Origin and Timing of the Horizontal Transfer of a Pgic Gene from Poa to Festuca Ovina

Available online at www.sciencedirect.com Molecular Phylogenetics and Evolution 46 (2008) 890–896 www.elsevier.com/locate/ympev Origin and timing of the horizontal transfer of a PgiC gene from Poa to Festuca ovina Pernilla Vallenback *, Maarit Jaarola, Lena Ghatnekar, Bengt O. Bengtsson Department of Cell and Organism Biology, Lund University, Genetics Building, So¨lvegatan 29, SE-223 62 Lund, Sweden Received 30 January 2007; revised 19 July 2007; accepted 26 November 2007 Available online 28 January 2008 Abstract A segregating second locus, PgiC2, for the enzyme phosphoglucose isomerase (PGIC) is found in the grass sheep’s fescue, Festuca ovina. We have earlier reported that a phylogenetic analysis indicates that PgiC2 has been horizontally transferred from the reproductively separated grass genus Poa. Here we extend our analysis to include intron and exon information on 27 PgiC sequences from 18 species representing five genera, and confirm our earlier finding. The origin of PgiC2 can be traced to a group of closely interrelated, polyploid and partially asexual Poa species. The sequence most similar to PgiC2 is found in Poa palustris with a divergence, based on synonymous substitutions, of only 0.67%. This value suggests that the transfer took place less than 600,000 years ago (late Pleisto- cene), at a time when most extant Poa and Festuca species already existed. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Horizontal gene transfer; Festuca ovina; Poa palustris; Cytosolic phosphoglucose isomerase; PgiC2 1. Introduction transposable elements. For example, Mutator-like elements, MULEs, are widespread among plants (Turcotte Eukaryotic organisms normally reproduce sexually and et al., 2001; Yu et al., 2000) and are known to sometimes horizontal transfer of genes, i.e. movement of genes incorporate nuclear fragments (Jiang et al., 2004; Yu between sexually well-separated species, is rare (Andersson, et al., 2000). The first unequivocal evidence of horizontal 2005; Keeling et al., 2005; Martin, 2005). During the last transfer of nuclear DNA in plants was recently reported years, some examples of horizontal gene transfer have, by Diao et al. (2006), who demonstrated that a MULE- however, been described between reproductively isolated sequence has been transferred between rice (Oryza)and plant species (Bergthorsson et al., 2003; Richardson and millet (Setaria) long after the separation of these lineages. Palmer, 2007). The majority of these studies concern genes However, as far as reported no active nuclear gene is asso- encoded by the mitochondria. Davis and Wurdack (2004), ciated with this transfer. for example, have described an instance of horizontal We have previously characterized an additional, second transfer of mitochondrial genes between two species in a locus for a functional nuclear gene specific for the grass host–parasite relationship, while Bergthorsson and col- sheep’s fescue, Festuca ovina (Ghatnekar et al., 2006). leagues (2004) have provided evidence for a massive trans- The gene, a cytosolic phosphoglucose isomerase denoted fer of mitochondrial genes into the basal angiosperm PgiC2, generates functional dimeric enzymes with the stan- Amborella trichopoda. dard locus of the species, PgiC1 (Ghatnekar, 1999). A phy- When horizontal transfer of nuclear genetic material has logenetic analysis based on exon sequences of 1.2 kb in been looked for in plants, most attention has been given to length indicated that PgiC2 has introgressed into F. ovina from the distant genus Poa (Ghatnekar et al., 2006). Two * Corresponding author. Fax: +46 46 147874. facts support the interpretation that this transgression is E-mail address: [email protected] (P. Vallenback). due to a recent horizontal transfer. First, the grass genera 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.11.031 P. Vallenback et al. / Molecular Phylogenetics and Evolution 46 (2008) 890–896 891 Poa and Festuca belong to different subtribes within Poeae some numbers, and the exons and introns sequenced. The (Poaceae) and are completely reproductively separated; in species were chosen based on our earlier results on fact, no sexual hybrid between them has ever been recorded sequence similarities and differences with PgiC2 (Ghatne- (Hegi, 1996; Knobloch, 1968). Second, PgiC2 is not fixed in kar et al., 2006). All plants were collected at sites in south- F. ovina but segregates in the species, with no trace of an ern Sweden. inactive gene copy in plants not expressing PgiC2. One sequence was obtained from each diploid species, Here we present a more detailed phylogenetic analysis of while two different gene copies were sequenced from each PgiC2. We include additional Poa species and base our plant of the four polyploid Poa species used in the analysis most complete study on a 3.7 kb long sequence containing (the very variable species P. pratensis was represented by both exons and introns. With this new information we do two plants). Thus, sequences from diploid plants are desig- not only corroborate our earlier findings, but can show nated without any numerals, while sequences from poly- from where within the speciose Poa genus the PgiC2 gene ploid species are designated first with the plant’s number has come. In addition, we estimate that the horizontal and then with the sequence’s number, see Table 1. Parts transfer took place in late Pleistocene, at a time when most of some of the sequences listed in Table 1 have earlier been extant Poa and Festuca species already existed. reported by Ghatnekar et al. (2006). Five of the analyzed species belong to the fine-leaved 2. Materials and methods festucoids, Aira praecox, F. tracophylla, F. rubra, F. polesica and F. ovina, while four belong to the broad-leaved 2.1. Plant material festucoids, F. altissima, F. pratensis, F. arundinacea and Lolium perenne (Catalan et al., 2004). Of the eight Poa spe- A total of 27 sequences of the gene for cytosolic phos- cies analyzed, P. annua, P. chaixii, P. trivialis, P. supina, P. phoglucose isomerase, PgiC, were investigated from plants nemoralis, P. palustris, P. pratensis and P. angustifolia, the belonging to five genera within subfamilies Poeae and Bro- last four belong to a well-characterized group of closely meae of the grass family Poaceae. Table 1 summarizes the interrelated, partially apomictic polyploids (Patterson species and subspecies analyzed, their recorded chromo- et al., 2005). Table 1 Data on the 27 sequences used in the analyses Species, subspecies Plant/sequence Chromosome number Sequenced parts Aira praecox 1–1 14 Exons 5–10, 16–21 Bromus sterilis 1–1 14 Exons 5–10, 16–21 Festuca altissima 1–1 14 Exons 5–10, 16–21 Festuca arundinacea 1–1 14 Exons 5–10, 16–21 Festuca ovina PgiC1 a 14 Exons and introns 5–21 Festuca ovina PgiC2 b 14 Exons and introns 5–21 Festuca polesica 1–1 14 Exons 5–10, 16–21 Festuca pratensis 1–1 14 Exons 5–10, 16–21 Festuca communtata 1–1 14 Exons 5–10, 16–21 Festuca rubra litoralis 1–1 14 Exons 5–10, 16–21 Festuca rubra rubra 1–1 14 Exons 5–10, 16–21 Festuca tracophylla 1–1 14 Exons 5–10, 16–21 Lolium perenne 1–1 14 Exons 5–10, 16–21 Poa angustifolia 1–1 46–63, 72 Exons and introns 5–21 Poa angustifolia 1–2 46–63, 72 Exons and introns 5–21 Poa annua 1–1 28 Exons 5–10, 16–21 Poa chaixii 1–1 14 Exons and introns 5–21 Poa nemoralis 1–1 28, 35, 42, 49–70 Exons and introns 5–21 Poa nemoralis 1–2 28, 35, 42, 49–70 Exons and introns 5–21 Poa palustris 1–1 21, 28–42 Exons and introns 5–21 Poa palustris 1–2 21, 28–42 Exons and introns 5–21 Poa pratensis 1–1 28–124 Exons and introns 5–21 Poa pratensis 1–2 28–124 Exons and introns 5–21 Poa pratensis 2–1 28–124 Exons and introns 5–21 Poa pratensis 2–2 28–124 Exons and introns 5–21 Poa supina 1–1 14 Exons 5–10, 16–21 Poa trivialis 1–1 14 Exons and introns 5–21 Chromosome number according to Lid (1979). a The earlier described sequence d from locus PgiC1 (see Ghatnekar et al., 2006). b The earlier described sequence c from locus PgiC2 (see Ghatnekar et al., 2006). 892 P. Vallenback et al. / Molecular Phylogenetics and Evolution 46 (2008) 890–896 2.2. DNA isolation, PCR amplification, cloning and DNA structed from multiple equally parsimonious trees. The ML sequencing analyses were carried out using the heuristic search approach and ‘‘as is” as well as ‘‘random” addition repli- Leaf material was ground to powder in liquid nitrogen cate. The hierarchical likelihood ratio test (hLRT) and and total genomic DNA was isolated using the Qiagen the Akaike information criterion (AIC), implemented in DNeasy Plant Mini Kit. PCR was performed using stan- the computer program MODELTEST version 3.7 (Posada dard techniques. Taq DNA polymerase (Roche) was used and Crandall, 1998), were used to establish the most appro- in all nested PCR amplifications and Expand Long Tem- priate model of DNA substitution for the NJ and ML anal- plate PCR System (Roche) was used for cloning purposes. yses. The MODELTEST analyses were performed with and Primers were constructed using Oligo version 6.0 without outgroup. We also used a simpler substitution (MedProbe). Information on primers can be obtained from model, the Kimura 2-parameter method (K2P; Kimura, the authors upon request. 1980), for comparison. Relative stability of MP, ML and Due to selfing or lack of population variation, PgiC NJ phylogenetic trees was assessed with bootstrap analyses sequences could be obtained from most plants by direct using 10,000 replicates, with exception of the ML analysis sequencing of PCR products.

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