DNA C-values and chromosome numbers in Fuchsia L. (Onagraceae) species and artificial hybrids

Transcription

1 Talluri & Murray C-values in Fuchsia 33 New Zealand Journal of Botany, 2009, Vol. 47: X/09/ The Royal Society of New Zealand 2009 DNA C-values and chromosome numbers in Fuchsia L. (Onagraceae) species and artificial hybrids R. S. Talluri B. G. Murray School of Biological Sciences The University of Auckland Private Bag Auckland Mail Centre Auckland 1142, New Zealand Abstract Chromosome numbers are reported for 13 species and four artificially produced interspecific hybrids of Fuchsia (Onagraceae). The chromosome number of F. encliandra is reported here for the first time and new chromosome numbers were obtained for F. triphylla and F. boliviana. An approximately 2.4-fold variation in DNA C-value was observed with a range of values from 1.46 pg to 3.44 pg per 2C nucleus. The lowest C-values were found in the species from New Zealand. The four polyploidy species had considerably lower 1Cx-values than most of the diploid species. The nuclear DNA content of the hybrids was, as expected, intermediate between those of the parental species. Keywords chromosome; DNA C-values; flow cytometry; Fuchsia; hybrids; polyploidy; propidium iodide INTRODUCTION Genome sizes (C-values) are now known for approximately 4500 angiosperm species (Bennett & Leitch 2004) and until recently the range of values was about 1000-fold (Doležel & Bartoš 2005). However, the recent paper by Greilhuber et al. (2006) has greatly extended this range, to approximately 2000-fold, following the discovery of ultra-small genomes in species of Lentibulariaceae. Their research B08039; Online publication date 11 March 2009 Received 13 October 2008; accepted 4 February 2009 highlights the need for more extensive sampling of angiosperm C-value diversity, from both a systematic and geographic perspective a theme of two workshops on plant genome size held over the past decade at the Royal Botanic Gardens, Kew, United Kingdom (see Bennett & Leitch 2005). The genus Fuchsia L. (Onagraceae) would appear to be a good candidate for such a survey as it is morphologically diverse and biogeographically unusual with a disjunct distribution between Central and South America (c. 105 species) and islands of the South Pacific (Tahiti and New Zealand; four species). In addition, there have been only 24 previous measurements of C-values in four genera (Clarkia, Ludwigia, Oenothera, and Epilobium) of the Onagraceae, some of which, like Epilobium palustre, have a C-value of only 0.30 pg/2c nucleus, despite the family containing over 650 species (Bennett & Leitch 2004). The mean DNA 2C-value for the family is 2.63 pg, with a range from 0.30 to 6.30 pg. No estimates of nuclear DNA content have been made previously in Fuchsia so this is the first report of C-values for the genus. MATERIAL AND METHODS The plant material used in this study is listed in Tables 1 and 2. The species were obtained as cuttings from Dr Keith Hammett, Massey, Auckland, New Zealand, and grown under glass at The University of Auckland. Controlled crosses to produce interspecific hybrids were made in an insect-free glasshouse and involved the emasculation of one of the parental species followed by the transfer of interspecific pollen to the recipient stigma once it was fully receptive. Chromosome numbers of all species and hybrids were determined using the Feulgen method and squashing the root tips in FLP orcein (Jackson 1973). For the measurement of C-values by flow cytometry, suspensions of intact nuclei were obtained by chopping c. 50 mg fresh, young leaf material with

2 34 New Zealand Journal of Botany, 2009, Vol. 47 Table 1 Species of Fuchsia with their chromosome numbers (2n), 2C-values, standard deviation (SD), and 1Cxvalues (in both pg and mega base pairs (Mbp)). 2C-value 1Cx-value 1Cx-value Species 2n (pg) SD (pg) (Mbp) F. arborescens Sims F. encliandra Steudel F. excorticata L.f F. fulgens DC F. microphylla H.B. & K F. minutiflora Hemsl F. procumbens R.Cunn ex A.Cunn F. splendens Zucc F. triphylla L F. boliviana Carrière F. glazioviana Taub F. hatschbachii P.E.Berry F. magellanica Lam Table 2 Artificial hybrids between Fuchsia species with their chromosome numbers (2n), 2C-values, standard deviation (SD), and expected C-values calculated from the C-values of their respective parental species. 2C-value Expected Hybrids 2n (pg) SD 2C-value F. triphylla F. arborescens F. triphylla F. glazioviana F. boliviana F. magellanica F. boliviana F. glazioviana a pair of new, single edged razor blades in 2 3 ml of ice-cold Galbraith s buffer (Galbraith et al. 1983) to which was added 3% (w/v) polyvinylpyrrolidone. About 20 mg of leaf tissue from seedlings of Hordeum vulgare Sultan was co-chopped with the Fuchsia leaves to provide an internal standard (2C = pg/2c nucleus; Bennett & Smith 1991). The suspension of released nuclei was filtered through a stainless steel mesh with a pore diameter of 32 µm, incubated with RNase for 30 min at 37 C, then stained with propidium iodide (PI, 100 µg/ ml) and left to equilibrate on ice for at least 30 min before being measured. A Coulter EPICS Elite ESP flow cytometer (Beckman-Coulter, Hialeah, Fl, USA) fitted with an air-cooled argon laser emitting light at 488 nm was used to measure the particle fluorescence emission. Approximately particles were measured per run and the C-values were calculated assuming a linear relationship between the measured mean fluorescent intensities of the 2C peaks of Fuchsia and Hordeum and their DNA content (Fig. 1). Five replicate nuclear preparations of each species and hybrid were measured but only single plants of each of these were available for analysis. To check for the presence of compounds in the leaves of Fuchsia that could affect the binding of PI to nuclear DNA samples of Hordeum nuclei, measurements were made on Hordeum nuclei on their own and then after co-chopping with leaves of F. procumbens, as suggested by Price et al. (2000). No significant difference was observed between the two sets of values for the Hordeum nuclei. The terms C-value [the DNA content of a holoploid genome (the whole chromosome complement) with chromosome number n] and Cx-value [the DNA content of a monoploid genome (one chromosome set of an organism) with chromosome base number x] have been used as proposed by Greilhuber

3 Talluri & Murray C-values in Fuchsia 35 RESULTS AND DISCUSSION Chromosome numbers The chromosome numbers of the plants that were studied are given in Tables 1 and 2. The species were either diploid, with 2n = 22 or tetraploid with 2n = 44. Amongst the artificial hybrids, the four combinations had the expected chromosome numbers. Previous reports on the chromosomes of Fuchsia species have been confined mainly to counts of chromosome number. The majority of the species in the genus reported by various workers are diploids with only 18 tetraploids and two octoploids out of some 110 species (Breedlove 1969; Breedlove et al. 1982; Berry 1982, 1985, 1989, 1995; Berry et al. 1988; Hoshino & Berry 1988, 1989; Berry & Breedlove 1996). Only diploid numbers have been recorded in South Pacific Fuchsia; the polyploids were found in the American species. Chromosome numbers have been published for the majority of the species that we have studied (see above) but the number for F. encliandra (2n = 22) is reported here for the first time. In two other species, our observations differ from those published previously: in F. triphylla, Berry (1982) found that 2n = 44, whereas our plants were 2n = 22. The reverse was the case in F. boliviana, which we found to be tetraploid, but it has previously been reported to be diploid (Berry 1982; Breedlove et al. 1982; Bernadello et al. 1990). There would appear to be diploid and tetraploid chromosome races in both of these species, as has been found in other Fuchsia species such as F. alpestris Gardner and F. regia (Vellozo) Munz subsp. regia (Hoshino & Berry 1989). Fig. 1 Flow cytometric analysis of nuclei isolated by chopping young leaves of two Fuchsia species and an interspecific hybrid together with Hordeum vulgare Sultan stained with propidium iodide. The peaks numbered 1 and 2 are the 2C and 4C peaks of the Fuchsia samples; those numbered 3 and 4 are the 2C and 4C peaks of the Hordeum standard. A, F. microphylla; B, F. arborescens; C, F. triphylla F. arborescens. et al. (2005). Values in pg were converted to mega base pairs (Mbp) using the formula 1 pg = 978 Mbp (Doležel et al. 2003). Nuclear DNA amounts Fuchsia species showed an approximately 2.4-fold range of C-values. The lowest value, 1.46 pg/2c, was found in F. excorticata and the highest, 3.44 pg/2c, in F. splendens (Table 1). The same range was seen in the Cx-values but in this case it was F. boliviana that had the smallest value, 0.72 pg/1cx, and F. splendens had the highest, 1.72 pg/1cx (Table 1). All the tetraploid species F. boliviana, F. glazioviana, F. hatschbachii, and F. magellanica had C-values that were less that the highest value for a diploid and similarly their 1Cx-values were lower than those of most diploids, the exceptions being the New Zealand species F. excorticata and F. procumbens (Table 1). The range of C-values observed in these Fuchsia species is well within the range of values previously recorded for the family and do not include

4 36 any species with particularly low values (Bennett & Leitch 2004). However, with previous reports from only four genera and a total of 24 species, the current observations mean that Fuchsia is now the most extensively sampled genus in the family Onagraceae. There are at least two aspects of this survey that are interesting. Firstly, the two species from New Zealand, F. excorticata and F. procumbens, had markedly smaller C-values than all the other seven diploid species that we measured, which originate from South or Central America. These two species were also placed in separate sections of the genus: section Procumbentes contains just F. procumbens, and F. excorticata is in section Skinnera, by Berry et al. (2004). Amongst the American species there does not appear to be any obvious correlation between C-value and geographical distribution, and whether there is an ecological or evolutionary relationship with DNA content in Fuchsia is difficult to ascertain. There is variation in DNA amount across geographically different regions (New Zealand and South/Central America), but whether this is correlated with environment, as previously reported in many genera (Grime & Mowforth 1982; Bottini et al. 2000; Palomino & Sousa 2000), is unknown as detailed information on the ecology of the species is not readily available. The second interesting observation is that Fuchsia, like many other angiosperm genera (Leitch & Bennett 2004), shows that 1Cx-values are smaller in polyploidy species than those of the majority of diploid species. In Fuchsia this possible reduction is large as the tetraploid species had 1Cx-values that were approximately half those of the American Fuchsia species. Only F. excorticata and F. procumbens, the two diploid species with the lowest 1Cx-values, have comparable 1Cx-values to those of the tetraploids but, as indicated, these species are geographically and taxonomically removed from the tetraploid ones. However, it is also possible that there are diploid American Fuchsia species with 1Cx values as low as those of the New Zealand species, and if these were the parents of the polyploids then there would be no reduction in 1Cx values following chromosome doubling. As the genomic origin of our tetraploids is unknown, either of these explanations is equally valid at this stage. In all the hybrids where C-values were analysed, the observed values were very close to the expected mid-parental value (Table 2). It would appear that differences in C-value probably have little effect on hybridisation success in Fuchsia. New Zealand Journal of Botany, 2009, Vol. 47 ACKNOWLEDGMENTS We thank Keith Hammett for providing the plant material and Ross Ferguson and Jingli Zhang (Horticulture and Food Research Institute of New Zealand) for their help with the flow cytometry. This work is part of a PhD thesis submitted to The University of Auckland, New Zealand, by the first author and was supported by a Top Achievers Doctoral Scholarship from the Foundation for Research, Science and Technology and a Doctoral Scholarship from The University of Auckland. REFERENCES Bennett MD, Leitch IJ Angiosperm C-value database (release 5.0, December 2004). cvalues/homepage.html. Bennett MD, Leitch IJ Plant genome size research: a field in focus. Annals of Botany 95: 1 6. Bennett MD, Smith JB Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London B274: Bernardello LM, Stiefkens LB, Piovano MA Números cromosómicos en dicotiledóneas Argentinas. Boletín de la Sociedad Argentina de Botánica 26: Berry PE The systematics and evolution of Fuchsia sect. Fuchsia (Onagraceae). Annals of the Missouri Botanical Garden 69: Berry PE The systematics of the apetalous Fuchsias of South America, Fuchsia sect. Hemsleyella (Onagraceae). Annals of the Missouri Botanical Garden 72: Berry PE A systematic revision of Fuchsia sect. Quelusia (Onagraceae). Annals of the Missouri Botanical Garden 76: Berry PE Two new species of Fuchsia sect. Fuchsia (Onagraceae) from southern Ecuador. Novon 5: Berry PE, Breedlove DE New taxa of Fuchsia from Central America and Mexico. Novon 6: Berry PE, Stein BA, Carlquist S, Nowicke J Fuchsia pachyrrhiza (Onagraceae), a tuberous new species and section of Fuchsia from western Peru. Systematic Botany 13: Berry PE, Hahn WJ, Sytsma KJ, Hall JC, Mast A Phylogenetic relationships and biogeography of Fuchsia (Onagraceae) based on non-coding nuclear and chloroplast DNA data. American Journal of Botany 91:

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