J. Cell Sd. 63, 135-146 (1983)
135
Printed in Great Britain © The Company of Biologists limited 1983
C VALUE AND CELL VOLUME: THEIR SIGNIFICANCE
IN THE EVOLUTION AND DEVELOPMENT OF
AMPHIBIANS
HEATHER A. HORNER AND HERBERT C. MACGREGOR
Department of Zoology, University of Leicester, Leicester LEI 7RH, England
SUMMARY
Cell volume has been determined in 18 species of amphibian, ranging in C value from l-4pg to
62 pg DN A. There is a strong linear relationship between C value and both erythrocyte volume and
erythrocyte nuclear volume. We have collected data on the timing of early embryogenesis from
fertilization of the egg to the hatching tadpole in some amphibians ranging in C value from 1-4 pg
to 83 pg. The species with large genomes take up to 24 times longer to reach a comparable state of
development. Polyploid species develop faster than closely related diploid species. These data are
discussed in relation to genome expansion and increase in cell cycle time as factors in the evolution
of the Amphibia.
INTRODUCTION
Genes consist of DNA and clearly play a major role in determining most of the
characteristics that we can see, measure and define in living organisms. Yet the cells
of most eukaryotes contain much more of this hereditary material that is needed to
provide for all the structural and regulatory genes that we can presently conceive as
essential parts of a complete genetic system. Moreover, as has so often been said in
discussions of the so-called C value paradox, there would seem to be no obvious
relationship between the amount of DNA in the haploid chromosome set of an organism (its genome size) and the organic complexity of that organism. There are several
groups of animals, plants and micro-organisms in which this paradox is particularly
striking, but from the standpoint of development and evolution none is so interesting
and challenging as the Amphibia. Here we have a group of vertebrates representing
all the stages in the exciting and important transition from water to land, animals
whose development offers exceptional opportunities for experimental study, and
animals that show a range of genome sizes that is quite extraordinary by any standards.
In this paper we wish to re-examine the matter of genome size in amphibians and
discuss its evolutionary and developmental significance on the premise that even
though two animals may have precisely the same repertoire of structural and regulatory genes, it has to make a difference, and a substantial one at that, if one of them has
twice as much chromosomal DNA as the other. But in what sense does it make a
difference ?
We have done three things. First, we have measured the genome sizes and cell sizes
in certain species of amphibian, each selected for a particular reason in relation to one
136
H. A. Homer and H. C. Macgregor
of the questions covered in this paper. In this study we have used some methods and
equipment that were not available to earlier investigators. The use of a scanning and
integrating microdensitometer that is capable of making large numbers of accurate
and reproducible readings from Feulgen-stained nuclei is probably the best way of
determining the DNA content of cell nuclei, and the use of a Coulter Counter and
channelizer is probably the most accurate way of determining cell sizes, once again
generating highly reproducible data from a very large sample of cells.
Second, we have examined the matter of genome size in relation to cell cycle time.
Do amphibian cells from a species with a small genome divide more rapidly than a
corresponding type of cell from a species with a larger genome?
Third, with reference to another carefully selected range of amphibian species, we
have determined the time taken to progress from the moment of deposition of a fertile
egg to the moment of hatching of a tadpole.
Taking these four matters, genome size, cell size, cell cycle time, and time from
fertilization to hatching, we shall discuss whether genome size is of selective value and
evolutionary significance in so far as it may affect the rate at which an amphibian
species accomplishes the early and perhaps the most vulnerable stages of its development.
Our choice of species in this study was determined as follows. For our studies of
genome and cell size we chose Xenopus tropicalis and X. laevts because they are said
to be diploid and tetraploid, respectively (Thiebaud & Fischberg, 1977). Two species
olPlethodon were included since they have identical chromosome numbers but widely
different genome sizes (Mizuno & Macgregor, 1974). Several species of Triturus were
used mainly because we were also able to obtain data on cell cycle and development
time in this genus. Three species of Taricha were included because Olmo & Morescalchi (1975) have reported that these animals have widely different nucleus:
cytoplasm volume ratios in spite of having almost identical genomes, and we thought
the matter worth reinvestigating. The small ambystomid salamander Rhyachotriton
olympicus was included since it was available in our laboratory and we knew that it had
an unusually large genome: more than 20 times that oiX. laevts and two to three times
as large as that of Triturus.
For our studies of cell cycle times we had just two cell lines available. The first is
a fibroblast culture said to be derived from kidney ofX. laevis. The second is a similar
culture derived from skin of Triturus cristatus carnifex. Both these lines have been
established in culture for many years and both are quite aneuploid. The chromosome
numbers and amount of DNA per nucleus for the Triturus line indicates that it is very
nearly equivalent to tetraploid. We have recognized that these cell lines can at best
provide us with a rough guide as to the relative rates of division of cells with widely
different genomes.
Our choice of a study of development time was mainly dictated by the decision to
select only species that laid uncleaved eggs in water and then proceeded through a
normal developmental sequence inside a jelly capsule, finally hatching as a freeswimming tadpole. We also tried to include in our list as wide as possible a range of
genome sizes, some related diploid and polyploid species, and at least some of the
C value and cell volume in amphibians
137
species that we had used in other parts of our study. Some of our data in this section
are the product of our own studies; others are quoted from work carried out by other
investigators. The range of genome sizes in this section is about as wide as can be
within the Amphibia, the African bullfrog Pyxicephalus adspersus having one of the
smallest genomes of all amphibians andNecturus maculosus having one of the largest.
Ceratophrys ornata is an octoploid species (Bario & Rinaldi de Chieri, 1970), and
Ambystomajeffersonianum and A. platineum are somatic diploid and triploid, respectively (Uzzell, 1964).
MATERIALS
AND
METHODS
Altogether 14 species of amphibian were used in this study. Native British species (Tritunis
vulgaris and T. cristatus) were collected locally in Leicestershire in spring 1981. Triturus cristatus
camifex was purchased from Xenopus Ltd, (South Nutfield, Redhill, Surrey, England), having
been imported from Italy. X. laevis and X. tropicalis were laboratory-bred animals. The X.
tropicalis were kindly provided by Dr P. J. Ford of the Department of Molecular Biology, University of Edinburgh. Plethodon and Rhyachotriton were collected and supplied by Dr James Kezer of
the University of Oregon. P. adspersus and C. ornata were laboratory-bred and supplied by The
Centre for Reptile and Amphibia Propagation (1444 North Roosevelt, Fresno, California 93728).
Bombina species were captive-bred and were kindly provided by Mr R. J. Bray.
To obtain whole blood either for cell size measurements or for blood smears to be used in Feulgen
microdensitometry, the animals were killed or anaesthetized with ethyl m-aminobenzoate (MS222)
and blood was taken with a heparinized pipette from the heart, from the tip of the tail or from a vein
in the web of a foot.
Cell size was determined by measuring displacement of electrolyte volume employing equipment
kindly loaned to us by Coulter Electronics Ltd (Northwell Drive, Luton, Bedfordshire, England).
Heparinized blood was diluted with calcium-free Ringer and passed through a 100 fim aperture in
a Coulter Counter model ZBI. The data were sorted by a C1000 channelizer and plotted on an XY
recorder. For each species, commercially calibrated latex spheres with volumes comparable to those
of the blood cells being measured were plotted on the same chart. To obtain measurements of
nuclear volume whole blood was added to 0-01 M-citric acid plus 0-05 % Nonidet P40. After gentle
pipetting, the nuclear suspension was passed twice through a 25 gauge hypodermic needle and
examined with a microscope to check that all cytoplasm had been removed. The nuclear suspension
was centrifuged for 5 min at 500 g, the supernatant removed, and the nuclei resuspended in calciumfree Ringer. The nuclei were then passed through the Coulter Counter followed by calibrating latex
spheres of a known size.
Amounts of DNA per diploid cell were obtained by Feulgen microdensitometry of erythrocyte
nuclei or tissue-culture cell nuclei. For each species a blood smear was made at one end of a clean
microscope slide, it was quickly air-dried and then fixed for 10 min in fresh, ice-cold methanol/
glacial acetic acid (3:1). A 'control' smear was made iromX. laevis blood on the other end of every
slide and treated in an identical manner with regard to every step in fixing, staining, and preparation
for microdensitometry. Splash preparations were made from fixed tissue-culture cells. Slides were
processed through the Feulgen reaction using the method of Swift (1955). Hydrolysis was for 18 min
in 5 M-HCI at 18 °C, followed by staining in Schiff's reagent for 90 min and washing in several
changes of sulphite rinse. Previous stoichiometry had established the conditions of fixation,
hydrolysis and staining that gave the highest and most consistent values for Feulgen dye contents
of amphibian erythrocyte nuclei (Mizuno & Macgregor, 1974). The stained smears were air-dried
and mounted in immersion oil. Feulgen dye contents were measured on a Vickers M85 Scanning
and Integrating Microdensitometer employing light of wavelength 535 nm. Altogether 50 readings
were taken from each end of every slide.
Two amphibian transformed cells lines with widely different nuclear DNA contents were grown
in parallel to compare cell cycle times and growth rates. The Xenopus line is designated as XK and
the Triturus line as TCC. Cells were grown at 26°C in Eagle's Minimal Essential medium diluted
with water (4:1) and supplemented with 8% foetal calf serum and 4 % lactalbumin hydrolysate
138
H. A. Homer and H. C. Macgregor
(AMEM). Samples containing known numbers of cells were dispensed into 5 ml AMEM in
25 cm2 culture flasks. Cells were harvested at 24 h intervals with 0-05% trypsin in calcium-free
Ringer and counted in a haemocytometer. Three samples were counted from each flask, and three
flasks of each cell line were harvested each day.
RESULTS
The channelizer of the Coulter Counter sorted cells into 100 different size classes
and plotted the data on an XY recorder. Cell volume was plotted linearly along the
abscissa and the number of cells was recorded on the ordinate. The channelizer
stopped automatically when 1000 cells had been measured in any one channel. The
total number of cells counted was in the range 2200 to 3200. Of these, 60-80 % were
under the peak defined by 15-20 channels each containing more than 50 measurements. The absolute volumes for cells or nuclei from each species were determined
by reference to the peak produced by the calibration spheres. Cell and nuclear
volumes for each of the 12 species measured, as well as for Xenopus and Triturus
tissue-culture cells are shown in Table 1. A plot of these values (not including data
from the tissue-culture cells) against nuclear DNA amounts is shown in Fig. 1. In the
present study, the ratio of cytoplasm to nucleoplasm in the Taricha species is the
same, and not widely different as reported by Olmo & Morescalchi (1975).
Table 1. Volumes of whole erythrocytes and isolated erythrocyte nucleifrom six species
of anuran, 12 species of urodele, and two transformed amphibian cell lines, as
measured with a Coulter Counter
C value
(Pg)
Cell volume
Nuclear
volume
P. adspersus (PA)»
X. tropicalis (XT)
X. laevis (XL)
Bombina orientalis (BO)
B. variegata (BV)
C. ornata (CO)
1-4
1-5
3
5-9
6-9
13-4
120
122
250
750
650
450
_
3
10
50
60
S3
_
119
240
700
590
397
Plethodon cinereus (PC)
Triturus alpestris (TA)
Triturus cristatus carmfex (TC)
Triturus cristatus cristatus (TC)
Triturus marmoratus (TM)
Triturus vulgaris (TV)
Notophthatmus viridescens (NV)
Taricha granules a (TG)
Taricha torosa (TT)
Taricha rivularis (TR)
Plethodon dutmi (PD)
R. otympicus (RO)
22-5
24
24-5
24-5
24-9
25
38
35
38-6
38-9
47-5
62
1476
1350
1500
1615
1674
1663
2204
2090
2232
2520
3204
3714
144
190
210
176
208
176
168
272
240
232
304
488
1332
1160
1490
1439
1466
1487
2036
1818
1998
2312
2900
3226
XK cell cultures
TCC cell cultures
2-2
41
1926
9139
1-8
323
1924
8816
Species
• Abbreviations in parentheses are used in Fig. 1.
Cytoplasmic
volume
(^m3)
139
C value and cell volume in amphibians
..#
3000
2000
E
1000.
PAXLBOB^0 CO
AT
Fig. 1. Regression plot of cell volumes and nuclear volumes (/an3) as measured with a
Coulter Counter, against C value (pg) measured by Feulgen microdensitometry. Control
X. laevis, 3 0 pg. From the data in Table 1. P = > 0 0 0 1 . See Table 1 for abbreviations of
species.
Table 2. Parallel growth of cell cultures with widely different amounts of DNAper
nucleus: XK = 4-4pg, TCC = 82pg
XK cells per cm 2
Day
TCC cells per cm2
i
0
2
3
4
5
6
7
8
9
10
11
12
r
Mean
S.E. (%)
Log mean
Mean
S.E. (%)
Log mean
256600
244400
323 200
458800
636600
818000
993 300
1418 300
1747700
2327700
2397 700
3 233 300
11-6
5-456
5-388
5-509
5-662
5-804
5-913
5-997
6152
6-242
6-367
6-380
6-510
165 000
67700
137500
184300
233 300
286600
317500
406500
513 300
462200
528800
441600
15-7
1-2
3-4
2-6
2-7
2-7
60
5-0
2-9
6-4
40-7
0-2
5-217
4-881
5-138
5-266
5-368
5-457
5-502
5-609
5-710
5-665
5-723
5-645
0-4
3-6
18-8
3-7
4-5
1-9
11-2
8-0
14-5
6-5
0-4
140
H. A. Homer and H. C. Macgregor
Amounts of nuclear DNA per cell are also given in Table 1. These were obtained
by measuring 50 nuclei from the species in question and then taking 50 readings from
X. laevis erythrocytes on the same slide. Values in absorption units were converted
to picograms on the basis of a diploid nuclear DNA amount for A', laevis of 6-0 pg
(Dawid, 1965).
For construction of growth curves for tissue-culture cells of both A*, laevis and T.
c. camifex the number of cells per cm2 was calculated for each of the nine samples that
6-6-
6-4-
6-2 -
c
8
4>
"5
E
15-6
CD
CD
O
5-4-
5-2 -
50-
4-8
2
4
6
Time (days)
8
10
12
Fig. 2. Parallel growth of cell cultures with widely different amounts of DNA per nucleus:
(•) XK = 4-4pg; (•) TCC = 82pg. Regression plot from the data in Table 2. P =
>0-001.
8484(3C)(b)
A. platineum
18
18
("C)
Temperature
7
5
24
20
31
80
87
96
103
Blastula
Gastrula Neural fold Tail bud
Hatching
(N&F, st8) (N&F, s t l l ) (N&F, st17) (N&F, st30) (N&F, st40)
Time in hours after fertilization. T h e stages (N&F, st8 etc.) refer to Nieuwkoop & Faber (1967).
+ C values measured by us, except: (a) Olmo & Morescalchi, 1978; (b) Brodie & Tumbarello, 1978.
13'4(1Cl-@)
5656(2C)(b)
35
83 (a)
5.3 (a)
10.5 (a)
6.9
24.5
28 (b)
?
3.0
1.4
C value*
(~g)
B.
C. ornata
A. jeffenonianum
A. jeffenonianum
T. c. cantifex
Rana syloatica
R. plplP;ens
B. variegata
x.t m i
Species
Table 3. Early development of a selected range of amphibian species
E-
0
J. Brothers (as above)
Brodie & Tumbarello
(1978)
Brodie & Tumbarello
(1978)
Personal observation
Brodie & Tumbarello
(1978)
Rugh (1948)
Noble (1931)
4
'
3
1
s.
2
?
-C
Univ. of California,
Berkley (Personal
Q
communication)
Nieuwkmp & Faber (1967)
Rugh (1948)
n
%
Rugh (1948)
Personal observation
$
J. Brothers, Zoology Dept,
References for
embryological data
142
H. A. Homer and H. C. Macgregor
were taken every 24 h. The logarithms of the mean cell densities were then plotted
against time (Fig. 2). The data from which these curves were constructed are given
in Table 2. After an initial lag phase, the cells went into exponential growth, represented in Fig. 2 by straight lines. After the ninth or tenth day the line deviated, as growth
slowed on account of contact inhibition and starvation. The doubling time for X.
laevis cells was 2\ days as compared with 3i days for T. c. carnifex.
Our information on the times taken for embryos of various species of amphibian to
reach blastula, gastrula, neurula, tailbud, and hatching stages are given in Table 3.
In so far as we shall wish to attach significance to the relationship between genome size
and developmental rate, we have included in Table 3A only those species that are
known to be normal diploids. In Table 3B we have presented separately the comparative data for C. ornata; an octaploid species, and for triploid Ambystoma
platineum. We are aware that the laboratory temperatures used may not be the
optimum for each species. However, in general, species with low C values reach any
of the defined developmental stages faster than those with higher C values. The
polyploid species for which we were able to obtain data develop faster, particularly in
the earlier stages, than do their diploid relatives (A. platineum compared with A.
jeffersonianum, according to Brodie & Tumbarello, 1978), or other species that have
comparable nuclear DNA contents (C. ornata compared with B. variegata).
DISCUSSION
In this paper we have presented a number of sets of data and we have pointed out
certain situations that seem to be good correlations. In the first place, the larger the
genome the larger the cell and the longer the cell cycle time. Cell size and cycle time
are likely to be of importance, particularly in relation to development and generation
time as well as in other processes such as gamete production and wound healing. In
our view, the primary changeable element in this matter is most likely to be the
genome itself, and the tendency will be for it to grow, unless checked by positive
selective pressure to keep it small or at a level that is acceptable for the species. Most
of the mechanisms for quantitative changes in DNA that are currently known, including gene duplication, insertion and transposition of sequences and unequal sister
chromatid exchange, will, if left unchecked, lead to an accumulation of new DNA
sequences and expansion of the genome. In turn this will produce larger cells and
longer cell cycles. If then it is selectively advantageous for an organism to possess an
average cell cycle of a particular duration, producing after a definitive number of cell
divisions an adult body of a particular size, then natural selection must discriminate
against excessively high levels of the kinds of events that lead to genome expansion.
In essence, genomes will continue to expand unless there are positive selective
pressures that keep them in check.
Within this general concept there is room for some fine-tuning that does not
necessarily involve constraints on genome size. If, for example, the duration of
5-phase in mitosis is taken as one of the major limiting factors in the cell cycle, then
natural selection may discriminate in favour of a reduced cell cycle time by allowing
C value and cell volume in amphibians
143
Table 4. Replicon spacings in synthesizing cultured cells
Species
X. laevis
T. cristatus
Gallus domesticus
Cricetulus griseus
Homo sapiens (HeLa)
C value
(pg)
Replicon spacings
3
24-5
1-4
57-5 (mean)
100-350
63 (mean)
SO (mean)
30 (mean)
3-5
From Callan (1972); MacFarlane & Callan (1973).
an increase in the number of initiation points for chromosome replication, effectively
decreasing the distances between these points. That there is scope for this kind of
adjustment is evident from the studies of Callan (1972) and McFarlane & Callan
(1973) and others cited by Callan (1972), who have found quite wide differences in
replicon spacings from one species to another that bear little relationship to genome
size (Table 4). Callan (1972) also found wide differences in the duration of synthesis
between somatic and embryonic cells, the 5-phase in somatic cells being up to 50 times
longer than in embryonic cells. Since the replication rate, that is, the speed at which
replication proceeds outwards from an initiation point, is constant for a given temperature, the variable factor must be the number of initiation points available for
polymerase attachment.
Yet another means of reducing cell cycle time without changing genome size is to
become polyploid. This principle has been amply demonstrated in plants (Bennett,
1971, 1972, 1977; Bennett, Smith & Lewis, 1982) and it seems to hold also at least
for the two polyploid amphibians that we have considered in this paper. On the other
hand, stable polyploidy is a relatively rare occurrence amongst bisexual animals, and
we are inclined to think of it as having a rather limited adaptive significance. It seems
more likely that in species such as A. jeffersonianum polyploidy became established,
accompanied by a gynogenetic mode of reproduction, and proved to be selectively
neutral. The polyploids have larger cells, they develop into slightly larger adults than
their diploid relatives, they occupy the same general habitat as the diploids, and
whatever they gain in terms of accelerated embryonic development they lose again in
the sense that their period of larval development is longer than that of the diploids
(Brodie & Tumbarello, 1978). Our observations on Xenopus and Triturus tissueculture cell lines are of some interest in relation to the effect of polyploidy. The
Xenopus line is clearly aneuploid but has a mean chromosome number and nuclear
DNA content that are near to the normal diploid values. The Triturus line, on the
other hand, began as diploid, and was recorded as such by Rudak (1976). The
derivative with which we have worked is aneuploid, but has almost twice the normal
diploid number of chromosomes and an amount of DNA per nucleus that is almost
twice that of the C value for T. cristatus. We suggest that the near-polyploid condition
of the Triturus line may account for the fact that its cell cycle time is much shorter in
144
H. A. Homer and H. C. Macgregor
relation to that of the Xenopus line than would be expected on the basis of the relative
nuclear DNA contents of the two lines.
The significance of the rather striking correlation between genome size and time to
hatching is not at all easy to understand. Previous studies of this kind have considered
the whole development time right through to metamorphosis (Goin, Goin & Bachman, 1968). In our view this introduces too many complications and unknowns. A
survey of times from hatching to metamorphosis amongst frogs, toads and salamanders will quickly show how highly adaptive this period is and how unrelated it is to
genome size. It is for this reason that we chose to concentrate on early embryonic
development in a series of animals all of which use the same general, and typically
amphibian, reproductive strategy. But even when considering relationships between
genome size and development time in these animals, it is exceedingly hard to analyse
the kinds of selective forces that must be at work. Some authors have stressed the
ecological problems that face amphibians that reproduce in ephemeral waters in short
spans of time between dry seasons (e.g., see Goin et al. 1968). Such a concept is
undoubtedly applicable in some extreme cases, and it may even apply to a species such
as P. adspersus in certain parts of its range, the animal seizing the opportunity to breed
in very large numbers in a short wet season in some tropical and sub-tropical regions
of Africa. But P. adspersus are also abundant during the wet season in ponds that
remain full for much longer than it takes the animal to proceed right through and far
beyond metamorphosis, and it co-exists in the same ponds with several other species
of frog, including X. laevis, that have considerably larger genomes and correspondingly slower developmental rates.
Another example that might usefully be cited is that of a range of temperate species
of Rana, Bombina and Bufo, as compared to several species of Triturus. The newts
and frogs as often as not inhabit the same ponds and despite major differences in
genome size and developmental rate, each is successful according to its own particular
strategy of reproduction, a fact that is doubly remarkable in relation to crested newts,
which not only develop much more slowly and have long precarious larval lives, but
reproduce successfully despite the loss of half their embryos at an early developmental
stage (Macgregor & Horner, 1980). In short, ecological arguments as a means of
rationalizing differences in genome size and corresponding differences in developmental rate are, except at the extreme limits of the ranges, rather unconvincing.
In our view, one of the most interesting and least investigated matters emerging
from the kinds of correlations that we have shown in this paper relates to the timing
of differentiation in development and the production of an organism of a definitive size
that is characteristic of a particular species. The construction of a vertebrate requires
that an egg of a particular size goes through a certain number of divisions to produce
a population of cells that are at first remarkably similar with regard to cell cycle and
appearance. As embryogenesis proceeds, these cells gradually start to differentiate
and acquire characteristics that are specific for a given function. The cues for this kind
of differentiation are not well understood, but at least one group of investigators has
argued that they may be part of a programme of which an important component is the
number of divisions that a cell has passed through since the start of
C value and cell volume in amphibians
145
Table 5. Sizes of eggs in three species of amphibians
Species
X. tropicalis
X. laevis
T. c. carnifex
Diameter
(mm)
Volume
(/im3)
C value
(pg)
0-9
1-4
1-7 (1-6 X 1-8)
0-4
1-4
2-7
1-4
3
24-5
Variation was negligible.
a developmental process (Summerbell, Lewis & Wolpert, 1973). We wish to cite just
two examples here to illustrate our point and expose just a few questions that future
investigators may perhaps wish to try and answer.
First, a mature egg of T. cristatus is twice the volume of a mature egg from A", laevis
(Table 5). These eggs develop through to hatching without the input of any new
material; that is, the dry mass of the fertilized egg is the same as that of the newly
hatched tadpole. Triturus has eight times as much nuclear DNA as Xenopus, and
although the cells of its emerging tadpole may not actually be eight times the volume
of the corresponding cells in Xenopus, those that have differentiated fully are likely
to have reached the normal adult size and others must be substantially larger than their
Xenopus counterparts, if only to accommodate the larger mitotic spindles that are
needed to move the big Triturus chromosomes. So the Triturus tadpole may be
expected to have a quarter of the number of cells that are in the Xenopus tadpole, and
it follows that in the formation of the Triturus tadpole there must have been fewer
divisions than in the formation of the Xenopus one. The same may be said in principle
with regard t o X laevis andX tropicalis (see Table 5). The cues for cell differentiation must therefore be tuned appropriately.
Our second example comes from the genus Plethodon, a group of lungless salamanders that are totally terrestrial and have no free-swimming tadpole stage in their life
cycle. Large fertile eggs are laid in the ground and these develop through an encapsulated larval phase and metamorphosis, and emerge as fully formed miniature
salamanders. Two species, P. dnereus and P. vehiculum, are remarkably alike except
insofar as P. vehiculum has a genome that is nearly twice as large as that off. dnereus
(Mizuno & Macgregor, 1974); but they have the same chromosome number, the same
egg size, and approximately the same adult body size. Our response to this particular
situation is to propose that the Plethodon genome has shown a normal tendency to
expand, and in the Western representatives of the genus the process has remained
unchecked, reaching the present high level of 69-3 pg in P. vandykei (Mizuno &
Macgregor, 1974). In Eastern species such as P. dnereus there has been quite strong
selective pressure to maintain the plethodontid genome at around 20 pg. We cannot
identify the factors behind these selective pressures, but whatever they are the strong
likenesses between P. vehiculum and P. dnereus signify that it is not the adult form
of the animal that is affected by the 'extra' DNA but rather some features of its
development, growth, life cycle, or natural history.
146
H. A. Homer and H. C. Macgregor
This work was supported by Science and Engineering Research Council grants nos. GR/C/
05250 and B/SF/151.
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(Received 1 February 1983 -Accepted 11 March 1983)