Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephemeral...

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Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx

YMPEV 4939 No. of Pages 14, Model 5G

21 June 2014

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Polyphyly, gene-duplication and extensive allopolyploidy framed theevolution of the ephemeral Vulpia grasses and other fine-leaved Loliinae(Poaceae)

http://dx.doi.org/10.1016/j.ympev.2014.06.0091055-7903/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Escuela Politécnica Superior Huesca, Universidad deZaragoza, Ctra. Cuarte km 1, 22071 Huesca, Spain. Fax: +34 974 239 302.

E-mail address: [email protected] (P. Catalán).1 Present address: Instituto de Genética y Centro de Investigaciones en Biot-

ecnología Agrícola (CIBA), Facultad de Agronomía, Universidad Central de Venezuela,2101, Maracay, Aragua, Venezuela.

2 Present address: Department of Biology, University of Shahrekord, Shahrekord,Iran.

Please cite this article in press as: Díaz-Pérez, A.J., et al. Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephVulpia grasses and other fine-leaved Loliinae (Poaceae). Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.06.009

A.J. Díaz-Pérez a,1, M. Sharifi-Tehrani a,2, L.A. Inda a, P. Catalán a,b,⇑a Departamento de Ciencias Agrarias y del Medio Natural (Botánica), Escuela Politécnica Superior-Huesca, Universidad de Zaragoza, Ctra. Cuarte km 1, 22071 Huesca, Spainb Department of Botany, Institute of Biology, Tomsk State University, Lenin Av. 36, Tomsk 634050, Russia

a r t i c l e i n f o a b s t r a c t

30313233343536373839404142

Article history:Received 11 December 2013Revised 26 May 2014Accepted 9 June 2014Available online xxxx

Keywords:Fine-leaved LoliinaeInterspecific hybridizationMediterranean and American silver grassesNuclear (ITS, GBSSI) genesPolyploid speciationSpecies tree reconstruction

4344454647

The fine-leaved Loliinae is one of the temperate grass lineages that is richest in number of evolutionaryswitches from perennial to annual life-cycle, and also shows one of the most complex reticulate patternsinvolving distinct diploid and allopolyploid lineages. Eight distinct annual lineages, that have tradition-ally been placed in the genus Vulpia and in other fine-leaved ephemeral genera, have apparently emergedfrom different perennial Festuca ancestors. The phenotypically similar Vulpia taxa have been recon-structed as polyphyletic, with polyploid lineages showing unclear relationships to their purported diploidrelatives. Interspecific and intergeneric hybridization is, however, rampant across different lineages. Anevolutionary analysis based on cloned nuclear low-copy GBSSI (Granule-Bound Starch Synthase I) andmulticopy ITS (Internal Transcribed Spacer) sequences has been conducted on representatives of mostVulpia species and other fine-leaved lineages, using Bayesian consensus and agreement trees, networkingsplit graphs and species tree-based approaches, to disentangle their phylogenetic relationships and toidentify the parental genome donors of the allopolyploids. Both data sets were able to reconstruct a con-gruent phylogeny in which Vulpia was resolved as polyphyletic from at least three main ancestral diploidlineages. These, in turn, participated in the origin of the derived allopolyploid Vulpia lineages togetherwith other Festuca-like, Psilurus-like and some unknown genome donors. Long-distance dispersal eventswere inferred to explain the polytopic origin of the Mediterranean and American Vulpia lineages.

� 2014 Elsevier Inc. All rights reserved.

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1. Introduction

Reticulate evolution is a major phenomenon that has affectedthe speciation processes of many angiosperm lineages, and partic-ularly the grasses (Stebbins, 1956; Leitch and Bennet, 1997). ThePoaceae family constitutes a paradigmatic case of multiple radia-tions across its main lineages, driven in most instances by theconcurrence of interspecific hybridization and allopolyploidizationevents (Kellogg, 2001; Gaut, 2002). Recent studies, based oncomparative genomics of whole genomes, have evidenced theplausible existence of a paleo-duplication of an ancestral grass

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genome, followed by subsequent reductions and genomic rear-rangements that led to the current organization of the main BEPand PACMAD genome types (Feuillet and Keller, 2002; Salseet al., 2008). These investigations also supported the existence ofmore recent genome duplication events, involving the origins ofrecent allopolyploids (Salse et al., 2008). Allopolyploidy, asopposed to autopolyploidy, has been the largest source of grassspecies diversity, ranging from fully allopolyploid genera (e.g. themegadiverse Calamagrostis) to partially allopolyploid genera,broadly distributed in both the temperate and tropical Poaceaeclades (GPWG, 2001; GPWG II, 2012). Reticulation has been partic-ularly extensive in some tribes, such as Triticeae and Poeae, wherea large number of genera or species are of hybrid allopolyploid ori-gin (Dewey, 1984; Quintanar et al., 2007). This led some authors toconsider some of these reticulate groups as close homeologousgenome cases (e.g. Triticeae), where extensive hybridization wascaused by the absence of interspecific barriers, thereby favoringthe occurrence of multiple crosses and genome duplicationsthrough time, ending in a plethora of new allopolyploid lineages

emeral

http://dx.doi.org/10.1016/j.ympev.2014.06.009

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Fig. 1. Phylogeny of the fine-leaved Loliinae (FEVRE) groups studied (see Table 1).Summarized combined ITS/trnTF consensus tree (cf. Inda et al., 2008) withgeographical distributions of the main lineages. FEVRE lineages: Aulaxyper + Vulpia2x (violet), American II (pink), Festuca (dark blue), Wangenheimia (light blue),Narduroides (light blue), Loretia (dark green), Exaratae (light green), Micropyrum(yellow), Psilurus/Vulpia 4x–6x (orange), American Vulpia (red), American I(brown), Eskia (gray). The annual FEVRE lineages (underlined) have apparentlyevolved from more ancestral FEVRE perennial lineages. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

2 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx


21 June 2014

or taxa (Dvorak, 2009). Despite the extensive documentation accu-mulated for some of the economically important grass allopoly-ploid groups (e.g. Triticum, Hordeum), there is still a lack ofknowledge about the frequency, origin and time-scale of the allo-polyploidization events in other temperate grasses, such as thelarge pasture and fodder grass subtribe Loliinae, and how theyhave influenced the speciation rates of these lineages.

The Loliinae is one of the main groups of temperate Pooideaewhich includes some economically and ecologically important for-age and lawn grasses, such as red, sheep, meadow and tall fescuesand the ryegrasses (Catalán, 2006). The subtribe comprises thelarge paraphyletic and worldwide-distributed genus Festuca andseveral genera nested within it (Catalán et al., 2004). Successivephylogenetic studies based on plastid and nuclear ITS genes haveconfirmed the divergence of two major clades, broad-leaved andfine-leaved Loliinae, with some poorly resolved intermediate lin-eages placed between them (Catalán et al., 2004; Inda et al.,2008; and references therein). Dated analyses estimated a mid-Miocene origin (13 Ma) for the crown age of the Loliinae, andlate-Miocene ages for the respective splits of an ancestral broad-leaved (11.9 Ma) and more recently evolved fine-leaved (10.6–8.8 Ma) lineages, with recent divergences of more internal groupsspanning from the Pliocene to the early Pleistocene (Catalán,2006; Inda et al., 2008). Substitution rates of both nuclear (ITS)and plastid (trnLF) sequences are significantly higher in the fine-leaved group than in the broad-leaved one (Torrecilla et al.,2004; Catalán et al., 2007). Catalán et al. (2007) further showedthat Vulpia and other fine-leaved FEVRE (FEstuca + Vul-pia + RElated) ephemeral lineages had the highest evolutionaryITS rates of all the Loliinae. Those accelerated rates were correlatedto the Minimum-Generation-Time (MGT) of these annuals and tothe high speciation rate of the group, based on its high numberof taxonomically distinct annual genera (e.g., Ctenopsis, Mycropy-rum, Narduroides, Psilurus, Vulpia, Wangenheimia).

Combined phylogenetic studies of fine-leaved Loliinae con-curred in successive divergences of moderately to highly supportedlineages (Eskia, American I, American Vulpia, Psilurus + Vulpia 4x–6x, Exaratae, Loretia, Festuca + Wangenheimia, American II, andAulaxyper + Vulpia 2x; Fig. 1), which were recovered by bothnuclear and plastid trees in most cases (Catalán, 2006; Inda et al.,2008). Concordant with previous studies, the most striking findingwas the polyphyletic origin of Vulpia (Torrecilla et al., 2004;Catalán et al., 2004; Inda et al., 2008). In contrast to the monophy-letic origin of Lolium and all the other minor Loliinae genera, Vulpiataxa were reconstructed into four non-related fine-leaved lineages(Inda et al., 2008). Furthermore, the Loretia ‘assemblage’ includedrepresentatives of four (out of five) morphologically dissimilar Vul-pia sections (Loretia, Apalochoa, Monachne, Spirachne) (Stace, 1981,2005) plus Ctenopsis and F. plicata, whereas representatives of thefifth Vulpia section (Vulpia), which are morphologically similar toeach other, fell into three separate clades:Vulpia 2x, Psilurus + Vul-pia 4x–6x, American Vulpia (Inda et al., 2008).

The investigation of the polyphyly and polyploidy of Vulpia is anextraordinary case study. The disparate reconstructions of someMediterranean diploid (Vulpia 2x) and polyploid (Vulpia 4x–6x)Vulpia sect. Vulpia species (Catalán et al., 2004; Torrecilla et al.,2004) led Stace (2005) to suggest the potential existence ofunknown underlying reticulation processes among these ‘taxo-nomically undistinguisable’ taxa and other putative alien Loliinaespecies. The later discovery of a third independent lineage in theNew World (American Vulpia), with species morphologicallyresembling the Mediterranean ones, but apparently related to theAmerican Festuca clade (American I) (Inda et al., 2008), further sug-gested the existence of introgression or the potential evolutionaryconvergence of morphological traits in these annual lineages,originating from different polytopic ancestors. It has been

Please cite this article in press as: Díaz-Pérez, A.J., et al. Polyphyly, gene-duplicVulpia grasses and other fine-leaved Loliinae (Poaceae). Mol. Phylogenet. Evol.

acknowledged that Vulpia and all the ephemeral Loliinae generamight have derived from ancestral perennial Festuca lineages(Catalán, 2006, and references therein); however, the potentialexistence of multiple independent origins for the taxonomicallysimilar Vulpia sect. Vulpia taxa remains questionable (Stace,2005; Catalán et al., 2007).

Comparative cytogenetic analysis indicated that Vulpia taxahave a smaller nuclear genome size than that of the Festuca sect.Aulaxyper taxa (Smarda et al., 2008), and they lack telomeric het-erochromatin (Bailey and Stace, 1992; Catalán, 2006). Despite thelarge number of taxonomic and cytogenetic differences betweenVulpia and Festuca species, several spontaneous intergenet-ic � Festulpia hybrids have been described (Stace and Cotton,1974; Stace and Ainscough, 1984; Ainscough et al., 1986). All ofthose spontaneous hybrids have hexaploid Festuca sect. Aulaxyperprogenitors [F. rubra (6x), F. nigrescens (6x)] and different diploidand polyploid Vulpia progenitors [Vulpia sect. Vulpia: V. bromoides(2x), V. myuros (6x); sect. Monachne: V. fasciculata (4x)]. Addition-ally, the mostly sterile pentaploid hybrid � Festulpia hubbardii(V. fasciculata � F. rubra) and a wild fertile backcross between itand its male progenitor F. rubra, showed evidence of homeologousor heterogenetic pairing between Vulpia and Festuca chromosomes,confirming continuous introgression of V. fasciculata’s genome into

ation and extensive allopolyploidy framed the evolution of the ephemeral(2014), http://dx.doi.org/10.1016/j.ympev.2014.06.009


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A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx 3


21 June 2014

F. rubra’s through this stable backcross (Stace and Ainscough,1984; Bailey and Stace, 1992; Bailey et al., 1993). These results sug-gest a long history of genomic admixture between Vulpia and Fest-uca sect. Aulaxyper, even for those Vulpia lineages that are,apparently, distintly related to Aulaxyper (e.g., Loretia (V. fascicula-ta), Psilurus/Vulpia 4x–6x (V. myuros); Inda et al., 2008).

Our study aims to disentangle the complex reticulate evolution-ary history of Vulpia and other ephemeral fine-leaved Loliinae lin-eages using complementary information from the nuclear ITS(internal transcribed spacer) and GBSSI (granule-bound starch syn-thase I) genes. We wanted to investigate: (i) if diploid Vulpia lin-eages had a single (monophyletic) or multiple (polyphyletic)origin; (ii) if polyploid Vulpia lineages were allopolyploids derivedfrom crosses of diploid Vulpia lineages and alien genomes; (iii) ifnon-related ancestral Festuca lineages were involved in the originsof Vulpia lineages; (iv) if intergeneric hybridizations had repeatedlyoccurred at different evolutionary times; (v) if introgression waspolytopic or had a restricted geographic origin. These hypotheseswere tested through independent and complementary Bayesiananalyses of nuclear ITS and GBSSI genes, using networking and spe-cies-tree based approaches.

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2. Materials and methods

2.1. Sampling

Sampling of fine-leaved Loliinae included 32 species, coveringalmost all the worldwide-distributed species of Vulpia across itsfive sections [15 spp; sects. Apalochloa (1), Loretia (3), Monachne(3), Spirachne (1), Vulpia (7)], of the mostly monotypic Mediterra-nean ephemeral genera [Micropyrum (1), Narduroides (1), Psilurus(1), Wangenheimia (1)], and representatives of the main Festuca[sects. and subsects. Aulaxyper (3), Eskia (1), Exaratae (2), Festuca(2), and American I clade (1)] and Hellerochloa [American II clade](1) perennial lineages (Table 1; Fig. 1). For the GBSSI analysis, awider sampling of other Loliinae groups and of several outgroupswas included (Table 1; see also Expanded Material and Methods,Supplementary information). The ITS analysis was restricted tothe fine-leaved group, using the broad-leaved taxon Festuca praten-sis as outgroup.

2.2. DNA isolation, cloning and sequencing

DNA was extracted from silica gel-dried leaves collected fromone individual wild plant per species (Table 1). Genomic DNAwas isolated using the QIAGEN DNeasy kit method for the GBSSIanalysis and the CTAB method (Doyle and Doyle, 1987) for theITS analysis. Protocols for the amplification, cloning and sequenc-ing of the GBSSI and ITS regions are provided in Supplementaryinformation.

2.3. Molecular characterization and preprocessing of DNA sequences

We performed separate molecular characterization of both theexons and the introns of the GBSSI gene and of the 5.8S gene andthe ITS1 + ITS2 spacers of the ITS region. The range of sequencelengths, number of gaps and the expected number of base differ-ences per site (p-distance) were computed for each case usingMEGA v. 5 (Tamura et al., 2011) and Bioedit v. 7.0.9.0 (Hall, 1999).

In order to discard spurious variation originating from PCR(Polymerase Chain Reaction) artifacts, we followed a modified ver-sion of Sánchez-Ken and Clark (2010) to generate a consensussequence (or type) from closely related sequences for a single indi-vidual. All intraspecific sequences with a p-distance lower than0.01 base differences per site were collapsed into a single type


using the program Bioedit version 7.0.9.0. Each type was codedprogressively as shown in Table 1, except for some non-FEVRE spe-cies where some available types were included in the study.

2.4. GBSSI recombinants, GBSSI paralogs and ITS pseudogenes

Detection of recombination in the Loliinae GBSSI data set wasperformed using Rdp, Geneconv, Bootscan, Maxchi, Chimaera, Sis-can and 3Seq methods implemented in the RDP3 program (Martinet al., 2010) using the default settings in all cases. Only potentialrecombinant events detected by at least two methods were consid-ered significant. The Bonferroni multiple comparison correctiontest was performed to diminish the expected number of false posi-tive results. Masking of similar sequences was allowed in order toincrease the power of the recombination detection methods.

GBSSI sequences of some diploid FEVRE samples showed evi-dence of two divergent types that were assumed to be the productof a duplication event. Following a ‘‘species-overlap rule’’(Gabaldón, 2008), we inferred that a node was associated with aspeciation event if its branches had mutually exclusive sets of dip-loid species; in contrast, a node with overlapping sets of diploidspecies was associated with a duplication event. Polyploid sampleswere excluded from this analysis because homoeologous copiesfrom widely divergent parents might be placed on differentbranches, hence increasing the number of spurious duplicationevents. Paralogous groups were further characterized through theNei-Gojobori Z test method (Nei and Gojobori, 1986; Nei andKumar, 2000) to test for any sign of relaxation of selective con-strains. In addition, we tested the heterogeneity of substitutionrates among and within orthologous and paralogous FEVREsequences using Tajima’s relative rate test (Tajima, 1993).

We looked for putative non-functional sequences of the ribo-somal 5.8S gene using three complementary approaches (see Sup-plementary information). First, we checked for the absence of anyof the three conserved plant motifs M1 (CGATGAAGAACGyAGC),M2 (GAATTGCAGAAwyC) and M3 (TTTGAAyGCA) (Fig. S1, Supple-mentary information), that are present in functional copies(Harpke and Peterson, 2008). Second, we analyzed the inabilityof the ITS sequences to generate the conserved secondary structureof 5.8S as another indicator of non-functional sequences (Harpkeand Peterson, 2008). Third, the Bootstrap Hypothesis Testing(BHT) method (Bailey et al., 2003) was performed to detect pseu-dogenes assuming similar non-constrained selective substitutionrates between the 5.8S gene region and the ITS1 + ITS2 spacerregion.

2.5. Phylogenetic reconstruction of the fine-leaved Loliinae

A GBSSI Loliinae tree (LoliinaeGBSSI-tree) was computed, includ-ing both FEVRE samples and other Loliinae and outgroup samples(Table 1) to detect ancestral vs. recent GBSSI duplications accord-ing to the ‘‘species-overlap rule’’. We also computed two fine-leaved Loliinae GBSSI (FEVREGBSSII-tree) and ITS (FEVREITS-tree)trees to test for the monophyletic robustness of FEVRE and forthe polyphyly of Vulpia, to infer an approximate FEVREspecies-tree,and to detect the origin of homoeologous copies in the polyploidspecies. In addition, the FEVREITS-tree was able to recover addi-tional polyploidization events that were not detected with GBSSIalone (see Results). According to model selection based on theAikake criterion implemented in the program MrModel Test v.2.3 (Nylander, 2004), Bayesian Inference (BI) was performed byimposing the GTR + C (nst = 6 and rates = invgamma) substitutionmodel plus exon and intron partitions on the GBSSI data set (Lolii-naeGBSSI-tree and FEVREGBSSI-tree) and the GTR + C model on theITS data set (FEVREITS-tree) using MrBayes version 3.1(Huelsenbeck and Ronquist, 2001). Indels of GBSSI introns were



Table 1Studied Loliinae and outgroup samples, placed according to their phylogenetic and taxonomic rank. Geographic origin, ploidy level, consensus GBSSI and ITS sequences (= types)with number of clones per type (in parenthesis), and major GBSSI clades obtained in the Bayesian analysis (see text). Genbank accession numbers of newly generated GBSSI andITS sequences are indicated in Table S1 (see Supporting information).

Lineage tribe/subtribe/genus

Species Geographicorigin

Ploidy GBSSI Major GBSSI clades ITS (ITS1-5.8S-ITS2)

Loliinae Dumort.Fine-Leaved Loliinae (FEVRE)Festuca L. WFL1 + WFL2 + WFL4 + WFL5

Sect. AulaxyperDumort.

F. agustinii Linding. Spain: CanaryIslands

2x Fagus1(3),2(1),3(1) WFL1C + WFL1D Fagus1(5)

F. rivularis Boiss. Spain:Granada

2x Frivu1(3),2(1),3(1) WFL1A + WFL5 Frivu1(5)

F. rubra L. Romania(cultivar)

6x Frubr1(3),2(1),3(1) WFL1A + WFL1C + WFL5 Frubr1(2),2(2),3(1)

Sect. Festuca F. alpina Suter Spain: Huesca 2x Falpi1(8) WFL1E Falpi1(5)F. plicata Hack. Spain:

Granada2x Fplic1(2),2(3) WFL1A Fplic1(5)

Subsect. ExarataeSt-Yves

F. borderei (Hack.) K. Richt. Spain: Huesca 2x Fbord1(9) WFL2 Fbord1(5)

F. capillifolia Dufour ex Roem. &Schult.

Spain: Jaen 2x Fcapi1(6) WFL1B Fcapi1(5)

Sect. Eskia Willk. F. eskia Ramond ex DC. Spain: Huesca 2x Feski1(5) WFL4 Feski1(5)Inc.sed. F. chimboracensis E.B. Alexeev Ecuador:

Cotopaxi6x Fchim1(2),2(2),3(1) WFL1D + WFL2 + WFL4 Fchim1(10),2(1)

Vulpia C.C. Gmel. WFL1 + WFL2 + WFL3 �WParaph

Sect. Vulpia V. bromoides (L.) Gray Spain: Lugo 2x – Vbrom1(5)V. muralis (Kunth) Nees Spain:

Zaragoza2x Vmura1(4),2(1) WFL1B + WFL1D Vmura1(5)

V. ciliata Dumort Spain:Zaragoza

4x Vcili1(9),2g(1)* WFL1B Vcili1(10)

V. myuros (L.) C.C.Gmel. USA:Washington

6x Vmyur1(7) WFL1B Vmyur1(6),2(3),3(1)

V. microstachys (Nutt.) Munro USA:California

6x Vmicr1(2),2(2),3(1) WFL1D + WFL2 Vmicr1(5)

V. octoflora (Walter) Rydb. USA:Washington

2x – Vocto1(4)

V. australis (Nees) Blom Argentina:Entrerrios

2x – Vaust1***

Sect. Spirachne(Hack.) Boiss.

V. brevis Boiss. & Kotschy Cyprus:Nicosia

2x – Vbrev1(5)

Sect. Loretia(Duval-Jouve)Boiss.

V. alopecuros (Schousb.) Dumort. Portugal:Algarve

2x Valop1(5),2g(1)* WFL1B Valop1(5)

V. sicula (C.Presl.) Link France: Corse 2x – Vsicu1***

V. geniculata (L.) Link Spain: Sevilla 2x Vgeni1(3),2(3),3(1),4(1) WFL1D + WFL3-WParaph Vgeni1(4)Sect. Monachne

Dumort.V. fontqueriana Melderis & Stace Spain: Segovia 2x Vfont1(5) WFL1C Vfont1(5)

V. membranacea (L.) Dumort. Spain: Cádiz 2x Vmemb1(5) WFL1C Vmemb1(5)V. fasciculata (Forssk.) Samp. Spain:

Barcelona4x Vfasc1(5),2(1),3(1) WFL1B + WFL1C Vfasc1(5)

Sect. Apalochloa(Dumort.) Stace

V. unilateralis (L.) Stace Spain:Zaragoza

2x Vunil1(5) WFL1A Vunil1(5)

HellerochloaRauchert

H. fragilis (Luces) Rauschert Venezuela:Mérida

? Hfrag1(5) WFL2 Hfrag1(5)

Micropyrum Link M. tenellum (L.) Link Spain: Segovia 2x Mtene1(5) WFL1E Mtene1(5)Narduroides Rouy N. salzmanii (Boiss.) Rouy Spain: Madrid 2x Nsalz1(5) WFL2 Nsalz1(5)Psilurus Trin. P. incurvus (Gouan) Schinz & Thell. Spain: Huesca 4x Psinc1(5) WFL1B Psinc1(5)Wangenheimia

MoenchW. lima (L.) Trin. Spain:

Zaragoza2x Wlima1(2),2(2),3(1) WFL1E Wlima1(4),2(1)

Broad-leaved LoliinaeFestuca WBL + WBL-Paraph + WBL-

Dact + WOutgr

Subgen.DrymantheleV.I.Krecz. &Bobrov

Festuca drymeja Mert & W.D.J. Koch Hungary:Balaton

2x Fdrym1(10) WBL –

Lojaconoa Catalán& Joch. Müll.

F. coerulescens Desf. Spain: Cádiz 2x Fcoer1(4) WBL –

F. triflora Desf. Spain: Cádiz 2x Ftrif2(1) WOutgr –Sect. Scariosae

Hack.F. scariosa (Lag.) Asch. & Graebn. Spain: Almeria 2x Fscar1(4) WBL –

Sect. SubbulbosaeNyman exHack.

F. spadicea L. Spain: Lugo 6x Fspad1(5) WBL-Paraph –



21 June 2014

Please cite this article in press as: Díaz-Pérez, A.J., et al. Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephemeralVulpia grasses and other fine-leaved Loliinae (Poaceae). Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.06.009


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Table 1 (continued)

Lineage tribe/subtribe/genus

Species Geographicorigin

Ploidy GBSSI Major GBSSI clades ITS (ITS1-5.8S-ITS2)

Subgen. Leucopoa(Griseb.) Hack.

F. sclerophylla Boiss. ex Bisch. Azerbaijan:Ordubad

6x Fscle2(1) WBL-Dact –

Subgen.Schedonorus (P.Beauv) Peterm.

F. pratensis Huds. England:Wiltshire

2x Fprat1(10),2(1) WBL-Dact Fprat1(5)

F. simensis Hochst. ex A. Rich. Uganda:Echuya

4x Fsime1(3) WBL-Dact –

F. fenas Lag. Spain:Mallorca

4x Ffena3(1) WBL-Dact –

Lolium L. L. multiflorum Lam. Egypt (seedsPI 343155)

2x Lmult2(1) WBL-Dact –

L. remotum Schrank France (seedsPI 283611)

2x Lremo1(3) WBL-Dact –

MycropyropsisRomero Zarco& Cabezudo

M. tuberosa Romero Zarco &Cabezudo

Spain: Huelva ? Mtube1(2) WBL –

Parapholiinae/cynosurinaeParapholis C.E.

HubbardP. incurva (L.) C.E. Hubb. Spain: Cádiz 4x Parin2(1),4(1) WParaph + WBL-Paraph –

Hainardia Greuter H. cylindrica (Willd.) Greuter Spain:Zaragoza

2x–4x Hcyli1(4) WParaph –

Cutandia Willk. C. memphitica (Spreng.) K. Richt. WesternMediterranean

? Cmemp1(5) WParaph –

Dactylidinae Stapf.Dactylis L. D. hispanica Roth Spain:

Alicante2x Dhisp2(1) WBL-Dact –

Lamarckia Moench L. aurea (L.) Moench Spain:Zaragoza

2x Laure1(4) WBL-Dact –

OutgroupsPoinae – PuccinelliinaePoa L. P. infirma Kunth Spain:

Zaragoza2x Pinf1(2) WBL –

Puccinellia Parl. P. festuciformis Parl. Italy: Sardinia 6x Pfest1(2) WOutgr –

Aveneae Dumort.Deschampsia

Beauv.D.cespitosa (L.) P. Beauv. Spain: Leon 6x Dcesp3(2),4(1) WBL + WOutgr –

Koeleria Pers. K. loweana Quintanar, Catalán &Castrov. (syn. Parafestuca albida(Lowe) Alexeev)

Portugal:Madeira

16x Palbi1(4) WKoel –

Triticeae Dumort.Andropogon L. A. gerardi Vitman 6x Agera (AF079235)** WOutgr –Agropyron

GaertnerA. cristatum (L.) Gaertner 2x Acris (AF079271)** WOutgr –

Triticum L. T. monococcum L. 2x Tmono(AF079286)** WOutgr –Aegilops L. A. speltoides Tausch 2x Aspel (AF079267)** WOutgr –

Brachypodieae HarzBrachypodium P.

Beauv.B. distachyon (L.) P. Beauv. 2x Bdist(6) WOutgr –

a g = sequences highly similar to conspecific sequences according to p-distance, but with some different indel positions.b Mason-Gamer et al. (1998) sequences.c Direct PCR DNA sequences. inc.sed.: Incertae sedis.



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treated as missing data, whereas for the ITS binary indel partitiondata set we imposed the rates = gamma model. For all analyses, wecomputed six runs, each with 1,000,000 generations and 4 chains,sampling trees every 100 generations, and a burn-in option of 2500trees per run once stability in the likelihood values was attained. Amajority-rule consensus tree was used to summarize the posteriordistribution of trees (posterior probability support of branches).Bootstrap support for branches of the GBSSI and ITS trees was fur-ther estimated through a Maximum Likelihood hill-climbing algo-rithm, based on the same parameters as in the respective Bayesiansearches, using PHYML (Guindon and Gascuel, 2003). Following thecriterion of Minaya et al. (2013), clades with bootstrap support val-ues (BS) of 75–100% or Posterior Probability support values (PPS) of90–100% were considered moderately to strongly supported.

Because the Bayesian majority-rule consensus tree is a combi-nation of all partitions with probabilities greater than 0.5, we


expected deep multifurcations generated when low probabilitybinary partitions were combined into a single well-supported mul-tifurcating node (Cranston and Rannala, 2007). In order to resolvesuch polytomies, we used a second approach that searches foragreement subtrees from the posterior distribution of trees(Cranston and Rannala, 2007). These fully resolved subtrees arepresent in a larger proportion of the sampled trees than the treetopology with the highest posterior probability, or the maximuma posteriori tree. Searching for subtrees was performed using theThreshold Accepting (TA) algorithm implemented in the Mapminerprogram (Cranston and Rannala, 2007), which is a stochastic anal-ysis to detect the optimal subset of sequences that need to bepruned to generate large probability subtrees. For the GBSSI Lolii-nae dataset, we performed an exploratory analysis with k rangingfrom 20 to 60, where k is the number of sequences to be prunedfrom the Loliinae subtrees. Initial threshold t ranged between



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0.003 and 0.012, with a decreasing threshold Dt of 10% of the initialvalue each it-th iteration, it being set to 100. Though we found apositive association between the Loliinae-subtree probabilitiesand different values of k, where the highest value was k = 60, wechose a lower k = 49, representing approximately 1/3 of the initialnumber of sequences in the data set, to guarantee the presence ofat least one representative sequence of each major Loliinae-treelineage. Nine independent analyses (runs) were performed subse-quently using k = 49 (t = 0.1–0.2, Dt = 10%, it = 1000). We did notattempt to generate subtrees for the FEVREGBSSI-tree because thisinformation was contained in the LoliinaeGBSSI-subtrees. For theITS region, the parameters for the exploratory analysis were setto k = 1–20, t = 0.001–0.2, Dt = 10%, it = 100. Two k values wereselected through independent runs, k = 19 (approximately 1/3 ofthe initial sequences) involving FEVREITS-subtrees with higherprobabilities but showing incomplete sampling of the mainFEVREITS-tree clades, and k = 10, involving FEVREITS-subtrees withlower probabilities but showing an almost complete sampling ofthe main FEVREITS-tree clades. We performed five independentruns for k = 19 (t = 0.15–0.25, Dt = 10%, it = 1000) and eight fork = 10 (t = 0.02–0.055, Dt = 10%, it = 1000).

The TA algorithm generated both a set of subtrees showing over-lapped subsets of clades and a set of clades (among subtrees) show-ing mutually exclusive or overlapped subsets of sequences for theseparate GBSSI and ITS data sets. Given the stochastic nature ofthe TA algorithm and the restriction imposed by k on the final num-ber of sequences incorporated into the subtrees, the subtrees oftenshowed incomplete sampling of the main LoliinaeGBSSI-tree orFEVREITS-tree lineages and incomplete sampling of the whole setof sequences sampled in each lineage. To uncover those incompletesamplings of sequences and clades, we collapsed sequences for agiven clade into a single subtree leaf; then a supernetwork was com-puted using SplitsTree4 version 4.12.3 (Huson and Bryant, 2006) inorder to represent all leaves in a single graphical representation(Huson et al., 2010). Congruence or incongruence among treeswas shown as tree-like or net-like split graphs, respectively(McBreen and Lockhart, 2006). The Zrule, TreeSizeWeightedMean,Number of Runs = 1000 and the Rooted Equal Angle options wereused for the analyses of both GBSSI and ITS data sets.

Considering that hybridization has been reported several timeswithin the fine-leaved Loliinae (Stace and Cotton, 1974; Ainscoughet al., 1986; Catalán, 2006; Krahulee and Nesvadbová, 2007), wecomplemented the information yielded by the Bayesian trees witha split graph from the GBSSI sequences (LoliinaeGBSSI-graph; seeSupporting information), which is expected to generate a better-quality representation of incompatible and ambiguous signals inthe data set that likely resulted from hybridization, but also from

Table 2Molecular characterization of GBSSI and ITS nuclear regions for Fine-leaved Loliinae (FEVRsequences per group). Length, number of nucleotide positions of the unaligned sequences. %matrix also included non-FEVRE groups. p-distance, mean proportion of base differences b

N Exons/5.8S

Length (bp) % Indels p-Distance (range)

GBSSIFEVRE 45 579 1.5 (9/588) 0.0523 (0–0.0984)Vulpia 20 579 1.5 (9/588) 0.0448 (0–0.0881)k 16 579 1.5 (9/588) 0.0302 (0–0.0535)d 6 579 1.5 (9/588) 0.0324 (0.0052–0.0k vs. d 0.0452 (0.0230–0.0

ITSFEVRE 35a 163 0 (0/163) 0.0185 (0–0.0859)Vulpia 16a 163 0 (0/163) 0.0214 (0–0.0491)

a Vsicu1 was excluded from this analysis due to lack of cloned ITS sequences. Outgroregions.


incomplete lineage sorting, horizontal gene transfer or recombina-tion events (Huson and Bryant, 2006).

3. Results

3.1. Characteristics of the GBSSI and ITS data sets

The final aligned Loliinae GBSSI data matrix included 1314 posi-tions, 588 retrieved from exons and 726 from introns. Sequencealignment of the GBSSI gene was based on exons 10, 11, 12 and13 as described in Mason-Gamer et al. (1998). All studiedsequences (Table 1) showed exons easily aligned (Table 2). Fourintron regions were observed between exons 9 and 14: 9–10,11–12, 12–13 and 13–14. The intron 10–11 was absent in the stud-ied Loliinae and close Poeae s.l. ougroups (Parapholiinae/Cynosu-riinae, Dactylidinae, Poinae-Puccinelliinae, Aveneae). Intronswere difficult to align because they showed a high number of sin-gle-bp indels, ranging from 64.5% of the total aligned positions inVulpia to 70% in FEVRE (Table 2). The aligned intron matrix yielded726 positions, including the sequences from the close Poeae s.l.subtribes. In addition to length variation, introns also showed morenucleotide differences per site than exons (Table 2). The meannumber of differences between a random pair of sequences was30 (= 0.0523 � 579 bp) for exons and 46 for introns for FEVREand 26 (= 0.0448 � 579) and 40 for Vulpia, respectively.

The complete FEVRE ITS data matrix included 616 positions,163 corresponding to the 5.8S gene and 453 to the ITS1 + ITS2spacers (Table 2). The 5.8S gene showed no indels whereas theITS1 + ITS2 spacers showed 29 and 26 indels (6.4% and 5.7% ofthe total alignment) for the FEVRE and Vulpia data sets, respec-tively (Table 2). ITS percentage values of indels were approxi-mately one order of magnitude smaller than those observed inthe GBSSI introns, making ITS an easy-to-align region for FEVRE.The mean number of differences between a random pair ofsequences within the 5.8S and ITS1 + ITS2 subregions was 3 (=0.0185 � 163 bp) and 31 (= 0.0693 � 453) for FEVRE and 4 (=0.0214 � 163) and 36 (= 0.0802 � 453) for Vulpia, respectively(Table 2). P-distance estimates indicate that the 5.8S gene was lessvariable than the ITS1 + ITS2 spacers, showing an ITS1 + ITS2 to5.8S ratio of 3.75 for both the Vulpia and the FEVRE data sets.The ITS region was more conserved than GBSSI with respect tothe proportion of single indels and length variation. P-distancesalso indicated that ITS was less variable than GBSSI, for whichFEVRE and Vulpia showed approximately 74 (34 + 40) vs. 142(76 + 66) base differences, respectively.

A total of 141 GBSSI clones and 149 ITS clones were generatedfor the FEVRE data set (Table 1 and S1, Supplementary informa-

E), Vulpia and GBSSI k and d paralogs (see text). N, sampling size (number of studiedIndels, percentage of single base pair indels of the total aligned matrix; for GBSSI, theetween a random pair of sequences.

Introns/(ITS1 + ITS2)

Length (bp) % Indels p-Distance (range)

363–435 70.0 (508/726) 0.1309 (0–0.2081)365–424 64.5 (468/726) 0.1158 (0–0.2012)363–424 60.6 (440/726) 0.0849 (0–0.1429)

518) 378–401 49.0 (356/726) 0.0457 (0.0080–0.0916)674) 0.0968 (0.0541–0.1404)

433–438 6.4 (29/453) 0.0693 (0–0.1460)433–438 5.7 (26/453) 0.0802 (0–0.1460)

up sequences retrieved from Mason-Gamer et al. (1998) did not include the intron



Fig. 2. Bayesian LoliinaeGBSSI (A) and FEVREGBSSI (B) trees. Major clades are preceded by the letter ‘W’ [= ‘Waxy’ (GBSSI)]; FL and BL indicate Fine- and Broad-leaved Loliinaesequences, respectively. d and k represent paralogs. Recombinant (rec) sequences are shown in green and red letters; ‘M’ and ‘m’ indicate major and minor parental sequencesinvolved in the origins of the recombinants, respectively; T indicates ‘trace’ recombinant sequence and ? indicates uncertain parent sequence. Colored circles indicate theexpected phylogenetic relationships according to previous phylogenetic studies (see Fig. 1). Posterior probabilities (100�) and Bootstrap (%) support values are shownproximal to each node, respectively. Shaded areas indicate the phylogenetic origin of the homoeologous genomes. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)



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Please cite this article in press as: Díaz-Pérez, A.J., et al. Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephemeralVulpia grasses and other fine-leaved Loliinae (Poaceae). Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.06.009


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21 June 2014

tion). An average of 5.9 and 5.5 clones per individual wereretrieved, respectively, for each molecule. After pruning closelyrelated sequences, we obtained a manageable data set of 45 con-sensus sequences or types for GBSSI and 33 for ITS, averaging 1.9and 1.2 types per individual. A subset of 79 additional GBSSI cloneswas obtained from broad-leaved Loliinae and closely related Poeaes.l. subtribes (Tables 1 and S1, Supplementary information). Afterpruning, a total of 25 types were retained for the GBSSI analysis,with an average of 1.1 sequences per individual.

3.2. GBSSI recombinants, GBSSI paralogs and ITS pseudogenes

Two recombinant events were detected in some Loliinae GBSSIsequences (Table S2, Supplementary information), with parentaland recombinant sequences completely or partially associatedwith Festuca sect. Aulaxyper taxa. The origin of one recombinationevent only involved sequences from this section (Frivu2, Frubr3);however, that of the other event involved both Aulaxyper (Frivu2)and more basal Eskia (Feski1) sequences. Most recombinants werefound within the Aulaxyper lineage (Frivu1, Frivu3, Frubr2,Frubr3), but two relatively divergent sequences from the Festuca(Falpi1) and the Loretia (Fplic2) lineages also showed evidence ofrecombination (Tables 1 and S2, Supplementary information).

Divergent GBSSI types were found in the diploid species F.agustinii and V. muralis, suggesting the existence of paralogous cop-ies. We used the LoliinaeGBSSI-tree (Fig. 2A) to infer the phyloge-netic origin of the putative duplication events in FEVRE. Thenode ‘‘D’’ of the LoliinaeGBSSI and FEVREGBSSI trees (Fig. 2A and B)was the shallowest node whose descendant lineages comprisedeach paralogous copy. The sister lineages leading to WFL1D andWFL1B + WFL1C comprised d and k paralogs, respectively. Given thatD and its descendant nodes showed moderate to high supportaccording to the FEVREGBSSI-tree (posterior probability between0.97 and 1 and bootstrap support (BS) of 77%; Fig. 2B), we are con-fident of the clear-cut separation of the two paralogous groups.Despite putative reticulation events in FEVRE that could have dis-torted phylogenetic relationships in the LoliinaeGBSSI-tree, the Lolii-naeGBSSI-graph suggests a compact group of d and k paralogsdefined by the ‘‘D’’ split, although with low (<50%) bootstrap sup-port (Fig. S2, Supplementary information). We also observed thatthe two paralogous clades showed incomplete sets of speciessequences (e.g., k WFL1B + WFL1C (Fcapi, Psinc, Valop, Vcili, Vfasc,Vfont, Vmemb, Vmyur) vs. d WFL1D (Fchim, Vgeni, Vmicr); Fig. 2Aand B), suggesting that the PCR and/or the cloning process couldhave captured only one paralogous copy in some cases. We cannotrule out the possibility that additional d types might have beenundetected given the homogenizing effect of sampling bias(N = 6). Also, the Nei-Gojobori Z test indicated that exons from bothparalogs were subjected to purifying selection (P < 0.01) (Table S3,Supplementary information). The relative rate test did not detectdifferent evolutionary rates either within or among paralogs (TableS4, Supplementary information), except for four pairwise compar-isons that involved the V. alopecuros sequence Valop1 (Loretialineage).

Three ITS sequences from different FEVRE species (F. rubraFrubr3, F. chimboracensis Fchim2, W. lima Wlima2) failed to gener-ate the conserved secondary structure of the 5.8S gene, becausesome base pairings could not be formed in the B5, B6, B8a andB8b helices (Table S5, Supplementary information). Frubr3 showedthe largest deviation from the consensus secondary structure,yielding four helices with unpaired bases, the transition C ? U inthe B5 helix also being associated with a loss of the conservedfunctional motif M1. This sequence was related to the unique test-able branch according to the Bootstrap Hypothesis Testing method.However, substitution rates in the 5.8S gene (K5.8S) were signifi-cantly higher than the rate of the putative neutral ITS region (KITS),


suggesting that some sort of positive selection is operating on thissequence. Fchim2 and Wlima2 also showed unstable secondarystructure, indicating that the three sequences could correspondto ITS pseudogenes.

3.3. Phylogeny of Vulpia and other fine-leaved Loliinae

The largest phylogenetic reconstruction of Loliinae based onGBSSI sequences is shown in Fig. 2A (LoliinaeGBSSI-tree). In thistopology the studied FEVRE sequences (Table 1) nested into fivemajor clades with moderate to high posterior probability (PP)and poor to high BS, respectively (WFL1: 0.51, 70%; WFL2: 0.53,29%; WFL3 to WFL5: 1.0, 94–100%). WFL1 showed the largest propor-tion of FEVRE sequences, further subdivided into five subclades(A–E). The more restricted FEVREGBSSI-tree (Fig. 2B), whichexcludes the broad-leaved Loliinae and the outgroup samples, alsosupported the existence of the WFL2–5 clades with moderate to highPP and poor to high BS (0.7–1.0 and 38–100%, respectively); how-ever, the WFL1 sequences were not monophyletic, falling into threelineages that showed distinct relationships to the remaining FEVREsequences: WFL1E paraphyletic and sister (pro parte) to WFL4

(PP = 0.52; BS = 12%); WFL1A sister to WFL2,3,5 (PP = 1.0; BS = 46%);WFL1B,C,D sister to WFL1E (pro parte) (PP = 0.85; BS = 23%). The Lolii-naeGBSSI-graph (Fig. S2, Supplementary information) identified thesame major Loliinae and outgroup clades of the LoliinaeGBSSI-tree,associated with splits with moderate bootstrap support. Interest-ingly, the highest-supported (74.9%) split within the FEVRE graphcore was associated with the largest WFL1 group, indicating a cleardifferentiation between the latter and the rest of the Loliinae andoutgroup sequences. It strongly suggested a greater divergence ofthe WFL1B,C,D group from the WFL1A and WFL1E subclades and fromthe remaining GBSSI clades, more than any other split.

The ITS Bayesian majority-rule consensus FEVREITS-tree (Fig. 3)was resolved into six lineages (IFL1–IFL6). Four of the six clades(IFL1,2,5,6) showed high PP but poor to high BS, ranging between0.95 and 1 and between 33% and 87%, respectively. IFL1 was roughlyequivalent (sensu Catalán et al., 2004; Inda et al., 2008) to the Lore-tia assemblage, IFL2 to the Aulaxyper + Vulpia 2x clades, IFL5 to thePsilurus/Vulpia 4x–6x clade and IFL6 to the American Vul-pia + American I clades.

The GBSSI agreement subtrees computed using the Bayesianposterior probability trees generated nine independent 25-taxonLoliinaeGBSSI-subtrees after pruning 49 sequences (2/3) of thewhole Loliinae + outgroup data set. Posterior probabilities of thesesubtrees ranged between 0.58 and 0.80, representing values threeorders of magnitude higher than that of the Maximum a Posterioritree (0.000333). The consensus LoliinaeGBSSI agreement subtree,displayed as a supernetwork splits-graph, summarized the sto-chastic differences among 9 subtree topologies (Fig. 4A). It wasalmost tree-like, supporting congruent topologies among all theagreement subtrees except for a net-like portion that suggesteduncertainty in the phylogenetic relationships between the WBL-Par-

Cyn-1 and the WFL5 clades. Divergence order in the consensusagreement subtree implied the early split of the outgroup clade(WOutgr) followed by the successive divergences of close subtribesplus broad-leaved lineages (WBL-Dact, WBL-Paraph) and then themajor FEVRE lineages. Considering only the FEVRE clades, thesequence of divergence ranged from the basal-most clade WFL4 toWFL2, WFL3 + WFL5 and WFL1; however, FEVRE was not resolved asmonophyletic, given the unexpected positions of the WParaph andWBL clades within it (Fig. 4A). The consensus agreement subtreealso showed evidence of a potential duplication event circum-scribed by the terminal and monophyletic WFL1B,C,D subclades(Fig. 4A).

Most of the Vulpia (Vulpia 2x, Psilurus/Vulpia 4x–6x, Loretia)and other fine-leaved Loliinae (Aulaxyper, Exaratae, American I)



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Fig. 3. Bayesian FEVREITS tree. Major FEVRE clades are preceded by the letter ‘I’ [= ‘ITS’ (ITS1-5.8S-ITS2)]. Colored circles indicate the expected phylogenetic relationshipsaccording to previous phylogenetic studies (see Fig. 1). Posterior probabilities (100�) and Bootstrap (%) support values are shown proximal to each node, respectively. Shadedareas indicate the phylogenetic origin of the homoeologous genomes. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)



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group samples (sensu Catalán et al., 2004; Inda et al., 2008; Fig. 1)were included within the WFL1B,C,D subclades (Figs. 2B and S2, Sup-plementary information). Some noticeable inferences wereretrieved from WFL1B,C,D: (i) GBSSI types from the tetraploid V. fas-ciculata were associated with types from consectional Monanchnediploid species (V. membranacea, V. fontqueriana) (kC paralog subsetwith PP = 1.0 and BS = 99%) rather than to Loretia diploid species (V.geniculata, V. alopecuros) types (d paralogs in the WFL1D subclade);(ii) types from diploid (Vulpia 2x) and polyploid (Psilurus + Vulpia4x–6x) species of two putative polyphyletic lineages were closelyassociated (kB paralog subset; PP = 1.0 and BS = 100%); (iii) typesfrom American species of two putatively distinct lineages, V. micro-stachys (American Vulpia) and F. chimboracensis (American I), werealso closely associated among themselves and to other FEVRE line-age types (Aulaxyper, Vulpia 2x, Loretia) (d paralogs; PP = 1.0 and


BS = 97%); (iv) Vulpia unilateralis showed a rather striking basalposition with respect to members of its Loretia lineage, which wereplaced in the WFL1B,C,D subclade; by contrast, the close relationshipof F. plicata to V. unilateralis agreed with previous phylogeneticfindings.

A deep FEVREITS-tree polytomy (Fig. 3) involving clades IFL1 toIFL4 was resolved using agreement subtrees. Five initially indepen-dent analyses, pruning k = 19 taxa at a time, generated subtreeswith posterior probabilities between 0.79 and 0.82 that were sum-marized in the single supernetwork FEVREITS-subtree (Fig. S3, Sup-plementary information). In that subtree IFL1 and IFL2 were resolvedas sisters, however no information could be extracted regardingIFL3 and IFL4. To include all the Bayesian clades in the consensusagreement subtree, we reduced the number of pruned taxa fromk = 19 (representing approximately 2/3 of the original data set) to



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Fig. 4. Bayesian LoliinaeGBSSI (A) and FEVREITS (B) consensus agreement subtrees and hypothetical FEVRE species tree (C). Consensus agreement subtrees for major clades ofLoliinae (A, see Fig. 2) and FEVRE (B, see Fig. 3): 2x and Px indicate confirmed diploid and polyploid taxa; ? = unknown ploidy; acronyms of major groups, samples,recombinants and paralogous copies correspond to those indicated in Figs. 2 and 3, respectively. K is the number of pruned sequences and Pr is the posterior probability rangeof the original subtrees. Color designation follows that indicated in Fig. 1. ‘Rec. Event’ indicates major clades showing recombinant sequences. Asterisk = see text for details.Hypothetical FEVRE species tree (C). Major clades were inferred from the GBSSI and the ITS trees (Figs. 2 and 3) and subtrees (A and B). The major ‘W’ and ‘I’ clades used toinfer the basic topology and the taxonomic composition of each lineage are shown above each branch. Homoeologous parental genomes involved in the origins of fourputative allopolyploid FEVRE species are shown on the right side of the figure. Parental lineages: Loretia (dark green); Aulaxyper + Vulpia 2x (violet), ancestral Psilurus-type(orange), Exaratae (red), Eskia (gray). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)



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k = 10 (1/3). Eight independent k = 10 subtrees showed lower prob-abilities (0.22–0.30) than the k = 19 subtrees but included all theclades. We decided to use the latter set as both sets (k = 19 andk = 10) were topologically congruent (Fig. 4B and S3, Supplemen-tary information). The consensus FEVREITS subtree for k = 10(Fig. 4B) showed two major subclades within FEVRE (depicted bythe highlighted node with asterisk; Fig. 4B) that mainly comprisedthe IFL1 (Loretia s.l. clade, 1.0; 83%) and IFL2 (0.95; 93%), which wasfurther subdivided into the IFL2A (Vulpia 2x, 1.0; 87%) and IFL2B

(Aulaxyper, 0.87; 47%) subclades (Figs. 3 and 4B).IFL4 included sequences of Wangenheimia (W. lima), Narduroides

(N. salzmanii), and V. unilateralis (Vunil1), with the latter sequencelocated apart from the remaining Loretia assemblage sequences


(IFL1) (Figs. 3 and 4B). The IFL5 clade (PP = 1.0; BS = 87%) divergedearlier; most of the polyploid Psilurus/Vulpia 4x–6x sequences(V. myuros, V. ciliata, P. incurvus) were nested within this clade.Interestingly, one hexaploid V. myuros sequence (Vmyur2) fellwithin IFL2A, close to the diploid consectional species. Most of theAmerican-taxa ITS sequences fell within the highly supported sub-basal IFL6 clade (PP = 1.0; BS = 81%; Figs. 3 and 4B). This heteroge-neous group included sequences from the American Vulpia (V.octoflora, V. australis) and the American I (F. chimboracensis) andAmerican II (H. fragilis) Festuca s.l. lineages. Only the American V.microstachys (Vmicr1) sequence fell outside, though close to it.The Eskia (F. eskia) sequence was resolved as the earliest diverginglineage within the FEVRE phylogeny (Figs. 3 and 4B).



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3.4. FEVRE species tree and inferred origins of allopolyploids

We detected homeologous genomes in the consensus sequence-types from polyploid samples that fell separately into majorBayesian clades of the same tree (e.g., American 1 and American2 genomes for V. microstachys, and American 1, 2, 3 genomes forF. chimboracensis; FEVREGBSSI-tree, Fig. 2B) associated withsequence types from taxonomically close diploid relatives. In addi-tion, potential genome type donors participating in the ancestralhybridizations included sequences from polyploid samples thatwere nested in different phylogenetic positions in each tree [e.g.,loretia2 (FEVREGBSSI-tree) and loretia1 (FEVREITS-tree) genomesfor V. fasciculata (Figs. 2B and 3)]. When diploid sequences werenot detected, we considered polyploid sequences as potentialhomeologous genomes if they were placed in different phyloge-netic positions with respect to other types [e.g., psilurus1 withrespect to psilurus2 (FEVREITS-tree) for V. myuros (Fig. 3),American1, 3 (FEVREGBSSI-tree) with respect to American2(FEVREITS-tree) for F. chimboracensis (Figs. 2B and 3)]. We assumedthat homonymous GBSSI and ITS genomes reflected the samehomeologous copy, explaining why the GBSSI and ITS American2or psilurus2 genomes showed, for example, similar phylogeneticpositions and genetic compositions in their respective FEVREGBSSI

and FEVREITS trees. The joint inspection of these two trees sug-gested the existence of seven homeologous genomes within thepolyploids (Figs. 2B and 3), including two genomes, psilurus2(GBSSI k copy in clade WFL1B+C, Fig. 2B, and ITS IFL2, Fig. 3) andAmerican2 (clades GBSSI WFL2, Fig. 2B, and IFL6, Fig. 3) that wereobserved in both trees.

Based on these results, we propose a hypothetical FEVRE spe-cies-tree that explains the major allopolyploidization eventsdetected in this study (Fig. 4C). The topological position and thegenetic composition of the main clades reflected the phylogeneticagreement among independent GBSSI and ITS trees and subtrees.For example, a major Aulaxyper + Vulpia 2x + Psilurus/Vulpia 4x–6x + American Vulpia + American I clade was resolved as sister toa major Loretia clade based on separate and joint congruentphylogenetic relationships between the WFL1B,C,D and IFL2 clades(Figs. 2–4). In general, the species tree concurs with previous find-ings, suggesting a recently evolved Aulaxyper + Vulpia 2x clade, anintermediate Psilurus/Vulpia 4x–6x clade and a basal divergence ofthe Eskia lineage and of the diploid-to-polyploid members of theAmerican Vulpia + American I clades (Fig. 4C). Nonetheless, someunexpected relationships were also recovered; for example, thecloser relationship of the Aulaxyper + Vulpia 2x clade to the Loretiaclade than to either the American II (H. fragilis) or the Festu-ca + Wangenheimia clades. Furthermore, this relationship wasdetected in both paralogous copies of GBSSI (e.g., close k Fagus,Vmemb and Vfont and d Fagus, Vmura and Vgeni sequences;Fig. 2B).

According to the phylogenetic positions of the homeologousgenomes (Figs. 2B and 3) we inferred past hybridization eventsthat finally led to the formation of four potential allopolyploidspecies (Fig. 4C), the tetraploid V. fasciculata, and the hexaploidV. myuros, F. chimboracensis and V. microstachys. The tetraploidV. fasciculata likely originated from the parental diploid Vulpia sect.Spirachne (loretia1) and the diploid Vulpia sect. Monachne or thediploid-to-polyploid Festuca sect. Aulaxyper (loretia2) genomes.The hexaploid V. myuros resulted from two Vulpia sect. Vulpiaparental genomes, the diploid Vulpia 2x (psilurus2) and thepolyploid Psilurus/Vulpia 4x–6x (psilurus1) genomes. At least twogenomes (American2, American1) are involved in the origin ofthe allohexaploid V. microstachys; its potential parents could berelated to American Vulpia, Exaratae or American I (Festuca)lineages of unknown ploidy and to diploid Vulpia2x or Loretialineages, respectively. The hexaploid F. chimboracensis probably


accumulates three distinct genomes. Two of them (American1–2)likely originated from a polyploid American I and/or from dip-loid-to-polyploid American Vulpia (V. microstachys)-like genomedonors, whereas the third one was related to a basal diploidEskia-type (American3) genome (Fig. 4C).

The potential female and male nature of the parental genomedonors of the allopolyploid species was deduced from contrastedanalysis of the biparentally-inherited nuclear GBSSI + ITS speciestree (Fig. 4C) and maternally-inherited plastid trnTF (Fig. S4, Sup-plementary information) topologies. A diploid maternal Monachneparent was inferred for V. fasciculata, a tetraploid Psilurus/Vulpia4x–6x for V. myuros, a potential diploid-to-tetraploid AmericanVulpia, Exaratae or American I (American2) for V. microstachys,and a tetraploid V. microstachys-like for F. chimboracensis (Fig. 4C).

4. Discussion

4.1. Evolutionary dynamics of the duplicated GBSSI and of the ITSsequences in the fine-leaved Loliinae

Our phylogenetic and networking analyses of the GBSSIsequences have demonstrated the existence of at least one duplica-tion event for this gene within the most recently evolved FEVRElineages (Figs. 2, 4A, and S2, Supplementary information). Two dif-ferent GBSSI paralogs (k, d) have been detected within diploid spe-cies of the Aulaxyper + Vulpia 2x clade (F. agustinii, V. muralis) inthe Bayesian trees (Figs. 2 and 4A) and the split-network (Fig. S2,Supplementary information). According to the species overlap rule(Gabaldón, 2008), the WFL1B+C (k) and WFL1D (d) subclades thatemerged from the ‘‘D split’’ could be associated to a duplicationevent which gave rise to the paralogous Vmura1 vs. Vmura2 andFagus1 vs. Fagus2 + Fagus3 sequences (Fig. 2). The extent of theparalogy could be even larger in FEVRE, as incomplete samplingcould have occurred in the diploid V. membranacea, V. geniculataand V. alopecuros, for which only one type of paralogous copy perspecies (k or d) was detected (Fig. 2A).

The topological position of node D suggests that the duplicationevent took place after the divergence of the fine-leaved Loliinaelineage, localizing it to a relatively recent clade within FEVRE, assuggested by the phylogenetic position of WFL1B, WFL1C and WFL1D

in the LoliinaeGBSSI-subtree (Fig. 4A). Nonetheless, the unexpectedcloseness of some diploid FEVRE functional sequences (WFL3 subc-lade: Vgeni2, Vgeni3) to the less-related Parapholiinae/Cynosurii-nae ones (WParaph), and of some diploid broad-leaved functionalsequences (Ftrif2) to the distantly related (e.g., Puccineliinae, Triti-ceae) ones of the outgroup taxa (WOutgr) suggest the potentialadmixture of paralogous sequences within those taxa and, conse-quently, a more ancestral scenario for an earlier duplication ofthe GBSSI copies before the split of the Loliinae, and probablybefore that of the core-pooids (Figs. 4A and S2, Supplementaryinformation). Mason-Gamer et al. (1998) and Mahelka andKopecky (2010) considered GBSSI to be a single-copy gene in thePoaceae. This hypothesis has been refuted, however, by recentstudies that have suggested the occurrence of a GBSSI duplicationin the Chloridoideae (Fortune et al., 2007), the Panicoideae(Sánchez-Ken and Clark, 2010) and the Arundinoideae (Zhanget al., 2012). Our study provides evidence for the additional exis-tence of such duplication in the temperate Pooideae, supportingthe idea of an ancestral GBSSI duplication in the grasses(Sánchez-Ken, 2005). As the GBSSI paralogy has been found in bothdiploid and polyploid species from across the PACMAD and BEP lin-eages (Fortune et al., 2007; Sánchez-Ken and Clark, 2010; Zhanget al., 2012; current study), its origin could predate the PACMAD/BEP split and might be related to the ancient genome duplication



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event that resulted in the potential modern allopolyploid-derivedgrass ancestor (Salse et al., 2008).

Despite the existence of duplicated paralogous FEVREsequences, phylogenetic analysis of cloned GBSSI sequences,together with that of cloned ITS sequences, has identified thehomeologous parental-type sequences of four allopolyploid FEVREspecies (Figs. 2, 3, and 4C). In contrast to the paralogous GBSSIclones, most of the ITS clones were resolved as monophyletic forthe diploid species (Results not shown). This suggests a major con-certed evolutionary trend of potential intragenomic ribotypicdiversity to a single species-specific type (Álvarez and Wendel,2003; Nieto-Feliner and Rosselló, 2007). However, different paren-tal ITS copies have been detected in at least one allopolyploid spe-cies (V. myuros; Fig. 3), indicating longtime persistence of thedistinct genomic ribotypes in the derived hybrid or the existenceof inter-genomic constraints against ribotypic homogenization.The higher number of distinct homeologous GBSSI sequencesfound in the other three allopolyploid species (V. fasciculata, V.microstachys, F. chimboracensis; Fig. 2) by a similar cloning sam-pling effort agrees with the higher capability of low-copy genes,in contrast to ITS genes, in keeping the different parental copiesin the hybrid genomes largely intact (Catalán et al., 2012; López-Álvarez et al., 2012).

4.2. Polyphyly of diploid Vulpia lineages: a rapid radiation scenario ofephemeral FEVRE lineages

Phylogenetic analysis of nuclear low-copy GBSSI and multicopyITS sequences has successfully contributed to disentangling thecomplex reticulate evolutionary history of Vulpia and the mainFEVRE lineages (Figs. 2–4). The ITS sequences have identified threemain diploid Vulpia s.l. lineages, the Vulpia s.s. (IFL2A), the Loretiaassemblage (IFL1A+B) and the American Vulpia pro partim (IFL6)clades, with the former more closely related to the Aulaxyper clade(IFL2B), the second to F. plicata and the third to the American I clade(Fig. 3). This resolution agrees with that of the plastid data (Fig. S4,Supplementary information) and with previous findings (Catalánet al., 2004; Torrecilla et al., 2004; Inda et al., 2008). Howeverthe use here of cloned ITS sequences strengthens support for thehypothesis of independent origins of the Vulpia s.s., Loretia anddiploid American Vulpia lineages from different Festuca ancestors.This is also corroborated by the phenotypic differentiation of twomain groups (Cotton and Stace, 1976; Stace, 1981, 2005), suggest-ing different genomic compositions for the highly homomorphicand cleistogamous Vulpia s.s. taxa with respect to the highlydiverse and mostly chasmogamous Loretia group taxa. Nonethe-less, there is still uncertainty about the apparent independent ori-gin of the diploid American Vulpia taxa (V. octoflora, V. australis; cf.Bailey and Stace, 1984), which phenotypically ressemble the Vulpias.s. taxa. A fourth potential Vulpia s.l. diploid lineage, the morpho-logically atypical and chasmogamous Apalochloa (V. unilateralis),also emerges from the ITS tree, sister to Narduroides (Fig. 3). How-ever, V. unilateralis is reconstructed within the Loretia clade in theplastid tree (Fig. S4, Supplementary information), indicating thelikely participation of an ancestral maternal Loretia-type plastidgenome and a paternal Narduroides-type nuclear genome in thepresent diploid V. unilateralis. This suggests a more complex radia-tion scenario for the diploid Vulpia taxa and confirms the plausibleartificiality of the genus, with some of its current sections previ-ously described as separate genera (e.g. Loretia, Nardurus (= Apalo-chloa), Vulpia; Stace, 1981; Torrecilla et al., 2004).

In contrast to ITS, the GBSSI reconstructions tend to amalgam-ate the diploid Vulpia s.s. and Loretia group sequences (diploidAmerican Vulpia taxa were not sequenced for GBSSI), togetherwith some Aulaxyper ones, joining them into the ‘D split’ cladeand with representatives of each lineage falling together in the


respective paralogous subclades (WFL1B+Ck, WFL1Dd) (Fig. 2). Thissuggests a recent common ancestry of shared genomes amongthe three groups, which would explain the occurrence of spontane-ous intergeneric � Festulpia hybrids involving Aulaxyper and eitherVulpia s.s. or Loretia taxa (Ainscough et al., 1986; Krahulee andNesvadbová, 2007). Due to the potential extended presence ofrecombinant and paralogous copies, the reconstruction of the evo-lutionary history of FEVRE based on GBSSI sequences is lessstraightforward than when based on ITS. Like ITS, the GBSSI analy-sis also places V. unilateralis in a separate lineage, outside the ‘D’clade (Figs. 2 and 4A); however, it does not show a close relation-ship to Narduroides, which is nested within the other major splitgroup (Fig. S2, Supplementary information). Also, the unexpectednesting of two Loretia GBSSI sequences (Vgeni2, Vgeni4) withinthe less related Parapholiinae-Cynosuriinae outgroup clade(WParaph) (Fig. S2, Supplementary information) suggests the pres-ence of alien or more ancestral genomes in V. geniculata, or theputative detection of new paralogs. These features might not corre-spond, however, to recent genomes introgressed into the genomesof the studied species but to conserved reminiscent regions of theancestral grass genome (Salse et al., 2008; Mahelka and Kopecky,2010).

4.3. Multiple and polytopic origins of the allopolyploid Vulpia andFEVRE taxa

Our comparative GBSSI and ITS analysis has allowed us to iden-tify the parental genomes of four allopolyploid FEVRE taxa (Fig. 4C)and to infer dispersal routes for explaining the polytopic origins ofthe allopolyploid Vulpia lineages (Fig. 1). The GBSSI data aloneidentified the genome-donor lineages involved in the origin ofthe hexaploid F. chimboracensis and V. microstachys, whereas theITS data identified those of the hexaploid V. myuros (Fig. 3). Thejoint analysis of both data sets corroborated the independent find-ings and also identified the genome-donors of the tetraploid V. fas-ciculata (Figs. 2, 3, and 4C). The comparative analysis of the nuclear(Fig. 4C) vs. the plastid data (Fig. S4, Supplementary information)has facilitated the identification of the respective maternal andpaternal genome donors of the hybrids. Most hybridization eventsinvolved parental genomes from the Aulaxyper + Vulpia 2x or Lore-tia clades and the Psilurus/Vulpia 4x–6x, American or Eskia clades.Apparently, the Festuca + Wangenheimia lineage did not partici-pate in allopolyploid formation for the studied taxa.

The origin of the allotetraploid V. fasciculata can be traced backto a diploid genome donor from its consectional Monachne lineage(GBSSI, Fig. 2), which apparently acted as maternal parent (plastidDNA; Fig. S4, Supplementary information), and another from themorphologically and evolutionarily close diploid Spirachne lineage,which acted as paternal parent (ITS, Fig. 3), all nested within theLoretia clade. This scenario would explain the high phenotypicresemblances of V. fasciculata to taxa from these two groups(Cotton and Stace, 1977; Stace, 1981; Ainscough et al., 1986) andthe likely occurrence of the cross between these sister lineages(Torrecilla et al., 2004). Biogeographically, this would imply a dis-persal of the current eastern Mediterranean endemic Spirachnedonor to the West, and its mating with a likely western Mediterra-nean endemic diploid Monachne species, originating the currentwidespread western Mediterranean and European V. fasciculatatetraploid (Fig. 1). Alternatively, the GBSSI data also suggest thepotential implication of an ancestral diploid Aulaxyper lineage asthe potential parent of the species (Figs. 2B and 4C). This scenariocould explain the relatively common occurrence of spontaneousinterspecific crosses from maternal allotetraploid V. fasciculataand paternal allohexaploid F. rubra-like taxa, which origi-nated � Festulpia hubbardii Stace & R. Cotton individuals, and eventhe occasional backcross introgression of this highly sterile hybrid



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with its paternal F. rubra parent (Stace and Ainscough, 1984). Thiswould confirm the high crossability of the Aulaxyper taxa withother non-related FEVRE and Loliinae taxa (Catalán, 2006). How-ever, it could also be possible that the Aulaxyper-type GBSSI copiesfound in V. fasciculata might have been acquired from an oppositebackcross or from more recent hybridization episodes, increasingthe Aulaxyper genetic pool in a more ancestral Loretia-type hybrid.

The identification of the Vulpia 2x and the Psilurus/Vulpia 4x–6x lineages as the respective paternal and maternal genome donorsof the widespread allohexaploid V. myuros (Fig. 3) resolves theapparent contradiction between the separate phylogenetic recon-structions of the diploid and polyploid Vulpia s.s. lineages observedin the previous analyses (Catalán et al., 2004; Torrecilla et al., 2004;Inda et al., 2008) and their close morphological phenotypes (Stace,1981, 2005). Our results support Stace’s (2005) hypothesis on thelikely participation of a diploid V. muralis-like ancestor in the originof the taxonomically similar V. myuros. The FEVREGBSSI tree alsoindicates the implication of an unknown tetraploid Psilurus or Vul-pia-type genome as the maternal parent of this species (Fig. 2B),suggesting that the ancestral cross could have occurred in theircommon native western Mediterranean region (Cotton and Stace,1976; Fig. 1). The Psilurus/Vulpia 4x–6x clade is an allopolyploidlineage that might have arisen from an ancestral diploid Psilurus-type genome which has repeatedly crossed with the Vulpia 2x gen-ome to originate the current V. ciliata (4x) and V. myuros (6x) allo-polyploid species. The cumulative genomes of the allohexaploid V.myuros might have conferred on this Mediterranean endemicannual species a high adaptive capability, having colonized tem-perate ephemeral pastures on almost all the remaining continentswhere it is an invasive species (Catalán, 2006).

The disjunct distribution of the American Vulpia taxa (Figs. 1–3)and their surprisingly isolated phylogenetic position with respectto their morphologically close Mediterranean Vulpia 2x lineage(Inda et al., 2008) have been also clarified in our study. The annualAmerican Vulpia lineage, apparently close to a perennial AmericanI (Festuca) lineage (Inda et al., 2008; Fig. 3), consists of severalendemic North American (V. octoflora 2x, V. microstachys 6x) andSouth American (V. australis 2x, -studied here for the first time)taxa of diploid-to-allopolyploid origin (Bailey and Stace, 1984).Our hypothesis about the single origin of the homogeneous Vulpias.s. group has been partially confirmed by the GBSSI and ITS data(Figs. 2 and 3). However our results could not reject the alternativepolytopic origin hypothesis for the diploid American Vulpia taxa(cf. Inda et al., 2008), which suggested that this annual Americanclade could have originated from an American perennial Festucaclade, similar to the origin of the Mediterranean Vulpia 2x cladefrom the Mediterranean-Eurasian Aulaxyper clade. The FEVREGBSSI

tree (Fig. 2B) and FEVRE species tree (Fig. 4C) show that V. micro-stachys 6x is an allopolyploid species which likely derived fromthe cross between a Mediterranean Vulpia 2x, Aulaxyper or Lore-tia-type paternal parent and an American Vulpia,American I orExaratae-type maternal parent (Figs. 2B and S4, Supplementaryinformation). The strong relationship of the American Vulpia taxato the American I Festuca taxa, recovered by the ITS data (Fig. 3),supports the American origin of the maternal ancestor of V. micro-pyropsis; however, all of its possible paternal ancestors had a Med-iterranean origin. Phenotypic similarity suggests a Vulpia 2x-typeannual ancestor. This hybridizing scenario implies a long-distancedispersal of the Mediterranean diploid paternal ancestor to Amer-ica and its subsequent cross with the maternal Festuca-derivedspecies. Inda et al. (2008) inferred successive colonization eventsof ancestral Mediterranean-Southwest Asian fine-leaved Loliinaelineages to North America and then to South America from the lateMiocene to the early Pliocene, which originated the American I andAmerican Vulpia lineages. Our current results support this scenarioof different dispersal waves and subsequent hybridizations


between more ancestral and more recent colonizers on the Amer-ican continent.

A similar, though more complex, biogeographic pattern has tobe invoked to explain the origin of the allohexaploid American F.chimboracensis (Fig. 4C). In contrast to the primary diploid Mediter-ranean Loliinae lineages, most of the American lineages (includingall the Southern Hemisphere ones) are of secondary polyploid ori-gin (Catalán, 2006). Two of the three GBSSI homeologous copiesdetected in F. chimboracensis had the same phylogenetic origin asthose of V. microstachys, suggesting a similar biogeographical sce-nario for the origin of the maternal tetraploid ancestor of this spe-cies; however, the third homeologous copy came from a moreancestral diploid Eskia-type paternal genome (Fig. 2B). This relicFEVRE lineage probably colonized the American continent fromthe Old World at an earlier stage (Inda et al., 2008) but hybridizedto the more recent allotetraploid V. microstachys-type genomelater.

Acknowledgments

We thank Clive Stace for his insightful comments and fruitfuldiscussions on the systematics of Vulpia, Robert Soreng and ananonymous referee for their valuable inputs, and Emily Lemondsfor linguistic assistance. This work has been supported by twoSpanish Ministry of Science and Innovation (CGL2009-12955-C02-01, CGL2012-39953-C02-01) research projects and by a Span-ish Aragón Government-European Social fund Bioflora grant. A.D.-P. was supported by a Universidad Central de Venezuela CDCHPh.D. fellowship. P.C. was supported by a Spanish Ministry of Edu-cation postdoctoral mobility grant.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2014.06.009.

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