280 AMERICAN JOURNAL OF BOTANY [Vol. 89 TABLE 1. Grass taxa used in the study (deposited at Kew) and details of associated voucher specimens. ID Taxon Voucher number 2 5 7 8 23 26 27 30 61 63 72 204 M. giganteus Greef & Deu. ex. Hodkinson & Renvoize Hodkinson s.n. 1990–381 M. sinensis Anderss. Hodkinson 40. 1978–1389 M. sinensis Anderss. ssp. condensatus (Hackel) T. Koyama Renvoize s.n. 1969–19091 M. giganteus Greef & Deu. ex. Hodkinson & Renvoize Gilbert s.n. 1969–19097 M. giganteus Greef & Deu. ex. Hodkinson & Renvoize a Cult. Kew 1780 (Holotype) M. giganteus Greef & Deu. ex. Hodkinson & Renvoize b ADAS MB93/01 M. sinensis Anderss. ‘Goliath’ ADAS MB93/02 M. sinensis Anderss. Hodkinson ADAS MB94/07 M. sacchariflorus (Maxim.) Benth. & Hook. ‘Purpurascens’ Hodkinson s.n. 1987–2727 M. sinensis Anderss. ‘Yakushimanum’ Hodkinson 21 1987–1148 M. floridulus (Labill.) Warb. ex. K. Schum. & Lauterb. Hodkinson 30 1978–1387 M. giganteus Greef & Deu. ex. Hodkinson & Renvoize a Hodkinson s.n. Harvey–1984 a These accessions were previously incorrectly named as M. sacchariflorus in our collection. b This accession was previously incorrectly named as M. sinensis ’Giganteus’ in our collection. line that has undergone subsequent cycles of sexual reproduction, the process of concerted evolution may homogenize copy types but sometimes favors one parental type over the other (Wendel, Schnabel, and Seelanen, 1995a, b; Cronn et al., 1996). In the case of a sterile hybrid such as M. giganteus, concerted evolution could not have occurred by unequal crossing over, and two copy types, corresponding to the two parental species, could be detectable. However, some degree of concerted evolution may have occurred by gene conversion. DNA sequences were used in a similar way by Gaut and Doebley (1997) to investigate the segmental allotetraploid origin of maize. Nuclear DNA sequences, such as ITS, are also subject to recombination and, following a number of generations, individual repeats ofthe ITS sequence cannot only vary from each other but can also become highly heterogeneous themselves. The repeat units can therefore become a mosaic of nucleotides from both parental types such that the original types are not easily distinguished (Wendel and Doyle, 1998). A well-documented example of recombination and concerted evolution is the case of tetraploid cotton, Gossypium L. (Wendel, Schnabel, and Seelanen, 1995a, b), in which two divergent ITS sequences have been combined in the allotetraploid nucleus. A further study involving 5S rDNA in allopolyploid Gossypium (Cronn et al., 1996) showed that concerted evolution had not homogenized this class of repeat. The 5S rDNA sequences are located near the centromere in Gossypium chromosomes (Hanson et al., 1996) and so may be unable to undergo unequal crossingover and thus concerted evolution (Leitch and Bennett, 1997). Concerted evolution has not homogenized the ITS regions of a number of other polyploids even when numerous generations have passed. In a review oftheuseof ITS for phylogenetic reconstruction, Soltis and Soltis (1998) reported that there were still two distinct copy types in polyploid Paeonia L. corresponding to the putative parental species, despite 1 10 6 yr of evolution (Sang, Crawford, and Steussy, 1995). Furthermore, they reported unpublished research by Koontz and coworkers showing that allotetraploid Tragopogon L. species of 80 yr had not experienced concerted evolution. All the studies of concerted evolution cited above involved fertile species. We are unaware of any studies that have examined concerted evolution andthe fate of parental repetitive sequences such as nrDNA in sterile allopolyploids such as M. giganteus that can reproduce by vegetative propagation. In our study the maternally inherited plastid trnL intron andthe trnL-F intergenic spacer (hereafter trnL-F; Taberlet et al., 1991) were sequenced to identify the maternal parent of M. giganteus. Plastid DNA regions have been used to discover the maternal parent of interspecific hybrids in other groups of grasses, such as Spartina Schreb. (Ferris, King, and Hewitt, 1997). The chromosome number and ploidy of seven Miscanthus accessions were also assessed using standard cytological techniques to help interpret the results ofthese other studies. Fluorescent in situ hybridization (including GISH) was used to assess the degree of homology at the repetitive DNA level between the different parental species in M. giganteus. Such techniques have proved useful for studying genome origin and organization in other plants groups (e.g., Bennett, Kenton, and Bennett, 1992; Leitch and Bennett, 1997; Takahashi et al., 1997). For example, Yang et al. (1999) successfully used FISH to study genome structure and evolution in the allohexaploid wild oat, Avena fatua L. MATERIALS AND METHODS Specimens—Specimens came from the Royal Botanic Gardens, Kew and ADAS (Agriculture Development and Advisory Service, which is a consultancy and research organization for agriculture and food, rural development, and environment in the UK and overseas) at the Arthur Rickwood Research Station, Cambridge, UK. Details of voucher specimens of each accession studied are listed in Table 1. DNA extraction—DNA was extracted from 0.5–1.0 g of fresh leaf material using a modified 2 CTAB procedure of Doyle and Doyle (1987) and precipitated using 100% ethanol for at least 48 h at 20C. The DNA was then centrifuged to a pellet, washed with 70% ethanol, and purified via cesium chloride/ethidium bromide (1.55 g/mL) gradient centrifugation with subsequent dialysis. DNA was stored in TE buffer (10 mmol/L Tris-HCl, 1 mM EDTA, pH 8.0) at 80C until use. DNA sequencingand cloning of PCR products—Two DNA regions were sequenced. The first was the ITS region of nuclear 18S–5.8S–25S ribosomal DNA that was amplified by the PCR using primers described by White, Bruns, and Taylor (1990). The second was the spacer and intron regions ofthe plastid trnL-F region that were amplified using the primers ‘‘c’’ and ‘‘f’’ described by Taberlet et al. (1991). The thermal cycling for all PCRs comprised 30 cycles, each with 1 min denaturation at 97C, 1 min annealing at 51C, and an extension of 3 min at 72C. A final extension of 7 min at 72C was included. Amplified, double-stranded DNA fragments were purified using Promega Wizard PCR mini-columns (Promega, Madison, Wisconsin, USA) and sequenced using Taq Dye-Deoxy Terminator Cycle Sequencing Kits (Perkin Elmer Applied Biosystems, Foster City, California, USA) on an ABI 373 or 377 automated DNA sequencer (Perkin Elmer Applied Biosystems). Sequence heterogeneity was found within ITS amplification products of M.
February 2002] HODKINSON ET AL.—ORIGINS OF ALLOPOLYPLOID MISCANTHUS (POACEAE) 281 TABLE 2. Selective amplification primer pairs/anchors used for AFLP analysis of Miscanthus species. EcoRI primer anchor Fluorescent label MseI primer anchor ACA ACT AAC ACC FAM (blue) FAM (blue) TAMRA (yellow) TAMRA (yellow) CAG CAA CTA CAT giganteus. A cloning step was therefore required to obtain clean sequences for this region. Cloning was performed using Promega’s pGem-T Easy Vector System (Promega), andthen the ITS region was reamplified from the transformed bacterial colonies by using a small portion of a colony as the PCR template. Amplified fragment length polymorphism—Amplified fragment length polymorphism is based on the selective amplification of restriction fragments from a digest of total DNA (see Vos et al.  or Ridout and Donini  for a full description ofthe method). Reactions were performed using standard protocols in the AFLP Plant Mapping Kit of ABI (Perkin Elmer Applied Biosystems). The DNA fragments were detected on an ABI 377 automated DNA sequencer with ABI GeneScan 2.02 and Genotyper version 1.1 software. Four primer pairs (see Table 2) were chosen for selective PCR based on a preliminary survey of 32 primer pairs. DNA fragments ranging from 50 to 500 base pairs (bp) from the AFLP analysis were scored. Neighbor joining (NJ) analysis was applied using Nei and Li genetic distances for restriction sites (Nei and Li, 1985; PAUP 4.0: Swofford, 1999). Internal support for groupings was assessed using the bootstrap procedure of Felsenstein (1985). Principal coordinates analysis (PCO) was performed with Le Progiciel R version 4.0d (Casgrain, 1999) using Dice distances (Dice, 1945). Chromosome preparations for Feulgen staining and fluorescent in situ hybridization—Actively growing root-tips were collected from potted Miscanthus plants and pretreated with either 0.2% colchicine at 18C for 5 h or ice cold water for 24 h. Roots were then fixed in 3 : 1 ethanol : acetic acid for a minimum of 1 h andthen hydrolyzed in 1 mol/L HCl at 60C for 11 min. This long hydrolysis time was required because Miscanthus roots are particularly fibrous. Hydrolyzed roots were stained in Feulgen solution (Schiff’s reagent) in the dark for 30 min before the cells were spread on a glass slide and visualized using light microscopy on a Zeiss photomicroscope (Thurnwood, New York, USA). Chromosome preparations for FISH were made according to Takahashi et al. (1997) and stored at 20C until use. Genomic probes—Total genomic DNA from M. sinensis and M. sacchariflorus was randomly sheared to 10 kb (kilobase) by vortexing for 5 min andthen passing it 50 times through a hypodermic needle (Sterican, 1.1 40 mm; B. Braun, Melsungen, AG). Ribosomal DNA probes—Nuclear 18S–5.8S–25S ribosomal DNA (18S– 25S rDNA) sequences were probed using the clone pTa71. This 9 kb clone contains the 5.8S, 18S, and 25S rRNA genes, their internally transcribed spacers, andthe nontranscribed intergenic spacer sequences isolated from wheat, Triticum aestivum (Gerlach and Bedbrook, 1979), and recloned into pUC19. Probe labelling and in situ hybridization—Probes (total genomic DNA and pTa71) were labelled with biotin-14-dATP by nick translation following the manufacturer’s instructions (GIBCO BRL BioNick Labelling System, Life Technologies, Eggenstein, Gebührenfreie Bestellungen, Germany). In all GISH experiments, unlabelled blocking DNA (100–250 bp) from either M. sinensis or M. sacchariflorus was prepared by autoclaving total genomic DNA for 5 min at 105C under 103 421 Pa. The appropriate blocking DNA was added to the genomic probe at a ratio of 1 : 100 (probe to block). Genomic in situ hybridization was performed on M. giganteus using probes from either M. sinensis or M. sacchariflorus according to the method of Takahashi et al. (1997, 1999). Probe hybridization was detected using the Fig. 1. Neighbor joining (NJ) tree of AFLP data for M. giganteus andits putative parental species M. sinensis and M. sacchariflorus. Numbers in brackets refer to identification number given in Table 1. Miscanthus giganteus is approximately equal in genetic distance from M. sinensis and M. sacchariflorus. It does have some unique markers not present in either ofthe putative parental species fingerprinted here; this can be attributed to intraspecific variation in the parental species (such as that seen between the two M. sinensis accessions). fluorochrome fluorescein isothiocyanate (FITC). Chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI), and propidium iodide. RESULTS Amplified fragment length polymorphism—A summary neighbor joining (NJ) tree from the data of four AFLP primers is illustrated in Fig. 1. Accessions of M. giganteus showed little genetic variation and to make the analysis of our results simpler, only two genotypes are presented. In the NJ analysis M. giganteus is approximately equidistant from both M. sinensis (distance of 0.37–0.40) and M. sacchariflorus (distance of 0.38–0.40) accessions. This contrasts with a genetic distance of 0.49–0.50 between M. sinensis and M. sacchariflorus. Miscanthus giganteus has some unique AFLP bands (as indicated by a genetic distance of 0.13 that separates M. giganteus from the branch joining the two putative parental species). The results ofthe PCO analysis are shown in Fig. 2. Miscanthus taxa are clearly separated using the first two axes ofthe PCO, andthese cumulatively account for 88.05% (55.63 and 32.42%, respectively) ofthe data variance; the third axis (not shown) represents 11.91% ofthe data variance. Miscanthus giganteus is approximately equidistant from M. sinensis and M. sacchariflorus on the first axis but is not intermediate on the second axis. DNA sequences—All sequences obtained from this study have been deposited in GenBank (see http://ajbsupp.botany. org/). The heterogeneity detected in the ITS sequences of M. giganteus (Fig. 3a) included sites that were otherwise unique in either M. sinensis or M. sacchariflorus (Fig. 3b, c). An examination of plastid DNA variation in the trnL-F region revealed that M. sacchariflorus and M. giganteus shared a number of nucleotide substitutions not found in any other species examined. Miscanthus giganteus and M. sacchariflorus