Karyotypes and nuclear DNA amounts in Polypodium L

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 .
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209
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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.
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