Clavicipitaceous entomopathogens: new species in Metarhizium and a new genus Nigelia

  • J. Jennifer Luangsa-ard
  • Suchada Mongkolsamrit
  • Donnaya Thanakitpipattana
  • Artit Khonsanit
  • Kanoksri Tasanathai
  • Wasana Noisripoom
  • Richard A. Humber
Original Article

DOI: 10.1007/s11557-017-1277-1

Cite this article as:
Luangsa-ard, J.J., Mongkolsamrit, S., Thanakitpipattana, D. et al. Mycol Progress (2017). doi:10.1007/s11557-017-1277-1
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Abstract

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.

Keywords

Clavicipitaceae Taxonomy Cordyceps Hypocreales Metarhizium Phylogeny Entomogenous fungi 

Introduction

There is a huge diversity of genera producing asexual reproductive forms associated with clavicipitoid families, and this association with the sexual morph is crucial in their classification, even after the reclassification of a genus. Some genera are important taxonomic determinants for genus and family. Linking the sexually reproductive forms to Akanthomyces, Beauveria, Isaria, Lecanicillium and Gibellula in the Cordycipitaceae, Hirsutella and Hymenostilbe in the Ophiocordycipitaceae and Aschersonia, Pochonia and Metarhizium in the Clavicipitaceae facilitates preliminary identification without necessarily having to resort to molecular tools.

It was decided during the last International Botanical Congress (IBC) in Melbourne that only one name can be applied for pleomorphic fungi (One Fungus = One Name; 1 F = 1 N). Some opinion papers that were published before or after the Melbourne IBC decision have presented the pros and cons of this new nomenclature (Gams et al. 2011; 2012b; Hawksworth et al. 2011). Committees of expert taxonomists have been formed to deal with many of the important taxonomic groups and establish ‘list-accepted’ and ‘list-demoted’ names (Gams et al. 2012a). Among the hypocrealean entomopathogens, it is only in the families Clavicipitaceae and Ophiocordycipitaceae where taxonomic recommendations and combinations have been proposed (Quandt et al. 2014; Kepler et al. 2014; Spatafora et al. 2015). In this study, we follow Kepler et al. (2014) in using Metarhizium for new Metacordyceps species in agreement with 1 F = 1 N on the basis of priority, being the genus first erected for this group of green-spored clavicipitaceous entomopathogens.

The Clavicipitaceae is the most heterogeneous and diverse group among the three families in the Hypocreales with entomogenous and phytopathogenic habits and to have both harmful or beneficial effects to humans and the environment. There are no distinct, synapomorphic morphological characters or ecological traits that define this family. There are genera that are pathogenic only to plants (Claviceps, Balansia) and genera pathogenic to a wide variety of invertebrates (Metarhizium, Pochonia, Rotiferophthora). Invertebrate pathogens within this family belong to seven sexually reproductive lineages - Aschersonia (Hypocrella), Conoideocrella, Orbiocrella, Regiocrella, Samuelsia and Moelleriella are genera pathogenic to scale insects and white flies (Hemiptera), while Metarhizium (Metacordyceps) is a genus with broad host associations, covering several insect orders.

The genus Metarhizium Sorokin was broadened in its definition by Kepler et al. (2014) to include the poorly understood Metacordyceps-like sexual stages due to the convergent nature of morphological characters with other genera (Sung et al. 2007a, b), as well as other anamorphic mononematous species such as Nomuraea rileyi, Paecilomyces carneus, P. marquandii and P. viridis. Paecilomyces-like anamorphs are found throughout Hypocreales (Luangsa-ard et al. 2004, 2005). Nomuraea was a polyphyletic genus with the green-spored species, N. rileyi, in Clavicipitaceae nested in the core Metarhizium clade and the pink-spored species Purpureocillium atypicolum (N. atypicola) in Ophiocordycipitaceae (Spatafora et al. 2015). Metarhizium includes species that typically parasitize insects, with some species growing in the rhizosphere of plants or free-living in soil but also infecting chameleons (Kepler et al. 2014).

In surveys of the forests over the years, specimens producing Metarhizium in culture and on the insect host were regularly collected, as well as specimens initially recorded as Cordyceps cf. martialis and Cordyceps cf. variegata due to their close resemblance to these species. Recent work on Metacordyceps placed Cordyceps martialis in the Clavicipitaceae under Metacordyceps (Kepler et al. 2012), which under 1 F = 1 N has been renamed Metarhizium martiale (Speg.) Kepler et al. (2014). The specimens collected in Thailand have the closest similarity to these two species based on their morphologies, their collection from Lepidoptera larvae, and the production of non-separating multicellular ascospores. The asexual stage of these fungi was never seen in the field.

The aims of this study were (1) to identify and to describe these commonly encountered but unknown invertebrate pathogens, (2) to determine their phylogenetic position among closely related invertebrate pathogenic fungi, and (3) to evaluate whether the morphological characters could be used in the delimitation of the genus.

Materials and methods

Collection and isolation of strains

Specimens were collected from natural forests in northern and central Thailand, but the majority of collecting sites were in the Khao Yai and Kaeng Krachan National Parks. Forest floors, leaf litter and the underside of leaves were examined for the emergence from any attached insects of brown, green or brightly colored stromata. Those specimens emerging from the leaf litter or ground were carefully excavated to reveal the host. Specimens were kept in small plastic boxes and transported to field house or laboratory for further examination. Primary isolations were made either in the field or within 2 days after returning to the laboratory. The stromata with perithecia were carefully suspended over the potato dextrose agar (PDA) plate so as to avoid contact of the stroma with the agar, to collect discharged ascospores. This was done in a moist chamber composed of a big plastic box covered with moist tissue paper to imitate forest conditions of high humidity. Discharged ascospores were transferred to fresh PDA plates and were examined daily for germination and also for fungal and bacterial contaminants. Isolations without contaminants were sub-cultured onto fresh PDA plates and then incubated at 25 °C in the laboratory for 8 weeks, before preparing for storage at −80 °C in 10% glycerol, and final deposition in the BIOTEC Culture Collection (BCC). For the purpose of comparison with other published Metarhizium species, Thai Metarhizium species were grown on SDYA/4 (Bischoff et al. 2009) to study their morphological characteristics, such as the size and shape of conidia and colony color in cultures (Table 1). The color of the freshly collected specimens were characterized with the color standard of Kornerup and Wanscher (1963), while the color of the cultures and their conidia were characterized using the Naturalist’s Color Guide (Smithe 1975) due to the absence of some colors in a single color chart. The reaction of the stroma in Metarhizium martiale with 3% potassium hydroxide (KOH) was observed as a colour reaction.
Table 2

List of specimens and their Genbank accession numbers used in this study

Code

Species

GenBank accession no.

References

SSU

LSU

EF1

RPB1

RPB2

BCC 8105

Aschersonia badia

DQ522537

DQ518752

DQ522317

DQ522363

DQ522411

Sung et al. (2007b)

BCC 7869

Aschersonia placenta

EF469121

EF469074

EF469056

EF469085

EF469104

Sung et al. (2007b)

A.E.G. 96-27a

Balansia henningsiana

AY545723

AY545727

AY489610

AY489643

DQ522413

Sung et al. (2007b)

A.E.G. 94-2

Balansia pilulaeformis

AF543764

AF543788

DQ522319

DQ522365

DQ522414

Sung et al. (2007b)

ARSEF 2567

Beauveria caledonica

AF339570

AF339520

EF469057

-

-

Sung et al. (2007b)

ATCC 26019

Claviceps fusiformis

DQ522539

U17402

DQ522320

DQ522366

-

Sung et al. (2007b)

ATCC 13892

Claviceps paspali

U32401

U47826

DQ522321

DQ522367

DQ522416

Sung et al. (2007b)

S.A. cp11

Claviceps purpurea

EF469122

EF469075

EF469058

EF469087

EF469105

Sung et al. (2007b)

AFTOL-ID 187

Clonostachys rosea

DQ862044

DQ862027

DQ862029

-

-

Sung et al. (2007b)

G.J.S. 90-227

Clonostachys rosea

AY489684

AY489716

AY489611

-

-

Sung et al. (2007b)

NHJ 11343

Conoideocrella luteorostrata

EF468995

EF468850

EF468801

EF468906

-

Sung et al. (2007b)

NHJ 12516

Conoideocrella luteorostrata

EF468994

EF468849

EF468800

EF468905

EF468946

Sung et al. (2007b)

NHJ 6293

Conoideocrella tenuis

EU369112

EU369044

EU369029

EU369068

EU369087

Johnson et al. (2009)

NHJ 345.01

Conoideocrella tenuis

EU369111

EU369045

EU369030

-

EU369088

Johnson et al. (2009)

EFCC 5886

Cordyceps kyusyuensis

EF468960

EF468813

EF468754

EF468863

EF468917

Sung et al. (2007b)

OSC 93623

Cordyceps militaris

AY184977

AY184966

DQ522332

DQ522377

AY545732

Sung et al. (2007b)

ARSEF 5689

Cordyceps scarabaeicola

AF339574

AF339524

DQ522335

-

-

Sung et al. (2007b)

ARSEF 5718

Cordyceps staphylinidicola

EF468981

EF468836

EF468776

-

-

Sung et al. (2007b)

NHJ12525

Hirsutella sp.

EF469125

EF469078

EF469063

EF469092

EF469111

Sung et al. (2007b)

OSC 128575

Hirsutella sp.

EF469126

EF469079

EF469064

EF469093

EF469110

Sung et al. (2007b)

GJS 89-104

Hypocrella sp.

-

DQ518772

DQ522347

DQ522393

DQ522448

Sung et al. (2007b)

TNM F10184

Metacordyceps atrovirens

JF415950

JF415966

-

JN049884

-

Kepler et al. (2012)

TNS F18494

Metacordyceps kusanagiense

JF415954

JF415972

JF416014

JN049890

-

Kepler et al. (2012)

TNS F16380

Metacordyceps pseudoatrovirens

-

JF415977

-

JN049893

JF415997

Kepler et al. (2012)

OSC 110996

Metacordyceps sp.

EF468974

EF468832

EF468773

EF468880

EF468928

Sung et al. (2007b)

ARSEF 5714

Metacordyceps taii

AF543763

AF543787

AF543775

DQ522383

DQ522434

Sung et al. (2007b)

ARSEF 6238

Metacordyceps taii

-

-

EU248857

EU248909

EU248937

Bischoff et al. (2009)

KS 50

Metacordyceps taii

GU979940

GU979949

GU979958

-

GU979972

Spatafora et al. (2007)

CBS 145.70

Metapochonia bulbillosa

AF339591

AF339542

EF468796

EF468902

EF468943

Sung et al. (2007b)

CBS 891.72

Metapochonia gonioides

AF339599

AF339550

DQ522354

DQ522401

DQ522458

Sung et al. (2007b)

CBS 464.88

Metapochonia rubescens

AF339615

AF339566

EF468797

EF468903

EF468944

Sung et al. (2007b)

ARSEF 324

Metarhizium acridum

-

-

EU248844

EU248896

EU248924

Bischoff et al. (2009)

ARSEF 7486

Metarhizium acridum

-

-

EU248845

EU248897

EU248925

Bischoff et al. (2009)

ARSEF 2082

Metarhizium album

DQ522560

DQ518775

DQ522352

DQ522398

DQ522452

Kepler et al. (2012)

ARSEF 7450

Metarhizium anisopliae

-

-

EU248852

EU248904

EU248932

Bischoff et al. (2009)

ARSEF 7487

Metarhizium anisopliae

-

-

DQ463996

DQ468355

DQ468370

Bischoff et al. (2009)

MY00896

Metarhizium blattodeae

HQ165657

HQ165719

HQ165678

HQ165739

HQ165638

This study

NHJ11597

Metarhizium blattodeae

HQ165662

HQ165724

HQ165683

HQ165743

HQ165643

This study

NHJ11618

Metarhizium blattodeae

HQ165663

HQ165725

HQ165684

HQ165744

HQ165644

This study

ARSEF 2107

Metarhizium brunneum

-

-

EU248855

EU248907

EU248935

Bischoff et al. (2009)

CBS 239.32

Metarhizium carneum

EF468988

EF468843

EF468789

EF468894

EF468938

Sung et al. (2007b)

CBS 399.59

Metarhizium carneum

EF468989

EF468842

EF468788

EF468895

EF468939

Sung et al. (2007b)

BCC19020

Metarhizium chaiyaphumense

HQ165654

HQ165716

HQ165675

HQ165737

HQ165635

This study

BCC19021

Metarhizium chaiyaphumense

HQ165655

HQ165717

HQ165676

HQ165738

HQ165636

This study

BCC78198

Metarhizium chaiyaphumense

KX369596

KX369593

KX369592

KX369594

KX369595

This study

RCEF 3632

Metarhizium cylindrosporae

JF415959

JF415982

JF416022

-

-

Kepler et al. (2012)

TNS 16371

Metarhizium cylindrosporae

JF415964

JF415987

JF416027

JN049902

-

Kepler et al. (2012)

CBS 218.56

Metarhizium flavoviride

-

-

KJ398787

KJ398598

-

Kepler et al. (2014)

ARSEF 2025

Metarhizium flavoviride

-

-

DQ464000

DQ468359

DQ468374

Bischoff et al. (2006)

ARSEF 4124

Metarhizium frigidum

-

-

DQ464002

DQ468361

DQ468376

Bischoff et al. (2009)

ARSEF 7445

Metarhizium frigidum

-

-

DQ464003

KJ398628

-

Bischoff et al. (2006), Kepler et al. (2014)

ARSEF 2596

Metarhizium globosum

-

-

EU248846

EU248898

EU248926

Bischoff et al. (2009)

CBS 258.90

Metarhizium guizhouense

-

-

EU248862

EU248914

EU248942

Bischoff et al. (2009)

TNS F18553

Metarhizium indigotica

JF415953

JF415968

JF416010

JN049886

JF415992

Kepler et al. (2012)

TNS F18554

Metarhizium indigotica

JF415952

JF415969

JF416011

JN049887

JF415993

Kepler et al. (2012)

BCC53581

Metarhizium kalasinense

KC011174

KC011182

KC011188

-

-

This study

BCC53582

Metarhizium kalasinense

KC011175

KC011183

KC011189

-

-

This study

BCC1376

Metarhizium khaoyaiense

KX983468

KX983462

KX983457

-

KX983465

This study

BCC14290

Metarhizium khaoyaiense

KX983469

KX983463

KX983458

-

KX983466

This study

BCC44287

Metarhizium khaoyaiense

KX983470

KX983464

KX983459

-

KX983467

This study

ARSEF 7488

Metarhizium lepidiotae

-

-

EU248865

EU248917

EU248945

Bischoff et al. (2009)

EFCC 1452

Metarhizium liangshanense

EF468962

EF468815

EF468756

-

-

Sung et al. (2007b)

EFCC 1523

Metarhizium liangshanense

EF468961

EF468814

EF468755

-

EF468918

Sung et al. (2007b)

ARSEF 1015

Metarhizium majus

-

-

EU248866

EU248918

EU248946

Bischoff et al. (2009)

ARSEF 1914

Metarhizium majus

-

-

EU248868

EU248920

EU248948

Bischoff et al. (2009)

CBS 182.27

Metarhizium marquandii

EF468990

EF468845

EF468793

EF468899

EF468942

Sung et al. (2007b)

ARSEF 2037

Metarhizium minus

AF339580

AF339531

DQ522353

DQ522400

DQ522454

Sung et al. (2007b)

NBRC 33258

Metarhizium owariense

HQ165669

HQ165730

HQ165689

HQ165747

-

Kepler et al. (2012)

ARSEF 7929

Metarhizium pingshaense

-

-

EU248847

EU248899

EU248927

Bischoff et al. (2009)

CBS 257.90

Metarhizium pingshaense

-

-

EU248850

EU248902

EU248930

Bischoff et al. (2009)

BCC47950

Metarhizium prachinense

KC011172

KC011180

KC011186

KC011184

-

This study

BCC47979

Metarhizium prachinense

KC011173

KC011181

KC011187

KC011185

-

This study

IndGH96

Metarhizium reniforme

HQ165670

HQ165732

-

-

HQ165649

This study

ARSEF 429

Metarhizium reniforme

HQ165671

HQ165733

HQ165690

-

HQ165650

This study

ARSEF 577

Metarhizium reniforme

HQ165672

HQ165734

HQ165691

-

HQ165651

This study

CBS 806.71

Metarhizium rileyi

AY624205

AY624250

EF468787

EF468893

EF468937

Sung et al. (2007b)

NBRC 8560

Metarhizium rileyi

HQ165667

HQ165729

HQ165688

-

-

This study

ARSEF 727

Metarhizium robertsii

-

-

DQ463994

DQ468353

DQ468368

Bischoff et al. (2009)

ARSEF 4739

Metarhizium robertsii

-

-

EU248848

EU248900

EU248928

Bischoff et al. (2009)

BCC17091

Metarhizium samlanense

HQ165665

HQ165727

HQ165686

-

HQ165646

This study

BCC17093

Metarhizium samlanense

HQ165666

HQ165728

HQ165687

HQ165746

HQ165647

This study

BCC30934

Metarhizium takense

HQ165658

HQ165720

HQ165679

HQ165740

HQ165639

This study

BCC30939

Metarhizium takense

HQ165659

HQ165721

-

HQ165741

HQ165640

This study

BCC30940

Metarhizium takense

HQ165660

HQ165722

HQ165681

HQ165742

HQ165641

This study

CBS 659.71

Metarhizium viride

HQ165673

HQ165735

HQ165692

-

HQ165652

This study

ARSEF 6927

Metarhizium viridulum

-

-

KJ398815

KJ398681

 

Kepler et al. (2014)

EFCC 2131

Metarhizium yongmunense

EF468977

EF468833

EF468770

EF468876

-

Sung et al. (2007b)

EFCC 2135

Metarhizium yongmunense

EF468979

EF468834

EF468769

EF468877

-

Sung et al. (2007b)

ARSEF 4358

Microhilum oncoperae

AF339581

AF339532

EF468785

EF468891

EF468936

Sung et al. (2007b)

BCC 14123

Moelleriella schizostachyi

DQ522557

DQ518771

DQ522346

DQ522392

DQ522447

Spatafora et al. (2007)

A.E.G 96-32

Myriogenospora atramentosa

AY489701

AY489733

AY489628

AY489665

DQ522455

Sung et al. (2007b)

BCC19950

Nigelia aurantiaca

GU979934

GU979943

GU979952

GU979961

GU979967

This study

BCC19475

Nigelia aurantiaca

GU979935

GU979944

GU979953

GU979962

GU979968

This study

BCC20179

Nigelia aurantiaca

GU979936

GU979945

GU979954

GU979963

GU979969

This study

BCC37621

Nigelia aurantiaca

GU979937

GU979946

GU979955

GU979964

GU979970

This study

BCC37627

Nigelia aurantiaca

GU979938

GU979947

GU979956

GU979965

-

This study

BCC13019

Nigelia aurantiaca

GU979939

GU979948

GU979957

GU979966

GU979971

This study

EFCC 6863

Nigelia martiale

-

JF415974

JF416015

-

-

Kepler et al. (2012)

HMAS 197472

Nigelia martiale

JF415956

JF415975

JF416016

JN049892

JF415995

Kepler et al. (2012)

TTZ070716-04

Nigelia martiale

JF415956

JF415975

JF416016

JN049892

JF415995

Kepler et al. (2012)

OSC 110987

Ophiocordyceps acicularis

EF468950

EF468805

EF468744

EF468852

-

Sung et al. (2007b)

OSC 110988

Ophiocordyceps acicularis

EF468951

EF468804

EF468745

EF468853

-

Sung et al. (2007b)

OSC 110999

Ophiocordyceps stylophora

EF468982

EF468837

EF468777

EF468882

EF468931

Sung et al. (2007b)

OSC 111000

Ophiocordyceps stylophora

DQ522552

DQ518766

DQ522337

DQ522382

DQ522433

Sung et al. (2007b)

NHJ 6209

Orbiocrella petchii

EU369104

EU369039

EU369023

EU369061

EU369081

Johnson et al. (2009)

NHJ 6240

Orbiocrella petchii

EU369103

EU369038

EU369022

EU369060

EU369082

Johnson et al. (2009)

CBS 504.66

Pochonia chlamydosporia

AF339593

AF339544

EF469069

EF469098

EF469120

Sung et al. (2007b)

CBS 101244

Pochonia chlamydosporia

DQ522544

DQ518758

DQ522327

DQ522372

DQ522424

Spatafora et al. (2007)

ARSEF 7682

Regiocrella camerunensis

-

DQ118735

DQ118743

DQ127234

-

Chaverri et al. (2005)

CBS 101437

Rotiferophthora angustispora

AF339584

AF339535

AF543776

DQ522402

DQ522460

Sung et al. (2007b)

EFCC 6279

Shimizuomyces paradoxus

EF469131

EF469084

EF469071

EF469100

EF469117

Sung et al. (2007b)

EFCC 6564

Shimizuomyces paradoxus

EF469130

EF469083

EF469072

EF469101

EF469118

Sung et al. (2007b)

Cultivation of fungi for molecular work

For DNA extraction purposes, starter cultures on PDA plates were prepared. After ca. 2 weeks, the plates were checked for contaminants and tiny agar blocks from pure cultures were inoculated into sterile Erlenmeyer flasks containing 50 ml Sabouraud Dextrose Broth (Difco) and incubated for 1–2 weeks at 25 °C, without shaking. Mycelium was then harvested by filtration and washed several times with sterile distilled water. Filtered mycelium was lyophilized and total genomic DNA was extracted as previously described (Luangsa-ard et al. 2004, 2005).

PCR and sequencing

Five strains of Cordyceps cf. martialis (MY 04790, MY 04873, MY 00628, MY 00626, MY 00634), one Cordyceps cf. variegata (NHJ 12082), fifteen Metarhizium from varying hosts, and three strains of Paecilomyces reniformis were sequenced. The nuclear regions sequenced for this study were from the small and large subunits of the ribosomal DNA (SSU and LSU), elongation factor 1-α (TEF), and the largest and second largest subunits of the RNA Polymerase II (RPB1 and RPB2). The PCR primers used to amplify the gene regions for this study were: NS1 and NS4 for SSU, LROR and LR7 for LSU (White et al. 1990), 983 F and 2218R for TEF, CRPB1 and RPB1Cr for RPB1, fRPB2-5 F2 and fRPB2-7cR for RPB2 (Castlebury et al. 2004). The 5 ́TEF and ITS fragments were analyzed separately in the PARB clade using primers EF1T and EF2T (Bischoff et al. 2009) for 5 ́TEF and ITS5, ITS4 for ITS rDNA (White et al. 1990). Amplifications were done in 50 μl volumes consisting of 1× PCR buffer, 200 μM of each of the four dNTPs, 2.5 mM MgCl2, 1 U SuperTaq Polymerase (HT Technologies, Cambridge, UK) and 0.5 μM of each primer. Sequencing primers used were the same as the amplification primers. PCR conditions were set as in Sung et al. (2007b). PCR amplicons were visualized by ethidium bromide staining after gel electrophoresis of 5 μl of the product in 0.8% agarose gel. Quantification of the PCR products was done using a standard DNA marker of known size and weight. Products with distinct bands were purified using Qiagen columns (QIAquick PCR Purification Kit Cat. No. 28106; Hilden, Germany). Purified PCR products were sent to Macrogen (South Korea) for sequencing. The BioEdit v.7.2.5 sequence alignment editor and sequence analysis program (Hall 2004) was used to compile the forward and reverse sequences generated from each template.

Phylogenetic analyses

Each sequence was checked for ambiguous bases and assembled in BioEdit v.7.2.5 and a multiple alignment was done using MUSCLE software (Edgar 2004). These were then submitted to GenBank (Tables 2 and 3). Proofed sequences were added to a set of sequence data, consisting mainly of known invertebrate pathogens and plant-associated fungi in the Clavicipitaceae, downloaded from GenBank. Maximum parsimony using PAUP v.4b10 (Swofford 2002) and Bayesian analyses using MrBayes v.3.0B4 (Ronquist and Huelsenbeck 2003) were first performed for each gene on individual datasets. Potential conflicts were assessed by comparing individual parsimony bootstrap trees. Sequences that grouped in clades with more than 70% bootstrap support passed this test and were included in the combined dataset (Wiens 1998).
Table 3

List of specimens and their Genbank accession numbers from the PARB clade used in this study

Code

Species

GenBank accession no.

References

5’TEF

ITS

BCC19950

Nigelia aurantiaca

KX823941

KY348780

This study

BCC19475

Nigelia aurantiaca

KX823942

KY348781

This study

BCC20179

Nigelia aurantiaca

KX823943

KY348782

This study

BCC37621

Nigelia aurantiaca

KX823940

KY348783

This study

ARSEF 324

Metarhizium acridum

EU248844

HM055449

Bischoff et al. (2009)

ARSEF 5748

Metarhizium acridum

EU248879

-

Bischoff et al. (2009)

ARSEF 7486

Metarhizium acridum

EU248845

HQ331458

Bischoff et al. (2009)

ARSEF 7450

Metarhizium anisopliae

EU248852

HQ331464

Bischoff et al. (2009)

ARSEF 7487

Metarhizium anisopliae

DQ463996

HQ331446

Bischoff et al. (2009)

NHJ11618

Metarhizium blattodeae

KX823951

HQ165704

This study

MY00896

Metarhizium blattodeae

KX823952

HQ165697

This study

ARSEF 2107

Metarhizium brunneum

EU248855

KC178691

Bischoff et al. (2009)

ARSEF 4152

Metarhizium brunneum

EU248853

HQ331452

Bischoff et al. (2009)

ARSEF 4179

Metarhizium brunneum

EU248854

HQ331451

Bischoff et al. (2009)

BCC19020

Metarhizium chaiyaphumense

KX823948

HQ165695

This study

BCC19021

Metarhizium chaiyaphumense

KX823949

HQ165696

This study

BCC78198

Metarhizium chaiyaphumense

KX823950

KX823950

This study

ARSEF 2133

Metarhizium flavoviride

DQ463988

NR131992

Bischoff et al. (2009)

ARSEF 4124

Metarhizium frigidum

DQ463978

HM055448

Bischoff et al. (2009)

ARSEF 6238

Metarhizium guizhouense

EU248857

HQ331447

Bischoff et al. (2009)

ARSEF 5714

Metarhizium guizhouense

EU248856

JN049829

Bischoff et al. (2009)

CBS 258.90

Metarhizium guizhouense

EU248862

HQ331448

Bischoff et al. (2009)

ARSEF 4321

Metarhizium guizhouense

EU248860

HQ331442

Bischoff et al. (2009)

ARSEF 4303

Metarhizium guizhouense

EU248859

HQ331465

Bischoff et al. (2009)

BCC53581

Metarhizium kalasinense

KX823944

KC011178

This study

BCC53582

Metarhizium kalasinense

KX823945

KC011179

This study

ARSEF 7488

Metarhizium lepidiotae

EU248865

HQ331456

Bischoff et al. (2009)

ARSEF 4628

Metarhizium lepidiotae

EU248863

-

Bischoff et al. (2009)

ARSEF 7412

Metarhizium lepidiotae

EU248864

HQ331455

Bischoff et al. (2009)

ARSEF 4566

Metarhizium majus

EU248869

HQ331441

Bischoff et al. (2009)

ARSEF 1946

Metarhizium majus

EU248867

HM055450

Bischoff et al. (2009)

ARSEF 1015

Metarhizium majus

EU248866

HQ331444

Bischoff et al. (2009)

ARSEF 1914

Metarhizium majus

EU248868

HQ331445

Bischoff et al. (2009)

ARSEF 4342

Metarhizium pingshaense

EU248851

HQ331454

Bischoff et al. (2009)

ARSEF 3210

Metarhizium pingshaense

DQ463995

HQ331449

Bischoff et al. (2009)

ARSEF 7929

Metarhizium pingshaense

EU248847

-

Bischoff et al. (2009)

CBS 257.90

Metarhizium pingshaense

EU248850

HQ331450

Bischoff et al. (2009)

BCC47950

Metarhizium prachinense

KX823939

KC011176

This study

BCC47979

Metarhizium prachinense

KX823938

KC011177

This study

ARSEF 7501

Metarhizium robertsii

EU248849

-

Bischoff et al. (2009)

ARSEF 4739

Metarhizium robertsii

EU248848

-

Bischoff et al. (2009)

ARSEF 727

Metarhizium robertsii

DQ463994

HQ331453

Bischoff et al. (2009)

ARSEF 6472

Metarhizium robertsii

EU248884

-

Bischoff et al. (2009)

BCC17091

Metarhizium samlanense

KX823947

HQ165709

This study

BCC17093

Metarhizium samlanense

KX823946

HQ165707

This study

BCC30934

Metarhizium takense

KX823953

HQ165698

This study

BCC30939

Metarhizium takense

KX823954

HQ165699

This study

Table 1

Phialide, conidial measurements and color of Metarhizium species in SDAY/4

Species

Substrate

Countries found

Media

Phialides (μm)

Conidia (μm)

Colony color

References

M. acridum

Orthoptera, Abandoned termite mound, Soil

Australia, Benin, Madagascar, Tanzania, Mexico, Niger, Senegal,

SDAY/4

4.5–13 × 2–4.5

5–7 × 2–4

Grayish yellow to grayish green

Bischoff et al. (2009)

M. anisopliae

Coleoptera, Hemiptera, Orthoptera

Australia, Colombia, Eritrea

SDAY/4

8–11.5 × 2–3

5–7 × 2–3.5

Grayish green

Bischoff et al. (2009)

M. brunneum

Coleoptera, Hemiptera, Soil

Australia, Argentina, Finland, Germany, Japan, Norway, USA

SDAY/4

6–18 × 2–5

4.5–8 × 2–3.5

Pale yellow to olive

Bischoff et al. (2009)

M. flavoviride

-

-

SDAY/4

5.5–9 × 2–2.5

4–5 × 2–2.5

-

Driver et al. (2000)

M. frigidum

Coleoptera

Australia

SDAY/4

-

4.5–7.5 × 2.5–3.5

Bright green

Bischoff et al. (2009)

M. globosum

Lepidoptera

India

SDAY/4

5–12 × 3–4

4–5 × 4–5

Grayish green

Bischoff et al. (2009)

M. guizhouense

Coleoptera, Lepidoptera, Diptera, Soil

Australia, China, Papua New Guinea

SDAY/4

6–20 × 2–3.5

5.5–9 × 2–3.5

-

Bischoff et al. (2009)

M. lepidiotae

Coleoptera, Hemiptera, Soil

Australia, Papua New Guinea

SDAY/4

6.5–12 × 2–3

5–7.5 × 2–3.5

Olive

Bischoff et al. (2009)

M. majus

Coleoptera, Lepidoptera, Soil

Australia, Japan, France, Philippines, Poland

SDAY/4

9–23.5 × 2.5–4.5

8.5–14.5 × 2.5–5

Olive to dark green

Bischoff et al. (2009)

M. pingshaense

Coleoptera, Isoptera

Australia, India, Solomon Islands

SDAY/4

7–17 × 2–3.5

4.5–8 × 2–3.5

Olive

Bischoff et al. (2009)

M. robertsii

Coleoptera, Orthoptera, Soil

Australia, Brazil, USA

SDAY/4

7–14.5 x 2–3.5

5–7.5 × 2–3.5

Olive to grayish green

Bischoff et al. (2009)

M. chaiyaphumense

Hemiptera (cicada)

Thailand

SDAY/4

10–12 × 2–3

Microconidia

3–6 × 2–3,

macroconidia

12–15 × 3–5

Leaf green

This study

M. blattoideae

Blattodeae

Thailand, Brazil

SDAY/4

-

No sporulation

White to yellow

This study

M. samlanense

Hemiptera (hopper)

Thailand

SDAY/4

5–7 × 2–3

3–5 in diameter

White

This study

M. takense

Hemiptera, cicada nymph

Thailand

SDAY/4

5–8 × 2–3

3–5 × 2–3

Greenish olive

This study

M. prachinense

Lepidoptera

Thailand

SDAY/4

2–5 × 2–2.5

3–5 × 2–3

white and sulphur yellow

This study

M. kalasinense

Coleoptera

Thailand

SDAY/4

8–12 × 2–3

6–8 × 2–3

Greenish olive and spectrum yellow

This study

M. pemphigi

Hemiptera(Pemphigus trehernei)

UK

SDAY/4

-

5–5.5 × 2–2.5

Light green

Driver et al. (2000)

Maximum parsimony analyses were performed using a heuristic search with 1000 random-addition sequence replicates using tree-bisection reconnection branch-swapping with the MulTrees option in effect. Only parsimony-informative characters were included in the analyses. Tree scores for the consistency index (CI), rescaled consistency index (RC), homoplasy index (HI) and retention index (RI) were computed. Relative support for the branches was obtained from bootstrap proportions using 1000 heuristic searches using the above parsimony settings and 10 random sequence additions per bootstrap replicate. Prior to conducting the Bayesian analysis the best nucleotide substitution model was determined by using MrModeltest v.2.2 (Nylander 2004). Bayesian analysis was then performed using MCMC with the suitable nucleotide substitution model. Four default chains were sampled every 100 generations and run for a total of 2 M generations. “Burn-in” trees for the Bayesian analyses were identified by assessment of the convergence of the log likelihood values from the 20 K Bayesian trees. Posterior probabilities were calculated from the posterior distribution of the retained Bayesian trees.

Maximum likelihood-based phylogenetic analyses were performed with RAxML v.8.2.8 (Stamatakis 2014) using the GTR-GAMMA model inferred from MrModel Test. Nodal support was assessed with nonparametric bootstrap using 1000 pseudoreplicates.

Results

Selection of taxa

Initial sequence data gathered for the specimens collected was the large subunit of the ribosomal DNA. A BLAST search and comparison with in-house database placed these strains in the Clavicipitaceae. The taxa included in this study were selected to represent the diversity of entomogenous species as well as the plant-associated fungi within the Clavicipitaceae. Representatives from the mainly entomopathogenic families within the Hypocreales (Ophiocordycipitaceae and Cordycipitaceae) were added to test the robustness of the dataset. We sampled 112 taxa of the three families within the Hypocreales for the overview of the phylogenetic relationships within the order: 98 from the Clavicipitaceae, 6 from the Ophiocordycipitaceae and 6 from the Cordycipitaceae. The taxon sampling within the Clavicipitaceae covers most of the asexual and sexual morphs known to be entomopathogens and some representatives of plant-associated genera. The list of taxa and their corresponding GenBank accession numbers are listed in Table 2.

Phylogenetic analysis

After initially examining individual trees for each gene region (SSU: tree length = 1022; parsimony informative characters = 125; CI = 0.580; RI = 0.811; RC = 0.470; HI =0.420; LSU: tree length = 878; parsimony informative characters = 184; CI = 0.440; RI = 0.786; RC = 0.346; HI = 0.560; EF1- α: tree length = 902; parsimony informative characters = 329; CI = 0.308; RI = 0.701; RC = 0.216; HI = 0.692; RPB1: tree length = 700; parsimony informative characters = 353; CI = 0.307; RI = 0.731; RC = 0.225; HI = 0.693; RPB2: tree length = 798; parsimony informative characters = 768; CI = 0.301; RI = 0.712; RC = 0.215; HI = 0.699), these gene regions were combined based on the topologies of the individual trees.

Phylogenetic conflict analyses did not detect any significant conflict between the individual gene datasets and allowed for combination of the five sets of gene sequences into a single combined data set (Wiens 1998). Of the 4293 characters in the combined alignment 2649 were constant and 1383 were parsimony-informative. Maximum parsimony analysis yielded 12 most parsimonious trees that differed only in the topologies of the terminal branches. The best tree is shown in Fig. 1 (tree length = 8306; CI = 0.313; RI = 0.708; RC = 0.222; HI = 0.687). The result of MrModeltest selected the General Time Reversible (GTR) model with proportion in invariable sites (I) and gamma distribution (G) (GTR+I+G; Lanave 1984) as the best-fit model by AIC in MrModeltest 2.2. The parameters included base frequencies—A = 0.2273, C = 0.2845, G = 0.2733, T = 0.2149—and the rate matrix for the substitution model: [A-C] = 1.0469, [A-G] = 3.8369, [A-T] = 1.1152, [C-G] = 0.8331, [C-T] = 6.9448, [G-T] = 1.000. For the among-site variation the proportion of invariable sites (I) was 0.4681, and the gamma distribution shape parameter was 0.5265. This model was used in MrBayes v.3.0B4 and RAxML v.8.2.8. Bayesian analyses resulted in 2 K “burn-in” trees; the consensus of the remaining 18 K trees resulted in identical topology (−lnL 43651.605203) as the Maximum Parsimony tree.
Fig. 1

Phylogenetic relationships inferred from the analyses of a combined data set for the partial genes LSU, SSU, TEF, RPB1 and RPB2 based on Maximum Parsimony, Bayesian analysis and RAxML. Nodal support for the three kinds of analyses are shown above the branches of the tree, first with Maximum Parsimony bootstrap support (MPBP), followed by the Bayesian posterior probabilities (PP) and Maximum Likelihood bootstrap support (MLBP). Bold lines mean support for the three analyses were 100%

The analyses showed three families that segregated corresponding to the recent phylogenetic classification of Cordyceps and related genera (Sung et al. 2007a, b). The family Cordycipitaceae comprises of the genus Cordyceps and its asexual morphs in the genera Isaria, Beauveria, and Microhilum with 100% MPBP, 100 PP and 100% MLBP. The family Ophiocordycipitaceae was represented by two asexual morphs Hirsutella and Hymenostilbe and by the sexually reproductive genus Ophiocordyceps with 100% bootstrap support while the majority of the unidentified strains including the five strains of Metarhizium cf. martiale and a Cordyceps cf. variegata grouped in the family Clavicipitaceae with 83% MPBP, 100 PP and 99% MLBP.

A core Metarhizium clade composed of the green-spored entomopathogens Metarhizium and Paecilomyces reniformis. Five new species including Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense and M. takense are nested in the core Metarhizium clade in all three analyses (MP, BP, RAxML; Fig. 1). The scale insect pathogens Hypocrella/Aschersonia, Orbiocrella, Conoideocrella, Regiocrella and the plant-associated Balansia, Claviceps and Myriogenospora as well as the seed-associated Shimizuomyces formed well-supported clades. This is the first group of scale insect pathogens that have independently formed in the Clavicipitaceae. A basal clade comprising Thai strains NHJ12082, MY00626, MY00628, MY00634, MY04873 that were previously labeled as Cordyceps cf. martialis and Cordyceps cf. variegata, together with Metarhizium martiale with 100% bootstrap support we describe here as a new genus, Nigelia.

A separate analysis of the Metarhizium pingshaense-anisopliae-robertsii-brunneum (PARB) clade consisting of a combined dataset consisting of 5 ́TEF and ITS rDNA sequence data using maximum parsimony of 47 taxa was carried out to confirm the presence and position of new species. The dataset was 894 bp long with 551 parsimony-informative characters and 71 variable and parsimony-uninformative characters (CI = 0.766, RI = 0.921, RC = 0.706, HI = 0.234), and the resulting consensus tree was obtained from a bootstrap analysis using 1000 heuristic searches. The list of taxa used for the PARB clade analysis is listed in Table 3. Bayesian analyses were performed with MrBayes v.3.0B4 (Ronquist and Huelsenbeck 2003) to determine posterior probabilities (PP). The result of MrModeltest selected the General Time Reversible + gamma distribution (GTR+G) as the best-fit model by AIC in MrModeltest 2.2 with parameters including the base frequencies A = 0.2312, C = 0.2711, G = 0.2375 and T = 0.2602. ML analysis of the same dataset resulted in a tree with a log likelihood of −7569.880048; Rate Matrix [A-C] = 1.192629, [A-G] = 2.646387, [A-T] = 0.875213, [C-G] = 0.654685, [C-T] = 2.896219 and [G-T] = 1.000000. The results showed clearly the segregation of the new species M. kalasinense, M. prachinburiense and M. samlanense that were different from other reported species (Fig. 2).
Fig. 2

Phylogenetic relationships of new species of Metarhizium with the core Metarhizium group, especially members of the M. anisopliae species complex inferred from ITS rDNA and 5 ́TEF sequences based on Maximum Parsimony, Bayesian analysis and RAxML. Nodal support for the three kinds of analyses are shown above the branches of the tree, first with Maximum Parsimony bootstrap support (MPBP), followed with the Bayesian posterior probabilities (PP) and Maximum Likelihood bootstrap support (MLBP)

In their search of establishing a phylogenetically well-supported taxonomic framework for Metarhizium and Metacordyceps that is consistent with 1 F = 1 N, Kepler et al. (2014) proposed Metarhizium based on priority, which included a core Metarhizium clade producing green-spored species and other species of Metacordyceps and Paecilomyces.

With the addition of scale insect pathogens Orbiocrella, Conoideocrella and Regiocrella to well-known species in the Clavicipitaceae comprising of plant-associated (Balansia, Claviceps, Myriogenospora and Shimizuomyces) and insect-associated (Hypocrella and Aschersonia) genera, our analyses gave a different topology of the phylogenetic relationships of Metarhizium martiale to other members of the core Metarhizium clade as presented and discussed by Kepler et al. (2014). The phylogenetic tree presented in this study is more consistent with the results that Kepler et al. (2013) has shown while proposing Polycephalomyces as another distinct genus from Ophiocordyceps in the Ophiocordycipitaceae. Since the monophyly of a group must be of foremost importance (Hennig 1950), including a broad coverage of the phylogenetic tree based on several genes as the underlying principles and guidelines for recognizing any taxonomic rank (Hennig 1950; Vellinga et al. 2015), we propose the following taxonomic additions and revisions to the Clavicipitaceae.

Taxonomy

Nigelia Luangsa-ard, Tasanathai & Thanakitpipattana, gen. nov.

Etymology: named after Dr. Nigel Hywel-Jones, who did pioneering work on invertebrate-pathogenic fungi research in BIOTEC

Stromata robust, solitary or several, simple or branched. Stipe tough, mustard yellow, orange (4B7) to reddish brown (9D8), cylindrical at the bottom and enlarging in the fertile part. Fertile part predominantly cylindrical, clavate to irregularly shaped. Perithecia completely immersed in the stroma with distinctly dark ostioles protruding from the stromatic surface in a manner making the surface appear to be spinose. Perithecia more or less separate or compactly aggregated, obliquely oriented to the stromatic surface, ovoid with curved, dark ostiole. Asci cylindrical, 8-spored. Ascospores filiform, septate, whole or fragmenting into part-spores, hyaline. In PDA cultures, conidiophores are verticillately or irregularly branched, bearing mostly solitary conidiogenous cells. Phialides variable in shape and size, smooth-walled, consisting of a cylindrical to globose basal portion tapering into a long neck or may proliferate and form 2–3 lateral necks.

Type species: Nigelia aurantiaca Luangsa-ard, Thanakitpipattana & Tasanathai

Note: Dried specimens of Nigelia aurantiaca show a wrinkled appearance and the surface of the fertile stomata appears spinous due to the protrusion of the bent dark perithecial ostioles. The surface of the fertile stroma in fresh specimens has a dense outer layer that is similar in texture to Metarhizium khaoyaiense. The hosts are Lepidoptera pupae and larvae.

Nigelia aurantiaca Luangsa-ard, Thanakitpipattana, Tasanathai, sp. nov. Fig. 3
Fig. 3

Nigelia aurantiaca.a Stroma arising from Lepidoptera larva (BBH 26484, BCC 37621); b stromata arising from Lepidoptera pupa (BBH 26497); c wrinkled and spinous appearance of fertile part of the stroma (BBH 26497); d oblique perithecial orientation; e perithecium; f asci tips; g foot of asci; h mature ascus; i whole ascospore; j whole ascospores showing septa (arrows); k colony on PDA at 20 °C after 45 days; ln phialides and conidia produced on PDA after 45 days. (d–n BBH 26484, BCC 37621). Bars (a, b) 5 mm, (c) 1 mm, (d) 500 μm, (e) 100 μm, (f) 2 μm, (g, h, m, n) 5 μm, (i, j, l) 20 μm, (k) 10 mm

MycoBank: MB808401

Etymology: from Latin aurantiaca, meaning orange-colored

Stromata erect, multiple, cylindric to clavate, simple or branched, on larvae or pupae of unidentified Lepidoptera, yellow-brown (5D8) to orange (6B8), 9–48 × 2–4 mm, becoming purple in 3% potassium hydroxide (KOH). Perithecia immersed, loose or compactly together, oblique in arrangement, ovoid with curved ostiole, 520–680 × 320–440 μm. Asci cylindrical, 8-spored, 210–400 × 2–5 μm; apical cap prominent, 2 × 2.5–3 μm. Ascospores filiform, with 15 septa but do not dissociate into part-spores, hyaline, 285–350 × 1 μm. The asexual morph was not seen in nature.

Culture characteristics: Colonies on PDA were relatively slow-growing, floccose with phialides variable in shape and size, smooth-walled, consisting of a cylindrical or globose basal portion tapering into a long neck or may proliferate and form 2–3 lateral necks and started to produce globose to subglobose white conidia 1.5–2 × 2 μm after 3 weeks in culture at 25 °C in the laboratory (Fig. 3m, n). The culture is whitish cream becoming light orange with age and did not turn green upon the production of conidia. Reverse of the culture is yellow orange turning dark brown with age.

Typus: THAILAND. On larva of unidentified Lepidoptera, 17 June 2009, collected by K. Tasanathai, P. Srikitikulchai, S. Mongkolsamrit, T. Chohmee, R. Ridkaew and N. L. Hywel-Jones (HOLOTYPE BBH 26484 [dried culture]: BCC 37621 ex-type).

Other specimens examined: Nakhon Ratchasima Province, Khao Yai National Park, 10 May 1994, N. L. Hywel-Jones (NHJ), R. Nasit (RN), S. Sivichai (SS), BBH 3483 (NHJ3678), 1 Sep. 2001, NHJ, (BBH 16836) 1 Oct. 2002, RN, W. Chaygate (WC) (BBH 8690), 9 Aug. 2005, SS, (BBH 14784), 24 Jul. 2006, K. Tasanathai (KT), S. Mongkolsamrit (SM), B. Thongnuch (BT), (BBH 18697), 12 Jun. 2007, SM, J.J. Luangsa-ard (JL), C. Chuaseeharonnachai (CC), L.T. Hung, S.S, (BBH 24293), Chao Ying Nature Trail, Khao Yai National Park, 2 Sept. 2008, BT, N.T. Toan, N.T. Vui, (BBH 24654), Mo Sing To, Khao Yai National Park, 20 May 2009, KT, P. Srikitikulchai (PS), P. Puyngain (PP), SM, T. Chohmee (TC), R. Ridkaew (RR), (BBH 27055), 17 Jun. 2009, KT, SM, PS, RR, TC, NHJ (BBH 26483, BBH 26754, BBH 26497, BBH 26326),12 Nov. 2009, KT, SM, TC, RR, M. Sudhadham (MS), A. Khonsanit (AK)(BBH 27158), 13 Nov. 2009, KT, SM, TC, RR, MS, AK, (BBH 27161); Nakhon Nayok Province, Pha Krachai Waterfall, Khao Yai National Park, 21 May 2008, KT, SM, PS, RR, BT, AK, (BBH 23839), 22 May 2008, KT, SM, PS, RR, BT, AK, (BBH 24253, BBH 25179); Chiang Mai Province, Doi Inthanon National Park, 20 Sep. 1994, NHJ, RN, SS, S. Thienhirun, A.J.S. Whalley, (BBH 5616, BBH 13161); 22 Sep. 1994, NHJ, RN, SS, S. Thienhirun, A.J.S. Whalley, (BBH 5619, BBH 4287); 22 Aug. 1995, NHJ (BBH 4711, BBH 12137, BBH 5465); 16 Jul. 1996, NHJ, (BBH 5477, BBH 5478, BBH 5479); 23 Aug. 2000, NHJ, RN, BT, (BBH 13447); Pong Dueat Pa Pae Geyser, Huai Nam Dang National Park, 5 Jul. 2008, KT, SM, PS, BT, AK, (BBH 23939); Prachuap Khiri Khan Province, Kaeng Krachan National Park, 5 Jun. 2000, NHJ, (BBH 16650), 27 May 2003, RN, (BBH 9248), 19 Apr. 2005, RN, (BBH 14521), 14 Nov. 2005, BT, KT, RR, WC, (BBH 15039, BBH 15040, BBH 15041, BBH 15042, BBH 15043, BBH 15044, BBH 15045, BBH 15046, BBH 15047, BBH 15048), 15 Nov. 2005, BT, KT, RR, WC, (BBH 15067, BBH 15068, BBH 15069, BBH 15070, BBH 15071, BBH 15072, BBH 15073, BBH 15074, BBH 15075, BBH 15076), 26 Apr. 2006, KT, JL, N. Boonyuen, RR, (BBH 18363, BBH 18364, BBH 17574). Nakhon Ratchasima Province, Pha Krachai Waterfall, Khao Yai National Park, 6 Aug. 1993, NHJ, RN, SS, R. Plomhan, (BBH 3389), 24 Aug. 1993, NHJ, RN, SS, L. Tangchit, (BBH 2862, BBH 2863), Heo Narok Waterfall, Khao Yai National Park, 2Sep. 1993, NHJ, RN, (BBH 5453), Km 29, Khao Yai National Park, 15 Jun. 1994, NHJ, RN, SS, (BBH 5460), Mo Sing To, Khao Yai National Park, 24 Aug. 1994, NHJ, RN, (BBH 5461), Pha Krachai Waterfall, Khao Yai National Park, 29 Sep. 1994, NHJ, (BBH 13166, BBH 5462, BBH 5463), Pha Kluaimai Waterfall, Khao Yai National Park, 17 Nov. 1995, NHJ, (BBH 4822).

Note: Nigelia aurantiaca can be predominantly found during the rainy season in June–September although specimens were also found in summer in April and during the cold dry season in November.

Nigelia martiale (Speg.) Luangsa-ard & Thanakitpipattana, comb. nov.

Cordyceps martialis Speg. Boletin, Academia nacional de Ciencias, Cόrdoba 11:535 (1889)

Metacordyceps martialis (Speg.) Kepler, G.-H Sung, & Spatafora. Mycologia 104 (1):182–197 (2012)

Metarhizium martiale (Speg.) Kepler, Rehner & Humber. Mycologia 106(4):811–829 (2014)

Notes: Cordyceps martialis was described by Spegazzini in 1889 as a new species on a larva of Cerambicidae, (long-horned beetle, Coleoptera) in Brazil. Kepler et al. (2012) sequenced 3 strains of M. martiale found on Lepidoptera larvae with bright red or orange stromata. Their assignment of this species in the Clavicipitaceae as Metarhizium was based on comparison of sequence data of several genetic loci, with Cephalosporium as the asexual morph. Nigelia aurantiaca looks morphologically similar to N. martiale but differs in the molecular data and in the type of ascospores. N. aurantiaca produces only whole (non-fragmenting) ascospores while N. martiale ascospores dissociate into part-spores (Mains 1958). New collections in Brazil, the type locality of N. martiale, should be done to compare these from specimens in Asia since the type specimen is no longer available for study.

New species ofMetarhizium

Metarhizium chaiyaphumense Tasanathai, Khonsanit, Thanakitpipattana, Mongkolsamrit & Luangsa-ard, sp. nov. Fig. 4
Fig. 4

Metarhizium chaiyaphumense. (BBH 41326, BCC 78198). a Stroma arising from head of host; b stroma showing immersed perithecia; c fungi on host; dM. chaiyaphumense on cicada adult (Hemiptera) (BBH 14968, BCC 19020); e perithecium; f asci; g ascospores showing septation; hm culture characteristics of sexual stage; h colonies on PDA; i part of conidiophores on PDA; j conidia on PDA; k colonies on SDYA/4; l part of conidiophores SDYA/4; m microconidia (red arrow) and macroconidia (yellow arrow) SDYA/4; ns culture characteristics of asexual stage; n colonies on PDA; o phialides with developing conidia on PDA; p mature conidia on PDA; q colonies on SDYA/4; r part of conidiophores on SDYA/4; s conidia on SDYA/4. Bars (a, d, h, k, n, q, s) 5 mm, (b) 0.5 mm, (c) 10 mm, (e) 100 μm, (f, g, o) 5 μm, (i, j, m, p, s) 10 μm, (l, r) 20 μm

MycoBank: MB818379

Etymology: M. chaiyaphumense is named after the place where the specimen was found – Chaiyaphum province.

Stromata arising from the head of the cicada nymphs, solitary or multiple, simple or 2–3 branched, grayish yellow (1C6) to yellowish olive green (1 F7), straight, cylindrical, 30–35 mm long; fertile area on the upper part of the stroma tapering or rounded, 10–15 mm long, 1.0–1.2 mm thick; terminal part mostly sterile, white to cream. Perithecia ovoid to obpyriform, immersed, 550–670 × 320–380 μm, oblique in arrangement. Asci cylindrical, 520–650 × 3–4 μm. Ascospores hyaline, filiform, 225–375 × 1 μm, smooth, multiseptate with cells 9–21.5 μm long, remaining whole after discharge (non-fragmenting). Asexual stage found only on adult cicadas of the genus Platypleura (Cicadidae, Hemiptera). Conidiophores arising all over the adult cicadas, at first white turning green due to the production of conidia.

Culture characteristics: Colonies relatively fast-growing on SDYA/4, attaining a diameter of 1.7 cm after 14 days at 25 °C, at first white becoming leaf green (No. 146). at 7 days in colony center due to the production of conidia. Vegetative hyphae smooth-walled. Conidiophores densely packed, terminating in branches with 2–5 phialides per branch. Phialides clavate, 10–12 × 2–3 μm. The colony is isolated from sexual morph and grown on the SDYA/4. Conidia catenulate, dimorphic; microconidia formed first, ovoid, ellipsoidal or subglobose, 3–6 × 2–3 μm; macroconidia formed later, mostly cylindrical, ellipsoidal, 12–15 × 3–5 μm. The colony is isolated from asexual morph and grown on the SDYA/4, conidia, globose 2–3 × 2–3 μm.

Culture on PDA. Colonies relatively fast-growing, attaining a diameter of 1.8 cm after 7 days at 25 °C, at first white turning to parrot green (No.60) due to heavy sporulation, velvety to floccose (Fig. 4h, n). Colony reverse cream to pale green. Mycelium hyaline, branched, septate, smooth-walled. The colony is isolated from sexual stage on PDA. Conidiophores consisting of divergent, terminal, often verticillate metulae, broadly clavate, or cylindrical, smooth-walled. Phialides hyaline, ovoid or ellipsoidal, appressed, 5–8 × 2–3 μm. Conidia catenulate, dimorphic; microconidia formed first, ovoid, ellipsoidal or sub-globose, 2–4 × 2–3 μm; macroconidia formed later, cylindrical, clavate, 4–9 × 2–3 μm. In colonies isolated from asexual stage conidia dimorphic, microconidia formed first, ovoid, ellipsoidal or subglobose, 5–10 × 2–4 μm; macroconidia formed later, cylindrical, clavate, 15–20 × 3–5 μm.

Notes: This species is morphologically very similar to M. owariense, M. takense and M. guniujiangense but differs in color of the stromata, which is dark green in M. takense, M. owariense and M. guniujiangense and olive green in M. chaiyaphumense. The size of the perithecia, asci and ascospores are also different among the four species (Table 4). Sterile terminal parts of the stromata are present in M. guniujiangense and M. chaiyaphumense, which is not seen in M. owariense.
Table 4

Comparisons between the sexual stages of Metarhizium and closely related taxa within the Clavicipitaceae

Species

Host

Stroma

Perithecia (μm)

Asci

(μm)

Ascospores (μm)

Anamorph

Phialides (μm)

Conidia (μm)

References

Nigelia aurantiaca

Lepidoptera

Cylindrical to clavate, yellow-brown to orange

9–48 × 2–4 mm

Embedded, oblique, ovoid

520–680 × 320–440

Cylindrical,

210–400 × 2–5

Whole spores, filiform,

285–350 × 1

Hirsutella-like

12–14 × 2–3

5–6 × 2–3

This study

Metarhizium martiale

Coleoptera

Cylindrical to clavate, brownish, red, orange-yellow, orange or cinnabar

10–30 × 2–4 mm

Embedded, oblique, Ovoid or conoid

650–840 × 240–450

Cylindrical,

300–510 × 3–4

Part-spores, filiform,

6–10 × 0.5–1

Not seen

-

-

Mains (1958)

Cordyceps variegata

Lepidoptera

Robust, simple or furcated, brown, 20 × 2 mm;

Fertile part claviform or fusoid

Pyriform, immersed,

350–750 × 250–450

Cylindrical, 260–550 × 3–5

Part-spores, 12.5–20 × 0.8–2.5

Not seen

-

-

Moureau (1949)

Cordyceps campsosterna

Elaterididae, Coleoptera

Greenish yellow, 16 cm long, simple, cylindrical

Pyriform to obovoid,

275–433 × 165–276

Cylindrical, 4-spored,

175–349 × 3.9,

Cap hemispherical 2.9–3.9 × 2

Filiform, part-spores,

2.9–5.9 × 1

Metarhizium

-

-

Zhang et al. (2004)

Metarhizium blattodeae

Blattaria

-

-

-

-

Metarhizium

3–5 × 1.5–2

Cylindrical, 4–6 × 2–3

This study

Metarhizium brittlebankisoides

Coleoptera

Pale green, cylindrical

Flask-shaped, ordinal immersed,

170–200 × 406–531

Cylindrical,

188–313 × 3–3.2

Cylindrical,

180–300 × 0.94

Metarhizium

-

-

Liu et al. (2001)

Metarhizium chaiyaphumense

Hemiptera (Cicada nymph)

Solitary, cylindrical, simple to 2-3 branched

Obliquely immersed,

550–670 × 320– 380

Cylindrical,

520–650 × 3–4

Whole spore, hyaline, filiform, 225–375 × 1

Metarhizium

5–8 × 2–3

Microconidia,

ovoid, subglobose 2–4 × 2–3; macroconidia 4–9 × 2–3

This study

Metarhizium cf. cylindrosporae

Hemiptera (Cicada adult)

-

-

-

-

Metarhizium

6–11 × 2.5–3

8–20 × 3–5

This study

Metarhizium guniujiangense

Hemiptera (Cicada nymph)

Stromata two, dark green, curving

Obliquely inserted, ampullaceous,

640–770 × 240–320

Cylindrical, 8-spored, 310–380 × 4–4.8

Whole spore, hyaline, filiform, 240–330 × 0.8–1

Metarhizium aff. cylindrosporae

4–5.5 × 2.8–3

5–6.5 × 3–4, 14.5–24 × 3–4

Li et al. (2010)

Metarhizium kalasinense

Coleoptera

Greenish yellow, 150 mm long

Obliquely immersed,

700–800 × 250–350

Cylindrical, hyaline

500–650 × 4–5

Whole spores, hyaline, filiform,

400–500 × 1–1.5

Metarhizium; yellow green

8–10 × 3–4

6–7 × 2–3

This study

Metarhizium liangshanense

Lepidoptera

Cylindrical to clavate, brown-dark brown

Ellipsoidal to ovoid, superficial,

400–740 × 300–450

Cylindrical,

260–480 × 8–12

Ascospore,

160–350 × 2.5–3.5, Part spores

10–20 × 2.5–3.5

 

-

-

Zang et al. (1982)

Metarhizium owariense

Hemiptera (Cicada nymph)

Fleshy, dark brown, straight, cylindrical, 5–7 cm long

Obliquely immersed, ampullaceous,

460–530 × 200–270

 

Part-spores,

4–4.3 × 2.4–2.8

Metarhizium

5–7 × 2–3

7–10 × 3–5

Kobayasi and Shimizu (1963)

Metarhizium prachinense

Lepidoptera larva

Several, cylindrical to clavate, white cream to pale brown 50–86 × 1–2 mm

Obliquely immersed, clavate to ovoid, pale brown to brown, 320–470 × 180–300

Cylindrical, hyaline with distinct apical cap, 100–271 × 3–5

Whole spores, hyaline, filiform, 94–107 × 1

Metarhizium;

White green

3–5 × 2

3–5 × 1.5–2.5

This study

Metarhizium samlanense

Hemiptera

-

-

-

-

Metarhizium

5–7 × 2–3

Globose, 3–4

This study

Metarhizium taii

Lepidoptera

Cylindrical, 20–35 × 2–5 mm

Obliquely immersed, flask-shaped,

230–380 × 810–1120

Cylindrical

300–490 × 3.4–5.7

Cylindrical part spores,

20–34.6 × 1.4–3.1

Metarhizium

  

Liang and Liu (1991)

Metarhizium takense

Hemiptera (Cicada nymph)

Simple, greenish brown, dark green, 70–130 mm

Oblique in arrangement, pale brown to brown, 510–550 × 250–350

Hyaline, cylindrical, 275–400 × 5

Filiform, hyaline, not fragmenting, 155–230 × 1.25

Metarhizium

3–5 × 2

Microconidia, 3–5 × 2–3; macroconidia, 8–16 × 3–4

This study

Metarhizium yongmunense

Lepidoptera

Clavate, white to pale yellow, perithecia brownish yellow to orange brown

Loosely embedded, fusiform to clavate, oblique, 550–800 × 450–500

Cylindrical, 205–260 × 5–7

Whole spores with distinct septa, filiform 180–345 × 1

-

-

-

Sung et al. (2007b)

Metarhizium cylindrospora

Hemiptera (Cicada adult

-

-

-

-

Nomuraea

5–8 × 3–4.2

Microconidia,ovoid, subglobose,

3.3–6.7 × 3.3–4.2;

macroconidia, cylindrical,

16.2–20.3 × 3.5-5

Tzean et al. (1993)

The presence of the sexual reproductive morph on one stage of the host (here on cicada nymphs) and the asexual morph on the other stage of the host (adult cicadas) is not a common phenomenon but has also been reported in Beauveria bassiana where most of the asexual stages were reported on adult insects and the sexual reproductive morph usually found on larval and pupal stages (Li et al. 2001).

Typus: THAILAND. On cicada nymph (Hemiptera), 13 August 2015, collected by S. Mongkolsamrit, A. Khonsanit, N. Kobmoo, D. Thanakitpipattana, W. Noisripoom, P. Srikittikulchai, S. Wongkanoun and R. Promhan in Phukhiao wild life sanctuary, Chaiyaphum province. (HOLOTYPE BBH 41326 [dried culture]: BCC 78198 ex-type.)

Other specimens examined: Chaiyaphum Province, Phukhiao wildlife sanctuary, 13 August 2015, S. Mongkolsamrit (SM), A. Khonsanit (AK), N. Kobmoo (NK), D. Thanakitpipattana (DT), W. Noisripoom (WS), P. Srikittikulchai (PS), S. Wongkanoun and R. Promhan BBH 40400 (MY10800), BBH 40401 (MY10801), BBH 40402 (MY10804), BBH 40403 (MY10805), BBH 40404 (MY10806), BBH 40405 (MY10807); 15 October 2005, K. Tasanathai, SM, R. Ridkaew and L. Hung, BBH 14968 (MY00521), BBH 14969 (MY00522), BBH 14496 (MY00524).

Metarhizium kalasinense Tasanathai, Khonsanit, Thanakitpipattana, Mongkolsamrit & Luangsa-ard, sp. nov. Fig. 5
Fig. 5

Metarhizium kalasinense. (BBH 53582, BCC 34590). a, b Stromata arising from Coleoptera larvae; c, d oblique perithecial orientation; e perithecium; f ascus; g ascus tip; h whole ascospore; i microcyclic conidiation of ascospores; j colonies on SDYA/4 at 25 °C after 14 days; k, l phialides and conidia on SDYA/4 at 25 °C; m colonies on PDA at 25 °C after 14 days; n, o phialides and conidia on PDA at 25 °C. Bars (a, j, m) 10 mm, (b) 50 mm, (c) 1.5 mm, (d) 200 μm, (e) 100 μm, (f) 50 μm, (g) 5 μm, (h) 40 μm, (i) 4 μm, (k, l, n, o) 10 μm

MycoBank: MB808402

Etymology: M. kalasinense is named after the place where the specimen was found – Kalasin province.

Stromata on Coleoptera larvae simple to sparingly branched, yellow brown (5D8) when young turning olive green (1 F7) to greenish brown (8 F3) with dark green (30 F3) ostioles (Fig. 5 a–c). Rhizoids flexuous, up to 30 cm buried deep in the ground. Stipe of the stroma brownish yellow (5C8) to olive green (1 F7), up to 0.5 cm broad. Sterile stipe emerging from the ground cylindrical, mustard yellow (4B7) to olive green (1 F7), 35 cm long, 0.5 cm wide. Fertile part clavate, 2530 × 35 mm. Perithecia flask-shaped, obliquely immersed (Fig. 5 d–e), 700800 × 250350 μm each wall with a layer of closely arranged parallel hyphae, 2030 μm thick. Asci hyaline, cylindrical, 500650 × 45 μm, apical cap prominent, 58 × 35 μm. Ascospores filiform, hyaline, without septations and not fragmenting in to part-spores, 400500 × 11.5 μm (Fig. 5h).

Culture characteristics: Colonies relatively fast growing on SDYA/4, attaining a diameter of 1 cm after 14 days at 25 °C, at first white becoming greenly pigmented after 5 days (Greenish Olive, No. 49) turning yellow (No. 55) in the middle and surrounding the colonies. Colonies floccose, with areas appearing powdery due to production of conidia. Conidiophores dense, terminating in branches, with 23 phialides per branch. Phialides clavate, 812× 23 μm (Fig. 5k). Conidia cylindrical with rounded ends, 6–8 × 2–3 μm.

Culture on PDA. Colonies relatively fast growing, attaining a diameter of 1 cm after 14 days at 25 °C, at first white turning pale yellow green (No. 58) with the production of conidia, velvety to funiculose (Fig. 5n). Colony reverse cream to yellow. Phialides cylindrical narrowing at the tip to lanceolate, forming long chains of cylindrical conidia with rounded ends, 6–7 × 2–3 μm (Fig. 5o).

This species is morphologically very similar to M. camptosterni but differs not only in having 8-spored asci but also in the size of the perithecia, asci and ascospores (see Table 4).

Typus: THAILAND. On an unidentified elaterid larva (Coleoptera), 15 June 2012, collected by K. Tasanathai, S. Mongkolsamrit, P. Srikittikulchai, A. Khonsanit and W. Noisripoom in Khok Pa Si Community Forest, Kalasin province. (HOLOTYPE BBH 34590 [dried culture]: BCC 53582 ex-type.)

Other specimens examined: Kalasin Province, Khok Pa Si Community Forest, Phu Si Than Wildlife Sanctuary, 15 June 2012, K. Tasanathai (KT), S. Mongkolsamrit (SM), P. Srikittikulchai (PS), A. Khonsanit (AK), W. Noisripoom (WS), BBH 53581 (MY07343), 26 June 2012, AK, WS, BBH 53629 (MY07440), 19 July 2012, KT, AK, WS, BBH 34586 (MY07510), BBH 34590 (MY07512), BBH 34592 (MY07513).

Metarhizium prachinense Tasanathai, Khonsanit, Thanakitpipattana, Mongkolsamrit & Luangsa-ard, sp. nov. Fig. 6
Fig. 6

Metarhizium prachinense.a, b Stroma arising from Lepidoptera larva (BBH 30600, BCC 47950 in A, C–P; BBH 30607 in B); c, d oblique perithecial orientation; e asci; f ascus; g asci tips; h whole ascospores; il colonies on SDYA/4 at 25 °C: i at 14 days; j, k conidiophores bearing phialides; l conidia; mp colonies on PDA at 25 °C: m after 14 days; n, o conidiophores bearing phialides; p conidia. Bars (a, b) 20 mm, (c) 1 mm, (d) 300 μm, (e) 40 μm, (f) 30 μm, (g, jl, np) 10 μm, (h) 15 μm, (i, m) 10 mm

MycoBank: MB808403

Etymology: named after the province where the specimen was found – Prachin Buri province.

Stromata usually branched, on larvae of Lepidoptera; 5086 × 12 mm, broad; stipe cylindrical, somewhat flat, pale yellow (1A3) to grayish yellow (4C6); fertile area cylindrical with pointed ends, white, pale yellow to grayish yellow, 0.81.7 × 1 mm. Rhizoids flexuous, up to 7 cm in the ground. Perithecia scattered or crowded, grayish yellow (4C6) to brown (6E5), oblique in arrangement, clavate to ovoid with slightly protruding, bent ostioles, 320–470 × 180300 μm (Fig. 6c–d). Asci hyaline, cylindrical, 100–271 × 3–5 μm, possessing a prominent apical cap, 1 × 2 μm. Ascospores filiform, hyaline, not fragmenting into part-spores, 94–107 × 1 μm.

Culture characteristics: Colonies relatively fast growing on SDYA/4, attaining a diameter of 1 cm after 10 days, at first white turning sulphur yellow (No. 57) in center of colony due to the production of conidia. Vegetative hyphae smooth-walled. Conidiophores erect, resembling Isaria but not having tapering long necks, bearing dense whorls of branches, each bearing 2–5 conidiogenous cells. Phialides ovoid to obpyriform with short distinct neck, 2–5 × 2–2.5 μm. Conidia hyaline subglobose, 3–5 × 2–3 μm.

Culture on PDA. Colonies grown at 25 °C on PDA initially colorless, turning green due to the production of green conidia after 7 days. Vegetative hyphae smooth walled. Conidiophores erect, resembling Isaria but not having tapering long necks, bearing dense whorls of branches, each bearing 3–5 conidiogenous cells. Phialides ovoid to obpyriform with short distinct neck, 3–5 × 2 μm. Conidia subglobose, green, 3–5 × 1.5–2.5 μm.

Typus: THAILAND. On larva of unidentified Lepidoptera, 2 June 2011, collected by K. Tasanathai, S. Mongkolsamrit, P. Srikittikulchai, A. Khonsanit and W. Noisripoom.

(HOLOTYPE BBH 30607 [dried culture]: BCC 47979 ex-type).

Other specimen examined: Prachin Buri Province, Km. 29, Khao Yai National Park, 12 May 2011, J.J. Luangsa-ard, K. Tasanathai, S. Mongkolsamrit, A. Khonsanit, MY06601 (BBH 30600).

Metarhizium reniforme (Samson & Evans) Luangsa-ard, Boucias & Hywel-Jones, comb. nov.

Paecilomyces reniformis Samson & Evans. Studies in Mycology 6: 43. (1974)

Notes: The conidiogenous structures of Metarhizium reniforme are reminiscent of those of Nomuraea rileyi consisting of a globose basal portion with a short neck and less compact conidiophores. It produces pale green conidia that are kidney-shaped, reminiscent of those of Isaria tenuipes (Paecilomyces tenuipes), hence the previous placement in Paecilomyces section Isarioidea by Samson (1974). Metarhizium reniforme appears to have a specificity for tettigoniid grasshoppers (Humber, personal communication) while Isaria tenuipes has been isolated mainly from lepidopteran larvae. The conidial shape of Metarhizium reniforme remains kidney-shaped after culture while that of Isaria tenuipes varies after subculturing from kidney shaped to fusoid or elongate fusoid. The kidney shape of the conidia of Isaria tenuipes is very distinct in the natural host.

Metarhizium samlanense Luangsa-ard, Thanakitpipattana, Tasanathai, Mongkolsamrit & Hywel-Jones, sp. nov. Fig. 7
Fig. 7

Metarhizium samlanense. (BBH 14640, BCC 17091). a Fungus on leaf hopper (Hemiptera); bf colonies on SDYA/4 at 25 °C: b at 14 days; c at 21 days; d, e conidiophores bearing phialides and conidia; f conidia; gj colonies on PDA at 25 °C: g at 14 days; h, i conidiophores bearing phialides and conidia; j conidia. Bars (a) 2 mm, (b, c, g) 10 mm, (d, e, h) 10 μm, (f, i, j) 5 μm

MycoBank: MB808404

Etymology: named after Samlan National Park, the type location for the specimen.

Specimens found only on leafhoppers (Hemiptera: Cicadellidae) on the underside of bamboo leaves up to 3 m above the ground. Host head and thorax covered with pale green (27A3) to green (27E8) mycelium covered by sporulating conidiophores.

Culture characteristics: Colonies fast growing on SDYA/4, attaining a diameter of 1 cm after 14 days, at first white turning green due to conidiation. Then cultures turn to yellow (No. 55) after ca. 21 days. Conidiophores erect, resembling Nomuraea in forming dense clusters of branches, each bearing 2–4 globose to subglobose conidiogenous cells without a distinct neck, around the stalk. Phialides are short and cylindrical, 5–7 × 2–3 μm. Conidia are green (No. 146), globose, 3–5 μm in diameter.

Culture on PDA. Colonies grown at 25 °C on PDA at first white turning green due to conidiation after 3 days. Vegetative hyphae are hyaline and smooth-walled, forming a basal felt bearing densely packed, erect conidiophores and often having a floccose marginal overgrowth of aerial mycelium. Conidiophores erect, resembling Nomuraea in forming dense clusters of branches, each bearing 2–4 globose to subglobose conidiogenous cells without a distinct neck, around the stalk. Phialides are short and cylindrical, 5–7 × 2–3 μm. Conidia globose, 3–4 μm in diameter.

Typus: THAILAND. On leafhopper (Hemiptera, Cicadellidae), 2 February 2005, collected by J.J. Luangsa-ard and S. Mongkolsamrit (HOLOTYPE BBH 14640 [dried culture]: BCC 17091 ex-type).

Other specimens examined: Kanchanaburi Province, Krathon Ruesi Nature Trail, Thung Yai Naresuan Wildlife Sanctuary (West), 2 December 2005, K. Tasanathai (KT), Wiwantanee Chaygate (WC), Boonchan Thongnuch (BT), Prasert Srikitikulchai (PS), MY00772 (BBH 15161), Kamphaeng Phet Province, Khlong Namlai Waterfall, Khlong Lan National Park, 12 October 2007, KT, SM, PS, BT, Rungpet Ridkaew (RR), Artit Khonsanit (AK), MY02604 (BBH 22653), MY02605 (BBH 22654), MY02606 (BBH 27998), Loei Province, Bird Watching Trail, Na Haeo National Park (Phu Suan Sai), 15 July 2008, KT, SM, PS, BT, AK, MY03408 (BBH 24005), Chiang Mai Province, Pong Dueat Pa Pae Geyser, Huai Nam Dang National Park, 5 September 2008, KT, SM, PS, WC, AK, MY03769 (BBH 24622), Kamphaeng Phet Province, Khun Lan Watershed Management Unit, Khlong Lan National Park, 26 September 2008, KT, PS, WC, AK, RR, MY03836 (BBH 25358), Nakhon Ratchasima Province, Mo Sing To Nature Trail, Khao Yai National Park, 10 December 09, KT, PS, SM, Tanapat Chohmee (TC), RR, AK, MY05786 (BBH 27327).

Metarhizium takense Tasanathai, Thanakitpipattana, Mongkolsamrit & Luangsa-ard, sp. nov. Fig. 8
Fig. 8

Metarhizium takense. (BBH 25192, BCC 30939). a Stroma arising from the head of cicada nymph; b part of stroma showing perithecia; c, d oblique perithecial orientation; e asci; f whole ascospores; g colonies on PDA at 25 °C; h phialides on PDA at 25 °C; i conidia on PDA at 25 °C; j colonies on SDYA/4 at 25 °C; k phialides on SDYA/4 at 25 °C; l conidia on SDYA/4 at 25 °C. Bars (a) 5 mm, (b) 0.5 mm, (c, d) 100 μm, (e) 50 μm, (f) 30 μm, (g, j) 15 mm, (h, k) 10 μm, (i) 15 μm, (l) 5 μm

MycoBank: MB818380

Etymology: named after the province where the specimen was found – Tak province.

Stromata simple, cylindrical, grayish green (27D5) to almost dark green (29 F5) arising from the head of the cicada nymphs, 70–130 × 0.6–1.8 mm. Terminal part of the stroma fertile, cylindrical. Perithecia flask-shaped, oblique in arrangement, 510–550 × 250–350 μm.Asci hyaline, cylindrical, 275–400 × 5 μm. Ascospores filiform, hyaline, not fragmenting into part-spores, 155–230 × 1.25 μm.

Culture characteristics: Colonies relatively fast growing on SDYA/4, attaining a diameter of 10 mm after 14 days at 25 °C, at first white becoming greenish olive (No. 49) at 7 days due to the production of conidia. Vegetative hyphae smooth walled. Conidiophores dense, terminating in branches with 2–3 phialides per branch. Phialides are fusiform to narrowly ovoid, 5–8 × 2–3 μm. Conidia catenulate, ovoid, ellipsoidal or subglobose, 3–5 × 2–3 μm.

Culture on PDA. Colonies relatively fast-growing, attaining a diameter of 10 mm after 10 days at 25 °C, at first white turning greenish olive (No. 49) due to the production of conidia in the middle of colony. Phialides fusiform to narrowly ovoid, 3–5 × 2 μm. Conidia catenulate, dimorphic; microconidia formed first, ovoid, ellipsoidal or sub-globose, 3–5 × 2–3 μm; macroconidia formed later, cylindrical, clavate, 8–16 × 3–4 μm.

Typus: THAILAND. On cicada nymph (Hemiptera), 24 June 2008, collected by J.J. Luangsa-ard, K.Tasanathai, S. Mongkolsamlit, P. Srikittikulchai, A. Khonsanit, B. Thongnuch (HOLOTYPE BBH 25192 [dried culture]: BCC 30939 ex-type).

Other specimen examined: Tak Province, Thi Lo Su Waterfall, Umphang Wildlife Sanctuary, 24 June 2008, J.J. Luangsa-ard, K. Tasanathai, S. Mongkolsamlit, P. Srikittikulchai, A. Khonsanit, B. Thongnuch, MY03255 (BBH 23894), MY03262 (BBH 23898).

Discussion

The Clavicipitaceae are a very heterogeneous family in terms of nutritional habits. Apart from the plant-associated and insect-pathogenic genera, some species have been isolated from the soil (Metarhizium carneum, M. marquandii) and from nematodes (Pochonia), rotifers (Rotiferophthora) and other invertebrates.

The presence of obliquely immersed perithecia has been reported in two clavicipitoid segregate families, Ophiocordycipitaceae and Clavicipitaceae. Ophiocordyceps species (Ophiocordycipitaceae) that have ascospores that dissociate into 64 part-spores and Hymenostilbe asexual morphs – e.g., Ophiocordyceps sphecocephala, O. oxycephala, O. irangiensis and O. myrmecophila show this kind of perithecial arrangement. The hosts of these species are predominantly social insects (Hymenoptera), but they also include other insect orders such as Diptera, Odonata and Hemiptera.

Oblique perithecial arrangements were noted by Mains (1958) in his description of Nigelia martiale (Cordyceps martialis) and by Moureau (1949) for Cordyceps variegata. The host of N. martiale is a coleopteran, while that of C. variegata has been reported to be larval lepidopterans, all of these taxa produce ascospores that dissociate into part-spores. Nigelia aurantiaca, on the other hand, has been found only from Lepidoptera larvae and produces whole (non-dissociating) ascospores (Table 4).

Oblique perithecia in the Clavicipitaceae are produced by several Metacordyceps-like species in Metarhizium sensu Kepler et al. (2014) and Nigelia although the perithecial ostioles of these fungi are usually found to be curved or bent to point upwards (Figs. 3d, e and 8c, d). The sexual reproductive morphs of the core Metarhizium clade have either long rhizomorphs (e.g., M. chaiyaphumense, M. kalasinense) or robust stipes (e.g., M. takense) and variable perithecial distribution. In contrast, oblique perithecia in Ophiocordyceps with Hymenostilbe asexual stages do not show this pronounced bending of the ostioles and the stipe could be pliant and wiry. The hosts of Hymenostilbe are mainly comprised of social insects (Hymenoptera), flies (Diptera), dragonflies (Odonata) and stinkbugs (Hemiptera). The hosts for Metarhizium species comprise an extremely broad range of insects found in the orders Coleoptera, Hemiptera, Lepidoptera, Orthoptera, and can also be found in spiders, chameleons, soil and plants (Kepler et al. 2014). However, the color of the stromata, size and shape of the perithecia and the asexual morph distinguish Metarhizium sexual morphs clearly from Nigelia (Table 4). M. yongmunense and M. taii have also been reported from Lepidoptera but only M. yongmunense produces whole ascospores, whereas the ascospores of M. taii and most sexual morphs with Metarhizium asexual morphs dissociate into cylindrical part-spores. Another species producing oblique perithecia and part-spores is M. owariense, whose stromata are yellowish green.

Although past classification schemes have heavily emphasized the position and placement of the perithecia and the types of ascospores (Mains 1958; Kobayasi 1941), it has been shown that these criteria are not taxonomically significant for the generic and specific circumscriptions of entomogenous fungi (Sung et al. 2007b). Morphological characters such as size, shapes and orientation of perithecia, ascus and ascospore are convergent and the most variable throughout all clavicipitoid fungi (Sung et al. 2007b). Perithecia may be scattered or crowded and partially to completely immersed in the stroma with an ordinal to oblique orientation. Miller and Huhndorf (2005) showed that some specific ascospore morphologies have evolved several times independently in the Sordariales. This study has shown that the oblique arrangements of perithecia have also apparently evolved independently at several times and in several families of the Hypocreales.

The ecology of the fungus also plays an important role in identification. M. marquandii and M. carneum are more commonly isolated from the soil than as a pathogen on an invertebrate. Morphologically similar taxa, even sharing similar hosts, collected in the tropics and in temperate countries could have different types of ascospores and may differ in their genotypic characters. Nigelia martiale in neotropical countries such as Brazil was found on a coleopteran larva while species identified as N. martiale (Metarhizium martiale) in the paleotropical countries are found on Lepidoptera larvae. Cordyceps pruinosa, which produces truncate cylindrical part-spores is commonly found in temperate regions parasitizing lepidopteran cocoons while Cordyceps caloceroides and C. ninchukispora show the same macromorphologies but produce whole ascospores (C. caloceroides) and bola ascospores (C. ninchukispora) in the tropics. Similarly, Cordyceps militaris in temperate regions produces part-spores while Cordyceps pseudomilitaris in Thailand produces whole ascospores. Attempts were made to find out the correlation of morphological features such as ascomatal ontogeny, and ascospore morphology with ascomycete phylogenies (Miller and Huhndorf 2005; Lumbsch and Huhndorf 2007; Dettman et al. 2001), but we have concluded that either the ascospore morphology is not phylogenetically informative or that the existing revised classifications based on these premises have either not yet included enough molecular characters or have used inappropriate molecular characters.

Phenotypic plasticity is common in fungi and makes it difficult to rely solely on morphology, ecology or host information but would additionally need molecular phylogenetic analyses to determine the most appropriate generic placement of many fungi and test the monophyly of genera. The plasticity of the asexual morphs and the presence of pleoanamorphs as in the case of Aschersonia insperata with a Hirsutella synanamorph or Gibellula with a Granulomanus synanamorph will make it difficult for even a well-trained taxonomist to identify it just to the generic level based on its morphological features (Liu et al. 2005).

Acknowledgements

The authors would like to thank Drs Lily Eurwilaichitr, Kanyawim Kirtikara (BIOTEC), Prof. Morakot Tanticharoen, and Khun Rungsima Tantalakha for the support of the project. This project was supported by Cluster and Program Management Office grant number P15-51452. Some of the collections were made by Dr Nigel Hywel-Jones, who is thanked for his early contributions to this project. We wish to thank Mr. Rungpet Ridkaew for his earlier contributions to this study. We also wish to thank Prof. Drion Boucias, for sending the Paecilomyces reniformis strains to study. We thank the Thailand Department of National Parks for the permission to collect fungi in the national parks. We are grateful to the anonymous reviewers who made valuable comments and suggestions to improve the manuscript.

Copyright information

© German Mycological Society and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • J. Jennifer Luangsa-ard
    • 1
  • Suchada Mongkolsamrit
    • 1
  • Donnaya Thanakitpipattana
    • 1
  • Artit Khonsanit
    • 1
  • Kanoksri Tasanathai
    • 1
  • Wasana Noisripoom
    • 1
  • Richard A. Humber
    • 2
  1. 1.Microbe Interaction and Ecology Laboratory (BMIE), BIOTEC, National Science and Technology Development Agency (NSTDA)Khlong LuangThailand
  2. 2.USDA, ARS Biological Integrated Pest Management Research Unit, Robert W. Holley Center for Agriculture and HealthIthacaUSA