evolutionary relationships among ascochyta …...from chickpea (ar), a. fabae f. sp. fabae (af) and...

Evolutionary relationships among Ascochyta species infecting wild and cultivatedhosts in the legume tribes Cicereae and Vicieae

T.L. Peever1

M.P. BarveL.J. Stone

Department of Plant Pathology, Washington StateUniversity, Pullman, Washington 99164-6430

W.J. Kaiser2

3394 Chickory Way, Boise, Idaho 83706

Abstract: Evolutionary relationships were inferredamong a worldwide sample of Ascochyta fungi fromwild and cultivated legume hosts based on phyloge-netic analyses of DNA sequences from the ribosomalinternal transcribed spacer regions (ITS), as well asportions of three protein-coding genes: glyceralde-hyde-3-phosphate-dehydrogenase (G3PD), translationelongation factor 1-alpha (EF) and chitin synthase 1(CHS). All legume-associated Ascochyta species hadnearly identical ITS sequences and clustered withother Ascochyta, Phoma and Didymella species fromlegume and nonlegume hosts. Ascochyta pinodes(teleomorph: Mycosphaerella pinodes [Berk. & Blox.]Vestergen) clustered with Didymella species and notwith well characterized Mycosphaerella species fromother hosts and we propose that the name Didymellapinodes (Berk. & Blox.) Petrak (anamorph: Ascochytapinodes L.K. Jones) be used to describe this fungus.Analysis of G3PD revealed two major clades amonglegume-associated Ascochyta fungi with members ofboth clades infecting pea (‘‘Ascochyta complex’’).Analysis of the combined CHS, EF and G3PD datasetsrevealed that isolates from cultivated pea (P. sati-vum), lentil (Lens culinaris), faba bean (Vicia faba)and chickpea (Cicer arietinum) from diverse geo-graphic locations each had identical or similarsequences at all loci. Isolates from these hostsclustered in well supported clades specific for eachhost, suggesting a co-evolutionary history betweenpathogen and cultivated host. A. pisi, A. lentis, A.fabae and A. rabiei represent phylogenetic speciesinfecting pea, lentil, faba bean and chickpea, re-spectively. Ascochyta spp. from wild relatives of peaand chickpea clustered with isolates from relatedcultivated hosts. Isolates sampled from big-flower

vetch (Vicia grandiflora) were polyphyletic suggestingthat either this host is colonized by phylogeneticallydistinct lineages of Ascochyta or that the hosts arepolyphyletic and infected by distinct evolutionarylineages of the pathogen. Phylogenetic speciesidentified among legume-associated Ascochyta spp.were fully concordant with previously describedmorphological and biological species.

Key words: Ascochyta blight, chitin synthase, DNAsequence, glyceraldehyde-3-phosphate-dehydrogenase,internal transcribed spacer, legumes, PCR, phylogeny,translation elongation factor alpha

INTRODUCTION

Species of the coelomycete genus Ascochyta Libertcause diseases known as Ascochyta blights of a num-ber of commercially important cool season legumespecies and their wild relatives. These diseases arecharacterized by tan-colored lesions on all above-ground parts of the plant which can result in severeyield losses under conducive environmental condi-tions (Nene 1982, Kaiser and Muehlbauer 1988,Bretag and Ramsey 2001). Lesions typically containconcentric rings of black pycnidia that exude cirrhiof one- or two-celled hyaline conidia, which aredispersed short distances via rain splash. Ascochytablights of chickpea (Cicer arietinum L.), lentil (Lensculinaris Medik.), faba bean (Vicia faba L.), vetches(Vicia spp.) and pea (Pisum sativum L.) areeconomically important diseases in all areas of theworld where these crops are grown. Most legume-associated species of Ascochyta examined to datehave teleomorphs that have been observed in thefield and are important in the biology and epidemi-ology of these diseases. Ascospores can be wind-borne, dispersed long distances by air and areconsidered important sources of primary inoculumin many areas (Trapero-Casas et al 1996, Kaiser1997). Most Ascochyta species have a bipolar, het-erothallic mating system (Barve et al 2003, Cherif etal 2006) and the sexual stage can be induced in thelaboratory (Wilson and Kaiser 1995, Kaiser et al1997). Ascospores are typically unequally two-celledwith a prominently constricted septum (Wilson andKaiser 1995, Kaiser et al 1997).

Ascochyta blights of chickpea, lentil, and faba beanare caused by the well characterized fungi Ascochytarabiei (Pass.) Labrousse, A. lentis Vassilevsky, and A.

Accepted for publication 6 October 2006.1 Corresponding author. E-mail: [email protected] Current address: Department of Chemical Sciences, Tata Instituteof Fundamental Research, Mumbai 400005, India.

Mycologia, 99(1), 2007, pp. 59–77.# 2007 by The Mycological Society of America, Lawrence, KS 66044-8897

59

fabae Speg., respectively. Three fungal species havebeen associated with Ascochyta blight of pea and arereferred to as the ‘‘Ascochyta complex’’ (Wallen1965). These include Ascochyta pisi Lib., Ascochytapinodes L.K. Jones (teleomorph: Mycosphaerella pi-nodes [Berk. & Blox.] Vestergen [syn. Didymellapinodes Berk. & Blox.] Petrak), and Phoma medicaginisMalbr. & Roum. var. pinodella (Jones) Boerema (syn.Ascochyta pinodella L.K. Jones and Phoma pinodella[L.K. Jones] Morgan-Jones & K.B. Burch). Ascochytaspecies have been isolated from other cool seasonlegumes in the ‘‘Vicioid clade’’ (Steele and Wojcie-chowski 2003) particularly wild Vicia spp. (vetches)but these fungi are poorly described (Leath 1994,T.L. Peever and W.J. Kaiser unpublished). SeveralAscochyta anamorphs have been connected to Didy-mella Saccardo teleomorphs (Muller and von Arx1962, Jellis and Punithalingam 1991, Kaiser 1997,Kaiser et al 1997); however, teleomorph connectionshave not yet been made for many of the species. Thefungi causing Ascochyta blight of faba bean, lentil andchickpea all have been connected to Didymellateleomorphs. Teleomorphs of A. fabae Speg., A. lentisVassilevsky and A. rabiei (Pass.) Labrousse are D. fabaeJellis & Punith. (Jellis and Punithalingam 1991), D.lentis Kaiser, Wang & Rogers (Kaiser and Hellier 1993,Kaiser et al 1997) and D. rabiei (Kovachevski) v. Arx(syn. Mycosphaerella rabiei Kovachevski) (Kovachevski1936), respectively. D. rabiei originally was placed inMycosphaerella by Kovachevski but subsequentlymoved to Didymella by von Arx (Muller and von Arx1962) based on pseudothecium size, ascospore sizeand septum constriction, and the presence of non-fasiculate asci and pseudoparaphyses. Ascochyta pi-nodes L.K. Jones (teleomorph: Mycosphaerella pinodes(Berk. & Blox.) Vestergen (syn. Didymella pinodes[Berk. & Blox.] Petrak), one of the fungi causingAscochyta blight of pea, has been connected to bothMycosphaerella and Didymella teleomorphs and thisfungus commonly is referred to as M. pinodes by plantpathologists (Onfroy et al 1999, Faris-Mokaiesh et al1996).

Experimental inoculation of Ascochyta sp. isolatesfrom lentil and faba bean on both hosts clearlydemonstrated their host specificity, but these host-specific taxa could not be differentiated by statisticalanalyses of conidium length, proportion of septateconidia and cultural morphology (Gossen et al 1986).Gossen et al (1986) proposed that these two fungi besynonymized under A. fabae using the formaespeciales designations A. fabae f. sp. fabae and A.fabae f. sp. lentis to denote their host specificity. Invitro genetic crosses made among isolates of A. rabieifrom chickpea (AR), A. fabae f. sp. fabae (AF) and A.fabae f. sp. lentis (AL) failed to produce pseudothecia

in the AR 3 AF and the AR 3 AL combinations(Kaiser et al 1997). However crosses between AF andAL were fertile and produced pseudothecia withviable ascospore progeny. Strong postzygotic matingeffects were observed in the AF 3 AL crossesincluding abnormal numbers of ascospores in eachascus, variable ascospore size and progeny isolatesthat grew abnormally in culture. All progeny isolatesfrom the AF 3 AL cross failed to infect either of theirparental hosts, lentil and faba bean. Kaiser et al(1997) also scored these isolates for RAPD markersand showed that the fungi from each host each haddistinct RAPD banding profiles and clustered sepa-rately in a UPGMA phenogram. The combination ofhost specificity, strong genetic differentiation inmolecular markers (i.e. lack of gene flow) andpostzygotic mating effects in the AL 3 AF crosseswere used to justify the elevation of A. fabae f. sp.lentis to A. lentis Vassilevsky (Kaiser et al 1997) andrepresents a rare example of application of thebiological species concept to plant-pathogenic fungi.By these criteria A. rabiei, A. fabae and A. lentis areconsidered biological species. Phylogenetic analysesof the cool season food legumes based on the plastidmatK gene have revealed that Cicer species (tribeCicereae) are highly diverged from Pisum, Vicia andLens species (tribe Vicieae) (Steele and Wojcie-chowski 2003). Within the Viciae, Vicia hirsuta andV. villosa were found in well supported clades distinctfrom other Vicia spp. Steele and Wojciechowski(2003) identified two subclades within the Vicieaeincluding Clade 1, which contained Pisum sativum,and Clade 2 which contained Lens culinaris and Viciagrandiflora.

Ascochyta pisi causes tan-colored lesions withdistinct dark margins on above-ground parts of pea(Allard et al 1993, Bretag and Ramsey 2001) andlesions are similar to those of Ascochyta blight ofchickpea and lentil. In contrast lesions caused by A.pinodes and A. pinodella are brown to tan with lack ofdistinct margin and can be found on both above- andbelow-ground parts of the plant. A. pinodes ishomothallic and readily produces pseudothecia insingle-ascospore or single-conidial cultures (Punitha-lingam and Holiday 1972, Onfroy et al 1999).Comparison of crosses made with single ascosporeversus pooled ascospore inoculum of A. pinodellademonstrated that this fungus is heterothallic (Bowenet al 1997). A. pisi has no reported teleomorph butthe pattern of PCR amplification of the conservedMAT1-2 HMG-box region of the MAT1-2 matingidiomorph among a collection of field isolates (HMGregion amplified from approximately half the iso-lates) was similar to known heterothallic Ascochytaspp. (T.L. Peever unpublished). A. pisi is morpho-

60 MYCOLOGIA

logically distinct from A. pinodes and A. pinodella inculture with the former having an orange/pinkcolony morphology and orange/red conidia and thelatter two having gray/black colony morphology andcream-colored conidia (Bretag and Ramsey 2001).The latter two fungi also produce chlamydospores inculture while A. pisi does not (Onfroy et al 1999,Bretag and Ramsey 2001, Fatehi et al 2003). Inaddition to morphological characters, A. pisi can bedistinguished easily from A. pinodes and A. pinodellawith molecular markers such as restriction fragmentlength polymorphism of the nuclear ribosomalintergenic spacer (IGS) (Faris-Mokaiesh et al 1996)and serological techniques (Madosingh and Wallen1968). Differentiating A. pinodes from A. pinodella hasbeen more difficult (Faris-Mokaiesh et al 1996, Jones1927, Onfroy et al 1999, Bretag and Ramsey 2001,Fatehi et al 2003). Fatehi et al (2003) were able toseparate A. pinodes and A. pinodella based onrestriction digestion of total mitochondrial DNA butnot digestions of ITS or partial beta-tubulin se-quences and thus considered A. pinodes and A.pinodella conspecific. Similarly, Barve et al (2003)were able to differentiate these fungi based onsequence data from the HMG motif of the MAT1-2mating gene but not with ITS sequence data. Basedon these studies, it appears that A. pinodes and A.pinodella are closely related and distinct from A. pisi.

The primary objective of this research was todetermine the evolutionary relationships amongAscochyta and Phoma species associated with cultivat-ed cool season legumes and related wild plants byperforming phylogenetic analyses with sequence datafrom multiple regions of the genome. We also wereinterested in testing the hypothesis that previouslyrecognized biological species could be consideredphylogenetic species. A secondary objective was to usephylogenetic analysis to identify closely related, host-specific fungi that might be used to study the geneticsof species-level host specificity and the mechanisms offungal speciation. A tertiary objective was to de-termine phylogenetic relationships among the threefungi associated with the ‘‘Ascochyta complex’’ of peaand to test the hypothesis that A. pinodes (tele-omorph: Mycosphaerella pinodes) is phylogeneticallydistinct from Mycosphaerella spp. associated withother plants.

MATERIALS AND METHODS

Sampling, fungal culture, morphology and DNA extraction.—Sixty-seven single-conidial isolates of Ascochyta spp., Phomaspp. and Didymella spp. from various wild and cultivatedlegume hosts were obtained from a culture collectionmaintained by the USDA Western Region Plant Introduc-

tion Station, Pullman, Washington (TABLE I). These isolateswere sampled over approximately 20 y by W.J. Kaiser andstored in an inert state on sterile, dry chickpea stems at 4 C.Isolates were selected from this collection to representdescribed morphological species from several cultivatedlegume hosts in diverse worldwide locations (TABLE I).Approximately 10 isolates per species were selected, andwhere possible, isolates that were deposited in the AmericanType Culture Collection were used. All isolates were single-spored, examined morphologically and assigned to species.Criteria used to classify each isolate to species included (i)original host of isolation, (ii) conidial dimensions andseptation, (iii) growth rate and colony morphology onpotato dextrose agar, (iv) pathogenicity tests and (v) pairingisolates with known mating type tester strains. Additionalfungi used in this study included isolates from wild Viciaspp. (vetches) collected 1983–2001 in Pullman, Washing-ton, and Aprilci, Bulgaria by T.L. Peever and W.J. Kaiser andisolates from wild and cultivated Cicer spp., Pisum spp. andVicia spp. collected by W.J. Kaiser in the Republic ofGeorgia in Jul 2004 (TABLE I). All isolates in this secondsample (labeled Georgia-XX) were single-spored but notextensively morphologically characterized and not assignedto species (TABLE I). Isolate AV11 from Vicia grandiflorapreviously was assigned to species by K.T. Leath (Leath1994). All isolates were grown 3–4 d at laboratory tempera-tures in liquid 2-YEG medium (10 g dextrose, 2 g yeastextract per liter) on a rotary shaker at 150 rpm andmycelium collected and lyophilized as described by Peeveret al (1999). Genomic DNA was extracted from 50 mglyophilized mycelium as described by Peever et al (1999)with modifications. One phenol/chloroform (1:1, vol/vol)extraction and one chloroform extraction were used. DNAconcentrations were estimated visually in 0.7% agarose gelscontaining 0.5 mg/mL ethidium bromide by comparingband intensity with known quantities of lambda DNA/HindIII markers (Promega, Madison, Wisconsin). DNA extrac-tions were diluted routinely to 20 ng/mL in sterile distilledwater for use as template DNA in PCR.

Nuclear ribosomal internal transcribed spacer (ITS) andglyceraldehyde-3-phosphate-dehydrogenease (G3PD) sequenc-ing.—Primers ITS1 and ITS4 (White et al 1990) were usedto amplify approximately 540 bp of ITS1, 5.8s and ITS2from all isolates. Twenty-five mL PCR reactions contained13 reaction buffer (Life Technologies, Carlsbad, Califor-nia), 0.4 mM each primer (Operon Technologies, Alameda,California), 200 mM dNTPs (Idaho Technologies, IdahoFalls, Idaho), 2.5 mM MgCl2 (Life Technologies), 20–40 ngof DNA and 1 unit of Taq polymerase (Life Technologies).PCR was carried out in a GeneAmp PCR System 9700thermocycler (PE Biosystems, Norwalk, Connecticut) andcycling conditions consisted of 94 C for 3 min followed by30 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 1 minfollowed by 5 min at 72C. Five hundred eighty-five bp of theG3PD gene was amplified from all isolates with gpd-1 andgpd-2 primers (Berbee et al 1999) and the same concentra-tions of PCR reagents and cycling conditions as describedabove for ITS. The amplified region corresponds to basepairs 793–1397 of the C. heterostrophus (Drechs.) Drechs.

PEEVER ET AL: RELATIONSHIPS AMONG ASCOCHYTA 61

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62 MYCOLOGIA

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ada

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ssen

——

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3839

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—

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um

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vu

mA

scoc

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apis

i—

AP

5Sa

skat

oo

n,

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ada

B.

Go

ssen

——

DQ

3839

65—

—

Pis

um

sati

vu

mA

scoc

hyt

apis

i—

AP

7Sa

skat

oo

n,

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ada

B.

Go

ssen

——

——

—

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um

sati

vu

mA

scoc

hyt

apis

i—

AP

8Sa

nta

Ro

sali

a,B

oli

via

W.J

.K

aise

r19

99—

——

—

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um

sati

vu

mA

scoc

hyt

apis

i—

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9M

on

teag

ud

o,

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livi

aW

.J.

Kai

ser

1999

——

——

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um

elati

us

Asc

ochyt

asp

.—

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rgia

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arts

khis

i,G

eorg

iaW

.J.

Kai

ser

2004

DQ

3839

55D

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3966

——

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um

elati

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Asc

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asp

.—

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rgia

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ten

i,G

eorg

iaW

.J.

Kai

ser

2004

——

—D

Q38

6495

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um

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us

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ochyt

asp

.—

Geo

rgia

-12

Ats

juri

,G

eorg

iaW

.J.

Kai

ser

2004

—D

Q38

3967

—D

Q38

6496

Vic

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.—

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W.J

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nn

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——

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lgar

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ser

1996

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rgia

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aro

.G

eorg

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.J.

Kai

ser

2004

—D

Q38

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DQ

3864

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6499

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scoc

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.—

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rgia

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edo

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Kai

ser

2004

—D

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3972

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3864

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.—

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rgia

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W.J

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iacf

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dif

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.—

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rgia

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i,G

eorg

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.J.

Kai

ser

2004

——

—D

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6502

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iasp

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rgia

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ten

i,G

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ser

2004

——

—D

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6503

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r20

04—

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um

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MP

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3976

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mA

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apin

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osphaer

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pin

odes

MP

3(2

0163

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oro

cco

—-

1989

——

——

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pin

odes

MP

8(2

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um

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odes

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pin

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MP

10N

ewZ

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nd

J.K

raft

1996

——

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um

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vu

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odes

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MP

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ain

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64 MYCOLOGIA

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W.J

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30Su

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livi

aW

.J.

Kai

ser

1999

——

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hom

am

edic

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MP

3(5

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ash

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SAW

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Kai

ser

1996

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am

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4(5

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ash

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ser

1996

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stra

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um

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vu

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hom

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edic

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nis

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MP

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un

isia

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Hal

ila

1996

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um

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vu

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Bo

livi

aW

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Kai

ser

1999

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stra

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1996

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um

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vu

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rgia

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shev

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Bu

lgar

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ser

2004

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66 MYCOLOGIA

G3PD coding sequence (GenBank Accession No. X63516)and includes two introns totaling 150 bp. ITS and G3PDamplicons were separated in 1.5% agarose gels (LifeTechnologies) with Hind III/EcoR1 digested lambda DNA(Promega) as size standards and viewed with a digitalimaging system (UltraViolet Products, Upland, California).Amplicons were direct sequenced on each strand with eachsequence reaction containing 40–90 ng DNA, 320 nMforward or reverse primer, 4 mL BigDye Terminator CycleSequencing Ready Reaction Mix (Applied Biosystems,Foster City, California), in 10 mL total volumes. Cyclesequence reactions were performed in a Hybaid Omn-Ethermal cycler and cycling conditions consisted of 25 cyclesof 15 s at 96 C, 15 s at 50 C, and 4 min at 60 C, following themanufacturer’s instructions. Products were purified withCentriflex Gel Filtration Cartridges (Edge BioSystems,Gaithersburg, Maryland), dried in a rotary evaporator andsequences were read in a PE Biosystems Model 3730Automated DNA Sequencer (Applera Corp., Norwalk,Connecticut). All sequencing was performed at the Labo-ratory for Biotechnology and Bioanalysis, School of Molec-ular Biosciences, Washington State University. ITS andG3PD sequences have been deposited under GenBankaccession Nos. DQ383949-DQ383979.

Chitin synthase (CHS) and translation elongation factor alpha(EF) sequencing.—A subset of isolates with variable G3PDhaplotype was selected for CHS and EF sequencing. Elevenisolates were selected, including AF1, AF8, AL1, AL11, AP2,AP4, AP5, AV1, AV8, AV11, and AV-01-1 as well as all isolatessampled from the Republic of Georgia (TABLES I, II).Ascochyta pinodes and A. pinodella isolates were notsequenced for CHS or EF. Primers CHS-79 and CHS-354(Carbone and Kohn 1999) were used to amplify 364 bp ofthe chitin synthase I gene. The amplified region corre-sponds to base pairs 1409–1772 of the Ampleomycesquisqualis Ces. ex Schltdl. CHS coding sequence (GenBankaccession No. X86802) and included one intron 55 bp long.Twenty-five mL PCR reactions contained 13 combinationPCR buffer/loading dye with 2 mM MgCl2 (Idaho Tech-nologies), 0.4 mM each primer, 200 mM dNTPs (MBIFermentas, Hanover, Maryland), 20–40 ng of DNA and 1unit of Taq polymerase (New England Biolabs, Beverly,Maryland). PCR was carried out in a Hybaid Omn-Ethermocycler (Hybaid, Ashford, Middlesex, UK) and cyclingconditions consisted of 97 C for 1 min followed by 35 cyclesof 96 C for 15 s, 62 C for 15 s, and 72 C for 15 s. PrimersEF1-728F and EF1-986R (Carbone and Kohn 1999) wereused to amplify 330 bp of the translation elongation factor 1alpha gene. The amplified region corresponds to base pairs716–1006 of the N. crassa Shear & Dodge EF1 gene(GenBank accession No. D45837) and includes approxi-mately 270 bp of intron sequence. PCR reactions werecarried out as described above with an annealing temper-ature of 50 C rather than 62 C. CHS and EF amplicons wereviewed, purified and sequenced as described above. CHSand EF sequences have been deposited under GenBankaccession Nos. DQ386480-DQ386506.

Data analysis.—Sequences were aligned with Clustal X 1.8(Thompson et al 1997). Maximum likelihood phylogenies

were estimated independently for each dataset (CHS, EF,G3PD) with unique sequences in both frequentist andBayesian statistical frameworks. Models of sequence evolu-tion were evaluated for each dataset and model parameterestimates obtained with Modeltest v.3.7 (Posada andCrandall 1998) implemented in PAUP v. 4.10b10 (Swofford2002). The Akaike information criterion (AIC), Bayesianinformation criterion (BIC) and hierarchical likelihoodratio tests were used to select models. When the same modelwas not selected under all three criteria, we chose the nextmost parameter-rich model selected by at least one of thecriteria. In all cases similar models were selected by all threecriteria. For the nuclear ribosomal internal transcribedspacer (ITS) dataset, the SYM+G+I model with equal basefrequencies, six substitution rate parameters (1.053, 1.673,2.054, 0.333, 4.864, 1.000), gamma distributed rates (shapeparameter 5 0.555), and a proportion of invariant sites(0.2501) was selected. The TIM+G model with unequal basefrequencies (A 5 0.262, C 5 0.314, G 5 0.230, T 5 0.193),four substitution rate parameters (1.000, 4.4137, 2.0745,2.7045, 7.7086, 1.000) and gamma distributed rates (shapeparameter 5 0.318) was chosen for the glyceraldehyde-3-phosphate dehydrogenase (G3PD) dataset. For the chitinsynthase (CHS) dataset, the HKY+G model with unequalbase frequencies (A 5 0.2992, C 5 0.2244, G 5 0.2816, T 5

0.1949), a transition:transversion ratio of 2.364 (two sub-stitution rate parameters) and gamma distributed rates(shape parameter 5 0.1355) was selected. For the trans-lation elongation factor alpha (EF) dataset, the HKY modelwith unequal base frequencies (A 5 0.2052, C 5 0.3213, G5 0.2339, T 5 0.2396) and a transition:transversion ratio of2.2724 was selected.

A ‘‘conditional data combination’’ approach (Huelsenbecket al 1996) was employed to determine whether the CHS, EF,and G3PD datasets (partitions) should be combined forphylogenetic analysis. Incongruence among partitions wasassessed using three approaches. First, topologies estimatedindependently for each partition were examined for wellsupported, conflicting nodes. Topologies were consideredincongruent if two different relationships (one monophyleticand the other nonmonophyletic) for the same set of taxaboth were supported by a 70% ML bootstrap value or 95%

Bayesian posterior probability (Binder and Hibbett 2002,Reeb et al 2004, Froslev et al 2005). The second approachinvolved Shimodaira-Hasegawa (SH) tests (Shimodaira andHasegawa 1999) implemented in PAUP. These tests com-pared the topology of the ML tree for each partition (usingthe best-fit model and parameters estimated for the partition)to the topology of the combined (concatenated) ML tree(using the best-fit model and parameters estimated forcombined dataset) when the former was used as theunconstrained tree and the latter as the constrained tree.The null distribution for the SH tests was generated with 1000nonparametric bootstrapped replicates with full likelihoodmaximization over each replicate dataset. The third approachemployed parametric bootstrapping (Huelsenbeck and Cran-dall 1997, Goldman et al 2000) and tested the null hypothesisthat the same topology underlay each dataset versus thealternate hypothesis that allows a different topology tounderlie each dataset (Huelsenbeck and Bull 1996, Huelsen-


beck and Crandall 1997). One hundred datasets weresimulated for each data partition (CHS, EF, G3PD) with theML trees, branch lengths and model parameters for thecombined dataset and for each partition in Seq-Gen v.1.3.2(Rambout and Grassly 1997). Parametric bootstrapping wasperformed with the combined ML tree (using the best-fitmodel and parameters estimated for the combined dataset)as the unconstrained tree and the ML tree for each partition(using the best-fit model and parameters estimated for eachpartition) as the constrained tree in PAUP. Heuristic searchesof the simulated datasets were conducted using full optimi-zation (ML parameters estimated on each simulated dataset),stepwise addition of taxa and TBR branch swapping. Log-likelihoods of unconstrained trees were subtracted from thoseof constrained trees to give a distribution of log-likelihooddifferences under the null hypothesis. The difference in log-likelihood value between the constrained and unconstrainedtrees for the actual data were compared to the distribution ofdifferences estimated from the simulated datasets. Theprobability of obtaining a larger difference under the nullhypothesis by chance was determined by enumerating thenumber of differences that were as large or larger than theactual difference. We considered topologies to be significantlydifferent when less than 5% of the simulated log-likelihooddifferences between constrained and unconstrained treeswere as large or larger than the log-likelihood differenceestimated from the actual data.

Maximum likelihood phylogenies were estimated inde-pendently for each data partition (CHS, EF, G3PD) and thecombined dataset using heuristic searches in PAUP. PAUPcurrently does not allow partitioned models (i.e. anindependent model of molecular evolution for each datapartition) so we used the best overall model selected for thecombined dataset in Modeltest. For the combined datasetthe TrN+G model with unequal base frequencies (A 5

0.2571, C 5 0.2957, G 5 0.2466, T 5 0.2006), sixsubstitution rate parameters (1.000, 3.8828, 1.000, 1.000,6.3065, 1.000), gamma-distributed rates (shape parameter5 0.2196) was selected. The reproducibility of clades wasassessed with heuristic searches of 1000 bootstrappeddatasets with ‘‘fast’’ stepwise addition of taxa and no branchswapping. Clades were inferred based on nodes with MLbootstrap values greater than or equal to 70%. Parametric

TABLE II. Sequence haplotypes identified among isolatesof Ascochyta and Didymella spp. sampled from legumes

Haplotype Isolates

ITS1 AF1, 4, 8, 14, 18, 22, 32, 43, 502 AL1, 2, 3, 6, 8, 11, 83, 84, 391, Georgia-2, 83 AP1, 2, 3, 4, 5, 7, 8, 9, AV11, Georgia-3, 7, 94 AR20, 19, 21, 628, 655, 735, 737, 823, 830, 844,

Georgia-155 AV1, 2, 8, 12, 19, 20, 22, 236 MP1, 2, 3, 10, 11, 14, 19, 21, 22, 27, 28, 30, PMP3,

4, 7, 9, 10, 13, 15, 26, 27, 29, Georgia-207 AR7388 Georgia-69 Georgia-10

G3PD1 AP42 AP1, 2, 3, 7, 8, 9, Georgia-123 AP54 AV11, Georgia-9, 135 Georgia-166 AF4, 8, 14, 18, 22, 32, 43, 507 AF18 Georgia-6, 79 Georgia-1210 Georgia-3, 411 AL1, 2, 3, 6, 8, 83, 84, 39112 AL1113 AV1, 2, 12, 19, 20, 23, AV-01-114 AV5, 8, 2215 Georgia-2, 816 AR20, 19, 21, 628, 655, 735, 737, 738, 823, 830,

844, Georgia-10, 15, 1917 Georgia-1118 MP1, 3, 8, 10, 22, 27, 28, 3019 MP220 MP1921 PMP3, 15, 26, Georgia-2022 PMP4, 7, 9, 10, 13

CHS1 AF1, 8, AP2, 4, 5, Georgia-6, 7, 9, 12, 13, 162 AL1, 113 AV1, 8, AV-01-1, Georgia-84 AV115 Georgia-26 Georgia-3, 47 AR20, 19, 735, 738, Georgia-10, 15, 198 Georgia-11

EF1 AF1, 82 Georgia-133 Georgia-94 AV115 Georgia-166 Georgia-37 Georgia-48 Georgia-7

Haplotype Isolates

9 AP2, 4, 5, Georgia-610 Georgia-1211 AL1, 5, 1112 Georgia-213 AV-1, 8, AV-01-114 Georgia-815 AR735, 1916 AR20, Georgia-1517 AR738, Georgia-1918 Georgia-1019 Georgia-11

TABLE II. Continued

68 MYCOLOGIA

bootstrapping also was used to test the hypothesis thatisolates sampled from big-flower vetch (V. grandiflora) andfrom wild and cultivated pea (Pisum elatius and P. sativum)were monophyletic (Huelsenbeck et al 1996, Goldman et al2000). One hundred datasets were simulated over thecombined ML tree (with best-fit model and parametersestimated for the combined tree) with Seq-Gen as describedabove. The first analysis forced all isolates sampled from big-flower vetch to be monophyletic and the second forced allisolates from wild and cultivated pea to be monophyletic.Constraint trees were generated in MacClade v.4.06(Maddison and Maddison 2003) and parametric boot-strapping was performed in PAUP as described above.Heuristic searches of simulated datasets and differences inlog-likelihoods between constrained and unconstrainedtrees were tested as described above. We rejected mono-phyly when less than 5% of the simulated log-likelihooddifferences between constrained and unconstrained treeswere as large as or larger than the log-likelihood differenceestimated from the actual data.

Maximum likelihood phylogenies also were estimated ina Bayesian framework with Markov chain Monte Carlo(MCMC) sampling in MrBayes version 3.1 (Huelsenbeckand Ronquist 2001). Flat Dirichlet probability densities wereused as priors for the substitution rate parameters andstationary nucleotide frequencies and uniform priors wereused for the shape and topology parameters and anexponential unconstrained prior was used for the branchlengths parameter. GTR models with or without gamma-distributed rates were employed and parameters wereestimated during each run. For analyses of the ITS andG3PD datasets, GTR models with six substitution rateparameters and gamma distributed rates were used. Foranalysis of the combined CHS, EF, and G3PD dataset,a partitioned model was implemented in MrBayes withevolutionary models and parameters estimated indepen-dently for each genomic region. Two substitution rateparameters were used with the CHS and EF datasets and sixsubstitution rate parameters with the G3PD dataset. Gammadistributed rates were applied to the CHS and G3PDdatasets but not the EF dataset. Each run of the samplerconsisted of 120 000 generations of the Markov chain. Sixchains were run in each analysis (one heated and five cold)with the temperature parameter set at 0.2 and randomchains swapped three times per generation. Six indepen-dent analyses (each of 120 000 generations) each werestarted from a random tree. Trees were sampled every 100generations and the first 20 000 generations (200 trees) ofeach analysis were discarded as burn-in. Average posteriorprobabilities were estimated for each node of the phylogenyacross all six runs (6000 trees from 600 000 total generationsof the MCMC). The ITS phylogeny was rooted by Myco-sphaerella punctiformis isolate CBS 724.79 (GenBank acces-sion No. AY490760), the G3PD phylogeny was rooted byPhaeosphaeria nodorum isolate S-82-13 (GenBank accessionNo. AY364464) and the combined phylogeny was unrooted.Clades were inferred based on posterior probabilitiesgreater than or equal to 95%.

Split decomposition analysis with uncorrected P distanceswas performed on the combined CHS, EF and G3PD dataset

with only the most closely related species (A. rabiei, A. pinodesand A. pinodella isolates not included) with SplitsTree v.4(Huson 1998). One thousand bootstrapped datasets wereused to estimate statistical support for the splits. Alignmentsof CHS, EF, G3PD and the combined dataset were evaluatedfor evidence of recombination using 10 recombination testsimplemented in RDP2 (Martin et al 2005). An incompatibil-ity matrix for the combined dataset was generated usingSNAP Workbench (Price and Carbone 2004) and recombi-nation hotspots identified with Recom58 (Griffiths andMarjoram 1996) implemented in SNAP Workbench.

RESULTS

Species identification.—Isolates were assigned to oneof six species, Ascochyta rabiei, A. fabae, A. lentis, A.pisi, A. pinodes and A. pinodella, based on fivemorphological and biological criteria outlined above(TABLE I). Ascochyta fabae, A. lentis, and A. pisi wereindistinguishable morphologically and could bedifferentiated only based on host of isolation,pathogenicity tests and mating tests. A. rabiei isolatesfrom cultivated and wild chickpea could be differen-tiated easily from A. lentis and A. fabae based onslower growth in culture and cultural morphology.Isolates sampled from the wild, perennial chickpeas(Cicer montbretii) in Bulgaria and (C. ervoides) in theRepublic of Georgia had identical cultural morphol-ogy to isolates from a worldwide collection fromcultivated chickpea (TABLE I). Isolates sampled fromVicia villosa, V. amoena, V. lathyroides, V. grandiflora,V. sepium and V. cordata had identical culturalmorphology and were indistinguishable from A.lentis, A. fabae and A. pisi. An isolate from cultivatedpea (Pisum sativum) in Republic of Georgia hadidentical cultural morphology to isolates of Phomamedicaginis from several cultivated legume hosts(TABLE I). Isolates from wild pea (P. elatius) in theRepublic of Georgia had identical cultural morphol-ogy to isolates from cultivated pea (TABLE I).

ITS phylogeny.—ITS sequences were edited to 470 bpto aid alignment with sequences downloaded fromGenBank. The alignment revealed 224 polymorphicsites of which 179 were parsimony informative whenGenBank data was included and 19 polymorphic sites,seven phylogenetically informative sites and ninehaplotypes among isolates sampled from wild andcultivated legume hosts for this study (i.e. excludingGenBank data) (TABLE II). Isolates sampled from thesame host in diverse worldwide locations and assignedto the same species generally had identical ITShaplotypes (TABLE II). The only exception was A.rabiei isolate AR738 which differed from other A.rabiei isolates at one nucleotide position. Someisolates sampled from different hosts had identical


ITS haplotypes. These included Haplotype 2 isolates,which were sampled from both Lens culinaris (AL1, 2,3, 6, 8, 11, 83, 391) and Vicia grandiflora (Georgia-2,-8); Haplotype 3 isolates, which were sampled fromPisum sativum (AP1, 2, 3, 4, 5, 7, 8, 9), Viciagrandiflora (AV11, Georgia-3), Vicia sp. (Georgia-9)and Pisum elatius (Georgia-7); and Haplotype 5isolates, which were sampled from several Vicia spp.including V. villosa (AV1, 2, 12, 22, 23), V. amoena(AV19) and V. lathyroides (AV8, 20). Two wellsupported (100% Bayesian PP, 100% ML bootstrap)clades were identified: a ‘‘Mycosphaerella’’ clade thatcontained the type species, M. punctiformis, as well asM. fijiensis and M. graminicola, and a ‘‘Pleosporales’’clade, which contained species of Pyrenophora,Cochliobolus, Alternaria, Leptosphaeria, Phaeosphaeriaas well as Ascochyta, Phoma and Didymella speciesfrom various legumes and M. pinodes from pea(FIG. 1). A ‘‘Didymella’’ clade was well supported byBayesian posterior probability (100%) but not by MLbootstrap (46%). The Didymella clade also containedDidymella bryoniae and D. cucurbitacearum fromcucurbits, Phoma macrostoma from Pinus sylvestris(pine), the type species P. herbarum from an un-known host, and P. glomerata from Platanus occiden-talis (sycamore). The branch separating the Myco-sphaerella clade from the Pleosporales clade was longand substantial pairwise homoplasy was evidentbetween taxa in each of these clades (data notshown). Despite the high levels of homoplasy, theITS phylogeny clearly demonstrated that M. pinodesfrom pea should not be considered a Mycosphaerellaspecies. Inclusion of several related ITS sequencesfrom GenBank in the phylogeny revealed that Phaeo-sphaeria spp. are the most likely sister taxon ofDidymella spp., mirroring the results of a previousstudy (Reddy et al 1998).

G3PD phylogeny.—Alignment of edited G3PD se-quences (524 bp) with indels removed (517 bp)revealed 114 polymorphic and 92 parsimony infor-mative sites among all isolates sampled from legumes(excluding outgroup Phaeosphaeria nodorumAY364464). The G3PD alignment revealed substan-tially more variation among legume-associated Asco-chyta spp. than did ITS and had approximately thesame level of phylogenetic resolution as HMGsequences of the MAT1-2 mating gene (Barve et al2003). G3PD sequence analysis with indels excludedrevealed 22 haplotypes among isolates sampled forthis study from wild and cultivated legume hosts.Isolates sampled from the same host in differentgeographic locations and assigned to the same speciesgenerally had similar or identical G3PD haplotypes(TABLE II). The G3PD phylogeny revealed two major

clades among legume-associated Ascochyta spp. witha long branch separating the Ascochyta pinodes/A.pinodella clade (MP and PMP isolates) from otherAscochyta spp. (FIG. 2). Within the A. pinodes/A.pinodella clade an A. pinodella subclade was sup-ported with ML bootstrap and posterior probabilityvalues just above our significance criteria. A. pinodesisolate MP19 appeared to be intermediate betweenthe two subclades. The G3PD phylogeny also revealeddifferentiation between Ascochyta rabiei sampled fromwild and cultivated chickpea and Ascochyta spp.sampled from other legume hosts, although an A.rabiei clade was not highly supported (FIG. 2). Twoadditional, well supported subclades were apparentwithin the A. rabiei/A. lentis/A. fabae/A. pisi clade,one consisting of isolates sampled from lentil (Lensculinaris) and another set of isolates sampled frombig-flower vetch (Vicia grandiflora) (FIG. 2).

Tests of incongruence among data partitions.—Nostrongly conflicting nodes (ML bootstrap values. 70% and/or Bayesian posterior probabilities. 95%) representing monophyletic and nonmono-phyletic relationships for the same set of taxa weredetected among the CHS, EF and G3PD phylogenies.Topologies were similar for each partition althoughCHS had substantially less phylogenetic resolutionthan either EF or G3PD (data not shown). Shimo-daira-Hasegawa (SH) tests and parametric bootstrap-ping similarly did not uncover evidence for significantconflict among the datasets (TABLE III). We wereunable to reject the null hypothesis of a difference intopology between the combined tree and treesestimated for any of the data partitions using theSH test, although P-values for the EF and G3PDpartitions came close to significance (P 5 0.074 and P5 0.054, respectively). We also were unable to rejectthe null hypothesis of the same topology underlyingeach of the data partitions with parametric boot-strapping (TABLE III). Differences between con-strained and unconstrained trees were not significantfor any of the partitions. These results were used tojustify combining the data and estimating a phylogenyfrom the combined dataset.

Combined CHS, EF and G3PD phylogeny.—Alignmentof CHS (329 bp, no indels) revealed 35 polymorphicsites and 18 parsimony informative sites among allisolates (MP and PMP isolates excluded). Alignmentof EF (305 bp) with three indels removed (303 bp)revealed 77 polymorphic sites and 58 parsimonyinformative sites among all isolates (MP and PMPisolates excluded). Eight haplotypes were identifiedfor CHS and 19 haplotypes for EF (TABLE III). Thecombined phylogeny revealed three major cladesamong isolates sampled from cultivated hosts

70 MYCOLOGIA

(FIG. 3). The A. rabiei clade contained isolates of A.rabiei from chickpea (Cicer arietinum), the A. lentisclade contained isolates of A. lentis from lentil (Lensculinaris) and the A. fabae/A. pisi clade contained A.fabae and A. pisi from faba bean (Vicia faba) and pea(Pisum sativum), respectively (FIG. 3). Within theA.fabae/A. pisi clade 2 subclades (V. faba clade, V.grandiflora/V. sepium clade) were evident and withinthe A. lentis clade 3 subclades (L. culinaris clade, V.villosa/V. lathyroides clade, V. grandiflora clade) wereevident (FIG. 3). Isolates from wild hosts weredistributed throughout the phylogeny. Isolate Geor-gia-11 from tiny vetch (V. hirsuta) was distinct andmight represent a phylogenetic species although wewere able to examine only a single isolate from thishost (FIG. 3). Isolates from wild, perennial chickpea(C. ervoides, C. montbretii) clustered with A. rabieiisolates from cultivated, annual chickpea (C. arieti-

num) and many had identical sequences to A. rabieiisolates for all three genomic regions (FIG. 3,TABLE II). Isolates from wild pea (Pisum elatius)clustered with A. pisi isolates from cultivated pea (P.sativum) but a ‘‘Pisum clade’’ was not highlysupported. The Shimodaira-Hasegawa test and para-metric bootstrapping analyses were unable to reject(P 5 0.336 and P 5 0.92, respectively) the hypothesisof monophyly of isolates AP2, 4, 5, Georgia-6, -7, -12sampled from wild and cultivated pea. Isolatessampled from wild pea (P. elatius), big-flower vetch(V. grandiflora), common vetch (V. cordata) weremost closely related to A. fabae and A. pisi while

FIG. 1. Maximum likelihood phylogeny estimated fromribosomal internal transcribed spacer sequence data (ITS)for Ascochyta and Didymella spp. sampled from legumes(shaded in gray) plus several reference ITS sequencesretrieved from GenBank. Phylogeny was rooted by M.punctiformis (GenBank accession No. AY490760). Onlyunique haplotypes were analyzed and presented anda complete list of isolates with each haplotype is provided(TABLE II). Upper numbers at major nodes indicateBayesian posterior probabilities of sampling the nodeamong 6000 trees (600 000 generations of the MCMCchain) and lower numbers indicate percent ML bootstrapvalues from 1000 bootstrap samples. Branch lengths areproportional to the inferred amount of evolutionary changeand the scale represents 0.1 nucleotide substitutions per site.

FIG. 2. Maximum likelihood phylogeny estimated fromglyceraldehyde-3-phosphate-dehydrogenase (G3PD) se-quence data for Ascochyta and Didymella spp. sampled fromvarious legume hosts. Phylogeny was rooted by Phaeo-sphaeria nodorum (GenBank accession No. AY364464). Onlyunique haplotypes were analyzed and presented anda complete list of isolates with each G3PD haplotype isgiven (TABLE II). Upper numbers at major nodes indicateBayesian posterior probabilities of sampling the nodeamong 6000 trees (500 000 generations of the MCMCchain) and lower numbers indicate percent ML bootstrapvalues from 1000 bootstrapped datasets. Clades inferredbased on ML bootstrap values greater than or equal to 70%

and posterior probabilities greater than or equal to 95%.Major clades are identified by open vertical bars andsubclades by solid-line boxes. Branch lengths are pro-portional to the inferred amount of evolutionary changeand the scale represents 0.1 nucleotide substitutions per site.


isolates from hairy vetch (V. villosa), spring vetch (V.lathyroides) and big-flower vetch (V. grandiflora) weremost closely related to A. lentis (FIG. 3). Isolates frombig-flower vetch (V. grandiflora) appeared to bepolyphyletic, falling out into three distinct, wellsupported clades. Supporting this the Shimodaira-Hasegawa test and parametric bootstrapping analysesstrongly rejected (P 5 0.000 and P 5 0.00, re-spectively) the hypothesis that isolates AV11, Georgia-2, -3, -8, -13 sampled from big-flower vetch weremonophyletic. Split decomposition analysis of thecombined dataset revealed a mostly bifurcating tree-like structure with topology similar to that estimatedusing maximum likelihood methods (FIG. 4). Twodistinct clades were evident, the ‘‘A. lentis clade’’containing A. lentis isolates AL1 and AL11 plusisolates AV1 from hairy vetch (V. villosa), AV8 fromspring vetch (V. lathyroides), and isolates Georgia-2and Georgia-8 from big-flower vetch (V. grandiflora).The other major clade (‘‘A. fabae/A. pisi clade’’)contained A. pisi isolates AP1, AP2, AP4, A. fabaeisolates AF1 and AF8 plus isolates from wild pea (P.elatius), common vetch (V. cordata), big-flower vetch(V. grandiflora) and bush vetch (V. sepium). Someincompatible splits resulting in loops in the phylog-eny were evident in the ‘‘A. fabae/A. pisi clade’’ butnot in the ‘‘A. lentis clade (FIG. 4). The ‘‘A. fabae/A.pisi clade’’ was also the region of the phylogeny thatshowed blocks of incompatible sites (data not shown).No putative intragenic or intergenic recombinationevents were detected within or between any of thegenomic regions with RDP2 (data not shown) butblocks of incompatible sites were evident in thecompatibility matrix for the combined dataset, whichsuggests a history of recombination among thesegenomic regions. Recombination was localized withRecom58 to the A. fabae/A. pisi clade and isolates

Georgia-6, Georgia-7 and Georgia-12 were identifiedas putative recombinants.

DISCUSSION

Ascochyta fungi sampled from wild and cultivatedlegumes worldwide form a monophyletic group thatalso includes Ascochyta, Phoma and Didymella spp.from nonlegume hosts. Taxa such as A. lentis, A. fabaeand A. rabiei have been connected formally toDidymella teleomorphs (Muller and von Arx 1962,Jellis and Punithalingam 1991, Kaiser et al 1997) butothers such as A. pisi and A. pinodella from pea(Pisum sativum), Ascochyta sp. from hairy vetch (V.villosa), V. amoena, bush vetch (V. seppium), commonvetch (V. cordata), spring vetch (V. lathyroides) andbig-flower vetch (V. grandiflora) have not. The resultsof the phylogenetic analyses presented here predictthat all legume-associated Ascochyta spp. likely will beconnected to Didymella teleomorphs in the future.The combined CHS, EF, and G3PD analysis revealedsix well supported clades among Ascochyta fungisampled from 12 legume species. Five of these cladeswere identified among Ascochyta spp. that weremorphologically indistinguishable and fungi in eachof these clades therefore may be considered ‘‘cryptic’’phylogenetic species. Three clades corresponded tothe described taxa A. rabiei, A. lentis and A. fabaefrom the cultivated legume hosts chickpea (Cicerarietinum), lentil (Lens culinaris) and faba bean(Vicia faba), respectively. Three additional cladescontained fungi sampled from various wild Vicia spp.,which either have never been described formally orhave been described under several names. Forexample a query of the Systematic Botany andMycology Laboratory (SMBL) host-fungus database(Farr et al 2005) for hairy vetch (Vicia villosa) yielded

TABLE III. Tests for incongruence among CHS, EF, and G3PD datasets using nonparametric and parametric bootstrapping

Partition Constrainta Score (-lnL) Difference (-lnL) PSHb PPB

c

CHS none 679.457 — — —combined 686.502 7.045 0.132 0.21

EF none 880.235 — — —combined 893.268 13.033 0.074 0.43

G3PD none 1109.853 — — —combined 1129.645 19.792 0.054 0.22

a Combined constraint tree is ML tree estimated for the concatenated dataset using model and model parameters estimatedfor the concatenated dataset.

b Probability that constrained and unconstrained trees are equally good explanations of the data. Null hypothesis tested usingthe Shimodaira-Hasegawa test (Shimodaira and Hasegawa, 1999) with full optimization over 1000 bootstrapped datasets.

c Probability of obtaining a larger log-likelihood difference under the null hypothesis of the same topology underlying eachdata partition. Distribution of the null hypothesis generated by Monte Carlo simulation of 100 datasets on the ML tree for thecombined dataset (estimated using model and parameters for each partition). Partition ML tree (estimated using model andparameters for each partition) was used as the constraint tree.

72 MYCOLOGIA

four taxonomic names of pathogens including A.pinodes, A. pisi, A. viciae-villosae and Mycosphaerellapinodes. It seems possible that all of these names referto the same species. Isolates sampled from hairy vetch(V. villosa) and spring vetch (V. lathyroides) for thisstudy formed a well supported, monophyletic groupand therefore should be considered a distinct phylo-

genetic species. Additional sampling of Ascochyta spp.from various legume species coupled with extensivemorphological, pathological and phylogenetic char-acterization needs to be completed to connect thesefungi to their teleomorphs and assign meaningfulnames to them.

Among the fungi sampled from cultivated hosts,each well supported clade was perfectly correlated tohost of isolation. Isolates from chickpea (Cicerarietinum), faba bean (Vicia faba), pea (Pisumsativum) and lentil (Lens culinaris) each formeda well supported monophyletic group suggesting thathost specificity has played an important role in theevolution of these fungi. Experimental inoculationsof various legume hosts with Ascochyta spp. hasconfirmed host specificity and supports this hypoth-esis (Gossen et al 1986, Kaiser et al 1997, Khan et al1999, Hernandez-Bello et al 2006). Isolates sampledfrom Pisum, Vicia and Lens fell into two wellsupported major clades that we have labeled the ‘‘A.fabae/A. pisi’’ and ‘‘A. lentis’’ clades. These fungi areidentical morphologically and a previous attempt todifferentiate A. lentis from A. fabae based onmorphological characters was unsuccessful (Gossenet al 1986). Our phylogenetic analyses revealed thatAscochyta fabae is most closely related to A. pisi and A.lentis is most closely related to Ascochyta sp. from V.villosa and V. lathyroides. These same relationshipswere observed in a phylogenetic analysis of HMGsequence data from many of the same taxa studied

FIG. 3. Maximum likelihood phylogeny estimated fromthe combined chitin synthase (CHS), translation elongationfactor alpha (EF) and glyceraldehyde-3-phosphate-dehydro-gense (G3PD) datasets for Ascochyta and Didymella spp.sampled from various legume hosts. Only unique haplo-types were analyzed and presented and a complete list ofisolates with each CHS, EF, and G3PD haplotype is given(TABLE II). Upper numbers at major nodes indicateBayesian posterior probabilities of sampling the nodeamong 6000 trees (600 000 generations of the MCMCchain) and lower numbers indicate percent ML bootstrapvalues from 1000 bootstrapped datasets. Clades inferredbased on ML bootstrap values greater than or equal to 70%

and posterior probabilities greater than or equal to 95%.Major clades are identified by open vertical bars and wellsupported subclades by solid-line boxes. Clades withbootstrap values and posterior probabilities below thesignificance criteria are indicated by dashed-line boxes.Branch lengths are proportional to the inferred amount ofevolutionary change and the scale represents 0.01 nucleo-tide substitutions per site. Host of isolation is indicated tothe right of the taxon labels and isolates sampled from Viciagrandiflora are indicated in gray.

FIG. 4. Split decomposition analysis of the combinedchitin synthase (CHS), translation elongation factor alpha(EF) and glyceraldehyde-3-phosphate-dehydrogense(G3PD) datasets. Only isolates from the A. fabae/A. pisiand A. lentis clades of the combined analysis (FIG. 3) wereincluded (i.e. no A. rabiei isolates or Georgia-11). Numbersindicate percent bootstrap support for each split among1000 bootstrapped datasets. Branch lengths are proportion-al to the inferred amount of evolutionary change and thescale represents 0.001 nucleotide substitutions per site. Thetwo major clades are indicated by gray textboxes


here (Barve et al 2003). Fungi within each of theseclades mate readily in the laboratory and culturalmorphology and in vitro growth rates of progeny arenormal (T.L. Peever unpublished). Amplified frag-ment length polymorphism (AFLP) marker segrega-tion ratios among the progeny from the ‘‘interspecif-ic’’ A. fabae 3 A. pisi cross were similar to theirrespective ‘‘intraspecific’’ crosses (i.e. crosses betweentwo A. pisi isolates or between two A. fabae isolates)indicating a lack of obvious intrinsic, postzygoticfertility barriers (Hernandez-Bello et al 2006). When120 progeny from this cross were inoculated on fababean and pea, only 3% of progeny isolates were ableto cause disease on pea and none were able to causedisease on faba bean (Hernandez-Bello et al 2006). Aprevious study employing progeny from the geneti-cally wider cross between A. lentis and A. fabae alsodemonstrated that progeny were unable to causedisease on either parental host (Kaiser et al 1997).The results of the above inoculation experiments withprogeny from ‘‘interspecific’’ crosses suggest thatpathogenicity on each legume host is likely controlledby multiple genetic loci. We speculate that hybridiza-tion of closely related, host-specific forms results indisruption of suites of alleles at loci controllingpathogenicity on each host. The resulting loss ofparasitic fitness of the progeny on both hosts mightconstitute a strong extrinsic isolating barrier (Kohn2005) and play an important role in isolating andpreventing gene flow among host-specific evolution-ary lineages on each host.

The phylogeny estimated for the Ascochyta spp.here correlates well with a plastid matK phylogeny ofthe hosts (Steele and Wojciechowski 2003), possiblysupporting the hypothesis of a co-evolutionary historyof pathogen and host and possible cospeciation. Thedifferentiation seen between Cicer species (tribeCicereae) and Pisum, Vicia and Lens species (tribeVicieae) in the host matK phylogeny is mirrored bythe pathogen phylogeny presented here. Within theVicieae, Vicia hirsuta and V. villosa were found in wellsupported clades distinct from other Vicia spp. Steeleand Wojciechowski (2003) identified two subcladeswithin the Vicieae including Clade 1 which containedPisum sativum and Clade 2 which contained Lensculinaris and Vicia grandiflora. Although there wasnot complete overlap in the hosts sampled for theSteele and Wojciechowski (2003) study and this study,there appears to be broad congruence betweenpathogen and host phylogenies with the Steele andWojciechowski (2003) Clade 1 corresponding to theA. pisi/A. fabae clade of our combined analysis andtheir Clade 2 to our A. lentis clade. Additional fast-evolving regions of the legume genome have beenidentified and currently are being used to resolve the

evolutionary relationships among members of theViciae and Cicerae tribes (Steele and Wojciechowski2003). Estimation of robust phylogenies for bothhosts and pathogens will provide some interestinginsights into the co-evolution of these pathosystems.Isolates sampled from each wild legume host dis-played much more sequence variation for all genomicregions compared to isolates from cultivated legumes.If our sampling had been restricted to fungi fromcultivated hosts we might have concluded that allAscochyta/legume interactions are characterized bya tight co-evolutionary relationship between pathogenand host. However the isolates from wild hosts did notfollow this same pattern with isolates from V.grandiflora distributed in three clades. There are atleast two hypotheses that might explain why theAscochyta fungi sampled from V. grandiflora werepolyphyletic. The first is that wild hosts, but notcultivated hosts, might be colonized by differentevolutionary lineages of Ascochyta pathogens. Thiswould imply that the apparent tight correlationbetween pathogen clade and host of origin seen withisolates from cultivated hosts is the result of selectionby each cultivated host for a single monophyleticlineage of pathogen. The alternative hypothesis is thatall Ascochyta fungi have tight co-evolutionary relation-ships with their hosts but the host taxa identified inthis study are polyphyletic. Host plants employed inthis study were identified morphologically and it ispossible that several distinct evolutionary lineageswere classified as V. grandiflora. There was uncertaintyin identification of at least two of the host taxasampled in this study because entries such as ‘‘Viciasp.’’ and ‘‘Vicia sp. (possibly V. grandiflora)’’ wererecorded in collecting notes at the time of sampling(TABLE I). To distinguish between these two hypoth-eses a more careful morphological analysis of thehosts needs to be performed as well as controlledinoculations of fungal isolates on the hosts.

Phylogenetic analyses revealed that A. rabiei, thepathogen of chickpea (Cicer arietinum), is distinctfrom the Ascochyta pathogens of pea, faba bean, wildvetches and lentil. A. rabiei is morphologically distinctwith slower growth in culture and darker colonymorphology relative to A. pisi, A. lentis and A. fabae.Isolates sampled from wild perennial Cicer spp. (C.montbretii and C. ervoides) had sequences that wereidentical or nearly identical to isolates from cultivatedannual chickpea (Cicer arietinum) for all loci. Cicerarietinum is an annual species that is most closelyrelated to the wild annual C. reticulatum and isgenetically distinct from the perennial species, C.montbretii and C. ervoides (Javadi and Yamaguchi2004, Sudupak et al 2004). The similarity of fungicolonizing annual and perennial Cicer hosts suggests

74 MYCOLOGIA

that the source of the Ascochyta blight fungus forepidemics on cultivated chickpea might be wild,perennial chickpea relatives. Isolates from wild pea(P. elatius) also clustered with isolates from cultivatedpea consistent with the hypothesis that the source ofAscochyta blight epidemics on cultivated pea mightbe Ascochyta-infected P. elatius, the presumed ances-tor of cultivated pea (Smartt 1990). We currently areperforming host inoculations of wild and cultivatedpea with fungi sampled from each of these hosts todetermine whether these fungi can cause disease onboth hosts. Sampling and phylogenetic analyses ofisolates from sympatric populations of cultivatedlegumes and their wild relatives will be required tocritically test these hypotheses.

The ITS phylogeny clearly demonstrated thatAscochyta pinodes (teleomorph: Mycosphaerella pi-nodes) is not closely related to true Mycosphaerellaspp. such as M. graminicola, M. fijiensis and M.punctiformis. M. pinodes was transferred to Didymellapinodes by Petrak in 1924 (Petrak 1924) but this namehas not been widely adopted by mycologists and plantpathologists. Our phylogenetic analyses indicate thatthis fungus is closely related to other Ascochytapathogens with Didymella teleomorphs and we rec-ommend that the name D. pinodes Petrak be appliedto these fungi. Phylogenetic analysis of G3PD in-dicated that the ‘‘Ascochyta complex’’ of pea iscaused by at least two, and possibly three, phyloge-netically distinct fungi and supported the results ofa previous phylogenetic analysis using mating geneHMG sequence data (Barve et al 2003). Isolatesidentified morphologically as Ascochyta pinodes and A.pinodella were related closely and highly divergentfrom the other member of the complex, A. pisi. Thisresult correlates well with previous morphological andmolecular studies (Madosingh and Wallen 1968, Fariset al 1995, Bretag and Ramsey 2001, Fatehi et al2003). Phylogenetic analysis of G3PD sequencesallowed differentiation of A. pinodes from A. pinodellausing our criterion of significantly supported nodeshaving bootstrap values and posterior probabilities of70% and 95%, respectively. However support for thenode separating A. pinodes from A. pinodella was onlymarginally above our cut-off criteria (71% and 95%,respectively). These taxa likely could be easilydifferentiated with additional sequence data frommore variable regions of the genome. Fatehi et al(2003) were unable to differentiate A. pinodes from A.pinodella based on restriction digestion of nuclearribosomal ITS or beta-tubulin regions and consideredthese fungi conspecific. A. pinodes and A. pinodellapreviously differentiated based on RAPD markers(Onfroy et al 1999) and restriction digestion ofmitochondrial DNA (Fatehi et al 2003). We did not

attempt to amplify CHS or EF from A. pinodes and A.pinodella but it seems likely that these regions wouldprovide increased support for monophyletic cladescorresponding to each morphological taxon.

ACKNOWLEDGMENTS

PPNS No. 0410, Project No. 0300, Department of PlantPathology, College of Agricultural, Human and NaturalResource Sciences, Washington State University, Pullman,Washington, 99164-6430. Research supported in part byUSDA-CSFL and the McKnight Foundation. The authorsthank T. Reynolds, T. Hughes and S. Salimath for technicalassistance.

LITERATURE CITED

Allard C, Bill L, Touraud G. 1993. L’anthracnose du pois:revue bibliographique et synthese. Agronomie Paris 3:5–24.

Barve MP, Arie T, Salimath S, Muehlbauer FJ, Peever TL.2003. Cloning and characterization of the mating type(MAT) locus from Ascochyta rabiei (teleomorph:Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp. Fung Genet Biol 39:151–167.

Berbee ML, Pirseyedi M, Hubbard S. 1999. Cochliobolusphylogenetics and the origin of known, highly virulentpathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences. Mycologia91(6):964–977.

Binder M, Hibbett DS. 2002. Higer-level phylogeneticrelationships of homobasidiomycetes (mushroom-forming fungi) inferred from four rDNA regions. MolPhylogenet Evol 22(1):76–90.

Bowen JK, Lewis BG, Matthews P. 1997. Discovery of theteleomorph of Phoma medicaginis var. pinodella inculture. Mycol Res 101:80–84.

Bretag TW, Ramsey M. 2001. Foliar Diseases caused byFungi: Ascochyta spp. In: Kraft JM, Pfleger FL., eds.Compendium of Pea Diseases. St Paul: AmericanPhytopathological Society Press. p 24–28.

Carbone I, Kohn LM. 1999. A method for designing primersets for speciation studies in filamentous ascomycetes.Mycologia 91(3):553–556.

Cherif M, Chilvers MI, Akamatsu H, Peever TL, Kaiser WJ.2006. Cloning of the mating type locus from Ascochytalentis (teleomorph: Didymella lentis) and developmentof a multiplex PCR mating assay for Ascochyta species.Curr Genet 50. (published online DOI: 10.1007/s00294-006-0085-y).

Faris MS, Corbiere R, Lyons NF, Spire D. 1995. Evaluationof an enzyme-linked immunosorbent assay for de-tection of Mycosphaerella pinodes in pea seeds. AnnAppl Biol 127(3):441–455.

Faris-Mokaiesh S, Boccara M, Denis JB, Derrien A, Spire D.1996. Differentiation of the ‘‘Ascochyta complex’’ fungiof pea by biochemical and molecular markers. CurrGenet 29:182–190.

Farr DF, Rossman AY, Palm ME, McCray EB. 2005. Fungal


Databases, Systematic Botany & Mycology Laboratory,ARS, USDA, USDA-ARS.

Fatehi J, Bridge PD, Punithalingam E. 2003. Molecularrelatedness within the ‘‘Ascochyta pinodes-complex’’.Mycopathologia 156:317–327.

Froslev TG, Matheny PB, Hibbett DS. 2005. Lower levelrelationships in the mushroom genus Cortinarius(Basidiomycota, Agaricales): a comparison of RBP1,RBP2, and ITS phylogenies. Mol Phylogenet Evol 37:602–618.

Goldman N, Anderson JP, Rodrigo AG. 2000. Likelihood-based tests of topologies in phylogenetics. Syst Biol49(4):652–670.

Gossen BD, Sheard JW, Beauchamp CJ, Morrall RAA. 1986.Ascochyta lentis renamed Ascochyta fabae f.sp. lentis. CanJ Plant Pathol 8:154–160.

Griffiths R, Marjoram P. 1996. Ancestral inference fromsamples of DNA sequences with recombination. JComp Biol 3:479–502.

Hernandez-Bello MA, Chilvers MI, Akamatsu H, Peever TL.2006. Host specificity of Ascochyta species infectinglegumes of the Viciae and Cicerae tribes and thepathogenicity of an interspecific hybrid. Phytopatho-logy 96:1148–1156.

Huelsenbeck JP, Bull JJ. 1996. A likelihood ratio test todetect conflicting phylogenetic signal. Syst Biol45(1):92–98.

———, ———, Cunningham CW. 1996. Combining data inphylogenetic analysis. Trend Ecol Evol 11(4):152–158.

———, Crandall KA. 1997. Phylogeny estimation andhypothesis testing using maximum likelihood. AnnRev Ecol Syst 28:437–466.

———, Hillis DM, Nielsen R. 1996. A likelihood ratio test ofmonophyly. Syst Biol 45(4):546–558.

———, Ronquist F. 2001. MrBayes: Baysian inference ofphylogenetic trees. Bioinformatics 17(8):754–755.

Huson DH. 1998. SplitsTree: analyzing and visualizingevolutionary data. Bioinformatics 14(1):68–73.

Javadi F, Yamaguchi H. 2004. Interspecific relationships ofthe genus Cicer L. (Fabaceae) based on trnT-Fsequences. Theo Appl Genet 109:317–322.

Jellis GJ, Punithalingam E. 1991. Discovery of Didymellafabae sp. nov., the teleomorph of Ascochyta fabae, onfaba bean straw. Plant Pathol 40:150–157.

Jones LK. 1927. Studies of the nature and control of blight,leaf and pod spot, and footrot of peas caused by speciesof Ascochyta. Bulletin of the New York State Agricul-tural Experimental Station 547:46.

Kaiser WJ. 1997. The teleomorph of Ascochyta rabiei and itssignificance in breeding chickpea. In: Udupa FS,Weigand M., eds. Application of DNA fingerprintingfor crop improvement: marker-assisted selection ofchickpea for sustainable agriculture in the dry areas.Aleppo: ICARDA. p 3–21.

———, Hellier BC. 1993. Didymella sp., the teleomorph ofAscochyta fabae f. sp. lentis on lentil straw. Phytopathol-ogy 83:692. (Abstract)

———, Muehlbauer FJ. 1988. An outbreak of Ascochytablight of chickpea in the Pacific Northwest. IntChickpea Newslet 18:16–17.

———, Wang B-C, Rogers JD. 1997. Ascochyta fabae and A.lentis: Host specificity, teleomorphs (Didymella), hy-brid analysis and taxonomic status. Plant Dis 81:809–816.

Khan MSA, Ramsey MD, Scott ES. 1999. Host range studieswith an Australian isolate of Ascochyta rabiei. AustralasiaPlant Pathol 28:170–173.

Kohn LM. 2005. Mechanisms of fungal speciation. Ann RevPhytopathol 43:279–308.

Kovachevski IC. 1936. The blight of chickpea (Cicerarietinum), Mycosphaerella rabiei n. sp. Sofia, Bulgaria:Ministry of Agriculture and National Domains.

Leath KT. 1994. Foliar blight of big-flower vetch caused byAscochyta fabae f. sp. vicia. Plant Dis 78:637–639.

Maddison DR, Maddison WP. 2003. MacClade 4: analysis ofphylogeny and character evolution. Sunderland, Mas-sachusetts: Sinauer Associates.

Madosingh G, Wallen VR. 1968. Serological differentiationof the Ascochyta species on peas. Can J Microbiol 14:449–451.

Martin DP, Williamson C, Posada D. 2005. RDP2: re-combination detection and analysis from sequencealignments. Bioinformatics 21(5):260–262.

Muller E, von Arx JA. 1962. Die gattungen der didymos-poren Pyrenomyceten. Beitr Kryptogamenfl Schweiz11:1–922.

Nene YL. 1982. A review of Ascochyta blight of chickpea.Tropic Pest Manage 28(1):61–70.

Onfroy C, Tivoli B, Corbiere R, Bouznad Z. 1999. Cultural,molecular and pathogenic variability of Mycosphaerellapinodes and Phoma medicaginis var. pinodella isolatesfrom dried pea (Pisum sativum) in France. Plant Pathol48:218–229.

Peever TL, Canihos Y, Olsen L, Ibanez A, Liu Y-C. 1999.Population genetic structure and host specificity ofAlternaria spp. causing brown spot of Minneola tangeloand rough lemon in Florida. Phytopathology 89:851–860.

Petrak F. 1924. Mykologische Notizen VII. Annal Mycol 22:1–182.

Posada D, Crandall KA. 1998. Modeltest: testing the modelof DNA substitution. Bioinformatics 14(9):817–818.

Price EW, Carbone I. 2004. SNAP: workbench managementtool for evolutionary population genetic analysis.Bioinformatics 21(3):402–404.

Punithalingam E, Holiday P. 1972. Mycosphaerella pinodes.Kew, UK: CMI Descriptions of Pathogenic Fungi. 340 p.

Rambout A, Grassly NC. 1997. Seq-Gen: an application forthe Monte Carlo simulation of DNA sequence evolu-tion along phylogenetic trees. Comp Appl Biosci13(3):235–238.

Reddy PV, Patel R, White JFJ. 1998. Phylogenetic anddevelopmental evidence supporting reclassification ofcruciferous pathogens Phoma lingam and Phomawasabiae in Plenodomus. Can J Bot 76:1916–1922.

Reeb V, Lutzoni F, Roux C. 2004. Contribution of RBP2 tomultilocus phylogenetic studies of the euascomycetes(Pezizomycotina, Fungi) with special emphasis on thelichen-forming Acarosporaceae and evolution of poly-spory. Mol Phylogenet Evol 32:1036–1060.

76 MYCOLOGIA

Shimodaira H, Hasegawa M. 1999. Multiple comparisons oflog-likelihoods with applications to phylogenetic in-ference. Mol Biol Evol 16:1114–1116.

Smartt J. 1990. Grain legumes: evolution and geneticresources. Cambridge, UK: Cambridge University Press.

Steele KP, Wojciechowski MF. 2003. Phylogenetic analysesof tribes Trifolieae and Vicieae based on sequences ofthe plastid gene, matK (Papilonoideae: Leguminosae).In: Klitgaard BB, Bruneau A, eds. Advances in LegumeSystematics. 10: higher level systemtics. Kew, UK: RoyalBotanic Gardens. p 355–370.

Sudupak MA, Akkaya MS, Kence A. 2004. Genetic relation-ships among perennial and annual Cicer speciesgrowing in Turkey assessed by AFLP fingerprinting.Theo Appl Genet 108:937–944.

Swofford DL. 2002. PAUP*: phylogenetic analysis usingparsimony (* and other methods). Sunderland, Massa-chusetts: Sinauer Associates.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,Higgins DG. 1997. The Clustal X windows interface:

flexible strategies for multiple sequence alignmentaided by quality analysis tools. Nuc Acid Res 24:4876–4882.

Trapero-Casas A, Navas-Cortes JA, Jimenez-Diaz RM. 1996.Airborne ascospores of Didymella rabiei as a majorsource of primary inoculum for Ascochyta blightepidemics in chickpea crops in southern Spain. EuroJ of Plant Patholol 102:237–245.

Wallen VR. 1965. Field evaluation and the importance ofthe Ascochyta complex on peas. Can J Plant Sci 45:27–33.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplificationand direct sequencing of fungal ribosomal RNA genesfor phylogenetics. In: Innis MA, Gelfand DH, SninskyJJ, White TJ, eds. PCR Protocols: a guide to methodsand applications. San Diego: Academic Press. p 315–322.

Wilson AD, Kaiser WJ. 1995. Cytology and genetics of sexualcompatibility in Didymella rabiei. Mycologia 87(6):795–804.


evolutionary relationships among ascochyta …...from chickpea (ar), a. fabae f. sp. fabae (af) and...

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