Molecular Phylogenetics and Evolution 79 (2014) 92–105

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Molecular Phylogenetics and Evolution

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Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephemeral Vulpia grasses and other fine-leaved Loliinae (Poaceae) ⇑ 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, Spain b Department of Botany, Institute of Biology, Tomsk State University, Lenin Av. 36, Tomsk 634050, Russia article info abstract

Article history: The fine-leaved Loliinae is one of the temperate grass lineages that is richest in number of evolutionary Received 11 December 2013 switches from perennial to annual life-cycle, and also shows one of the most complex reticulate patterns Revised 26 May 2014 involving distinct diploid and allopolyploid lineages. Eight distinct annual lineages, that have tradition- Accepted 9 June 2014 ally been placed in the genus Vulpia and in other fine-leaved ephemeral genera, have apparently emerged Available online 18 June 2014 from different perennial Festuca ancestors. The phenotypically similar Vulpia taxa have been recon- structed as polyphyletic, with polyploid lineages showing unclear relationships to their purported diploid Keywords: relatives. Interspecific and intergeneric hybridization is, however, rampant across different lineages. An Fine-leaved Loliinae evolutionary analysis based on cloned nuclear low-copy GBSSI (Granule-Bound Starch Synthase I) and Interspecific hybridization Mediterranean and American silver grasses multicopy ITS (Internal Transcribed Spacer) sequences has been conducted on representatives of most Nuclear (ITS, GBSSI) genes Vulpia species and other fine-leaved lineages, using Bayesian consensus and agreement trees, networking Polyploid speciation split graphs and species tree-based approaches, to disentangle their phylogenetic relationships and to Species tree reconstruction identify 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 diploid lineages. These, in turn, participated in the origin of the derived allopolyploid Vulpia lineages together with other Festuca-like, Psilurus-like and some unknown genome donors. Long-distance dispersal events were inferred to explain the polytopic origin of the Mediterranean and American Vulpia lineages. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction genome, followed by subsequent reductions and genomic rear- rangements that led to the current organization of the main BEP Reticulate evolution is a major phenomenon that has affected and PACMAD genome types (Feuillet and Keller, 2002; Salse the speciation processes of many angiosperm lineages, and partic- et al., 2008). These investigations also supported the existence of ularly the grasses (Stebbins, 1956; Leitch and Bennet, 1997). The more recent genome duplication events, involving the origins of Poaceae family constitutes a paradigmatic case of multiple radia- recent allopolyploids (Salse et al., 2008). Allopolyploidy, as tions across its main lineages, driven in most instances by the con- opposed to autopolyploidy, has been the largest source of grass currence of interspecific hybridization and allopolyploidization species diversity, ranging from fully allopolyploid genera (e.g. the events (Kellogg, 2001; Gaut, 2002). Recent studies, based on megadiverse Calamagrostis) to partially allopolyploid genera, comparative genomics of whole genomes, have evidenced the broadly distributed in both the temperate and tropical Poaceae plausible existence of a paleo-duplication of an ancestral grass clades (GPWG, 2001; GPWG II, 2012). Reticulation has been partic- ularly extensive in some tribes, such as Triticeae and Poeae, where a large number of genera or species are of hybrid allopolyploid ori- ⇑ Corresponding author at: Escuela Politécnica Superior Huesca, Universidad de gin (Dewey, 1984; Quintanar et al., 2007). This led some authors to Zaragoza, Ctra. Cuarte km 1, 22071 Huesca, Spain. Fax: +34 974 239 302. consider some of these reticulate groups as close homeologous E-mail address: [email protected] (P. Catalán). genome cases (e.g. Triticeae), where extensive hybridization was 1 Present address: Instituto de Genética y Centro de Investigaciones en Biot- ecnología Agrícola (CIBA), Facultad de Agronomía, Universidad Central de Venezuela, caused by the absence of interspecific barriers, thereby favoring 2101, Maracay, Aragua, Venezuela. the occurrence of multiple crosses and genome duplications 2 Present address: Department of Biology, University of Shahrekord, Shahrekord, through time, ending in a plethora of new allopolyploid lineages Iran. http://dx.doi.org/10.1016/j.ympev.2014.06.009 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 93 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 of knowledge about the frequency, origin and time-scale of the allo- polyploidization events in other temperate grasses, such as the large pasture and fodder grass subtribe Loliinae, and how they have influenced the speciation rates of these lineages. The Loliinae is one of the main groups of temperate Pooideae which includes some economically and ecologically important for- age and lawn grasses, such as red, sheep, meadow and tall fescues and the ryegrasses (Catalán, 2006). The subtribe comprises the large paraphyletic and worldwide-distributed genus Festuca and several genera nested within it (Catalán et al., 2004). Successive phylogenetic studies based on plastid and nuclear ITS genes have confirmed the divergence of two major clades, broad-leaved and fine-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, and late-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 groups spanning 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 showed that Vulpia and other fine-leaved FEVRE (FEstuca + Vul- pia + RElated) ephemeral lineages had the highest evolutionary ITS rates of all the Loliinae. Those accelerated rates were correlated to the Minimum-Generation-Time (MGT) of these annuals and to the high speciation rate of the group, based on its high number Fig. 1. Phylogeny of the fine-leaved Loliinae (FEVRE) groups studied (see Table 1). of taxonomically distinct annual genera (e.g., Ctenopsis, Mycropy- Summarized combined ITS/trnTF consensus tree (cf. Inda et al., 2008) with rum, Narduroides, Psilurus, Vulpia, Wangenheimia). geographical distributions of the main lineages. FEVRE lineages: Aulaxyper + Vulpia Combined phylogenetic studies of fine-leaved Loliinae con- 2x (violet), American II (pink), Festuca (dark blue), Wangenheimia (light blue), curred in successive divergences of moderately to highly supported Narduroides (light blue), Loretia (dark green), Exaratae (light green), Micropyrum (yellow), Psilurus/Vulpia 4x–6x (orange), American Vulpia (red), American I lineages (Eskia, American I, American Vulpia, Psilurus + Vulpia 4x– (brown), Eskia (gray). The annual FEVRE lineages (underlined) have apparently 6x, Exaratae, Loretia, Festuca + Wangenheimia, American II, and evolved from more ancestral FEVRE perennial lineages. (For interpretation of the Aulaxyper + Vulpia 2x; Fig. 1), which were recovered by both references to color in this figure legend, the reader is referred to the web version of nuclear and plastid trees in most cases (Catalán, 2006; Inda et al., this article.) 2008). Concordant with previous studies, the most striking finding was 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, Vulpia acknowledged that Vulpia and all the ephemeral Loliinae genera taxa were reconstructed into four non-related fine-leaved lineages might have derived from ancestral perennial Festuca lineages (Inda et al., 2008). Furthermore, the Loretia ‘assemblage’ included (Catalán, 2006, and references therein); however, the potential representatives of four (out of five) morphologically dissimilar Vul- existence of multiple independent origins for the taxonomically pia sections (Loretia, Apalochoa, Monachne, Spirachne)(Stace, 1981, similar Vulpia sect. Vulpia taxa remains questionable (Stace, 2005) plus Ctenopsis and F. plicata, whereas representatives of the 2005; Catalán et al., 2007). fifth Vulpia section (Vulpia), which are morphologically similar to Comparative cytogenetic analysis indicated that Vulpia taxa each other, fell into three separate clades:Vulpia 2x, Psilurus + Vul- have a smaller nuclear genome size than that of the Festuca sect. pia 4x–6x, American Vulpia (Inda et al., 2008). Aulaxyper taxa (Smarda et al., 2008), and they lack telomeric het- The investigation of the polyphyly and polyploidy of Vulpia is an erochromatin (Bailey and Stace, 1992; Catalán, 2006). Despite the extraordinary case study. The disparate reconstructions of some large number of taxonomic and cytogenetic differences between Mediterranean diploid (Vulpia 2x) and polyploid (Vulpia 4x–6x) Vulpia and Festuca species, several spontaneous intergenet- Vulpia sect. Vulpia species (Catalán et al., 2004; Torrecilla et al., ic  Festulpia hybrids have been described (Stace and Cotton, 2004) led Stace (2005) to suggest the potential existence of 1974; Stace and Ainscough, 1984; Ainscough et al., 1986). All of unknown underlying reticulation processes among these ‘taxo- those spontaneous hybrids have hexaploid Festuca sect. Aulaxyper nomically undistinguisable’ taxa and other putative alien Loliinae progenitors [F. rubra (6x), F. nigrescens (6x)] and different diploid species. The later discovery of a third independent lineage in the and polyploid Vulpia progenitors [Vulpia sect. Vulpia: V. bromoides New World (American Vulpia), with species morphologically (2x), V. myuros (6x); sect. Monachne: V. fasciculata (4x)]. Addition- resembling the Mediterranean ones, but apparently related to the ally, the mostly sterile pentaploid hybrid  Festulpia hubbardii (V. American Festuca clade (American I) (Inda et al., 2008), further sug- fasciculata  F. rubra) and a wild fertile backcross between it and gested the existence of introgression or the potential evolutionary its male progenitor F. rubra, showed evidence of homeologous or convergence of morphological traits in these annual lineages, orig- heterogenetic pairing between Vulpia and Festuca chromosomes, inating from different polytopic ancestors. It has been confirming continuous introgression of V. fasciculata’s genome into 94 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105

F. rubra’s through this stable backcross (Stace and Ainscough, using the program Bioedit version 7.0.9.0. Each type was coded 1984; Bailey and Stace, 1992; Bailey et al., 1993). These results sug- progressively as shown in Table 1, except for some non-FEVRE spe- gest a long history of genomic admixture between Vulpia and Fest- cies where some available types were included in the study. uca sect. Aulaxyper, even for those Vulpia lineages that are, apparently, distintly related to Aulaxyper (e.g., Loretia (V. fascicula- 2.4. GBSSI recombinants, GBSSI paralogs and ITS pseudogenes ta), Psilurus/Vulpia 4x–6x (V. myuros); Inda et al., 2008). Our study aims to disentangle the complex reticulate evolution- Detection of recombination in the Loliinae GBSSI data set was ary history of Vulpia and other ephemeral fine-leaved Loliinae lin- performed using Rdp, Geneconv, Bootscan, Maxchi, Chimaera, Sis- eages using complementary information from the nuclear ITS can and 3Seq methods implemented in the RDP3 program (Martin (internal transcribed spacer) and GBSSI (granule-bound starch syn- et al., 2010) using the default settings in all cases. Only potential thase I) genes. We wanted to investigate: (i) if diploid Vulpia lin- recombinant events detected by at least two methods were consid- eages had a single (monophyletic) or multiple (polyphyletic) ered significant. The Bonferroni multiple comparison correction origin; (ii) if polyploid Vulpia lineages were allopolyploids derived test was performed to diminish the expected number of false posi- from crosses of diploid Vulpia lineages and alien genomes; (iii) if tive results. Masking of similar sequences was allowed in order to non-related ancestral Festuca lineages were involved in the origins increase the power of the recombination detection methods. of Vulpia lineages; (iv) if intergeneric hybridizations had repeatedly GBSSI sequences of some diploid FEVRE samples showed evi- occurred at different evolutionary times; (v) if introgression was dence of two divergent types that were assumed to be the product polytopic or had a restricted geographic origin. These hypotheses of a duplication event. Following a ‘‘species-overlap rule’’ were tested through independent and complementary Bayesian (Gabaldón, 2008), we inferred that a node was associated with a analyses of nuclear ITS and GBSSI genes, using networking and spe- speciation event if its branches had mutually exclusive sets of dip- cies-tree based approaches. loid species; in contrast, a node with overlapping sets of diploid species was associated with a duplication event. Polyploid samples were excluded from this analysis because homoeologous copies 2. Materials and methods from widely divergent parents might be placed on different branches, hence increasing the number of spurious duplication 2.1. Sampling events. Paralogous groups were further characterized through the Nei-Gojobori Z test method (Nei and Gojobori, 1986; Nei and Sampling of fine-leaved Loliinae included 32 species, covering Kumar, 2000) to test for any sign of relaxation of selective con- almost all the worldwide-distributed species of Vulpia across its strains. In addition, we tested the heterogeneity of substitution five sections [15 spp; sects. Apalochloa (1), Loretia (3), Monachne rates among and within orthologous and paralogous FEVRE (3), Spirachne (1), Vulpia (7)], of the mostly monotypic Mediterra- sequences using Tajima’s relative rate test (Tajima, 1993). nean ephemeral genera [Micropyrum (1), Narduroides (1), Psilurus We looked for putative non-functional sequences of the ribo- (1), Wangenheimia (1)], and representatives of the main Festuca somal 5.8S gene using three complementary approaches (see [sects. and subsects. Aulaxyper (3), Eskia (1), Exaratae (2), Festuca Supplementary information). First, we checked for the absence of (2), and American I clade (1)] and Hellerochloa [American II clade] any of the three conserved plant motifs M1 (CGATGAAGAACGyAGC), (1) perennial lineages (Table 1; Fig. 1). For the GBSSI analysis, a M2 (GAATTGCAGAAwyC) and M3 (TTTGAAyGCA) (Fig. S1, Supple- wider sampling of other Loliinae groups and of several outgroups mentary information), that are present in functional copies was included (Table 1; see also Expanded Material and Methods, (Harpke and Peterson, 2008). Second, we analyzed the inability Supplementary information). The ITS analysis was restricted to of the ITS sequences to generate the conserved secondary structure the fine-leaved group, using the broad-leaved taxon Festuca praten- of 5.8S as another indicator of non-functional sequences (Harpke sis as outgroup. and Peterson, 2008). Third, the Bootstrap Hypothesis Testing (BHT) method (Bailey et al., 2003) was performed to detect pseu- 2.2. DNA isolation, cloning and sequencing dogenes assuming similar non-constrained selective substitution rates between the 5.8S gene region and the ITS1 + ITS2 spacer DNA was extracted from silica gel-dried leaves collected from region. one individual wild plant per species (Table 1). Genomic DNA was isolated using the QIAGEN DNeasy kit method for the GBSSI 2.5. Phylogenetic reconstruction of the fine-leaved Loliinae analysis and the CTAB method (Doyle and Doyle, 1987) for the ITS analysis. Protocols for the amplification, cloning and sequenc- A GBSSI Loliinae tree (LoliinaeGBSSI-tree) was computed, includ- ing of the GBSSI and ITS regions are provided in Supplementary ing both FEVRE samples and other Loliinae and outgroup samples information. (Table 1) to detect ancestral vs. recent GBSSI duplications accord- ing to the ‘‘species-overlap rule’’. We also computed two fine-

2.3. Molecular characterization and preprocessing of DNA sequences leaved Loliinae GBSSI (FEVREGBSSII-tree) and ITS (FEVREITS-tree) trees to test for the monophyletic robustness of FEVRE and for We performed separate molecular characterization of both the the polyphyly of Vulpia, to infer an approximate FEVREspecies-tree, exons and the introns of the GBSSI gene and of the 5.8S gene and and to detect the origin of homoeologous copies in the polyploid the ITS1 + ITS2 spacers of the ITS region. The range of sequence species. In addition, the FEVREITS-tree was able to recover addi- lengths, number of gaps and the expected number of base differ- tional polyploidization events that were not detected with GBSSI ences per site (p-distance) were computed for each case using alone (see Results). According to model selection based on the MEGA v. 5 (Tamura et al., 2011) and Bioedit v. 7.0.9.0 (Hall, 1999). Aikake criterion implemented in the program MrModel Test v. In order to discard spurious variation originating from PCR 2.3 (Nylander, 2004), Bayesian Inference (BI) was performed by (Polymerase Chain Reaction) artifacts, we followed a modified ver- imposing the GTR + C (nst = 6 and rates = invgamma) substitution sion of Sánchez-Ken and Clark (2010) to generate a consensus model plus exon and intron partitions on the GBSSI data set (Lolii- sequence (or type) from closely related sequences for a single indi- naeGBSSI-tree and FEVREGBSSI-tree) and the GTR + C model on the vidual. All intraspecific sequences with a p-distance lower than ITS data set (FEVREITS-tree) using MrBayes version 3.1 0.01 base differences per site were collapsed into a single type (Huelsenbeck and Ronquist, 2001). Indels of GBSSI introns were A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 95

Table 1 Studied 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 and ITS sequences are indicated in Table S1 (see Supporting information).

Lineage tribe/ Species Geographic Ploidy GBSSI Major GBSSI clades ITS (ITS1-5.8S-ITS2) subtribe/genus origin Loliinae Dumort. Fine-Leaved Loliinae (FEVRE)

Festuca L. WFL1 +WFL2 +WFL4 +WFL5

Sect. Aulaxyper F. agustinii Linding. Spain: Canary 2x Fagus1(3),2(1),3(1) WFL1C +WFL1D Fagus1(5) Dumort. Islands

F. rivularis Boiss. Spain: 2x Frivu1(3),2(1),3(1) WFL1A +WFL5 Frivu1(5) Granada

F. rubra L. Romania 6x Frubr1(3),2(1),3(1) WFL1A +WFL1C +WFL5 Frubr1(2),2(2),3(1) (cultivar)

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

F. plicata Hack. Spain: 2x Fplic1(2),2(3) WFL1A Fplic1(5) Granada

Subsect. Exaratae F. borderei (Hack.) K. Richt. Spain: Huesca 2x Fbord1(9) WFL2 Fbord1(5) St-Yves

F. capillifolia Dufour ex Roem. & Spain: Jaen 2x Fcapi1(6) WFL1B Fcapi1(5) Schult.

Sect. Eskia Willk. F. eskia Ramond ex DC. Spain: Huesca 2x Feski1(5) WFL4 Feski1(5)

Inc.sed. F. chimboracensis E.B. Alexeev Ecuador: 6x Fchim1(2),2(2),3(1) WFL1D +WFL2 +WFL4 Fchim1(10),2(1) Cotopaxi

Vulpia C.C. Gmel. WFL1 +WFL2 +WFL3 À WParaph Sect. Vulpia V. bromoides (L.) Gray Spain: Lugo 2x – Vbrom1(5)

V. muralis (Kunth) Nees Spain: 2x Vmura1(4),2(1) WFL1B +WFL1D Vmura1(5) Zaragoza * V. ciliata Dumort Spain: 4x Vcili1(9),2g(1) WFL1B Vcili1(10) Zaragoza

V. myuros (L.) C.C.Gmel. USA: 6x Vmyur1(7) WFL1B Vmyur1(6),2(3),3(1) Washington

V. microstachys (Nutt.) Munro USA: 6x Vmicr1(2),2(2),3(1) WFL1D +WFL2 Vmicr1(5) California V. octoflora (Walter) Rydb. USA: 2x – Vocto1(4) Washington V. australis (Nees) Blom Argentina: 2x – Vaust1*** Entrerrios Sect. Spirachne V. brevis Boiss. & Kotschy Cyprus: 2x – Vbrev1(5) (Hack.) Boiss. Nicosia * Sect. Loretia V. alopecuros (Schousb.) Dumort. Portugal: 2x Valop1(5),2g(1) WFL1B Valop1(5) (Duval-Jouve) Algarve Boiss. 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 V. fontqueriana Melderis & Stace Spain: Segovia 2x Vfont1(5) WFL1C Vfont1(5) Dumort.

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

V. fasciculata (Forssk.) Samp. Spain: 4x Vfasc1(5),2(1),3(1) WFL1B +WFL1C Vfasc1(5) Barcelona

Sect. Apalochloa V. unilateralis (L.) Stace Spain: 2x Vunil1(5) WFL1A Vunil1(5) (Dumort.) Stace Zaragoza

Hellerochloa H. fragilis (Luces) Rauschert Venezuela: ? Hfrag1(5) WFL2 Hfrag1(5) Rauchert Mérida

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 W. lima (L.) Trin. Spain: 2x Wlima1(2),2(2),3(1) WFL1E Wlima1(4),2(1) Moench Zaragoza Broad-leaved Loliinae

Festuca WBL +WBL-Paraph +WBL-Dact

+WOutgr

Subgen. Festuca drymeja Mert & W.D.J. Koch Hungary: 2x Fdrym1(10) WBL – Drymanthele Balaton V.I.Krecz. & Bobrov

Lojaconoa Catalán F. coerulescens Desf. Spain: Cádiz 2x Fcoer1(4) WBL – & Joch. Müll.

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

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

Sect. Subbulbosae F. spadicea L. Spain: Lugo 6x Fspad1(5) WBL-Paraph – Nyman ex Hack.

(continued on next page) 96 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105

Table 1 (continued)

Lineage tribe/ Species Geographic Ploidy GBSSI Major GBSSI clades ITS (ITS1-5.8S-ITS2) subtribe/genus origin

Subgen. Leucopoa F. sclerophylla Boiss. ex Bisch. Azerbaijan: 6x Fscle2(1) WBL-Dact – (Griseb.) Hack. Ordubad

Subgen. F. pratensis Huds. England: 2x Fprat1(10),2(1) WBL-Dact Fprat1(5) Schedonorus (P. Wiltshire Beauv) Peterm.

F. simensis Hochst. ex A. Rich. Uganda: 4x Fsime1(3) WBL-Dact – Echuya

F. fenas Lag. Spain: 4x Ffena3(1) WBL-Dact – Mallorca

Lolium L. L. multiflorum Lam. Egypt (seeds 2x Lmult2(1) WBL-Dact – PI 343155)

L. remotum Schrank France (seeds 2x Lremo1(3) WBL-Dact – PI 283611)

Mycropyropsis M. tuberosa Romero Zarco & Spain: Huelva ? Mtube1(2) WBL – Romero Zarco Cabezudo & Cabezudo Parapholiinae/cynosurinae

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

Hainardia Greuter H. cylindrica (Willd.) Greuter Spain: 2x–4x Hcyli1(4) WParaph – Zaragoza

Cutandia Willk. C. memphitica (Spreng.) K. Richt. Western ? Cmemp1(5) WParaph – Mediterranean Dactylidinae Stapf.

Dactylis L. D. hispanica Roth Spain: 2x Dhisp2(1) WBL-Dact – Alicante

Lamarckia Moench L. aurea (L.) Moench Spain: 2x Laure1(4) WBL-Dact – Zaragoza

Outgroups Poinae – Puccinelliinae

Poa L. P. infirma Kunth Spain: 2x Pinf1(2) WBL – Zaragoza

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

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

Koeleria Pers. K. loweana Quintanar, Catalán & Portugal: 16x Palbi1(4) WKoel – Castrov. (syn. Parafestuca albida Madeira (Lowe) Alexeev) Triticeae Dumort. ** Andropogon L. A. gerardi Vitman 6x Agera (AF079235) WOutgr – ** Agropyron A. cristatum (L.) Gaertner 2x Acris (AF079271) WOutgr – Gaertner ** Triticum L. T. monococcum L. 2x Tmono(AF079286) WOutgr – ** Aegilops L. A. speltoides Tausch 2x Aspel (AF079267) WOutgr – Brachypodieae Harz

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

* g = sequences highly similar to conspecific sequences according to p-distance, but with some different indel positions. ** Mason-Gamer et al. (1998) sequences. *** Direct PCR DNA sequences. inc.sed.: Incertae sedis. treated as missing data, whereas for the ITS binary indel partition expected deep multifurcations generated when low probability data set we imposed the rates = gamma model. For all analyses, we binary partitions were combined into a single well-supported mul- computed six runs, each with 1,000,000 generations and 4 chains, tifurcating node (Cranston and Rannala, 2007). In order to resolve sampling trees every 100 generations, and a burn-in option of 2500 such polytomies, we used a second approach that searches for trees per run once stability in the likelihood values was attained. A agreement subtrees from the posterior distribution of trees majority-rule consensus tree was used to summarize the posterior (Cranston and Rannala, 2007). These fully resolved subtrees are distribution of trees (posterior probability support of branches). present in a larger proportion of the sampled trees than the tree Bootstrap support for branches of the GBSSI and ITS trees was fur- topology with the highest posterior probability, or the maximum ther estimated through a Maximum Likelihood hill-climbing algo- a posteriori tree. Searching for subtrees was performed using the rithm, based on the same parameters as in the respective Bayesian Threshold Accepting (TA) algorithm implemented in the Mapminer searches, using PHYML (Guindon and Gascuel, 2003). Following the program (Cranston and Rannala, 2007), which is a stochastic anal- criterion of Minaya et al. (2013), clades with bootstrap support val- ysis to detect the optimal subset of sequences that need to be ues (BS) of 75–100% or Posterior Probability support values (PPS) of pruned to generate large probability subtrees. For the GBSSI Lolii- 90–100% were considered moderately to strongly supported. nae dataset, we performed an exploratory analysis with k ranging Because the Bayesian majority-rule consensus tree is a combi- from 20 to 60, where k is the number of sequences to be pruned nation of all partitions with probabilities greater than 0.5, we from the Loliinae subtrees. Initial threshold t ranged between A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 97

0.003 and 0.012, with a decreasing threshold Dt of 10% of the initial incomplete lineage sorting, horizontal gene transfer or recombina- value each it-th iteration, it being set to 100. Though we found a tion events (Huson and Bryant, 2006). positive association between the Loliinae-subtree probabilities and different values of k, where the highest value was k = 60, we 3. Results chose a lower k=49, representing approximately 1/3 of the initial number of sequences in the data set, to guarantee the presence of 3.1. Characteristics of the GBSSI and ITS data sets at least one representative sequence of each major Loliinae-tree lineage. Nine independent analyses (runs) were performed subse- The final aligned Loliinae GBSSI data matrix included 1314 posi- quently using k=49 (t=0.1–0.2, Dt=10%, it = 1000). We did not tions, 588 retrieved from exons and 726 from introns. Sequence attempt to generate subtrees for the FEVREGBSSI-tree because this alignment of the GBSSI gene was based on exons 10, 11, 12 and information was contained in the LoliinaeGBSSI-subtrees. For the 13 as described in Mason-Gamer et al. (1998). All studied ITS region, the parameters for the exploratory analysis were set sequences (Table 1) showed exons easily aligned (Table 2). Four to k=1–20, t = 0.001–0.2, Dt=10%, it = 100. Two k values were intron regions were observed between exons 9 and 14: 9–10, selected through independent runs, k = 19 (approximately 1/3 of 11–12, 12–13 and 13–14. The intron 10–11 was absent in the stud- the initial sequences) involving FEVREITS-subtrees with higher ied Loliinae and close Poeae s.l. ougroups (Parapholiinae/Cynosurii- probabilities but showing incomplete sampling of the main nae, Dactylidinae, Poinae-Puccinelliinae, Aveneae). Introns were FEVREITS-tree clades, and k = 10, involving FEVREITS-subtrees with difficult to align because they showed a high number of single- lower probabilities but showing an almost complete sampling of bp indels, ranging from 64.5% of the total aligned positions in Vul- the main FEVREITS-tree clades. We performed five independent pia to 70% in FEVRE (Table 2). The aligned intron matrix yielded runs for k =19(t = 0.15–0.25, Dt=10%, it = 1000) and eight for 726 positions, including the sequences from the close Poeae s.l. k =10(t = 0.02–0.055, Dt=10%, it = 1000). subtribes. In addition to length variation, introns also showed more The TA algorithm generated both a set of subtrees showing over- nucleotide differences per site than exons (Table 2). The mean lapped subsets of clades and a set of clades (among subtrees) show- number of differences between a random pair of sequences was ing mutually exclusive or overlapped subsets of sequences for the 30 (= 0.0523 Â 579 bp) for exons and 46 for introns for FEVRE separate GBSSI and ITS data sets. Given the stochastic nature of and 26 (= 0.0448 Â 579) and 40 for Vulpia, respectively. the TA algorithm and the restriction imposed by k on the final num- The complete FEVRE ITS data matrix included 616 positions, ber of sequences incorporated into the subtrees, the subtrees often 163 corresponding to the 5.8S gene and 453 to the ITS1 + ITS2 showed incomplete sampling of the main LoliinaeGBSSI-tree or spacers (Table 2). The 5.8S gene showed no indels whereas the FEVREITS-tree lineages and incomplete sampling of the whole set ITS1 + ITS2 spacers showed 29 and 26 indels (6.4% and 5.7% of of sequences sampled in each lineage. To uncover those incomplete the total alignment) for the FEVRE and Vulpia data sets, respec- samplings of sequences and clades, we collapsed sequences for a tively (Table 2). ITS percentage values of indels were approxi- given clade into a single subtree leaf; then a supernetwork was com- mately one order of magnitude smaller than those observed in puted using SplitsTree4 version 4.12.3 (Huson and Bryant, 2006)in the GBSSI introns, making ITS an easy-to-align region for FEVRE. order to represent all leaves in a single graphical representation The mean number of differences between a random pair (Huson et al., 2010). Congruence or incongruence among trees of sequences within the 5.8S and ITS1 + ITS2 subregions was 3 was shown as tree-like or net-like split graphs, respectively (= 0.0185 Â 163 bp) and 31 (= 0.0693 Â 453) for FEVRE and 4 (McBreen and Lockhart, 2006). The Zrule, TreeSizeWeightedMean, (= 0.0214 Â 163) and 36 (= 0.0802 Â 453) for Vulpia, respectively Number of Runs = 1000 and the Rooted Equal Angle options were (Table 2). P-distance estimates indicate that the 5.8S gene was less used for the analyses of both GBSSI and ITS data sets. variable than the ITS1 + ITS2 spacers, showing an ITS1 + ITS2 to Considering that hybridization has been reported several times 5.8S ratio of 3.75 for both the Vulpia and the FEVRE data sets. within the fine-leaved Loliinae (Stace and Cotton, 1974; Ainscough The ITS region was more conserved than GBSSI with respect to et al., 1986; Catalán, 2006; Krahulee and Nesvadbová, 2007), we the proportion of single indels and length variation. P-distances complemented the information yielded by the Bayesian trees with also indicated that ITS was less variable than GBSSI, for which a split graph from the GBSSI sequences (LoliinaeGBSSI-graph; see FEVRE and Vulpia showed approximately 74 (34 + 40) vs. 142 Supporting information), which is expected to generate a better- (76 + 66) base differences, respectively. quality representation of incompatible and ambiguous signals in A total of 141 GBSSI clones and 149 ITS clones were generated for the data set that likely resulted from hybridization, but also from the FEVRE data set (Table 1 and S1, Supplementary information).

Table 2 Molecular characterization of GBSSI and ITS nuclear regions for Fine-leaved Loliinae (FEVRE), Vulpia and GBSSI k and d paralogs (see text). N, sampling size (number of studied sequences per group). Length, number of nucleotide positions of the unaligned sequences. % Indels, percentage of single base pair indels of the total aligned matrix; for GBSSI, the matrix also included non-FEVRE groups. p-distance, mean proportion of base differences between a random pair of sequences.

N Exons/5.8S Introns/(ITS1 + ITS2) Length (bp) % Indels p-Distance (range) Length (bp) % Indels p-Distance (range) GBSSI FEVRE 45 579 1.5 (9/588) 0.0523 (0–0.0984) 363–435 70.0 (508/726) 0.1309 (0–0.2081) Vulpia 20 579 1.5 (9/588) 0.0448 (0–0.0881) 365–424 64.5 (468/726) 0.1158 (0–0.2012) k 16 579 1.5 (9/588) 0.0302 (0–0.0535) 363–424 60.6 (440/726) 0.0849 (0–0.1429) d 6 579 1.5 (9/588) 0.0324 (0.0052–0.0518) 378–401 49.0 (356/726) 0.0457 (0.0080–0.0916) k vs. d 0.0452 (0.0230–0.0674) 0.0968 (0.0541–0.1404) ITS FEVRE 35a 163 0 (0/163) 0.0185 (0–0.0859) 433–438 6.4 (29/453) 0.0693 (0–0.1460) Vulpia 16a 163 0 (0/163) 0.0214 (0–0.0491) 433–438 5.7 (26/453) 0.0802 (0–0.1460)

a Vsicu1 was excluded from this analysis due to lack of cloned ITS sequences. Outgroup sequences retrieved from Mason-Gamer et al. (1998) did not include the intron regions. 98 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105

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 Loliinae sequences, respectively. d and k represent paralogs. Recombinant (rec) sequences are shown in green and red letters; ‘M’ and ‘m’ indicate major and minor parental sequences involved in the origins of the recombinants, respectively; T indicates ‘trace’ recombinant sequence and ? indicates uncertain parent sequence. Colored circles indicate the expected phylogenetic relationships according to previous phylogenetic studies (see Fig. 1). Posterior probabilities (100Â) and Bootstrap (%) support values are shown proximal to each node, respectively. Shaded areas indicate the phylogenetic origin of the homoeologous genomes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 99

An average of 5.9 and 5.5 clones per individual were retrieved, suggesting that some sort of positive selection is operating on this respectively, for each molecule. After pruning closely related sequence. Fchim2 and Wlima2 also showed unstable secondary sequences, we obtained a manageable data set of 45 consensus structure, indicating that the three sequences could correspond sequences or types for GBSSI and 33 for ITS, averaging 1.9 and to ITS pseudogenes. 1.2 types per individual. A subset of 79 additional GBSSI clones was obtained from broad-leaved Loliinae and closely related Poeae 3.3. Phylogeny of Vulpia and other fine-leaved Loliinae s.l. subtribes (Tables 1 and S1, Supplementary information). After pruning, a total of 25 types were retained for the GBSSI analysis, The largest phylogenetic reconstruction of Loliinae based on with an average of 1.1 sequences per individual. GBSSI sequences is shown in Fig. 2A (LoliinaeGBSSI-tree). In this topology the studied FEVRE sequences (Table 1) nested into five 3.2. GBSSI recombinants, GBSSI paralogs and ITS pseudogenes major clades with moderate to high posterior probability (PP)

and poor to high BS, respectively (WFL1: 0.51, 70%; WFL2: 0.53, Two recombinant events were detected in some Loliinae GBSSI 29%; WFL3 to WFL5: 1.0, 94–100%). WFL1 showed the largest propor- sequences (Table S2, Supplementary information), with parental tion of FEVRE sequences, further subdivided into five subclades and recombinant sequences completely or partially associated (A–E). The more restricted FEVREGBSSI-tree (Fig. 2B), which with Festuca sect. Aulaxyper taxa. The origin of one recombination excludes the broad-leaved Loliinae and the outgroup samples, also event only involved sequences from this section (Frivu2, Frubr3); supported the existence of the WFL2–5 clades with moderate to high however, that of the other event involved both Aulaxyper (Frivu2) PP and poor to high BS (0.7–1.0 and 38–100%, respectively); how- and more basal Eskia (Feski1) sequences. Most recombinants were ever, the WFL1 sequences were not monophyletic, falling into three found within the Aulaxyper lineage (Frivu1, Frivu3, Frubr2, lineages that showed distinct relationships to the remaining FEVRE

Frubr3), but two relatively divergent sequences from the Festuca sequences: WFL1E paraphyletic and sister (pro parte) to WFL4 (Falpi1) and the Loretia (Fplic2) lineages also showed evidence of (PP = 0.52; BS = 12%); WFL1A sister to WFL2,3,5 (PP = 1.0; BS = 46%); recombination (Tables 1 and S2, Supplementary information). WFL1B,C,D sister to WFL1E (pro parte) (PP = 0.85; BS = 23%). The Lolii- Divergent GBSSI types were found in the diploid species naeGBSSI-graph (Fig. S2, Supplementary information) identified the F. agustinii and V. muralis, suggesting the existence of paralogous same major Loliinae and outgroup clades of the LoliinaeGBSSI-tree, copies. We used the LoliinaeGBSSI-tree (Fig. 2A) to infer the phyloge- associated with splits with moderate bootstrap support. Interest- netic origin of the putative duplication events in FEVRE. The node ingly, the highest-supported (74.9%) split within the FEVRE graph

‘‘D’’ of the LoliinaeGBSSI and FEVREGBSSI trees (Fig. 2A and B) was the core was associated with the largest WFL1 group, indicating a clear shallowest node whose descendant lineages comprised each para- differentiation between the latter and the rest of the Loliinae and logous copy. The sister lineages leading to WFL1D and WFL1B +WFL1C outgroup sequences. It strongly suggested a greater divergence of comprised d and k paralogs, respectively. Given that D and its the WFL1B,C,D group from the WFL1A and WFL1E subclades and from descendant nodes showed moderate to high support according to the remaining GBSSI clades, more than any other split. the FEVREGBSSI-tree (posterior probability between 0.97 and 1 The ITS Bayesian majority-rule consensus FEVREITS-tree (Fig. 3) and bootstrap support (BS) of 77%; Fig. 2B), we are confident of was resolved into six lineages (IFL1–IFL6). Four of the six clades the clear-cut separation of the two paralogous groups. Despite (IFL1,2,5,6) showed high PP but poor to high BS, ranging between putative reticulation events in FEVRE that could have distorted 0.95 and 1 and between 33% and 87%, respectively. IFL1 was roughly phylogenetic relationships in the LoliinaeGBSSI-tree, the Lolii- equivalent (sensu Catalán et al., 2004; Inda et al., 2008) to the Lore- naeGBSSI-graph suggests a compact group of d and k paralogs tia assemblage, IFL2 to the Aulaxyper + Vulpia 2x clades, IFL5 to the defined by the ‘‘D’’ split, although with low (<50%) bootstrap sup- Psilurus/Vulpia 4x–6x clade and IFL6 to the American Vul- port (Fig. S2, Supplementary information). We also observed that pia + American I clades. the two paralogous clades showed incomplete sets of species The GBSSI agreement subtrees computed using the Bayesian sequences (e.g., k WFL1B +WFL1C (Fcapi, Psinc, Valop, Vcili, Vfasc, posterior probability trees generated nine independent 25-taxon Vfont, Vmemb, Vmyur) vs. d WFL1D (Fchim, Vgeni, Vmicr); Fig. 2A LoliinaeGBSSI-subtrees after pruning 49 sequences (2/3) of the and B), suggesting that the PCR and/or the cloning process could whole Loliinae + outgroup data set. Posterior probabilities of these have captured only one paralogous copy in some cases. We cannot subtrees ranged between 0.58 and 0.80, representing values three rule out the possibility that additional d types might have been orders of magnitude higher than that of the Maximum a Posteriori undetected given the homogenizing effect of sampling bias tree (0.000333). The consensus LoliinaeGBSSI agreement subtree, (N = 6). Also, the Nei-Gojobori Z test indicated that exons from both displayed as a supernetwork splits-graph, summarized the sto- paralogs were subjected to purifying selection (P < 0.01) (Table S3, chastic differences among 9 subtree topologies (Fig. 4A). It was Supplementary information). The relative rate test did not detect almost tree-like, supporting congruent topologies among all the different evolutionary rates either within or among paralogs agreement subtrees except for a net-like portion that suggested (Table S4, Supplementary information), except for four pairwise uncertainty in the phylogenetic relationships between the comparisons that involved the V. alopecuros sequence Valop1 WBL-ParCyn-1 and the WFL5 clades. Divergence order in the consensus (Loretia lineage). agreement subtree implied the early split of the outgroup clade

Three ITS sequences from different FEVRE species (F. rubra (WOutgr) followed by the successive divergences of close subtribes Frubr3, F. chimboracensis Fchim2, W. lima Wlima2) failed to gener- plus broad-leaved lineages (WBL-Dact,WBL-Paraph) and then the ate the conserved secondary structure of the 5.8S gene, because major FEVRE lineages. Considering only the FEVRE clades, the some base pairings could not be formed in the B5, B6, B8a and sequence of divergence ranged from the basal-most clade WFL4 to B8b helices (Table S5, Supplementary information). Frubr3 showed WFL2,WFL3 +WFL5 and WFL1; however, FEVRE was not resolved as the largest deviation from the consensus secondary structure, monophyletic, given the unexpected positions of the WParaph and yielding four helices with unpaired bases, the transition C ? Uin WBL clades within it (Fig. 4A). The consensus agreement subtree the B5 helix also being associated with a loss of the conserved also showed evidence of a potential duplication event circum- functional motif M1. This sequence was related to the unique test- scribed by the terminal and monophyletic WFL1B,C,D subclades able branch according to the Bootstrap Hypothesis Testing method. (Fig. 4A).

However, substitution rates in the 5.8S gene (K5.8S) were signifi- Most of the Vulpia (Vulpia 2x, Psilurus/Vulpia 4x–6x, Loretia) cantly higher than the rate of the putative neutral ITS region (KITS), and other fine-leaved Loliinae (Aulaxyper, Exaratae, American I) 100 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105

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 relationships according to previous phylogenetic studies (see Fig. 1). Posterior probabilities (100Â) and Bootstrap (%) support values are shown proximal to each node, respectively. Shaded areas 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 web version of this article.) group samples (sensu Catalán et al., 2004; Inda et al., 2008; Fig. 1) BS = 97%); (iv) Vulpia unilateralis showed a rather striking basal were included within the WFL1B,C,D subclades (Figs. 2B and S2, position with respect to members of its Loretia lineage, which were Supplementary information). Some noticeable inferences were placed in the WFL1B,C,D subclade; by contrast, the close relationship retrieved from WFL1B,C,D: (i) GBSSI types from the tetraploid V. fas- of F. plicata to V. unilateralis agreed with previous phylogenetic ciculata were associated with types from consectional Monanchne findings. diploid species (V. membranacea, V. fontqueriana)(kC paralog subset A deep FEVREITS-tree polytomy (Fig. 3) involving clades IFL1 to with PP = 1.0 and BS = 99%) rather than to Loretia diploid species (V. IFL4 was resolved using agreement subtrees. Five initially indepen- geniculata, V. alopecuros) types (d paralogs in the WFL1D subclade); dent analyses, pruning k = 19 taxa at a time, generated subtrees (ii) types from diploid (Vulpia 2x) and polyploid (Psilurus + Vulpia with posterior probabilities between 0.79 and 0.82 that were

4x–6x) species of two putative polyphyletic lineages were closely summarized in the single supernetwork FEVREITS-subtree (Fig. S3, associated (kB paralog subset; PP = 1.0 and BS = 100%); (iii) types Supplementary information). In that subtree IFL1 and IFL2 were from American species of two putatively distinct lineages, V. micro- resolved as sisters, however no information could be extracted stachys (American Vulpia) and F. chimboracensis (American I), were regarding IFL3 and IFL4. To include all the Bayesian clades in the con- also closely associated among themselves and to other FEVRE line- sensus agreement subtree, we reduced the number of pruned taxa age types (Aulaxyper, Vulpia 2x, Loretia) (d paralogs; PP = 1.0 and from k = 19 (representing approximately 2/3 of the original data A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 101

Fig. 4. Bayesian LoliinaeGBSSI (A) and FEVREITS (B) consensus agreement subtrees and hypothetical FEVRE species tree (C). Consensus agreement subtrees for major clades of Loliinae (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 range of 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 to infer the basic topology and the taxonomic composition of each lineage are shown above each branch. Homoeologous parental genomes involved in the origins of four putative 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.)

set) to k = 10 (1/3). Eight independent k = 10 subtrees showed (IFL1)(Figs. 3 and 4B). The IFL5 clade (PP = 1.0; BS = 87%) diverged lower probabilities (0.22–0.30) than the k = 19 subtrees but included earlier; most of the polyploid Psilurus/Vulpia 4x–6x sequences all the clades. We decided to use the latter set as both sets (k =19 (V. myuros, V. ciliata, P. incurvus) were nested within this clade. and k = 10) were topologically congruent (Fig. 4BandS3, Supple- Interestingly, one hexaploid V. myuros sequence (Vmyur2) fell mentary information). The consensus FEVREITS subtree for k =10 within IFL2A, close to the diploid consectional species. Most of the (Fig. 4B) showed two major subclades within FEVRE (depicted by American-taxa ITS sequences fell within the highly supported sub- the highlighted node with asterisk; Fig. 4B) that mainly comprised basal IFL6 clade (PP = 1.0; BS = 81%; Figs. 3 and 4B). This heteroge- the IFL1 (Loretia s.l. clade, 1.0; 83%) and IFL2 (0.95; 93%), which was neous group included sequences from the American Vulpia further subdivided into the IFL2A (Vulpia 2x, 1.0; 87%) and IFL2B (Aul- (V. octoflora, V. australis) and the American I (F. chimboracensis) axyper, 0.87; 47%) subclades (Figs. 3 and 4B). and American II (H. fragilis) Festuca s.l. lineages. Only the American

IFL4 included sequences of Wangenheimia (W. lima), Narduroides V. microstachys (Vmicr1) sequence fell outside, though close to it. (N. salzmanii), and V. unilateralis (Vunil1), with the latter sequence The Eskia (F. eskia) sequence was resolved as the earliest diverging located apart from the remaining Loretia assemblage sequences lineage within the FEVRE phylogeny (Figs. 3 and 4B). 102 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105

3.4. FEVRE species tree and inferred origins of allopolyploids accumulates three distinct genomes. Two of them (American1–2) likely originated from a polyploid American I and/or from dip- We detected homeologous genomes in the consensus sequence- loid-to-polyploid American Vulpia (V. microstachys)-like genome types from polyploid samples that fell separately into major donors, whereas the third one was related to a basal diploid Bayesian clades of the same tree (e.g., American 1 and American Eskia-type (American3) genome (Fig. 4C). 2 genomes for V. microstachys, and American 1, 2, 3 genomes for The potential female and male nature of the parental genome

F. chimboracensis; FEVREGBSSI-tree, Fig. 2B) associated with donors of the allopolyploid species was deduced from contrasted sequence types from taxonomically close diploid relatives. In addi- analysis of the biparentally-inherited nuclear GBSSI + ITS species tion, potential genome type donors participating in the ancestral tree (Fig. 4C) and maternally-inherited plastid trnTF (Fig. S4, hybridizations included sequences from polyploid samples that Supplementary information) topologies. A diploid maternal Mona- were nested in different phylogenetic positions in each tree [e.g., chne parent was inferred for V. fasciculata, a tetraploid Psilurus/ loretia2 (FEVREGBSSI-tree) and loretia1 (FEVREITS-tree) genomes Vulpia 4x–6x for V. myuros, a potential diploid-to-tetraploid for V. fasciculata (Figs. 2B and 3)]. When diploid sequences were American Vulpia, Exaratae or American I (American2) for V. micro- not detected, we considered polyploid sequences as potential stachys, and a tetraploid V. microstachys-like for F. chimboracensis homeologous genomes if they were placed in different phyloge- (Fig. 4C). netic positions with respect to other types [e.g., psilurus1 with respect to psilurus2 (FEVREITS-tree) for V. myuros (Fig. 3), Ameri- can1, 3 (FEVREGBSSI-tree) with respect to American2 (FEVREITS- 4. Discussion tree) for F. chimboracensis (Figs. 2B and 3)]. We assumed that hom- onymous GBSSI and ITS genomes reflected the same homeologous 4.1. Evolutionary dynamics of the duplicated GBSSI and of the ITS copy, explaining why the GBSSI and ITS American2 or psilurus2 sequences in the fine-leaved Loliinae genomes showed, for example, similar phylogenetic positions and genetic compositions in their respective FEVREGBSSI and Our phylogenetic and networking analyses of the GBSSI FEVREITS trees. The joint inspection of these two trees suggested sequences have demonstrated the existence of at least one duplica- the existence of seven homeologous genomes within the polyp- tion event for this gene within the most recently evolved FEVRE loids (Figs. 2B and 3), including two genomes, psilurus2 (GBSSI k lineages (Figs. 2, 4A, and S2, Supplementary information). Two dif- copy in clade WFL1B+C, Fig. 2B, and ITS IFL2, Fig. 3) and American2 ferent GBSSI paralogs (k, d) have been detected within diploid spe- (clades GBSSI WFL2, Fig. 2B, and IFL6, Fig. 3) that were observed in cies of the Aulaxyper + Vulpia 2x clade (F. agustinii, V. muralis)in both trees. the Bayesian trees (Figs. 2 and 4A) and the split-network (Fig. S2, Based on these results, we propose a hypothetical FEVRE spe- Supplementary information). According to the species overlap rule cies-tree that explains the major allopolyploidization events (Gabaldón, 2008), the WFL1B+C (k) and WFL1D (d) subclades that detected in this study (Fig. 4C). The topological position and the emerged from the ‘‘D split’’ could be associated to a duplication genetic composition of the main clades reflected the phylogenetic event which gave rise to the paralogous Vmura1 vs. Vmura2 and agreement among independent GBSSI and ITS trees and subtrees. Fagus1 vs. Fagus2 + Fagus3 sequences (Fig. 2). The extent of the For example, a major Aulaxyper + Vulpia 2x + Psilurus/Vulpia 4x– paralogy could be even larger in FEVRE, as incomplete sampling 6x + American Vulpia + American I clade was resolved as sister to could have occurred in the diploid V. membranacea, V. geniculata a major Loretia clade based on separate and joint congruent phylo- and V. alopecuros, for which only one type of paralogous copy per genetic relationships between the WFL1B,C,D and IFL2 clades (Figs. 2– species (k or d) was detected (Fig. 2A). 4). In general, the species tree concurs with previous findings, sug- The topological position of node D suggests that the duplication gesting a recently evolved Aulaxyper + Vulpia 2x clade, an interme- event took place after the divergence of the fine-leaved Loliinae diate Psilurus/Vulpia 4x–6x clade and a basal divergence of the lineage, localizing it to a relatively recent clade within FEVRE, as

Eskia lineage and of the diploid-to-polyploid members of the suggested by the phylogenetic position of WFL1B,WFL1C and WFL1D American Vulpia + American I clades (Fig. 4C). Nonetheless, some in the LoliinaeGBSSI-subtree (Fig. 4A). Nonetheless, the unexpected unexpected relationships were also recovered; for example, the closeness of some diploid FEVRE functional sequences (WFL3 subc- closer relationship of the Aulaxyper + Vulpia 2x clade to the Loretia lade: Vgeni2, Vgeni3) to the less-related Parapholiinae/Cynosurii- clade than to either the American II (H. fragilis) or the Festu- nae ones (WParaph), and of some diploid broad-leaved functional ca + Wangenheimia clades. Furthermore, this relationship was sequences (Ftrif2) to the distantly related (e.g., Puccineliinae, Triti- detected in both paralogous copies of GBSSI (e.g., close k Fagus, ceae) ones of the outgroup taxa (WOutgr) suggest the potential Vmemb and Vfont and d Fagus, Vmura and Vgeni sequences; admixture of paralogous sequences within those taxa and, conse- Fig. 2B). quently, a more ancestral scenario for an earlier duplication of According to the phylogenetic positions of the homeologous the GBSSI copies before the split of the Loliinae, and probably genomes (Figs. 2B and 3) we inferred past hybridization events before that of the core-pooids (Figs. 4A and S2, Supplementary that finally led to the formation of four potential allopolyploid spe- information). Mason-Gamer et al. (1998) and Mahelka and cies (Fig. 4C), the tetraploid V. fasciculata, and the hexaploid V. Kopecky´ (2010) considered GBSSI to be a single-copy gene in the myuros, F. chimboracensis and V. microstachys. The tetraploid V. fas- Poaceae. This hypothesis has been refuted, however, by recent ciculata likely originated from the parental diploid Vulpia sect. Spir- studies that have suggested the occurrence of a GBSSI duplication achne (loretia1) and the diploid Vulpia sect. Monachne or the in the Chloridoideae (Fortune et al., 2007), the Panicoideae diploid-to-polyploid Festuca sect. Aulaxyper (loretia2) genomes. (Sánchez-Ken and Clark, 2010) and the Arundinoideae (Zhang The hexaploid V. myuros resulted from two Vulpia sect. Vulpia et al., 2012). Our study provides evidence for the additional exis- parental genomes, the diploid Vulpia 2x (psilurus2) and the tence of such duplication in the temperate Pooideae, supporting polyploid Psilurus/Vulpia 4x–6x (psilurus1) genomes. At least two the idea of an ancestral GBSSI duplication in the grasses genomes (American2, American1) are involved in the origin of (Sánchez-Ken, 2005). As the GBSSI paralogy has been found in both the allohexaploid V. microstachys; its potential parents could be diploid and polyploid species from across the PACMAD and BEP lin- related to American Vulpia, Exaratae or American I (Festuca) eages (Fortune et al., 2007; Sánchez-Ken and Clark, 2010; Zhang lineages of unknown ploidy and to diploid Vulpia2x or Loretia et al., 2012; current study), its origin could predate the PACMAD/ lineages, respectively. The hexaploid F. chimboracensis probably BEP split and might be related to the ancient genome duplication A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 103

event that resulted in the potential modern allopolyploid-derived respective paralogous subclades (WFL1B+Ck,WFL1Dd)(Fig. 2). This grass ancestor (Salse et al., 2008). suggests a recent common ancestry of shared genomes among Despite the existence of duplicated paralogous FEVRE the three groups, which would explain the occurrence of spontane- sequences, phylogenetic analysis of cloned GBSSI sequences, ous intergeneric  Festulpia hybrids involving Aulaxyper and either together with that of cloned ITS sequences, has identified the Vulpia s.s. or Loretia taxa (Ainscough et al., 1986; Krahulee and homeologous parental-type sequences of four allopolyploid FEVRE Nesvadbová, 2007). Due to the potential extended presence of species (Figs. 2, 3, and 4C). In contrast to the paralogous GBSSI recombinant and paralogous copies, the reconstruction of the evo- clones, most of the ITS clones were resolved as monophyletic for lutionary history of FEVRE based on GBSSI sequences is less the diploid species (Results not shown). This suggests a major con- straightforward than when based on ITS. Like ITS, the GBSSI analy- certed evolutionary trend of potential intragenomic ribotypic sis also places V. unilateralis in a separate lineage, outside the ‘D’ diversity to a single species-specific type (Álvarez and Wendel, clade (Figs. 2 and 4A); however, it does not show a close relation- 2003; Nieto-Feliner and Rosselló, 2007). However, different paren- ship to Narduroides, which is nested within the other major split tal ITS copies have been detected in at least one allopolyploid spe- group (Fig. S2, Supplementary information). Also, the unexpected cies (V. myuros; Fig. 3), indicating longtime persistence of the nesting of two Loretia GBSSI sequences (Vgeni2, Vgeni4) within distinct genomic ribotypes in the derived hybrid or the existence the less related Parapholiinae-Cynosuriinae outgroup clade of inter-genomic constraints against ribotypic homogenization. (WParaph)(Fig. S2, Supplementary information) suggests the pres- The higher number of distinct homeologous GBSSI sequences ence of alien or more ancestral genomes in V. geniculata, or the found in the other three allopolyploid species (V. fasciculata, V. putative detection of new paralogs. These features might not corre- microstachys, F. chimboracensis; Fig. 2) by a similar cloning sam- spond, however, to recent genomes introgressed into the genomes pling effort agrees with the higher capability of low-copy genes, of the studied species but to conserved reminiscent regions of the in contrast to ITS genes, in keeping the different parental copies ancestral grass genome (Salse et al., 2008; Mahelka and Kopecky´ , in the hybrid genomes largely intact (Catalán et al., 2012; López- 2010). Álvarez et al., 2012). 4.3. Multiple and polytopic origins of the allopolyploid Vulpia and 4.2. Polyphyly of diploid Vulpia lineages: a rapid radiation scenario of FEVRE taxa ephemeral FEVRE lineages Our comparative GBSSI and ITS analysis has allowed us to iden- Phylogenetic analysis of nuclear low-copy GBSSI and multicopy tify the parental genomes of four allopolyploid FEVRE taxa (Fig. 4C) ITS sequences has successfully contributed to disentangling the and to infer dispersal routes for explaining the polytopic origins of complex reticulate evolutionary history of Vulpia and the main the allopolyploid Vulpia lineages (Fig. 1). The GBSSI data alone FEVRE lineages (Figs. 2–4). The ITS sequences have identified three identified the genome-donor lineages involved in the origin of main diploid Vulpia s.l. lineages, the Vulpia s.s. (IFL2A), the Loretia the hexaploid F. chimboracensis and V. microstachys, whereas the assemblage (IFL1A+B) and the American Vulpia pro parte (IFL6) clades, ITS data identified those of the hexaploid V. myuros (Fig. 3). The with the former more closely related to the Aulaxyper clade (IFL2B), joint analysis of both data sets corroborated the independent find- the second to F. plicata and the third to the American I clade ings and also identified the genome-donors of the tetraploid V. fas- (Fig. 3). This resolution agrees with that of the plastid data ciculata (Figs. 2, 3, and 4C). The comparative analysis of the nuclear (Fig. S4, Supplementary information) and with previous findings (Fig. 4C) vs. the plastid data (Fig. S4, Supplementary information) (Catalán et al., 2004; Torrecilla et al., 2004; Inda et al., 2008). How- has facilitated the identification of the respective maternal and ever the use here of cloned ITS sequences strengthens support for paternal genome donors of the hybrids. Most hybridization events the hypothesis of independent origins of the Vulpia s.s., Loretia and involved parental genomes from the Aulaxyper + Vulpia 2x or Lore- diploid American Vulpia lineages from different Festuca ancestors. tia clades and the Psilurus/Vulpia 4x–6x, American or Eskia clades. This is also corroborated by the phenotypic differentiation of two Apparently, the Festuca + Wangenheimia lineage did not partici- main groups (Cotton and Stace, 1976; Stace, 1981, 2005), suggest- pate in allopolyploid formation for the studied taxa. ing different genomic compositions for the highly homomorphic The origin of the allotetraploid V. fasciculata can be traced back and cleistogamous Vulpia s.s. taxa with respect to the highly to a diploid genome donor from its consectional Monachne lineage diverse and mostly chasmogamous Loretia group taxa. Nonethe- (GBSSI, Fig. 2), which apparently acted as maternal parent (plastid less, there is still uncertainty about the apparent independent ori- DNA; Fig. S4, Supplementary information), and another from the gin of the diploid American Vulpia taxa (V. octoflora, V. australis; cf. morphologically and evolutionarily close diploid Spirachne lineage, Bailey and Stace, 1984), which phenotypically ressemble the Vulpia which acted as paternal parent (ITS, Fig. 3), all nested within the s.s. taxa. A fourth potential Vulpia s.l. diploid lineage, the morpho- Loretia clade. This scenario would explain the high phenotypic logically atypical and chasmogamous Apalochloa (V. unilateralis), resemblances of V. fasciculata to taxa from these two groups also emerges from the ITS tree, sister to Narduroides (Fig. 3). How- (Cotton and Stace, 1977; Stace, 1981; Ainscough et al., 1986) and ever, V. unilateralis is reconstructed within the Loretia clade in the the likely occurrence of the cross between these sister lineages plastid tree (Fig. S4, Supplementary information), indicating the (Torrecilla et al., 2004). Biogeographically, this would imply a dis- likely participation of an ancestral maternal Loretia-type plastid persal of the current eastern Mediterranean endemic Spirachne genome and a paternal Narduroides-type nuclear genome in the donor to the West, and its mating with a likely western Mediterra- present diploid V. unilateralis. This suggests a more complex radia- nean endemic diploid Monachne species, originating the current tion scenario for the diploid Vulpia taxa and confirms the plausible widespread western Mediterranean and European V. fasciculata artificiality of the genus, with some of its current sections previ- tetraploid (Fig. 1). Alternatively, the GBSSI data also suggest the ously described as separate genera (e.g. Loretia, Nardurus (= Apalo- potential implication of an ancestral diploid Aulaxyper lineage as chloa), Vulpia; Stace, 1981; Torrecilla et al., 2004). the potential parent of the species (Figs. 2B and 4C). This scenario In contrast to ITS, the GBSSI reconstructions tend to amalgam- could explain the relatively common occurrence of spontaneous ate the diploid Vulpia s.s. and Loretia group sequences (diploid interspecific crosses from maternal allotetraploid V. fasciculata American Vulpia taxa were not sequenced for GBSSI), together and paternal allohexaploid F. rubra-like taxa, which origi- with some Aulaxyper ones, joining them into the ‘D split’ clade nated  Festulpia hubbardii Stace & R. Cotton individuals, and even and with representatives of each lineage falling together in the the occasional backcross introgression of this highly sterile hybrid 104 A.J. Díaz-Pérez et al. / Molecular Phylogenetics and Evolution 79 (2014) 92–105 with its paternal F. rubra parent (Stace and Ainscough, 1984). This between more ancestral and more recent colonizers on the Amer- would confirm the high crossability of the Aulaxyper taxa with ican continent. other non-related FEVRE and Loliinae taxa (Catalán, 2006). How- A similar, though more complex, biogeographic pattern has to ever, it could also be possible that the Aulaxyper-type GBSSI copies be invoked to explain the origin of the allohexaploid American F. found in V. fasciculata might have been acquired from an opposite chimboracensis (Fig. 4C). In contrast to the primary diploid Mediter- backcross or from more recent hybridization episodes, increasing ranean Loliinae lineages, most of the American lineages (including the Aulaxyper genetic pool in a more ancestral Loretia-type hybrid. all the Southern Hemisphere ones) are of secondary polyploid ori- The identification of the Vulpia 2x and the Psilurus/Vulpia 4x– gin (Catalán, 2006). Two of the three GBSSI homeologous copies 6x lineages as the respective paternal and maternal genome donors detected in F. chimboracensis had the same phylogenetic origin as of the widespread allohexaploid V. myuros (Fig. 3) resolves the those of V. microstachys, suggesting a similar biogeographical sce- apparent contradiction between the separate phylogenetic recon- nario for the origin of the maternal tetraploid ancestor of this spe- structions of the diploid and polyploid Vulpia s.s. lineages observed cies; however, the third homeologous copy came from a more in the previous analyses (Catalán et al., 2004; Torrecilla et al., 2004; ancestral diploid Eskia-type paternal genome (Fig. 2B). This relic Inda et al., 2008) and their close morphological phenotypes (Stace, FEVRE lineage probably colonized the American continent from 1981, 2005). Our results support Stace’s (2005) hypothesis on the the Old World at an earlier stage (Inda et al., 2008) but hybridized likely participation of a diploid V. muralis-like ancestor in the origin to the more recent allotetraploid V. microstachys-type genome of the taxonomically similar V. myuros. The FEVREGBSSI tree also later. indicates the implication of an unknown tetraploid Psilurus or Vul- pia-type genome as the maternal parent of this species (Fig. 2B), Acknowledgments suggesting that the ancestral cross could have occurred in their common native western Mediterranean region (Cotton and Stace, We thank Clive Stace for his insightful comments and fruitful 1976; Fig. 1). The Psilurus/Vulpia 4x–6x clade is an allopolyploid discussions on the systematics of Vulpia, Robert Soreng and an lineage that might have arisen from an ancestral diploid Psilurus- anonymous referee for their valuable inputs, and Emily Lemonds type genome which has repeatedly crossed with the Vulpia 2x gen- for linguistic assistance. This work has been supported by two ome to originate the current V. ciliata (4x) and V. myuros (6x) allo- Spanish Ministry of Science and Innovation (CGL2009-12955- polyploid species. The cumulative genomes of the allohexaploid V. C02-01, CGL2012-39953-C02-01) research projects and by a myuros might have conferred on this Mediterranean endemic Spanish Aragón Government-European Social fund Bioflora grant. annual species a high adaptive capability, having colonized tem- A.D.-P. was supported by a Universidad Central de Venezuela CDCH perate ephemeral pastures on almost all the remaining continents Ph.D. fellowship. P.C. was supported by a Spanish Ministry of where it is an invasive species (Catalán, 2006). Education postdoctoral mobility grant. The disjunct distribution of the American Vulpia taxa (Figs. 1–3) and their surprisingly isolated phylogenetic position with respect Appendix A. Supplementary material to their morphologically close Mediterranean Vulpia 2x lineage (Inda et al., 2008) have been also clarified in our study. The annual Supplementary data associated with this article can be found, in American Vulpia lineage, apparently close to a perennial American the online version, at http://dx.doi.org/10.1016/j.ympev.2014. I(Festuca) lineage (Inda et al., 2008; Fig. 3), consists of several 06.009. endemic North American (V. octoflora 2x, V. microstachys 6x) and South American (V. australis 2x, -studied here for the first time) References taxa of diploid-to-allopolyploid origin (Bailey and Stace, 1984). 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