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Genetics |
Genome Evolution and Speciation Laboratory. CNRS UMR 6553 University of Rennes 1 Campus Scientifique de Beaulieu, Bât. 141 35042 Rennes Cedex (France)
Received for publication 17 September 2007. Accepted for publication 6 February 2008.
ABSTRACT
The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.
Key Words: allopolyploidy • Bromus • gene duplication • molecular phylogeny • Poaceae • Waxy
Plant evolution is characterized by the prominence of polyploid speciation (Cui et al., 2006
). In the recent years, much progress has been accomplished regarding the contribution of molecular data to the understanding of polyploid genome origin and evolution (Soltis and Soltis, 1999; Soltis et al., 2004a; Wendel and Doyle, 2004). Using a combination of uniparentally inherited chloroplast DNA and biparentally inherited nuclear genes, numerous researchers have documented either multiple recurrent origins (Sharbel and Mitchell-Olds, 2001; Abbott and Lowe, 2004; Doyle et al., 2004; Soltis et al., 2004b) or unique origin (Ainouche et al., 2004; Jakobsson et al., 2006) of polyploid species. Nuclear genes duplicated by polyploidy (homeologues) may be used to identify diploid progenitors (Doyle et al., 2000; Popp and Oxelman, 2001; Petersen and Seberg, 2004; Petersen et al., 2006). Additionally, incongruence among multiple gene phylogenies has been used as an important signal to detect hybrid origins of polyploids (e.g., Baumel et al., 2002; Cronn et al., 2003; Mansion et al., 2005; Popp et al., 2005; Lihova et al., 2006).
Bromus L. represents a large and complex genus of about 160 annual and perennial species (Acedo and Llamas, 2001) in the Pooid clade of the Poaceae family (Soreng and Davis, 2000). This genus is distributed in all continents, and it is notorious for being taxonomically complex (Smith, 1970, 1980; Acedo and Llamas, 1999) because of important morphological variation, plasticity, and hybridization. This considerable heterogeneity has resulted in many different taxonomic treatments. Polyploidy has played a major role in the diversification of this genus (Stebbins, 1981; Armstrong, 1991), ranging from diploids (2n = 14) to decaploids (2n = 70).
Genus Bromus can be divided into six sections according to Smith (1970): Bromus, Genea Dumort., Pnigma Dumort., Ceratochloa (P. Beauv.) Griseb., Neobromus Shear., and Nevskiella V. Krecz and Vved. This study will focus on a group of related annual self-fertilizing species from section Genea Dumort. (= subgen. Stenobromus sensu Stebbins 1981) that originated in the Mediterranean region and Southwest Asia (Sales, 1993, 1994) and that have various ploidy levels from diploid to octoploid. This group includes the diploid B. sterilis L., B. tectorum L., and B. fasiculatus Presl.; the tetraploid B. madritensis L. and B. rubens L.; the hexaploid B. rigidus Roth.; and the octoploid B. diandrus Roth.
Bromus sterilis and B. tectorum are very common and morphologically distinct species (Sales, 1993), with Euro-Siberian and Irano-Touranian range. Bromus sterilis is characterized by a loose and dropping panicle with long peduncles bearing laterally compressed spikelets. Bromus tectorum is easily distinguished by its slender one-sided panicle (Sales, 1991). The third diploid B. fasciculatus has a more restricted distribution in the eastern Mediterranean and is listed as an endangered species in the Mediterranean islands (e.g., Corsica). The depauperate forms of this latter species have some similarities with the tetraploid B. madritensis from which they are usually distinguished by their fan-shaped spikelet (Portal, 1995). The specific status of the polyploid species has been debated because of continuous morphological variation and plasticity (Esnault, 1984; Esnault and Huon, 1985). For instance, controversies arose as whether the tetraploid B. rubens and B. madritensis should be considered as subspecies (Sales, 1994) or as separate species (Oja, 2002b). These species occur commonly in the south and west Europe, the Mediterranean region, and North Africa; they extend eastward until Iran. The hexaploid B. rigidus and the octoploid B. diandrus are common in the Mediterranean region and in South and West Europe. They have very similar morphology and a complex history of taxonomical nomenclature (reviewed in Acedo and Llamas, 1999). These taxa are still considered alternatively as separate species (e.g., Smith, 1980; Böcker et al., 1990), subspecies (e.g., Bolos et al., 1987), varieties (Sales, 1993), or as a polyploid complex (Oja and Laarman, 2002). Both diploid and polyploid species have been introduced into other continents where they occur as aggressive weeds (Roy et al., 1991). The cleistogamous B. tectorum is considered as a particular noxious weed invading open, disturbed and dry areas in North America (Novak et al., 1991; Novak and Mack, 1993), and altering grassland ecosystems (Sperry et al., 2006). Bromus rigidus and B. diandrus have been introduced in Australia, where they are competing with crops (Gill and Carstairs, 1988; Kon and Blacklow, 1990).
In spite of abundant literature on the taxonomical ambiguity, species delimitation problems, and ecological impacts of the invasive species, very few studies have attempted to elucidate the evolutionary relationships among these taxa and the origin of the polyploids using recent genomic approaches. The first molecular phylogeny in Bromus (Pillay and Hilu, 1995) involved chloroplast (cp) DNA restriction site analysis to examine the relationships among representatives of different Bromus sections. This study included two diploids (B. sterilis and B. tectorum) and two tetraploids (B. madritensis and B. rubens) from section Genea that displayed genomic affinity with the species of section Bromus. No hypothesis could be inferred regarding the diploid–polyploid relationships. Thus, there is a great need for a comprehensive analysis including all the diploid and the polyploid species belonging to this section.
By combining the phylogenetic information from two sequence data sets from the maternally inherited chloroplast genome (the trnT-trnL spacer and the trnL-trnF region) and two biparentally inherited nuclear genes (the ribosomal ITS region and the Waxy gene), we aimed with this study to unravel the history and formation of the polyploid species of section Genea. The ITS sequences have already proved their phylogenetic utility in examining diploid–polyploid relationships in section Bromus (Ainouche and Bayer, 1997; Ainouche et al., 1999). The Waxy gene encodes GBSSI (granule-bound starch synthase I) and is considered to be a single copy gene within the Poaceae (Mason-Gamer et al., 1998). Waxy contains 13 exons and 12 introns (Olsen and Purugganan, 2002), the latter often providing sufficient signal for phylogenetic investigations within genera in the Poaceae (Mason-Gamer et al., 1998; Mason-Gamer, 2001; Ingram and Doyle, 2003). Waxy data have been successfully employed to examine the origin and reticulate evolution of polyploid species (Baumel et al., 2002; Ingram and Doyle, 2003, 2004; Smedmark et al., 2003; Mason-Gamer, 2004; Winkworth and Donoghue, 2004; Fortune et al., 2007).
More specifically, we asked the following questions: (1) What are the phylogenetic relationships among species of section Genea, and what are the diploid lineages involved in the formation of the polyploid taxa? (2) Which maternal lineage(s) could have contributed to the present-day polyploids? (3) How congruent are the different gene trees from the nuclear and chloroplast genomes? (4) How many homeologous copies may be distinguished in the nuclear genome of the polyploid taxa that might help identifying their auto- or allopolyploid origin?
MATERIALS AND METHODS
Plant material
The Bromus samples from section Genea included in this study are presented in Table 1. We are interested in unraveling the phylogenetic relationships among the tetraploid species B. madritensis and B. rubens, the hexaploid B. rigidus, the octoploid B. diandrus and their potential diploid progenitors B. fasciculatus, B. tectorum and B. sterilis. Diploid representatives from other Bromus sections have been introduced for comparisons: B. pseudolaevipes (section Pnigma), B. danthoniae, B. pseudanthoniae, B. briziformis, B. japonicus (section Bromus). No diploid species are known in section Ceratochloa, so we included the hexaploid B. catharticus. Three diploid taxa closely related to Bromus in the Pooideae subfamily were used as the outgroup: Hordeum vulgare, Pseudoregneria stipifolia, and Critesion californicum.
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30–50 mg of fresh leaves from each individual using the CTAB extraction buffer from Doyle and Doyle (1987). Fresh leaves were ground in liquid N2, incubated for 1 h at 65°C in 300 µL of 2% CTAB solution with 1% β-mercaptoethanol, and subsequently cleaned with chloroform: isoamyl alcohol (24:1). DNA was precipitated with 90% ethanol, then washed with 70% ethanol, and resuspended in 150 µL Tris-EDTA buffer.
Amplification
PCR was used to amplify Waxy from the 3' end of exon 8 to the 5' end of exon 10 with F-for (5'-TGCGAGCTCGACAACATGCG-3', Mason-Gamer et al., 1998) and K-bac (5'-GCAGGGCTCGAAGCGGCTGG-3', Mason-Gamer et al., 1998) primers. The ITS region of the rDNA was amplified with ITS1 (5' -TCCGTAGGTGAACCTGCGG-3', White et al., 1990) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', White et al., 1990) primers. The chloroplast trnT-trnL spacer was amplified with the a (5'-CATTACAAATGCGATGCTCT-3') and b (5'-TCTACCGATTTCGCCATATC-3') primers, and the chloroplast trnL-trnF spacer was amplified using the c (5'-CGAAATCGGTAGACGCTACG-3') and f (5'-ATTTGAACTGGTGACACGAG-3') primers of Taberlet et al. (1991). Reactions were conducted in 50-µL volumes containing 5 µL 10x RedTaq PCR buffer (Sigma, Saint-Quentin Fallavier, France), 5 µL of 2 mM each dNTPs (Eurogentec, Angers, France), 1 µL 5 pM primer F-for, 1 µL 5 pM primer K-bac, 2.5 U RedTaq DNA polymerase (Sigma, Saint-Quentin Fallavier, France), and 1 µL of genomic DNA solution. PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of 1 min at 94°C, 1 min at annealing, and 2 min elongation at 68°C; with a final extension cycle at 68°C for 10 min. Annealing temperature was 65°C for Waxy and 48°C for ITS, trnT-trnL and trnL-trnF.
Cloning
Cleaned PCR products (using Nucleospin Extract II, Macherey-Nagel, Düren, Germany) were cloned into pGEM-T (pGEM-T Vector System I, Promega, Madison, Wisconsin, USA) following instructions of the manufacturer. After insertion of the PCR product in the plasmid, 2 µL of ligation products were used to transform 50 µL of competent cells (Escherichia coli DH5
) by electroporation (1800 V). Transformed cells were incubated 1 h at 37°C in 1 mL Luria-Bertani (LB) broth (Sigma, Saint-Quentin Fallavier, France), plated on LB agar (Sigma) with X-Gal (Promega), isopropyl-beta-thio-galactoside (IPTG; Promega), and ampicillin (Sigma) for blue/white clone selection. Plates were incubated at 37°C overnight. Multiple colonies from each individual were selected for recombinant plasmids and were further grown in 5 mL LB broth with 100 µg/mL ampicillin. Plasmids were extracted using the Wizard Plus Minipreps kit (Promega). Purified plasmids were tested for presence of the correct insert by PCR under the conditions described. All procedures (PCR, ligation, cloning) were performed twice to minimize technical biases (Cronn et al., 2002; Doyle et al., 2002).
Sequencing
Waxy inserts were sequenced from purified plasmids using the primers described. Because heterogeneous amplicon pools can be expected, 15 inserts were sequenced for diploid species, 20 for tetraploids, 25 for the hexaploid, and 30 for the octoploid as recommended by Small et al. (2004). We also sequenced five ITS inserts per species. The chloroplastic loci were sequenced directly after PCR because no heterogeneity was expected. DNA sequencing was conducted by Macrogen (Seoul, South Korea), using the BigDye terminator method on ABI automated sequencers (Applied Biosystems, Foster City, California, USA). Because a given Waxy sequence type was often recovered several times in a single genomic DNA extract, only one sequence per group of Waxy sequences was used in the final phylogenetic analysis for clarity. In this study, we are primarily interested in identifying divergent sequences among the diploid species that could help the understanding of their phylogenetic relationships and the identification of homeologous copies in the polyploids, rather than exploring the potential allelic variation that may exist at a given orthologous gene within species.
Sequence alignment
Multiple sequence alignment was performed with the program ClustalX (Thompson et al., 1997) and manually corrected in SeqApp (ftp.bio.indiana.edu). The resulting data matrix was imported in the program McClade (Maddison and Maddison, 1992). To examine the potential phylogenetic information contained in insertion–deletion zones, we coded gaps according to the procedure of Barriel (1994).
Phylogenetic analyses
Two phylogenetic reconstruction methods were employed: parsimony analysis and maximum likelihood, both performed with the program PAUP* version 4.0b10 (Swofford, 2001).
Parsimony analyses were conducted with heuristic searches and the default search options. Several exploratory analyses were performed, combining different outgroup taxa. Bootstrap analyses used 10000 replicates for parsimony analyses. Parsimony analyses were conducted on separate data sets. Congruence among the obtained topologies for the ITS and chloroplast data sets was checked with the SLPT test (Templeton, 1983). Maximum likelihood analyses were conducted with heuristic searches as described in Harrison and Langdale (2006). The choice of the model of sequence evolution was performed using the program Modeltest 3.7 (Posada and Crandall, 1998) and Modeltest WebServer (http://darwin.uvigo.es) with all options set to default. Several exploratory analyses were performed on the coding and noncoding portions of the sequences for Waxy. Both exon and intron sequences of Bromus were retained, and Modeltest returned the TrN+G model (Posada and Crandall, 1998) as the best fit model for our Waxy data (unequal base frequencies, proportion of invariable site: 0, gamma distribution shape parameter: 0.4638), the GTR+G model for the ITS (unequal base frequencies, proportion of invariable site: 0, gamma distribution shape parameter: 0.9124) and the TVM+I+G model for the chloroplast spacers (unequal base frequencies, proportion of invariable site: 0.6815, gamma distribution shape parameter: 1.1234). Bootstrap analyses used 1000 replicates for maximum likelihood analyses.
To depict reticulate relationships detected among species, we used multilabelled-tree representation in the PADRE software (Huber et al., 2006) with the Newick most parsimonious tree obtained with PAUP* on the Waxy data set.
Sequence variation
Sequence variation within Genea was examined for the nuclear and chloroplast sequences. To evaluate the influence of genome duplication on individual genes in the polyploid species, we examined the molecular divergence among homologous and homeologous copies encountered for the nuclear Waxy gene. The numbers of substitutions and indels between the different cloned copies of Waxy encountered in the polyploids were tabulated. Distances between the different Waxy sequences in section Genea were computed with the Kimura-2-parameter correction. Coding and noncoding regions were determined by comparison with previously published reading frames of Waxy in Bromus (B. tectorum, NCBI accession AY362757) and checked against the Oryza sativa mRNA sequence (NCBI accession X62134). Coding sequences were translated to check for stop codons. To examine the potential relaxation of selective constraints on the coding portions of the homeologous Waxy copies, we computed substitution rates (synonymous: Ks, nonsynonymous: Ka) using the program DNAsp (Rozas et al., 2003). Neutral sequence evolution is reflected in ratios near 1, whereas constrained sequences have significantly more synonymous than nonsynonymous substitutions (Ka/Ks < 1) and positive selection may lead to Ka/Ks ratios greater than 1 (Yang and Belawski, 2000).
Substitution rate heterogeneity among sequences was examined using Tajima's relative rate test (Tajima, 1993). These analyses were conducted using the program Mega 3.1 (Kumar et al., 2004) with B. catharticus as the outgroup.
RESULTS
Sequence characteristics within section Genea
Sequences characteristics were computed for the different data sets.
trnT-trnL spacer
Length of the sequenced amplicons ranged from 575 to 592 bp for the species of the Genea section and from 575 to 598 for all Bromus species. Accession numbers for these sequences range from EU036136 to EU036162. Alignment of the Bromus sequences and the other Triticeae required the inference of 23 insertion/deletion events (indels) ranging from 1 to 116 nucleotides; this resulted in a data set of 745 aligned nucleotide sites. When the indels are excluded and converted into coded characters, the new data matrix contained 531 characters, of which 438 were invariant and 60 were phylogenetically informative. Pairwise divergence among Genea sequences (including coded gapped sites) varied from 0.6 (B. rigidus–B. diandrus) to 3.1% (B. tectorum–B. fasciculatus), while the same type of pairwise comparison between all the Bromus species varied from 0 (B. pseudodanthoniae–B. japonicus) to 7.1% (B. tectorum–B. catharticus).
trnL-trnF region
Within the Genea section, length variation of the targeted region ranged from 970 to 980 bp, whereas it ranged from 964 to 986 for all Bromus species (GenBank accessions EU036163–EU036188. Alignment of the Bromus sequences and the other Triticeae required the inference of 42 indels ranging from 1 to 113 nucleotides; this resulted in a data set of 1062 aligned nucleotide sites. Exclusion and conversion of the indels into coded characters resulted in a new data matrix of 944 characters (826 invariant, 59 phylogenetically informative). Pairwise divergence varied from 0.2 (B. rigidus–B. diandrus) to 2.6% (B. tectorum–B. fasciculatus) among Genea sequences, and from 0.2 (B. rigidus–B. diandrus) to 3.5% (B. fasciculatus–B. catharticus) between all the Bromus species.
ITS
The five clones per species were found to be almost identical (up to two differential substitutions). ITS sequences used have GenBank accession numbers from EU036189 to EU036208 and from U82325 to U83386 (Ainouche and Bayer, 1997). Length variation of the sequenced region ranged from 414 to 415bp for the species of the Genea section and from 414 to 418 for all Bromus species. The alignment of Genea sequences with the other Bromus and Triticeae sequences required 14 indels of 1–4 nucleotides, resulting in a data set of 426 aligned nucleotide sites. Coding the indels resulted in a new data matrix with 430 characters (275 invariant, 95 phylogenetically informative). Pairwise divergence among Genea sequences ranged from 0.2 (B. rigidus–B. diandrus) to 3.2% (B. rubens–B. madritensis), while the same type of pairwise comparison between all the Bromus sequences varied from 0.2 (B. rigidus–B. diandrus) to 11.3% (B. danthoniae–B. sterilis).
Waxy
We sequenced and analyzed 230 clones for the different Bromus species with 125 cloned sequences for the samples from section Genea. Two very divergent sequence types were encountered in the tetraploid B. madritensis and B. rubens, three in the hexaploid B. rigidus, and four and the octoploid B. diandrus, as expected from their ploidy level. Only one sequence type was found in the diploid species B. tectorum, B. sterilis, and B. fasciculatus as well as in all the other diploid species (B. briziformis, B. danthoniae, B. japonicus, B. pseudodanthoniae, B. pseudolaevipes, H. vulgare, P. stipifolia, C. californicum). Only one sequence type was encountered in the hexaploid species B. catharticus over the five clones sequenced. GenBank accession numbers for the Waxy sequences range from EF656580 to EF656602.
Length variation of the targeted region ranged from 544 to 548 bp for the species of section Genea and from 536 to 555 for all Bromus species. Alignment of Genea sequences and the outgroup sequences from other Bromus species and other Triticeae was straightforward for the exon regions. In the introns, alignment required the inference of 14 insertion/deletion events ranging from 1 to 32 nucleotides; this resulted in a GBSSI data set of 587 aligned nucleotide sites. Among the latter, 331 sites correspond to exon sequence. When the indels are excluded and converted into coded characters, the new data matrix contained 540 characters, of which 359 were invariant and 95 were phylogenetically informative. Among these, 57 sites were in introns and 38 were in the exons, including 14, 4, and 20 sites at the first, second, and third codon positions, respectively. Overall, 11 synonymous and 27 nonsynonymous substitutions were observed at these exon sites. Pairwise divergence among Genea sequences (including coded gapped sites) varied from 0.4 (B. diandrus T–B. rigidus T) to 5.8% (B. rigidus X–B. madritensis F), while the same type of pairwise comparison between all the Bromus sequences varied from 0.4 (B. diandrus T–B. rigidus T) to 9.9% (B. rigidus X–B. catharticus). GC content of the Waxy copies in section Genea ranged from 57.0 to 58.5%.
Phylogenetic analyses
Separate analyses were performed on the different data sets.
Chloroplast DNA sequences
We first performed preliminary analyses of the two chloroplast data sets separately. Because no incongruence was observed among the topologies obtained, we combined the two data sets to maximize the potential information from the chloroplast genome. The maximum parsimony analysis was performed on a data matrix of 1475 characters among which 1259 were constant and only 119 were parsimony informative. The analysis resulted in 15 equally parsimonious trees with a length of 282 (CI = 0.791, including autapomorphies). The strict consensus tree obtained (Fig. 1A) reveals that B. catharticus (section Ceratochloa) is sister to the remainder of the genus, followed subsequently by B. pseudolaevipes (section Pnigma). The other species fall in two well-supported clades. The first one includes the species of section Bromus (bootstrap 97%) where all the phylogenetic relationships appear unresolved in a polytomy. The second clade corresponds to section Genea (bootstrap 80%) and splits in two well-supported subclades: one group including B. fasciculatus, B. rubens, and B. madritensis in a polytomy (bootstrap 75%) and the other one (bootstrap 93%) including the sister species B. tectorum and B. sterilis (bootstrap 98%) and the sister species B. rigidus and B. diandrus (bootstrap 100%). The maximum likelihood tree presented in Fig. 1B displays the same phylogenetic relationships.
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ITS sequences
The maximum parsimony analysis yielded four equally parsimonious trees with a length of 201 (CI = 0.851). The strict consensus tree is presented in Fig. 2A. Genus Bromus displays a basal polytomy including B. pseudolaevipes from section Pnigma and two well-supported clades. The first one (bootstrap 100%) is composed of the species from section Bromus in which B. japonicus is sister to the remaining taxa. The second clade (bootstrap 99%) includes B. catharticus from section Ceratochloa at a basal position. Section Genea is monophyletic (bootstrap 93%). In section Genea, B. fasciculatus and B. madritensis form a clade that is sister to the remainder of the section (bootstrap 87%); a clade of B. tectorum and B. rubens (bootstrap 94%) is then sister to a clade of B. sterilis plus the sister species B. diandrus and B. rigidus (bootstrap 74%).
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Waxy gene
As different copies of Waxy were found in the polyploids, each copy was named "S", "F", or "T" according to its phylogenetic relationship with a copy from a given diploid species, B. sterilis, B. fasciculatus, or B. tectorum, respectively. "X" refers to a copy of unknown origin that is not closely related to any of the extant diploid species studied here.
The maximum parsimony analysis resulted in a single most parsimonious tree (Fig. 3A) with a length of 240 (CI = 0.792). In this tree, B. catharticus is sister to the remainder of the genus. Then the genus splits into two clades. The first one (bootstrap 75%) is divided in two subclades: one containing the S copy of B. diandrus (bootstrap 52%) and the S copy of B. madritensis as sister to B. sterilis, and the other one (bootstrap 93%) containing the sequence of B. tectorum as sister to the T copy of B. rubens (bootstrap 89%) and the T copy of B. rigidus as sister to the T copy of B. diandrus (bootstrap 92%). In the second clade, the X copies of B. rigidus and B. diandrus diverge first as sisters followed by B. pseudolaevipes. The other sequences fall into two subclades. One subclade (bootstrap 100%) contains the remaining sequences of section Genea. In this subclade, B. fasciculatus diverges first as sister to the F copy of B. rubens (bootstrap 95%) followed by the F copy of B. madritensis (bootstrap 62%) and the F copy of B. diandrus sister to the F copy of B. rigidus (bootstrap 67%). The second subclades corresponds to section Bromus in which B. pseudodanthoniae diverges first followed by B. briziformis (bootstrap 95%) and the sister species B. japonicus and B. danthoniae. The maximum likelihood tree is presented on Fig. 3B, and it confirms the phylogenetic relationships among the Waxy copies from section Genea suggested by the parsimony analyses.
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DISCUSSION
The aim of this paper was to investigate the origin of the polyploid species belonging to section Genea using a molecular phylogeny approach based on nuclear and chloroplast DNA sequences.
Comparison of data sets and monophyly of section Genea
The nuclear ITS region and the chloroplast DNA sequences support section Genea as a monophyletic group in the parsimony analyses, whereas this section appears paraphyletic when considering the nuclear Waxy gene phylogeny. In the latter analysis, the F sequences related to the diploid B. fasciculatus appear as sister to species from section Bromus, and the X sequences found in B. rigidus and B. diandrus fall in a basal clade to this group (Fig. 3A). This result would suggest a shared history among species from these two sections of annual bromes in the Old World. Relationships and reticulate events between these two sections have been previously suspected for some tetraploid species belonging to section Bromus and displaying intermediate morphologies such as the African B. pectinatus complex (Scholtz, 1981) or the Australian endemic B. arenarius (Stebbins, 1981), a hypothesis not supported by the homogenized ITS sequence repeats found in these species (Ainouche and Bayer, 1997). A restriction-site-based analysis of chloroplast DNA indicated a derived position of B. tectorum and B. sterilis in a clade including various species from section Bromus and the tetraploid B. rubens and B. madritensis as basal in this clade (Pillay and Hilu, 1995). However, this analysis did not include the third diploid of section Genea (B. fasciculatus), nor the hexaploid and octoploid taxa.
B. fasciculatus and origin of the tetraploid species
In our analyses, the diploid B. fasciculatus appears distantly related to the other diploids B. sterilis and B. tectorum, which have sister relationships in section Genea for the chloroplast sequences. The chloroplast genome data reveal that both tetraploids (B. rubens and B. madritensis) share a maternal lineage related to the diploid B. fasciculatus. This species has a relatively high number (11) of autopomorphic substitutions in the otherwise slowly evolving chloroplast data set we examined (Fig. 1A), suggesting that the actual maternal progenitor of the tetraploids is a more ancient "fasciculatus" type than the present-day species. The nuclear genes provide complementary information regarding both evolutionary gene dynamics in polyploids and phylogenetic relationships. The low copy, nuclear Waxy gene indicates that the tetraploid B. madritensis has retained two homeologous copies closely related to B. fasciculatus and B. sterilis, respectively. The other tetraploid B. rubens has a Waxy copy sister to B. tectorum and another copy related to the B. fasciculatus–madritensis clade. The ITS sequences also strongly support a sister relationship between B. rubens and B. tectorum. All together, these data reveal that B. rubens and B. madritensis are both allotetraploids (Fig. 4A) deriving from the same maternal lineage (represented by B. fasciculatus) and different paternal species (B. tectorum and B. sterilis, respectively). Accordingly, we suggest the following genomic composition: FF SS for B. madritensis and FF TT for B. rubens.
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). Our chloroplast DNA data indicate that this diploid represents the lineage that has provided the maternal genome in both hybridizations. The nuclear gene phylogenies for both maternal and paternal progenitors agreed with the isozyme data of Oja (2002a) in which more similarities were found between B. madritensis and B. sterilis and between B. rubens and B. tectorum and with the morphological data that led Tsvelev (1976) to suggest B. sterilis and B. rubens as potential progenitors of B. madritensis. The tetraploid B. rubens and B. madritensis display a large amplitude of variation and phenotypic plasticity (Wu and Jain, 1978) that resulted in various taxonomic treatments (e.g., Scholtz, 1981). Multivariate analysis of morphological data among tetraploid specimen collected all over the world led Sales (1994) to the conclusion that the range of variation falls into one species with two subspecies: B. madritensis subsp. madritensis and B. madritensis subsp. rubens. These two taxa have different ecologies, both being ruderal, but B. rubens is usually confined to more xeric, desert areas in both the Old World (its native range) and the New World where it has been introduced; these two tetraploids also have different isozyme phenotypes (Oja, 2002b). Our data unambiguously support the independent formation of these two allopolyploids from different paternal progenitors and confirm the recognition of two genomically distinct species.
Origin of the hexaploid and octoploid species
The hexaploid B. rigidus and the octoploid B. diandrus that are very similar in morphology are closely related in our phylogenetic trees. They share a common maternal genome that could not be identified among the analyzed diploid species with our chloroplast sequence data set but that seems closely related to the cytotypes of B. sterilis and B. tectorum. The nuclear Waxy gene is the most informative regarding the diploid genomes involved in the parentage of these taxa because both contain three homeologous copies related to the diploid B. tectorum (T clade), to the diploid B. fasciculatus (F clade), and to an unidentified third ancestor (X clade), which could have been the cytotoplasmic genome donor. In the present state of our data, it is unclear whether the hexaploid has formed independently or whether it originated from hybridization between a tetraploid ancestor (related to B. rubens) and an unidentified (X) parent.
Bromus diandrus has an additional Waxy copy that is closely related to that of B. sterilis. This result would suggest that the octoploid derived from hybridization between the hexaploid B. rigidus (as maternal genome donor) and the diploid B. sterilis, which agrees with the early hypotheses from morphology, experimental crosses (Cugnac and Camus, 1931) and isozyme data (Oja and Jaaska, 1996; Oja and Laarman, 2002). Cytogenetic investigations (Kon and Blacklow, 1990) revealed that both hexaploid (B.rigidus) and octoploid (B. diandrus) have predominantly bivalent chromosomes and a few quadrivalents at meiosis. Based on these observations and on genetic segregation at a morphological marker locus (palea hairiness), they suggested that the genomic formula for B. diandrus was either AABBCCDD (disomic inheritance) or AAAABBCC (disomic and tetrasomic inheritance). Our data support the presence of at least three different genomes in both the octoploid and the hexaploid species (Fig. 4B). The present state of our hypotheses regarding the reticulate origins of the tetraploid, hexaploid, and octoploid species as suggested by the nuclear and chloroplast DNA phylogeny is summarized in Fig. 5.
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; Feliner and Rossello, 2007). In a polyploidy context, sequence heterogeneity is expected as a consequence of biparental inheritance in recently formed species of hybrid origin (e.g., Ainouche et al., 2004; Soltis et al., 2004a;), with various degrees of repeat homogenisation and concerted evolution (Wendel et al., 1995; Ainouche and Bayer, 1997; Rauscher et al., 2002; Kovarik et al., 2004). In the polyploid bromes we have analyzed here, this homogenization seems to have occurred either toward maternal (e.g., in B. rubens) or paternal (e.g., in B. madritensis or B. diandrus) repeats.
The nuclear Waxy gene copies duplicated by polyploidy have been retained in the tetraploid, the hexaploid, and the octoploid Bromus species examined in this study. The substitution rates and Ka/Ks ratios in the coding portion examined suggest that the duplicated homeologues are evolving under selective constraint, which may be related to the functional role of the granule bound starch synthase (Olsen and Purugganan, 2002). Low Ka/Ks ratios for Waxy were also encountered in polyploid Spartina species (Fortune et al., 2007). However, in this latter case, differential loss of duplicated copies occurred in different polyploid lineages that may be a concern for organismal phylogeny inference (Baumel et al., 2002). Interestingly, the homeologous (T and F) Waxy copies of the octoploid B. diandrus had significant rate heterogeneity, which may suggest some degree of evolutionary rate acceleration at higher ploidy levels.
In conclusion, the cytoplasmic and nuclear DNA sequences employed here provided significant information to elucidate the reticulate origins of polyploid annual bromes from section Genea and to identify the parental lineages involved in the formation of the concerned species. Additional nuclear gene and taxon sampling in species from other Bromus sections could provide further insights into the deepest history of Bromus species and perhaps shed light on the unidentified X lineage involved in the hexaploid and octoploid species.
FOOTNOTES
1 The authors thank L. Gicquiaud, M. A. Esnault, R. Amirouche, K. Tielbörger, C. Lampei, L. Janeway, L. Ahart, K. Schierenbeck, and A. M. Chèvre for collecting Bromus samples and F. Ebert for assistance in flow cytometry analyses. This work was funded by UMR CNRS 6553 Ecobio, University of Rennes 1 and the ANR "Polyploidy and Biodiversity" collaborative research project. 
2 Author for correspondence (e-mail: malika.ainouche{at}univ-rennes1.fr), phone: +33 2 23 23 51 11, fax: +33 2 23 23 50 47
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