Differential Usefulness of Nine Commonly Used

ORIGINAL RESEARCH published: 03 October 2018 doi: 10.3389/fmicb.2018.02334

Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species Xiao Yang* and Chuanxue Hong Hampton Roads Agricultural Research and Extension Center, Virginia Tech, Virginia Beach, VA, United States

Edited by: Hector Mora Montes, Universidad de Guanajuato, Mexico Reviewed by: Henrik R. Nilsson, University of Gothenburg, Sweden Bernardo Franco, Universidad de Guanajuato, Mexico *Correspondence: Xiao Yang [email protected] Specialty section: This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology Received: 03 August 2018 Accepted: 12 September 2018 Published: 03 October 2018 Citation: Yang X and Hong C (2018) Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species. Front. Microbiol. 9:2334. doi: 10.3389/fmicb.2018.02334

The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and β-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade. Keywords: oomycetes, plant disease diagnosis, plant pathology, genetics, plant destroyers

INTRODUCTION The genus Phytophthora currently consists of approximately 200 formal and provisional species with many high-impact plant pathogens (Erwin and Ribeiro, 1996; Yang et al., 2017). For example, P. infestans and P. sojae are major threats to potato and soybean production, respectively (Erwin and Ribeiro, 1996). Phytophthora ramorum (Goheen et al., 2002; Rizzo et al., 2002, 2005) and P. cinnamomi (Zentmyer, 1980; Shearer et al., 2004) are destructive forest pathogens causing tree decline in the U.S. and Australia, respectively. Identifying Phytophthora isolates to species is the first and critical step to support plant biosecurity. This process is now done primarily by DNA sequencing. Concerted efforts have been made to identify genetic markers and improve the accuracy of DNA sequence-based identification. As a result, a variety of markers have been identified and utilized (Cooke et al., 2000; Martin and Tooley, 2003; Kroon et al., 2004; Blair et al., 2008; Robideau et al., 2014). Meanwhile, many signature sequences from ex-types (type-derived cultures) and authentic isolates (representative isolates designated by the originators of the respective species) have been generated (Cooke et al., 2000; Kroon et al., 2004; Blair et al., 2008; Martin et al., 2014; Yang et al., 2017), although their

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1

October 2018 | Volume 9 | Article 2334

Yang and Hong

Evaluating Markers for Identifying Phytophthoras

The mass was blotted dry on sterile tissue paper, transferred to a R -24 (MP Biomedicals, garnet bead tube and lysed in a FastPrep R 16 Santa Ana, CA). gDNA was purified using a custom Maxwell R FFS nucleic acid extraction kit in combination with a Maxwell Rapid Sample Concentrator (Promega, Madison, WI). A pair of primers including the forward primer ITS6 and reverse primer ITS4 (Cooke et al., 2000) was used to amplify the ITS region. The cox1 fragment was amplified with the primer pair COXF4N and COXR4N (Kroon et al., 2004). PCR reaction mixtures were prepared with Takara Taq DNA polymerase (Takara Shuzo, Shiga, Japan) according to the manufacturer’s instructions. Each cox1 PCR reaction mixture contained an additional 2-µL 25 mM MgCl2 and 0.25-µL Bovine serum albumin (BSA) per 25-µL. Thermal cycling protocols were described previously (Cooke et al., 2000; Kroon et al., 2004). All PCR products were evaluated for successful amplification using agarose gel electrophoresis. Sequencing reactions were run in both directions with the same primer pairs used for amplification at the University of Kentucky Advanced Genetic Technologies Center (Lexington, KY) or Eton Bioscience Inc. (Durham, NC). Results were viewed in Finch TV version 1.4.0 (Geospiza, Seattle, WA), aligned using Clustal X (Larkin et al., 2007), and edited manually to correct obvious sequencing errors and code ambiguous sites according to the International Union of Pure and Applied Chemistry (IUPAC) nucleotide ambiguity codes to produce a consensus sequence. All sequences produced in this study have been deposited in GenBank (Table 1). Rates of PCR success for all nine genetic markers were estimated by calculating the percentage of successful amplifications over all PCR reactions performed by the authors for each marker during the past 6 years.

availability in public repositories depends upon species (Kang et al., 2010). These two lines of advancement have raised several questions of practical importance. What genetic markers are most useful? Is their resolution dependent upon (sub)clade? How many markers are required to identify Phytophthora isolates within a respective (sub)clade to species? Answers to the above and other related questions will help identifying Phytophthora species accurately in the timeliest fashion and at the lowest cost. To this end, Martin et al. (2012) indicated that a set of genetic markers may be required for the most accurate identification. These included the internal transcribed spacer region (ITS), 60S ribosomal protein L10 (60S), beta-tubulin (β-tub), elongation factor 1 alpha (EF-1α), enolase (ENL), heat shock protein 90 (Hsp90), 28S ribosomal DNA (28S), tigA gene fusion protein (tigA), cytochrome-c oxidase 1 and 2 (cox1 and cox2), subunit 9 of NADH dehydrogenase (nad9), ribosomal protein S10 (rps10), and SecY protein (secY) coding regions. Correspondingly, reference sequences from various markers have been compiled for many known Phytophthora species (Cooke et al., 2000; Kroon et al., 2004; Blair et al., 2008; Grünwald et al., 2011; Park et al., 2013; Martin et al., 2014; Yang et al., 2017). In separate studies, Martin et al. (2014) and Martin and Tooley (2003) provided the average pairwise species distances for the concatenated nuclear and mitochondrial genes, and five mitochondrial markers, namely cox1&2, nad9, rps10, and secY. The objectives of this study were to evaluate nine commonly used genetic markers against more than 170 Phytophthora taxa and identify the most informative markers for individual (sub)clades.

MATERIALS AND METHODS Genus-Wide Distance Analyses

Sequence Selection

All nine genetic markers were analyzed for overall species distances resolved across the genus Phytophthora. Sequence datasets of each marker were aligned using the MUSCLE version 3.7 (Edgar, 2004) in MEGA version 7.0.26 (Kumar et al., 2016). Alignments were manually modified when obvious errors were present. The alignment of each marker was then trimmed to an equal size and question marks were inserted to represent missing data at both ends of short sequences. DNA sequence distances were calculated using the Kimura 2-parameter (K2P) distance model (Kimura, 1980) to explore the maximum, minimum and mean distances across the genus.

Nine common genetic markers, namely ITS, cox1, 60S, β-tub, EF-1α, ENL, Hsp90, 28S, and tigA, were evaluated. Sequences of 180 Phytophthora isolates representing 154 described and 17 provisionally named species were analyzed. These included 116 ex-types and 28 authentic isolates (Table 1). Eight taxa were represented by two or three isolates due to the lack of sequence data for all regions of individual isolates. The majority of 60S, β-tub, EF-1α, ENL, Hsp90, 28S, and tigA sequences originated from two previous studies (Blair et al., 2008; Yang et al., 2017). ITS and cox1 sequences of 90 and 79 Phytophthora species, respectively, were downloaded from GenBank (Benson et al., 2018). Sequences from P. sp. ohioensis (ST18-37) were obtained from the Phytophthora Database (Park et al., 2013). Seventy-nine and 86 isolates were sequenced for ITS and cox1, respectively in this study as described below to fill the signature sequence gaps in current public repositories.

Distance Analyses Within Individual (Sub)Clades Four selected markers that had relatively high mean species distances across the genus (cox1, ITS, tigA, and β-tub) were analyzed for distances within individual (sub)clades. Phylogenetic (sub)clade assignments for each species were identified according to the recent study by Yang et al. (2017). Sequence datasets within individual (sub)clades of each marker were aligned and edited as described above. Maximum, minimum, and mean distances within individual (sub)clades of each marker were calculated as described above.

DNA Extraction, Amplification, and Sequencing To extract genomic DNA (gDNA), a 5 × 5 mm agar plug was cut from the actively growing edge of a fresh culture and transferred to 20% clarified V8 broth. Cultures were incubated at room temperature (c. 23◦ C) for 7–14 d to produce a mycelial mass.


2



3

30G8

P. tentaculata

2b

554.88

122779

62B4

42B2

45G4

41B7

P. glovera

P. mengei

P. mexicana

P. siskiyouensis

MYA-4187

MYA-4554 46731

121969

22F4

P. capsici

15399, MYA-4034

92550 P15122

P0646

P11685

133865

65B8

P. terminalis

P. amaranthi

101557

65B9

P. occultans

T

T

T

A

T

T

T

Nepal

Hawaii, USA

Virginia, USA

Taiwan

The Netherlands

The Netherlands

Brazil

1995

1948

2008

2010

1998

1987

Stream water

Solanum lycopersicum

Oregon, USA

Argentina

2003

n.a.

Persea americana California, USA n.a.

Nicotiana tabacum

Capsicum annum New Mexico, USA

Amaranthus tricolor

Pachysandra terminalis

Buxus sempervirens

2005

2005

2000

1966

North Carolina, n.a. USA

Hevea brasiliensis India

219.88

61J9

P. meadii

129185

Quercus leucotricophora

128767

61G2

P. himalsilva

T

Colocasia esculenta

n.a.

1999

1992

2001

2004

1969

1985

1975

1987

2001

n.a.e

Year

Delaware, USA 2000

Mexico

Hevea brasiliensis Malaysia

35D3

T

P. colocasiae

P3425 Irrigation water

581.69

62C6

Nicotiana tabacum

03E5

15410, MYA-4037

P. botryosa

2a

22F9

P. citrophthora

P. nicotianae

Phaseolus lunatus

Mirabilis jalapa

23B4

A

P. phaseoli

P3006

30C1

Mexico

Ecuador

Germany

Iran

P. mirabilis

Solanum brevifolium

Argyranthemum frutescens

Solanum melongena

Australia

Oregon, USA

Ipomoea Mexico longipedunculata

T

A

T

T

Trifolium subterraneum

Psendotsuga menziesii

31B5

P10225

P13365

P3882

T

T

Scotland, UK

P. ipomoeae

136915

158964

287317

P3943

P10339

Rubus idaeus

The Netherlands

Ohio, USA

Location

Solanum tuberosum

64069, MYA-4062

MYA-3655

60237

58713, 60438 278933

331662

T A

Viburnum sp.

Rhododendron sp.

Host or substrate

27A8

109229

374.72

347.86

52938

313728 313727

T

Typec

P. infestans

P. andina

P. andina

61J4

32G1

P. iranica

P. clandestina

P. clandestina

1

1c

1b

P. pseudotsugae

MYA-4065

P6767

971.95

34D4

P. idaei

P. idaei

P19523

111725

62A5

P. hedraiandra

WPC P10194

IMI

22E6

ATCC

P. cactorum

CBS

1a

CH

Species

(Sub) cladea

Isolate identificationb

TABLE 1 | Information and GenBank accession numbers of isolates used in this study.

KF317102

MH620024

MH620023

MH620022

KF317094

n.a.

JX978168

MH620021

AY564192

MH620020

KF317097

KF317096

MH620019

KF317091

MH620018

MH620017

MH620016

KC733447

AY564160

MH620015

AY564189

AY564172

AY564199

AY564185

AY769115

MH620014

cox1

KF317081

MH620111

EU748545

MH620110

KF317073

GU111585

JX978167

JX978155

MH620109

MH620108

KF317076

KF317075

MH620107

KF317070

MH620106

MH620105

MH620104

KC733443

FJ801734

MH620103

MH620102

MH620101

FJ802112

FJ801946

AY707987

MH620100

ITS

KX250677

KX250670

KX250656

KX250649

KX250635

n.a.

KX250607

KX250600

KX250593

KX250572

KX250565

KX250544

KX250537

KX250509

KX250495

KX250481

EU080830

KX250474

EU080182

KX250453

KX250439

EU079866

EU080426

EU080129

KX250397

KX250369

60S

KX250678

KX250671

KX250657

KX250650

KX250636

KJ179949

KX250608

KX250601

KX250594

KX250573

KX250566

KX250545

KX250538

KX250510

KX250496

KX250482

EU080831

KX250475

EU080183

KX250454

KX250440

EU079867

EU080427

EU080130

KX250398

KX250370

β-tub

KX250679

KX250672

KX250658

KX250651

KX250637

n.a.

KX250609

KX250602

KX250595

KX250574

KX250567

KX250546

KX250539

KX250511

KX250497

KX250483

EU080832

KX250476

EU080184

KX250455

KX250441

EU079868

EU080428

EU080131

KX250399

KX250371

EF-1α

KX250680

KX250673

KX250659

KX250652

KX250638

n.a.

KX250610

KX250603

KX250596

KX250575

KX250568

KX250547

KX250540

KX250512

KX250498

KX250484

EU080833

KX250477

EU080185

KX250456

KX250442

EU079869

EU080429

EU080132

KX250400

KX250372

ENL

GenBank accession no.d

KX250681

KX250674

KX250660

KX250653

KX250639

n.a.

KX250611

KX250604

KX250597

KX250576

KX250569

KX250548

KX250541

KX250513

KX250499

KX250485

EU080834

KX250478

EU080186

KX250457

KX250443

EU079870

EU080430

EU080133

KX250401

KX250373

Hsp90

KX250682

KX250675

KX250661

KX250654

KX250640

n.a.

KX250612

KX250605

KX250598

KX250577

KX250570

KX250549

KX250542

KX250514

KX250500

KX250486

EU080835

KX250479

EU080187

KX250458

KX250444

EU079871

EU080431

EU080134

KX250402

KX250374

28S

(Continued)

KX250683

KX250676

KX250662

KX250655

KX250641

n.a.

KX250613

KX250606

KX250599

KX250578

KX250571

KX250550

KX250543

KX250515

KX250501

KX250487

EU080836

KX250480

EU080188

KX250459

KX250445

EU079872

EU080432

EU080135

KX250403

KX250375

tigA

Yang and Hong Evaluating Markers for Identifying Phytophthoras



4

4

3

2e

2d

2c

(Sub) cladea

MYA-4084

T

Quercus robur

784.95

30A5

P. quercina

Theobroma cacao Soil

MYA-4038

Theobroma cacao

15C7

T

Soil

Soil

P. quercetorum

202077

Western Australia, Australia

Germany

Germany

Oregon, USA

n.a.

n.a.

2012

2009

Germany

1995

South Carolina, 1997 USA

Costa Rica

Cameroon



n.a.

1995

1997

2008

KF358241

KF358240

KF317108

MH620035

KJ396688

MH620034

KF317106

KF358239

KF317105

MH620033

KF317104

California, USA n.a.

KF317103

MH620032

KF317098

MH620031

JX524159

2006

n.a.

2001

2004

MH620030

MH620029

KF317101

KF317100

MH620028

FJ237508

KF317095

MH620027

MH620026

n.a.

MH620025

cox1

n.a.

The Netherlands

Virginia, USA

The Netherlands

Eucalyptus dunnii South Africa

Quercus robur

Quercus robur

Rainwater

Lithocarpus densiflorus

Ilex aquifolium

Stream water

Cymbidium sp.

2005

1998

1925

2008

2007

1987

n.a.

2010

1969

n.a.

Year

North Carolina, 1999 USA

South Africa


Minnesota, USA

UK


Taiwan

South Africa

Italy

Brazil

Hawaii, USA

Location

Eucalyptus smithi South Africa

Soil

Fragaria × ananassa

Agathosma betulina

Kalmia latifolia

Pinus resinosa

Acuba japonica

Soil

Citrus sinensis

Curtisia dentata

Acer pseudoplatanus

Theobroma cacao

Macadamia integrifolia

22G9

42100

T

A

T

T

T

T

T

A

T

T

T

T

A

A

T

T

T

T

T

T

A

T

P. palmivora

238.83

P16948

P10410

P10117

P0716

P1819

P0630

WPC

Host or substrate

61J5

138637

502404

21173

IMI

Typec

P. megakarya

P. boodjera

127950

55C2

P. arenaria

803.95

29J5

P. psychrophila

121939

MYA-4222

30A8

P. pseudosyringae

47G5

MYA-4930

111772

60B3

P. pluvialis

P. alticola

MYA-2948

41C4

MYA-4577

P. nemorosa

114348

62A7

38J5

P. taxon aquatilis

545.96

P. ilicis

29E3

47G8

P. multivesiculata

P. frigida

125799

61F2

P. taxon emzansi

55C4

22E9

P. plurivora

P. elongata

45F1

P. pini

122081

61H7

P. pachypleura

31E6

MYA-3657

124094

55C5

P. multivora

P. bisheria

64532

128319

221.88

62C1

33H8

P. capensis 60440

46705

76651, MYA-4218

ATCC

P. citricola

133931

434.91

35C8

61H1

CBS

CH


P. acerina

P. sp. brasiliensis

P. tropicalis

Species

TABLE 1 | Continued

KF358229

KF358228

KF317086

MH620121

KJ372244

MH620120

KF317084

KF358227

KF317083

MH620119

KF317082

JX524158

FJ666126

MH620118

KF317077

MH620117

MH620116

MH620115

KF317080

KF317079

MH620114

FJ237521

KF317074

MH620113

JX951285

GU259388

MH620112

ITS

KX252654

KX251062

KX251055

KX251034

n.a.

KX251013

KX251006

KX250992

KX250978

KX250971

KX250964

KX250950

KX250929

EU080065

KX250915

KX250894

EU080741

KX250859

KX250817

KX250810

KX250789

KX250775

KX250747

KX250726

KX250712

EU080419

KX250698

60S

KX252655

KX251063

KX251056

KX251035

KJ372283

KX251014

KX251007

KX250993

KX250979

KX250972

KX250965

KX250951

KX250930

EU080066

KX250916

KX250895

EU080742

KX250860

KX250818

KX250811

KX250790

KX250776

KX250748

KX250727

KX250713

EU080420

KX250699

β-tub

KX252656

KX251064

KX251057

KX251036

n.a.

KX251015

KX251008

KX250994

KX250980

KX250973

KX250966

KX250952

KX250931

EU080067

KX250917

KX250896

EU080743

KX250861

KX250819

KX250812

KX250791

KX250777

KX250749

KX250728

KX250714

EU080421

KX250700

EF-1α

KX252657

KX251065

KX251058

KX251037

KJ396738

KX251016

KX251009

KX250995

KX250981

KX250974

KX250967

KX250953

KX250932

EU080068

KX250918

KX250897

EU080744

KX250862

KX250820

KX250813

KX250792

KX250778

KX250750

KX250729

KX250715

EU080422

KX250701

ENL


KX252658

KX251066

KX251059

KX251038

KJ396710

KX251017

KX251010

KX250996

KX250982

KX250975

KX250968

KX250954

KX250933

EU080069

KX250919

KX250898

EU080745

KX250863

KX250821

KX250814

KX250793

KX250779

KX250751

KX250730

KX250716

EU080423

KX250702

Hsp90

KX252659

KX251067

KX251060

KX251039

n.a.

KX251018

KX251011

KX250997

KX250983

KX250976

KX250969

KX250955

KX250934

EU080070

KX250920

KX250899

EU080746

KX250864

KX250822

KX250815

KX250794

KX250780

KX250752

KX250731

KX250717

EU080424

KX250703

28S

(Continued)

KX252660

KX251068

KX251061

KX251040

n.a.

KX251019

KX251012

KX250998

KX250984

KX250977

KX250970

KX250956

KX250935

EU080071

KX250921

KX250900

EU080747

KX250865

KX250823

KX250816

KX250795

KX250781

KX250753

KX250732

KX250718

EU080425

KX250704

tigA




6b

6a

5

(Sub) cladea

5 MYA-4456

131653

132023

61G8

60B2

P. bilorbang

P. borealis

140357

129424

127951

554.67

66D1

55B6

62B8

34A8

P. crassamura

P. fluvialis

P. gibbosa

P. gonapodyides

P. chlamydospora

131652

61G6

P. amnicola

60351

P6872

T

T

T

T T

MYA-4881 28765

T

T

A

P16851

T A A

389736

P11555

P. taxon walnut

40A7

47J1

P. sp. personii

P. sp. personii

P. rosacearum



Spain

Taiwan

The Netherlands




Malaysia

Hawaii, USA

Taiwan

New Zealand

Ohio, USA

Location

2013

2010

1996

1976

1999

2008

2011

2011

n.a.

1990

n.a.

2006

2006

Year

Reservoir water

Soil

Stream water

Soil

Soil

Prunus sp.

Stream water

Soil

Stream water

Irrigation water

Nicotiana tabacum

2008

2010

2009

2006

n.a.



Italy


1967

2009

KC733448

MH620046

MH620044 MH620045

2012

HQ012878

MH620043

MH620042

MH620041

MH620040

MF326861

MH620039

MF326858

MF326847

KF112863

KF112862

MH620038

HQ012881

MF326843

MF326863

AY564182

MH620037

AY564190

MH620036

n.a.

cox1

2009

1995

United Kingdom 1971

Alaska, USA



Virginia, USA

North Carolina, n.a. USA

Malus domestica California, USA n.a.

Soil

143061

P. pseudorosacearum

Olea sp. Soil

T

Soil

Zostera marina

143060

390121

T

A

P. kwongonina

30J3

200.81

32F8

P. humicola

P. inundata

123382

Soil

143062

P. cooljarloo

46H1

Soil

143059

P. condilina

P. gemini

Soil

143058

Heavae sp.

Cocos nucifera

Soil

Agathis australis

Soil

Host or substrate

P. balyanboodja

P3826

T

52179, MYA-4080

T 180616

22J1

296.29

67D6

T T

P. heveae

P15175

ST18-37 A

WPC

P. cocois

325914

IMI

67D5 36818

ATCC

61J7

587.85

CBS

Typec

P. agathidicida

CH


P. castaneae

P. sp. ohioensis

Species

TABLE 1 | Continued

60S β-tub

EF-1α

ENL

Hsp90

28S tigA

KF112854

MH620130

MH620129

KP863493

AF541890

MH620128

MH620127

MH620126

MH620125

FJ801604

MH620124

KJ372267

JN547636

KF112856

KF112855

FJ217680

HQ012957

KJ372262

KJ372258

MH620123

KP295304

MH620122

KP295308

KX251236

KX251222

KX251208

KX251201

JF273125

KX251187

KX251181

KX251167

KX251452

EU080312

KX251445

n.a.

n.a.

KX251153

KX251139

KX251125

n.a.

n.a.

n.a.

KX251111

KX251104

KX251097

KX251076

KX251237

KX251223

KX251209

KX251202

KF750602

KX251188

KX251182

KX251168

KX251453

EU080313

KX251446

MF326827

MF326824

KX251154

KX251140

KX251126

MF326816

MF326814

MF326806

KX251112

KX251105

KX251098

KX251077

KX251238

KX251224

KX251210

KX251203

n.a.

KX251189

n.a.

KX251169

KX251454

EU080314

KX251447

n.a.

n.a.

KX251155

KX251141

KX251127

n.a.

n.a.

n.a.

KX251113

KX251106

KX251099

KX251078

KX251239

KX251225

KX251211

KX251204

n.a.

KX251190

KX251183

KX251170

KX251455

EU080315

KX251448

n.a.

n.a.

KX251156

KX251142

KX251128

n.a.

n.a.

n.a.

KX251114

KX251107

KX251100

KX251079

KX251240

KX251226

KX251212

KX251205

HQ012922

KX251191

KX251184

KX251171

KX251456

EU080316

KX251449

MF326878

MF326876

KX251157

KX251143

KX251129

HQ012925

MF326869

MF326892

KX251115

KX251108

KX251101

KX251080

KX251241

KX251227

KX251213

KX251206

GU594846

KX251192

KX251185

KX251172

KX251457

EU080317

KX251450

n.a.

n.a.

KX251158

KX251144

KX251130

n.a.

n.a.

n.a.

KX251116

KX251109

KX251102

KX251081

(Continued)

KX251242

KX251228

KX251214

KX251207

n.a.

KX251193

KX251186

KX251173

KX251458

EU080318

KX251451

n.a.

n.a.

KX251159

KX251145

KX251131

n.a.

n.a.

n.a.

KX251117

KX251110

KX251103

KX251082

Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Database Database Database Database Database Database Database Database

ITS





6

7b

7a

6

(Sub) cladea

43F3

P. × stagnum

52854

45F3

31E7

60A4

P. melonis

P. niederhauserii

P. pisi

582.69

44388

45F7

133347

61H3

P. cajani

46719, MYA-4076

P. asiatica

P. × multiformis

141207

141209

67C1

67C2

P. × heterohybrida

22F6

P. × cambivora

P. × incrassata

47A7

P. × alni

109054

392316

392314

P10617

P3105

P16202

P10413

T

A

T

T

T

T

T

T

T

62A3

P. uliginosa

P. uniformis

T T

67B9

46C7

P. intricata

P. rubi

T P6231

T

209.46

67C4

61J3

P. formosa

P. fragariae

T T

109049

62A2

67C3

P. europaea

T

A

T

T

T

T

T

T

T

T

T

T

T

P. flexuosa

P6306

P16100

P3599

P10337

WPC

141199

181417

32035

389725

IMI

67C5

90442

MYA-4826

MYA-4926

MYA-4882

MYA-4946

58817

ATCC

Typec

P. attenuata

P. sp. sulawesiensis

132095

127954

55C1

P. thermophila

62C4

132024

60B1

P. riparia

P. asparagi

140647

122924

66D2

47H1

57J3

P. mississippiae

P. ornamentata

402.72

62C7

P. megasperma

P. pinifolia

127953

127952

62B9

55B9

CBS

CH


P. litoralis

P. lacustris

P. gregata

Species

TABLE 1 | Continued

Taiwan

England, UK

Taiwan

Taiwan

France

Taiwan

Indonesia

Japan

India

Japan

The Netherlands

Taiwan

Taiwan

Oregon, USA

1998

n.a.

n.a.

2005

1994

2013

2013

n.a.

1994

1996

Pea

Sweden

2009

Thuja occidentalis North Carolina, 2001 USA

Cucumis sativus

Cajanus cajani

Pueraria lobata

Abies sp.

Stream water

Stream water

Abies sp.

UK

Sweden

Alnus sp. Alnus sp.

Poland

Soil

n.a.

2013

n.a.

2013

2013

1998

2013

1989

MH620066

MH620065

MH620064

MH620063

MH620062

KU681018

KU517150

KU517145

MH620061

KU681017

KU681019

MH620060

DQ674736

MH620059

MH620058

MH620057

MH620056

MH620055

MH620054

HQ261458

MH620053

KC631619

MH620052

2004

2007

MH620051

JN935960

MH620050

KF112860

MH620049

MH620048

JF896561

MH620047

cox1

2006

2007

2012

2012

1931

2008

1972

2009

Year

Michigan, USA 2006

Virginia, USA


Oregon, USA

Chile

Italy

Mississippi, USA

Washington DC. USA


England, UK


Location

Rubus idaeus cv. Scotland, UK Glen Clova

Soil


Soil

Soil

Soil

Castanopsis carlesii rhizosphere soil

Syzygium aromaticum

Asparagus officinalis

Irrigation water

Soil

Stream water

Pinus radiata

Soil

Irrigation water

Althaea rosea

Soil

Salix matsudana

Soil

Host or substrate

KT183042

MH620144

KT183041

MH620143

MH620142

AF139368

KU517156

KU517151

KT183037

MH620141

AF139367

MH620140

HQ643340

KU517155

MH620139

KU517153

KU517152

MH620138

KU517154

EF590257

MH620137

n.a.

MH620136

MH620135

MH620134

KP863496

KF112852

MH620133

MH620132

AF266793

MH620131

ITS

KX251735

KX251728

KX251707

KX251686

KX251665

n.a.

KX251644

KX251637

KX251494

KX251588

n.a.

EU080011

KX251564

KX251630

KX251543

KX251623

KX251616

KX251522

KX251609

EU080345

KX251473

KX251375

KX251354

KX251347

KX251333

KX251319

KX251305

KX251285

KX251278

EU080530

KX251250

60S

KX251736

KX251729

KX251708

KX251687

KX251666

KU899239

KX251645

KX251638

KX251495

KX251589

KU899260

EU080012

KX251565

KX251631

KX251544

KX251624

KX251617

KX251523

KX251610

n.a.

KX251474

KX251376

KX251355

KX251348

KX251334

KX251320

KX251306

KX251286

KX251279

EU080531

KX251251

β-tub

KX251737

KX251730

KX251709

KX251688

KX251667

n.a.

KX251646

KX251639

KX251496

KX251590

n.a.

EU080013

KX251566

KX251632

KX251545

KX251625

KX251618

KX251524

KX251611

EU080346

KX251475

KX251377

KX251356

KX251349

KX251335

KX251321

KX251307

KX251287

KX251280

EU080532

KX251252

EF-1α

KX251738

KX251731

KX251710

KX251689

KX251668

n.a.

KX251647

KX251640

KX251497

KX251591

n.a.

KX251571

KX251567

KX251633

KX251546

KX251626

KX251619

KX251525

KX251612

EU080347

KX251476

KX251378

KX251357

KX251350

KX251336

KX251322

KX251308

KX251288

KX251281

EU080533

KX251253

ENL


KX251739

KX251732

KX251711

KX251690

KX251669

KU899396

KX251648

KX251641

KX251498

KX251592

KU899417

KX251572

KX251568

KX251634

KX251547

KX251627

KX251620

KX251526

KX251613

EU080348

KX251477

KX251379

KX251358

KX251351

KX251337

KX251323

KX251309

KX251289

KX251282

EU080534

KX251254

Hsp90

KX251740

KX251733

KX251712

KX251691

KX251670

n.a.

KX251649

KX251642

KX251499

KX251593

n.a.

EU080015

KX251569

KX251635

KX251548

KX251628

KX251621

KX251527

KX251614

EU080349

KX251478

KX251380

KX251359

KX251352

KX251338

KX251324

KX251310

KX251290

KX251283

EU080535

KX251255

28S

(Continued)

KX251741

KX251734

KX251713

KX251692

KX251671

n.a.

KX251650

KX251643

KX251500

KX251594

n.a.

KX251573

KX251570

KX251636

KX251549

KX251629

KX251622

KX251528

KX251615

EU080350

KX251479

KX251381

KX251360

KX251353

KX251339

KX251325

KX251311

n.a.

KX251284

EU080536

KX251256

tigA




7

8c

8b

8a

7d

7c

(Sub) cladea

121655

270.31

22H1

22H9

32G2

P. hibernalis

P. lateralis

P. ramorum

131246

49J8

61G1

P. taxon parsley MYA-3638 36906

P10974

P7517, P19521

A

MYA-3898

60352

P6871 A

T

A

A

T

T

T

T

A

T

T

T

T

T

T

T

T

T

A

T

T

Indiana, USA

West Virginia, USA

Mississippi, USA

Lactuca sativa

Daucus carota

Oregon, USA

Portugal

Tennessee, USA

Greece

Sweden

n.a.

1931

2004

2006

1995

2013

1997

2001

2009

2004

Camellia japonica South Carolina, n.a. USA

Chamaecyparis lawsoniana

Citrus sinensis

Rhododendron sp.

Petroselinum crispum


Japan

Germany

Greece

France

Cichorium intybus The var. foliosum Netherlands

1986

1998

n.a.

n.a.

n.a.

n.a.

Ecuador USA

2006

n.a.

n.a.


Ohio, USA

Ireland

Brassica oleracea The Netherlands

Abies concolor

Trifolium sp.

Glycine sp.

Zantedeschia aethiopica

Solanum marginatum

Isopogon buxifolius

Medicago sativa

Solanum tuberosum

MH620090

MH620089

MH620088

EU124918

MH620087

MH620086

n.a.

KF358238

MH620085

MH620084

MH620083

MH620082

MH620081

MH620080

MH620079

MH620078

KP288342

KF358236

MH620077

MH620076

MH620074

MH620073

MH620072

MH620071

MH620070

MH620069

MH620068

MH620067

cox1

Beta vulgaris var. California, USA n.a. altissima

1968

2005

2008

2011

1922

n.a.

1959

1986

Year

MH620075

Ireland

Japan

Japan

Virginia, USA

Italy

Indonesia

n.a.

Ontario, Canada

Iran

Location

n.a.

Solanum lycopersicum

Rosa sp.


Ilex glabra “Shamrock”

Arbutus unedo

Cinnamomum burmannii

Glycine max

Glycine max

Pistacia vera

Lactuca sativa

P. foliorum

61E7

P. taxon castitis

MYA-4162

MYA-4455

P3103

P1693

P1087

P1738

P2110

T

137103

29E9

P. primulae

P. pseudolactucae

61F4

P. lactucae

34684

60353, 46734 325930

52402

MYA-3900

46724

180615

22938

WPC

Host or substrate

Primula acaulis

127102

61E5

P. dauci

46735 46671

131375

386658

IMI

Typec

620.97

115029

62A8

P. cichorii

24A7

P. sp. kelmania

117687

179.87

62A9

61J8

47H3

P. sansomeana

P. trifolii

240.3

139749

P. brassicae

45F5

P. richardiae

P. pseudocryptogea

P. pseudocryptogea

23A4

129.23

61J2

P. erythroseptica

P. medicaginis

292.35

23J5

P. drechsleri

133248

113.19

61H5

61H9

P. nagaii

P. cryptogea

135747

P. sp. ax

61H4

132772

66C8

46H5

P. parvispora

P. fragariaefolia

144.22

45G6

P. vignae

61J1

16705, MYA-3899

P. cinnamomi

MYA-4082

312.62

22D8

ATCC

33D6

CBS

P. sojae

CH


P. pistaciae

Species

TABLE 1 | Continued

MH620166

MH620165

KT183039

MH620164

MH620163

MH620162

AB894388

KF358226

MH620161

MH620160

MH620159

MH620158

MH620157

MH620156

MH620155

MH620154

KP288376

KF358223

MH620153

MH620152

MH620151

MH620150

MH620149

MH620148

KC478667

MH620147

MH620146

MH620145

KT183043

ITS

KX252147

KX252133

KX252119

KX252112

KX252105

KX252098

n.a.

KX252063

KX252042

KX252014

KX252007

KX252000

KX251986

KX251958

KX251930

KX251923

EU080626

KX251902

KX251895

KX251888

KX251867

KX251860

KX251853

KX251846

KX251839

KX251811

KX251776

KX251762

KX251748

60S

KX252148

KX252134

KX252120

KX252113

KX252106

KX252099

n.a.

KX252064

KX252043

KX252015

KX252008

KX252001

KX251987

KX251959

KX251931

KX251924

EU080627

KX251903

KX251896

KX251889

KX251868

KX251861

KX251854

KX251847

KX251840

KX251812

KX251777

KX251763

KX251749

β-tub

KX252149

KX252135

KX252121

KX252114

KX252107

KX252100

n.a.

KX252065

KX252044

KX252016

KX252009

KX252002

KX251988

KX251960

KX251932

KX251925

EU080628

KX251904

KX251897

KX251890

KX251869

KX251862

KX251855

KX251848

KX251841

KX251813

KX251778

KX251764

KX251750

EF-1α

KX252150

KX252136

KX252122

KX252115

KX252108

KX252101

n.a.

KX252066

KX252045

KX252017

KX252010

KX252003

KX251989

KX251961

KX251933

KX251926

EU080629

KX251905

KX251898

KX251891

KX251870

KX251863

KX251856

KX251849

KX251842

KX251814

KX251779

KX251765

KX251751

ENL


KX252151

KX252137

KX252123

KX252116

KX252109

KX252102

n.a.

KX252067

KX252046

KX252018

KX252011

KX252004

KX251990

KX251962

KX251934

KX251927

EU080630

KX251906

KX251899

KX251892

KX251871

KX251864

KX251857

KX251850

KX251843

KX251815

KX251780

KX251766

KX251752

Hsp90

KX252152

KX252138

KX252124

KX252117

KX252110

KX252103

n.a.

KX252068

KX252047

KX252019

KX252012

KX252005

KX251991

KX251963

KX251935

KX251928

EU080631

KX251907

KX251900

KX251893

KX251872

KX251865

KX251858

KX251851

KX251844

KX251816

KX251781

KX251767

KX251753

28S

(Continued)

KX252153

KX252139

KX252125

KX252118

KX252111

KX252104

n.a.

KX252069

KX252048

KX252020

KX252013

KX252006

KX251992

KX251964

KX251936

KX251929

n.a.

KX251908

KX251901

KX251894

KX251873

KX251866

KX251859

KX251852

KX251845

KX251817

KX251782

KX251768

KX251754

tigA



Frontiers in Microbiology | www.frontiersin.org MYA-4457 MYA-4945

23J7

58A7

47C3

46A2

47C8

P. irrigata

P. macilentosa

P. parsiana

P. virginiana

P. aff. parsiana G1

8

10 MYA-3893 TSD-7

111474

50A1

22G7

45B7

P. gondwanensis

P. intercalaris

140632

291.29

45F9

46J2

P. fallax

125801

38789

46733

P. gallica

55C3

P. constricta

142610

691.79

407.48

P. boehmeriae

46H7

49J9

38E1

46C4

P. captiosa

P. pseudopolonica

P. polonica

9a (cluster P. insolita 9a3)

P. quininea

P. macrochlamydospora-33D5 G2

P. macrochlamydosporaG1 340618

180614

288805

P16826

P6950

P10722

P10719

P15005

T

T

T

T

T

T

A

T

T

Stream water

n.a.

Quercus robur

1994

2005

Year

New Zealand


New Zealand

Poland

Taiwan

Peru

The Netherlands

New South Wales, Australia

New South Wales, Australia

Ecuador

Ecuador

Iran

Iran

Iran

Iran

Virginia, USA

Iran

Mississippi, USA

Virginia, USA

Virginia, USA

Virginia, USA

Japan

Virginia, USA

Mississippi, USA

Virginia, USA

Ohio, USA

France

2007

n.a.

1998

1927

1997

2006

1992

2006

1980

n.a.

1927

1994

n.a.

n.a.

1993

n.a.

1992

1992

n.a.

2007

1991

2012

2000

2000

2007

2000

2006

2012

California, USA n.a.

Germany

Argentina

Location

Boehmeriae nivea Taiwan

Eucalyptus delegatensis

Soil

Eucalyptus saligna

Soil

Soil

Cinchona officinalis

Zantedeschia aethiopica

Glycine max

n.a.

P10267

T

n.a.

Pistacia vera

Pistacia vera

Pistacia vera

Pistacia vera

Irrigation water

Ficus carica

Irrigation water

Irrigation water

Irrigation water

Irrigation water

Chrysanthemum × morifolium

Irrigation water

Irrigation water

Citrus sp.

Soil

Austrocedrus chilensis

Host or substrate

Glycine max

P8217

P. sp. lagoariana

A

A

A

A

T

T

T

T

T

T

T

T

T

T

T

Typec

P10264

P8213

P8618

P0649

WPC

P. sp. cuyabensis

9a (cluster P. macrochlamydospora-33E1 9a2) G1

9b

60353

47D8

P. aff. parsiana G3

60B5

395328 395330

47C5

P. aff. parsiana G2

395329

IMI

P. aff. parsiana G1

240.3

MYA-4460

05D1

P. hydropathica

MYA-4927

MYA-4919

46A3

P. hydrogena

123163

61F1

MYA-4578

MYA-4944

P. chrysanthemi

58A1

34002

MYA-4074

ATCC

40A6

P. stricta

129273

122911

CBS


9a (cluster P. aquimorbida 9a1)

8

60E9

21H9

41B6

P. obscura

P. austrocedrae

8d

CH

P. syringae

Species

(Sub) cladea

TABLE 1 | Continued

KT163315

KT183046

KF317112

KT183047

KC733451

KC733450

KC733449

n.a.

KC733456

AY564188

MH620099

MH620098

KC733454

MH620097

n.a.

MH620096

MH620095

MH620094

KC295546

KC733455

KF192708

KC733453

KC733452

KC249962

MH620093

GQ294536

KF192702

MH620092

MH620091

KF358233

cox1

KT163268

KT183035

KF317090

KT183036

MH620176

MH620175

MH620174

KY707115

KF358225

GU111612

MH620173

MH620172

KC733445

MH620171

FJ802118

n.a.

MH620170

MH620169

KC295544

KC733446

KF192700

EU334634

EU583793

KC249959

KT183038

FJ666127

KF192694

MH620168

MH620167

KF358220

ITS

KX252610

KX252603

KX252589

EU080161

KX252568

KX252561

EU079658

n.a.

EU080256

EU080175

EU079802

KX252516

KX252510

KX252503

EU080664

KX252463

KX252433

KX252397

KX252378

KX252357

KX252343

KX252315

KX252294

KX252280

KX252266

KX252238

KX252210

KX252196

KX252175

KX252168

60S

KX252611

KX252604

KX252590

EU080162

KX252569

KX252562

EU079659

KY707104

KX252546

EU080176

EU079803

KX252517

KX252511

KX252504

EU080665

KX252464

KX252434

KX252398

KX252379

KX252358

KX252344

KX252316

KX252295

KX252281

KX252267

KX252239

KX252211

KX252197

KX252176

KX252169

β-tub

KX252612

KX252605

KX252591

EU080163

KX252570

KX252563

EU079660

KY787198

EU080258

EU080177

EU079804

KX252518

KX252512

KX252505

EU080666

KX252465

KX252435

KX252399

KX252380

KX252359

KX252345

KX252317

KX252296

KX252282

KX252268

KX252240

KX252212

KX252198

KX252177

KX252170

EF-1α

KX252613

KX252606

KX252592

EU080164

KX252571

KX252564

EU079661

n.a.

EU080259

EU080178

KX252524

n.a.

EU080007

KX252506

EU080667

KX252466

KX252436

KX252400

KX252381

KX252360

KX252346

KX252318

KX252297

KX252283

KX252269

KX252241

KX252213

KX252199

KX252178

KX252171

ENL


KX252614

KX252607

KX252593

EU080165

KX252572

KX252565

EU079662

n.a.

EU080260

EU080179

EU079805

KX252519

KX252513

KX252507

EU080668

KX252467

KX252437

KX252401

KX252382

KX252361

KX252347

KX252319

KX252298

KX252284

KX252270

KX252242

KX252214

KX252200

KX252179

KX252172

Hsp90

KX252615

KX252608

KX252594

EU080166

KX252573

KX252566

EU079663

n.a.

EU080261

EU080180

EU079806

KX252520

KX252514

KX252508

EU080669

KX252468

KX252438

KX252402

KX252383

KX252362

KX252348

KX252320

KX252299

KX252285

KX252271

KX252243

KX252215

KX252201

KX252180

KX252173

28S

(Continued)

KX252616

KX252609

KX252595

EU080167

KX252574

KX252567

EU079664

n.a.

EU080262

EU080181

EU079807

KX252521

KX252515

KX252509

EU080331

n.a.

n.a.

EU080201

KX252384

KX252363

KX252349

KX252321

KX252300

KX252286

KX252272

KX252244

KX252216

KX252202

KX252181

KX252174

tigA



phylogenetic (sub)clade as indicated by the concatenated-sequence tree (TreeBASE S22998). identification: CH, Chuanxue Hong laboratory at Virginia Polytechnic Institute and State University, Virginia Beach, VA, USA; CBS, Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht, The Netherlands; ATCC, American Type Culture Collection, Manassas, VA, USA; IMI, CABI Biosciences, UK; WPC, the World Phytophthora Genetic Resource Collection at University of California, Riverside, USA. c Ex-types (T) or authentic (A) isolates (designated as representative isolates by the originators of the respective species). d Marker: cox1, cytochrome-c oxidase 1 gene; ITS, internal transcribed spacer region; 60S, 60S Ribosomal protein L10; β-tub, beta-tubulin; EF-1α, elongation factor 1 alpha; ENL, enolase; Hsp90, heat shock protein 90; 28S, 28S ribosomal DNA; tigA, tigA gene fusion protein. e n.a., not available.

Pythium aphanidermatum

101728 Outgroup Elongisporangium undulatum

a Molecular

AY564163 337230

32199 60173 135746

357.52 P. sp. boehmeriae-like 45F8

P. lilii n.a.

121982 62B5 P. morindae

46C8 P. kernoviae


Comparison of Individual-Marker Trees With Concatenated-Sequence Tree Each marker tree for all four selected markers (cox1, ITS, tigA, and β-tub) included a set of identical 150 Phytophthora taxa, plus two outgroup taxa: Elongisporangium undulatum (basionym: Pythium undulatum) was used as the outgroup taxon for ITS, tigA, and β-tub, while Pythium aphanidermatum (Uzuhashi et al., 2010) was used for the mitochondrial marker cox1. The sequence dataset of each marker was aligned in MEGA 7 and edited as described above. Then, the four alignments were combined in MEGA 7 to produce a concatenated sequence alignment. Phylogeny reconstructions including four individualmarker trees and a concatenated-sequence tree were carried out using both Maximum likelihood (ML) and Neighbor joining (NJ) methods with the K2P model and 1000 bootstrap replications in MEGA 7. Alignments and phylogenetic trees have been deposited in TreeBASE (S22998). To validate the accuracy of the concatenated-sequence trees, the clade affiliation of individual species was compared with those presented in previous phylogenetic studies. The overall topological scores between the concatenated-sequence trees and individual-marker trees were calculated using Compare2Trees version September 2011 (Nye et al., 2006).

RESULTS PCR Consistency, Amplifications, and Sequence Alignments The ITS region was the easiest genetic marker to amplify (Table 2). The rates of PCR amplification success for β-tub, 28S, 60S, Hsp90, and EF-1α were also high (>90%; Table 2). Markers tigA, ENL, and cox1 (using primer pair COXF4N and COXR4N) had relatively low success rates (≤80%) with the tigA being the most difficult (Table 2). Sequences could not be obtained from 11 taxa for 60S, 2 for β-tub, 12 for EF-1α, 12 for ENL, 3 for Hsp90, 11 for 28S, 16 for tigA, 2 for ITS, and 6 for cox1 (Table 2). These taxa were excluded from distance analyses of individual markers. Eleven taxa missing any of cox1, ITS, tigA, or β-tub sequences were also excluded in the comparison of individual-marker trees with concatenated-sequence tree. Sequence lengths were consistent for all markers except for the ITS, in spite of missing data at either or both ends of short sequences. Length of ITS sequences varied from 744 bases (P. hydrogena in cluster 9a1 of subclade 9a) to 848 bases (P. intercalaris in clade 10). Among the nine markers, the aligned length was the shortest for 60S and longest for Hsp90 (Table 2). The aligned length of concatenated sequences (cox1, ITS, tigA, and β-tub) of 150 Phytophthora taxa plus the outgroup was 4,714 bases.

Genus-Wide Distance Analyses The mean species distance of cox1 was the highest among the nine markers (Table 2). ITS had the highest genus-wide resolution among the nuclear markers, followed by tigA and βtub (Table 2). ENL, 28S, 60S, Hsp90, and EF-1α had lower species

b Isolate

EU080445 EU080441 FJ802126 1989 Scotland, UK Larix sp. P10342

T

KX252646

AB856800 AB856797

KX252645 KX252644

AB856794 AB856791

KX252643 KX252642

AB856788 AB856782

KX252641 KX252640 KF317089

MG865523

1939

KF317111

Japan

Argentina Citrus sinensis

Lilium sp. T

P1378

A

1987

AB856786

AB856779

KX252639 KX252638 KX252637 KX252636 KX252635 KX252633 MH620178 KT183050 2005 Hawaii, USA Morinda citrifolia var. citrifolia T

P10956

WPC IMI ATCC CH

CBS

Isolate identificationb Species (Sub) cladea

TABLE 1 | Continued

KX252634

KX252631 EU080046 EU080045 EU080044 EU080043 EU080042 MH620177 KT183048

Typec

Host or substrate

Rhododendron ponticum

Location

England, UK

Year

2004

60S ITS

EU080041

ENL EF-1α


cox1

β-tub


Hsp90

28S

tigA

Yang and Hong

9


Yang and Hong


TABLE 2 | PCR consistency and overall species distance across the genus Phytophthora by genetic marker. Markera

Internal primers usedb

Aligned length (bases)

No. of amplifications

No. of speciesd

Rate of PCR success (%)c

Mean distancee

Distance range

cox1 ITS

No

867

456

165

75

0.092 ± 0.0003

0–0.256

No

1,039

408

169

99

0.082 ± 0.0003

0–0.182

tigA

Yes

1,670

284

155

71

0.058 ± 0.0001

0–0.110

β-tub

No

1,136

502

169

96

0.043 ± 0.0001

0–0.087

ENL

No

1,168

345

159

80

0.035 ± 0.0001

0–0.097

28S

No

1,273

323

160

97

0.035 ± 0.0002

0–0.088

60S

No

496

344

160

97

0.030 ± 0.0001

0–0.085

Hsp90

Yes

1,758

330

168

95

0.024 ± 0.0001

0–0.116

EF-1α

No

1,015

299

159

98

0.008 ± 0.0000

0–0.024

a Marker: cox1, cytochrome-c oxidase 1 gene; ITS, internal transcribed spacer region; 60S, 60S Ribosomal protein L10; β-tub, beta-tubulin; EF-1α, elongation factor 1 alpha; ENL, enolase; Hsp90, heat shock protein 90; 28S, 28S ribosomal DNA; tigA, tigA gene fusion protein. b Internal primers for sequencing tigA and Hsp90 are listed in Blair et al. (2008). c Rate of successful PCR amplification for each marker done by the authors during the past 6 years. d Number of species (one isolate per species) included in the sequence alignment of each marker. e Overall species distance (mean ± standard error) calculated using the Kimura 2-parameter (K2P) distance model in MEGA 7.

2014; Yang et al., 2017) except that the placement of P. quercina was ambiguous. All trees from sequences of the three nuclear markers had similar topologies (score = 75.3–81.7%) to those of the concatenated-sequences trees in both ML and NJ analyses. In contrast, cox1 sequences produced trees of distinct topologies (TreeBASE S22998). The overall topological similarities to the concatenated-sequences trees were approximately 45% lower than those of nuclear markers in both analyses (Table 5).

distances (mean distance