Phylogeny of the festucoid grasses of subtribe Loliinae and allies

MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 31 (2004) 517–541

EVOLUTION

www.elsevier.com/locate/ympev

Phylogeny of the festucoid grasses of subtribe Loliinae and allies(Poeae, Pooideae) inferred from ITS and trnL–F sequences

Pilar Catal�an,a,* Pedro Torrecilla,a,1 Jose �Angel L�opez Rodr�ıguez,a

and Richard G. Olmsteadb

a Department of Agriculture (Botany), University of Zaragoza, 50013 Zaragoza, Spainb Department of Biology, Washington University, USA

Received 10 March 2003; revised 22 August 2003

Abstract

Analyses of ribosomal ITS and chloroplast trnL–F sequences provide phylogenetic reconstruction for the festucoids (Poeae:

Loliinae), a group of temperate grasses with morphological and molecular affinities to the large genus Festuca. Parsimony and

Bayesian analyses of the combined ITS/trnL–F dataset show Loliinae to be monophyletic but unresolved for a weakly supported

clade of �broad-leaved Festuca,� a well-supported clade of �fine-leaved Festuca,� and Castellia. The first group includes subgenera

Schenodorus, Drymanthele, Leucopoa, and Subulatae, and sections Subbulbosae, Scariosa, and Pseudoscariosa of Festuca, plus

Lolium and Micropyropsis. The second group includes sections Festuca, Aulaxyper, Eskia, and Amphigenes of Festuca, plus Vulpia,

Ctenopsis, Psilurus, Wangenheimia, Cutandia, Narduroides, and Micropyrum. Subtribes Dactylidinae and Cynosurinae/Parapholii-

nae are sister clades and are the closest relatives of Loliinae. Vulpia is polyphyletic within the �fine-leaved� fescues as revealed by the

two genome analyses. Lolium is resolved as monophyletic in the ITS and combined analyses, but unresolved in the trnL–F based

tree. Conflict between the ITS and the trnL–F trees in the placement of several taxa suggests the possibility of past reticulation

events, although lineage sorting and possible ITS paralogy cannot be ruled out.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Festucoids; Loliinae; Dactylidinae; Cynosurinae; Parapholiinae; ITS; trnL–F; Phylogeny and systematics

1. Introduction

The grass subtribe Loliinae is one of the largest of

tribe Poeae (Pooideae, Poaceae). It encompasses the

broad genus Festuca, which accounts for more than 500

species distributed in the holarctic region and in tem-perate zones of the Southern hemisphere (Kerguel�en and

Plonka, 1989; Watson and Dallwitz, 1992), as well as its

satellite genera (Clayton and Renvoize, 1986; Watson

and Dallwitz, 1992). Festuca species are characterized by

their dorsally rounded lemma and linear hilum, whereas

species of Poa (subtribe Poinae) are distinguished by

their keeled lemmas and round to oval hilum. Clayton

and Renvoize (1986) speculated on the evolution of the

* Corresponding author. Fax: +34-976-762488.

E-mail address: [email protected] (P. Catal�an).1 Present address: Department of Botany, Faculty of Agronomy,

University of Central Venezuela, Venezuela.

1055-7903/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2003.08.025

main Poeae lines, suggesting the evolution of Mediter-

ranean annuals from mountain-grassland perennials.

These authors pictured Lolium, Vulpia, and other small

genera (Micropyropsis, Micropyrum, Castellia, Psilurus,

Wangenheimia, Cynosurus, and Lamarckia, among oth-

ers) as derived groups of Festuca, and Puccinellia andother small genera (Dactylis, Desmazeria, Sclerochloa,

Cutandia, Sphenopus, and Parafestuca, among others) as

close allies of Poa. The circumscription of the festucoid

lineage agrees in part, though not completely, with the

systematic proposal of Tzvelev (1982), who restricted

subtribe Festucinae to nine genera (Festuca, Lolium,

Vulpia, Nardurus, Loliolum, Scleropoa, Cutandia,

Sphenopus, and Bellardiochloa). Nomenclatural priorityfavours Loliinae Dumort. over Festucinae C. Presl

(Soreng and Davis, 2000) as the correct subtribe name

for the festucoids.

Festuca, in contrast to Poa, is a complex genus that

has been divided into several subgenera and sections

mail to: [email protected]

518 P. Catal�an et al. / Molecular Phylogenetics and Evolution 31 (2004) 517–541

(Alexeev, 1977, 1978, 1980, 1981, 1986; Hackel, 1882,1887, 1906; Krechetovich and Bobrov, 1934; Krivotu-

lenko, 1960; Piper, 1906; Tzvelev, 1971). Conversely,

several segregates of Festuca, which were in the past

included within this genus (Vulpia, Schedonorus, Dry-

mochloa, and Leucopoa), have been recognized as inde-

pendent genera at different times (Cotton and Stace,

1977; Gmelin, 1805; Holub, 1984, 1998; Soreng and

Terrell, 1998, in preparation; Stace, 1981; Tzvelev, 1999,2000). Phylogenetic studies of Festuca sensu lato based

on analysis of chloroplast RFLP data (Darbyshire and

Warwick, 1992) and on ribosomal ITS sequences

(Charmet et al., 1997; Gaut et al., 2000; Torrecilla and

Catal�an, 2002) demonstrated that Festuca was a para-

phyletic lineage and that Lolium and Vulpia were nested

within it. Torrecilla and Catal�an (2002) distinguished

two separate lineages within Festuca, which were re-spectively called the �broad-leaved� and �fine-leaved�Festuca. The closeness of Lolium to the broad-leaved

Festuca of subgen. Schedonorus and that of Vulpia to the

fine-leaved Festuca confirmed previous findings based

on chromosome analyses and on artificial crosses

(Ainscough et al., 1986; Barker and Stace, 1982; Jenkin,

1933; Malik and Thomas, 1966). Further investigations

on the relationship of Vulpia, Festuca, and their closestallies based on independent and simultaneous analyses

of ITS and trnL–F sequences (Torrecilla et al., 2003b)

have extended the range of paraphyly of the �fine-leaved�fescues, a group that includes not only the polyphyletic

Vulpia lineages but also six other Mediterranean annual

genera (Psilurus, Ctenopsis, Narduroides, Wangenheimia,

Cutandia, and Micropyrum).

This work is part of a broader investigation aimed tocontinue exploring the phylogeny and evolutionary

trends of the festucoids, one of the largest groups of

tribe Poeae. We have extended our present survey to

those subgenera and sections of �broad-leaved� Festucathat were under-represented in our previous studies and

to other genera of Poeae considered to be close to this

lineage (cf. Clayton and Renvoize, 1986; Soreng and

Davis, 2000; Tzvelev, 1982). Our taxon sampling in-cludes some of the most valuable forage grasses of cold

and temperate climates of the Northern hemisphere, like

the meadow and tall fescues (Festuca pratensis and F.

arundinacea complexes of Festuca subgen. Schedonorus),

the �ovina� and �red� fescues (Festuca ovina and F. rubra

groups of Festuca subgen. Festuca), the rye-grass genus

Lolium, and the orchardgrass or cock�s-foot genus

Dactylis, together with a large number of taxa endemicto the Western Mediterranean region, the likely centre

of diversification of several of these lineages. We have

used sequences from the ribosomal ITS regions and

from the chloroplast trnL–F region, two differently in-

herited molecules which have proved to be useful in

phylogenetic analyses of angiosperms and in recon-

structing the phylogeny of this group of grasses (Hsiao

et al., 1995a; Torrecilla and Catal�an, 2002; Torrecillaet al., 2003a,b).

Conflicts between topologies recovered from different

genomes have been interpreted in different ways, such as

lineage sorting or reticulation (Wendel and Doyle,

1998). The existence of past hybridization events can

create problems for phylogenetic reconstruction in an-

giosperms and is of particular concern within the tem-

perate grasses (Davis and Soreng, 1993; Kellogg et al.,1996; Mason-Gamer and Kellogg, 2000; Soreng and

Davis, 2000). Chloroplast capture has been considered a

possible explanation for the otherwise unexpected

placements of some Aveneae taxa within some Poeae

clades and vice versa (Soreng and Davis, 2000), whereas

lineage sorting coupled with hybridization and allo-

polyploidy are hypotheses invoked to explain the failure

to reconcile topologies recovered from different genomes(nuclear vs. chloroplast) in tribe Triticeae (Kellogg et al.,

1996; Mason-Gamer and Kellogg, 1996, 1997). Potential

conflict also may arise within a single dataset as a result

of extended paralogy (Buckler et al., 1997); for example

pseudogene copies of ITS sequences have been detected

in Lolium (Gaut et al., 2000). Mason-Gamer and Kel-

logg (1997) indicated putative incongruences within ITS

sequences of Triticeae based on the low resolution andpoor bootstrap support of the clades.

Because some of our previous studies indicated an

increasing conflict between rival ITS and trnL–F to-

pologies derived from a larger taxon sampling of rep-

resentatives of the festucoids, we have traced potential

past hybridizations events within our ingroup taxa by

comparing bootstrap support for lineages resolved by

the independent topologies and by the combined treesand taking into account documented records of present

introgression events within Loliinae.

2. Materials and methods

Sampling was designed to represent the taxonomic,

geographic, and phenotypic diversity in tribe Loliinaeand to build upon previous studies (Torrecilla and Ca-

tal�an, 2002; Torrecilla et al., 2003b). This study included

109 accessions of Poeae, three members of the sister

tribe Aveneae (Avena barbata, A. eriantha, and Des-

champsia cespitosa), and one representative each of

tribes Triticeae (Secale cereale) and Brachypodieae

(Brachypodium distachyon), for a total of 139 samples.

Sampling of Poeae covers 120 representatives of subtribeLoliinae (¼Festucinae), 5 of subtribe Poinae (Poa,

Puccinellia, Sclerochloa), 4 of subtribe Dactylidinae

(Dactylis, Lamarckia), 2 of subtribe Cynosurinae (Cy-

nosurus), 5 of subtribe Parapholinae (Monerma, Parap-

holis, Catapodium, Sphenopus), and one each of

subtribes Sesleriinae (Sesleria argentea), and Psilurinae

(Psilurus). Within the festucoids (Loliinae), 71 samples

P. Catal�an et al. / Molecular Phylogenetics and Evolution 31 (2004) 517–541 519

corresponded to taxa of Festuca sensu lato, representing5 subgenera and 12 sections, 18 corresponded to taxa of

Vulpia, representing the 5 sections of this genus, 10

corresponded to taxa of Lolium, and 10 corresponded to

genera that have been considered or demonstrated to be

more or less related to Festuca (Micropyrum, Narduro-

ides, Wangenheimia, Cutandia, Ctenopsis, Castellia, Mi-

cropyropsis, Hellerochloa, Parafestuca). The list of taxa

with authorities, localities, herbarium vouchers, ploidylevels, and GenBank accession numbers is shown in

Table 1.

Fresh material and herbarium vouchers were col-

lected for this molecular survey. Eighty samples ana-

lyzed in our previous studies (Torrecilla and Catal�an,2002; Torrecilla et al., 2003b) were used in this survey

along with 45 new samples. Both ITS and trnL–F se-

quences obtained for those 45 new samples were de-posited in GenBank (Table 1). ITS sequences of

A. barbata (Moore and Field, 2002) and of Lolium

remotum, L. temulentum, L. subulatum, and L. rigidum

(Charmet et al., 1997) were retrieved from GenBank,

incorporated to the data matrix and used for the phy-

logenetic analyses.

DNA isolation from fresh leaves followed the pro-

cedures stated in Torrecilla and Catal�an (2002) consist-ing of a modified CTAB protocol for minipreparations

(Doyle and Doyle, 1987); DNA from herbarium

vouchers was extracted with the DNAeasy Plant Mini

Kit (Qiagen) used according to manufacturer�s instruc-

tions. Amplification of the ribosomal ITS region (ITS1-

5.8S-ITS2) and further sequencing were performed as

indicated in Torrecilla and Catal�an (2002) using the

external primers KRC (forward) and ITS4 (reverse).PCR amplification and sequencing of the trnL–F region

was accomplished as indicated in Torrecilla et al.

(2003a) using the external pair primers �c� and �f� (Tab-erlet et al., 1991), or the combined pair �fern� (forward)and �f� (reverse). Ninety-five out of the 110 taxa studied

have been sequenced for both the ITS and the trnL–F

region. Partial sequences of the ITS region of Festuca

nevadensis (ITS1 spacer, 5.8 gene) and of the trnL–Fregion of L. rigidum 1 (trnL intron, trnL30 exon) and of

Festuca coromotensis (trnL intron, trnL30 exon, partialtrnL–trnF spacer) were included in the analyses, because

of their potential phylogenetic information.

The ITS and trnL–F sequence data matrices were

aligned visually with the help of the program Se-Al v.

1.0 alpha 1 (Rambaud, 1996). The boundaries of the ITS

region (ITS1-5.8-ITS2) and of the trnL–F region (trnLintron–trnL 30exon–trnL–F spacer) were determined

according to those established by Torrecilla et al.

(2003a,b) for Festuca and related genera. Gaps were

coded as binary characters by their presence/absence;

only those gaps that were unambiguous and potentially

informative were added to the corresponding sequence

matrix and used for the parsimony-based analyses.

Phylogenetic analyses were performed on each indi-vidual dataset and the combined dataset with PAUP* ver.

4.0 beta 10 (Swofford, 2002), based on maximum parsi-

mony, and with MRBAYES v. 3.0 (Huelsenbeck and

Ronquist, 2002), based on Bayesian inference. Parsimony

analysis was first conducted on each independent data

matrix following the strategies described in Catal�an et al.

(1997). The ITS dataset consisted of 118 sequences and

the trnL–F dataset consisted of 112 sequences, with thecombined dataset consisting of the 101 accessions in

common between the two individual datasets. Each data

set was subjected to two heuristic searches aimed to find

putative islands of equally parsimonious trees. An initial

search was completed after 500 replicates of random-or-

der-entry-starting trees (TBR, MULPARS ON) saving

nomore than five trees per replicate with length greater or

equal than 5. All parsimonious trees found from thissearch were used to compute a strict consensus tree that

was used as a negative constraint for a second search of

500 replicates (random-order-entry, TBR, MULPARS

ON) saving no more than two trees per replicate. This

second search did not find any other island of equally

parsimony for any of the two datasets, thus indicating

that a strict consensus tree of themost-parsimonious trees

that were found with each data set should represent theresolution available in the data, even if all shortest trees

were not found. In all parsimony analyses B. distachyon

was used to root the trees.

Branch support for the optimal trees found under the

parsimony criterion was estimated through 1000 boot-

strap replicates (Felsenstein, 1985) using the TBR-M

(Tree Bisection Reconstruction swapping, MULPARS

OFF) strategy ofDeBry andOlmstead (2000) as amethodto reduce computational efforts in those resampling

analyses.

The Bayesian inference search was performed for the

separate ITSand trnL–Fdatamatrices.A test of goodness

of fit for 56 nucleotide substitutions models was previ-

ously conducted on each individual dataset using the

likelihood ratio test statistic included in the program

Model Test ver. 3.06 (Posada and Crandall, 1998). Thetwo independent datasets showed the sameoptimalmodel

(GTR+G+ I, 4 gamma rate categories); this model was

imposed in the subsequent Bayesian analyses. Bayesian

inference on the combined dataset used separate substi-

tution models for each data matrix in the simultaneous

analysis using the partition options provided in MRBA-

YES 3.1. The Bayesian analysis of each separate data set

was first performed through 1,000,000 generations by theMarkov chain Monte Carlo (MCMC) sampling trees

every 100 generations and allowing the program to esti-

mate the respective likelihood parameters (nucleotide

frequencies, nucleotide substitution rates, gamma shape,

proportion of invariable sites). Phylogenies sampled from

their posterior probability distribution were analyzed

in order to observe the number of generations of trees

Table 1

List of taxa included in the phylogenetic ITS and trnL–F study of festucoid grasses of subtribe Loliinae and allies

Taxaa and ploidy levelb Source GenBank Accession No.

ITS trnL–F

Festuca L.

Subgen. Festuca

Sect. Festuca

Subsect. Festuca (‘‘F. ovina complex’’)

Festuca alpina Suter (2x) Torrecilla and Catal�an (2002); Spain:

Huesca: Pyr�en�ees: Vallibierna

AF303415 AF478522

Festuca clementei Boiss. (2x) UZ, PC-JALR-PT 81.2000; Spain: Granada:

Sierra Nevada: Veleta

AF478482 AF478524

Festuca filiformis Pourr. (2x) Charmet et al. (1997) AJ240160 —

Festuca frigida (Hackel) K. Richter (2x) UZ, PC-JALR-PT 80.2000; Spain: Granada:

Sierra Nevada: Laguna Aguas Verdes

AF478481 AF478521

Festuca glacialis Mi�egev. ex Anon. (2x) Torrecilla and Catal�an (2002); Spain: Huesca:

Pyr�en�ees: Cotiella

AF303428 AF478523

Festuca hystrix Boiss. (2x) UZ, PC-JALR-PT 31.2000; Spain: Almer�ıa:

Sierra de G�ador: Morr�on del Observatorio

AF478480 AF478520

Festuca idahoensis Elmer (4x) BW 533; USA: Oregon: Deschutes County, — AF533064

Festuca indigesta Boiss. subsp. indigesta (6x) UZ, PC-JALR-PT 43.2000; Spain: Granada,

Sierra Nevada

— AF478519

Festuca indigesta Boiss. subsp. aragonensis (Willk.)

(1) Kergu�elen (1) (4x)

UZ, JALR 01174(A); Spain: Zaragoza:

Moncayo

AF519975 AF495884

Festuca indigesta Boiss. subsp. aragonensis (Willk.)

(2) Kergu�elen (2) (4x)

UZ, PC 17.2002; Spain: Zaragoza:

Moncayo

— AF533062

Festuca longiauriculata Fuerte,

Ort�unez and Ferrero (2x)

UZ, PC-JALR-PT 59.2000; Spain: Almer�ıa:

Sierra de los Filabres: Calar Alto

AF478479 AF478518

Festuca ovina L. (2x) UZ, JM 6879; Germany: Th€uringen:

Saale-Holzland-Kreis

AF532959 AF533063

Festuca plicata Hackel (2x) UZ, PC-JALR-PT 86.2000; Spain:

Granada: Sierra Nevada: Dornajo

AF478483 AF478525

Subsect. Exaratae St-Yves

Festuca borderei (Hackel) K. Richter (2x) Torrecilla and Catal�an (2002); Spain:

Huesca:Vallibierna

AF303403 AF478510

Festuca capillifolia Dufour (2x) Torrecilla and Catal�an (2002); Spain: Ja�en: Cazorla AF303419 AF478511

Festuca querana Litard. (4x) UZ, JALR 1326; Spain: Lugo: Monforte de Lemos AF532957 AF533057

Sect. Aulaxyper Dumort. (‘‘F. rubra complex’’)

Festuca ampla Hackel (4x) UZ, JALR 01155; Spain: C�adiz, Grazalema — AF543516

Festuca heterophylla Lam. (1) (4x) UZ, JM 7600; Germany: Bayern: Kreis Kelheim AF532958 —

Festuca heterophylla Lam. (2) (4x) Charmet et al. (1997) AJ240159 —

Festuca iberica (Hackel) K. Richter (6x) UZ, PC-JALR-PT 77.2000; Spain: Granada:

Sierra Nevada: Borreguiles de San Juan

AY118087 AF478516

Festuca juncifolia St.-Amans (8x) UZ, JALR 01366; Spain: Lugo: Viveiro:

Brieiro: Arenales de Area

AF478478 AF478515

Festuca nevadensis (Hackel)

Markgr.-Dannenb. (10x)

UZ, PC-JALR-PT 69.2000; Spain: Granada:

Sierra Nevada: Dornajo

AF478477 AF478514

Festuca pyrenaica Reuter (4x) Torrecilla and Catal�an (2002); Spain:

Huesca: Pyr�en�ees: Cotiella

AF303423 AF478517

Festuca rivularis Boiss. (2x) UZ, PC-JALR-PT 78.2000; Spain: Granada:

Sierra Nevada: Borreguiles de San Juan

AF478475 AF478512

Festuca rothmaleri (Litard.)

Markgr.-Dannenb. (8x)

UZ, JALR 01227B; Spain: Madrid: Lozoya AF478476 AF478513

Festuca rubra L. (1) (6x) Torrecilla and Catal�an (2002); Romania AF303422 AY118098

Festuca rubra L. (2) subsp. megastachys Gaudin (8x) JM, 8060; Switzerland: Valais: Desses sse Ferret AY118088 AY118099

Sect. Eskia Willk. p. p.

Festuca burnatii St.-Yves (2x) UZ, PC-PT 44.2001; Spain: Cantabria:

Picos de Europa

AY099007 AY099002

Festuca eskia Ramond ex DC (2x) Torrecilla and Catal�an (2002); Spain:

Huesca: Pyr�en�ees: BenasqueAF303412 AF478508

Festuca gautieri (Hackel) K. Richter (2x, 4x) Torrecilla and Catal�an (2002); Spain:

Gerona: Pyr�en�ees: Nuria

AF303414 AF478507

Festuca quadriflora Honck. (1) (2x) Torrecilla and Catal�an (2002); France:

Pyr�en�ees: Col de Baroude

AF303413 AF478506


Table 1 (continued)


ITS trnL–F

Festuca quadriflora Honck. (2) (2x, 4x) UZ, JM 8011; Switzerland: Valais: Alps,

Le Cartogne nw. Orsi�eres

AF519983 AF519988

Sect. Pseudatropis Krivot.

Festuca elegans Boiss. (2x, 4x) Torrecilla and Catal�an (2002); Spain: Granada: Baza AF303406 AF478509

Sect. Scariosae Hack.

Festuca scariosa (Lag.)

Ascherson and Graebner (2x)

UZ, PC-JALR-PT 62.2000; Spain:

Almeria: Ser�on: Sierra Filabres: Las Menas

AF519978 AY098999

Festuca mairei St.-Yves (4x) UAM, CC, JF,and AR 4064; Morocco:

Marrakech: Oukaimeden

AF303424 AY098996

Sect. Pseudoscariosa Krivot.

Festuca pseudeskia Boiss. (2x) UZ, PC-JALR-PT 73.2000; Spain: Granada:

Sierra Nevada: Collado del Diablo

AF519979 AY099000

Sect. Amphigenes Janka

Festuca agustinii (C. M. Smith ex Link)

Linding (2x)

AS 25-8-01; Spain: Tenerife: Near El Bailadero

(Anaga)

AY099005 AY099003

Festuca carpatica Dietr. (4x) KM s/n; Slovak Republic: Vysok�e Tatry Mts.:

Tisovnice

AY099006 AY099001

Festuca dimorpha Guss. (1) (4x) Herb. JM 10969, Korneck s.n.: France:

Alpes de Haute-Provence: Col des

Champs

AF519982 AF519987

Festuca dimorpha Guss. (2) (4x) W, K. Ronniger s/n; France: Alpes Maritimes AF532955 —

Festuca pulchella Schrader subsp. pulchella (2x) UZ, JM 7807; Switzerland: Bern AF519980 AF519985

Festuca pulchella Schrader subsp.

jurana (Gren.) Markgr.-Dann. (2x)

UZ, JM 8421; Italy: Trento: Passo di Sella n.

Canazei

AF519981 AF519986

Festuca spectabilis Jan (6x) Herb. JM 8229; Italy: Lombardia:

Bergamo: Passo della Presolana

AF519977 AF519984

Sect. Subbulbosae Hack.

Festuca coerulescens Desf. (2x) UZ, PC-JALR-PT 91.2000; Spain: C�adiz:

Jerez de la Frontera

AF538363 AF533051

Festuca triflora Desf. (2x) UZ, PC_JALR_PT 95.2000; Spain:

C�adiz, Grazalema: Barranco de Ballesteros

AF538362 AF533052

Festuca paniculata (L.)

Schinz and Thell. subsp. paniculata (2x)

UAM, CC and AR s. n.; France:

Mont Aigoual

AF303407 AF533046


Schinz and Thell. subsp. spadicea (L.) Litard. (4x)

UZ, JALR 01346; Spain: Lugo,

Folgoso do Caurel

— AF533048


Schinz and Thell. subsp. baetica (Hack.)

Markgr.-Dann.

UAM, CC and AR s. n.; Spain: C�adiz:

Sierra de la Palma

AF303405 AF533049

Festuca paniculata (L.) Schinz and Thell. subsp.

baetica (Hack.) Markgr.-Dann. Var.

moleroi Cebolla and Rivas Ponce

UZ, PC-JALR-PT 65.2000; Spain:

Granada, Sierra Nevada: Dornajo.

— AF543515

Festuca durandoi Clauson (2x) UZ, JALR 6-6-00; Spain: Segovia: Riaza AF543514 AF533047

Subgen. Drymanthele Krechetovich and Bobrov

Festuca altissima All. (2x) Torrecilla and Catal�an (2002); France:

Pyr�en�ees: Aspe

AF303411 AF478505

Festuca drymeja Mert and Koch (2x) LEI s. n.; Hungary: Balaton AF303425 AY098997

Festuca lasto Boiss. (2x) LEI s. n.; Spain: C�adiz: Sierra Bermeja AF303418 AY098998

Subgen. Subulatae (Tzvel.) Alexeev

Festuca subulata Trin.

(2x, 4x)

BW 10512; USA: Oregon: Clatsop County:

Saddle Mountain

AF532953 AF533056

Subgen. Leucopoa (Griseb.) Hackel

Sect. Leucopoa (Griseb.) Kriv.

Festuca kingii (S. Watson)

Cassidy (8x)

UZ, PC 1.93; USA: Colorado:

Boulder Co: Flat Irons

AF303410 AY099004

Sect. Breviaristatae Kriv.

Festuca altaica Trin. (4x) RS 5996; Canada: Yukon Territory: Teslin Lake AF532952 AF533055

Festuca californica Vasey (4x, 8x) BW 7014; USA: Oregon: Benton County AF532956 AF533054


Table 1 (continued)


ITS trnL–F

Subgen. Schedonorus (P. Beauv.) Peterm.

Sect. Schedonorus (P. Beauv.) W.D.J. Koch

Festuca arundinacea Schreber subsp.

arundinacea (1) (6x)

UZ, JALR 1081; Spain: Lugo:

L�ancara: Santa B�arbaraAF519976 AY098995


arundinacea (2) (6x)

Charmet et al. (1997) AJ240153 —


atlantigena (St-Yves) Auquier (8x)



fenas (Lagasca) Arcangeli (4x)

UZ, JALR s/n; Spain: Segovia:

Condado de Castilnovo

AF532951 AF533042

Festuca arundinacea Schreber var.

glaucescens Boiss. (4x)

Charmet et al. (1997) AJ240157 AF533045

Festuca arundinacea Schreber var. letourneuxiana

(St-Yves) Torrecilla and Catal�an (10x)


Festuca fontqueri St.-Yves (2x) Torrecilla and Catal�an (2002);

Morocco: Rif Mountains

AF303404 AF533044

Festuca pratensis Huds. subsp. pratensis (1) (2x) Torrecilla and Catal�an (2002);

England: Wilshire:Calne

AF303421 AF478503

Festuca pratensis Huds. subsp. pratensis (2) (2x) US, RS 6025; USA: Alaska AF532948 —

Festuca apennina De Not (2x) UZ, JM 7965; Switzerland:

Valais: Gletsch (N Oberwald)

AF548028 AF533041

Sect. Plantynia (Dumort.) Tzvelev

Festuca gigantea (L.) Villars (6x) Torrecilla and Catal�an (2002);

Spain: Navarra: Arce

AF303416 AF533043

Incertae sedis:

Festuca elviae Brice~no MERC, PC s/n; Venezuela: M�erida:Laguna de Coromoto

— AF543517

Festuca coromotensis Brice~no MERC, PC s/n; Venezuela: M�erida:

Laguna de Coromoto

— AF543518

Hellerochloa Fourn.

Hellerochloa fragilis (Luces) Rauschert MERC, PC s/n; Venezuela: M�erida:

P�aramo de Piedras Blancas

AF532960 AF533059

Vulpia Gmelin

Sect. Vulpia

Vulpia bromoides (L.) S.F. Gray (2x) UZ, JALR 01080; Spain: Lugo:

L�ancara

AF478485 AF487616

Vulpia ciliata Dumort. (1) (4x) UZ, PC-SP 10.2000; Spain: Zaragoza:

Actur

AF478486 AF478527

Vulpia ciliata Dumort. (2) (4x) UZ, PC 19.2002; Spain: Zaragoza: Vedado de

Pe~naflor

AY118094 AY118104

Vulpia ciliata Dumort. (3) (4x) JACA, 37694; Spain: Burgos: Solduengo:

Navas de Bureba

— AY118105

Vulpia muralis (Kunth) Nees (1) (2x) UZ, PC-SP 11.2000; Spain: Zaragoza: Actur AF478484 AF478526

Vulpia muralis (Kunth) Nees (2) (2x) UZ, PC 1.2002; Spain: Sevilla:

Sierra Morena: Alto del Palancar

AY118091 AY118102

Vulpia myuros (L.) C.C. Gmelin (1) (6x) UZ, PC 54.2001; USA: WA: King

Co: Seattle: Lake Forest Park

AY118092 AY118103

Vulpia myuros (L.) C.C. Gmelin (2) (6x) Charmet et al. (1997) AJ240162 —

Sect. Loretia (Duval-Jouve) Boiss.

Vulpia alopecuros (Schousboe) Dumort. (2x) LEI, CAS; Portugal: Algave: Meia Praia:

Lagos

AF478491 AF487617

Vulpia geniculata (L.) Link (2x) JACA, J29397; Spain: Sevilla: Constantina AF478490 AF478531

Vulpia sicula (C. Presl) Link (2x) JACA, 366589; France: Corse: Ponte Leccia AY118089 AY118100

Sect. Monachne Dumort.

Vulpia fasciculata (Forsk.) Samp. (1) (4x) UZ, SP 15.2000; Spain: Barcelona: Vilanova AF478487 AF478528

Vulpia fasciculata (Forsk.) Samp. (2) (4x) Torrecilla and Catal�an (2002); Greece:

Kikladhes: Paros

AF303402 —

Vulpia fontquerana Melderis and Stace (2x) UZ, JALR 16-6-2000; Spain: Segovia:

Lastras de Cuellar: Nava del Pobo

AF478488 AF478529


Table 1 (continued)


ITS trnL–F

Vulpia membranacea (L.) Dumort. (2x) UZ, PC 8.2002; Spain: C�adiz:

Sanlucar de Barrameda: La Algaida

AY118090 AY118101

Sect. Apalochloa (Dumort.) Stace (=Nardurus (Reichenb.) Stace)

Vulpia unilateralis (L.) Stace (1) (2x) UZ, PC 18.2002; Spain: Zaragoza:

Vedado de Pe~naflorAY118095 AY118106

Vulpia unilateralis (L.) Stace (2) (2x) JACA, 22694; Spain: Huesca: Laspu~na AY118096 AY118107

Sect. Spirachne (Hack. Boiss.

Vulpia brevis Boiss and Kotschy (2x) LEI, CAS; Cyprus: SE Nicosia AF478489 AF478530

Castellia Tineo

Castellia tuberculosa Tineo UZ, JALR s/n; Spain: C�adiz: Algaida. AF532954 AF533053

Catapodium Link

Catapodium rigidum (L.) C.E. Hubbard UZ, JALR s/n; Spain: Segovia:

Sep�ulveda

AF532940 AF533034

Ctenopsis De Not

Ctenopsis delicatula (Lag.) Paunero (2x) UZ, JALR s/n; Spain: Madrid:

Garganta de Los Montes

AF478499 AF478537

Cutandia Willk.

Cutandia maritima (L.) W. Barbey (2x) UZ, JALR 01050; Spain: Coru~na:Ribeira: Arenales del Castro

AF478496 AF487618

Cynosurus L.

Cynosurus echinatus L. (2x) JACA 40698; Spain: Soria: Monte Valonsadero AF532937 AF533031

Cynosurus cristatus L. (2x) UZ, JALR 230; Spain: Segovia: Riaza AF532938 AF533032

Dactylis L.

Dactylis glomerata L. subsp. glomerata (4x) Torrecilla and Catal�an (2002); Spain:

Zaragoza: Moncayo

AF393013 AF533028

Dactylis glomerata L. subsp.

hispanica (Roth) Nyman (2x)

Torrecilla and Catal�an (2002); Spain:

Zaragoza: Pe~naflor

AF393014 AF533027

Lamarckia Moench

Lamarckia aurea (L.) Moench (1) (2x) UZ, PC 14.2000; Spain: Zaragoza: Puente Almozara AF532935 AF533029

Lamarckia aurea (L.) Moench (2) (2x) UZ, SP 1.2000; Spain: Zaragoza: Cogullada AF532936 —

Monerma P. Beauv.

Monerma cyl�ındrica (Willd.) Cosson

and Durieu (4x)

UZ, SP 21-5-00; Spain: Zaragoza:

Parque Tio Jorge

AF532941 AF533035

Micropyrum Link

Micropyrum tenellum (L.) Link (2x) UZ, s/n; Spain: Segovia: Navafr�ıa AF478494 AF478534

Micropyrum patens (Brot.) Rothm. (2x) UZ, JALR 01194; Spain: Madrid:

Cadalso de los Vidrios

AF478495 AF495885

Micropyropsis Romero Zarco and Cabezudo

Micropyropsis tuberosa Romero Zarco

and Cabezudo

CRZ s/n; Spain: Huelva: Almonte (cultivated) AF532943 AF 533037

Narduroides Rouy

Narduroides salzmanii (Boiss.) Rouy (2x) UZ, JALR 01007; Spain: Madrid:

Dehesa de Arganda

AF478497 AF478535

Parapholis C.E. Hubbard

Parapholis incurva (L.) C.E. Hubbard (4x) UZ, PC-SP-PT 23.2000; Spain: Zaragoza:

Vedado de Pe~naflorAF532942 AF533036

Psilurus Trin.

Psilurus incurvus (Gouan) Schinz and Thell (4x) JACA, 236098; Spain: Huesca: Estopi~nan AF478493 AF478533

Wangenheimia Moench

Wangenheimia lima (L.) Trin. (2x) UZ, PC-SP-PT 17.2000; Spain: Zaragoza,

Vedado de Pe~naflor

AF478498 AF478536

Lolium L.

Lolium canariense Steud. (2x) UZ, AS s.n.: Spain: Canary isles: Tenerife:

Las Ca~nadas

AY228161 AY228162


Table 1 (continued)


ITS trnL–F

Lolium multiflorum Lam. (1) (2x) UZ, JALR 01092; Spain: Lugo: Sarria AF532946 AF533038

Lolium multiflorum Lam. (2) (2x) UZ, PC-PT 2001; Spain: Cantabria:

Santillana del Mar

AF532945 —

Lolium perenne L. (2x) Torrecilla and Catal�an (2002); England (cv.) AF303401 AF478504

Lolium remotum Schrank (2x) Charmet et al. (1997) AJ240146 —

Lolium rigidum Gaudin (1) (2x) UZ, PC-SP-PT 18.2000; Spain: Zaragoza,

Vedado de Pe~naflor

AF532944 AF533039

Lolium rigidum Gaudin (2) (2x) UZ ; PC 2002 ; Spain: Huelva: El Rocio AF532947 AF533040

Lolium rigidum Gaudin (3) (2x) Charmet et al. (1997) AJ240143 —

Lolium subulatum Vis. (2x) Charmet et al. (1997) AJ240148 —

Lolium temulentum L. (2x) Charmet et al. (1997) AJ240145 —

Poa L.

Poa bulbosa L. UZ, PC 14.2000; Spain: Huesca: Cuarte — AF533025

Poa infirma Kunth (2x) Torrecilla and Catal�an (2002); Spain:

Zaragoza: La Jota

AF393012 AF488773

Poa trivialis L. (2x, 4x) UZ, JALR 01090, Spain: Lugo: Sarria. AF532932 —

Puccinellia Parl.

Puccinellia distans (L.) Parl. JACA, J207897; Spain: Navarra: Lazagurr�ıa AF532934 AF533024

Sclerochloa Beauv.

Sclerochloa dura (L.) Beauv. (2x) UZ, JALR s/n; Spain: Segovia: Sep�ulveda AF532933 AF533023

Sphenopus Trin.

Sphenopus divaricatus (Gouan) Reichenb. UZ, PC-SP-PT22.2000; Spain: Zaragoza:

Vedado de Pe~naflor

AF532939 AF533033

Parafestuca Alexeev

Parafestuca albida (Lowe) Alexeev UZ, MS 4033A; Portugal: Madeira:

Pico do Arieiro

AF532930 AF533022

Sesleria Scop.

Sesleria argentea (Savi) Savi (4x) UZ, PC 21; Spain: Navarra: Araxes AF532931 AF533030

Avena L.

Avena barbata Link Moore and Field (2002) AY 093613 —

Avena eriantha Durieu UZ, JALR 032001; Spain: Madrid: Chinch�on — AF533021

Deschampsia Beauv.

Deschampsia cespitosa (L.) Beauv. UZ, PC s/n; USA: Colorado: Boulder Co.:

Rocky Mnt.

AF532929 AF533026

Secale L.

Secale cereale L. (2x) Torrecilla and Catal�an (2002); USA (cv.) AF303400 AF478501

Brachypodium P. Beauv.

Brachypodium distachyon (L.) P.Beauv. (2x) Torrecilla and Catal�an (2002); Slovenia: Ljubljana AF303399 AF478500

Sources, ploidy levels, and GenBank accession numbers. Abbreviations: JACA, Herbario del Instituto Pirenaico de Ecolog�ıa de Jaca; LEI,

Leicester University Herbarium; MERC, Herbario de la Universidad de M�erida (Venezuela) Ciencias; US, US National Herbarium (Smithsonian);

UZ, Herbario de la Universidad de Zaragoza; BW, Barbara Wilson collection; CAS, Clive A. Stace collection; CRZ, Carlos Romero-Zarco

collection; JALR, Jos�e�Angel L�opez-Rodr�ıguez collection; JM, Jochen M€uller collection; KM, Karol Marhold collection; MS, Miguel Sequeira

collection; PC, Pilar Catal�an collection; PT, Pedro Torrecilla collection; RS, Robert Soreng collection; SP, Samuel Pyke collection; W, Naturhis-

torisches Museum Wien Herbarium; As, Arnoldo Santos collection.aNumbers in parentheses indicate different accessions of the same taxon.bSources. Cotton and Stace (1976); Fuente et al. (2001); Torrecilla and Catal�an (2002); Torrecilla et al. (2003b). Ploidy levels are indicated in

parentheses.


needed to converge to a stable likelihood value for each

separate data set (Huelsenbeck and Ronquist, 2002).

Stationarity was achieved when the plotting of the log-

likelihood scores of sample points against generation time

reached a stable equilibrium value (Leach�e and Reeder,

2002). Sampled points from generations previous to sta-

tionarity were discarded using the burn-in option of

MRBAYES 3.0 and new Bayesian searches of 1,000,000

MCMCgenerationswere conducted for each separate dat

set. All trees sampled from these new Bayesian searches


were used to construct the respective 50% majority-ruleconsensus trees where the percentage of times a clade is

recovered is interpreted as an estimation of robustness.

The Bayesian analyses of each separate data set were

performed twice to confirm the congruence of the result-

ing posterior-probability-based consensus trees.

Topological conflicts between the ITS and the trnL–F

parsimony based trees were first analyzed visually;

conflicts were found for at least 10 lineages of Loliinaeand external taxa (Sesleriinae, Aveneae). The Incon-

gruence Length Difference (ILD) test of Farris et al.

(1994) was calculated to determine if the two datasets

(ITS and trnL–F) were significantly different from ran-

dom subsets of the same size.

3. Results

3.1. The ITS region

The ITS region comprises 644 aligned nucleotide (nt)

positions of which 237 correspond to the ITS1 spacer,

165 to the 5.8 gene, and 242 to the ITS2 spacer. Also 401

of the 644 total positions are variable (63%) and 294 of

them are parsimony informative (46% of the total). Gaplength ranged from 1 to 11 nt. Eleven gaps are poten-

tially informative and were used for parsimony analysis;

5 of those gaps are synapomorphic for the Dactylidinae

(Dactylis, Lamarckia) clade. The heuristic search found

148 equally shortest trees of 1585 steps (CI¼ 0.442;

RI¼ 0.724). The strict consensus tree of all most parsi-

monious trees is shown in Fig. 1A.

Resolution is low, in general terms, for the internalbranches of the tree depicted in Fig. 1A though many of

the named lineages show high bootstrap support. Four

basal lineages, the single taxon Deschampsia cespitosa,

an Avena–Sesleriinae clade (84% bootstrap support),

which also includes Parafestuca, a Poinae clade (91%),

and a clade comprising the Loliinae and allies (86%)

collapse in a polytomy at the base of the tree. Within the

last group the Dactylidinae (100%) and the Cynosuri-nae/Parapholiinae (99%) clades form an unresolved

polytomy with five lineages of broad-leaved Festuca and

with the clade of fine-leaved Festuca (93%). Resolution

is complete and fully supported for the sister genera

Dactylis (Dactylis glomerata/D. hispanica) and Lamarc-

kia within Dactylidinae. The Cynosurinae taxa appear

as a paraphyletic basal lineage with respect to the more

recently evolved Parapholiinae; two basal Cynosurus

taxa (Cynosurus cristatus, C. echinatus) collapse with a

clade of Sphenopus, Catapodium, and the sister taxa

Monerma/Parapholis (73%).

Within the broad-leaved Festuca, the Schedono-

rus+Lolium clade (80%) includes Micropyropsis tube-

rosa and Festuca apennina in its European subclade

(97%) and Festuca fenas in its Mahgrebian (northwest

Africa) subclade (100%) (Torrecilla and Catal�an, 2002).Lolium is monophyletic (94%). Another monophyletic

and well-supported lineage is that of subgenera Leuco-

poa+Subulatae (87%) with Festuca pulchella and Fest-

uca spectabilis collapsing at its base along with a clade of

Festuca kingii and the sister taxa Festuca altaica and

Festuca subulata. The remaining lineages of Subbulbosae

p. p. (90%), Festuca paniculata group (99%), and a

mixed group of representatives of subgen. Drymanthele

and sects. Scariosae and Pseudoscariosa (49%) are sim-

ilar to those described in Torrecilla and Catal�an (2002),

except for the novel presence of Castellia tuberculosa

within the last clade.

The fine-leaved Festuca clade is unresolved at its base

where a subclade of representatives of sects. Eskia and

Amphigenes p. p. plus Festuca californica collapses with

two individual species of sect. Eskia (Festuca burnatii, F.elegans) and the Festuca s. s. plus related taxa core.

Within the last group the South American taxon Hel-

lerochloa fragilis appears to be sister to the remaining

taxa which form a polytomy of several lineages previ-

ously described in Torrecilla et al. (2003b). The best

supported clades of this group are those corresponding

to the Aulaxyper+Vulpia p. p. (2x) +Micropyrum sub-

group (61%), the Festuca s. s. subgroup (81%), thePsilurus+Vulpia p. p. (4x, 6x) subgroup (100%), and the

Monachne-Spirachne-Loretia+Cutandia+Festuca pli-

cata subgroup (64%).

The Bayesian analysis sampled 9931 trees which

reached a stable likelihood value after the burn-in of 537

trees; the 50% majority rule consensus tree of all sam-

pled trees is shown in Fig. 1B. Resolution increases in

the consensus tree generated through the Bayesiananalysis, though the length of the internal branches is

usually short. Phylogenetic relationships are similar for

the main clades of the tree, which also show high levels

of support, like those of the clades Dactylidinae (100%),

Lolium+Micropyropsis+Schedonorus (83%), Leuco-

poa+Subulatae (100%), Drymanthele+Pseudoscari-

osa+Scariosae (100%), Psilurus+Vulpia p. p. (4x–6x)

(100%), and Festuca (100%). The Dactylidinae clade andthe F. paniculata group clade appear in this tree as the

successive sister groups of the fine-leaved Festuca.

3.2. The trnL–F region

The sequenced chloroplast trnL–F region encom-

passes 1089 aligned nucleotide positions, 548 correspond

to the trnL intron, 51 to the conserved trnL 30 exon, and490 to the trnL–trnF intergeneric spacer. The trnL–F

region is less variable than the nuclear ITS region for the

studied samples; only 466 out of 1089 positions are

variable (43% of the total) and of them only 227 (21%)

are parsimony informative. Gaps are larger and more

frequent than in ITS, ranging from 1 to 33 nt, with a

gap of 71 nt shared by Sclerochloa dura and Puccinellia

Fig. 1. ITS trees: (A) strict consensus tree of 148 equally parsimonious trees (L ¼ 1585, CI¼ 0.442 excluding uninformative characters, RI¼ 0.724);

(B) Bayesian 50% MR consensus tree of 9931 trees. Bootstrap and posterior probability percentages are indicated on the corresponding branches.


Fig. 1. (continued)


Fig. 2. trnL–F trees: (A) strict consensus tree of 40 equally parsimonious trees (L ¼ 915, CI¼ 0.656 excluding uninformative characters, RI¼ 0.807);

(B) Bayesian 50% MR consensus tree of 9681 trees. Bootstrap and posterior probability percentages are indicated on the corresponding branches.


Fig. 2. (continued)



distans and a deletion of 252 nt shared by Parafestuca

albida and Avena eriantha. Fifteen shared gaps were

used as characters for the phylogenetic analyses. Two of

those gaps are synapomorphies for the Schedono-

rus+Micropyropsis+Lolium group. One hundred and

nineteen positions of the trnL–F alignment that were too

ambiguous to be acceptable were excluded from the

phylogenetic analysis. The heuristic search rendered 40

trees that were 915 steps long (CI¼ 0.656, RI¼ 0.807).The strict consensus tree of these equally parsimonious

trees is depicted in Fig. 2A.

Phylogenetic relationships are resolved at the deepest

branches of the trnL–F tree, though with low support

for most clades, except for the Loliinae (77%) (Fig. 2A).

Resolution is poor for most of the recently evolved

lineages, consistent with the conserved nature of this

molecule relative to the more variable ITS region. Ac-cording to the tree shown in Fig. 2A, the Dactylidinae

(Dactylis/Lamarckia) (100%) and Deschampsia cespitosa

collapse in a polytomy with the Loliinae, whereas the

Cynosurinae/Parapholiinae clade is sister to the Lolii-

nae/Dactylidinae/Deschampsia group. Resolution within

the Cynosurinae/Parapholiinae is greater here than in

the ITS tree and most of the branches show high

bootstrap values). The odd relationship of Sesleria tothis Cynosurinae/Parapholiinae clade lacks bootstrap

support >50%. The basalmost lineages of the trnL–F

trees are those corresponding to a Parafestuca/Avena

clade (100%), a Scleropoa/Puccinellia clade (100%), and

a Poinae (Poa infirma/P. bulbosa) clade (93%), which in

turn is sister to the group of Loliinae and related allies.

Within monophyletic Loliinae further resolution is

obtained for two separate lineages that correspond,roughly, to the well-supported clades of broad-leaved

Festuca (86%) and fine-leaved Festuca (85%) in a poly-

tomy with two small lineages of broad-leaved taxa (F.

pulchella and the Castellia/Festuca californica clade).

The broad-leaved Festuca clade includes a clade

comprised of Schedonorus+Lolium taxa (93%), which

also includes Micropyropsis and Festuca apennina within

its European subclade (81%); otherwise there is littleresolution. Lolium does not appear as a monophyletic

group in this trnL–F topology; all representatives of this

taxon collapse in a large polytomy with representatives

of the F. pratensis complex, Festuca fontqueri, F. gi-

gantea, and Micropyropsis (Fig. 2A).

The true �fine-leaved� Festuca clade is also unresolved

at its base, where intermingled lineages of representa-

tives of Festuca sects. Eskia and Amphigenes plus F.

subulata and F. altaica collapse with a clade of remain-

ing taxa. Within this last clade resolution of groups is

mainly concordant with that indicated in Torrecilla et al.

(2003a,b), especially for the best supported groups

Festuca+Wangenheimia (90%), Psilurus+Vulpia p. p.

(4x–6x) (70%), and Aulaxyper+Vulpia p. p. (2x) (41%).

The most noticeable finding is the close relationship

shown by a group of Southern American taxa (H. fra-

gilis, Festuca elviae, F. coromotensis) to the clade of red

fescues (Festuca sect. Aulaxyper).

The Bayesian search sampled 9681 trees which

reached a stable likelihood value after the burning of

300 trees; the 50% majority rule consensus tree of all

sampled trees is shown in Fig. 2B. Relationships within

Loliinae are much the same as in the parsimony con-

sensus tree depicted in Fig. 2A. The two main clades of�broad-leaved� Festuca (99%) and �fine-leaved� Festuca(68%) show topological similarities, but better resolu-

tion is seen in this tree than in the parsimony-based one.

Support for the latter clade also decreases by the odd

position of the intermediate broad-leaved taxa F. pul-

chella subspp., C. tuberculosa and F. californica whereas

the true �fine-leaved� clade is highly supported (99%).

3.3. Combined analysis

Because the phylogenetic signal recovered from both

datasets is mostly concordant and there are no strongly

conflicting placements within the ingroup, we assumed

that the two sequenced molecules have evolved in par-

allel along the lineages that resulted in the present day

taxa and that their respective data matrices could becombined onto a single dataset and used for simulta-

neous analysis. However, we first conducted a test of

homogeneity for the separate datasets (ILD test) in the

combined data matrix of accessions that were sequenced

for both the ITS and trnL–F regions (100 samples). The

resulting p value of this test was significant (p < 0:01),meaning that the two datasets are different from random

partition of the same size as the original partitions. Al-though resolution of the ITS and trnL–F trees is mostly

congruent for the main groups of Loliinae and their

close allies our present survey detected 10 cases of po-

tential conflict between ITS and trnL–F topologies

(Figs. 1 and 2). Two of them correspond to external taxa

of tribe Aveneae (Deschampsia) and subtribe Sesleriinae

(Sesleria) and the remaining eight cases to festucoid taxa

(Castellia, F. pulchella, F. californica, F. subulata, F. al-taica, Hellerochloa, Micropyrum, and Festuca quadrifl-

ora2). In order to investigate if the conflicting lineages

mentioned above in the ITS and trnL–F trees could have

caused incongruence, we removed the sequences of those

10 species from the combined ITS/trnL–F dataset and

conducted a further ILD test and the result was also

significant (p < 0:01).The heuristic search conducted on the combined ITS/

trnL–F dataset rendered 136 most parsimonious trees of

2411 steps (CI¼ 0.514 and RI¼ 0.708). The strict con-

sensus tree of all those equally parsimonious trees is

shown in Fig. 3A. The Bayesian search sampled 6216

trees after the burnin of 2476 trees; the 50% majority

rule consensus tree of all these trees is shown in Fig. 3B.

The most noticeable features of these combined trees

Fig. 3. Combined ITS/trnL–F trees: (A) strict consensus tree of 136 equally parsimonious trees (L ¼ 2411, CI¼ 0.514 excluding uninformative

characters, RI¼ 0.708); (B) Bayesian 50% MR consensus tree of 6216 trees. Bootstrap and posterior probability percentages are indicated on the

corresponding branches.


Fig. 3. (continued)



(Figs. 3A and B), with respect to the separate ITS andtrnL–F topologies, are the following: (i) the festucoid

lineage (Loliinae) is resolved as monophyletic with

moderate (bootstrap) to high (posterior probability)

support; (ii) the sister group of Loliinae is (Dactylidinae

(Cynosurinae/Parapholiinae)), which form a well-sup-

ported clade with the Loliinae; (iii) the �fine-leaved�Festuca are monophyletic and strongly supported; (iv)

the �broad-leaved� Festuca are also monophyletic thoughweakly supported; (v) Castellia shows an unresolved

intermediate placement between the clades of broad and

fine-leaved fescues in the parsimony based tree (Fig. 3A)

but is resolved as sister to the fine-leaved fescues in the

Bayesian-based tree (Fig. 3B); (vi) subgen. Leucopoa and

sects. Subbulbosae and Amphigenes of Festuca are

polyphyletic; (vii) Hellerochloa is sister to the clade of

red fescues (Aulaxyper+Vulpia p. p. (2x).

4. Discussion

4.1. Phylogeny of festucoids within tribe Poeae. Closest

subtribes of Loliinae

The importance of taxonomic sampling is crucial torecover past evolutionary relationships for any group of

living organisms (Hillis and Wiens, 2000; Lecointre

et al., 1993). Our present study provides a good example

of this within subtribe Loliinae, which has been subject

to a series of molecular studies with progressively

greater sampling. Topologies obtained for Festuca and

the genera Lolium and Vulpia in the works of

Lehv€aslaiho et al. (1987) and Darbyshire and Warwick(1992) based on chloroplast RFLP analysis, and of

Charmet et al. (1997), Gaut et al. (2000), and Torrecilla

and Catal�an (2002) based on ITS analysis were largely

congruent. However, unanticipated relationships among

some lineages were not explored in these previous

studies due to limited taxon sampling. A substantial

extension of the phylogenetic boundaries of Festuca s. l.

has been achieved after the recent molecular studies ofTorrecilla et al. (2003a,b) and the present survey. Pa-

raphyletic Festuca encompasses not only Vulpia and

Lolium, but also a number of other genera that are

nested either within the �fine-leaved� Festuca (Micropy-

rum, Wangenheimia, Ctenopsis, Narduroides, Cutandia,

Psilurus, Hellerochloa), within the �broad-leaved� Fest-uca (Micropyropsis), or in an unresolved position be-

tween them (Castellia).Obtaining an accurate phylogeny at the tribal level is

also affected by taxon sampling. The simple sister rela-

tionship of clades Poeae and Aveneae recovered in early

molecular studies (Catal�an et al., 1997; Hsiao et al.,

1995a; Soreng et al., 1990) was obscured when a larger

dataset of combined chloroplast RFLP and structural

characters was subjected to phylogenetic analysis (Sor-

eng and Davis, 2000). Intertribal admixture betweenseveral lineages of Poeae and Aveneae was found in the

combined tree recovered by the latter authors. Kellogg

and Watson (1993) predicted similar levels of extended

polytomies for trees recovered from DNA sequences as

found in those reconstructed from morphological data if

taxonomic sampling becomes large enough. In spite of

the higher resolution provided by the DNA sequence

characters over the morphological ones within Loliinae(present study and unpublished data), larger taxon

sampling has increased instances of homoplasy for the

weakly supported clades and the �unstable� taxa; con-versely, well-supported clades are maintained when

sampling of taxa of those groups is enlarged.

Our separate ITS and combined ITS and trnL–F

analyses indicate that subtribe Loliineae and its close

relatives Dactylidinae and Cynosurinae/Parapholiinaeform a monophyletic and well-supported group within

the Poeae (Figs. 1 and 3). The combined ITS/trnL–F

analysis further distinguishes (Dactylidinae (Cynosuri-

nae/Parapholiinae)) as the sister lineage of the festucoids

(Fig. 3). This sister relationship contradicts, in part, the

finding of Soreng and Davis (2000), who showed Lolii-

nae to be sister to a Puccinellia/Sclerochloa clade and

together they are sister to a Cynosurinae/Parapholinaeclade. Resolution within the last group was much the

same as the one recovered here. However, outgroup

sampling in this study is limited and, while sufficient for

rooting the ingroup tree, is not designed for inferring

relationships among the outgroups.

Parafestuca is a monotypic genus endemic to the

Madeira archipelago. Its single species (Parafestuca

albida) was first classified within Festuca by Lowe (1831)and later separated from it by Alexeev (1985) based on

its strongly carinate and trinerviate lemma and upper

glume and its oval hilum. Relationships of Parafestuca

to other genera of Pooideae has not been studied before.

Our combined ITS/trnL–F tree shows Parafestuca to be

nested within an Avena–Sesleria clade with 100% boot-

strap support for the sister relationship of Parafestuca

albida and Avena eriantha (Fig. 3). The unexpected linkof this strongly perennial plant to the annual Avena is

mostly based on chloroplast sequence data; the two taxa

share several nucleotide synapomorphies and a deletion

of 253 bp. The ITS sequences reveal a strongly sup-

ported sister relationship of Parafestuca to an Avena/

Sesleria clade. Despite these minor inconsistencies, it is

now clear that Parafestuca is neither connected to

Festuca or to other festucoids.

4.2. The broad-leaved Festuca

Attempts to clarify the phylogeny of the broad-leaved

Festuca have been hampered by the unresolved rela-

tionships recovered for the main lineages of the group in

the ITS tree and the succession of polytomies observed


in the trnL–F tree (Figs. 1 and 2). Resolution increasesgreatly in the combined ITS/trnL–F tree, though most of

its deep internal branches (except that of the Schedon-

orus+Micropyropsis+Lolium clade), are short (Fig. 3B)

or lack strong bootstrap support (Fig. 3A). Dichotomy

between typical lineages of �broad-leaved� and �fine-leaved� fescues (Torrecilla and Catal�an, 2002) is less

apparent in this study with greater sampling (Figs. 1–3),

because some �broad-leaved� taxa are placed either inintermediate positions between the two main groups or

within the clade of �fine-leaved� taxa. Reinterpretation of

the new topologies suggests a basal paraphyly of the

�broad-leaved� lineages of Festuca s. l. with respect to the

more recently evolved �fine-leaved� ones. However, a

clade of broad-leaved taxa is still recovered in the

combined ITS/trnL–F tree (Fig. 3), though it shows only

moderate bootstrap support.Representatives of six out of nine subgenera of

Festuca recognized by Clayton and Renvoize (1986)

have been included in the present study; four of them

correspond to broad-leaved fescues (subgen. Schedono-

rus, Leucopoa, Drymanthele, and Subulatae). Resolution

for some of these lineages is concordant with that pre-

sented in Torrecilla and Catal�an (2002) and Torrecilla

et al. (2003a). The Asian–North American subgen.Leucopoa, the European sect. Amphigenes, and the

Western Mediterranean sect. Subbulbosae are resolved

as polyphyletic taxa. The Eurasian and Mediterranean

subgen. Drymanthele appears to be paraphyletic, with F.

scariosa and F. pseudeskia (subgen. Festuca) derived

from within it. Also, the Eurasian and Mediterranean

subgen. Schedonorus forms a monophyletic group when

Lolium andMicropyropsis are included within it. Festucasubgen. Leucopoa, Drymanthele, and Subulatae, as well

as sect. Amphigenes share several foliar characters re-

lated to the �broad-leaved� syndrome (extravaginal

shoots, presence of cataphylls, convolute leaf-vernation,

complete sclerenchyma girders). Leucopoa (sect. Leuco-

poa) differs from the others in being dioecious. On the

other hand, Festuca sects. Subbulbosae, Scariosae, and

Pseudoscariosa show intermediacy in their characters,with mixed intravaginal and extravaginal shoots, con-

volute to conduplicate leaf-vernation, presence or ab-

sence of cataphylls, and incomplete sclerenchyma

girders. Interpretation of changes of characters on the

broad-leaved Festuca clade of the ITS/trnL–F trees de-

picted in Fig. 3 indicates that the �broad-leaved� foliarsyndrome could be plesiomorphic and also could have

evolved several times towards the opposite trend alongthe clade; however, the presence of old lineages of �in-termediate� taxa in most subclades raises doubts about

their �ancestry.�Subgenus Leucopoa is polyphyletic in the combined

ITS/trnL–F tree. F. kingii (sect. Leucopoa) is resolved as

the sister taxon of F. spectabilis (subgen. Festuca, sect.

Amphigenes) in an intermediate clade of the broad-

leaved subtree, F. altaica (sect. Breviaristatae) falls outas sister to F. subulata (subgen. Subulatae) in a basal

clade of the subtree, and F. californica (sect. Breviari-

statae) is nested within the �fine-leaved� Festuca clade

(Fig. 3). In the ITS-based tree (Fig. 1), all Leucopoa s. l.

taxa (F. kingii, F. altaica, F. spectabilis, and F. pulchella)

join F. subulata in a well-supported clade. Phylogenetic

inconsistencies of subgen. Leucopoa were also detected

by Darbyshire and Warwick (1992) in their chloroplastRFLP study; these authors found a close relationship of

sect. Breviaristatae to subgen. Subulatae and of sect.

Leucopoa (F. kingii) to subgen. Schedonorus. They pro-

posed to separate sect. Breviaristatae from subgen.

Leucopoa. Representatives of sect. Breviaristatae (F.

altaica, F. californica), subgen. Subulatae (F. subulata),

and sect. Amphigenes p. p. (F. pulchella, F. carpatica, F.

dimorpha) included in our study could be lineages ofhybrid origin that show intermediate placements be-

tween the �broad-leaved� and �fine-leaved� Festuca clades

(Figs. 2 and 3). None of them is included in the well-

supported clade of true �broad-leaved� Festuca recovered

in the chloroplast based tree (Fig. 2).

Relationships for the remaining members of the

�broad-leaved� Festuca are similar to those described in

Torrecilla and Catal�an (2002) for clades representingSubbulbosae p. p., and the Drymanthele, Scariosae,

and Pseudoscariosa taxa and for the �European� and

�Mahgrebian� subclades of the more recently diverged

Schedonorus group. Leucopoa s. s. (F. kingii and F.

spectabilis) and the F. paniculata complex (F. paniculata,

F. durandoi, F. baetica) are resolved here as consecutive

sister groups to the Schedonorus/Lolium/Micropyropsis

clade. Festuca mairei, a taxon formerly classified withinsect. Scariosae, is nested within the Schedonorus �Mah-

grebian� clade (Figs. 1–3). Torrecilla and Catal�an (2002)

proposed to transfer this taxon to subgen. Schedonorus.

Micropyropsis has been resolved as a member of the

Schedonorus+Lolium clade in this study; its close rela-

tionship with Schedonorus is strongly supported by both

the ITS and the trnL–F datasets (Figs. 1 and 2). This

monotypic genus endemic to SW Spain and N Morocco(Devesa and Romero-Zarco, 1996; Romero-Zarco and

Cabezudo, 1983) was described based on its engrossed

base of the culm and racemose inflorescence (M. tube-

rosa; Romero-Zarco and Cabezudo, 1983). Micropyr-

opsis shows noticeable morphological similarities with

the Schedonorus species and with Lolium. All of them

plus C. tuberculosa share falcate auricles, which are

otherwise not found within Loliinae. Micropyropsis alsopossesses a subracemose inflorescence, an intermediate

trait between the paniculate one of Schedonorus and the

reduced spike of Lolium.

Monophyly of Lolium generally has been accepted

after phylogenetic studies based on analysis of ITS se-

quences (Charmet et al., 1997; Gaut et al., 2000; Tor-

recilla and Catal�an, 2002). However, Gaut et al. (2000)


indicated the odd placement of one F. pratensis acces-sion within their large Lolium clade, questioning the

monophyly of Lolium. These authors also showed an

intraspecific polyphyly for the highly variable L. rigi-

dum. Four Lolium taxa (Lolium perenne, L. multiflorum,

L. rigidum, L. canariense) presented here were unre-

solved in the trnL–F tree depicted in Fig. 2. However, a

larger sampling of ITS sequences retrieved from Gen-

Bank and incorporated into our dataset resolved ahighly supported clade for Lolium (Fig. 1). The com-

bined ITS/trnL–F tree also recovers a monophyletic

group for the four species of Lolium (Fig. 3). Evidence

from the separate analyses suggests that Lolium is of

recent origin and probably evolved from a European

Schedonorus ancestor, as demonstrated by its present

ability to intercross with Festuca pratensis and F. arun-

dinacea (Jenkin, 1933). An evolutionary trend in re-duction of life-cycle, habit, and reproductive organs can

be traced from the perennial, robust, and paniculate

Schedonorus taxa, through the perennial, slender, su-

bracemose Micropyropsis taxon, to the mostly annual,

ephemeral, racemose and single glumed taxa of Lolium.

4.3. The ‘fine-leaved’ Festuca

In contrast to the �broad-leaved� Festuca, the �fine-leaved� fescues are resolved as monophyletic in both

separate and combined analyses of ITS and trnL–F se-

quences (Figs. 1–3). The clade shows moderate to high

bootstrap support in the ITS and trnL–F based trees

(Figs. 1 and 2). Lack of support of the ITS data might

originate from the �intermediate� taxa F. altaica (Brev-

iaristatae), F. subulata (Subulatae), and F. carpatica andF. dimorpha (Amphigenes p. p.) and from Castellia. In-

ternal clades are better resolved and similarly recovered

from the two independent datasets within the �fine-leaved� Festuca. Resolution of the main groups corre-

spond to that described by Torrecilla et al. (2003b) for

the Festuca+Vulpia+Related Ephemerals (FEVRE)

group with the addition of a group of South American

taxa (H. fragilis, F. elviae, F. coromotensis) that areshown to belong to the clade of red fescues (Festuca sect.

Aulaxyper) (Fig. 2). Four moderately to well-supported

clades are distinguished within the fine-leaved Festuca:

the Festuca (+Wangenheimia) clade, the Aulaxy-

per+Vulpia p. p. (2x) clade, the Psilurus+Vulpia p. p.

(4x–6x) clade, and the Spirachne-Monachne-Lore-

tia +Cutandia +Festuca plicata clade (Figs. 1–3). The

combined ITS/trnL–F tree shows a greater resolutionfor the deep internal branches of the tree, though they

have either short branches or weak bootstrap support

(Fig. 3). According to this hypothesis, the oldest lineages

within the fine-leaved fescues are those belonging to taxa

of sects. Amphigenes s. s. (F. carpatica, F. dimorpha) and

of sect. Eskia plus F. californica. F. elegans (sect.

Pseudatropis) is resolved as the sister taxon of the core

Festuca s. s. clade. Within the core Festuca s. s. a di-chotomy separates the �F. ovina� and �F. rubra� groupsplus related annuals (Vulpia s. s., Psilurus, Micropyrum,

Narduroides) from a large group of Vulpia taxa and al-

lies (Spirachne-Monachne-Loretia-Apalochloa+Cutan-

dia+Festuca plicata+Ctenopsis) plus Festuca subsect.

Exaratae.

A novel finding within this �fine-leaved Festuca� cladeis the unexpected relatedness recovered for the threeAndean taxa to the Aulaxyper+Vulpia 2x clade. Helle-

rochloa has been classified either within Festuca (e.g.,

Alexeev, 1980, as subgen. Helleria), or as a genus of its

own based on a monoecious breeding system and long

protruding glumes. F. elviae and F. coromotensis were

described from the Venezuelan Andean region by

Brice~no and Morillo (1994). The evolutionary placement

of these taxa within Festuca has not been determinedyet; however, Stancik and Peterson (2002) have recently

classified F. coromotensis in subgen. Subulatae (sect.

Subulatae) based on their shared flat leaves, extravaginal

innovations without cataphylls and large branched

panicles, and have also tentatively attributed F. elviae to

this section. Based on this taxonomic treatment, sect.

Subulatae would constitute another polyphyletic group

of the broad-leaved fecues. The three taxa have beensequenced for trnL–F, though only Hellerochloa was

sequenced for ITS. The chloroplast based tree recovers a

weakly supported clade (51% bootstrap) that shows a

basal placement of the three taxa with respect to the

Aulaxyper+Vulpia p. p. (2x) clade (Fig. 2A) and a

similar topological placement in the Bayesian-based

tree. On the other hand,Hellerochloa is resolved as sister

of the FEVRE group in the ITS based trees (Fig. 1). Thecombined ITS/trnL–F tree places Hellerochloa close to

the Aulaxyper group (70% bootstrap and 100% posterior

probability percentages, Fig. 3). H. fragilis bears capil-

late conduplicate leaves and resembles the �fine-leaved�fescues in most vegetative features. However, F. elviae

and F. coromotensis exhibit �broad-leaved� habit. The

close topological placement of these three samples in

the trnL–F tree indicates either convergent evolutionof the broad-leaved syndrome or horizontal transfer of

their chloroplast genome.

Discussion of the polyphyly of Vulpia, the circum-

scription of its clades, and its paraphyly with respect to

genera Cutandia and Ctenopsis and to F. plicata has been

detailed in Torrecilla et al. (2003b). Psilurus shows a

strongly supported relationship to polyploid taxa of sect.

Vulpia (V. ciliata, V. myuros). Psilurus was included in itsown subtribe Psilurinae by Pilger (1954) based on unique

traits related to its extremely reduced inflorescence, but

this genus shares some floral synapomorphies with V.

ciliata. According to our phylogenetic results Psilurus

should be classified within subtribe Loliinae.Narduroides

was in turn placed within tribe Hainardieae (¼Moner-

meae) by Clayton and Renvoize (1986) based on its


indurate andbroad scariosemargin lemmas.Our analysesshow thatNarduroides is not related to the Parapholiinae

clade but to the �fine-leaved� FEVRE group.

4.4. Unstable taxa. Causes of conflict between topologies

Topologies recovered from the separate ITS and

trnL–F analyses are congruent for the resolution of the

best supported groups that include the clades Dacty-lidinae, Cynosurinae/Parapholiinae, Schedonorus+Mi-

cropyropsis+Lolium, Festuca, Aulaxyper+Vulpia p. p.

(2x), and Psilurus+Vulpia p. p. (4x–6x); partial con-

gruence also has been found for the Spirachne-Mona-

chne-Loretia+Cutandia+Festuca plicata clade in both

trees (Figs. 1 and 2). Thus, the results from the nuclear

and chloroplast based trees are mostly in agreement for

the closest relatives of Loliinae and for several lineagesof fine-leaved Festuca, and to a lesser extent for the

broad-leaved Festuca. Disagreements affect the place-

ment of 10 independent lineages (Deschampsia, Sesleria,

Castellia, Hellerochloa, Micropyrum, F. pulchella, F.

californica, F. subulata, F. altaica, and F. quadriflora2)

which have different positions in the ITS and trnL–F

trees. These conflicts occur among outgroups (Des-

champsia, Sesleria) or among taxa that occupy relativelybasal positions in the broad-leaved and fine-leaved

groups and, in many cases, there is only weak support

for the conflicting placements in one or both trees.

The ILD test conducted on both the complete ITS/

trnL–F dataset (100 samples) and the �pruned� ITS/trnL–F dataset (excluding the 10 conflicting taxa) was

significant in either case. Heterogeneity detected by the

ILD test between two different sequence datasets hasbeen used as a counter-indication for the combination of

data partitions for simultaneous analysis (Johnson and

Soltis, 1998), though a significant value of the ILD test

has been interpreted as irrelevant in terms of combin-

ability by other authors (Barker and Lutzoni, 2002;

Yoder et al., 2001). The latter authors (Barker and

Lutzoni, 2002) further emphasized the poor predicting

value demonstrated by the ILD test for dataset com-binability intended to increase phylogenetic accuracy

even at very low critical values (0.01–0.001). The ILD

test is a useful means to explore the source of hetero-

geneity in the data, but probably should not, by itself, be

the arbiter of whether or not to combine data. Given

that the congruent portions of the trees still give a sig-

nificant ILD test, the incongruence may have arisen

from differences in the characteristics of the two data-sets, rather than from their phylogenetic histories.

Conflicts between ITS and trnL–F topologies could

be explained by the existence of past hybridization

events. However, the persistent heterogeneity detected

through the ILD test after the removal of the �misplaced�taxa in the �pruned� ITS/trnL–F data matrix favors other

potential causes. Alternatively, the two molecules could

have been affected by lineage sorting if the ancestrallineages of the festucoids and their allies were diversi-

fying faster than the fixation of different gene copies in

separate lineages. This scenario, hypothesized for other

groups of grasses, is impossible to distinguish from the

reticulate one (Kellogg et al., 1996). Trees with short

basal internodes and poor bootstrap support, like our

trnL–F trees (Fig. 2), might have resulted from either

past lineage sorting or reticulation. Finally, a thirdpossible explanation derives from the stochastic nature

of mutations and the possibility that incongruence can

arise by chance whenever a finite data set is used.

Hybridization is considered to be one of the main

evolutionary processes operating in temperate Poeae

grasses, based on the existence of ahigh percentage of taxa

of hybrid origin (Soreng and Davis, 2000), and has been

suggested to be a potential risk for phylogenetic inference.The extent of reticulation within the Loliinae was assayed

in the last century by the study of spontaneous hybrids

and through artificial crosses between different Festuca

lineages and other close allies (Ainscough et al., 1986;

Barker and Stace, 1982, 1984, 1986; Borrill, 1972; Borrill

et al., 1977; Jauhar, 1993; Jenkin, 1933, 1955a,b). The

resulting data established the limits of reproductive bar-

riers and assessed the degree of genomic relatednessamong lineages, which were regarded in some cases as a

measure of evolutionary relatedness (Borrill, 1972; Borrill

et al., 1977; Jauhar, 1993). A summary of the hybridiza-

tion documented within the festucoids is presented in

Fig. 4. Intergeneric hybrids between Festuca and Lolium

(�Festulolium) (Lewis, 1975) and between Festuca and

Vulpia (�Festulpia) (Willis, 1975) occur spontaneously in

the wild, although all of the progeny are highly sterile.Introgression towards the Festuca sect. Aulaxyper parent

has been documented from the �Festulpia taxa (Ains-

cough et al., 1986) indicating potential gene-pool re-

cruitment into the F. rubra-complex parental line.

Reticulation may be involved in the origin of some mi-

crospecies of Festuca subgen. Schedonous (Jenkin, 1933;

Jauhar, 1993), subgen. Festuca sects. Festuca and Aul-

axyper (Jenkin, 1955a,b), sect. Eskia (Gutierrez-Villariasand Homet, 1984; Saint-Yves, 1924), subgen. Drymant-

hele (Borrill, 1972; Borrill et al., 1977), and Lolium (Jen-

kin, 1933; Terrell, 1968), giving rise to more or less fertile

descendants. However, most of these hybridizations oc-

cur within closely related groups of species (Fig. 4). For

those that imply a cross between less related groups (i.e.,

F. rubra andLolium) spontaneous progeny are scarce and

totally infertile (Nilsson, 1933). In this respect, somehighly polyploid festucoid lineages of recent origin, like

the red fescues (6x–10x) and theMahgrebianSchedonorus

taxa (4x–10x) could be more affected at present by re-

current introgression. Nonetheless, it seems to us the

differences observed between the ITS and the trnL–F trees

have to domore with sequence sampling in a finite dataset

and possibly with lineage sorting than with hybridization

Fig. 4. Mapping of structural characters, hybridization events, and biogeographical distributions of Loliinae taxa onto the combined MP strict

consensus tree. Branch shadows indicate character-states associated with the �broad-leaved syndrome� (flat leaves, schlerenchyma girders, extra-

vaginal shoots, convolute to supervolute vernation). Thickness of vertical bars indicate increasing levels of ploidy. Connecting arrows indicate

present spontaneous and artificial crosses between different Loliinae groups (black and dotted lines, respectively). Boxes summarize the geographical

range of distribution of the studied groups (Am, American; Eur, Eurasian; Hol, Holartic; Med, Mediterranean; asterisk indicates the presence of a

South-American lineage sister to the Holartic-Mediterranean Festuca sect. Aulaxyper).


per se, since many of the observed topological incongru-

ences are near the base of weakly supported branches of

the ITS and trnL–F trees.Paralogy and gene conversion are frequent inmultiple-

copy genes like those of the ribosomal ITS families

(Buckler et al., 1997). The existence of ITS intragenomic

paralogy has been reported in calamoid palms (Baker

et al., 2000) where sequences from the same individual are

not resolved as monophyletic. Equally active paralogs,

recombinant ribotypes, and pseudogenes have been

found in different grasses likeZea (Buckler andHoltsford,

1996a,b), Tripsacum (Buckler et al., 1997), and Lolium

(Gaut et al., 2000). The latter authors attributed some ITSsequences of Lolium canariense (Charmet et al., 1997) to

pseudogenes based on their low folding energies. Paral-

ogy that goes undetected throughPCRsequencing studies

would increase instances of homoplasy and could lead to

erroneous phylogenies (Buckler et al., 1997; Sanderson

and Doyle, 1992). Thus, the general acceptance that the

ITS region is homogenized through concerted evolution


inmost of the angiosperms (Baldwin et al., 1995) has beenbrought into question and should not be assumed to be

the case. Concerted ITS changes are assumed to occur in

the grass family (Hsiao et al., 1995a, 1999), even across

different ITS arrays of monogenomic Triticeae, because

most taxa sampled by more than one representative show

a unique ITS sequence (Hsiao et al., 1995b; Kellogg et al.,

1996; Mason-Gamer and Kellogg, 1997). However, a

larger sampling of intraspecific representatives of Festucasubgen. Schedonorus and Lolium taxa in the ITS-based

study of Gaut et al. (2000) manifested the presence

of polyphyletic and paraphyletic lineages for F. mairei,

F. pratensis, F. arundinacea, L. rigidum, and L. multiflo-

rum. In our present survey most of the intraspecific

sequences analyzed are shown to be monophyletic except

for V. ciliata and F. quadriflora.

Taxa of hybrid origin may also experience geneconversion towards one of their parental ITS ribotypes

(Buckler et al., 1997). Gaut et al. (2000) detected the

existence of different F. arundinacea (6x) ITS sequences,

some of which were identical to a group of F. pratensis

(2x) ITS sequences, whereas others were distinct from

them, pointing towards another different unknown

parent. Biased concerted evolution of ITS sequences in

hybrid taxa would obscure phylogenetic reconstructionsand would increase conflicts between ITS and chloro-

plast-based trees. Mason-Gamer and Kellogg (1997)

tested conflicts among nuclear and chloroplast datasets

in the tribe Triticeae through different analytical meth-

ods. They conclude that ILD test and Tree comparison

approaches are useful for initial comparisons; these

authors established a bootstrap cut off of 70% for

comparing conflicting clades on the trees. Other authorshave followed similar tree comparison approaches for

Bayesian reconstructed topologies based on posterior

probability values >95% (Leach�e and Reeder, 2002). We

have followed the 70% bootstrap criterion of Mason-

Gamer and Kellogg (1997) to examine potential cases of

reticulation for some of the conflicting taxa detected in

our present study which is mostly coincident with the

95% posterior probability criterion for these taxa.Deschampsia cespitosa, classified as a member of the

Aveneae, falls outside the clade of Loliinae and allies in

the ITS-based tree, but is resolved as sister to the

Dactylidinae group in the trnL–F based tree (Figs. 1 and

2). The unexpected placement of D. cespitosa within a

clade of Poeae taxa is in agreement with the previous

finding of Catal�an et al. (1997) based on analysis of

chloroplast ndhF sequences but contradicts that ofSoreng and Davis (2000) based on simultaneous analysis

of cpDNA RFLP and morphological data. Thus, the

unresolved placement of this taxon in the combined ITS/

trnL–F tree (Fig. 3) reflects the present controversy

about its phylogeny. Support for the placement of

Deschampsia is <70% in both the ITS and trnL–F trees

(Figs. 1A and 2A), although the support for the

Dactylidinae plus Loliinae clade in ITS is >70%(Fig. 1a). Thus, there is no support of >70% for the two

conflicting placements of Deschampsia.

Sesleria argentea is resolved as a member of a

strongly supported Sesleria+Avena+Parafestuca clade

(>80%) in the ITS based tree, but as the weakly-sup-

ported sister taxon of the Cynosurinae/Parapholinae

clade in the trnL–F based tree. Thus, there is no strong

conflict in the two trees. This taxon was resolved assister to Poa in the chloroplast study of Catal�an et al.

(1997). Shortage of sampling of other representatives of

Sesleriinae, Poinae, and Aveneae prevents speculation

on the phylogenetic relationships of Sesleria. The

stronger support of the ITS data indicates that S. ar-

gentea is not a close ally of the Loliinae.

Castellia tuberculosa represents one of the most strik-

ing paradoxes of Loliinae, because this taxon is mor-phologically close to Schedonorus and Lolium, but is

resolved as close to the Drymanthele+Scariosa+

Pseudoscariosa clade (�broad-leaved� Festuca) in the ITS

tree and to F. californica (�fine-leaved� Festuca) in the

trnL–F tree. There is little support for including Castellia

in the fine-leaved clade in trnL–F, but conflicting evidence

from ITS. The combined ITS/trnL–F analysis places

Castellia in an unresolved intermediate position betweenthe two large lineages. C. tuberculosa was classified as a

monotypic genus based on its tuberculate lemmas; it has

been considered to share morphological affinities with a

Desmazeria group of annual grasses (Stace, 1981). Cas-

tellia shares synapomorphic falcate auricles with the

Schedonorus+Micropyropsis+Lolium group. Its unex-

pected placements in both ITS and trnL–F trees cannot be

explained satisfactorily on the basis of morphology.Other misplaced lineages in the ITS vs. trnL–F trees

are those of the �intermediate� taxa F. pulchella, F.

subulata, F. altaica, and F. californica (Figs. 1A and 2A).

These �broad-to-fine� leaved Festuca represent a transi-

tion between the two main groups. All of these taxa have

strong conflict (>70%) in both datasets. F. californica,

though classified within Leucopoa sect. Breviaristatae,

represents an unusual case, since it bears conduplicateleaves with incomplete sclerenchyma girders and mixed

extravaginal-intravaginal shoots, features that ally it

with the fine-leaved Festuca. Its placement within the

fine-leaved Festuca clade (Fig. 3A), though unresolved,

agrees with its general morphology. F. pulchella, F. al-

taica, and F. subulata are members of a strongly sup-

ported Leucopoa s. l. clade in the ITS based tree, but are

mostly unresolved within the �fine-leaved� Festuca cladein the trnL–F based tree. Their placement in a basal and

well-supported clade within the �broad-leaved� Festucaclade in the combined ITS/trnL–F tree decreases the

bootstrap value of the �broad-leaved� group. The three

taxa might represent descendant species from a rela-

tively ancient case of introgression showing a nuclear

affinity to the Leucopoa genome and an unknown


chloroplast inherited genome, or three parallel cases ofintrogression. Alternatively, this may represent a case of

lineage sorting among taxa that diverged near the base

of the broad and fine-leaved clades.

Micropyrum and Hellerochloa may constitute further

examples of reticulation within the �fine-leaved� Festucagroup. Micropyrum shows a close and moderately sup-

ported affinity (61%) to the Aulaxyper+Vulpia p. p. (2x)

clade in the ITS based tree (Fig. 1A), but is weakly re-solved as the sister group of the Psilurus/Vulpia p. p.

(4x–6x) clade in the trnL–F based tree (Fig. 2A). The

combined analysis places Micropyrum close to this last

group. Because the two species of Micropyrum link in a

fully supported clade in all independent and combined

trees, one explanation could be that this diploid genus is

of hybrid origin presenting a maternal Vulpia-like

chloroplast genome and a paternal Festuca sect. Aul-

axyper+Vulpia p. p. (2x) nuclear one. Hellerochloa

shows the opposite trend; its sister relationship to the

core of �fine-leaved� fescues in the ITS based tree is

weakly supported as is its chloroplast affinity to the

Aulaxyper+Vulpia p. p. (2x) clade in the trnL–F tree.

However, the combined analysis resolves Hellerochloa

as sister to the last clade with moderate bootstrap sup-

port. Hellerochloa might bear a Festuca sect. Aulaxyper/Vulpia p. p. (2x) type chloroplast. There is no strong

conflict in either dataset for the placement of these taxa.

Two sampled accessions of F. quadriflora from differ-

ent mountain ranges (F. quadriflora1¼Pyrenees, F.

quadriflora2¼Alps) have sequence differences for both

the ITS and the trnL–F regions. Since sequences from

both ITS and trnL–F molecules differentiate the two ac-

cessions of F. quadriflora and resolve them in close vi-cinity, but not as sister taxa, it is presumed they represent

molecular vicariants of a geographically split taxon or

these two sequences represent two distinct species and we

are unable to detect any morphological differences.

4.5. Biogeographic history

Some insights about the biogeographical patternsshown by the studied Loliinae groups can be concluded

from the combined phylogenetic tree (Fig. 4). The Med-

iterranean region appears as the likely primary place of

speciation for both broad-leaved and fine-leaved lineages,

since most diploid representatives are endemic to this re-

gion. Diploid ephemeral taxa (Lolium, Vulpia, Narduro-

ides, Wangenheimia, Ctenopsis, Micropyrum, Cutandia)

are native to the Mediterranean area. Diploid perenniallineages (Subulbosae, Drymanthele, Scariosae, and

Pseudoscariosa within the broad-leaved clade, and Eskia

and Exaratae within the fine-leaved clade) are also dis-

tributed in theMediterranean region and, to some extent,

in Eurasia. A secondary radiation within the more re-

cently evolved perennial lineages favors the postglacial

colonization of new territories resulting in their larger

present distribution areas, like those of the EurasiaticSchedonorus and the holartic Festuca and Aulaxyper lin-

eages. Nonetheless, for most of these lineages their basal

diploid taxa are also Mediterranean in origin. Festucoid

groups that are not present in the Mediterranean region

have presumably had different geographical origins.

However, karyological reports indicate that most of the

studied American and Southern hemisphere fescues are

polyploids, whereas diploids are confined to the Medi-terranean area and to Asia (Dubcovsky and Mart�ınez,1992). Thus, it is likely that the Mediterranean and Eur-

asian region was the center of origin for the older Festuca

lineages and that different migration routes allowed the

more aggressive polyploid lineages to colonize other

continents where successive radiations increased the

spectrum of the present known taxa. A similar evolu-

tionary biogeographical scenario has been postulated forthe worldwide distributed grass genus Brachypodium

(Schippmann, 1991). With respect to the present phy-

logeny of Festuca, the highly polyploid Aulayper group is

closely related to a group of South American taxa in the

chloroplast trnL–F tree (Figs. 3 and 4). Further investi-

gations should focus on the potential relationships be-

tween the red fescues and polyploid lineages from the

Southern hemisphere.

Acknowledgments

We thank Clive A. Stace, Jochen M€uller, and two

anonymous referees for their valuable comments on themanuscript, to David Posada for his helpful advise on

Bayesian analysis, to Jochen M€uller, Arnoldo Santos,

Carlos Romero-Zarco, and Miguel Sequeira, Karol

Marhold, Samuel Pyke, Robert Soreng for providing us

fresh and silicagel dried materials of some taxa, the

curatorial staffs of BC, COLO, G, JACA, MA, ORT,

PRC, SEV, W, and WA for sending us herbarium

vouchers of the taxa under study for analysis. This workhas been subsidized by a Spanish Ministry of Science

and Technology grant (BOS2000-0996 project) to P.C.

and supported by a Central University of Venezuela

(CDCH) doctorate fellowship to P.T.

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Phylogeny of the festucoid grasses of subtribe Loliinae and allies

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