Morphometric and molecular variation in concert: taxonomy and genetics of the reticulate Pyrenean...

Morphometric and molecular variation in concert:taxonomy and genetics of the reticulate Pyreneanand Iberian alpine spiny fescues (Festucaeskia complex, Poaceae)

PEDRO TORRECILLA1,2†, CARMEN ACEDO3†, ISABEL MARQUES1,ANTONIO JOSÉ DÍAZ-PÉREZ1,2, JOSÉ ÁNGEL LÓPEZ-RODRÍGUEZ1,VICTORIA MIRONES1, ANA SUS1, FÉLIX LLAMAS3, ALICIA ALONSO3,ERNESTO PÉREZ-COLLAZOS1, JUAN VIRUEL1, ELVIRA SAHUQUILLO4,MARIA DEL CARMEN SANCHO5, BENJAMIN KOMAC6, JOSÉ ANTONIO MANSO1,JOSÉ GABRIEL SEGARRA-MORAGUES7, DAVID DRAPER8, LUIS VILLAR5 andPILAR CATALÁN1*

1Escuela Politécnica Superior de Huesca, Universidad de Zaragoza, Ctra Cuarte km 1, 22071Huesca, Spain2Facultad de Agronomía, Universidad Central de Venezuela, Av El Limón s. n., Maracay, Venezuela3Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s. n.,24071 León, Spain4Facultade de Ciencias, Universidade da Coruña, Campus da Zapateira s. n., 15071 A Coruña, Spain5Instituto Pirenaico de Ecología, IPE-CSIC, Av. Na Sa de la Victoria 12, 22700 Jaca, Spain6CENMA – Institut d’Estudis Andorrans, Avinguda Rocafort 21-23, 600 Sant Julià de Lòria, Andorra7Centro de Investigaciones sobre Desertificación (CIDE-CSIC-UV-GV), Carretera Moncada-Náquera km4.5, 46113 Moncada, Spain8Instituto de Ecología, Universidad Técnica Particular de Loja, San Cayetano Alto s. n., 1101608 Loja,Ecuador

Received 25 March 2013; revised 3 June 2013; accepted for publication 8 August 2013

The Iberian mountain spiny fescues are a reticulate group of five diploid grass taxa consisting of three parentalspecies and two putative hybrids: F. × souliei (F. eskia × F. quadriflora) and F. × picoeuropeana (F. eskia × F. gau-tieri). Phenotypic and molecular studies were conducted with the aim of determining the taxonomic boundaries andgenetic relationships of the five taxa and disentangling the origins of the two hybrids. Statistical analyses of 31selected phenotypic traits were conducted on individuals from 159 populations and on nine type specimens.Molecular analyses of random amplified polymorphic DNA (RAPD) markers were performed on 29 populations. Thephenotypic analyses detected significant differences between the five taxa and demonstrated the overall interme-diacy of the F. × picoeuropeana and F. × souliei between their respective parents. The RAPD analysis corroboratedthe genetic differentiation of F. eskia, F. gautieri and F. quadriflora and the intermediate nature of the two hybrids;however, they also detected genetic variation within F. × picoeuropeana. These results suggest distinct origins forF. × picoeuropeana in the Cantabrian and Pyrenean mountains, with the sporadic Pyrenean populations havingpotentially resulted from recent hybridizations and the stabilized Cantabrian ones from older events followed bypotential displacements of the parents. © 2013 The Linnean Society of London, Botanical Journal of the LinneanSociety, 2013, ••, ••–••.

*Corresponding author. E-mail: [email protected]†These authors contributed equally to the paper.

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Botanical Journal of the Linnean Society, 2013, ••, ••–••. With 9 figures

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, ••, ••–•• 1

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ADDITIONAL KEYWORDS: cleaved amplified polymorphic sequences (CAPS) – Festuca × picoeuropeana –Festuca × souliei – Festuca gautieri – Festuca quadriflora – grasses – homoploid hybrids – morphoanatomy –random amplified polymorphic DNA (RAPD) – restriction fragment length polymorphism (RFLP).

INTRODUCTION

The Mediterranean mountains are among the mostdiverse areas of the world (Médail & Quézel, 1997).The high diversity of species currently distributedin these areas is the consequence of their variableand intricate multi-ecosystem landscapes and theirhistory as refugia for populations and lineages duringthe Tertiary and Quaternary climatic oscillations(Pauli et al., 2003; Kadereit, Griebeler & Comes,2004). Several Mediterranean mountains have beenclassified as hotspots of plant diversity based on theirrichness in endemic species (Médail & Diadema,2009). The type of endemism varies from narrowlyendemic plants, which are more typical in southernMediterranean ranges (Nieto-Feliner, 2011), to morewidespread endemics, which are commonplace innorthern Mediterranean ranges, such as the sharedspecies distributed in the Pyrenees, the Cantabrianmountains and the Alps (Vargas, 2003; Kropf, Comes& Kadereit, 2006, 2008). Part of the accumulatedspecies diversity in these mountains derives fromhybrid zones; for example, the areas where secondarycontacts of previously isolated species or lineages tookplace during the warm interglacial and postglacialperiods of the Pleistocene and the Holocene (Hewitt,1988, 2000). Altitudinal migrations and contactsduring favourable climatic phases led to an admixtureof divergent lineages, giving rise to a variety of plantand animal hybrids and hybrid swarms (Hewitt,1999; Gutiérrez-Larena, Fuertes-Aguilar & Nieto-Feliner, 2002). Various hybrid zones have beendetected in the mountain passes of the central Pyr-enean axis (Ritchie, Butlin & Hewitt, 1989; Hewitt,1993) and the Cantabrian mountains (Bella et al.,2007), where lineages previously isolated on thenorthern and southern sides met and crossed, produc-ing new hybrid lineages.

In the southern European mountains, the subal-pine and alpine belts show the highest rates of plantendemism (Kadereit et al., 2004). These altitudinalzones were recurrently covered by ice during succes-sive glacial phases and then recolonized by foundersfrom putative lowland, peripheral or nunatak refugiain the interglacial phases (Hewitt, 2000; Holderegger& Thiel-Egenter, 2009). This would explain therecent divergence detected among recently estab-lished subalpine and alpine populations of someendemic Pyrenean plants, as opposed to the longisolation and large divergence observed in their

closely related Pre-Pyrenean mountain populations(Segarra-Moragues & Catalán, 2008). Broad land-scape genetic studies of alpine and subalpine plantsconducted in the Alps and the Carpathians haveconcluded that species diversity is not necessarilyconnected with genetic diversity, pointing to thecumulative effects of availability and distribution ofisolated source refugia and the adaptive success ofthe colonizers as the main drivers shaping thecurrent genetic composition of the floristic diversity(Thiel-Egenter et al., 2009). A high percentage ofalpine and subalpine plants are polyploids, as innorthern Eurasian latitudes (Brochmann et al.,2004); however, in contrast to the northern territo-ries, a considerable amount of the diversity is alsoaccounted for by endemic diploids that survived theglaciations in these southern refugia (Nieto-Feliner,2011). Most of the mountain endemic plants of hybridorigin are allopolyploids (Stebbins, 1984, 1985;Catalán et al., 2006), but homoploid hybrid speciationhas also contributed to the origin of new alpine andsubalpine hybrid plants (Mallet, 2007).

Grassland ecosystems currently dominate thealpine and subalpine landscapes of the Pyrenean andIberian mountains, where different plant communi-ties are adapted to distinct climatic and edaphic con-ditions (Braun-Blanquet, 1948). Nonetheless, thesecommunities coexist in suture zones, providing a sitefor new potential hybridization events among closelyrelated taxa (Benito-Alonso, 2012). The Iberian andPyrenean mountain spiny fescues (Festuca eskiaRamond ex DC. complex, Poaceae) are a group ofthree closely related alpine and subalpine grasses andtwo putative hybrids that characterize some of thetypical pastures of the Pyrenean and Cantabrianranges (Catalán, 2006). These predominantly diploidspecies show different altitudinal, ecological and geo-graphical ranges, although they also grow in sympa-try in some ecotonal areas. The silicicolous F. eskia isdistributed in the subalpine and low alpine belts ofthe Pyrenean and Cantabrian mountains, whereasthe calcicolous species F. gautieri (Hackel) K.Richterand F. quadriflora Honckeny are distributed, respec-tively, in the montane and subalpine belts of thePyrenean and eastern Iberian mountains and in thealpine belt of the Pyrenees, Jura mountains and Alps(Fuente & Ortúñez, 2001; Catalán, 2006). Two puta-tive hybrids have been recognized and thought tohave been formed by crosses between F. eskia and itstwo close congeners; however, whereas F. × souliei

2 P. TORRECILLA ET AL.

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, ••, ••–••

Saint-Yves (F. eskia × F. quadriflora) is a rare taxonthat only occurs in the central Pyrenees in a fewlocalities where the two putative parents co-occur,F. × picoeuropeana Nava (synonym F. × picoeuropeanaGutiérrez-Villarías & Homet) (F. eskia × F. gautieri) ismore common, being distributed in some ecotonalzones of the central Pyrenees, and also in broad zonesof the Cantabrian mountains where its purportedparents are not currently present (Catalán, 2006).

The systematics of the F. eskia complex has been acontroversial issue. Torrecilla et al. (2003) first cir-cumscribed the taxonomic and evolutionary bounda-ries of Festuca section Eskia Willk., an earlydiverging lineage of the fine-leaved Loliinae clade towhich the F. eskia complex belongs. Phylogeneticstudies of Catalán et al. (2004) and Inda et al. (2008)recognized a strong F. eskia/F. gautieri sister relation-ship and a close but less-related link of F. quadriflorato them. The estimated recent Pleistocene–Pliocenedivergences of F. eskia and F. gautieri (1.87 Mya) andof F. eskia and F. quadriflora (c. 4 Mya) from theirrespective common ancestors (M. Minaya & P.Catalán, unpubl. data) would explain the feasibilityand likely success of their respective interspecificcrosses. The distribution of the common F. × picoeuro-peana is, however, striking. Despite the narrow andscarce occurrence of its two parental species in thecalcareous Cantabrian Picos de Europa, where theynever grow in sympatry (F. eskia has only been foundin a few places in the central and western massifs,and F. gautieri only occurs in a few sites in theeastern massif), F. × picoeuropeana is widespread inall three massifs where it was originally described(Gutiérrez-Villarías & Homet, 1985a; Nava, 1985). Incontrast, despite the wide distribution of F. eskia andF. gautieri throughout the Pyrenees and the extensiveoccurrence of contact zones where the two taxacoexist, the hybrid is only known in the central Pyr-enees, where it is scarce and always grows in sym-patry with its two parents (Catalán, 2006).

Taxonomically, all the infraspecific rank diversitydescribed in F. eskia (Nègre, 1975) and F. gautieri(Hackel, 1882) has been disregarded by recentauthors, who have demonstrated that the morphologi-cal features used to differentiate the infraspecific taxawere inconsistent (Fuente & Ortúñez, 1988, 2001).However, phenotypic overlap between F. gautieri andF. × picoeuropeana has been reported in the easternmassif of the Cantabrian Picos de Europa, and anadditional hybrid taxon, F. × jierru Nava, hypotheti-cally resulting from the backcross of F. × picoeurope-ana to one of its progenitors, F. gautieri, was firstdescribed (Nava, 1985) and later disclaimed as havingbeing confused with specimens of both F. gautieri andF. × picoeuropeana (Gutiérrez-Villarías & Homet,1985b; Nava, 1988).

Because of the current uncertainty regarding thetaxonomic limits between the parental and hybridtaxa of the F. eskia complex and the unpredicteddistribution of some of the hybrids, we conducted aphenotypic and molecular study of representatives ofthe five taxa using morphological traits that weresuccessfully used to discriminate species of Festucasection Eskia (Torrecilla et al., 2003), and hypervari-able molecular markers that have demonstrated theirvalue in discriminating infraspecific diversity andhybrid taxa (De Greef & Triest, 1999;Segarra-Moragues & Catalán, 2003, 2005). The aimsof our study were to: (1) characterize phenotypicallythe five species of the complex and identify the traitsthat could significantly discriminate them; (2) analyseinter- and infraspecific morphometric variation of theparental and putative hybrid taxa and the degree ofpotential overlap among them; (3) investigate themolecular distinctiveness of the three parentalspecies and the intermediacy or closeness of thehybrids between the parents or to one parent; (4) testthe potential occurrence of various hybridizationevents that gave rise to the Cantabrian vs. Pyreneanpopulations of F. × picoeuropeana; and (5) decipherthe historical scenarios that could explain the currentexistence of the ‘orphan’ populations of F. × picoeuro-peana in the Cantabrian Picos de Europa mountains.

MATERIAL AND METHODSPLANT SAMPLING

Representatives of the five studied taxa of the F. eskiacomplex were sampled throughout their distributionareas in the Pyrenees, the Cantabrian Mountains andthe remaining Iberian mountains (Table 1, Fig. 1). Atotal of 623 individuals corresponding to 58 popula-tions of F. eskia, 67 of F. gautieri and 37 ofF. × picoeuropeana were sampled in the Iberian Pen-insula and the Pyrenees. Twenty-five individuals fromseven populations of the uncommon (in the Pyreneanrange) F. quadriflora, sampled in the Pyrenees andthe Alps, and ten of the extremely scarce F. × souliei,sampled in one of the six known Pyrenean popula-tions, were also included in the study for a total of 31additional samples. Most of the samples were col-lected in the field, but some populations were sampledfrom herbarium vouchers. The analysed samples havebeen deposited in the EPSHU-UZA, FCO, JACA andLEB herbaria. Type specimens of the main specificand infraspecific taxa [nine types: six holotypes ofF. eskia Ramond ex DC. var. eskia (G00169255),F. picoeuropeana Nava (FCO10880) F. × picoeurope-ana Gutiérrez-Villarías & Homet (FCO10875), F. ×jierru Nava (FCO 10877), F. × souliei (G-Herb.St-Yves s.n.), F. eskia var. tenufolia Nègre (JACA

TAXONOMY AND GENETICS OF FESTUCA ESKIA 3


Table 1. List of Festuca eskia complex samples used in the morphological and molecular analyses. Codes and localitiesof provenance are indicated for each population. Type specimens are indicated as follows: HT, holotype; LT, lectotype; NT,neotype. PEDRAJA (O. Sanchez-Pedraja), MÜLLER (J. Müller), UZ (University of Zaragoza) herbaria.

Population code LocalityMorphometricstudy

Molecularstudy Voucher

Festuca eskiaFE01 Spain: Pyrenees; Huesca; Panticosa x UZ 16.97FE02 Spain: Pyrenees; Huesca; Benasque; Baños

de BenasqueUZ 57.97

FE03 Spain: Pyrenees; Huesca; Canfranc; Anayet x UZ 24.96FE04 Spain: Cantabrian Mountains; Asturias;

Somiedox UZ 21.97

FE10 Spain: Cantabrian Mountains; Cantabria;TresMares

x x UZ 35.97

FE12 Spain: Cantabrian Mountains; Leon; Picosde Europa, Central Massif; Colladinas

x JACA 110885

FE14 Spain: Pyrenees; Huesca; Canfranc x UZ 28.96FE15 Spain: Pyrenees; Huesca; Somport x UZ 24.96FE16 Spain: Pyrenees; Huesca; Ordesa;

Custodia 1x UZ 20.96

FE17 Spain: Pyrenees; Girona; Toses x x UZ 31.96FE18 Spain: Cantabrian Mountains; Asturias;

Leitariegos; Arbasx x UZ 20.97

FE19 Spain: Pyrenees; Huesca; Cutas x UZ 14.97FE20 Spain: Pyrenees; Huesca; Cutas x UZ 14.97FE21 Spain: Cantabrian Mountains; Asturias;

Somiedo; Corniónx x UZ 22.97

FE22 Spain: Cantabrian Mountains; León;Valdeón

x UZ 31.97

FE23 Spain: Cantabrian Mountains; León;Pandetrave 1

x UZ 32.97

FE24 Spain: Cantabrian Mountains; León;Pandetrave 2

x UZ 33.97

FE26 Spain: Pyrenees; Girona; Nuria 1 x UZ 43.97FE27 Spain: Pyrenees; Girona; Nuria 2 x UZ 45.97FE28 Andorra: Pyrenees; Ports d’Envalira 1 x UZ 47.97FE29 Andorra: Pyrenees; Ports d’Envalira 2 x UZ 48.97FE40 Andorra: Pyrenees; Ports d’Envalira 3 x UZ 48.97FE30 Spain: Pyrenees; Lleida; Aigüestortes; San

Mauricix x UZ 50.97

FE31 Spain: Pyrenees; Lleida; Bonaigua x UZ 52.97FE32 Spain: Pyrenees; Lleida; Baqueira x UZ 53.97FE33 Spain: Pyrenees; Huesca; Benasque; Llanos

del Hospitalx x UZ 52.97

FE34 Spain: Pyrenees; Huesca; Benasque x UZ 57.97FE35 Spain: Pyrenees; Huesca; Ordesa; Cutas 1 x x UZ 70.97FE33 Spain: Pyrenees; Huesca; Ordesa; Cutas 2 x UZ 60.97FE36 Spain: Pyrenees; Huesca; Custodia 2 x UZ 61.97FE39 Spain: Pyrenees; Huesca; Bielsa; Barrosa x x UZ 13.98FE41 Spain: Pyrenees; Huesca; Cutas 3 x UZ 59.97FE44 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massif;Espinama

x JACA 277188

FE45 Spain: Cantabrian Mountains; León; Mahon x JACA 478984FE47 Spain: Pyrenees; Huesca; Portalet x JACA 217064AFE48 France: Pyrenees; Ossau x JACA 340480



Table 1. Continued



FE49 Spain: Pyrenees; Huesca; Plan x JACA 405180FE50 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massifx PEDRAJA 5839

FE52 Spain: Cantabrian Mountains; Palencia;Cervera Pisuerga

x PEDRAJA 6052

FE53 Spain: Pyrenees; Huesca; Port Picada x MA 12512,MA 157831

FE54 Andorra: Pyrenees; Tristaina x MA 514188FE55 France: Pyrenees; Hautes Pyrenées x UZ FE.B6FE56 France: Pyrenees; Gavarnie x UZ FE.B7FE57 France: Pyrenees; Auge x MA 12497FE58 France: Pyrenees; Col Aubesque x MA 286742FE59 Spain: Cantabrian Mountains; León; Mahon x MA 363134FE60 Spain: Cantabrian Mountains; León; Mahon x MA 378022FE61 Spain: Cantabrian Mountains; León; Mahon x MA 265469FE62 Spain: Cantabrian Mountains; León;

PicoHuevox MA 489539

FE63 Spain: Pyrenees; Huesca; Sin x MA 581234FE64 Spain: Pyrenees; Huesca; Pardinas x MA 581468FE65 Spain: Pyrenees; Huesca; Panticosa x MA 363136,

MA 12509FE66 Spain: Pyrenees; Huesca; Eriste x MA 391939FE67 Spain: Pyrenees; Huesca; Benasque x MA 446723FE68 Spain: Pyrenees; Huesca; Bielsa x MA 265470FE69 Spain: Pyrenees; Huesca; Brazato x MA 714128FE70 France: Pyrenees; Hautes Pyrenées x UZ FE.B30FE71 Spain: Cantabrian Mountains; Palencia;

Pista hacia el Espigüetex LEB 66551

FE72 Spain: Cantabrian Mountains; León; Picosde Europa, Central Massif; ColladoJermoso

x JACA 233182

FE73 Spain: Pyrenees; Huesca; Sayerri x JACA 413370FE-LT France: Pyrenées x G00169255FET-HT Spain: Palencia; Espigüete; Collado de los

Arranx JACA 614072

Festuca gautieriFG01 Spain: Pyrenees; Huesca; San Juan de la

Peñax UZ 12.97

FG02 Spain: Pyrenees; Huesca; Panticosa x UZ 15.97FG06 Spain: Pyrenees; Girona; Nuria x x UZ 41–97FG07 Spain: Prepyrenees; Huesca; Guara; Tozal

de Guarax JACA 4007

FG11 Spain: Pyrenees; Huesca; Canfranc x UZ 27.96FG10 Spain: Pyrenees; Girona; Toses x UZ 40.97FG14 Spain: Pyrenees; Girona; Toses x UZ 32.96FG15 Spain: Pyrenees; Girona; Toses x UZ 33.96FG18 Spain: Pyrenees; Girona; Nuria x UZ 46.97FG19 Andorra: Pyrenees; Encamp x UZ 49.97FG20 Spain: Pyrenees; Lleida; Aigüestortes; Sant

Mauricix x UZ 51.97

FG21 Spain: Pyrenees; Huesca; Benasque; Bañosde Benasque

x x UZ 54.97



Table 1. Continued



FG22 Spain: Pyrenees; Huesca; Benasque; Llanosdel Hospital

x UZ 56.97

FG23 Spain: Pyrenees; Huesca; Ordesa; Cutas x x UZ 58.97FG25 Spain: Pyrenees; Huesca; Custodia x UZ 64.97FG26 Spain: Pyrenees; Huesca; Canfranc; Ip x JACA 254585FG27 Spain: Pyrenees; Huesca; Cotiella x x UZ 5.98FG28 Spain: Pyrenees; Huesca; Bielsa; Barrosa x UZ B26FG29 Spain: Pyrenees; Huesca; Bisaurin x JACA 635067FG30 Spain: Pyrenees; Huesca; Acher x JACA 428374FG31 Spain: Pyrenees; Huesca; Anso x JACA 494573FG32 Spain: Pyrenees; Huesca; Hecho x JACA

10077674FG33 Spain: Pyrenees; Huesca; Hecho x JACA 411773FG34 Spain: Pyrenees; Huesca; Collarada x JACA 169376FG35 Spain: Pyrenees; Huesca; Collarada x JACA 576467FG36 Spain: Pyrenees; Huesca; Ip x JACA 569167FG37 Spain: Pyrenees; Huesca; Astún x JACA 306873FG38 Spain: Pyrenees; Huesca; Canfranc x JACA 3613 79FG39 Spain: Pyrenees; Huesca; Benasque x JACA 99494FG40 Spain: Pyrenees; Huesca; Torla x JACA 8700FG41 France. Pyrenees; Pas Escalé x JACA 223585FG42 Spain: Pyrenees; Huesca; Tella x JACA 2389FG43 Spain: Pyrenees; Huesca; Guara x JACA 383974FG44 Spain: Pyrenees; Huesca; Guara x JACA 400772FG45 Spain: Pyrenees; Huesca; Canfranc x JACA 255585FG46 Spain: Pyrenees; Huesca; Panticosa x JACA 207280FG47 Spain: Pyrenees; Huesca; Linás de Broto x JACA 238773FG48 Spain: Pyrenees; Huesca; Laspuña x JACA 481374FG49 Spain: Pyrenees; Huesca; Laspuña x JACA 403375FG50 Spain: Pyrenees; Huesca; Fanlo x JACA 372484FG51 Spain: Pyrenees; Huesca; Plan x JACA 396780FG52 Spain: Pyrenees; Huesca; Tella x JACA 571286FG53 Spain: Pyrenees; Huesca; Aneto x JACA 204484FG54 Spain: Pyrenees; Lleida; Salás Pallars 1 x JACA 573887FG55 Spain: Pyrenees; Lleida; Salás Pallars x JACA 569987FG56 Spain: Pyrenees; Lleida; Ruda x JACA 246492FG57 Andorra: Pyrenees; Santa Coloma x JACA Soc. Éch.

7214FG58 Spain: Pyrenees; Girona; La Molina x JACA 9550AFG59 France: Pyrenees; Aude x JACA 125683FG60 France: Pyrenees; Aude x JACA 122083FG61 France: Pyrenees; Ariège x JACA 131483FG62 Spain: Pyrenees; Huesca; Canciás x JACA 80687FG63 Spain: Iberian System; Teruel; Javalambre x MA 363092FG64 Spain: Pyrenees; Girona; Cerdagna x MA 466654FG65 Andorra: Pyrenees; Sta Coloma x MA 363093,

MA286739FG66 France: Pyrenees; Ariège x JACA 131483FG67 Spain: Levantean System; Castellón;

Fredesx MA 463002

FG68 Spain: Levantean System; Castellón;Villafranca

x MA 546140



Table 1. Continued



FG69 Spain: Levantean System; Castellón;Peñagolosa

x MA 511437

FG70 Spain: Iberian System; Soria; Santa Inés x MA 363084,MA363146

FG71 Spain: Pyrenees; Lleida; SalarsPallars x MA 581467FG72 Spain: Levantean System; Tarragona;

Puertos de Beceitex MA 440411

FG73 Spain: Pyrenees; Huesca; Fiscal x MA 478498FG74 Spain: Pyrenees; Huesca; Bara x MA 478499FG75 Spain: Iberian System; Teruel; Alcalá de la

Selvax MA 421787,

MA151182FG76 Spain: Pyrenees; Huesca; Ordesa x MA 53785FG77 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massif; Ándarax MA 363092

FG78 Spain: Betic System; Granada; La Sagra;Bajo el collado de las Víboras

x UZ 35.2000

FG79 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif; Ándara;Traviesas

x x JACA 3764

FG80 Spain: Pyrenees; Huesca; Ordesa; Cutas x JACA CA 3691FG81 Spain: Pyrenees; Huesca; Ordesa; Custodia x MA 312672FG82 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massif; Ándarax JACA 297888

FGG-LT France: Eastern Pyrenees; Coll de Nourry;Valle del Maulet

x W 14728

FGS-LT France: Pyrenees; Hautes Pyrenées;Gavarnie

x W-Rchb.1889–0104051

Festuca × picoeuropeanaFP01 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Western Massif; PorruBolu

x UZ FP1

FP02 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Western Massif: LaFragua

x x UZ 23.97

FP04 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif; Ándara

x UZ 25.97

FP05 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif; Ándara;Traviesas

x UZ 26.97

FP07 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif; Ándara;Valdom

x UZ 27.97

FP10 Spain: Cantabrian Mountains; León; Picosde Europa; Central Massif; Colladinas

x x UZ 17.96

FP15 Spain: Pyrenees; Huesca; Somport x UZ 25.96FP16 Spain: Pyrenees; Huesca; Somport x UZ 13.97FP18 Spain: Pyrenees; Huesca; Canfranc; Tobazo x x UZ 20.96FP19 Spain: Pyrenees; Huesca; Tobazo x UZ 30.96FP20 Spain: Pyrenees; Huesca; Custodia x UZ 22.96FP21 Spain: Pyrenees; Huesca; Cutas x UZ 6.97FP22 Spain: Pyrenees; Huesca; Cutas x UZ 7.97FP24 Spain: Pyrenees; Huesca; Ordesa; Cutas x UZ 7.97FP25 Spain: Pyrenees; Huesca; Custodia x UZ 62.97



Table 1. Continued



FP26 Spain: Huesca; Acumuer; Bucuesa x UZ FP26FP32 Spain: Pyrenees; Huesca; Custodia x UZ 63.97FP33 Spain: Pyrenees; Huesca; Benasque x UZ 5597FP34 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massif; Ándarax PEDRAJA 7291


x PEDRAJA 281


x PEDRAJA 6652

FP37 Spain: Cantabrian Mountains; León;Valdeón

x PEDRAJA 7045


x PEDRAJA 7292

FP39 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif; Ándara;Peñarrubia

x JACA 297888

FP40 Spain: Cantabrian Mountains; León; Picosde Europa, Central Massif; Colladinas

x PEDRAJA 4821

FP41 Spain: Pyrenees; Huesca; Ref. Blancas x JACA 521471FP43 Spain: Cantabrian Mountains; Cantabria;

Picos de Europa, Eastern Massif;Espinama

x JACA 290988

FP44 Spain: Cantabrian Mountains; León; Picosde Europa, Central Massif; Valdeón

x JACA 861185

FP45 Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif;Espinama

x JACA 281488


x JACA 299188

FP48 Spain: Cantabrian Mountains; León; Picosde Europa, Central Massif; Colladinas

x JACA 110785

FP-HT Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Western Massif; Torrede los Traviesos

x FCO 10880

FxJ-HT Spain: Cantabrian Mountains; Cantabria;Picos de Europa, Eastern Massif;Samelar

x FCO 10877

FxP-HT Spain: Cantabrian Mountains; Picos deEuropa, Central Massif; Collada de lasNieves

x FCO10875

Festuca quadrifloraFQ01 Spain: Pyrenees; Huesca; Bielsa; Barrosa x x JACA 127598,

UZ 15.98FQ02 Spain: Pyrenees; Huesca; Aísa x JACA 317785FQ03 Italy: Alps; Bolzano x JACA 294989FQ04 Italy: Alps; Bolzano x JACA 267687FQ05 France. Pyrenees; Valais x MA 490135FQ06 Italy: Alps; Bolzano x MA 465173FQ07 Suisse: Alps x MÜLLER 8011FQ-NT Suisse: Alps; Vaud; Mount Taveyannaz x Z000017975



Table 1. Continued



Festuca × soulieiFS01 Spain: Pyrenees; Huesca; Bielsa; Barrosa x x JACA 127498FS-HT France: Htes. Pyrenees; Aragnouet; Port de

Barroudex G-Herb.

ST.-YVES s.n.

Numbers in the ‘Locality’ column correspond to different populations for the same taxon from the same locality.

Figure 1. Geographical distribution of the studied taxa of the Festuca eskia complex in their native Pyrenean and IberianPeninsula mountains. Black continuous, black dotted, and white continuous lines represent the distribution areas ofF. eskia (Spain, France, Andorra), F. gautieri (Spain, France, Andorra) and F. × picoeuropeana (Spain), respectively,whereas those of F. quadriflora (Spain, France) and F. × souliei (Spain, France) are represented by symbols. Symbols mapthe sampled localities of the studied populations of each taxon (see Table 1).



614072), two lectotypes of F. gautieri (Hack.) K.Richt.subsp. gautieri (W 14728) and F. gautieri subsp. sco-paria (A.Kern. & Hack.) Kerguélen (W-Rchb. 1889-0104051) and the neotype of F. quadriflora Honck.(Z000017975)] were studied and used in the pheno-typic analysis (Table 1).

MORPHOLOGICAL ANALYSES

Morphometric analyses were based on a selection of31 potentially informative phenotypic traits thatcould be used to analyse the inter- and infraspecificvariation and to separate and identify the five taxa ofthe F. eskia complex (Table 2). These characters werechosen according to their diagnostic value for dis-criminating among the five studied taxa (Fig. 2)(cf. Saint-Yves, 1924; Gutiérrez-Villarías & Homet,1985a, b; Nava, 1985; Fuente & Ortúñez, 1988, 2001;Torrecilla et al., 2003) and for detecting within-species and within-hybrid morphological and ana-tomical variation (Metcalfe, 1960). Twenty-one of thecharacters were quantitative and ten were qualita-tive. Whenever possible, five individuals per sampledpopulation were measured, aiming at uncovering themaximum phenotypic diversity of the wild individualsin the populations. Morphological data were alsoscored from the nine type specimens and were incor-porated into the statistical analyses.

Exploratory descriptive analyses were applied to allsamples to eliminate outliers and non-significantvariables. In the end, five variables were eliminatedfrom the analyses as they showed no significant dif-ferences between species (P > 0.05). Simple descrip-tive statistics of the intra- and interspecific pheneticdiversity (mean, range, standard deviation, box plotsof median, range and percentiles) were calculatedfrom the data. Interspecific response variables thatcomplied with requirements of normality were esti-mated through one-way analysis of variance (ANOVA)F-tests. All character values were standardized beforetheir use in multivariate analyses to reduce the influ-ence of allometry on the results (Sneath & Sokal,1973). To avoid redundancy in the data set, five vari-ables showing high correlation coefficients with leafdiameter (R > 0.98, P < 0.05) were removed from theanalyses, resulting in a total matrix of 21 variablesfor the final study. A principal component analysis(PCA) of the log-transformed variables wasalso performed to assess the level of covariation invariables. To facilitate the interpretation of the mul-tivariate pattern described by the PCA analysis,maintaining at the same time the orthogonality in thedata set, varimax rotation was used (Rencher, 2002).

A classification discriminant analysis (DA, cross-validation) was conducted with all the variables todetermine the highest probability membership group

of the samples (Legendre & Legendre, 1998). Theidentification of the more discriminating variableswas carried out by means of Fisher’s coefficient(Fisher, 1936) at the significant threshold value of0.05. The posterior probability of classification of eachsample and the Wilks’ Lambda value of each discri-minant function were calculated (Wilks, 1932). AWilks’ Lambda value closer to zero indicated a betterdiscrimination between the predefined groups.

The relationships among samples were furtherassessed based on the combination of quantitativeand qualitative morphoanatomical data. Resem-blances between all pairs of samples were quantifiedusing Gower’s coefficient. The standardized datamatrix was used to compute a distance matrix usingthe Euclidean distance algorithm, and represented bymeans of an unweighted pair group method witharithmetic mean (UPGMA). Gower’s similarity coeffi-cient was estimated with SYNTAX 2000 (Podani,2001). All the remaining statistical analyses wereconducted using SPSS 21.0.0.

MOLECULAR SAMPLING

Various molecular analyses were conducted with asubset of the samples used in the morphoanatomicalanalyses. Because of the close relationships of thesister taxa F. eskia and F. gautieri and of the closeF. quadriflora, which show a low divergence in theirinternal transcribed spacer (ITS) DNA sequences (cf.Inda et al., 2008) and could share some plastid DNAhaplotypes (I. Marques, J. G. Segarra-Moragues, D.Draper & P. Catalán, unpubl. data), a pilot study wasconducted first with a representation of 22 samples ofF. eskia (seven), F. gautieri (six) and F. × picoeurope-ana (nine) from different geographical locations(Table 1). These samples were used for restrictionfragment length polymorphism (RFLP) analysis ofnuclear ribosomal NOR genes and a subset (eightsamples) was used for cleaved amplified polymorphicsequences (CAPS) analysis of nuclear ribosomal 5Sgenes and plastid trnL-F genes (Table 1), aimed atdetecting potential species-specific markers that couldserve to discriminate the parents and to confirm thederived origin of their hybrids. However, as none of theassayed restriction enzymes detected any RFLP orCAP polymorphism (see RESULTS), random amplifiedpolymorphic DNA (RAPD, Williams et al., 1990) analy-sis was conducted on a larger representation of 29populations of the five studied species (ten of F. eskia,nine of F. gautieri, seven of F. × picoeuropeana, one ofF. quadriflora and one of F. × souliei), with the aim ofdiscriminating the three parental species and the twoputative hybrids using these highly variable markers.The molecular RAPD sampling covered the distribu-



Tab

le2.

Sta

tist

ical

desc

ript

ors

of31

mor

phoa

nat

omic

alch

arac

ters

anal

ysed

insa

mpl

esof

F.e

skia

,F

.gau

tier

i,F

.qu

adri

flor

a,F

.xpi

coeu

rope

ana

(in

clu

din

gF.

xji

erru

)an

dF

.xso

uli

ei

F.ga

uti

eri

F.×

pico

euro

pean

aF.

eski

a

NM

inim

um

Max

imu

mM

ean

SD

Var

ian

ceN

Min

imu

mM

axim

um

Mea

nS

DV

aria

nce

NM

inim

um

Max

imu

m

CH

719.

5445

.07

24.8

87.

9262

.68

3110

.92

39.2

321

.43

6.56

42.9

758

14.7

64.4

5C

W71

0.36

0.85

0.67

0.11

0.01

310.

480.

970.

700.

100.

0158

0.53

1.18

LiL

710.

351.

730.

820.

280.

0831

0.57

2.68

1.74

0.58

0.34

582.

716.

58In

LL

711.

1610

.19

4.77

1.86

3.46

313.

3010

.49

6.08

1.87

3.49

584.

1024

.65

ILD

710.

300.

600.

500.

060.

0031

0.40

0.90

0.61

0.12

0.01

580.

602.

00IL

712.

806.

984.

840.

980.

9531

3.40

7.83

5.47

1.01

1.02

583.

0011

.05

IW71

0.50

2.52

1.05

0.30

0.09

310.

781.

801.

120.

230.

0558

0.93

1.98

LB

L71

0.80

4.10

2.51

0.64

0.42

302.

106.

903.

170.

880.

7758

2.20

5.50

LIN

L71

0.93

2.64

1.61

0.43

0.18

311.

002.

251.

620.

310.

1058

0.75

3.11

SL

715.

0311

.00

8.65

1.08

1.17

317.

2411

.00

8.91

0.84

0.71

585.

5010

.96

PeL

710.

935.

623.

421.

041.

0728

1.72

5.61

3.36

1.12

1.26

571.

015.

71L

GL

712.

145.

503.

910.

630.

3931

3.21

5.16

4.03

0.53

0.28

583.

245.

61L

GW

700.

601.

330.

810.

130.

0231

0.67

1.50

0.85

0.16

0.03

570.

601.

21U

GL

713.

106.

004.

800.

530.

2831

4.13

6.00

5.04

0.53

0.28

583.

506.

38U

GW

700.

731.

911.

430.

200.

0431

1.19

2.14

1.49

0.22

0.05

571.

012.

12L

mL

714.

557.

256.

070.

580.

3431

5.44

7.55

6.43

0.50

0.25

583.

757.

69L

mW

701.

213.

001.

740.

290.

0928

1.48

2.30

1.83

0.24

0.06

581.

302.

41A

L71

0.00

1.26

0.34

0.33

0.11

310.

001.

500.

590.

400.

1658

0.00

1.19

PaL

713.

186.

575.

340.

640.

4129

4.18

6.56

5.57

0.59

0.35

583.

127.

31P

aW69

0.43

1.37

0.86

0.14

0.02

280.

641.

520.

890.

190.

0457

0.39

1.50

An

L63

0.45

4.96

2.84

0.59

0.34

281.

743.

532.

760.

410.

1744

1.50

3.53

NN

C71

12

10

031

13

21

058

14

NN

R71

37

51

031

37

51

158

47

NL

B71

13

21

031

12

21

058

15

NS

I71

619

113

631

722

133

958

1056

NS

LB

712

73

11

311

64

11

582

12N

FS

713

65

11

313

65

11

582

7N

LR

711

31

00

313

75

11

585

13N

LV71

24

20

031

28

61

258

814

NL

N71

57

61

131

711

71

158

918

AL

S71

01

00

031

11

10

058

11



Tab

le2.

Con

tin

ued

F.es

kia

F.×

sou

liei

F.qu

adri

flor

a

Mea

nS

DV

aria

nce

NM

inu

mu

nM

axim

um

Mea

nS

DV

aria

nce

NM

inim

um

Max

imu

mM

ean

SD

Var

ian

ce

CH

26.6

698.

2949

68.8

052

17.0

017

.19

17.1

00.

140.

028

5.01

18.4

411

.16

4.41

19.4

2C

W0.

810.

120.

022

0.87

0.90

0.89

0.02

0.00

80.

480.

870.

700.

150.

02L

iL4.

280.

800.

642

1.55

2.25

2.05

0.70

0.50

80.

451.

520.

860.

350.

12In

LL

8.91

3.67

13.5

02

2.10

4.10

3.10

1.42

2.01

81.

936.

823.

851.

943.

75IL

D1.

020.

210.

042

0.65

0.70

0.68

0.04

0.00

80.

450.

580.

480.

040.

00IL

6.82

1.39

1.95

24.

004.

504.

250.

350.

128

2.32

3.85

3.04

0.52

0.27

IW1.

360.

260.

072

1.00

1.09

1.04

0.06

0.00

80.

851.

301.

040.

170.

03L

BL

3.42

0.74

0.55

21.

502.

101.

790.

410.

178

0.90

2.00

1.39

0.38

0.15

LIN

L1.

650.

480.

232

0.89

0.90

0.89

0.01

0.00

80.

471.

300.

910.

300.

09S

L8.

741.

141.

302

7.85

9.50

8.67

1.17

1.37

75.

288.

637.

041.

151.

31P

eL3.

071.

021.

042

2.63

2.63

2.63

0.00

0.00

72.

009.

753.

882.

667.

05L

GL

4.04

0.50

0.25

23.

463.

743.

600.

200.

048

2.50

3.76

3.28

0.45

0.20

LG

W0.

850.

130.

022

1.05

2.25

1.65

0.85

0.72

70.

781.

080.

880.

100.

01U

GL

4.90

0.56

0.31

24.

474.

594.

530.

080.

018

3.43

4.80

4.22

0.50

0.25

UG

W1.

470.

200.

042

1.47

1.47

1.47

0.00

0.00

71.

472.

701.

700.

450.

20L

mL

6.10

0.72

0.52

25.

235.

485.

350.

180.

038

4.03

5.63

4.98

0.50

0.26

Lm

W1.

890.

280.

082

1.40

1.41

1.40

0.01

0.00

71.

501.

971.

680.

200.

04A

L0.

670.

280.

082

0.50

0.90

0.70

0.28

0.08

80.

511.

250.

910.

250.

06P

aL5.

390.

720.

522

4.52

5.48

5.00

0.68

0.46

83.

214.

884.

320.

580.

33P

aW0.

890.

180.

032

0.99

0.99

0.99

0.00

0.00

70.

751.

030.

840.

100.

01A

nL

2.72

0.48

0.23

22.

353.

002.

670.

460.

217

1.72

3.04

2.42

0.54

0.30

NN

C2

10

21

11

00

81

21

10

NN

R6

10

25

55

00

84

65

11

NL

B1

11

22

22

00

81

21

10

NS

I19

862

210

1111

11

87

1710

311

NS

LB

52

32

44

40

08

23

30

0N

FS

51

02

45

40

08

35

41

1N

LR

91

22

57

61

28

13

21

1N

LV10

12

26

66

00

82

43

11

NL

N13

23

27

77

00

85

76

11

AL

S1

00

21

11

00

80

00

00

CH

,cu

lmh

eigh

t(c

m);

CW

,cu

lmw

idth

(mm

);L

iL,

ligu

lele

ngt

h(m

m);

InL

L,

inn

ovat

ion

leaf

len

gth

(cm

);IL

D,

inn

ovat

ion

leaf

diam

eter

(mm

);IL

,in

flor

esce

nce

len

gth

(cm

);IW

,in

flor

esce

nce

wid

th(c

m);

LB

L,

low

erbr

anch

len

gth

(cm

);L

INL

,lo

wer

inte

rnod

ele

ngt

h(c

m);

SL

,sp

ikel

etle

ngt

hfr

omth

eba

seto

the

apex

ofth

e4t

hle

mm

a,w

ith

out

awn

s(m

m);

PeL

,pe

dice

lle

ngt

h(m

m);

LG

L,

low

ergl

um

ele

ngt

h(m

m);

LG

W;

low

ergl

um

ew

idth

(mm

);U

GL

,u

pper

glu

me

len

gth

(mm

);U

GW

,upp

ergl

um

ew

idth

(mm

);L

mL

,lem

ma

len

gth

from

the

seco

nd

basa

lfl

oret

(mm

);L

mW

,lem

ma

wid

th(m

m);

AL

,aw

nle

ngt

h(m

m);

PaL

,pal

eale

ngt

h(m

m);

PaW

,pal

eaw

idth

(mm

);A

nL

,an

ther

len

gth

(mm

);N

NC

,nu

mbe

rof

nod

esof

culm

;NN

R,n

um

ber

ofn

odes

ofra

chis

;NL

B,n

um

ber

oflo

wes

tbr

anch

es;N

SI,

nu

mbe

rof

spik

elet

spe

rin

flor

esce

nce

;N

SL

B,

nu

mbe

rof

spik

elet

sof

low

est

bran

ch;

NF

S,

nu

mbe

rof

flow

ers

per

spik

elet

;N

LR

,n

um

ber

ofle

afri

bs;

NLV

,n

um

ber

ofle

afva

lley

s;N

LN

,n

um

ber

ofle

afn

erve

s;A

LS

,ab

axia

lle

afsc

lere

nch

yma

[in

ari

ng

(1)

orin

sepa

rate

bloc

ks(0

)].



Table 3. Significance tests of mean values of the 31 analysed characters. Mean ± SD and ANOVA F-tests of variablesused for comparisons among taxa (d.f. 4). Superscript letters (a, b, c) denote Tukey HSD pairwise comparisons betweentaxa; means with the same letter do not differ significantly (P < 0.05). FE, F. eskia; FG, F. gautieri; FP, F. × picoeuropeana;FQ, F. quadriflora; FS, F. × souliei

Variables that significantly discriminate F. eskia vs. F. gautieri + F. quadriflora vs. F. × picoeuropeana + F. × souliei

LiL NLR NLV

FG 0.8165 ± 0.27653c 1.127 ± 0.476c 2.127 ± 0.476c

FP 1.7441 ± 0.57975b 5.258 ± 0.8551b 5.806 ± 1.4473b

FE 4.2812 ± 0.79887a 9.369 ± 1.4989a 10.401 ± 1.3876a

FS 2.05 ± 0.70711c 6 ± 1.4142c 6 ± 0c

FQ 0.862 ± 0.35226b 1.591 ± 0.9052b 2.604 ± 0.9081b

F 316.390 537.114 481.166

Variable that significantly discriminates F. gautieri + F. quadriflora vs. remaining taxa

ALS

FG 0.37 ± 0.485a

FP 1 ± 0b

FE 1 ± 0b

FS 1 ± 0b

FQ 1 ± 0a

F 50.058

Variables that significantly discriminate F. eskia vs. remaining taxa

ILD NLN

FG 0.5049 ± 0.06272b 6.219 ± 0.9403b

FP 0.6148 ± 0.11584b 7.452 ± 1.15b

FE 1.0166 ± 0.20599a 12.592 ± 1.8635a

FS 0.675 ± 0.03536b 7 ± 0 b

FQ 0.48 ± 0.04472b 5.75 ± 0.8864b

F 120.981 194.427

Variable that significantly discriminates F. quadriflora vs. remaining taxa

SL

FG 8.6525 ± 1.08309b

FP 8.9127 ± 0.84261b

FE 8.7357 ± 1.13997b

FS 8.6732 ± 1.16932b

FQ 7.0352 ± 1.14609a

F 4.566

Variable that significantly discriminates F. × souliei vs. remaining taxa

LGW

FG 0.8059 ± 0.12816b

FP 0.8505 ± 0.16419b

FE 0.8514 ± 0.13366b

FS 1.6497 ± 0.84895a

FQ 0.8786 ± 0.10492b

F 15.341



tion ranges of the five taxa in the Pyrenees and thenorthern Iberian mountains (Table 1).

DNA ISOLATION

Fresh leaves from sampled individuals were dried onsilica gel (Chase & Hills, 1991) and used for DNAisolation. Up to ten individuals per population weresampled and used to prepare bulked DNA samples.DNA was extracted following the cetyltrimethylammonium bromide (CTAB) protocol of Doyle & Doyle(1987). Approximately, 10 μg of DNA per sample wereused for the restriction enzyme-based analyses. DNAsamples were diluted to a final concentration ofc. 5 ng μL−1 for the RAPD amplification.

RFLP ANALYSIS

Restriction enzyme digestion, gel electrophoresis andSouthern analyses were conducted following thestandard procedures indicated in Shi, Draper &Stace (1993), with some modifications. DNA sampleswere separately digested with a set of six digestionenzymes [three 6-bp restriction site enzymes(BamIII, EcoRI and HindIII) and three 4-bp restric-tion site enzymes (AluI, HaeIII and RsaI)] accordingto the manufacturer’s instructions. RFLP of the ribo-somal NOR genes were examined using the pTa71probe, which contains a 9-kb EcoRI fragment of therepeat unit of 25S-5.8S-18S rDNA isolated from Triti-cum aestivum L. (Gerlach & Bedbrock, 1979). ThepTa71 probe was labelled with digoxigenin-16-dUTP(Roche) by nick translation. The probe mixture con-tained 50% (v/v) formamide, 20% (w/v) dextran sul-phate, 2 × saline sodium citrate buffer (SSC),10–25 ng probe, 20 μg salmon sperm DNA and 0.3%sodium dodecyl sulphate (SDS). Hybridization took

place overnight at 42 °C and the most stringent washwas carried out with 20% (v/v) formamide and0.1 × SSC at 60 °C. After hybridization and strin-gency washes, an alkaline phosphatase (AP) conju-gated anti-DIG antibody was incubated with themembrane for 30 min, and then with a chemilumi-nescent substrate (CDP-Star) for 5 min for detectionof the DIG-labelled probe and the scoring of thehybridized fragments.

CAPS ANALYSIS

CAPS analysis was performed following the proce-dures of Konieczny & Ausubel (1993). The nuclearribosomal 5S gene and the plastid trnL-F intergenicspacer were amplified using the primers and protocolsof Shi (1991) and Taberlet et al. (1991), respectively.The amplified 5S and trnL-F fragments were digestedwith four (BamIII, HinfI, MboI, RsaI) and two (EcoRI,AluI) restriction enzymes, respectively. The digestedproducts were separated by electrophoresis in 1%agarose gels and the fragments were visualized withethidium bromide staining.

RAPD AMPLIFICATION

RAPD amplifications were carried out using 20unique primers (Operon Technologies, OPA-01 toOPA-20), following a modified version of the Williamset al. (1990) protocol. Amplifications were carried outin 20 μL total volume containing 1 × buffer (Ecogen),2.5 mM magnesium chloride (MgCl2), 100 μM eachdeoxynucleotide (dNTP), 0.5 μM of primer, 1.0 unitTaq DNA polymerase, and 2 ng template DNA. PCRwas programmed for an initial cycle of 4 min at 94 °C,followed by 40 cycles each of 94 °C for 1 min, 39 °C for1 min and 72 °C for 1.5 min, ending with an elonga-

Table 3. Continued

Variable that significantly discriminates F. quadriflora and F. × souliei vs. remaining taxa

LINL

FG 1.6088 ± 0.42869b

FP 1.6247 ± 0.31097b

FE 1.6465 ± 0.47665b

FS 0.8927 ± 0.01036a

FQ 0.9139 ± 0.29706a

F 6.848

LiL, ligule length (mm); NLR, number of leaf ribs; NLV, number of leaf valleys; ALS, abaxial leaf sclerenchyma [in aring (1) or in separate blocks (0)]; ILD, innovation leaf diameter (mm); NLN, number of leaf nerves; SL, spikeletlength from the base to the apex of the fourth lemma, without awns (mm); LGW, lower glume width (mm); LINL,lower internode length (cm).



tion step of 72 °C for 7 min. Eleven primers out of the20 that were assayed and rendered amplicons thatwere reproducible after three rounds of amplifications(< 5% error; Bonin, Ehrich & Manel, 2007) wereselected for the genetic study. The amplified productswere electrophoresed in 2% agarose gels stained withethidium bromide; electrophoresis was set at 100 Vfor 4 h in 0.5 × Tris/borate/EDTA (TBE) buffer. RAPDbands were visualized with ultraviolet (UV) transmit-ted light and gel images were captured with Gel Doc1000 (Bio-Rad) for subsequent band scoring.

MOLECULAR STATISTICAL ANALYSES

RFLP and CAPS data did not produce any polymor-phism and were not further analysed (see RESULTS).RAPD bands were scored by their presence/absence,resulting in a binary data matrix. This data matrixwas used to analyse the genetic relationships amongthe 29 surveyed populations. A matrix of pairwisedifference distances of RAPD phenotypes amongpopulations was computed with ARLEQUIN v. 3.11(Excoffier, Laval & Schneider, 2005). The genetic rela-tionships among populations were visualized using aneighbor joining tree constructed with MEGA 5.0(Tamura et al., 2011), where the statistical robustnessof the groupings was assessed by a 1000-replicatebootstrap analysis (Felsenstein, 1985) conducted withPAUP* (Swofford, 2002) and by a principal coordinateanalysis (PCO) conducted with NTSYS (Rohlf, 2002).A minimum spanning tree (MST) was constructed andprojected onto the PCO plot.

RESULTSMORPHOLOGY OF THE FESTUCA ESKIA COMPLEX

All the studied morphoanatomical characters showedboth inter- and intraspecific diversity in the studiedindividuals (Table 2, Figs 3, A1; see Appendix). Nineof the 31 traits [ligule length (LiL), innovation leafdiameter (ILD), lower internode length (LINL), spike-let length (SL), lower glume width (LGW), number ofleaf ribs (NLR), number of leaf valleys (NLV), numberof leaf nerves (NLN), abaxial leaf sclerenchyma(ALS)] were useful in significantly discriminatinggroups of taxa from each other (Table 3). The remain-ing traits did not significantly differentiate betweenthe taxa. For most of the discriminating variables, themean values of F. eskia tended to be the highest andthose of F. gautieri and F. quadriflora the lowest.F. × picoeuropeana and F. × souliei showed intermedi-ate values. Ligule length (F = 316.390, P < 0.001),innovation leaf diameter (F = 120.981, P < 0.001),lower internode length (F= 6.848, P <0.001), spikelet length (F = 4.566, P = 0.002), lowerglume width (F = 15.341, P < 0.001), number of leafT

able

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LU

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0.66

9±

0.11

335

4.76

99±

1.85

935

1.04

65±

0.29

512

4.84

13±

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524

1.04

65±

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512

2.50

7±

0.64

493.

4198

±1.

0360

83.

9111

±0.

6252

4.79

85±

0.53

041

1.43

29±

0.20

256

6.06

56±

0.58

052

FP

0.69

5±

0.09

978

6.07

61±

1.86

896

1.12

28±

0.23

328

5.47

4±

1.01

083

1.12

28±

0.23

328

3.16

7±

0.87

613.

3595

±1.

124

4.02

98±

0.53

101

5.03

96±

0.53

231

1.48

89±

0.22

362

6.43

41±

0.50

35F

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8122

±0.

1224

28.

9096

±3.

6741

21.

3617

±0.

2593

26.

819

±1.

3944

91.

3617

±0.

2593

23.

415

±0.

7388

3.06

55±

1.01

844

4.03

52±

0.49

968

4.90

09±

0.55

725

1.46

53±

0.20

238

6.09

89±

0.72

386

FS

0.88

56±

0.02

038

3.10

17±

1.41

658

1.04

39±

0.06

205

4.25

2±

0.35

069

1.04

39±

0.06

205

1.79

3±

0.41

372.

6289

±0.

0016

3.59

9±

0.19

663

4.52

99±

0.08

471.

4682

±0.

0025

55.

3527

±0.

1801

FQ

0.69

57±

0.15

253

3.84

76±

1.93

527

1.04

44±

0.17

274

3.04

16±

0.51

933

1.04

44±

0.17

274

1.38

9±

0.38

313.

8793

±2.

6553

93.

2778

±0.

4513

24.

2202

±0.

495

1.69

71±

0.44

539

4.98

11±

0.50

468

F8.

831

13.9

5922

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35.9

1912

.056

24.0

591.

431

3.66

34.

184

2.42

09.

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Lm

WA

LP

aLP

aWA

nL

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NR

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BN

SI

NS

LB

NF

S

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1.73

52±

0.29

319

0.34

47±

0.32

704

5.34

42±

0.63

604

0.85

76±

0.14

097

2.84

17±

0.58

693

1.35

2±

0.48

14.

845

±0.

5769

1.64

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9910

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5283

3.39

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0.76

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0.89

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±0.

956

1.51

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812

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±2.

9935

3.69

8±

0.92

584.

651

±0.

7779

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1.88

82±

0.27

626

0.67

25±

0.27

886

5.39

41±

0.72

142

0.88

79±

0.17

607

2.72

2±

0.47

817

1.62

1±

0.67

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517

±0

1.37

9±

0.76

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±7.

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324.

891

±0.

643

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1.40

36±

0050

30.

7±

0.28

284

5.00

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0.67

543

0.99

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353

2.67

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0.46

265

1±

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6554

2±

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0.70

713.

811

±0.

4962

4.35

±0.

1701

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1.67

56±

0.00

504

0.90

8±

0.24

778

4.31

58±

0.57

629

0.83

9±

0.10

315

2.42

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0.54

402

1.37

5±

0.51

755

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0.75

591.

375

±0.

5175

10.3

44±

3.27

582.

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0.37

4.44

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0.72

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7.72

21.

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20.8

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ribs (F = 537.114, P < 0.001), number of leaf valleys(F = 481.166, P < 0.001), number of leaf nerves(F = 194.427, P < 0.001) and abaxial leaf scleren-chyma (F = 50.058, P < 0.001) showed a significantincrease from the lowest mean values of F. gautieriand F. quadriflora, through the intermediate onesof F. × picoeuropeana and F. × souliei to the highestones of F. eskia. These results clearly indicatedthat three groups of taxa of the Festuca eskiacomplex (F. eskia vs. F. gautieri + F. quadriflora vs.F. × picoeuropeana + F. × souliei) could be morpho-logically differentiated. Different sets of traitssignificantly discriminated F. eskia vs. F. gautieri +F. quadriflora vs. F. × picoeuropeana + F. × souliei(ligule length, number of leaf ribs, number of leafvalleys) and F gautieri + F quadriflora (abaxial leafsclerenchyma), F quadriflora + F × souliei (lowerinternode length), F. eskia (innovation leaf diameter,number of leaf nerves), F. quadriflora (spikeletlength) and F. × souliei (lower glume width) from the

rest (Table 3). The type specimen of F. × jierru,described as potentially resulting from the backcrossof F. gautieri and F. × picoeuropeana (Nava, 1985),overlapped with F. × picoeuropeana. Our results castdoubt on the taxonomic identity of F. × jierru, agree-ing with Gutiérrez-Villarías & Homet (1985b), whoalso disregarded it. The remaining type specimens ofthe five recognized taxa were consequently used astaxonomic reference samples in the PCA, DA andUPGMA analyses.

The PCA identified two axes with eigenvalues > 1(Table A1, see Appendix), which altogether accountedfor 66.96% of the observed morphological variation. Aneat cluster of samples of F. eskia separates from theother taxa along axis I, whereas the respective ofF. gautieri and F. quadriflora are located on oppositeextremes of axis II (Fig. A2; see Appendix). Theremaining clusters of the F. × picoeuropeana andF. × souliei taxa were generally intermediate withrespect to the parental species.

Figure 2. Morphological distinctiveness of the five studied taxa of the Festuca eskia complex. A–B, F. gautieri. C–D,F. eskia. E–F, F. quadriflora. G–H, F. × picoeuropeana. I–J, F. × souliei. The hybrid taxa show intermediacy in theanatomical leaf section and ligule features between those of their respective parents.



The standard discriminant analysis classificationmethod based on the whole set of analysed characters(31) resulted in the correct classification of 95% of theF. eskia, F. gautieri and F. quadriflora samples intotheir respective predefined groups. For F. × picoeuro-peana, 82.4% of the samples were correctly classified;only two, three and one samples were attributed tothe F. eskia, F. gautieri and F. quadriflora groups,respectively. The few studied samples of F. × souliei(two) could not be properly classified. In the bidimen-sional discriminant analysis scatterplot (Fig. 4), theF. eskia samples vs. the remaining samples clusteredon opposite sides of the first discriminant function,which accounted for 90.7% of the total variation,whereas the F. gautieri samples separated (althoughnot totally) from the F. quadriflora ones along thesecond discriminant function, which accounted for

6.2% of the variance. The F. × picoeuropeana andF. × souliei samples clustered at intermediate posi-tions between the clusters of their respective parentsin the space determined by the two discriminantfunctions (Fig. 4). The discriminant analysis con-firmed that the morphological separation of the fivetaxa is supported by nine characters (ligule length,innovation leaf diameter, lower internode length,spikelet length, lower glume width, number of leafribs, number of leaf valleys, number of leaf nerves,abaxial leaf sclerenchyma). Wilks’ lambda values ofthe first and second discriminant functions were0.019 and 0.311, respectively. The lowest value,obtained for the first discriminant function, supportedthe clear phenetic separation between F. eskia andthe remaining taxa. However, the low Wilks’ Lambdavalue obtained for the second discriminant function

Figure 3. Box plots of simple statistics values (mean, percentiles, range) of nine morphological characters analysed inindividuals from the 169 studied populations plus nine types of the five Festuca eskia complex taxa (Table 1). Variablesthat significantly discriminate among some of the five taxa: FE, F. eskia; FG, F. gautieri; FQ, F. quadriflora; FP,F. × picoeuropeana; FS, F. × souliei. Abbreviations of variables: LiL, ligule length (mm); ILD, innovation leaf diameter(mm); LINL, lower internode length (cm); SL, spikelet length from the base to the apex of the fourth lemma, without awns(mm); LGW, lower glume width (mm); NLR, number of leaf ribs; NLV, number of leaf valleys; NLN, number of leaf nerves;ALS, abaxial leaf sclerenchyma (in a ring (1) or in separate blocks (0).



that separated F. gautieri and F. quadriflora also sup-ported their phenetic differentiation.

The UPGMA dendrogram based on the Gower simi-larity index (Fig. 5) showed two clearly distinct hier-archical groups formed by the F. eskia cluster and acluster of the remaining taxa. Further substructuringwas recovered in the latter group that showed theneat differentiation of the F. gautieri and F. quadri-flora (Alpine samples, pro parte) subgroups. Most ofthe F. × picoeuropeana samples clustered in a groupclose to F. gautieri. Some Alpean and Pyreneansamples of the variable F. quadriflora clustered,however, in the separate F. × picoeuropeana andF. gautieri subgroups. The F. × souliei samples alsoclustered in an intermediate position between theparental species, although closer to F. quadriflora andnested within the F. × picoeuropeana subgroup(Fig. 5).

RFLP, CAPS AND RAPD DATA

The RFLP analysis conducted on the 22 studiedsamples of F. eskia, F. gautieri and F. × picoeuropeanadid not show any polymorphism in the restrictionsites or the fragment lengths for any of the six

assayed enzymes. BamHI showed three restrictionsites in the ribosomal DNA repeat unit of thesefescues, resulting in three digested fragments(c. 10.0 kbp, 8.0 kbp, 4.5 kbp); EcoRI showed two(c. 10 kbp, 8 kbp); and HindIII one (c. 10 kbp).However, none was species- or population-specific(results not shown). Similarly, the 4-bp restrictionenzymes RsaI, AluI and HaeIII, did not produce anypolymorphic RFLP patterns in the studied popula-tions or taxa. RsaI showed five restriction sites(c. 2.0 kbp, 1.5 kbp, 1.0 kbp and two of < 0.5 kbp),AluI nine (all of them < 2 kbp) and HaeIII five(< 1 kbp); all were homomorphic across the studiedsamples (results not shown).

The CAPS analysis carried out on a restricted sam-pling of F. eskia, F. gautieri and F. × picoeuropeanaalso failed to detect any polymorphic variation forthe five assayed enzymes. The amplification of thenuclear multicopy ribosomal 5S gene produced asmall 300-bp fragment in all the scrutinized samples.BamHI, HinfI and RsaI showed no restriction sites inthis region. MboI showed one restriction site thatresulted in one homomorphic digested fragment(c. 150 bp) in the three taxa. The amplification of theplastid trnL-F region produced a large c. 1 kbp in all

Figure 4. Two-dimensional scatterplot of classification discriminant analysis of Festuca eskia complex samples fortaxonomic phenetic differentiation of the five studied taxa. The first and second canonical discriminant functionsexplained 90.7 and 6.2% of the interspecific taxonomic variation, respectively. FE, F. eskia; FG, F. gautieri; FP,F. × picoeuropeana; FQ, F. quadriflora; FS, F. × souliei. Open squares indicate the respective group centroids.



the studied samples. AluI showed three restrictionsites in the region, revealed as four digested frag-ments, and EcoRI showed two, revealed as threefragments, all of which were also homomorphic(results not shown).

In contrast to the more conservative restrictionenzyme analyses, RAPD markers detected geneticvariability at the inter- and infraspecific levels. The11 primers used produced 108 markers for the 29surveyed populations (Table 1). Five (4.63%) markerswere exclusive to F. gautieri, and two markers each(1.85%) were exclusive to F. eskia and F. × picoeuro-peana; however, none of these bands was fixed.Ninety-one (91.66%) of the markers were sharedbetween two or more taxa. Overall, the F. × picoeuro-peana populations shared 60% of the bands withF. gautieri and 40% with F. eskia, whereas F. × soulieishared 38 (95%) of its markers with F. eskia and 26(65%) with F. quadriflora. The 11 primers provided adistinct RAPD profile for each of the studied samples.The genetic distances between samples were higherbetween species than within them, thus supportingtheir divergence.

Principal coordinate analysis showed a clear differ-entiation between the F. eskia and F. gautieri samplesthat clustered separately in the space delimited bythe first two axes, which accounted for 29.74% of

the variance (Fig. 6). The single representativesample of F. quadriflora separated from these twogroups along the second axis of the plot. Most of theF. × picoeuropeana and F. × souliei samples clusteredat intermediate plot distances between those of theirhypothesized parents. The MSP showed uniqueconnections between the F. eskia–F. gautieri andthe F. eskia–F. quadriflora groups through theF. × picoeuropeana and the F. × souliei samples,respectively (Fig. 6). Nonetheless, the heterogeneousF. × picoeuropeana samples were differently resolved;all the Pyrenean samples clustered between F. eskiaand F. gautieri, but the Cantabrian Picos de Europasamples were closer to F. eskia (western Picos), toF. gautieri (eastern Picos) or intermediate betweenthem (central Picos).

The neighbor joining tree constructed from pairwisedifference distances between the 29 populations(Fig. 7) also revealed the differentiation of two mainclusters, corresponding to the F. eskia and F. gautierigroups, when F. quadriflora was used to root the tree.Most of the relationships were poorly supported. TheF. × picoeuropeana and the F. × souliei groups also fellin intermediate positions between those of theirhypothesized parents. However, one subgroup of Can-tabrian western Picos de Europa F. × picoeuropeanapopulations fell in the F. eskia group, nesting in a

Figure 5. Unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on pairwise on Gowerdissimilarity values calculated from a matrix of 170 samples and 16 discriminant and variable morphological characters(see Table 3). FE (blue), F. eskia; FG (orange), F. gautieri; FP (green), F. × picoeuropeana; FQ (brown), F. quadriflora; FS(grey), F. × souliei. Type specimens are indicated by T.



Cantabrian subgroup, and one sample from the Can-tabrian central Picos de Europa fell in the F. gautierigroup, showing a strong sister relationship to aneastern Picos de Europe population. The F. eskiagroup was substructured into eastern Pyrenees,western Pyrenees and Cantabrian Mountains sub-groups, but with low support.

DISCUSSIONMORPHOLOGICAL AND MOLECULAR DIFFERENTIATION

OF THE IBERIAN AND PYRENEAN FESTUCA ESKIA

COMPLEX TAXA: TAXONOMIC IMPLICATIONS

Our morphological and molecular analyses have sta-tistically demonstrated the existence of phenotypicand genetic divergences among the five studied taxaof the Iberian and Pyrenean F. eskia complex, thussupporting their separate taxonomic status. Morpho-metric and RAPD data clearly differentiate the threeparental species, F. eskia, F. gautieri and F. quadri-flora, and the hybrids, F. × souliei and F. × picoeuro-peana, from their respective parents (Figs 2, 3;Tables 2, 3). Complementary analysis of highly vari-able molecular RAPD markers and quantitative mor-phological traits had been previously demonstrated to

be useful tools for detecting taxonomic structureamong closely related Arctic polyploid Festuca spp.(Fjellheim, Elven & Brochmann, 2001). Here, agree-ment between molecules and morphology has beenconvincingly demonstrated in this group of diploidAlpine fescues, in which homoploid hybrid speciationhas probably occurred at least twice. The phenotypicdistinction found among F. eskia, F. gautieri andF. × picoeuropeana, and among F. eskia, F. quadrifloraand F. × souliei corroborates previous results byGutiérrez-Villarías & Homet (1985a, b) and Fuente &Ortúñez (1988, 2001) and by Saint-Yves (1924) andFuente & Ortúñez (1988, 2001), respectively. Theintermediacy of the phenotypic traits of F. × soulieibetween those of F. eskia and F. quadriflora and ofF. × picoeuropeana between those of F. eskia andF. gautieri supports their purported hybrid nature.However, the morphometric and molecular variationdetected within F. × picoeuropeana also suggest dif-ferent hybridization events for the Pyrenean andCantabrian hybrids and also among the Cantabrianones (see comments below).

Morphologically, F. eskia is the most distinct taxonof the three parental species; five phenotypic traits(ligule length, innovation leaf diameter, number ofleaf ribs, number of leaf valleys, number of leaf

Figure 6. Principal coordinate analyses (PCO) based on pairwise difference distance of random amplified polymorphicDNA (RAPD) phenotypes, showing the relationships among populations of Festuca eskia (circles, up triangles), F. gautieri(squares, down triangles), F. quadriflora (black stars) and their putative hybrids F. × picoeuropeana (diamonds) andF. × souliei (grey stars). A minimum spanning tree (MST) was superimposed on the PCO plot. The symbols and coloursindicate geographical areas: black circles, black squares, black diamonds and black stars, Central Pyrenees; white circlesand white squares, eastern Pyrenees; black triangles, Central Cantabrian; open triangles and open diamonds, westernCantabrian; grey down triangles, eastern Cantabrian; grey diamonds, eastern + central Cantabrian.



Figure 7. Neighbor joining tree based on pairwise difference distance of random amplified polymorphic DNA (RAPD)phenotypes, showing the relationships among populations of Festuca eskia (circles, up triangles), F. gautieri (squares,down triangles), F. quadriflora (black stars), and their putative hybrids F. × picoeuropeana (diamonds) and F. × souliei(grey stars). Festuca quadriflora was used to root the tree. Bootstrap values obtained from 1000 permutations overpopulations are shown above branches of the neighbor joining tree when higher than 40%. The symbols and coloursindicate geographical areas: Black circles, black squares, black diamonds and black stars, Central Pyrenees; white circlesand white squares, eastern Pyrenees; black triangles, Central Cantabrian; open triangles and open diamonds, westernCantabrian; grey down triangles, eastern Cantabrian; grey diamonds, eastern + central Cantabrian.



nerves) (Table 3; Figs 2, 3) significantly discriminatethis species from the rest, and the discriminant analy-sis and PCA plots show a unique separate clusteringfor this group (Figs 4, A1). The F. eskia plants aremore robust and have longer ligules (LiL > 3 mm) andlonger and thicker innovation leaves (ILD > 1 mmdiameter) than the other taxa. The studied type speci-men of F. eskia var. eskia and the other samples areclassified in the F. eskia cluster in the discriminantanalysis (Fig. 4), corroborating the taxonomic adscrip-tion of the wild studied samples to this taxon. Thetype of F. eskia var. tenuifolia also falls in the F. eskiarange defined by the discriminant analysis coordi-nates (Fig. 4) and the UPGMA clustering (Fig. 5),indicating that this variety represents part of thenatural variation of the species. The RAPD markersalso discriminate F. eskia from the remaining taxa inthe PCO plot (Fig. 6). Furthermore, the neighborjoining tree suggests a geographical substructuring ofthe molecular variation of the species, distributedamong the eastern Pyrenean, central Pyrenean andCantabrian ranges, with moderate support for thesister relationship of the westernmost Cantabrianpopulations (Fig. 7).

The two other parental species, F. gautieri andF. quadriflora, show a close resemblance in severalmorphological traits; only one trait (spikelet length;Table 3, Fig. 3) significantly discriminates betweenthem. However, their respective clusters separatesomehow along the second axis in the discriminantanalysis plot (Fig. 4). The two taxa have short ligules(LiL < 1 mm) and a similar anatomical leaf-sectionpattern (Fig. 2); however, F. gautieri differs fromF. quadriflora in its longer spikelet. The two studiedtype specimens of F. gautieri (subsp. gautieri andsubsp. scoparia) and the type specimen of F. quadri-flora are classified in the F. gautieri and F. quadrifloragroups in the discriminant analysis analysis, respec-tively (Fig. 4), corroborating the taxonomic adscrip-tion of the wild studied samples to these species.Additionally, this placement corroborates the lack ofmorphological differentiation between the two F.gautieri subspecies, suggesting, in agreement withFuente & Ortúñez (1988, 2001), that the two taxashould be regarded as synonyms and subsumed intothe same specific rank. The RAPD analysis supportedthe molecular differentiation of the F. gautieri andF. quadriflora samples, which showed a close butseparated clustering in the two-dimensional PCO plotand in the neighbor joining tree (Figs 6, 7). All theanalysed F. gautieri samples, representing its distri-bution across the eastern Iberian mountains (fromthe Pyrenees up to the southern Iberian Betic systemand the eastern Picos de Europa massif, Fig. 1), fell inthe same compact group, supporting the taxonomicrobustness of this species and the advocated syn-

onymy of different nomenclatural names attributed toits geographical races (Fuente & Ortúñez, 1988). Con-cordantly, the RAPD markers distinguished littlegeographical substructuring in the studied set ofpopulations of F. gautieri.

Despite the fact that the two hybrids were classifiedin intermediate positions between those of theirparents in the phenotypic multivariate PCA and dis-criminant analyses, they are similar to each otherand cluster in the same space in the bidimensionaldiscriminant analysis and PCA plots (Figs 4, A1) andclose to each other in the UPGMA dendrogram(Fig. 5). Furthermore, the studied type specimens ofFestuca × souliei and F. × picoeuropeana (includingF. × jierru) also fall in their respective groups (Figs 4,5). These results, however, point to the low resolutionof morphological traits alone to discriminate betweenthe two hybrids. The F. × souliei and F. × picoeurope-ana samples overlap for most of the analysed mor-phological traits. Their similar intermediate staturesand other intermediate morphological [e.g. ligulelength (1.5–2.5 mm)], anatomical (e.g. leaf-sectionpatterns) and inflorescence characters, betweenF. eskia/F. quadriflora and F. eskia/F. gautieri (Figs 2,3), led to taxonomical misattribution of F. × picoeuro-peana to F. × souliei (Catalán, 1990). The morphologi-cal resemblances of the two hybrids could beexplained by the shared inheritance of traits from acommon parent (F. eskia) and by the distinctly inher-ited but phenotypically similar traits acquired fromthe second parent (F. quadriflora and F. gautieri,respectively).

In contrast to their high phenotypic similarity,which makes them almost indistinguishable in someanalyses and in the field, the two hybrids have beenclearly differentiated by RAPD markers. The PCOanalysis discriminated the distinct molecular natureof F. × souliei and F. × picoeuropeana, which clusteredseparately in the intermediate spaces between theirparents (Fig. 6). The single link of the F. × soulieipopulation between F. quadriflora and F. eskia in theminimum spanning tree supported its hybrid-derivedorigin between these taxa; by contrast, the F.× picoeuropeana populations distinctly join to F. eskiaand F. gautieri, although most of them connectedthese two taxa, supporting their derived origin fromthem (Fig. 6). Those findings were further confirmedby the neighbor joining analysis (Fig. 7). In this tree,the F. × souliei lineage shows an intermediate resolu-tion between those of its F. quadriflora and F. eskiaparents, as expected from these biparentally inher-ited nuclear markers, which show an additive paren-tal profile pattern in the hybrids (Segarra-Moragues et al., 2007b). In contrast, the F. × picoeuro-peana populations showed different resolutions, withall the Pyrenean samples clustered in a monophyletic



group nested between the F. eskia and F. gautieripopulations, and with the western Picos de Europasamples nested in the F. eskia group and the easternPicos de Europa populations nested in the F. gautierigroup (Fig. 7). These results suggest a potential poly-phyletic origin of F. × picoeuropeana.

DIFFERENT HYBRIDIZATION EVENTS IN THE

PYRENEES AND THE CANTABRIAN MOUNTAINS

The extent and frequency of hybridization betweenclosely related taxa largely depends on the life historytraits and the evolutionary history of individuals andpopulations (Thompson, 2005) and on the possibilityof occurrence of suitable habitats for mating(Nieto-Feliner, 2011). South-western European moun-tains harbour a large diversity of ecological nichesthat probably provided sites for secondary contact(admixture and hybridization) of lineages followingsome degree of isolation (Nieto-Feliner, Gutiérrez-Larena & Fuertes-Aguilar, 2004; Fuertes, Gutiérrez-Larena & Nieto-Feliner, 2011; Nieto-Feliner, 2011).The existence of hybrid zones in the Pyrenees hasbeen widely documented (Ritchie, Butlin & Hewitt,1989; Hewitt, 1993, 1999; Segarra-Moragues et al.,2007a); more recently, hybrid or secondary-contactzones have also been detected in the Cantabrianmountains and in other southern Iberian ranges(Bella et al., 2007). The role played by these zones inthe Cantabrian and Pyrenean ranges probablyallowed for the origination of the new mountain spinyfescue hybrid taxa during recent Quaternary times.

Festuca × souliei and F. × picoeuropeana are prob-ably homoploid hybrids that resulted from crosses ofF. eskia with F. quadriflora and F. gautieri, respec-tively (Ortúñez & Fuente, 2004; Catalán, 2006;current study). Karyological data indicate that all thestudied samples of F. eskia and F. × picoeuropeana,and most of the F. gautieri and F. quadriflorasamples, are diploids (2n = 2x = 14) (Ortúñez &Fuente, 2004). Tetraploid individuals of F. quadrifloraand F. gautieri have only been recorded in the Alpsand in a few localities of the eastern Pyrenees, respec-tively, in areas where the hybrids do not exist or havenot been detected (Ortúñez & Fuente, 2004).Although no chromosomal studies have been carriedout on F. × souliei so far, this hybrid is probably alsoa diploid, resulting from the cross between its diploidPyrenean parents (cf. Ortúñez & Fuente, 2004).Current genome size analyses conducted on Pyreneanand Cantabrian populations of F. eskia, F. gautieriand F. × picoeuropeana (T. Garnatje, C. Acedo & P.Catalán, unpubl. data) have confirmed the diploidnature of these species. The reticulate history of thisgroup of close diploid taxa and their derived homop-loid hybrids is unusual in the fescues, as most of the

known hybrid taxa of Festuca, fine- or broad-leaved,are allopolyploids (Jauhar, 1975; Catalán, 2006). Thisgroup, however, constitutes a clear example of initialhomoploid hybrid speciation, in which the speciationprocesses might have been driven by ecological adap-tation (Gross & Rieseberg, 2005), as demonstrated bythe shift of hybrids to ecotonal zones distinct fromthose occupied by the parents. Further research,however, would be required in order to differentiatethis effect from other potential speciation events suchas chromosomal or fertility isolation (Gross &Rieseberg, 2005).

The existence of these two types of homoploidhybrids could be explained by the close relationshipsbetween their parents, which probably favouredhybridizations, and by the existence of suitablecontact zones in the Pyrenean and Cantabrian moun-tains during the Pleistocene. The possibility of veg-etative propagation, a common feature in thesefescues, might also help in the establishment andgrowth of hybrid populations. A close genetic affinitybetween F. eskia and F. gautieri and, consequently,their derived hybrid, could be deduced from the lackof any polymorphic variation in the assayed RFLPand CAPS analyses of nuclear ribosomal NOR and 5Sand plastid trnL-F genes. Although digestions werecarried out with a range of different restrictionenzymes, all detected homomorphic RFLP and CAPSpatterns across the studied samples. This contrastswith the extensive length variation in rDNA RFLPsfound among or within annual species of Triticum L.,Hordeum L. or Oryza L., but agrees with the largehomomorphy detected in recently evolved perennialspecies of Brachypodium P.Beauv. (cf. Shi, Draper &Stace 1993). The recent divergences of the threeparental F. eskia, F. gautieri and F. quadrifloraspecies were also supported by Bayesian relaxed-clockdating analysis of nuclear and plastid DNA-basedphylogenetic analyses of Loliinae (M. Minaya & P.Catalán, unpubl. data). These analyses inferred arecent early Pleistocene split of the sister F. eskia andF. gautieri lineages (c. 1.87 Mya) and a Pliocene diver-gence of the F. quadriflora clade from an earlierancestor (c. 4.0 Mya), suggesting a probable timeframe for the two types of hybridizations from theearly–mid Pleistocene onwards.

The scarce occurrence of Festuca × souliei is prob-ably a consequence of the restricted distribution ofsympatric areas for the parents. Although F. quadri-flora is broadly distributed in the Alps and the Jura,it only coexists with F. eskia in a narrow area of thecentral Pyrenees (Fig. 1). This would explain the scar-city of localities (six) where the hybrid populationshave been detected, in schist-limestone ecotones ofthe central Pyrenean alpine belt where F. × soulieialways grows in close sympatry with its two parents



(Saint-Yves, 1924; Catalán et al., 1999). The estab-lishment of the hybrid populations must necessarilyhave occurred during the postglacial phases, once theice sheet had retreated from this high altitudinal belt(González-Sampériz et al., 2006; Pallàs et al., 2006;García-Ruiz et al., 2010), but the hybrid could havebeen formed earlier. Nonetheless, the extreme rarityand small size of the F. × souliei populations(Saint-Yves, 1924; Catalán et al., 1999), and the factthat they always overlap with those of F. eskia andF. quadriflora, suggest a recent Late Pleistocene–Holocene occurrence of this hybridization process. IfF. × souliei is a rare but extant hybrid, the apparentnon-existence of the other potential hybrid in thistriangular complex, derived from the cross betweenF. quadriflora and F. gautieri, could have differentexplanations. It could be assumed that the hybridmight exist but could have passed undetected becauseof the close morphological resemblance of the twopotential parents (e.g. Figs 2–5). This would likelyhave made it undistinguishable from one or other ofthe parental species. However, an alternative ecologi-cal niche-based hypothesis for the non-existence ofsuch a taxon could be invoked to explain best theevidence found to date. Despite the shared prefer-ences of alpine F. quadriflora and montane–subalpineF. gautieri for calcareous substrates, their currentaltitudinal ranges are different and do not overlap(Catalán, 2006). This would probably have preventedthe cross between these two taxa during recentPleistocene–Holocene times, impeding the potentialorigin of a new taxon.

The most intriguing hybrid case is, however, thatof Festuca × picoeuropeana. Our morphological andmolecular data have shed light on the potentialcauses of its disjunct distribution in the Pyreneanand the Cantabrian mountains and on the presenceof the large number of ‘orphan’ populations in thelatter range (Catalán, 2006). Both analyses supportthe admixed nature of all the Pyrenean F. ×picoeuropeana samples, which cluster in an interme-diate position between those of F. eskia and F. gaut-ieri in the morphological discriminant analysis andPCA plots, the UPGMA dendrogram (Figs 4, 5, A1)and the RAPD PCO plots (Fig. 6). This intermediacyis similar to that observed in F. × souliei and wouldalso support a potential homoploid hybrid speciationorigin driven by ecological selection (Gross &Rieseberg, 2005), given that these F. × picoeuropeanapopulations grow in ecotonal F. eskia–F. gautierizones in the subalpine Pyrenean belt. By contrast,different alternative scenarios of disparate origins,taxonomic misidentifications or multiple hybridiza-tions or introgressions should be invoked to explainthe different resolutions detected among the subal-pine Cantabrian F. × picoeuropeana samples.

The central Picos de Europa massif populationsthat cluster together with the Pyrenean populationsin the morphometric and RAPD plots (Fig. 6)might have had a similar derived origin from aF. eskia × F. gautieri cross. However, the eastern Picosde Europa massif samples that fall in the F. gautiericluster (Fig. 6) could represent introgressed formswith this parent. Our phenetic analyses have shownthat the F. × jierru type clusters in the F. × picoeuro-peana group in the discriminant analysis scatterplot(Fig. 4), thus rejecting its taxonomic distinction.Nonetheless, the close relationships observed betweenthe eastern F. × picoeuropeana samples and theF. gautieri samples from the same massif in themolecular PCO plot and neighbor joining tree andtheir divergence with respect to other F. gautierisamples (Figs 6, 7) suggest a common but separateorigin from the rest. This might have been caused bygeographical isolation of the eastern Picos de Europamassif F. gautieri populations from the remainingeastern Iberian populations, followed by local hybridi-zation with F. eskia and then by introgression of thehybrids with these Cantabrian F. gautieri stocks.Conversely, the western Picos de Europa massifF. × picoeuropeana samples that show a close link tothe Cantabrian F. eskia samples (Figs 6, 7) could rep-resent the reverse introgression of a local hybrid withits F. eskia parent.

Alternative hypotheses/scenarios involving biologi-cal traits, ecological competition and life historytraits could be added to explain the strikingly widedistribution of the orphan F. × picoeuropeana popula-tions across the three Cantabrian Picos de Europamassifs, whereas the two parents do not grow inclose vicinity in any of them (Fig. 1). Except for a fewscattered localities where F. × picoeuropeana coexistswith one or other of its parents, its populations growalone. The long-term viability of these widely spreadpopulations was first questioned, as these plantswere considered to be highly sterile interspecifichybrids because of their low rates of pollen viability(e.g. 18%; cf. Gutiérrez-Villarías & Homet, 1985a).However, they were later regarded as belonging to astabilized hybridogenous species (Gutiérrez-Villarías,Nava & Homet, 1992). Current analyses indicatethat the pollen viability rate of the CantabrianF. × picoeuropeana individuals, although low, couldrange up to 30% (M. C. Sancho, C. Acedo, F. Llamas,I. Marques & P. Catalán, unpubl. data), suggestingthat these individuals could occasionally reproducesexually. In addition, these plants, and F. gautieriand F. eskia, show a pseudorrhizomatose clonalpropagation system (P. Catalán, pers. observ.), whichallows them to spread. These biological attributes,together with the probable past history of the Can-tabrian F. × picoeuropeana populations, could explain



the current predominant distribution of this taxon inthe Picos de Europa massifs. In contrast to the Pyr-enean populations, which grow in ecotonal schist-calcareous substrates in soils with neutral pH values(B. Komac, E. Sahuquillo, J. Viruel & P. Catalán,unpubl. data), the Cantabrian F. × picoeuropeanaindividuals are adapted to rocky limestone substratesin soils with highly basophilous pH values. Thishabitat is similar to that occupied by F. gautieri inthe Pyrenees. We propose a hypothesis of potentialmultiple origins of the Cantabrian F. × picoeuropeanapopulations followed by the displacement of F. gaut-ieri. Ecological niche models suggest that F. gautiericould have been more widely distributed in the past,during the Last Glacial Maximum, in the Picos deEuropa (D. Draper, unpubl. data). This species couldhave hybridized with F. eskia, giving rise to thehybrid descendants. The more robust habit of thehybrids could have resulted in more successfullyadapted individuals than those of the basophilousparent. A feasible and rapid clonal propagation,coupled with occasional sexual crossing, would havefavoured the spread of the F. × picoeuropeana‘orphan’ populations in the three massifs and theretreat of F. gautieri to its current narrow range inthe eastern massif. Displacement of less competitiveparents by highly vigorous sterile, but largely clonalhybrids has been documented in other Iberianorphan populations (Marques et al., 2010). Furthergenetic research is currently underway aiming todissect the hybrid nature of the distinct F. × picoeuro-peana populations.

TAXONOMY OF THE PYRENEAN AND CANTABRIAN

SPINY FESCUES (FESTUCA ESKIA COMPLEX)

1. Ligule of the innovation leaf > 4 mm long, acute;transversal section of leaf elliptical in outline,c. 1 mm in diameter, with nine to 11 adaxial ribsreinforced with sclerenchyma, and more than tendeep valleys. Abaxial sclerenchyma of the leafin a homogeneously thick and continuous ring..............................................................F. eskia

1. Ligule of the innovation leaf < 3 mm long, trun-cate; transversal section of leaf polygonal or sub-elliptical in outline, up to 0.7 mm in diameter, withup to seven adaxial ribs and six valleys. Abaxialsclerenchyma different ................................... 2

2. Transversal section of the innovation leaf polygo-nal in outline, 0.3–0.6 mm in diameter, withshallow adaxial valleys (up to one quarter of theleaf thickness). Adaxial schlerenchyma absent;ligule, in general, < 1 mm long ....................... 3

2. Transversal section of the innovation leaf subellip-tical in shape, 0.6–0.9 mm in diameter, with deep

adaxial valleys (up to one third of the leaf thick-ness). Adaxial schlerenchyma present; ligule2–3 mm long ................................................. 4

3. Leaf thin, thread-like. Spikelet 6.0–8.1 mm long,transversal section of the innovation leaf with verysmall blocks of abaxial sclerenchyma, one adaxialrib and two valleys ...................... F. quadriflora

3. Leaf thick, non-thread-like. Spikelet 7.5–10.0 mmlong. Abaxial sclerenchyma forming a continuousbut irregular ring, or distributed in several decur-rent blocks; three adaxial ribs and four shallowvalleys ............................................. F. gautieri

4. Innovation leaf non-stiff with slightly spiny apex.Lower glume 3.4–4.0 × 1.2–3.0 mm .... F. × souliei

4. Innovation leaf stiff with sharp spiny apex. Lowerglume 3.2–5.2 × 0.6–1.5 mm ... F. × picoeuropeana

Festuca eskia Ramond ex DC. in Lam. et DC., Fl.Franç. 3: 52, 1805.

= F. eskia var. orientalis Nègre, Candollea 30: 318,1975.

Culm 14–65 cm × 0.5–1.2 mm, one to four nodes.Innovation leaf 4.1–24.7 cm, ligule 2.7–6.6 mm long.Inflorescence 3–11 × 0.9–2.0 cm wide; four to sevennodes, lower internode of rachis 0.7–3.2 cm long; oneto five branches in the lowest node, lower branch2.2–5.5 cm. Spikelets 10–56, 2–12 in the lowestbranch. Spikelet 5.5–11.0 mm long. Pedicel 1.0–5.7 mm long, two to seven fertile flowers per spikelet.Lower glume 3.2–5.6 × 0.6–1.2 mm; upper glume 3.5–6.6 × 1.0–2.2 mm; lemma of the second basal floret3.7–7.7 × 1.3–2.4; awn up to 1.2 mm; palea 3.1–7.3 × 0.4–1.5 mm. Anther 1.5–3.6 mm. Anatomy of theinnovation leaf blade: elliptical in section, diameter0.9–1.2 mm; abaxial sclerenchyma in a thick, continu-ous and homogeneous ring, five to 13 ribs, eight to 14leaf valleys and nine to 18 nerves.

Festuca quadriflora Honck., Verz. Gew. Teutschl.:271, 1782.

≡ F. varia Haenke subsp. pumila (Chaix) Hack.,Bot. Centralbl. 8: 408, 1881.

= F. pumila Chaix, Pl. Vapinc.: 12, 1785.; Chaix inVill., Prosp. Hist. Pl. Dauphiné: 316, 1786.

Culm 5.1–18.5 cm × 0.4–0.9 mm, one or two nodes.Innovation leaf 1.9–6.9 cm, ligule 0.4–1.6 mm long.Inflorescence 2.3–3.9 × 0.8–1.3 cm; four to six nodes,lower internode of rachis 0.4–1.3 cm long; one or twobranches in the lowest node, lower branch 0.9–2.0 cm.Spikelets seven to 17, two to three in the lowestbranch. Spikelet 5.25–8.70 mm (excluding awns) fromthe base to the apex of the fourth lemma. Pedicel2–10 mm, three to five fertile flowers per spikelet.Lower glume 2.5–3.8 × 0.7–1.1 mm; upper glume 3.4–4.8 × 1.4–2.7 mm; lemma of the second basal floret4–5.7.0 × 1.5–2.0 mm; awn 0.50–1.25 mm; palea 3.2–



4.9 × 0.7–1.0 mm. Anther 1.7–3.1 mm. Anatomy of theinnovation leaf blade: polygonal in outline, diameter0.4–0.6 mm; abaxial sclerenchyma in small separateblocks, one to three ribs, two to four leaf valleys andfive to seven nerves.

Festuca gautieri (Hack.) K.Richt., Pl. Eur. 1: 105,1890.

≡ F. varia subsp. scoparia var. gautieri Hack.,Monogr. Festuc. Eur.: 181, 1882.

= F. varia subsp. scoparia A.Kern. & Hack, inHack., Monogr. Festuc. Eur.: 181, 1882.

Culm 9.50–45.07 cm × 0.36–0.85 mm, with one ortwo nodes. Innovation leaf 1.1–10.2 cm, ligule trun-cate, 0.3–1.8 mm long. Inflorescence 2.8–7.0 × 0.5–2.6 cm; three to seven nodes; lower internode of rachis0.9–2.7 cm long; one to three branches in the lowestnode; lower branch 0.8–4.1 cm. Spikelets six to 19,two to seven in the lowest branch; spikelet 5–11 mmlong; pedicel 0.9–5.7 mm long; three fertile flowersper spikelet. Lower glume 2.1–5.5 × 0.6–1.4 mm;upper glume 3.1–6 × 0.7–1.9 mm; lemma of thesecond basal floret 4.5–7.3 × 1.2–3.0 mm; awn up to1.3 mm, palea 3.1–6.6 × 0.4–1.4 mm. Anther 4–5 mm.Anatomy of the innovation leaf blade: polygonal intransverse section, diameter 0.3–0.6 mm; abaxialsclerenchyma in a ring or in separate but decurrentblocks, one to three ribs, two to four leaf valleys andfive to seven nerves.

Festuca × souliei St.-Yves, Bull. Soc. Bot. France 71:126, 1924.

= Festuca eskia Ramond ex DC. × F. quadrifloraHonck.

Culm 17–17.2 cm × 0.8–0.9 mm, 1 node. Innovationleaf 2.1–4.1 cm, ligule 1.5–2.3 mm long. Inflorescence4–4.5 × 1.0–1.1 cm wide; c. five nodes, lower internodeof rachis 0.8–0.9 cm; two branches in the lowest node,lower branch 1.5–2.1 cm. Spikelets ten or 11, four inthe lowest branch. Spikelet 7.8–9.5 mm from the baseto the apex of the fourth lemma (excluding awns).Pedicel 2.6 mm; four or five fertile flowers per spike-let. Lower glume 3.4–3.7 × 1–2.3 mm; upper glume4.4–4.6 × 1.4 mm; lemma of the second basal floret5.2–5.5 × 1.4 mm; awn 0.5–0.9 mm; palea 4.5–5.5 × 1.4 mm. Anther 2.3–3.0 mm. Anatomy of theinnovation leaf blade: diameter 0.6–0.7 mm abaxialsclerenchyma in a continuous ring, five to seven ribs,six valleys and seven nerves.

Festuca × picoeuropeana Nava, Fontqueria 7: 23,Jan 1985, pro sp.

= F. eskia Ramond ex DC. × F. gautieri (Hack.) K.Richt.

= F. × picoeuropeana Gutiérrez Villarías et Homet,Bol. Ci. Nat. I.D.E.A. 34: 146, Mar 1985, nom. illeg.

= F. × jierru Nava, Fontqueria 7: 24, 1985 p.p.Culm 10–40 cm × 0.5–1 mm, one to three nodes.

Innovation leaf 3.3–10.5 cm, ligule 0.55–2.70 mmlong, truncate. Inflorescence 3.4–7.9 cm × 0.7–1.8 cmwide; three to seven nodes, one or two branches in thelowest node, lower internode of rachis 1.0–1.3 cmlong; lower branch 2.1–7.0 cm. Spikelets seven to 22,one to six in the lowest branch. Spikelet 7.2–11.0 mm.Pedicel 1.7–5.7 mm long. Three to six fertile flowersper spikelet. Lower glume 3.2–5.2 × 0.6–1.5 mm;upper glume 4–6 × 1.1–2.2 mm; lemma of the secondbasal floret 5.4–7.6 × 1.4–2.3 mm; awn up to 1.5 mmlong; palea 4.1–6.6 × 0.6–1.6 mm. Anther 1.7–3.6 mm.Anatomy of the innovation leaf blade: diameter 0.4–0.9 mm; abaxial sclerenchyma in a continuous irregu-lar ring, three to seven ribs, two to eight valleys andseven to 11 nerves.

ACKNOWLEDGEMENTS

We thank Victor Sorribas, Shi Ying and Clive Stacefor their advice and help with the RFLP and CAPSanalyses, the authorities of the Spanish NationalParks of Ordesa-Monte Perdido, Aigüestortes i Estanyde Sant Maurici and Picos de Europa for the permitsto sample the Festuca materials in these protectedareas, the curators of the G, FCO, JACA, LEB, MA,W, Z and Jochen Müller and Oscar Sánchez-Pedraja(private) herbaria for facilitating the loan of voucherspecimens, Jochen Müller for fruitful nomenclaturaldiscussion and Emily Lemonds for linguistic assis-tance. This work was supported by a Spanish Minis-try of the Environment – National Parks Organismresearch project (059/2009) and by an Instituto deEstudios Altoaragoneses and Aragón Government-European FEDER Bioflora grant. P.T. and A.J.D.-P.were supported by Venezuelan CDCH postdoctoraland PhD grants, respectively. J.G.S.-M. was sup-ported by two consecutive Aragon Government ‘Araid’and Spanish Ministry of Science and Innovation‘Ramón y Cajal’ postdoctoral contracts. P.C. was sup-ported by an Aragón CAI-Programa Europa XXI fel-lowship.

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APPENDIXTable A1. Eigenvalues and variance accumulated by the first components of the principal component analysis (PCA)analysis

Eigenvalue % Total variance Cumulative eigenvalue Cumulative %

1 3.55 43.94 3.51 43.942 1.84 23.02 5.35 66.963 0.85 10.68 6.20 77.604 0.59 7.45 6.80 85.005 0.47 5.96 7.28 91.026 0.34 4.26 7.62 95.287 0.21 2.71 7.84 98.008 0.15 1.99 8.00 100.00



Figure A1. See caption on next page.



Figure A1. Box plots of simple statistics values (mean, percentiles, range) of 22 discriminant morphoanatomicalcharacters analysed (Table 2) in individuals from the 170 studied populations plus nine types of the five Festuca eskiacomplex taxa (Table 1). Variables that do not significantly discriminate among the studied taxa: FE, F. eskia; FG,F. gautieri; FQ, F. quadriflora; FP, F. × picoeuropeana; FS, F. × souliei. Abbreviations of variables: CH, culm height (cm);CW, culm width (mm); InLL, innovation leaf length (cm); IL, inflorescence length (cm); IW, inflorescence width (cm); LBL,lower branch length (cm); PeL, pedicel length (mm); LGL, lower glume length (mm); UGL, upper glume length (mm);UGW, upper glume width (mm); LmL, lemma length from the second basal floret (mm); LmW, lemma width (mm); AL,awn length (mm); PaL, palea length (mm); PaW, palea width (mm); AnL, anther length (mm); NNC, number of nodes ofculm; NNR, number of nodes of rachis; NLB, number of lowest branches; NSI, number of spikelets per inflorescence;NSLB, number of spikelets of lowest branch; NFS, number of flowers per spikelet.

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Figure A2. Bidimensional scatterplot of principal component analysis (PCA) of Festuca eskia complex samples fortaxonomic phenetic differentiation of the five studied taxa. The first and second canonical discriminant functionsexplained 66.96% of the interspecific taxonomic variation. FE, F. eskia; FG, F. gautieri; FP, F. × picoeuropeana; FQ,F. quadriflora; FS, F. ×souliei.



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