A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis)

Dhanushka Udayanga & Xingzhong Liu &

Pedro W. Crous & Eric H. C. McKenzie &

Ekachai Chukeatirote & Kevin D. Hyde

Received: 12 June 2012 /Accepted: 12 July 2012 /Published online: 16 August 2012# Mushroom Research Foundation 2012

Abstract The genus Diaporthe (Phomopsis) includes im-portant plant pathogenic fungi with wide host ranges andgeographic distributions. In the present study, phylogeneticspecies recognition in Diaporthe is re-evaluated using amulti-locus phylogeny based on a combined data matrix ofrDNA ITS, and partial sequences from the translation elon-gation factor 1-α (EF 1-α), β tubulin (TUB) and calmodulin(CAL) molecular markers. DNA sequences of available ex-type cultures have been included, providing a multi-locusbackbone tree for future studies on Diaporthe. Four utiliz-able loci were analyzed individually and in combination,and ITS, EF 1-α and multi-locus phylogenetic trees arepresented. The phylogenetic tree inferred by combined anal-ysis of four loci provided the best resolution for species as

compared to single gene analysis. Notes are provided fornine species previously known in Phomopsis that aretransferred to Diaporthe in the present study. Theunraveling of cryptic species complexes of Diaporthebased on Genealogical Concordance PhylogeneticSpecies Recognition (GCPSR) is emphasized.

Keywords Ex-type culture . Host diversity . Mating types .

Molecular systematics . New combination . Phytopathogen .

Species recognition . Taxonomy

Introduction

The genus Diaporthe Nitschke (anamorph Phomopsis(Sacc.) Bubák) includes phytopathologically important taxawith wide host ranges and geographic distributions (Uecker1988; Crous and Groenewald 2005; Rossman et al. 2007).Diaporthe species have also been reported as endophytes inhealthy leaves and stems, saprobes on decaying wood andleaf litter, and even parasites in humans and other mammals(van Warmelo et al. 1970; Sutton et al. 1999; Garcia-Reyneet al. 2011; Iriart et al. 2011; Botella & Diez 2011; Sun et al.2011; Rocha et al. 2011). The host specificity and geograph-ic distributions of most phyopathogenic species ofDiaporthe are unknown, hindering the international ex-change of agricultural commodities (Udayanga et al. 2011;Cowley et al. 2012; Sun et al. 2012). Studies on phytopath-ogenic Diaporthe species are therefore particularly impor-tant to plant pathologists working on wide range of cropdiseases (e.g. grapes, sunflower, soybean and various dis-eases associated with fruit and ornamental trees). DNAsequence comparisons have made it possible to reliablyconnect sexual and asexual states of the species of pleomor-phic genus Diaporthe. Being the older name, Diaporthe haspriority over Phomopsis and should be the generic nameadopted for these taxa in future studies (Santos et al. 2010,

D. Udayanga : E. Chukeatirote :K. D. HydeInstitute of Excellence in Fungal Research,Mae Fah Luang University,Chiang Rai 57100, Thailand

D. Udayanga (*) : E. Chukeatirote :K. D. HydeSchool of Science, Mae Fah Luang University,Chiang Rai 57100, Thailande-mail: udayasun55@yahoo.com

D. Udayanga :X. Liu (*)State Key Laboratory of Mycology, Institute of Microbiology,Chinese Academy of Sciences,No 3 1st West Beichen Road, Chaoyang District,Beijing 100101, People’s Republic of Chinae-mail: liuxz@im.ac.cn

P. W. CrousCBS-KNAW Fungal Biodiversity Centre,Uppsalalaan 8,3584 CT, Utrecht, The Netherlands

E. H. C. McKenzieLandcare Research,Private Bag 92170,Auckland, New Zealand

Fungal Diversity (2012) 56:157–171DOI 10.1007/s13225-012-0190-9

2011; McNeill et al. 2011; Crous et al. 2011; Hawksworth2011; Wingfield et al. 2012). In exceptional cases, where thename Phomopsis is used in this study, it is used explicitly toidentify the two morphs and to distinguish between existingnames that are not yet been formally transferred toDiaporthe.

Species recognition criteria in Diaporthe have historicallybeen based on morphology, culture characteristics and hostaffiliation (Wehmeyer 1933; van der Aa et al. 1990; Rehnerand Uecker 1994; Mostert et al. 2001; van Niekerk et al. 2005;Santos and Phillips 2009). The current status of taxonomicknowledge of Diaporthe effectively means that strains can beidentified to species level only if molecular techniques areemployed (Castlebury et al. 2003; Castlebury 2005; Crous2005; Crous and Groenewald 2005; Santos et al. 2010;Udayanga et al. 2012). rDNA ITS, partial sequences of trans-lation elongation factor 1-α (EF 1-α) and mating type genes(MAT) have commonly been used in contemporary moleculartaxonomic studies of the genus (van Niekerk et al. 2005; vanRensburg et al. 2006; Santos et al. 2010, 2011; Udayanga et al.2011; Sun et al. 2012). In the current study, we infer the firstmulti-locus phylogeny of Diaporthe using combined sequen-ces of ITS, and partial sequences of EF 1-α, TUB and CALgenes. Establishing a well-resolved phylogenetic basis for thegenus is important not only for validating diagnostic methodsand resolving cryptic species (Udayanga et al. 2011), but alsofor interpreting the evolutionary history of various genetictraits of interest, such as pathogenicity (De Guido et al.2003; Kanematsu et al. 2007; Garcia-Guzman and Morales2007; Catalano et al. 2012), host diversity, geographic distri-bution (Rehner and Uecker 1994) and mating types (Santos etal. 2010).

Genealogical Concordance Phylogenetic SpeciesRecognition (GCPSR), which uses the concordance of morethan one gene genealogy in various combinations, has beenshown to provide better resolution for species as compared tospecies concepts based on morphology and reproductive be-haviour (Avise et al. 1987; Templeton 1989; Hudson andCoyne 2002; Taylor et al. 2000). Protein-coding genes arewidely used in fungal phylogenetics both for higher-leveltaxonomy and species level diagnostics with the addition ofnew molecular markers to the fungal taxonomists' toolbox(Einax and Voigt 2003; Hofstetter et al. 2007; Schmitt et al.2009; Walker et al. 2012). Multi-locus phylogenetic analyseshave become a routine procedure to identify novel fungalspecies, especially in those genera that lack distinctive mor-phological characters, and to resolve species complexes whereconventional taxonomy has resulted in confusion (Rokas et al.2003a, b; Lumbsch et al. 2005; James et al. 2006; Alves et al.2006; Schoch et al. 2006; Cai et al. 2011a, b; Manamgodaet al. 2011; Udayanga et al. 2012).

The objectives of this study were (1) to compare theeffectiveness of individual and combined gene analyses to

resolve species boundaries and relationships withinDiaporthe and (2) to provide a backbone phylogenetic treefor future studies in Diaporthe based on available ex-typecultures using a multi-gene analysis and (3) to introducenew species combinations for the well resolved species inDiaporthe based on multi-locus phylogeny, and observa-tions of ex-type cultures and specimens.

Materials and methods

Collection and isolation

Plant pathogenic and endophytic strains of Diaporthe werecollected in field surveys in different locations from varioushosts in Chiang Rai and Chiang Mai Provinces in northernThailand (Table 1). Specimens with disease symptoms wereobserved using a stereo microscope and sporulating fruitingbodies were used for single spore isolation by a modifiedspore suspension method as described for different fungalgroups (Choi et al. 1999; Chomnunti et al. 2011). Thesporulating pycnidia or ascomata were excised using a ster-ile needle, crushed with a few drops of sterile distilled waterand spore suspension was then transferred to water agar(WA) plates. The inoculated WA plates were incubated for24 h and germinating single spores were then transferred tomalt extract agar (MEA) plates and incubated at 25 °C in thedark. Endophytic fungi from leaves were isolated using theprotocol outlined by Murali et al. (2006). All fresh cultureswere deposited in Mae Fah Luang University CultureCollection (MFLUCC) and herbarium material in MFLU.Duplicate cultures are deposited in BCC and CBS, the latterunder Material Transfer Agreement (MTA: C27/2011).Details of nomenclatural novelties and new combinationswere added to MycoBank (Crous et al. 2004). Ex-type andex-epitype cultures were obtained from CBS (Utrecht,Netherlands), BRIP (Queensland, Australia), and directlyfrom authors of recently described new species ofDiaporthe (Table 1).

DNA extraction, gene amplification and sequencing

Isolates were grown on potato-dextrose agar (PDA) overlaidwith sterilized cellophane for 5 days at 25 °C (Murali et al.2006) and total genomic DNA was extracted from 0.05 to0.10 g of axenic mycelium scraped from the edge of thegrowing culture (Wu et al. 2001). Mycelium was groundwith half volume of PVP (polyvinylpyrrolidone), sterilequartz sand and 200 μl of 2 % CTAB buffer using asterilized glass pestle in micro centrifuge tubes. Then,400 μl of CTAB was added and incubated in 65 °C forabout 40 min and centrifuged at 12,000 rpm for 10 min. Thesupernatant was subjected to phenol/chloroform extraction

158 Fungal Diversity (2012) 56:157–171

Tab

le1

Isolates

used

inthisstud

y,thegenessequ

encedandGenBankaccessions

CollectionCod

eIdentity

Host

Cou

ntry

ofOrigin

Collector

GenBankAccession

numbers

Detectio

nof

mating

type

genes

ITS

EF1-α

TUB

CAL

MAT1-1-1

MAT1-2-1

CBS43

9.82

TD.cotoneastri

Coton

easter

sp.

UK,Scotland

HButin

FJ889

450

GQ25

0341

JX27

5437

JX19

7429

-+

DNP12

8T

D.castan

eae-

mollissimae

Castaneamollissima

China

SX

Jiang

JF95

7786

JX27

5401

JX27

5438

JX19

7430

++

DNP12

9D.castan

eae-

mollissimae

Castaneamollissima

China

SX

Jiang

JQ61

9886

JX27

5402

JX27

5439

JX19

7431

++

CBS16

0.32

TD.vaccinii

Oxycoccus

macrocarpus

USA

HFBain

AF31

7578

GQ25

0326

JX27

5436

n.d.

+-

CBS1132

01T

D.vticola

Vitis

vinifera

Portugal

AJL

Phillips

AY48

5750

GQ25

0327

JX27

5454

JX19

7445

--

DNP08

6-g1

D.vticola

Vitis

vinifera

Italy

XZLiu

JQ61

9896

JX27

5412

JX27

5455

JX19

7446

--

DNP08

6-g2

D.vticola

Vitis

vinifera

Italy

XZLiu

JQ61

9896

JX27

5413

JX27

5456

JX19

7447

--

CBS1134

87T

D.au

stralafrican

aVitis

vinifera

Sou

thAfrica

LMostert

AF23

0744

n.d.

JX27

5457

JX19

7448

--

MFLUCC10

-05

76aT

D.thun

bergii

Thu

nbergialaurifo

liaThailand

DSManam

goda

JQ61

9893

JX27

5409

JX27

5449

JX19

7440

-+

MFLUCC10

-05

76b

D.thun

bergii

Thu

nbergialaurifo

liaThailand

SCKarun

arathn

aJQ

6198

94JX

2754

10JX

2754

50JX

1974

41-

+

MFLUCC10

-05

76c

D.thun

bergii

Thu

nbergialaurifo

liaThailand

DUdayang

aJQ

6198

95JX

2754

11JX

2754

51JX

1974

42-

+

CBS12

6679

TD.am

ygda

liPrunu

sdu

lcis

Portugal

EDiogo

GQ28

1791

JX27

5400

JX27

5435

JX19

7428

-+

CBS1140

16T

D.neoviticola

Vitis

vinifera

France

PLarigno

nAF23

0751

GQ25

0351

JX27

5452

JX19

7443

+-

CBS10

9745

TD.perjun

cta

Ulmus

glab

raAustria

WJaklitsch

AY48

5785

GQ25

0323

JX27

5453

JX19

7444

+-

BRIP

4508

9aT

P.em

icis

Emex

australis

Auatralia

RG

Shivas

JF95

7784

JX27

5414

JX27

5458

JX19

7449

-+

BRIP

4508

9bP.

emicis

Emex

australis

Auatralia

RG

Shivas

JQ61

9898

JX27

5415

JX27

5459

JX19

7450

-+

CBS16

1.64

TD.ph

oenicicola

Areca

catechu

India

HCSivastava

FJ889

452

GQ25

0349

JX27

5440

JX19

7432

-+

MFLUCC10

-060

9Diapo

rthe

sp.

Man

gifera

sp.

Thailand

SCKarun

arathn

aJQ

6198

92JX

2754

08JX

2754

46JX

1974

37-

+

MFLUCC10

-058

7Diapo

rthe

sp.

Tecton

agran

dis

Thailand

DUdayang

aJQ

6198

90JX

2754

06JX

2754

44JX

1974

36-

+

MFLUCC10

-059

0Diapo

rthe

sp.

Cassiaspectabilis

Thailand

DUdayang

aJQ

6198

91JX

2754

07JX

2754

45n.d.

-+

MFLUCC10

-058

0aT

D.pterocarpicola

Pterocarpus

indicus

Thailand

DUdayang

aJQ

6198

87JX

2754

03JX

2754

41JX

1974

33-

+

MFLUCC10

-058

0bD.pterocarpicola

Pterocarpus

indicus

Thailand

NFWulandari

JQ61

9888

JX27

5404

JX27

5442

JX19

7434

-+

MFLUCC10

-058

3Diapo

rthe

sp.

Tecton

agran

dis

Thailand

DUdayang

aJQ

6198

89JX

2754

05JX

2754

43JX

1974

35-

+

CBS1171

69T

D.aspa

lathi

Aspalathu

slin

earis

Sou

thAfrica

JCJvanRensberg

DQ28

6275

DQ28

6249

JX27

5447

JX19

7438

-+

CBS16

2.33

TD.crotalariae

Crotalariaspectabilis

unkn

own

GFWeber

FJ889

445

GQ25

0307

JX27

5448

JX19

7439

--

MFLUCC10

-057

1D.pterocarpi

Pterocarous

indicus

Thailand

DUdayang

aJQ

6198

99JX

2754

16JX

2754

60JX

1974

51-

+

MFLUCC10

-057

5D.pterocarpi

Pterocarous

indicus

Thailand

NFWulandari

JQ61

9901

JX27

5418

JX27

5462

JX19

7453

-+

MFLUCC10

-058

8D.pterocarpi

Mag

nolia

sp.

Thailand

DUdayang

aJQ

6199

00JX

2754

17JX

2754

61JX

1974

52-

+

CBS18

7.27

TD.neotheicola

Cam

ellia

sinensis

Italy

MCurzi

DQ28

6287

DQ28

6261

JX27

5463

n.d.

--

CBS12

3208

TD.neotheicola

Foeniculum

vulgare

Portugal

AJL

Phillips

EU81

4480

GQ25

0315

JX27

5464

n.d.

++

MFLUCC10

-060

8D.ph

aseolorum

Hylocerus

unda

tus

Thailand

DUdayang

aJQ

6198

75JX

2753

89JX

2754

24JX

1974

18-

+

MFLUCC10

-060

3D.ph

aseolorum

Hylocerus

unda

tus

Thailand

DUdayang

aJQ

6198

76JX

2753

90JX

2754

25JX

1974

19-

+

Fungal Diversity (2012) 56:157–171 159

Tab

le1

(con

tinued)

CollectionCod

eIdentity

Host

Cou

ntry

ofOrigin

Collector

GenBankAccession

numbers

Detectio

nof

mating

type

genes

ITS

EF1-α

TUB

CAL

MAT1-1-1

MAT1-2-1

CBS50

7.78

TD.melon

isCucum

ismelo

USA

LBerha

FJ889

447

GQ25

0314

JX27

5423

JX19

7417

++

CBS1140

15T

D.am

bigu

aPyrus

commun

isSou

thAfrica

SDenman

AF23

0767

GQ25

0299

JX27

5434

JX19

7427

++

CBS59

2.81

TD.helia

nthi

Helianthu

san

nuus

Serbia

MMun

tano

laCvetkov

icAY70

5842

GQ25

0308

JX27

5465

JX19

7454

+-

CBS1115

92T

D.an

gelicae

Heracleum

spho

ndylium

Austria

WJaklitsch

AY19

6779

GQ25

0302

n.d.

n.d.

--

CBS19

3.36

TD.stew

artii

Cosmos

bipinn

atus

unkn

own

ALHarrison

FJ889

448

GQ25

0324

JX27

5421

JX19

7415

-+

CBS1174

99T

D.cupp

atea

Aspalathu

slin

earis

Sou

thAfrica

JCJvanRensberg

AY33

9322

AY33

9354

JX27

5420

JX19

7414

+-

CBS12

3212

TD.lusitanicae

Foeniculum

vulgarae

Portugal

JMSantos

GQ25

0190

GQ25

0311

JX27

5422

JX19

7416

+-

CBS29

6.67

TD.sclerotio

ides

Cucum

issativus

Netherlands

HAVan

derKesteren

AF43

9626

GQ25

0350

JX27

5426

JX19

7420

+-

CBS19

4.36

TD.strumella

Ribes

sp.

Canada

LEWehmeyer

FJ889

449

GQ25

0325

JX27

5427

n.d.

+-

MFLUCC10

-060

1Diapo

rthe

sp.

Coffeaarab

ica

Thailand

DUdayang

aJQ

6199

02JX

2754

19JX

2754

66JX

1974

55-

+

MFLUCC10

-058

4Diapo

rthe

sp.

Tecton

agran

dis

Thailand

DUdayang

aJQ

6198

84JX

2753

98n.d.

n.d.

-+

MFLUCC10

-058

2Diapo

rthe

sp.

Aeschynan

thus

radicans

Thailand

SCKarun

arathn

aJQ

6198

85JX

2753

99JX

2754

33JX

1974

26-

+

MFLUCC10

-057

0Diapo

rthe

sp.

Deadwoo

d-un

know

nThailand

DUdayang

aJQ

6198

77JX

2753

91JX

2754

28JX

1974

21-

+

DEN

009

Diapo

rthe

sp.

Tecton

agran

dis

Thailand

DUdayang

aJQ

6198

82JX

2753

97JX

2754

32JX

1974

25-

+

MFL

UCC10-0573a

TD.siam

ensis

Dasym

aschalon

sp.

Thailand

DUdayang

aJQ

6198

79JX

2753

93JX

2754

29JX

1974

23-

+

MFLUCC10

-057

3bD.siam

ensis

Dasym

aschalon

sp.

Thailand

NFWualand

ari

JQ61

9880

JX27

5395

JX27

5430

JX19

7423

-+

MFLUCC10

-057

3cD.siam

ensis

Dasym

aschalon

sp.

Thailand

DUdayang

aJQ

6198

81JX

2753

96JX

2754

31JX

1974

24-

+

MFLUCC10

-058

9Diapo

rthe

sp.

Mag

nolia

sp.

Thailand

DUdayang

aJQ

6198

78JX

2753

92n.d.

n.d.

-+

MFLUCC10

-058

1Diapo

rthe

sp.

Rha

pissp.

Thailand

DUdayang

aJQ

6198

83JX

2753

94n.d.

n.d.

-+

MFLUCC:Mae

Fah

Luang

University

Culture

CollectionCBS:Centraalbureauvo

orSchim

melcultu

res,Utrecht,The

Netherlands

BRIP:Australianplantpathog

encultu

recollection,

Queensland

DNP/DEN:Firstauthor’sperson

alcollection(depositedin

MFLUCC),T:ex-typ

e/ex-epitype

isolates

(+):Matingtype

gene

present,(−):matingtype

genesabsent,n.d.

:no

tdeterm

ined,JX

,JQ

prefixes

ofaccessionnu

mbers:sequ

encesgeneratedin

thisstud

y,Referencesforothersequ

encesas

listedin

Udayang

aet

al.20

11

160 Fungal Diversity (2012) 56:157–171

followed by precipitation of DNA from the aqueous phasewith ice-cold iso-propanol at 4 °C for 1 h. Precipitated DNAwas recovered by centrifugation of 12,000 rpm for 10 minand washed with 70 % ethanol, air dried, dissolved in 50 μlof TE buffer and stored at −20 °C until use for amplificationreactions.

Six loci were sequenced including rDNA ITS (White etal. 1990), EF 1-α, CAL (Carbone and Kohn 1999), TUB(Glass and Donaldson 1995), MAT 1-1-1 and MAT 1-1-2(Santos et al. 2010). The primers, references and PCR pro-tocols are summarized in Table 2. The 50 μl reaction vol-ume (1×PCR buffer, 0.2 mM dNTP, 0.4 μM of each primer;1.5 mM MgCl2, 2 % foramide/1 % DMSO (variable), 0.8units Taq Polymerase and 10 ng template DNA), was usedfor each of the reaction with the adjustments of componentswhen needed.

The PCR products, spanning approximately 300–500 bp(ITS, EF 1-α, TUB, CAL) were visualized on 1 % agarosegels stained with Goldview (Geneshun Biotech, China) withD2000 DNA ladder (Realtimes Biotech, Beijing, China).For the MAT 1-1-1 and MAT 1-2-1 genes (200 and300 bp, respectively), the amplicons were subjected to si-multaneous electrophoresis in 1.5 % agarose gels with a100 bp DNA ladder (Realtimes Biotech, Beijing, China) in100 V for 45 min and visualized in GelDoc image system(Bio-Rad) with modifications as described in Santos et al.(2010). All the PCR products were then purified accordingto the company protocols and DNA sequencing was per-formed using the above-mentioned primers in an AppliedBiosystem 3730 DNA analyzer at the SinogenomaxCompany, Beijing, China.

Sequence alignment and phylogenetic analyses

Sequence homologies for the assembled consensus sequen-ces were analyzed using the BLAST search engine of theNational Center for Biotechnology Information (NCBI) andfor the rough identification of fresh isolates used in theanalyses. Sequences of the available ex-type cultures were

obtained from GenBank (Table 1) listed in Udayanga et al.(2012). The consensus sequences for each gene were initiallyaligned by Clustal-W as implemented in Bioedit (Thompsonet al. 1994) and improved in MAFFTv6 (Katoh et al. 2002;Katoh and Toh 2008), online sequence alignment editor underthe default settings (mafft.cbrc.jp/alignment/server/) andoptimized manually when needed. Ambiguously alignedregions were excluded from all the analyses and con-firmed that there is not conflict in datasets. A partitionhomogeneity test (PHT0ILD0Incongruency LengthDifference Test, Farris et al. 1994) was applied as imple-mented in PAUPv4.0b10 (Swofford 2002) to evaluate thefeasibility of combining datasets. PAUPv4.0b10 wasused to conduct the parsimony analysis to obtain thephylogenetic trees. Trees were inferred using the heuris-tic search option with 1000 random sequence additions.Maxtrees were unlimited, branches of zero length werecollapsed and all multiple parsimonious trees weresaved. Descriptive tree statistics for parsimony (TreeLength [TL], Consistency Index [CI], Retention Index[RI], Relative Consistency Index [RC] and HomoplasyIndex [HI] were calculated for trees generated underdifferent optimality criteria. Kishino-Hasegawa tests(KHT) (Kishino & Hasegawa 1989) were performed inorder to determine whether trees were significantly dif-ferent. Trees were figured in Treeview (Page 1996). Intotal, five data matrices were analyzed and compared:based on rDNA ITS sequences (data matrix I), EF 1-αsequences (data matrix II), TUB sequences (data ma-trix III), CAL sequences (data matrix IV), combinedITS, TUB, EF 1-α, CAL genes (data matrix V). Forthe Data matrix V, Bayesian analysis was performed asdescribed in Phillips et al. (2007), setting burn-in at2000 generations. The amplification reactions for MAT1-1-1 and MAT 1-2-1 genes were performed, but thesequences were not used in phylogenetic analysis. Theresults of the detection of mating type genes via simul-taneous electrophoresis as described in Santos et al.(2010) are presented in Table 1.

Table 2 Genes/loci used in the study with PCR primers, references and protocols

Gene/loci ITS CAL EF 1-α TUB MAT 1-1-1 MAT 1-2-1

PCR primers (for/rev) ITS 1/ITS4

CAL228F/CAL737R

EF1-728F/EF1-986R

Bt2a/Bt2b MAT1-1-1-FW/MAT1-1-1RV

MAT1-2-1FW/MAT1-2-1RV

References for primers used White et al.1990

Carbone andKohn 1999

Carbone andKohn 1999

Glass andDonaldson1995

Santos et al. 2010 Santos et al. 2010

PCR: thermal cycles: a

(Annealing temp. in bold)(95 °C : 30 s, 55 °C:50 s,72 °C:1 min) ×40 cycles

(95 °C: 30 s, 58 °C:50 s,72 °C:1 min) ×40 cycles

(94 °C : 30 s, 50 °C (94 °C; 30 s, 56 °C :

Same conditions areapplicable to both markers

Same conditions are applicableto both markers

30 s, 72 °C:1 min)×40 cycles

30 s, 72 °C:1 min)×40 cycles

a All the PCR thermal cycles include Initiation step of 95 °C: 5 min, and final elongation step of 72 °C: 10 min and final hold at 4 °C

Fungal Diversity (2012) 56:157–171 161

Results

One hundred and fifty new sequences were generated in thisstudy (Table 1) from 23 ex-type cultures and fresh collec-tions of Diaporthe from northern Thailand and elsewhere.Other sequences were downloaded from GenBank and usedin the phylogenetic analysis.

PCR optimization and phylogenetic performance of selectedloci

PCR conditions at optimum annealing temperature andreagent concentrations were optimized to develop effectiveamplification of ITS, EF, TUB and CAL loci. The optimumannealing temperatures were recognized to amplify ITS,CAL (55 °C) and EF, TUB (58 °C). This procedure reducesthe time required for working with multiple strains ofDiaporthe to generate the multiple DNA sequences. TwoPCR thermal cycling conditions can be used for the ampli-fication of four loci in separate systems. Phylogenetic per-formance of each locus was compared with the multilocusphylogeny based on alignment properties of data matricesand selected characters in parsimony analysis (Table 3).

rDNA ITS sequences in species delimitation

The rDNA ITS phylogenetic tree (Fig. 1) consists of sequen-ces derived from fresh isolates and ex-type, ex-epitype andauthentic sequences as indicated in the ITS backbone phy-logenetic tree (Udayanga et al. 2011). The ITS data matrixcontains 52 taxa including the outgroup and averaging 522characters (including gaps); 45 characters are excluded inthe parsimony analysis. The resulting statistics for the par-simony analysis revealed that 309 characters are constant,95 characters are parsimony informative and 73 variablecharacters are parsimony-uninformative. The parsimonyanalysis yielded 50 equally parsimonious trees and the first

tree (Fig. 1) was recognized as the best tree and presentedhere as the basic identification guide to the isolatesused in this study (TL0368, CI00.563, RI00.797,RC00.448, HI00.438).

The phylogenetic tree (Fig. 1) includes ex-type culturesfrom a wide range of hosts and various geographic locationswhile the fresh isolates were chiefly from northern Thailand(Table 1). Although the terminal nodes differentiate eachtaxon included in the analysis, the bootstrap support valueswere inconclusive in most cases and unable to distinguishcryptic taxa.

EF 1-α sequences in species delimitation

The EF 1-α data matrix contains 52 taxa including theoutgroup and averaged 484 characters (including gaps) 45characters were excluded in the parsimony analysis. Thestatistics for the parsimony analysis revealed that 80 char-acters are constant, 254 characters are parsimony informa-tive, while 67 variable characters are parsimony-uninformative. The parsimony analysis of the alignmentyielded six equally parsimonious trees and the first tree(Fig. 2) was recognized as best tree and presented here asthe basic identification guide to the isolates used in thisstudy (TL01378, CI00.464, RI00.745, RC00.346, HI00.536). The amplified segment contains part of EF 1-α genespanning an entire intron (with more variable characters)and partial sequences of the flanking exons. Some isolatesidentified to be the same species based on EF 1-α phylog-eny (with 100 % bootstrap similarity), were further tested inITS, TUB, CAL and MAT 1-2-1 phylogenetic trees (data notshown). We observed that the groups of isolates identified assiblings in terminal clades in the EF 1-α phylogeny showphylogenetic variability when employing different proteincoding genes used in this study. Therefore we recognizedthat there may be distinct phylogenetic species that areobscured in the analysis of the EF 1-α gene.

Table 3 Comparison of PCR success and alignment properties of the parsimony analysis of genes/loci used in phylogenetic analysis

Genes/loci ITS EF TUBa CALa Combined ITS/EF/TUB/CAL

PCR success 100 % 99 % 95 % 95 % -

Alignment strategy (MAFFT v6) FFT-NS-I FFT-NS-I+manual FFT-NS-I FFT-NS-I -

Characters included (with gaps) 522 484 479 555 2040

Characters excluded in parsimony analysis 45 45 - 54 144

Invariable characters 309 80 226 211 826

Parsimony informative characters (%) 95 (18 %) 254 (52 %) 167 (35 %) 245 (44 %) 761 (37 %)

Uninformative polymorphic characters 73 67 86 43 269

Number of branches >70 % bootstrap inparsimony analysis

15 24 25 23 32

a Phylogenetic trees are not shown

162 Fungal Diversity (2012) 56:157–171

Combined analysis of ITS, EF1-α, CAL and β- tubulingenes

The combined gene data matrix contains 52 taxa includingthe outgroup and an average of 2040 characters; 184

characters were excluded in parsimony analysis. The statis-tics for the parsimony analysis revealed that 826 charactersare constant, 761 characters are parsimony informative,while 269 variable characters are parsimony-uninformative.The parsimony analysis of the alignment yielded four equally

Fig. 1 The phylogram inferredfrom a parsimony analysis ofrDNA ITS sequences ofDiaporthe from ex-type and (inbold), and newly generatedsequences from fresh isolates.MP bootstrap values >70 % inthe 1000 replications are shownabove or below the branches.The existing names ofPhomopsis (P), acceptednames, new combinations andfresh collections are named asDiaporthe (D). *: novel combi-nations in this publication, **:taxa newly described inUdayanga et al. 2012 (in press).The tree is rooted with Valsaambiens.

Fungal Diversity (2012) 56:157–171 163

parsimonious trees and the first tree (Fig. 3) was recog-nized as the best tree and presented here as the basicidentification guide to the isolates used in this study(TL03460, CI00.492, RI0, 0.747, RC00.368, HI0

0.508). Based on the combined phylogenetic tree, werecognized that most of the ex-type derived taxa areplaced in terminal clades and highly supported, withoutconflict between well-recognized taxa. However the

Fig. 2 The phylogram inferredfrom a parsimony analysis ofEF1-α sequences from ex-type,ex-epitype (in bold) and newlygenerated sequences ofDiaporthe. MP bootstrapvalues >70 % in the 1000 rep-lications are shown above orbelow the branches. The exist-ing names of Phomopsis (P),accepted names, new combina-tions and fresh collections arenamed as Diaporthe (D). *:novel combinations, **: taxanewly described in Udayanga etal. 2012 (in press). The tree isrooted with Valsa ambiens.

164 Fungal Diversity (2012) 56:157–171

branch lengths of several sub-clades are shorter, indicating thespeciation occurred in short time frames. The terminal branchsupport values and additional internal branch support values(>70 %) has been increased by combining the four genescompared to ITS and EF 1-α phylogenetic trees.

Taxonomy

In this section we transfer nine species of Phomopsis toDiaporthe based on multi-locus DNA sequence data, anno-tated with the details of ex-type and ex-epitype isolates and

Fig. 3 Phylogram inferredfrom parsimony analysis of fourcombined genes (rDNA ITS,EF 1-α, CAL and TUBsequences). The existing namesof Phomopsis (P), acceptednames, new combinations andfresh collections are named asDiaporthe (D). Ex-type and ex-epitype cultures are in bold. Thebootstrap support values >70 %from 1000 replicates are shownbelow or above the branchesfollowed by Bayesian posteriorprobabilities. *: new speciescombinations proposed in thisstudy, **: taxa newly describedin Udayanga et al. 2012 (inpress). The tree is rooted withValsa ambiens.

Fungal Diversity (2012) 56:157–171 165

specimens. Brief notes are given where the novel combina-tion is not straight forward.

Diaporthe amygdali (Delacr.) Udayanga, Crous & K.D.Hyde, comb. nov.MycoBank 800722

≡ Phomopsis amygdali (Delacr.) J.J. Tuset & M.T. Portilla,Can. J. Bot. 67(5): 1280 (1989)≡ Fusicoccum amygdali Delacr., Bull. Soc. mycol. Fr. 21:280 (1905)

Specimen examined: PORTUGAL, Trás-os-Montes,Mirandela, on twigs of Prunus dulcis, Sept. 2005, E.Diogo (CBS-H 20420, epitype), ex-epitype culture: CBS126679, ex-epitype sequence: GQ281791 (ITS).

Diaporthe castaneae-mollisimae (S.X, Jiang & H.B. Ma)Udayanga, Crous & K.D. Hyde, comb. nov.MycoBank 800702

≡ Phomopsis castaneae-mollisimae S.X. Jiang & H.B. Ma,Mycosystema 29: 467 (2010)

Specimen examined: CHINA, Shangdong Province, onleaves of Castanea mollissima, April 2006, S.X. Jiang(CLS-0612, holotype), ex-type culture: DNP 128, ex-typesequence: JF 957786 (ITS).

Diaporthe cotoneastri (Punith.) Udayanga, Crous & K.D.Hyde, comb. nov.MycoBank 800697

≡ Phomopsis cotoneastri Punith., Trans. Br. mycol. Soc. 60(1): 157 (1973)

Specimen examined: SCOTLAND, Ayr, on Cotoneaster sp.,May 1982, H. Butin (CBS-H 7633: isotype), ex-isotypeculture: CBS 439.82, ex-isotype sequence: FJ889450(ITS).

Diaporthe cuppatea (E. Jansen, Lampr. & Crous)Udayanga, Crous & K.D. Hyde, comb. nov.MycoBank 800698

≡ Phomopsis cuppatea E. Jansen, Lampr. & Crous, Stud.Mycol. 55: 72 (2006)

Specimen examined: SOUTH AFRICA, Western CapeProvince, on Aspalathus linearis, 2006, J. Janse vanRensburg (CBS H-19687, holotype), ex-type culture: CBS117499, ex-type sequence: AY339322 (ITS).

Diaporthe neotheicola A.J.L. Phillips & J.M. Santos,Fungal Diversity 34: 120 (2009)

0 Phomopsis theicola Curzi, Atti Ist. bot. R. Univ. Pavia, 3Sér. 3: 65 (1927)

Notes: The sexual morph of Phomopsis theicola was orig-inally proposed as a distinct taxon from Diaporthe theicolaCurzi, and was described as Diaporthe neotheicola inSantos and Phillips (2009). The multi-locus phylogeny(Fig. 3), however, reveals that the ex-type isolates of P.theicola and D. neotheicola represent the same taxon as alsostated by Santos and Phillips (2009). Because the nameDiaporthe theicola is already occupied, we opt to use thename D. neotheicola. Further study and epitypification of D.theicola Curzi is needed.Specimens examined: PORTUGAL Évora, on Foeniculumvulgare, Nov. 2007, A.J.L. Phillips (CBS-H 20131, holo-type), ex-type culture: CBS 123208, ex-type sequence:EU814480 (ITS).

Diaporthe phoenicicola (Traverso & Spessa) Udayanga,Crous & K.D. Hyde, comb. nov.MycoBank 800699

≡ Phomopsis phoenicicola Traverso & Spessa, Bolm Soc.broteriana, Coimbra, sér. 1 25: 177 (1910)

0 Subramanella arecae H.C. Srivastava., Zakia &Govindar., Mycologia 54(1): 7 (1962)Notes: The sequences available as P. phoenicicola are de-rived from an isotype of Subramanella arecae which is alater synonym. An ex-type culture of P. phoenicicola doesnot exist, and therefore the ex-isotype of Subramanellaarecae will serve as the representative strain for this taxonuntil revisited.Specimen examined: INDIA, on fruit of Areca catechu, Feb.1964, H.C. Srivastava (CBS H-7808, isotype), ex-isotypeculture: CBS 161.64, ex-isotype sequence: FJ889452(ITS).

Diaporthe sclerotioides (Kesteren) Udayanga, Crous &K.D. Hyde, comb. nov.MycoBank 800700

≡ Phomopsis sclerotioides Kesteren, Neth. Jl Pl. Path. 73:115 (1967)

Specimen examined: NETHERLANDS, Maarssen, on rootsof Cucumis sativus, June 1967, H.A. Van der Kesteren (IMI151828, PD 68/690, holotype), ex-type culture: CBS296.67, ex-type sequence: AF439626 (ITS).

Diaporthe neoviticola Udayanga, Crous & K.D. Hyde,nom. nov.MycoBank 800717

≡ Phoma viticola Sacc., Michelia 2: 92 (1880)≡ Phomopsis viticola (Sacc.) Sacc., Ann. Mycol. 13: 118(1915)0 Fusicoccum viticolum Reddick, Cornell Univ. Agr. Exp.Sta. Bull. 263: 331 (1909)

166 Fungal Diversity (2012) 56:157–171

≡ Phomopsis viticola (Reddick) Goid., Atti R. Accad. Naz.Lincei 26: 107 (1937)0 Phomopsis viticola Sacc. var. ampelopsidis Grove, Bull.Misc. Inf. (Kew) 4: 183. (1919)0 Phomopsis ampelina (Berk. & Curt.) Grove, Bull. Misc.Inf. (Kew) 4: 184 (1919)

Notes: Diaporthe viticola Nitschke (1870) is a well-known,distinct taxon thus the epithet is occupied (Mostert et al.2001, van Niekerk et al. 2005). Mostert et al. (2001) neo-typified P. viticola (CBS 114016), for which D. neoviticolais proposed as new name.Specimen examined: FRANCE, Bordeaux, Naujan-et-Postiac, on Vitis vinifera (Cabernet Sauvignon grapevine),May 1998, P. Larignon (PREM 56460, BPI 871304: neo-type), ex-neotype culture: CBS 114016, ex-neotype se-quence: AF2390751 (ITS).

Discussion

Individual genes versus combined genes in resolvingspecies within Diaporthe

We evaluated the phylogenetic species recognition ofDiaporthe based on the four utilizable loci individuallyand in combination to establish robust concept to circum-scribe species in the genus.

rDNA ITS as a phylogenetic marker of Diaporthe

The rDNA ITS phylogenetic tree generated here is based onthe phylogenetic backbone tree presented in Udayanga et al.(2011) as a rough and quick identification guide for freshisolates of Diaporthe species. Analyses of rDNA ITS cou-pled with morphology, pathogenicity or EF 1-α sequencedata have been used in successful taxonomic revisions incontemporary molecular phylogenetic studies (Farr et al.2002a; van Rensburg et al. 2006; Santos and Phillips2009; Diogo et al. 2010; Santos et al. 2011; Thompson etal. 2011). ITS sequences provide persuasive evidence forspecies delineation with a few distantly related taxa ana-lyzed (e.g.; species associated with the diseases of soybean,and sunflower; Jurković et al. 2007; Thompson et al. 2011;Santos et al. 2011), but confusion occurs when large numb-ers of species from a wide range of host species are ana-lyzed. Typically, branches in phylogenetic trees arebifurcate. Any node that has only two intermediate dece-dents is said to be resolved. The internal nodes are polyto-mous (more than two descendents i.e., sister taxa), whererelationships are unclear. This can be seen in ITS phylo-grams of Diaporthe when large numbers of taxa are incor-porated. A large amount of homoplasy (similarity of

sequences among species of different ancestry) across thegenus may have resulted in a large number of most parsimo-nious trees using ITS sequence data (Farr et al. 2002a). Theseproblems can be eliminated in the combined gene analysis wehave used in this study. Morphological and culture-basedstudies have revealed that there may be different species thatare not well resolved using only ITS sequence data in prelim-inary analyses (Farr et al. 2002a, b; van Rensburg et al. 2006;Thompson et al. 2011). Different gene genealogies are there-fore needed to resolve the cryptic species of Diaporthe byeither individual or combined gene analysis.

In the barcoding initiatives of a wide range of fungi, ITSsequence data can reliably identify 73 % of taxa studiedacross kingdom Fungi (Schoch et al. 2012). The ITS regionhas also been used to develop molecular markers, species-specific probes and other alternative and comparative assaysin the detection of pathogens of Diaporthe which is signif-icantly important in rough and quick identification for plantpathogens (Zhang et al. 1997, 1998; Moleleki et al. 2002).

Multiple protein coding genes as phylogenetic markers

An approximately 350 base pair region of the translationelongation factor-1 alpha gene (EF 1-α) has been used, tobetter resolve the species in Diaporthe in several consecu-tive studies (Castlebury et al. 2001; Castlebury 2005; vanRensburg et al. 2006; Santos et al. 2010). The speciesresolved by the EF 1-α gene is congruent with the taxaidentified by MAT gene genealogies. We compared individ-ual gene analysis of rDNA ITS and EF 1-α, TUB, CAL andcombined analysis. Species resolved in the terminal nodesof EF 1-α and combined gene analyses were congruent witha few exceptions. For instance, Diaporthe pterocarpicola isnot clearly differentiated as a distinct species using EF 1-αanalysis. However this was resolved when we combined therDNA ITS, EF 1-α, TUB, CAL genes, where the phyloge-netic species, D. pterocarpicola is distinguished from D.phoenicicola (Udayanga et al. 2012).

The TUB sequences yielded concordant support for thespecies recognized in EF 1-α phylogenies with higherbranch support values in terminal clades (phylogram notshown). Therefore, the TUB gene could also be used as aphylogenetic marker for Diaporthe to assess diversity and todelineate taxonomic units where the confusion occurs insingle gene analysis with ITS and EF 1-α gene sequences.When compared to rDNA ITS and EF 1-α sequence data-sets, the TUB data matrix contains fewer ambiguouslyaligned regions and less homoplasy across the genus, andshould be considered as secondary phylogenetic marker forthe genus. The CAL sequences yielded less resolution forsome of the cryptic species, although the overall data matrixcontains a higher percentage of variable characters. Thiswould be, due to ambiguous alignment in the second intron

Fungal Diversity (2012) 56:157–171 167

of the CAL data matrix, thus technically eliminated in all theanalyses. For instance D. viticola and D. australafricanacould not be reliably distinguished when employing onlyCAL genes in the phylogenetic analysis. However, D. viti-cola and D. australafricana, two species associated withgrapevines in Europe, Africa and Australia are distinct taxaand represent the ex-type sequence data incorporated in theanalysis. Therefore care should be taken when interpretingthe circumscription of some of the cryptic species ofDiaporthe based on CAL gene genealogies. However, theCAL gene sequences are also able to resolve the speciessimilarly congruent with EF, and TUB, and are thus recom-mended in the combined gene analysis.

This study confirms that all four gene regions used in thisstudy are useful markers to assess diversity and identifyspecies boundaries and relationships of Diaporthe.However, the combined gene analysis provides robust sup-port to delineate cryptic species at the terminal nodes, and torecognize sub-clades of closely related taxa across thegenus.

Genealogical concordance phylogenetic speciesrecognition: impact on accurate identificationof species

The multi-locus phylogenetic tree presented here has basi-cally resolved the problems of phylogenetic species inDiaporthe. However, we have observed a high correlationin some of the monophyletic sub-clades (closely relatedtaxa), which should be revised in future studies.

The adoption of Genealogical Concordance PhylogeneticSpecies Recognition (GCPSR) has profound implicationsfor accurate species recognition and resolution of complexesof cryptic taxa (Taylor et al. 2000; Cai et al. 2011a, b).Cryptic species of common plant pathogenic genera (e.g.Armillaria, Coetzee et al. 2005; Fusarium, Aoki et al. 2005,Summerell et al. 2010, Summerell and Leslie 2011;Calonectria, Lombard et al. 2010; Plagiostoma, Mejia etal. 2011; Phyllosticta, Glienke et al. 2011; Wikee et al. 2011,Wang et al. 2011; Cercospora, Groenewald et al. 2005,Crous et al. 2006; Colletotrichum, Crouch et al. 2009;Phoulivong et al. 2010; Damm et al. 2012), and Diaporthe(present study) have been resolved using a combined geneanalysis. Therefore, GCPSR has become a tool to unravelthe cryptic species complexes especially, where there is adearth of distinguishing morphological characters (Hyde etal. 2011; Shivas and Cai 2011). The multi-locus phylogenyof Diaporthe would serve as a fundamental tool for consol-idating the evolutionary biology, host diversity, biogeo-graphic structure and ecology of this non-modal organism.Therefore future studies are needed based on collectionsfrom various geographical locations worldwide as shownin Diaporthe and other taxa in Diaporthales (Rehner and

Uecker 1994, Zhang et al. 1998, Zhang 2002, Walker et al.2012, Mejia et al. 2011a, b).

In conclusion, the backbone phylogenetic tree compris-ing type-derived sequences presented here provides an ad-ditional resource for accurate species identification, resolvecryptic species complexes of Diaporthe in future studies.The multi-locus phylogeny and observations of ex-typecultures and specimens has made it possible to reliablyconnect the sexual and asexual states of Diaporthe withrequired changes in nomenclature concerning one namefor one biological species. More ex-type sequences need tobe added to the data matrix to increase its validity, thusaiding identification of species and avoiding misapplicationof names.

Acknowledgements This project is supported by the State Key Lab-oratory of Mycology, Institute of Microbiology by grant NFSCY2JJ011002 and Thailand Research fund BRG 52800002. DhanushkaUdayanga thanks the State Key Laboratory of Mycology, Institute ofMicrobiology, Chinese Academy of Sciences, Beijing and the Mush-room Research Foundation, Chiang Mai, Thailand for a postgraduatescholarship. Lei Cai (CAS-Beijing), Nilam F. Wulandari (Chiang MaiUniversity, Thailand) and Samantha C. Karunarathna, Dimuthu S.Manamgoda (Mae Fah Luang University, Thailand) are thanked forproviding fungal specimens

References

Alves A, Correia A, Phillips AJL (2006) Multi-gene genealogies andmorphological data support Diplodia cupressi sp. nov., previouslyrecognized as D. pinea f. sp. cupressi, as a distinct species. FungalDivers 23:1–15

Aoki T, O'Donell K, Scandiani MM (2005) Sudden death syndrome ofsoybean in South America is caused by four species of Fusarium:Fusarium brasiliense sp. nov., F. cuneirostrum sp. nov., F. tucu-maniae, and F. virguliforme. Mycoscience 46:162

Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE,Reeb CA, Saunders NC (1987) Intraspecific Phylogeography: theMitochondrial DNA Bridge between Population Genetics andSystematics. Ann Rev Ecol Syst 18:489–522

Botella L, Diez JJ (2011) Phylogenic diversity of fungal endo-phytes in Spanish stands of Pinus halepensis. Fungal Divers47:9–18

Cai L, Giraud T, Zhang N, Begerow D, Cai G, Shivas RG (2011a) Theevolution of species concepts and species recognition criteria inplant pathogenic fungi. Fungal Divers 50:121–133

Cai L, Udayanga D, Manamgoda DS, Maharachchikumbura SSN, LiuXZ, Hyde KD (2011b) The need to carry out re-inventory oftropical plant pathogens. Trop Plant Pathol 36:205–213

Carbone I, Kohn L (1999) A method for designing primer sets forspeciation studies in filamentous ascomycetes. Mycologia91:553–556

Castlebury L (2005) The Diaporthe vaccinii complex of fruit patho-gens. Inoculum 56:12

Castlebury LA, Farr DF, Rossman AY (2001) Phylogenetic distinctionof Phomopsis isolates from cucurbits. Inoculum 52:25

Castlebury LA, Farr DF, Rossman AY, Jaklitsch WJ (2003) Diaportheangelicae comb. nov., a modern description and placement ofDiaporthopsis in Diaporthe. Mycoscience 44:203–208

168 Fungal Diversity (2012) 56:157–171

Catalano V, Rekab D, Firrao G, Vannacci G, Vergara EM (2012) Anendopolygalacturonase gene of Diaporthe helianthi. PhytopatholMediterr 51:23–36

Choi YW, Hyde KD, Ho WH (1999) Single spore isolation of fungi.Fungal Divers 3:29–38

Chomnunti P, Schoch CL, Aguirre-Hudson B, Ko-Ko TW, HongsananS, Jones EBG, Kodsueb R, Phookamsak R, Chukeatirote E,Bahkali AH, Hyde KD (2011) Capnodiaceae. Fungal Divers51:103–134

Coetzee MPA, Wingfield BD, Bloomer P, Wingfield MJ (2005) Phy-logenetic analyses of DNA sequences reveal species partitionsamongst isolates of Armillaria from Africa. Mycol Res109:1223–1234

Cowley RB, Ash GJ, J Harper JDI, Luckett DJ (2012) Evaluation ofresistance to Phomopsis stem blight (caused by Diaporthe toxica)in Lupinus albus. Eur J Plant Pathol. 133:631–644

Crouch JA, Clarke BB, Hillman BI (2009) What is the value of ITSsequence data in Colletotrichum systematics and species diagno-sis? A case study using the falcate-spored graminicolous Colleto-trichum group. Mycologia 101:648–656

Crous PW (2005) Impact of molecular phylogenetics on the taxonomyand diagnostics of fungi. Bull OEPP/EPPO 35:47–51

Crous PW, Groenewald JZ (2005) Hosts, species and genotypes:opinions versus data. Australas Plant Pathol 34:463–470

Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004)MycoBank: an online initiative to launch mycology into the21st century. Stud Mycol 50:19–22

Crous PW, Groenewald JZ, Groenewald M, Caldwell P, Braun U,Harrington TC (2006) Species of Cercospora associated with greyleaf spot of maize. Stud Mycol 55:189–197

Crous PW, Groenewald JZ, Shivas RG, Edwards J, Seifert KA et al(2011) Fungal planet description sheets: 69–91. Persoonia26:108–156

Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir BS, TanYP, Shivas RG, Crous PW (2012) The Colletotrichum boninensespecies complex. Stud Mycol 73:1–36

de Guido MA, Pollastro S, Carlucci A, de Miccolis Angelini RM,Faretra F (2003) Phomopsis viticola is easily transformed withhph and Bmlr genes. J Plant Pathol 85:43–52

Diogo ELF, Santos JM, Phillips AJL (2010) Phylogeny, morphologyand pathogenicity of Diaporthe and Phomopsis species onalmond in Portugal. Fungal Divers 44:107–115

Einax E, Voigt K (2003) Oligonucleotide primers for the universalamplification of β-tubulin genes facilitate phylogenetic analyses.Organ Divers Evol 3:185–194

Farr DF, Castlebury LA, Rossman AY, Putnam ML (2002b) A newspecies of Phomopsis causing twig dieback of Vaccinium vitis-idaea (lingonberry). Mycol Res 106:745–752

Farr DF, Castlebury LA, Rossman AY (2002b) Morphological andmolecular characterization of Phomopsis vaccinii and additionalisolates of Phomopsis from blueberry and cranberry in the easternUnited States. Mycologia 94:494–504

Farris JS, Kallersjo M, Kluge AG, Bult C (1994) Testing significanceof incongruence. Cladistics 10:315–320

Garcia-Guzman G, Morales E (2007) Life-history strategies of plantpathogens: distribution patterns and phylogenetic analysis. Ecol-ogy 88:589–596

Garcia-Reyne A, López-Medrano F, Morales JM, García Esteban C,Martín I, Eraña I, Meije Y, Lalueza A, Alastruey-Izquierdo A,Rodríguez-Tudela JL, Aguado JM (2011) Cutaneous infection byPhomopsis longicolla in a renal transplant recipient from Guinea:first report of human infection by this fungus. Transpl Infect Dis13:204–207

Glass NL, Donaldson GC (1995) Development of primer sets designedfor use with the PCR to amplify conserved genes from filamen-tous ascomycetes. Appl Environ Microbiol 61:1323–1330

Glienke C, Pereira OL, Stringari D, Fabris J, Kava-Cordeiro V,Galli-Terasawa L, Cunnington J, Shivas RG, Groenewald JZ,Crous PW (2011) Endophytic and pathogenic Phyllostictaspecies, with reference to those associated with Citrus BlackSpot. Persoonia 26:47–56

Groenewald M, Groenewald JZ, Crous PW (2005) Distinct speciesexist within the Cercospora apii morphotype. Phytopathology95:951–959

Hawksworth DL (2011) A new dawn for the naming of fungi: impactsof decisions made in Melbourne in July 2011 on the futurepublication and regulation of fungal names. IMA Fungus 2:155–162

Hofstetter V, Miadlikowska J, Kauff F, Lutzoni F (2007) Phylogeneticcomparison of protein-coding versus ribosomal RNA-codingsequence data: a case study of the Lecanoromycetes(Ascomycota). Mol Phylogenet Evol 44:412–426

Hudson RR, Coyne JA (2002) Mathematical consequences of thegenealogical species concept. Evolution 56:1557–1565

Hyde KD, McKenzie EHC, KoKo TW (2011) Towards incorporatinganamorphic fungi in a natural classification— checklist and notesfor 2010. Mycosphere 2:1–88

Iriart I, Binois R, Fior R, Blanchet R, Berry A, Cassaing S, Amazan E,Papot E, Carm B, Aznar C, Couppié P (2011) Eumycetomacaused by Diaporthe phaseolorum (Phomopsis phaseoli): a casereport and a mini-review of Diaporthe/Phomopsis spp invasiveinfections in humans. Clin Microbiol Infec 17:1492–1494

James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ,Celio G, Gueidan C, Fraker E, Miadlikowska J, Lumbsch HT,Rauhut A, Reeb V et al (2006) Reconstructing the early evo-lution of Fungi using a six-gene phylogeny. Nature 443:818–822

Jiang SX, Ma HB (2010) A new species of Phomopsis on Castaneamollisima. Mycosystema 29:467–471

Jurković D, Vrandečić K, Ćosić J, Riccioni L, Duvnjak T (2007)Morphological identification of Diaporthe/Phomopsis sp. iso-lated from Xanthium italicum. Poljoprivreda (Osijek) 13:10–14

Kanematsu S, Adachi Y, Ito T (2007) Mating-type loci of heterothallicDiaporthe spp. homologous genes are present in opposite mating-types. Curr Genet 52:11–22

Katoh K, Toh H (2008) Recent developments in the MAFFT multiplesequence alignment program. Brief Bioinform 9:276–285

Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novelmethod for rapid multiple sequence alignment based on fast Four-ier transform. Nucleic Acids Res 30:3059–3066

Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihoodestimate of the evolutionary tree topologies from DNA sequencedata, and the branching order in Hominoidea. J Mol Evol 29:170–179

Lombard L, Crous PW, Wingfield BD, Wingfield MJ (2010) Phylog-eny and systematics of the genus Calonectria. Stud Mycol 66:31–69

Lumbsch HT, Schmitt I, Lindemuth R, Miller A, Mangold A, Fernan-dez F, Huhndorf S (2005) Performance of four ribosomal DNAregions to infer higher-level phylogenetic relationships of inoper-culate euascomycetes (Leotiomyceta). Mol Phylogenet Evol34:512–524

Manamgoda DS, Cai L, Bahkali AH, Chukeatirote E, Hyde KD (2011)Cochliobolus: an overview and current status of species. FungalDivers 51:3–42

McNeill J, Turland NJ, Monro A, Lepschi BJ (2011) XVIII Interna-tional Botanical Congress: preliminary mail vote and report ofCongress action on nomenclature proposals. Taxon 60:1507–1520

Mejía LC, Castlebury LA, Rossman AY, Sogonov MV, White JF Jr(2011) A systematic account of the genus Plagiostoma

Fungal Diversity (2012) 56:157–171 169

(Gnomoniaceae, Diaporthales) based on morphology, host-associations, and a four-gene phylogeny. Stud Mycol 68:211–235

Mejia LC, Rossman AY, Castlebury LA, White JF Jr (2011) Newspecies, phylogeny, host-associations, and geographic distributionof the genus Cryptosporella (Gnomoniaceae, Diaporthales).Mycologia 103:379–399

Moleleki N, Preisig O, Wingfield MJ, Crous PW, Wingfield BD (2002)PCR-RFLP and sequence data delineate three Diaporthe speciesassociated with stone and pome fruit trees in South Africa. Eur JPlant Pathol 108:909–912

Mostert L, Crous PW, Kang JC, Phillips AJL (2001) Species ofPhomopsis and a Libertella sp. occurring on grapevines withspecific reference to South Africa: morphological, cultural, mo-lecular and pathological characterization. Mycologia 93:146–167

Murali TS, Suryanarayanan TS, Geeta R (2006) Endophytic Phomop-sis species: host range and implications for diversity estimates.Can J Microbiol 52:673–680

Page RDM (1996) TREEVIEW: an application to display phylogenetictrees on personal computers. Comput Appl Biosci 12:357–358

Phillips AJL, Crous PW, Alves A (2007) Diplodia seriata, the ana-morph of Botryosphaeria obtusa. Fungal Divers 25:141–155

Phoulivong S, Cai L, Chen H, McKenzie EHC, Abdelsalam K,Chukeatirote E, Hyde KD (2010) Colletotrichum gloeospor-ioides is not a common pathogen on tropical fruits. FungalDivers 44:33–43

Rehner SA, Uecker FA (1994) Nuclear ribosomal internal transcribedspacer phylogeny and host diversity in the coelomycetes Pho-mopsis. Can J Bot 72:166–167

Rocha ACS, Garcia D, Uetanabaro APT, Carneiro RTO, Isabela S,Araújo IS, Carlos Mattos CRR, Góes-Neto A (2011) Foliar endo-phytic fungi from Hevea brasiliensis and their antagonism onMicrocyclus ulei. Fungal Divers 47:75–84

Rokas A, Williams BL, King N, Carroll SB (2003a) Genome scaleapproaches to resolving incongruence in molecular phylogenies.Nature 425:798–804

Rokas A, King N, Finnerty JR, Carroll SB (2003b) Conflicting phylo-genetic signals at the base of the metazoan tree. Dev Evol 5:346–359

Rossman AY, Farr DF, Castlebury LA (2007) A review of the phylog-eny and biology of the Diaporthales. Mycoscience 48:135–144

Santos JM, Phillips AJL (2009) Resolving the complex of Diaporthe(Phomopsis) species occurring on Foeniculum vulgare in Portu-gal. Fungal Divers 34:111–125

Santos JM, Correia VG, Phillips AJL (2010) Primers for mating-typediagnosis in Diaporthe and Phomopsis: their use in teleomorphinduction in vitro and biological species definition. Fungal Biol114:255–270

Santos JM, Vrandečić K, Ćosić J, Duvnjak T, Phillips AJL (2011)Resolving the Diaporthe species occurring on soybean in Croatia.Persoonia 27:9–19

Schmitt I, Crespo A, Divakar PK, Fankhauser JD, Herman-Sackett E,Kalb K, Nelsen MP, Nelson NA, Rivas-Plata E, Shimp AD,Widhelm T, Lumbsch HT (2009) New primers for promisingsingle copy genes in fungal phylogenetics and systematic.Persoonia 23:35–40

Schoch CL, Shoemaker RA, Seifert KA, Hambleton S, Spatafora JW,Crous PW (2006) A multigene phylogeny of the Dothideomy-cetes using four nuclear loci. Mycologia 98:1041–1052

Schoch CL, Seifert KA, Huhndorf S et al (2012) Nuclear ribosomalinternal transcribed spacer (ITS) region as a universal DNA bar-code marker for Fungi. P Natl Acad Sci 109:6241–6246

Shivas RG, Cai L (2011) Cryptic fungal species unmasked. Microbi-ology Australia 33:36–37

Summerell BA, Leslie JF (2011) Fifty years of Fusarium: how werenine species ever enough? Fungal Divers 50:135–144

Summerell BA, Laurence MH, Liew ECY, Leslie JF (2010) Biogeog-raphy and phylogeography of Fusarium: a review. Fungal Divers44:3–13

Sun X, Guo LD, Hyde KD (2011) Community composition of endo-phytic fungi in Acer truncatum and their role in decomposition.Fungal Divers 47:85–95

Sun S, Van K, Kim MY, Min KH, Lee YW, Lee SH (2012) Diaporthephaseolorum var. caulivora, a causal agent for both stem cankerand seed decay on soybean. Plant Pathol J 28:55–59

Sutton DA, Timm WD, Morgan-Jones G, Rinaldi MG (1999)Human phaeohyphomycotic osteomyelitis caused by the coe-lomycete Phomopsis Saccardo 1905: criteria for identifica-tion, case history, and therapy. J Clin Microbiol 37:807–811

Swofford DL (2002) PAUP 4.0b10: phylogenetic analysis using parsi-mony. Sinauer Associates, Sunderland

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,Fisher MC (2000) Phylogenetic species recognition and speciesconcepts in fungi. Fungal Genet Biol 31:21–32

Templeton AR (1989) The meaning of species and speciation: a geneticperspective. In: Otte D, Endler JA (eds) Speciation and its con-sequences. Sinauer, Sunderland, pp 3–27

Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improvingthe sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties andweight matrix choice. Nucleic Acids Res 22:4673–4680

Thompson SM, Tan YP, Young AJ, Neate SM, Aitken EA, Shivas RG(2011) Stem cankers on sunflower (Helianthus annuus) in Aus-tralia reveal a complex of pathogenic Diaporthe (Phomopsis)species. Persoonia 27:80–89

Udayanga D, Liu XX, McKenzie EHC, Chukeatirote E, Bahkali AH,Hyde KD (2011) The genus Phomopsis: biology, applications,species concepts and names of common phytopathogens. FungalDivers 50:189–225

Udayanga D, Liu XX, Crous PW, McKenzie EHC, Chukeatirote E,Hyde KD (2012) Multilocus phylogeny of Diaporthe reveals threenew cryptic species from Thailand. Cryptogamie Mycologie. InPress

Uecker FA (1988) A world list of Phomopsis names with notes onnomenclature, morphology and biology. Mycol Mem 13:1–231

van der Aa HA, Noordeloos ME, Gruyter J (1990) Species con-cepts in some larger genera of the coelomycetes. Stud Mycol32:3–19

van Niekerk JM, Groenewald JZ, Farr DF, Fourie PH, Halleen F, CrousPW (2005) Reassessment of Phomopsis species on grapevine.Australas Plant Path 34:27–39

van Rensburg JCJ, Lamprecht SC, Groenewald JZ, Castlebury LA,Crous PW (2006) Characterisation of Phomopsis spp. associatedwith die-back of rooibos (Aspalathus linearis) in South Africa.Stud Mycol 55:65–74

van Warmelo KT, Marasas WFO, Adelaar TF, Kellerman TS, vanRensburg IBJ, Minne JA (1970) Experimental evidence that lupi-nosis of sheep is a mycotoxicosis caused by the fungus Phomop-sis leptostromiformis (Kuhn) Bubak. J S Afr Vet Med Assoc41:235–247

Walker DM, Castlebury LA, Rossman AY, White JF (2012) New molec-ular markers for fungal phylogenetics: two genes for species levelsystematics in the Sordariomycetes (Ascomycota). Molecular Phy-logenetics and Evolution. Accepted. Manuscript MPE-11-256

Wang XH, Chen GQ, Huang F, Zhang JZ, Hyde KD, Li HG (2011)Phyllosticta species associated with citrus diseases in China.Fungal Divers 52:209–224

Wehmeyer LE (1933) The genus Diaporthe Nitschke and itssegregates. University of Michigan Studies Scientific Series9:1–34

White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics. In:

170 Fungal Diversity (2012) 56:157–171

Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR proto-cols: a guide to methods and applications. New York AcademicPress. pp 315–322

Wikee S, Udayanga D, Crous PW, McKenzie EHC, Chukeatirote E,Bahkali AH, Hyde KD (2011) Phyllostica- an overview of currentstatus of species recognition. Fungal Divers 51:46–60

Wingfield MJ, de Beer ZW, Slippers B, Wingfield BD, Groenewald JZ,Lombard L, Crous PW (2012) One fungus one name promotesprogressive plant pathology.Molecular Plant Pathology 13:604–613

Wu ZH, Wang TH, Huang W, Qu YB (2001) A simplified method forchromosome DNA preparation from filamentous fungi. Mycosys-tema 20:575–577

Zhang N (2002) Molecular phylogeny ofMelanospora and Diaporthales,and population genetics of dogwood anthracnose fungus. PhD The-sis. Department of Biological Sciences, Lousiana State University

Zhang AW, Hartman GL, Riccioni L, Chen WD, Ma RZ, PedersenWL (1997) Using PCR to distinguish Diaporthe phaseolorumand Phomopsis longicolla from other soybean fungal patho-gens and to detect them in soybean tis-sues. Plant Dis 81:1143–1149

Zhang AW, Riccioni L, Pedersen WL, Kollipara KP, Hartman GL(1998) Molecular identification and phylogenetic grouping ofDiaporthe phaseolorum and Phomopsis longicolla isolates fromsoybean. Phytopathology 88:1306–1314

Fungal Diversity (2012) 56:157–171 171