Botanical J'ourrral oj- the Linnean Socieiy , 1985). 90, 209-216. With 6 figures Karyotypes and nuclear DNA amounts in Polypodium L. (Polypodiaceae) BRIAN G. MURRAY, F.L.S.* Department o f Botany and Biochemistry, Westjield College, University o f London, London NW3 7ST Received Fehruay 1985, accepted,Jor publication March 1985 MURRAY, B. G., 1985. Karyotypes and nuclear DNA amounts in Polypodium L. (Polypodiaceae). Karyotype studies in several species of Polypodium show that telocentric chromosomes are the most common with acrocentrics forming the remainder of the complement. T h e relative numbers of these chromosome types can be used as indicators of species relationships although direct comparisons are difficult to make due to the large number of similar-sized chromosomes. T he karyotype dat a support the theory that P. interjecturn is a polyploid derived from the hybridization of P. australa and P . ziulgare. Measurements of nuclear DNA content show that the four diploid species P. austmle, P. scouleri, P. uirginianum and P. glycyrrhiza all have ver>- similar amounts of DNA. T h e trtraploid P. uulgare has one-and-a-half times the DNA content of the diploids and the hexaploid P. intejectum has two times the DNA content of the diploids. The chromosomes of the tetraploid and hexaploid are smaller than those of the diploids and evolution in Po&odium appears to have been accompanied by either a loss or gain of nuclear DNA; the dirrction of the change cannot be ascertained by the present study. ADDI'I'I0S:IL KEY LVORDS: -Natural hybrid ~ nuclear DNA variation polyploidy. - C ONT EN TS . Introduction . Material and methods Results . . Discussion . . . Acknowledgements References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 . . 210 211 2 14 2 15 215 IUTRODLCTION The use of chromosome studies to elucidate patterns of ebolution among the ferns has been confined largely to observations of chromosome pairing at meiotic metaphase in species and natural and artificial hybrids. Karyotype analyses are rare, attributable to factors such as high chromosome number, low mitotic index and the general hardness of the roots, all interacting to make well-spread mitotic metaphase figures difficult to obtain. However, where karyotypes ha\ e been studied useful information about species relationships can be obtained, as * Present addrev Departmrnt of Botan\ ITni\ersit\ of .4uckland, Privatr Bag 4uckland Ye\+ Lealand 0024 407.1 85 010209 f 0 8 SO 3 00 0 209 0 1985 The Linnran Socien of London 210 B. G . MURRAY Walker (1984) has demonstrated in Blechnum. I n other cases, such as the work of Tatuno & Yoshida (1967) and Tatuno & Kawakami (1969), features of the karyotype have been used to support the argument that many diploid ferns are really ancient polyploids since the chromosome complements can be divided into two duplicate sets. Among the angiosperms, differences in nuclear DNA content between related species with the same chromosome number can be very large (Rees & Jones, 1972; Bennett & Smith, 1976; Bennett, Smith & Heslop-Harrison, 1982). I n Lolium, for example, the two diploid species L. perenne and L. temulentum have vastly different DNA amounts, the latter having 35% more than the former (Rees & Jones, 1967). Such differences in DNA content have been used to investigate the relationships of diploid and polyploid species in many genera such as Triticum (Rees & Walters, 1965), Brassica (Verma & Rees, 1974) and Nicotiana (Narayan & Rees, 1974), and when differences in DNA density and amount of heterochromatin are taken into account these studies have been useful in identifying or confirming the diploid progenitors of polyploid species. Direct measurements of nuclear DNA amounts in ferns are few and in many cases only a single species has been studied (Ophioglossum petiolatum: Price et al., 1972; Ceratopteris thalictroides: Polito, 1980). An exception is the work of Vida & Mohay (1980) who examined five species of Cystopteris, several of which show intra-specific polyploidy. Their study shows that within ploidy levels DNA values between species are almost identical and the polyploids contain direct multiples of the diploid values. Thus, in Cystopteris little information about evolutionary relationships can be obtained from DNA values. The aim of this study was to see whether karyotype analyses and nuclear DNA measurements could be used to study evolutionary relationships within the Polypodium vulgare species complex. The complex consists of diploid, tetraploid and hexaploid species in Europe and diploid and tetraploid species in N America. Lovis (1977) has clearly summarized the established and hypothetical relationships of these species. Polypodium vulgare L. sensu stricto is thought to be the allotetraploid derivative of diploid P. uirginianum L. and P. glycyrrhiza (DC) Eat., and the hexaploid P. interjectum Shivas is thought to have arisen by hybridization of this tetraploid with the genomically unrelated diploid P. australe Fte followed by chromosome doubling. Manton (1950) and Shivas (1961a) also showed that P. vulgare and P. interjectum hybridize to produce a pentaploid hybrid. DNA values have been measured in these taxa and in one other diploid member of the complex, P. scouleri Hook. & Grev. These are presented here together with some karyotype data. MATERIAL AND METHODS The plants of Polypodium used in this study are listed in Table 1. For karyotype studies, actively growing roots were obtained by growing the plants in a greenhouse at 20IfI5"C and keeping them well watered. The root tips were pretreated in a saturated solution of paradichlorobenzene for 20 h at 4°C. They were then fixed in ice-cold 4% formaldehyde in M/15 phosphate buffer, pH 7, for 1 h. After fixation they were washed in running water for 1 h before hydrolysis in 5 M HCl at room temperature for a further 1 h. They were then stained in Feulgen for 1 h before the terminal 1-2 mm of the root was macerated KARYOTYPES AND DNA IS POLYPODILT,\I 211 Table 1. Place of origin, source and ploidy level of the Polypodium species used in this study Species Ploidy levrl Source and place of origin ~~ P. australe P. scoulerz P. r,irginianum P. glycyrrhiza P. aulgare P. ~'ulgarex P. zntrrjectum P. interjectuni ~~ x X X X 4x 5x 6x University of London Botanical Supply Unit; origin unknown. Dr .M. Gibby, Chelsea Physic Garden; origin unknown. Dr A. Sleep, University of Leeds; Halifax, Nova Scotia, Canada Dr A. Sleep, University of Leeds; Duncan, BC, Canada. B.G.M. nr Colchester, Essex and nr Buxton, Derbyshirc. University of London Botanical Supply Unit; origin unknown. Dr M. Gibby, Chelsea Physic Garden; Cornwall Dr A. Sleep, University of Leeds; Cornwall. with a brass rod in a drop of aceto-orcein. A cover slip was then applied, the cell suspension heated and finally squashed between layers of filter paper. Measurements of total chromosome lengths were made from camera lucida drawings using a map measurer. With the exception of P. inteyectum, where only five cells were measured, measurements were made on 10 well-spread metaphase cells. Nuclear DKA4measurements were made on root tip nuclei that were fixed as above but for a period of 2 h. The material was washed overnight in running water and then hydrolysed in 5 M HC1 (Analar) for 60 min at 20°C. After washing for 1 min in distilled water the roots were stained for 1 h in Feulgen, pH 3.6. The root tips were then washed three times in SO,-water and finally in distilled water before being squashed in 50°, glycerine on a slide. Measurements were made on 25 presumed 2C nuclei and five presumed 4C nuclei. On11 single plants of P. gbcyrrhiza, P. uirginianum, P. scouleri and the pentaploid hybrid were available for study, but for the other three species at least two different plants were studied. A4tleast thrce sets of densitometry readings were made for each of the species. The conversion of densitometry readings to absolute DNA amounts was carried out by including Allium cepa (2C DNA content = 33.55 pg) root tips as a control with each batch of fern roots. This was done after it had been established that with this staining schedule the optimum hydrolysis and staining times were the same for the two types of material. Measurements of nuclear DNA and nuclear area were made with a Vickers M85 integrating microdensitometer. RESULTS The karyot) pes of four of the species are illustrated in Figs I , 2, 5 & 6. In all four species the majority of the chromosomes are telocentric with acrocentrics making up the rest of the complement. In P. australe, 50 out of the total of 74 chromosomes are telocentrics and they range in size fi-om 4.5 to 2.2 pm. The acrocentrics are of similar size and range from 5 to 3 pm in length. Polypodzum scouleri, the other diploid studied, has a similar karyotype but uith 54 telocentrics and 20 acrocentrics which are of a size similar to those of P. nushale, although P. scoulerz does appear to have one pair of telocentrics thdt is significantly smaller than the others. The tetraploid P. fiulgart and hexaploid 212 B. G. MURRAY Figures 1-4. Fig, 1. Mitotic metaphase in P. australe (2n = 74). Fig. 2. Mitotic metaphase in P. uulgare (2n = 148). Fig. 3. Interphase nucleus in P. australe. Fig. 4. Interphase nucleus in P . interjecturn. Scale bar = 10 pm. P. interjecturn also have karyotypes made up of telocentric and acrocentric chromosomes. P. uulgare (Fig. 6A) has 40 acrocentrics and 108 telocentrics while P. interjecturn (Fig. 6B) has 64 acrocentrics and 158 telocentrics. In the pentaploid hybrid the maximum number of acrocentrics observed was 39 with 146 telocentrics making up the rest of the complement. In all the species there is KAR YOTYPES AND DNA IN POLYPOD!Df A 213 iii«.ttu••n •t••''" lltta••••• •.. •..,•..,,.,,.........,...... . "...,.... I,., ~ B IJIJifll UlttllllfiiCIIJU ,,. ''' IJC ,. ,., •• , lltUiti»C.IUI ,........... FigureS. KarYotvpes of two diploid Polypodium species (2n = 74). A. P. scouleri. B. P. australe. Scale bar= IOJ.lm. an almost continuous gradation in chromosome size in both the aero- and telocentrics. ~Ieasurements of total chromosome length (Table 2) show that this is very similar in the two diploids. In P. vulgare and P. interjectum the total chromosome length is, respectively, much less than twice and three times that of ...................................................... , ..... ..•.••........... •............•.•.., s IIIIJJIUUtJUIIItiiU Jill I nt liUIIUI u'"n uuu n' 111n UIJIJJU tUUUI~Jttant UUUIIUIIUIUIU IU&.IIUUI lltJ)fUIIUIUU.,' UU tiUUU U) III II lf,UU t JIU .,, U uu IIUfUUUUUtU UUI"CUfUUttltltiUtiU••• Figure 6. Karnltypes. :\. P. 1·ulgare (2n = 148). B. P. interjectum (2n = 222 . Scale bar = 10 J.lm B. G. MURRAY 214 Table 2. Nuclear DNA content, total chromosome length, nuclear area and DNA density in six species and one interspecific hybrid of Polypodium. Nuclear area and total chromosome length are given in arbitrary units DNA content (pg/LC nucleus) Total chromosome length Nuclear area (2C) density 34.2 k 2.5 45.3 0.45 148 185 20.15 20.50 20.66 2 1.40 30.75 35.65 33.5 k 3.8 58.5 & 7.3 74.7+ 13.8 47.2 58.9 70.1 0.45 0.52 0.51 222 39.32 92.2 k 5.8 72.6 0.54 Species 2n P. scouleri P. g l y y r h i z a P. uirginianurn P. australe P. vulgare P. uulgare x P. interjecturn P. interjecturn 74 74 74 74 ~ - ~ DNA ~ - the diploids. The value for the pentaploid hybrid is midway between those of the tetraploid and hexaploid species. Measurements of nuclear DNA amount were made on P. glycyrrhiza and diploid P. uirginianum as well as the other four species and the pentaploid hybrid mentioned above. The results are presented in Table 2 and show that all four diploid species have approximately equal amounts of nuclear DNA (20 pg/2C nucleus). However, tetraploid P. vulgare has only one-and-a-half times the DNA of the diploids and the hexaploid only two times the DNA of the diploid. The DNA content of the pentaploid hybrid is more-or-less midway between that of the tetraploid and hexaploid. Measurements of nuclear area were made and these, together with DNA density (DNA amount/unit area), are also given in Table 2. The values for DNA density are similar for all the species although they do show a slight increase with increasing ploidy level. Observations on interphase nuclei show that their organization is very similar in all the species (Figs 3 & 4). Conspicuous chromocentres were not observed and the nuclei appear to be relatively homogeneous. DISCUSSION An interesting feature of the karyotype of these Polypodium species is the presence of such a large number of telocentric chromosomes. Among the angiosperms, telocentrics are uncommon but in several less-advanced groups of plants such as the gymnosperms (Hair & Beuzenberg, 1958) and cycads (Marchant, 1968) as well as in other genera of ferns such as Blechnum (Walker, 1984), they have been reported to be common. This apparent difference in frequency could be used to argue in support of the primitive nature of the telocentric chromosome, but as Jones (1970, 1978) has pointed out chromosomes undergo cycles of change and there now seems to be little evidence, or indeed any need, to propose the primitive or advanced nature of any particular chromosome type. The information gained from these karyotype studies appears to support previous conclusions based on morphology and genome analysis (Manton, 1950; Shivas, 1961a, b) that hexaploid P. interjectum combines the genomes of tetraploid P. uulgare with that of the diploid P. australe. Polypodium interjectum has 64 acrocentrics and 158 telocentrics, which represents KARYOTYPES AND DNA IN POLYPOD/U.'vf 215 the combined totals of these two chromosome types from P. vulgare and P. australe. However, care should be taken not to overemphasize this sort of evidence since these chromosomes are both small and numerous and it can be difficult to decide whether a chromosome is truly telocentric. In addition, the aero- and telocentric chromosomes in each of the species show a continuous range in size, and although the largest members of each group are twice the size of the smallest it is almost impossible to group the chromosomes into mutually exclusive size classes. Finally, it must also be remembered that chromosomes evolve and that karyotype analysis by matching patterns does not prove that morphologically similar chromosome sets are homologous. There is also the problem of the pentaploid hybrid. Only one plant of this hybrid was available for study. This had 39 aero- and 146 telocentric chromosomes, numbers or proportions that seem to bear no clear relationship with the expectation that it is a hybrid between P. vulgare and P. inteljectum. Such a hybrid should have 51 acrocentrics and 134 telocentrics. It is possible that there has been considerable repatterning of the karyotype in this hybrid or that hybridity alters centromere 'expression' or makes them more difficult to identify. Nuclear DNA values are also of little use in unravelling species relationships in Polypodium. As in C':Jslopteris (Vida & Mohay, 1980) the diploid species of Polypodium all have similar DNA amounts and therefore these data cannot be used to help identify putative genome donors. In Polypodium there is also the unexpected result that the polyploid species do not contain exact or even very similar multiples of the DNA values of the diploids. The polyploids have component genomes smaller than those of the diploids, a factor that is also reflected in the observation that the polyploids have a relatively smaller total chromosome length than the diploids. Since nuclear organization in the interphase nuclei appears similar in all the species, differences in DNA density (which are in any case small in Polypodium) would not appear to be an important factor distorting densitometry readings, as has been found in Brassica and Nicotiana (Narayan & Rees, 1974; Verma & Rees, 1974). Thus, it appears that changes in genome size have occurred with time in Polypodium, but whether there has been a loss of DNA in the polyploids or a gain in the diploids cannot be ascertained at present. It is interesting to note at this stage that although only limited data are available, there seems to be remarkably little DNA variation within ploidy levels in the ferns. This situation is in complete contrast to other plant and animal groups, and it therefore seems unlikely that measurements of DNA amount will be of much use in unravelling evolutionary relationships in the ferns. ACKNOWLEDGEMENTS I would like to thank Dr Mary Gibby of the British Museum (Natural History) and Dr Anne Sleep of the University of Leeds for the loan of Po(vpodium plants. REFEREXCES ~1. D. & S:\llTH.J. B.. 1976. :\'uclear DNA amounts in angiospnms. Phi!oJophical TJrlli'tiCiiJJIIJ of the Royal Sociel)' of London, Serie.1 B. 274: 228-274. BENNETT, 216 B. G. MURRAY BENNETT, M. D., SMITH, J. B. & HESLOP-HARRISON, J . S., 1982. Nuclear DNA amounts in angiosperms. Proceedings o f t h e Royal Society of London, Series B, 216: 179-199. HAIR. 1. B. & BEUZENBERG, E. -I., 1958. Chromosome evolution in the Podocarpaceae. Nature, London, 181.' 1584-1586. "IONES., K.., 1970. Chromosome changes: reliable indicators of evolution? 'Taxon, 19: 172-1 79. JONES, K., 1978. Aspects of chromosome evolution in higher plants. Aduances in Botanical Research, 6: 119-194. LOVIS, J. D., 1977. Evolutionary patterns and processes in ferns. Aduances in Botanical Research, 4: 229-415. MANTON, I., 1950. Problems of Cytology and Evolution in the Pteridophyta. Cambridge: Cambridge University Press. MARCHANT, C. J., 1968. Chromosome patterns and nuclear phenomena in the cycad families Stangeriaceae and Zamiaceae. Chromosoma (Berlin), 24: 100-134. NARAYAN, R. K . J. & REES, H., 1974. Nuclear DNA, heterochromatin and phylogeny of N c t o t i ~ n a amphidiploids. Chromosoma (Berlin), 47: 75-84. POLITO, V. S., 1980. DNA microspectrophotometry of shoot apical meristem populations in Ceratopteris thalictroides (Filicales). American Journal of B o t a g , 67: 274-277. PRICE, H. J., LEVIS, R . W., COGGINS, L. W. & SPARROW, A. H., 1972. High DNA content ofSprekelia formosissima Herbert (Amaryllidaceae) and Ophioglossnm petiolatum Hook. (Ophioglossaceae). Experimental Cell Research, 73: 187-191. REES, H. &JONES, R. N., 1967. Chromosome evolution in Lolium. Heredity, 22: 1-18. REES, H. & JONES, R . N., 1972. The origin of the wide species variation in nuclear DNA content. International Review of Cytology, 32: 53-92. REES, H. & WALTERS, M. R., 1965. Nuclear DNA and the evolution of wheat. Heredity, 20: 73-82. SHIVAS, M. G., 1961a. Contributions to the cytology and taxonomy of species of Polypodium in Europe and America. I. Cytology. Journal o f the Linnean Society of London (Botany), 58: 13-24. SHIVAS, M. G., 1961b. Contributions to the cytology and taxonomy of species of Pobpodium in Europe and America. 11. Taxonomy. Journal o f t h e Linnean Society of London (Botany), 58: 27-38. TATUNO, S. & KAWAKAMI, S., 1969. Karyological studies on Aspleniaceae I. Karyotypes of three species of Asplenium. Botanical Magazine ( Tojgo),82: 436-444. TATUNO, S. & YOSHIDA, H., 1967. Karyological studies on Osmundaceae 11. Chromosome of the genus Osmundastrum and Plenasium in Japan. Botanical Magasine ( T o k y o ) , 80: 130-138. VERMA, S. C. & REES, H., 1974. Nuclear DNA and the evolution of allotetraploid Brasicae. HerediQ, 33; 61-68. VIDA, G. & MOHAY, J., 1980. Cytophotometric DNA studies in polyploid series of the fern genus Cystopteris Bernh. Acta Botanica Academiae Scientarum Hungaricae, 26: 455-46 1. iVALKER, T. G., 1984. Chromosomes and evolution in pteridophytes: 103-141. In A. K. Sharma & A. Sharma (Eds), Chromosomes in Euolution of Eukaryotic Groups, vol. 11. Boca Raton, Florida: CRC Press. Y
© Copyright 2026 Paperzz