar canal; g. peridium; h. paraphyses; i–l. asci; m–o. ascospores; p. germinating ascospore; q, r. cultures on PDA from above and below after 6 weeks. Scale bars: 500 µm (b–d), 200 µm (e), 100 µm (f), 20 µm (g–l), 5 µm (m–p).

Culture characteristics – Colonies on PDA, reaching 21 mm diam. after 2 weeks at 20–25oC, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish-white at margin, yellow to brown at centre; mycelium yellowish.

Etymology

The specific epithet guizhouensis refers to the locality in which the fungus was collected.

Notes

Paraeutypella guizhouensis resembles P. vitis, which comprises stromata that are erumpent through bark, with elongated perithecial necks and allantoid, slightly to moderately curved ascospores (Glawe and Jacobs 1987). However, P. guizhouensis differs from P. vitis in having comparatively longer ostiolar necks and longer asci (55–80 × 5–9 μm), while P. vitis has comparatively shorter ostiolar necks and shorter asci (40–46 × 6–8 μm) (Glawe and Jacobs 1987). Paraeutypella vitis (UCD2428TX) differs phylogenetically from our new taxon in 14% (80/576) base pairs in the ITS and 10% (42/405) base pairs in β-tubulin. Thus, P. guizhouensis is introduced as a new species in Paraeutypella, based on its morphology, base pair differences and phylogenetic analyses (94% ML, Fig. 1).

Paraeutypella citricola (Speg.,) L.S. Dissan., Wijayaw., J.C. Kang & K.D. Hyde, comb. nov. ≡ Eutypella citricola Speg., in Anales del Museo Nacional de Buenos Aires 6: 245, (1898)

Nomenclature

= Eutypella citricola Syd. & P. Syd., Hedwigia 49: 80 (1909), nom. illegit., Art. 53.1

Materials    Download as CSV 
Holotype:
  1. institutionID:
    LPS-2120
Paratype:
  1. kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Sordariomycetes
    ; order:
    Xylariales
    ; family:
    Diatrypaceae
    ; genus:
    Paraeutypella
    ; specificEpithet:
    citricola
    ; country:
    China
    ; county:
    Guiyang
    ; locationAccordingTo:
    Guizhou University Garden (South)
    ; year:
    2019
    ; month:
    October
    ; day:
    5
    ; habitat:
    on a dead branch of Acer sp.
    ; recordedBy:
    Nalin N. Wijayawardene
    ; identifiedBy:
    L.S.Dissanayake
    ; type:
    paratype
    ; institutionID:
    HMAS 290660, HMAS 290659
    ; collectionID:
    culture KUMCC 20–0024, KUMCC 20–0023
    ; institutionCode:
    Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium
    ; collectionCode:
    Kunming Institute of Botany Culture Collection

Description

Saprobic on dead twigs of Acer palmatum (Fig. 4). Sexual morph: Stromata immersed in bark of dead branches, erumpent, solitary or aggregated, circular to irregular in shape, superficial, carbonaceous. Ascomata 410–430 × 430–470 μm (x̅ = 420 × 450 µm, n = 10), perithecial, with groups of 4–6 perithecia arranged in a valsoid configuration, black, subglobose, clustered, immersed in ascostroma with ostiolar neck. Necks 360–390 μm long (x̅ = 375 µm, n= 10), papillate, sulcate, central ostiolar canal filled with paraphyses. Peridium 27–40 μm wide, composed of two layers of textura angularis; inner layer cells hyaline, outer layer cells dark brown to black. Hamathecium composed of 3–7 μm wide (x̅ = 5 µm, n= 10), hyaline, paraphyses arising from base of perithecia, composed of long, narrow, unbranched, septate, guttulate, narrowing and apically truncate. Asci 70–75 × 5–8 μm (x̅ = 72.5 × 6.5 μm, n = 20), 8-spored, unitunicate, thin-walled, clavate to cylindrical clavate, long pedicellate (40–50 μm), J- apical ring. Ascospores 7–10 × 2–3 μm (x̅ = 8.5 × 2.5 μm, n = 30), overlapping biseriate, allantoid, hyaline to light brown, smooth, aseptate, usually with guttules. Asexual morph: Undetermined.

Figure 4.  

Paraeutypella citricola (HMAS 290660) a–c. stromata on substrate; d. cross section of stroma; e. vertical section through stroma showing ostiolar necks and perithecia; f. ostiolar canal; g. peridium; h. paraphyses; i–k. asci; l–n. ascospores; o. germinating ascospore; p, q. culture on PDA after 6 weeks from above and below. Scale bars: 500 µm (b–d), 200 µm (e–g), 20 µm (gl), 5 µm (m–o).

Culture characteristics – Colonies on PDA, reaching 21 mm diam. after 2 weeks at 20–25oC, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish-white at margin, yellow to brown at centre; mycelium yellowish.

Notes

Eutypella citricola was described by Spegazzini (1898) from Citrus in Argentina and has since been reported to cause dieback on various woody plants in warm temperate and tropical regions (Farr and Rossman 2020). Eutypella citricola strains have previously been isolated from hosts such as Citrus limon, C. sinensis, C. paradisi, Salix spp., Schinus molle, Ulmus procera and Vitis vinifera (Trouillas et al. 2011, Mehrabi et al. 2016). In our study, we provide additional information for P. citricola from dead stems of Acer (Sapindaceae) in China. In morphology, our collection (HMAS 290660) resembles Eutypella, thus having pustulate stromata with stout, converging ostiolar necks and asci with eight spores. According to phylogenetic analysis, KUMCC 20–0024 closely groups with a collection of E. citricola (IRAN 2349C), which was collected on dead branches of Salix sp. (Salicaceae) in Gilan Province, Iran (Mehrabi et al. 2016) (Fig. 1). However, the IRAN 2349C strain is slightly different from our strain in having stromata with groups of 6–25 perithecia in a valsoid configuration and short ostiolar necks (100–300 µm), while our collection comprises stromata with groups of 4–6 perithecia in a valsoid configuration with a longer neck (356–385 μm). Based on phylogenetic analysis, both strains grouped in Paraeutypella sensu stricto (Fig. 1). Hence, the name Eutypella citricola is placed in Paraeutypella as P. citricola.

Paraeutypella guizhouensis, the type of Paraeutypella, morphologically resembles P. citricola both having immersed stromata, perithecia each with a long ostiolar neck and allantoid, aseptate ascospores with an oil droplet at each end. However, Paraeutypella citricola differs from P. guizhouensis by the number of perithecia within a stroma (4–6 vs. 6–12). A comparison of the ITS 1.0% (6/576) and β-tubulin 1.2% (5/406) between KUMCC 20-0024 and IRAN 2340C, ITS 1.0% (6/576) and β-tubulin 1.0% (5/406) between KUMCC 20-0024 and HVGRF01, HVVIT07 has been made.

Paraeutypella vitis (Schwein.,) L.S. Dissan., J.C. Kang & K.D. Hyde, comb. nov. ≡ Sphaeria vitis Schwein., in Schr. Naturf. Ges. Leipzing 1: 39 (1822)

Nomenclature

= Valsa vitis (Schwein.) Fuckel, Jb. Nassau. Ver. Naturk. 23-24: 199 (1870)

= Engizostoma vitis (Schwein.) Kuntze, Revis. Gen. pl. (Leipzig) 3 (3): 475 (1898)

= Eutypella vitis (Schwein.) Ellis & Everh., The North American Pyrenomycetes: 490 (1892)

Notes

Eutypella vitis was collected from young shoots of grape vines in New York and was introduced by Ellis and Everhart (1982). According to our phylogenetic analyses, our new strain which represents Eutypella vitis (UCD 2291AR, USE2428TX) grouped as the sister clade (bootstrap support: 78% ML) to Paraeutypella citricola within Paraeutypella sensu stricto. Hence, in this study, we introduce the new combination, Paraeutypella vitis. Paraeutypella vitis shares similar morphologies to Paraeutypella species, such as having erumpent stromata through bark, 3–4 sulcate, long ostiolar necks, clavate asci, allantoid, slightly to moderately curved ascospores with several oil droplets in each end.

Identification keys

Key to species similar to Diatrypella longiasca

1 Ascospores 4–5 μm long on average Diatrypella major
Ascospores 6–10 μm long on average 2
2 Entostroma yellowish or olive-green 3
Entostroma white 4
3 Asci larger, 120–150 × 15.5–21.5 μm D. tectonae
Asci smaller, 40 × 8–12 μm D. frostii
4 Stromata small, up to 2 mm diam. 5
Stromata larger than 2 mm 6
5 1–4 ascomata per stromata, on twigs of Hevea brasiliensis D. heveae
3–4 ascomata per stromata, on seed pods of Delonix regia D. delonicis
6 4–8 ascomata per stromata, (0.25–0.45 mm) with obscure ostiolar necks D. vulgaris
2–5 ascomata per stromata, (0.5–0.7 mm) with prominent ostiolar necks D. longiasca

Key to species of Paraeutypella

1 Stromata immersed Paraeutypella citricola
stromata erumpent 2
2 Short ostiolar neck and longer asci (55–80 × 5–9 μm) P. vitis
Long ostiolar neck and shorter asci (40–46 × 6–8 μm) P. guizhouensis

Analysis

Phylogenetic analyses

The combined ITS and β-tubulin matrix comprises 79 sequences that represents the genera in Diatrypaceae including the outgroup taxa. The best scoring RAxML tree is shown (Fig. 1) with a final ML optimisation likelihood value of -12709.069416. The matrix had 784 distinct alignment patterns, with 28.77% undetermined characters or gaps. Estimated base frequencies were: A = 0.226868, C = 0.263622, G = 0.232845, T = 0.27666; substitution rates AC = 1.218567, AG = 2.693651, AT = 1.272423, CG = 0.850048, CT = 3.427431, GT = 1.000000; proportion of invariable sites I = 0.100328; gamma distribution shape parameter α = 0.775027. All trees (ML and BYPP) were similar in topology and did not differ in generic relationships, which are in agreement with multi-gene phylogenies of previous studies.

Species of Eutypella are polyphyletic in our phylogram, while new isolates KUMCC 20-0023 and KUMCC 20-0024 grouped in a clade that comprises Eutypella citricola Syd. & P. Syd. and Eutypella vitis (Schwein.) Ellis & Everh. (Fig. 1). KUMCC 20-0016 and KUMCC 20-0017 formed a separate clade basal to E. vitis with high statistical support (94% ML) (Fig. 1). These species form a separate clade from the Eutypella clade. A novel genus is needed to accommodate these species, hence we introduce Paraeutypella.

Our new strains KUMCC 20-0021 and KUMCC 20-0022 are accommodated within Diatrypella with high statistical support (96% ML, 1.00 BYPP) as a distinct lineage.

Discussion

This study introduces a new genus, Paraeutypella and accepts 22 genera in Diatypaceae. According to the previous analyses of combined ITS and β-tubulin sequence data, the genus Eutypella has been often identified as polyphyletic in Diatrypaceae (Trouillas et al. 2011, Mehrabi et al. 2016, Mehrabi et al. 2019, Dayarathne et al. 2016, Dayarathne et al. 2020a, Dayarathne et al. 2020b) and determined in our study as well (Fig. 1). The type of Eutypella, E. cerviculata (Fr.) Sacc. grouped with E. semicircularis S. Chacón & M. Piepenbr., E. persica Mehrabi et al. and E. quercina Mehrabi et al.

Eutypella citricola groups separately from Eutypella sensu stricto with Eutypella vitis and our newly-generated strains. These new strains are introduced as a new genus, Paraeutypella with three species viz. P. citricola, P. guizhouensis and P. vitis. We studied the morphological characteristics of the species belonging to this clade and found considerable morphological differences from Eutypella sensu stricto. The differences include stromata with 4–25 groups of perithecia in a valsoid configuration, 3–6 sulcate, long ostiolar necks; thus, we consider them to belong in a distinct genus from the Eutypella and hence, we introduce the novel Paraeutypella.

There does not appear to be any host-specificity since members of Diatypaceae are found on a wide range of hosts in various habitats. Diatypaceae species frequently have been identified as saprobes on the decaying wood of angiosperms. Few endophytes, such as Diatrypella frostii Peck and Peroneutypa scoparia (Schwein.) Carmarán & A.I. Romero, have been reported (de Errasti et al. 2010, Vieira et al. 2011, Grassi et al. 2014). Therefore, the family may have the potential for switching nutritional modes during the degradation of plant material (de Errasti et al. 2010, Grassi et al. 2014). Several species have been reported as pathogens, such as Cryptosphaeria populina (Pers.) Sacc., C. pullmanensi Glawe and Eutypella parasitica R.W. Davidson & R.C. Lorenz, causing canker disease (Glawe and Rogers 1984, Rappaz 1987, Ma et al. 2016), Cryptovalsa ampelina (Nitschke) Fuckel causing grapevine trunk disease (Luque et al. 2006), Eutypa lata (Pers.) Tul. & C. Tul. causing canker and dieback disease (Lardner et al. 2005) and E. leptoplaca (Durieu & Mont.) Rappaz contributing to the dieback of grapevines (Trouillas and Gubler 2004, Catal et al. 2007).

In our phylogenetic analyses, some species of Diatrypella: D. favacea (Fr.) Ces. & De Not., D. iranensis Mehrabi & Hemmati, D. macrospora Mehrabi et al. and D. pulvinata Nitschke formed a distinct lineage (Fig. 1) in Diatrypella. Similarly, some species of Eutypella (E. caricae (De Not.) Berl., E. parasitica R.W. Davidson & R.C. Lorenz and E. virescens Wehm.) often form distinct lineages within Diatrypaceae (Fig. 1). This may be due to lack of single-copy nuclear genes like β-tubulin or misidentified species.

Acknowledgements

This work was funded by grants of the National Natural Science Foundation of China (NSFC Grants Nos. 31670027 & 31460011). Dr. Shaun Pennycook is thanked for the nomenclatural advice. Nalin N. Wijayawardene gratefully acknowledges Natural Science Foundation of China (grant No. NSFC 31950410558) and grant FAMP201906K provided by the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University. Dong-Qin Dai thanks the National Natural Science Foundation of China (NSFC 31760013) and the Thousand Talents Plan, Youth Project of Yunnan Provinces for support. Monika C. Dayarathne would like to thank National Natural Science Foundation of China (No. 31972222, 31560489). Lakmali S. Dissanayake would like to thank Ms. D.S. Marasinghe and Ms. S. N. Wijesinghe for valuable suggestions and guidance.

References