A8STRACT
m.sc.
Lillian Janette Taper
Siology
THE RELATIONSHIP SETWEEN CHROmOSOmE SIZE AND
DEOXYRIBONUCLEIC ACID CONTENT IN SIRCH
(SETULA) SPECIES
An analysis
cytophotometric
of chromosome
measurements
nucleic acid (DNA) content
ploid,
pentaploid
carried out.
morphology
of
for
the
and Feulgen
nuclear deoxyribo-
diploid, triploid, tetra-
and hexaploid
species
A direct correlation
of
Setula was
was found between the
observed DNA absorbance and the chromosome number
for the
28-, 42-, 56- and 70-chromosome plants.
density
value
for the 84-chromosome plants
approximately
equivalent
chromosome plant.
to
that
The DNA
was calculated
to be
of a
theoretical 61-
The average DNA value
per unit length
of chromosome was 0.283 units for the 84-chromosome plants
in contrast
for each
to 0.350 units
per unit length of chromosome
of the other euploid levels.
length for each level of ploidy
Total
complement
increased with increasing
chromosome number, although not in direct proportion.
observed
reduction
in
nuclear
DNA
content
The
in the 84-
chromosome plants did not appear to be directly related to
any change in chromosome size.
THE RELATIONSHIP BETlliEEN CHRomosomE SIZE AND
DEOXYRIBONUCLEIC ACID CONTENT IN
BIRCH (BETULA) SPECIES
by
Lillian Janette Têper
A thesis presented ta the Fêculty of Grêduête
Studies and Research
in partiê: fulfil~ent
of the requirements for the degree of
ffiaster of Science
8iology Departnent
mcGill University
ifion creel
@
I.ill; an
Janet.te Taper
1971
Shor t ti tle
SIZE AND DNA CONTENT OF BrRCH CHRomosomES
Lillian Janette Taper
ABSTRACT
ffi.Sc.
Lillian Janette Taper
Biology
THE RELATIONSHIP BETWEEN CHROmOSOmE SIZE AND
DEOXYRIBONUCLEIC ACID CONTENT IN BIRCH
(BETULA) SPECIES
An analysis
cytophotometric
of chromosome
measurements
nucleic acid (DNA) content
ploid,
pentaploid
carried out.
morphology
of
for
the
and Feulgen
nuclear deoxyribo-
diploid, triploid, tetra-
and hexaploid
species
A direct correlation
of
Betula was
was found between the
observed DNA absorbance and the chromosome number
for the
The DNA
density
28-, 42-, 56- and 70-chromosome plants.
value
for the 84-chromosome plants
approximately
Equivalent
chromosome plant.
to
was calculated
to be
that
of a
theoretical 61-
The average DNA
~alue
per unit length
of chromosome was 0.283 units for the 84-chromosome plants
in contrast
for each
to 0.350 units
per unit length of chromosome
of the other euploid levels.
length for each level of ploidy
Total
complement
increased with increasing
chromosome number, although not in direct proportion.
observed
reduction
in
nuclear
DNA
content
The
in the 84-
chromosome plants did not appear to be directly related to
any change in chromosome size.
ACKNOWLEOGEmENTS
The author
W.
wishes ta express her gratitude
ta Or.
F. Grant, Professor of Genetics, Oepartment of Biology,
ffiacdonald Campus
throughout
the
of mcGill University,
course
of this study
for
and
his guidance
for
his help
during the preparation of the manuscript.
She would also like ta thank Dr. H. Tyson,
ment of Biology,
Centre,
and
mcGill University,
statistical analysis;
for
Professor J.
mr.
for
D. Burrage,
their assistance
DepartComputer
in the
E. Gyapay and ffir. Paul Choo-Foo
their technical assistance;
and
mrs. m. Couture for
typing the manuscript.
The author gives her special thanks ta her parents,
Dr. and ffirs. C. D. Taper for their help and encouragement.
TABLE OF CONTENTS
Page
ACKNOWLEDGEmENTS
i
. . . . . . . .
LIST OF TABLES
LIST OF FIGURES
iv
vi
INTRODUCTION
l
LITERATURE REVIEW .
4
A. The Genus Betula •
1. History of the study of the genus .
II. North American species of Betula used in
this study • • • • • • • . . • . • •
III. Variability within the genus Betula •
B. Cytophotometry . • . .
. •.••.•
1. Cytophotometry in the study of nucleic
acids .
.
.
.
.
.
4
4
6
18
23
23
II. Cytophotometric studies of nucleic acids
III. Assessment of the cytophotometric technique
ffiATERIALS AND ffiETHODS •
24
34
35
1. Plant material
. ••
•..
II. Stomatal measurements • . • •
••.
III. Somatic chromosome number determinations
and preparation of idiograms . . • .
•
IV. Analysis of the deoxyribonucleic acid (DNA)
content of the root tips • • . . . . • . •
ii
35
38
38
42
Page
RESULTS • • • . •
44
A. The Stomatal measurements
44
B. The Karyotypes • • • • . • • • • • • . • • . • •
1. The karyological description of the species
a. Diploid species
. • • •
b. Triploid species •
. • .
c. Tetraploid species . . . • • . •
d. Pentaploid species • . • • .
e. Hexaploid species
.•••
II. Statistical analyses
. . •
a. A comparison of the percentage total
complements (%TCL) and the long arml
short arm (Lis) ratios of the six
diploid species • • . • • . . • • •
b. A linear regression analysis of the
total complement lengths for diploid,
triploid, tetraploid, pentaploid and
hexaploid species
. . • • • • •
51
67
67
71
72
C. The Cytophotometric Comparison of the Species
1. A comparison of the ~uclear DNA content of
six diploid, one triploid, five tetraploid,
five pentaploid and five hexaploid species
79
of Betula .
.
.
.
•
.
•
.
.
..
..
..
..
..
.
....
II. The relationship between chromosome number
and DNA content for diploid, triploid,
tetraploid, pentaploid and hexaploid
species of Betula • • • • . • • . • . •
III. The relationship between chromosome length
and DNA content for diploid, triploid,
tetraploid, pentaploid and hexaploid
species of Betula • • • • . • • • . . •
73
73
74
74
76
79
101
107
109
DISCUSSION
A. Stomatal measurements
B. Karyotype Studies
C. Cytophotometric measurements - Nuclear DNA
Content . . . . . . . . .
• .••
109
110
114
summA RY • • •
.. . .. .. . . .. . . .. . .. . .. . .. .. .
123
LlïERATURE CliED
.. . .. .. .. .. .. .. .. .. .. .. .. . .. .. .. ..
127
iii
LIST OF TA8LES
Table
1.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
Page
The species studied, their accession number,
source, and chromosome number
• • .
•••
36
measurements of the stomatal guard cells for
diploid, triploid, tetraploid, pentaploid and
hexaploid species of 8etula
. . . . .
45
Analysis of linear regression in Figure 2
50
Karyotype analyses of the somatic chromosomes
for ten 8etula species . • • . • • • •
60
Analysis of variance and Duncan's test for
(A) the percentage total complement length
(%TCL) and (8) the long-short arm ratio (LiS)
for the diploid species of 8etula • • • . . .
75
Analysis of the linear regression in Figure 10
76
Analysis of variance of the DNA absorbance
values for the standard species, ~. populifolia (633)
•....•.••......
79
ffiean nuclear DNA values (in arbitrary units)
for 2C nuclei of 8etula species • • • . • . •
81
Analysis of variance and Duncan's test of DNA
variation of 2C nuclei between diploid,
triploid, tetraploid, pentaploid and hexaploid
species of Betula • • . • . • . • • . . • . .
100
DNA absorbance for different levels of ploidy
in 8etula relative to the absorbance of the
standard species, ~. populifolia (2~
28)
given the value of 1.00
•••.•.•. . . •
102
test for the relationship between chromosome number and DNA density value for diploid,
tetraploid and pentaploid species of Betula
103
=
XI.
~tfl
iv
Table
XII.
XIII.
Page
nt" test for the relationship between chromosome number and DNA density value for
hexaploid species of Betula • • • • • .
104
The relationship between chromosome length and
DNA content in diploid, triploid, tetraploid,
pentaploid and hexaploid species of Betula.
108
v
LIST Of fIGURES
figure
1.
Page
feulgen stain bound by chromosomes of Betula
species after hydrolysis in N HCl at 600C
for various intervals of time
•• . • . .
40
The relationship between the mean stomatal
length and chromosome number in Betula species
48
Somatic chromosomes from root tip cells of
diploid, tetraploid, pentaploid and hexaploid
species of Betula • • • • • . . • • • • . •
52
7.
Idiograms of the diploid species of Betula
54
8.
Idiograms of triploid, tetraploid and
pentaploid species of Betula . • •
56
Idiogram of a hexaploid species of Betula
58
The relationship between the total complement
length and the chromosome number in Betula
species
77
2.
3-6.
9.
10.
11-18. Histograms of the distribution of DNA amounts
estimated in sixt Y 2C nuc1ei of Betula species
19.
The relationships bet~een the theoretically
expected DNA absorbance and the observed DNA
absorbance for diploid, triploid, tetraploid,
pentaploid and hexaploid species of Betula • .
vi
83
105
INTRODUCTION
Photometry is the measurement of radiant energy in
the form of visible and ultraviolet light.
By comparing
the intensity of radiation entering a substance with its
Emergent intensity following passage through the substance,
the amount of radiation absorbed can be measured.
Cyto-
photometry allows the cytogeneticist to investigate cells
from a chemical approach.
Various morphological characters such as stomatal
and pollen grain sizes have proven reliable for the
detection and separation of polyploids in certain cases
(Grant, 1954).
It has been suggested that Feulgen cyto-
photometry may also be a reliable method for differentiating
between species by measuring differences in the DNA content
of their nuclei.
Birch (Betula) species have somatic chromosome
numbers of 28, 56, 70, and 84, plus sorne natural hybrids
with 42 somatic chromosomes.
and difficult to count.
ïhese chromosomes are small
The ability ta separa te individu-
als into different levels of ploidy without actually
determining the chromosome nurnber would require less
l
2
precision and less time than the laborious procedure of
counting these chromosomes.
Brittain and Grant (l967b) analysed the stomatal
guard cells of birch species.
They found that the mean
length of the guard cells for diploid species was
significantly different from that for a polyploid species,
being shorter, but that there was a considerable overlapping of the individual measurements.
Therefore, unless
a very large number of measurements was made, it would be
difficult ta ascertain the true ploidy of an individual by
this method.
A scanning integrating microdensitometer has been
shawn ta detect minute differences in DNA quantities in
Equivalent nuclei between individual specimens of birch.
Grant (1969) undertook a study ta determine if cytophotometry could actually be used ta distinguish the
various eup10id leveis for individual birch plants whose
chromosome numbers were not known.
He found that the
average of a number of readings was close ta the theoretical
expected value for the 42, 56, and 70 euploid leveis.
However, birch plants with 84 somatic chromosomes showed a
DNA absorbance value Equivalent ta the theo=etical
expected value for plants with 63 chromosomes.
Grant has
suggested that hybridization and polyploidization between
different birch species, or individuals, with different
3
basic absorption values (based on different chromosome
sizes of the individual chromosomes which make up the 84chromosome plants) could have occurred ta bring about the
DNA value observed for the 84-chromosome plants.
Alternatively, a failure of DNA synthesis prior ta, or
during, polyploidization in a 42-chromosome plant would
give rise ta a 42-chromosome gamete with half the normal
DNA content.
The union of such a gamete with a normal
42-chromosome gamete would give rise ta a plant with 84
soma tic chromosomes but with a DNA content equivalent ta
that of a 63-chromosome plant.
The present study was carried out along the same
lines as the above ta test the relationship between the
amount of DNA and the chromosome number and the size of
the chromosomes and the chromosome number in various
Canadian species of birch.
LITERATURE REVIEW
A.
The Genus Betula
The family Betulaceae contains six genera, the
genus Betula L. being the largest.
This genus consists of
about fort y species of trees and shrubs growing in the
coo1er northern regions of Europe, Asia, and North America.
I. History of the study of the genus
As early as 1753 Linnaeus had described two
American species, Betula 1enta and
(1785) gave a description of
~.
~.
nigra.
marshal1
papyrifera from North
America and later michaux (1803) discussed the economic
value of this species.
Hooker (1838) described eight
species of Betula inc1uding a new species, 8. occidentalise
Regel (1861, 1865, 1868) described man y North American
species.
Sargent (1896) described six tree birches from
North America, and later (Sargent, 1905) described nine
arborescent species.
Fernald (1902) suggested that North American and
European species could be grouped together.
Howeve~,
he
changed this opinion (Fernald, 1945) and suggested that
most of the North American species are Endemie.
4
5
Butler (1909), following a study of western North
American species, concluded that the birches of the west
are mostly distinct from those of the East, and stated
.that only a few of the Eastern species reach regions west
of the Great Plains.
The ide a of regarding the western
forms as varieties, or even as hybrids of the Eastern
species, appeared to him to have no scientific basis.
He
recognized 17 western species; however, several of these
have since been shown to be of hybrid origine
The genus Betula contains species and varieties
having interesting chromosome numbers:
35 pairs, 42 pairs, etc.
14 pairs, 28 pairs,
There is polymorphism within
sorne of the Betula species, due in part at least to the
extensive occurrence of hybridization intraspecifically
between different individuals.
This variability in
chromosome number as weIl as in other characteristics has
made this genus very difficult ta classify taxonomically.
Dugle (1966) suggested that although the widely diverging
classifications proposed by Fernald (1902) and Butler
(1909) might be acceptable to sorne taxonomists, neither
one mas satisfactory and perhaps an intermediate position
should be adopted.
6
II. North American species of Betula
used in this study
Among the North American species are
marsh.,
g.
resinifera Britt.,
8. caerulea Blanchard,
g.
g.
g.
papyrifera
cordifolia Regel,
caerulea-grandis Blanchard,
8. eopulifolia ffiarsh., and B. kenaica Evans.
g.
papyrifera, the native white or paper birch, was
first described by marshall (1785).
It isfound in
locations stretching From Eastern to western North America.
Butler (1909) described the western form of this species
as Ua forest tree 15-25 meters high with smooth, chalky
white bark, easily separable into thin paper-like layers;
branchlets more or less pubescent, densely so on the young
shoots; leaf blades 4-8 cm long, 2-5 cm wide, narrowly
ovate to oval, and rounded, sometimes truncate, finely or
coarsely serrate, sometimes slightly lobed, hairy when
young, becoming glabrous; fruiting aments 2-5 cm long on
slender, resinous stalks."
He stated that "the Eastern
form of this species has densely resin-dotted leaves, and
the bark peels more readily th an that of the western form."
Brittain and Grant (1965a) carried out a morphological and cytological study on
collected in Nova Scotia,
Ne~
g.
papyrifera individuals
Brunswick, and Quebec.
ïhey
found somatic chromosome numbers of 56, 70, and 84 for these
Eastern collections.
ïhe presence of three collections
7
having different chromosome numbers from a single parental
tree indicated that there is Iittle barrier to cross
fertilization between plants with different leveis of
ploidy.
They suggested that such hybridization between
plants with different levels of polyploidy, and subsequent
chromosomal and gene segregation, are the major causes of
the polymorphism found in this species.
Woodworth (1931)
reported chromosome numbers of 56, 70, and 84 in varieties
ofB. papyrifera.
numbers of 2n
~.
Johnsson (1945) reported chromosome
= 70,
71, 72, 75, 77, 80, 82, 83, and 84 for
papyrifera individuals.
were 2n = 84.
Nearly half of these counts
Clausen (1962b, 1963) suggested that a
chromosome number higher than 2,!l
B. papyrifera.
= 84
might exist in
This has not been confirmed.
A similar study was carried out by Brittain and
Grant (1966) using specimens of
British Columbia.
li.
papyrifera collected in
As with the Eastern collections,
these
individuals showed considerable variation in minor
characteristics.
However, 8rittain and Grant did not feel
that there was enough variation in morphological characters
to justify the separation of the western form of this
species into two varieties, 8. papyrifera var.
(Regel) Fern.
Sarge
and~.
paovrifera var. subcordata (Rydb.)
ïhe only difference between these two
their bark color,
co~mutata
vari~ties
var. commutata being darker than var.
is
8
subcordata.
One population was shown to have a somatic
chromosome number of 56.
The remaining populations were
highly variable, and had chromosome numbers of 56, 70, and
84.
The high variability is presumed by Brittain and
Grant to be due to cross pollination.
A further study by Brittain and Grant (1968a) showed
that~.
papyrifera collected in the Rocky mountains, mainly
in Alberta, were large trees and differed From the very
large Pacifie Coast specimens in crown shape and bark color.
The coastal specimens possess a characteristic compact
rounded crown with slender branches and very dark brown bark.
The Rocky mountain specimens have bronzy, close bark
although a few specimens have lighter colored, freely
exfo~iating
bark.
Soma tic chromosome numbers of 70 and 84
were found.
A similar study concerning
~.
papyrifera individuals
collected in northwestern Canada along the mackenzie River
(arittain and Grant, 1968b) revealed the same variability in
morphological characters displayed by populations From other
parts of the country.
The occurrence of resinous glands on
the branchlets of mature specimens and seedlings was
observed more frequently than in eastern specimens.
The
soma tic chromosome number that predominated in these northweste~n
specimens mas 2n
= 84
in contrast to Eastern
specimens where soma tic chromosome numbers of 56 and 70
were represented in higher frequencies.
9
In other studies carried out on western individuals
of B. papyrifera, Johnsson (1945) reported chromosome
numbers
~f
2n
=
70-84 for individuals from British
Columbia with a concentration at 2n
= 75
and 2n
Dugle (1966) reported chromosome numbers of 2n
= 84.
=
ca.56,
ca.62, ca.63, ca.64, ca.68, ca.72, ca.74, ca.75, ca.77, 78,
ca.79, ca.80, 84, and n
= 42
for individuals collected in
western Canada.
Betula resinifera Britt., the Alaskan birch, is
found in bogs, on sandhills, and sunny slopes in northern
British Columbia and Alberta.
Rocky mountains.
It is not found in the
It is found in northern Saskatchewan and
manitoba; northward in the Northwest Territories, the
Yukon, and Alaska to the tree line.
In the south its
margins reach to southern Alberta, Saskatchewan, and
manitoba.
This species was described by Britton and
Rydberg (1901) as fla tree 10-15 meters tall with erect,
spreading branches.
The branchlets are reddish brown,
covered with resinous glands during the first year and
sometimes remaining until the second and third years.
The
bark is thin, reddish brown ta almost white in color, and
marked with dark, elongated lenticels.
The leaves are
dark green on the upper surface, pale ta yellow green on
the lower surface.
They are 3-8 cm long, 2-6 cm wide,
have a slender midrib and pubescent (ultimately glabrous)
10
primary veins.
2-3 mm long."
2n
= 28.
The fruit is a samara, 1-2 mm wide and
They reported the chromosome number to be
Woodworth (1930) reported the chromosome number
of B. resinifera as n
= 14.
In a cytological study of
B. resinifera Brittain and Grant (1968b) determined the
chromosome number to be 2n
= 28,
although among the diploid
progeny of one collection of B. resinifera a triploid
seedling (2n
= 3~ =
42) was found.
Specimens which Dugle (1966) considered to be
g.
hybrids between B. resinifera and
designated
g.
x winteri.
papyrifera were-'
She determined the chromosome
number of these plants to be 2n
tion of individuals at 2n
= 56.
=
28-84 with a concentra-
Brittain and Grant (1968b)
found that specimens which possessed characteristics of
both
g.
papyrifera and
g.
resinifera, and which were
considered to be hybrids between these two species, had a
chromosome number of 2n
= 56.
Betula cordifolia Regel, the mountain white birch,
is an Eastern Canadian species.
It is common in maritime
regions with short growing seasons and low summer temperatures and has been collected in the Atlantic provinces;
northern New Brunswick and Newfoundland, and in the Cape
Breton area of Nova Scotia.
It has been collected as far
west as northwestern Ontario, but apparently does not enter
the prairie region.
Its range overlaps
~ith
that of
Il
g.
papyrifera in the northern portion of the latterls
range.
Brittain and Grant (1967b) described B. cordifolia
as "a tree, the seedlings showing an averagé growth of
1.02 m in five years.
The first leaves are small and
dark, finely pubescent, appearing Iribbed l due to the
presence of darker veins.
The leaves are mostly cordate
with the widest point approximately 1/3 distance From the
base.
The branchlets are smooth.
reaches an average length of 5 cm.
pistillate catkin is 8 mm long,
in smooth curves.
wide."
to be 2n
28.
The bract of the
the lateral lobes ascending
The achene is 2.75 mm long and 2 mm
The chromosome number of B.
=
The female catkin
cordifolia was found
Brittain and Grant (1967b) also found a
few specimens with a chromosome number of 2n
they considered to be tetraploid collections.
some number of 2n
=
= 56.
These
A chromo-
56 was also found in sorne specimens
which were clearly hybrids between B. cordifolia and
g.
papyrifera.
These latter specimens were collected in
sites where the two species were intimately associated.
llioodworth (1929, 1931) reported a chromosome number of
n
=
28 for B. cordifolia.
showed considerable
~eiotic
He found that su ch plants
irregularities.
He suggested
that thesé plants might have been of recent polyploid
origin and,
therefore, did not represent the majority of
B. cordifolia plants.
12
Fernald (1945) suggested that~. cordifolia should
not be regarded as a species distinct From
since the variety
~.
~.
papyrifera
papyrifera var. macrostachya is a
transitional variety between B. papyrifera and B. cordifolia Regel.
Therefore, he suggested that B. cordifolia
be designated as B. papyrifera var. cordifolia (Regel)
Fern.
Brittain and Grant (1965b), in a morphological and
cytological study of
~.
papyrifera and the varieties
macrostachya Fern. and cordifolia, showed that macrostachya
was closer morphologically to
~.
papyrifera than to var.
cordifolia, and had soma tic chromosome numbers of 70 and 84
(similar to B. papyrifera) as opposed to a soma tic chromosorne number of 28 for var. cordifolia.
that~.
They recommended
papyrifera var. cordifolia be reinstated to a
specifie rank, namely,
that of
~.
cordifolia Regel.
Blanchard (1904a) described two newly discovered
species of birch, the blue birch (~. caerulea) and the
large blue birch
(~.
caerulea-grandis).
The leaves of
both were described as being bluish in color, having long
slender petioles, being thin and long-pointed and glabrous
on both sides.
into sheets.
The bark of these trees separated easily
~.
caerulea-grandis was the larger of the
two species, being as large
as~.
papyrifera, and having
--
laroer leaves and fruitino catkins than B. caerulea.
-
second paper Blanchard (1904b) classified the two as
In a
13
B. caerulea and B. caerulea var. grandis, seemingly
indicating that he did not believe the latter to be a
distinct species as he originally had.
However, in a
footnote, he added that he believed his original classification to be the correct one.
Sargent (1905) renamed the large blue birch
B. caerulea var. Blanchardii.
In 1922 he suggested that
both tree forms had originated as hybrids between B.
papyrifera and
g.
populifolia.
Fernald (1922) stated that he believed B. caeruleagrandis to be a distinct species, while B. caerulea was a
hybrid between li. populifolia and
g.
caerulea-grandis.
Woodworth (1929) agreed with Fernald, stating that
Sargent's interpretation was impossible, as he had found
somatic chromosome numbers of 2n
2n
= 28
and 2n
= 28
for li.caerulea-grandis, 2n
=
for
= 28
g.
caerulea,
for B. populifolia,
70 for B. papyrifera.
Erskine (1960) reported on specimens of these
plants found in Prince Edward Island and stated that their
characteristics suggested a hybrid origin between
g.
populifoli? and B. papyrifera.
Brayshaw (1965) stated that the b1ue birches are
found in groups scattered throughout a region which
coincides with the overlap in the ranges of the white
14
birch (~. papyrifera) and the grey birch (~. cordifolia).
He stated that the blue birches differed from the white
and gray birches in the bluish col or of their foliage, but
in other characteristics were similar to either the white
or the grey birches, or were intermediate to them.
After examining specimens of the blue birch and the
large blue birch more closely and comparing their characteristics with those of specimens of grey and white birch,
Brayshaw concluded that the blue birches constitute a
hybrid swarm between the white and grey birches as Sargent
had suggested in 1922.
Brayshaw suggested that this swarm
was of quite recent origin and had established itself in
areas of human disturbance, pastures, cleared roadsides,
etc.; habitats which differed from those of the parent
species and where hybrid seedlings could establish themselves rapidly.
He designated members of this swarm as
B. x caerulea Blanchard.
Brittain and Grant (1967a) carried out a morphological and cytological study on specimens of the blue
birch and the large blue birch, collected in the three
maritime provinces and eastern Ouebec.
that since
~.
papyrifera is the first species to show signs
of growth in the spring, followed by
8. cordifolia,
They suggested
and~.
~.
caerulea-grandis,
Dopulifolia in that order, pollen
discharge would allow cross-pollination to occur readily
15
between
~.
and
~.
caerulea-grandis and either
populifolia.
~.
~.
Cross-pollination between
cordifolia or
~.
populifolia
papyrifera would be difficult due to the
growth fl pattern.
~time
of
Although no hybrids of 8. caerulea-
grandis with 8. cordifolia have been recorded, seedlings
collected in Nova Scotia From one specimen thought to
represent this cross are now growing in the Morgan Arboretum.
A preliminary examination of these seedlings showed no
clear division line between 8. caerulea and 8. caeruleagrandis.
Two other specimens examined were designated as
8. caerulea-grandis.
2n
= 2B.
However,
80th had a chromosome number of
the young leaves and stems of one of
the specimens were characteristic of 8. caerulea-grandis,
while those of the other were characteristic of B. cordifolia.
Brittain and Grant (l.~.) feel that the latter
specimen con tains an Element of B. cordifolia.
Another specimen, grown From seed given to the
Arnold Arboretum by Blanchard, did not appear to be a true
~.
caerulea-grandis, as was believed by 81anchard.
The
description of this plant given by Brittain and Grant
(1967a) difFers From that given for 8. caerulea-grandis.
The soma tic chromosome number for this specimen was found
to be 2n
= â2.
The morphological characteristics
indicated that this tree con tains an Element of 8. cordifolia.
Brittain and Grant suggested that the most probable
16
explanation for this hybrid would be a cross between
Blanchard's parent tree
and a tetraploid
~.
(~.
caerulea-grandis),
cordifolia,
2~
= 56.
2~
=
28,
Brittain and
Grant (1965b) described plants of the latter type.
However,
they are not convinced that the tetraploid B.
cordifolia specimens which they described are pure
cordifolia and they suggest that they may have arisen From
a cross between
2n
=
28.
~.
papyrifera,
2~
= 84,
and 8. cordifolia,
Therefore, the above specimen would be a tri-
hybrid From the cross (~. papyrifera x~. cordifolia) x
B. caerulea-grandis.
Brittain and Grant (1967a) do not agree with Sargent
that~.
~.
papyrifera is involved in the
~.
caerulea-
caerulea-grandis complex, as the somatic chromosome
number of aIl specimens of
caerulea-grandis and
~.
B. populifolia, including possible hybrids, has been found
ta be
2~
=
28, which would not be expected in crosses with
B. papyrifera.
between
~.
~.
Seedlings produced by artificial crosses
papyrifera
and~.
populifolia do not resemble
caerulea-grandis seedlings.
Also the synchronous
blossoming period and common chromosome number of
~.
cordifolia suggest that it may be involved in the
B.
caerulea-~.
caerulea-grandis complexe
later Brittain and Grant (1959) showed in a morphological and cytological study that Ê. cordifo1ia is cleaLly
17
distinct From
g.
8. caerulea is
8. cordifolia.
g. pOEulifolia and that
intermediate to g. populifolia and
They consider g. caerulea and 8. caeruleapapyrifera and
grandis to share a common parentage and to be different
extremes of introgressants between
8. cordifolia.
g.
populifolia and
They feel that it is meaningless to give
names to different introgressants and, therefore, suggest
that all individuals sharing a common parentage between
g.
populifolia and
g.
cordifolia be designated as
x 8. caerulea 81anchard.
They showed in a study of hybrid
indices that 8. caerulea introgresses towards both
g.
populifolia and 8. cordifolia.
The degree of intro-
gression varies in different regions and hence in one area
there may be more
with
g.
g.
caerulea types (greater introgression
populifolia) or more
g.
caerulea-grandis types
(greater introgression with 8. cordifolia).
8etula kenaica Evans is an Alaskan species found
along the northwestern coast.
red or black birch.
Locally it is known as the
Evans (1899) described it as "a tree
up to 13 m in height with thin, dark brown bark separating
into layers.
The twigs are red and shiny.
The leaf b1ades
are 6 cm long, nearly as wide, ovate, slightly hairy wh en
young, becoming g1abrous.
They are dul1 dark green abovE
and paler green underneath, being acute at the apex,
obtuse at the base, sharply, coarsely,
~nd
irregu1arly
18
serrate.
The fruiting aments are 2 cm long and 5 cm wide.
The samara wings are about as wide as the small oblong
nut."
Woodworth (1930) gave the chromosome number of
B. kenaica as n
= 35.
Brittain and Grant (1968b) determined
a somatic chromosome number of 2n
B. kenaica.
=
84 for a collection of
Hultén (1944) reported that hybridization of
B. kenaica with B. resinifera occurs between these two
species in Alaska.
Backcrosses of such hybrids with
B. kenaica may also occur as suggested by the chromosome
number reported for this species by Woodworth.
(1964) reported that some of her specimens of
possessed characteristics of
~.
Dugle
~.
kenaica
papyrifera suggesting that
hybridization also occurs between these two species.
III. Variability within the
genus Betula
It has been shown that the species and varieties of
birch possess different chromosome numbers.
Species
formation in genera such as Betula, where the species do
possess different chromosome numbers, could be due to gene
mutations and other structural changes within individual
chromosomes, to hybridization between varieties and
species, and to duplication of individual chromosomes
o~
chromosome sets and to the recombination of such sets from
different species.
19
As early as 1865 Regel discussed hybridization in
the genus Betula and gave descriptions of sorne hybrid
birches.
Ostenfeld (1910) suggested that hybridization
was probably responsible for the formation of new species
in polymorphie genera.
Helms and Jorgensen (1925) made a
study of hybrid birches from northern Europe and suggested
that sorne of the polymorphism in the genus Betula is due
to hybridization.
Winkler (1904) named fourteen natural
hybrids of the genus Betula and described eight of them.
In 1930 he pointed out that hybridization had played an
important role in the genus Betula and was directly
responsible for much of the variability so characteristic
of this genus.
Woodworth (1929, 1930, 1931) did cytological
studies on several birch species and hybrids to try to find
a reason for the marked variability within the genus.
He
found that hybrid birches representing crosses between
species with unlike chromosome numbers showed meiotic
irregularities, whereas those representing crosses between
species with like chromosome numbers showed few, if any,
meiotic irregularities.
Univalents, abortive pollen grains,
and dyad and monad pollen grains,
irregularities reported.
~re
sorne of the
The production of diploid and
tetraploid pollen grains has been recorded mainly from
heterozygou~
types.
He further suggested that such
abnormal gametes are instrumental in the production of
higher polyploids.
20
Woodworth concluded from his studies that
1. Polymorphie groups of plants are consistently proving
to be groups of plants containing species which
hybridize rapidly.
2. A wealth of Evidence indicates hybridization is the
cause, and polymorphism, the effect.
3. Setula is a highly polymorphie genus.
4.
Polymorphie groups usually show polyploidy.
5. Setula is a polyploid genus containing diploid,
triploid,
tetraploid, pentaploid, hexaploid, and
dysploid species and hybrids.
6. Polymorphism in Setula is apparently due to the
readiness with which the species cross in nature.
7. It follows that Setula is another genus in which the
multiplication of species has come about,
partly at
least, by hybridization.
Johnsson (1940), after a systematic and cytological
study of Swedish birches, concluded that the great variability of sorne birch species is not due to hybridization
but to genetical heterogeneity.
Regel (1865) pointed out that FI hybrids often have
sterile pollen, and are, therefore, more likely to be
pollinated by either of the parent species.
He stated
that such backcross progenies resemble the recurrent
parent more closely, rather than being interrnediate to the
parental species.
21
Cases of introgression have been reported by
Froiland (1952) who found Evidence of introgressive
hybridization of
g.
g.
occidentalis Hook. into
papyrifera
in a hybrid swarm near Boulder, Colorado.
Clausen (1962)
reported Evidence of introgression between
g.
glandulifera Regel and B. papyrifera.
pumila var.
He proposed the
hypothesis that gene flow is in the direction of higher
degree of ploidy.
Dugle (1966) reported that introgressive
hybridization was a common phenomenon among western Canadian
species.
She found cases where introgression was towards
the parent with the lower chromosome number.
Introgressive hybridization leads to the establishment of populations containing a few genes of one species
on the genetic background of another.
These populations
may be better suited ta new ecological niches than either
parent, and introgression may thus play an important raIe
in the Evolution of ecotypes.
Clausen (1966) made a study ta test the compatibility in the genus Betula.
Information on inter-
specifie compatibility may lead to a clearer understanding
of the phylogeny and Evolution within the genus.
that
~.
populifolia
appea~s
He found
to be nearly self-incompatible.
Only a few of his intraspecific crosses succeeded and
germination was less than
2%
in aIl cases.
ïhree out of
four 8. papyrifera trees set seed which ranged From 6-11%
22
germination.
Thus a certain amount of self-compatibility
seems ta be present in this species.
crosses he found that
~.
papyrifera.
g.
~.
alleghaniensis crossed weIl with
pendula x
reciprocal were successful.
~.
In interspecific
g.
papyrifera and its
Crosses of B. pendula with
populifolia produced few viable seeds and small seed-
ling populations.
Clausen concluded that self-incompatibility is
common in Betula species, but that the degree of incompatibility varies with individual plants.
Interspecific
crosses showed that most Betula species can be crossed
without difficulty but that sorne incompatibility occurs.
Reproductive isolation between Betula species is not
complete.
The differences in chromosome numbers between
different species of Betula may complicate interspecific
compatibility patterns.
However, not enough information
is available ta tell whether compatibility increases with
increasing ploidy levels.
There are indications that
crosses succeed more readily when made in the direction of
low ploidy female ta high ploidy male.
23
8. Cytophotometry
1. Cytophotometry in the study
of nucleic acids
The nucleic acids are highly polymerized substances
which stay in the cell after fixation.
Cytochemical
methods used to study nucleic acids are dependent upon the
properties of three components of the nucleotide:
phosphoric aCid, carbohydrates, and the purine and pyrimidine bases.
The carbohydrate deoxyribose present in DNA
is responsible for the Feulgen reaction, specifie for this
nucleic acid.
The technique for the Feulgen reaction was
developed by Feulgen and Rossenbeck (1924).
Fixed tissue
is submitted to a mild hydrolysis and then treated with
Schiff's reagent.
The hydrolysis frees aldehyde bases in
the purines (adenine and guanine) of the DNA molecule, and
these then react with the Schiff's reagent to form a purple
compound.
Cytochemical organization of the cell nucleus can
be studied by applying qualitative and quantitative
methods of photometrie analysis ta microscopie work.
Nucleic acids have a characteristic absorption spectrum in
visible, ultraviolet, or infrared light.
The degree of
absorption is re1ated to the concentration of the absorbing
material.
Caspersson (1936) used photometrie techniques
ta measure the ultraviolet absorption of substances in
24
tissue sections.
He showed that under certain conditions,
and in connection with a sensitive photometer,
the micro-
scope could be used as a microspectrophotometer.
Using
quartz lenses developed by Koller and an appropriate light
source he found that the maximum absorption for nucleic
acids was at 2600A.
The height of such an absorption
curve is proportional ta the amount of absorbing material
and thus permitted both qualitative and quantitative
analysis of certain cellular compounds.
The specifie absorption of nucleic acid (at 2600A)
is due ta the presence of purine and pyrimidine bases.
This absorption is similar for RNA and DNA.
cytophotometry,
Ultraviolet
therefore, permits localization of both
types of nucleic acid, while the nuclear reaction of DNA
with Schiff's reagent reveals the presence of DNA.
The
Feulgen nuclear reaction mas adapted ta quantitative
determinations of DNA by Di Stefano (1948) using monochroma tic light of 550 mu corresponding to the maximum
absorption of this stain.
II. Cytophotometric studies of
nucleic acids
Early studies, using cytophotometrical techniques
to assess the relationships between karyotypes of related
species, were carried out by the Schraders (Hughes-Schrader,
25
1953, 1958; Schrader and Hughes-Schrader, 1956, 1958).
Hughes-Schrader (1953) undertook a study of species in the
Mantid genus Liturgousa.
L. maya
(a
(a
2rr
= 17), h.
In the three species studied,
actuosa
(a
2n
= 23),
and
h.
cursor
2n = 33), an inverse relationship between chromosome
size and number suggests that structural rearrangements
not involving polyploidy have brought about the diversity
of the karyotypes.
This non-po1yploid relationship
suggested by the chromosome morpho1ogy was confirmed by
microspectrophotometric de termina tian of the nuclear DNA.
Liturgousa maya, with eight pairs of autosomes, had an
identical nuclear DNA content ta that of L. cursor with 16
pairs of autosomes.
Liturgousa actuosa, intermediate in
chromosome number and size ta
1.
maya and
half as much again DNA per nucleus.
h.
cursor, had
A fourth Liturgousa
species designated as species ~ (a 2n
= 21)
was shawn ta
be separable morphologically from L. actuosa but ta have
an Equivalent nuclear DNA value.
similar morphologically ta
1.
Liturgousa sp. n was
maya and
1.
cursor but had a
DNA value one and a half times as great.
The fact that the total length oF all the chromosomes of the complement is approximately the same in all
four species supports Hughes-Schrader's conclusion that
the karyotypes of species
~,
maya, and actuQsa have
differentiated by structural rearrangements of chromosomal
26
parts involving little or no change in amount but accompanied by loss or gain of from two to six centromeres.
In
the differentiation of maya and cursor, however, the
entire complement of chromosomes has been involved.
Therefore, a large change in DNA content of the
nucleus can occur in the process of evolutionary differentiation with no corresponding and detectable effect
on karyotypes (Hughes-Schrader, 1953).
How does one explain the relationship between maya
and cursor on the one hand, and species
the other?
~
and actuosa on
Here there is a change in relative nuclear DNA
amount while the total chromosome length remains the same.
Assuming that the direction of evolution is from a
lower to a higher DNA value, Hughes-Schrader suggests that
a ratio of 1:1.5 might suggest a differential polyteny in
the two haploid sets of one species.
by the cytological Evidence.
This is not supported
Hughes-Schrader feels that a
more agreeable suggestion is that of differential polyteny
among individual chromosomes or of regional differences in
DNA synthesis along the length of the individual chromosomes.
Hughes-Schrader and Schrader (1956) studied eleven
species of the tribe
Pentato~
of the order Hemiptera.
of one of the sub-farnilies
They found extreme diversification
in ONA values of the species
c~mbined
with constancy in
27
chromosome number and uniformity of chromosome complement.
They suggested in such cases, where closely related species
have chromosome complements which show no evidence of
revolutionary change in number and structure and yet display large differences in DNA content, that polyteny may
be the most likely explanation for this difference.
Wahrman and O'Brien (1956) measured the DNA content
of two species of the genus Ameles.
have five different karyotypes.
These two species
The nuclear DNA content,
as determined by Feulgen cytophotometry, was almost
identical for different karyotypes within the same species.
DNA values between the two species showed a slight but
significant difference.
These results support a Robertson-
ian equivalence inferred From a morphological analysis.
The differences in karyotypes are due to translocations.
Slight structural rearrangements, undetectable cytologically,
su ch as duplications and deletions,
seem to be the most
probable cause of differences in DNA content, but Wahrman
and O'Brien suggest that the interspecific difference in
DNA content could also be related to differences in chromosome diameter.
fficLeish (1962) studied relationships betmeen RNA
and DNA in the nuclei of diploid,
tetraploid, and hexaploid
Allium species and diploid and tetraploid forms or
Tradescantia ohioensis, Vicia faba,
Pisum
~
-
sa~~vum,
and
28
Zaa mays.
He found large differences between the nuclear
DNA contents of these species un~ccompanied by a corresponding increase in RNA.
RNA and DNA should show parallel
variations with differences in chromosome size and number
due to polyteny and polyploidy.
increase with higher ploidy.
DNA content was found to
RNA content did note
This
was true not only for distantly related species but for
closely related ones also.
It was true for a polyploid
series within one genus; for diploid and tetraploid forms
of a single species; and for the closely related V.
faba
and P. sativum where differences in chromosome size and
DNA content may be due to polyteny.
In no case did
mcLeish find any correlation between DNA content and total
nuc1ear RNA.
Ha1kka (1964) undertook a photometrie study of six
species representing the three subgenera of the genus
Luzula.
Feulgen cytophotometry showed that the DNA content
of tetraploid and hexaploid forms was respectively two and
three times the DNA content of the diploid level.
mello-
Sampayo (1961) had concluded that there were three lines
of Llromosomal evolution in the genus Luzula:
1. Ordinary
polyp10idization, 2. DifferentiaI polyteny or longitudinal
partition, and 3. Agmatoploidy or transverse fragmentation.
Halkka found that the amount of DNA increased
~ith
the
degree of true polyploidy but remained constant with
30
cellular DNA content and cell size in polyploid series and
between total chromosome length and cell size.
This
suggested that an increase in DNA content automatically
results in increased cell size.
Interspecific variations
in DNA content could arise by polyploidy, aneuploidy, or
by differential polynemy.
Martin proposed a hypothesis
stating that selection acts on a variant with increased
DNA through its effect on cell size, i.e., variations in
DNA per cell may have evolved by selection for variations
in cell size.
Christensen (1966) studied the DNA content of diploid
and polyp1oid Enchytraeidae.
He found that in 45 species
the relative DNA content varied From a value of 0.40 to
one of 4.85.
0.40 and 1.12.
The range in dip10id species was between
Sorne genera showed wide variation both in
chromosome number and DNA content whereas others showed
only a small variation.
Species of one genus showed a
wide variation in DNA content but a constancy in chromosome number.
Out of ten po1yploid species studied, he
found that nine showed chromosome numbers and DNA contents
which were multiples of those values found in related
diploids or lower polyp1oids.
Polyploidy, therefore, is
an evolutionary mechanism which opera tes in lower animals
as weIl as in the plant kingdom.
31
Beçak (1967) measured DNA values in three species
of South American frogs belonging ta the family Ceratophrydidae:
Odontophrynus cultripes (2n = 22),
Q. americanus (2n
104).
= 44),
and Ceratophryus dorsata (2n
=
He used nuclei from the erythrocytes, liver, and
pancreas for his measurements.
The relative DNA values of
the three species conformed to the 1:2:4 ratio expected if
one assumes polyploid evolution in this family.
Southern (1967) used cytophotometry to study the
relationships of diploid and polyploid species belonging
to the sub-section Eriostemones of the genus Tu1ipa.
He
found that the range of DNA values for diploid species is
relatively small, but that they do differ significant1y.
Southern found that the se values provided little assistance
in ascertaining the pattern of p10idy invo1ved in the
evolution of this group, but demonstrated that cytophotometry could be used as a means of differentiating between
diploid, triploid, and tetraploid species of Tulipa.
Keyl (1965) developed a high1y refined technique
which permitted a comparison of the DNA content of chromosomes or chromosome segments rather than that of the entire
nucleus.
In a study of primary spermatocytes and salivary
gland nuclei he found that Chironomus thummi thummi had
27% more DNA than C. thummi piger.
He concluded that this
difference depends on the fact that certain bands in the
32
polytene chromosomes of thummi thummi contain 2, 4, 8, or
16 times the amount of DNA present in homologous bands of
thummi piger.
DNA content.
This is a localized method for increasing
He felt that there were no grounds to sustain
the argument that a variation in lateral multiplicity of
DNA strands accounts for DNA changes.
Ullerich (1966) studied the differences in DNA
content between three species of the genus Bufo.
He found
that the relative total DNA values per genome were:
B. bufo, 1.49;
~.
viridis, 1.07; and
g.
calamita, 1.00.
He concluded that these differences were due to sorne type
of localized duplication, as described by Keyl,
this time
in the large chromosomes of B. viridis and in all the
chromosomes of
g.
bufo, during the evolutionary process.
John and Hewitt (1966), in a study of the karyotypes of nine species of Acrididae, found that on the
average those species having 23 chromosome arms in the
male karyotype had less DNA than those species having 17
chromosome arms in the male karyotype.
They suggested
that these differences in DNA content may be due to
localized duplication of certain bands in the polytene
~
chromosome number through the production of metacentric
members.
Rees et~.
three genera:
(1966) studied dip10id species in the
Lathyrus, Vicia, and Lolium.
They found
33
that a correspondence in chromosome number did not
indicate a correspondence in DNA value.
They discussed
changes in chromosome material assbciated with polyploidy
stating that increases in chromosome number due to polyploidy bring about an increase in the number of linkage
groups, which may be detrimental to the organisme
Increases independent of polyploidy would not have this
effect.
They concluded that for lolium, at least, pachy-
tene loops found in FI hybrids between species with smal!
and large chromosomes imply variation in DNA content is
due to lengthwise duplication of chromosome segments.
Rees and Jones (1967) favored the idea of lengthwise incorporation or loss of a chromosomal segment to
account for DNA changes observed in species of Allium.
They stated that the difference in chromosome size and in
chromosomal DNA between
~.
cepa and A.fistulosum is due
entirely to lengthwise incorporation or loss of chromosome
segments.
Jones and Rees (1968) found that widespread changes
in nuclear DNA content accompanied the divergence and
evolution of species within the genus Allium.
Such varia-
tion was largely independent of change in the basic
chromosome number but they found that nuclear DNA content
was proportional to chromosome volume.
The DNA changes
were found to be highly localized within the individual
34
chromosomes, the variation being due to lengthwise
duplication or loss of chromosome segments.
III. Assessment sf the cytophotometrie technique
It has been found that related species with
markedly different chromosome numbers have the same DNA
content; species in the same genus have shown a 2:1 ratio
in chromosome number, but a 1:1 ratio in DNA content,
indicative of differential polynemy; or a 1:1 ratio in
chromosome number accompanied by a 2:1 ratio in DNA content, indicative of differential polyteny; different
sub-species of the same species have shown geometrical
differences in DNA content of the same cytological locus,
i.e., a specifie polytene band.
DNA comparisons may be
capable of reflecting evolutionary changes that may pass
undetected from a simple comparison of chromosome
morphology.
Thus, the measurement of the relative
quantity of DNA in equivalent nuclei of related species
pre vides a method for assessing relationships between
karyotypes in related species.
mATERIALS AND mETHODS
I. Plant material
The plants used in this study represent a wide
variety of species collected from various locations across
Canada.
They are listed in Table l along with their
sources and chromosome numbers.
The plants were grown in
six-inch plastic pots and were kept outside in cold frames
during the summer.
During September the plants remained
in the cold frames but the photoperiod was lengthened to
twenty-four hours using artificial lighting.
In October
the plants were moved into the greenhouse where they were
kept at a temperature of 55-59 o F.
An electric heating pad
fitted with a thermostat was placed on the bench un der the
pots to keep the soil temperature at 6S o F.
Irradiation
from an HO type, 400 watt mercury-vapor lamp above the
plants lengthened the photoperiod to twenty-four hours in
an attempt to prevent
~he
period during the winter.
plants from going into a dormant
The plants were watered each
day, after the root tips had been collected, with a solution of .40 9 of 10-52-10 (N-P-K) in 1 liter of water.
35
36
TA BLE I.
The species studied,
their accession number,
source, and chromosome number. Authorities for the species
are given in the Introduction
------------------------------------------------------------------------------------------------------------------Species
Accession
number
Scurce
Soma tic
chromosome
number
(2.!J)
Diploids
B. caerulea
6
Val cartier
Quebec
28
B. caerulea-grandis
7
Charlottetown
P.LI.
28
B. caerulea-grandis
7G
Newton, near
Guysboro, N. S.
Morgan Arboretum
Quebec
28
Val cartier
Quebec
28
B. pendula
21
B. caerulea-grandis
65
28
B. resinifera
107
Remples Road
Alberta
28
B. resinifera
110
Swan Hills
Alberta
28
B. cordifolia
180
Catfish Lake
Ontario
28
B. populifolia
633
Lac Carré, Quebec
28
Arnold Arboretum
masse
42
Culloden, Digby
Co. , fil. S.
ffiorgan Arboretum
Ouebec
Fish Lake Raad
B.C.
56
Triploids
B. caerulea-grandis
7AA
(hybrid)
Tetraploids
B. papyr ifera
Il
8. papyrifera x
populi fel ia
B. species
81
ë.
(table continued)
119
56
56
37
TABLE 1. (continued)
==========================================================
Species
Accession
number
Soma tic
chromosome
number
(2n)
Source
Tetraploids (continued)
B. papyrifera
150
st. John,
N.B.
56
B.
329
Fish Lake Road
B.C.
56
4
Hall's Harbor
N.S.
70
8
Pictou, N.S.
70
species
Pe:ïtaploids
B.
pap~rifera
papyrifera
-B.
var. macrostach;ta
B. pap;trifera
19
ffiorgan Arboretum
Quebec
70
B. kenaica
91A
Arnold Arboretum
maSSe
70
B. pap;trifera
132
Alexandria
Br idge, B.C.
B.
pap~rifera
149
Fredericton
N.B.
70
8.
pap~rifera
227
ffianitoulin l s.
Ontario
70
Br idg ewa ter, N.S.
84
Hexap10ids
B. paE:irifera
1
B.
pap~rifera
15
Hal1's Harbor
N.S.
84
B.
paE:trifera
52
Brier Is., N.S.
84
B. pap:trifera
53
Brier l s. , N.S.
84
B. E;!8E:trifera
56
Jasper National
Park, Alberta
84
39
which suppresses nuclear division by arresting mitosis at
metaphase) for one hour at room temperature (20-22 0 C).
The root tips were fixed in Carnoy's fluid (absolu te
alcohol-glacial acetic acid, 3:1, v/v) for a minimum of
twenty-two hours at room temperature.
In preparation for
staining, the root tips were washed in running distilled
water for fifteen minutes.
They were th en hydrolysed.
After testing hydrolysis periods of 6, 8, 10, 12, 14 and
16 minutes, it was found that the chromosomes stained
deepest with a hydrolysis time of 12 minutes in N HCl at
60 o C.
These results are presented graphically in Figure 1.
Hydrolysis was arrested by washing the root tips in cold
distilled water for thirty minutes.
The root tips were
stained in basic fuchsin (Feulgen technique) for two hours
and then partially decolorized with three rinses of 50
2
~ater
(S ml of N HCl, S ml of 10% Na 2 5 2 0 S ' 100 ml of
distilled water) for ten minutes each.
After staining,
the root tips were placed in 4% pectinase in distilled
water to break down the pectin in the middle lamella of
the cell wall so that the cells would separate weIl mhen
squashed.
The root tips were squashed on a glass slide in
4S% ace tic acid.
The slides were made serni-permanent by
sealing the coverslips with clear nail-polish.
Using slides prepared in the above manner,
tan
metaphase cells in mhich the chromosomes were very weIl
Figure 1.
Feulgen stain bound by chromosomes
of Betula species after hydrolysis in N HCl at 60 0 C
for various intervals of time.
41
3
....
-..
."
2
a
~
~
.a
C
a
1
c
=-:
ca
2
4
6
a
10
12
14
16
18
20
42
spread, were examined for each of the plants and karyotypes were made.
Using a camera lucida the chromosomes
were drawn in outline and later were measured using a
millimeter ruler and a set of calipers.
Idiograms of the
chromosome complements measured in this manner were
prepared uSing the total complement length (TeL) as the
ordinate, the chromosomes being arranged in order of
decreasing size of the small arm of the chromosome,
the
centromere being given a constant spacing.
sis of the deox ribonucleic acid
content of the root ti s
IV.
The density of nuclear deoxyribonucleic acid (DNA)
was calculated using a Barr and Stroud Integrating microdensi tometer.
before.
Root tips were collected and cleaned as
However,
the pretreatment in 8-hydroxyquinoline
was omitted as it was not necessary to arrest mitosis at
metaphase.
The root tips mere fixed in Carnoy's fluid and
washed, hydrolysed, and stained in the same manner as
above.
Root tips from each plant were processed in the
same vial with root tips from a plant cho$en as the
standard, the length of the root tip distinguishing the
standard from the root tip with which it was being
compar-ed.
To minimize any errors due to imperfections
between slides, root tip
~eristems
fro~
bath plants
(standard and sample) were squashed in 45% ace tic acid on
43
the same slide at different locations.
The relative DNA
content of the nuclei in telophase was measured.
Three
readings per nucleus and three corresponding background
readings were taken and the readings averaged.
The
relative absorption was found by subtracting the average
background reading from the average abject reading.
Sixt Y
chromosome fields (three series with twenty readings per
series) were examined for each of six diploid, one triploid,
five tetraploid, five pentaploid and five hexaploid species.
RESULTS
A. The Stomatal measurements
The stomatal guard cells were measured in nine
diploid, one triploid, five tetraploid, se ven pentaploid
and se ven hexaploid plants comprising a total of twentynine different accession numbers and representing nine
different species.
The species studied and the results of
the measurements are presented in Table II.
The mean
stomatal lengths (p) for each level of ploidy are
summarized graphically in Figure 2.
was calculated and analysed.
are presented in Table III.
A linear regression
The results of the analysis
The F value obtained was not
significant and therefore, a straight line does satisfactorily explain the relationship between mean stomatal
length and chromosome number in species of Betula.
44
45
TABLE II.
ffieasurements of the stomatal guard cells for
diploid,
triploid,
tetraploid,
pentaploid and hexaploid
species of Betula
==========================================================
ffiean
stomatal
length Cll)
Range of
Accession based
Species
on
50
stomatal
number
measurements lengths Cll)
from each of
1-3 plants
Diploids
B. caerulea
6
1. 37.84
2. 34.14
3. 37.41
28.4 - 48.4
24.4 - 46.0
28.4 - 52.0
B. caerulea-grandis
7
1. 48.67
3. 39.80
39.6 - 60.4
28.4 - 48.0
30.0 - 54.0
1. 39.52
2. 39.63
3. 45.32
32.0 - 56.0
32.0 - 56.0
36.4 - 56.0
1.
2.
3.
1.
2.
1.
2.
3.
20.0
28.0
28.0
36.0
32.0
34.9
28.4
28.4
24.4
29.0
29.0
30.0
22.0
27.6
2. 37.74
B. caerulea-grandis
7G
B. pendula
21
B. caerulea-grandis
65
B. resinifera
107
B. resinifera
110
25.98
37.94
38.64
43.38
37.53
42.38
39.00
37.15
1. 33.98
2. 35.82
3. 35.30
B. cordifolia
B. populifolia
180
1. 37.02
633
2.
3.
1.
2.
3.
30.76
32.82
38.37
37.63
37.90
-
44.4
52.4
52.4
56.0
48.0
44.0
48.0
51.6
44.4
40.4
44.4
44.4
36.4
40.4
30.0 - 44.4
30.0 - 44.4
28.0 - 46.0
Triploids
~.
-
caerulea-Jrandis
(hybrid
(table continued)
7AA
1. 38.26
32.4 - 42.0
46
TA8LE II.
(continued)
==========================================================
Mean
stomata1
Range of
1ength ClJ)
Accession based
Species
on
50
stomata1
number
measurements 1engths ClJ)
From each of
1-3 plants
Tetrap10ids
8. papyrifera
8. papyrifera x
8. popu1ifo1ia
11
81
8. species
119
8. papyrifera
8. species
150
329
-
1. 39.59
1. 38.34
2. 38.61
1. 47.14
2. 45.32
1. 35.19
1. 42.02
2. 41.71
52.4
44.4
32.4
34.0
44.4
32.0 - 68.4
24.0 - 66.0
28.0 - 44.4
50.0
31.6
31.6 - 50.0
1.
1.
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
1.
39.02
39.15
44.38
47.04
48.07
34.14
37.14
35.93
40.90
44.80
41.69
44.44
37.30
48.4
30.0
50.0
22.0
32.4
52.0
38.0
60.0
38.0
60.0
29.4
40.4
28.4
43.6
28.4 - 40.4
32.0
59.6
32.4
56.0
64.0
32.4
36.4
60.4
44.4
30.0
1. 41. 70
2. 42.55
3. 43.84
24.0 - 60.0
28.0 - 60.0
40.0 - 60.4
30.0
Pentap10ids
8. papyrifera
4
8. papyrifera
8. papyrifera
8
19
8. kenaica
91A
papyr i fera
132
8. papyrifera
8. oapyrifera
149
227
8-.
--
-
--
-
-
Hexaploids
8. oapyrifera
(table continued)
l
47
TA8LE II.
(continued)
-------------------------------------------------------------------------------------------------------------------
Species
Accession
number
Mean
stomatal
length (p)
based on 50
measurements
From each of
1-3 plants
Range of
stomatal
lengths (p)
Hexaploids (continued)
8. papyrifera
5
1. 49.88
2. 47.14
30.6
40.0
8. papyrifera
9
8. papyrifera
15
8. papyrifera
52
8. papyrifera
53
32.4
40.0
40.4
36.4
39.6
34.0
36.0
35.6
35.6
8. papyrifera
56
1.
1.
2.
3.
1.
2.
3.
1.
2.
1.
2.
3.
38.73
51.44
52.03
50.32
46.73
43.60
44.25
43.28
44.24
39.49
39.07
39.66
31.6
31.6
31.6
-
64.4
64.4
- 56.1
60.4
- 60.4
60.4
62.0
48.4
60.0
60.0
60.4
- 48.4
- 48.4
46.0
---
-
-
Figure 2.
The relationship between the
mean stomatal length and chromosome number in
Betula species.
Theoretical expected values are
represented by open circles. Observed values are
represented by solid circles.
)
49
45
44
43
--=
--=
42
41
t:.O
C.I
-'
-
40
CI:
~
39
E
0
en
=
CI:
C.I
::
38
37
36
35
14
28
42
Chromosome
56
70
84
Namber (2a)
TABLE III.
Analysis of linear regression in Figure 2
==================================c==============================================
Sourco of variation
Degre8s of freedom
Sum of squares
Mean square
F value
Rogression
l
22.6099
22.6099
-3.69
Residual
3
-6.1335
-2.0445
U1
o
51
B. The Karyotypes
Representative photographs of the chromosome complements of one diploid, one tetraploid, one pentaploid
and one hexaploid species of Betula are shown in Figures
Idiograms of the chromosome complements for six
3-6.
diploid, one triploid, one tetraploid, one pentaploid and
one hexaploid species of Betula are shown in Figures 7-9.
The measurements of the somatic chromosomes taken from
ten metaphase cells for each of the ten species are
presented in Table IV.
The numbers 1-14 for the diploid
species, 1-21 for the triploid species, 1-28 for the
tetraploid species, 1-35 for the pentaploid species and
1-42 for the hexaploid species, representing the pairs of
homologous chromosomes, are arranged in descending order
of the length of the chromosomes, chromosome pair number 1
being the longest pair of chromosomes in the complement.
The total chromosome complements (TeL) and arm ratios
(L/S) were calculated in the following manner:
L
=
length of long arm
S
=
=
=
length of short arm
n
N
number of chromosomes
number of cells (here, N
= 10)
The total length of a chromosome
The ratio of long arm to short arm
=
IIN (L
=
+
lIN (L/S)/N
The total length of the chromosome complement
I1N[I1n(L
+
S)]/N
S)/N
=
Figures 3-6.
Somatic chromosomes from
root tip ceIIs of diploid, tetraploid, pentapioid
and hexapioid species of Betula.
Fig. 3.
ffietaphase of
B. populifolia, 2n = 28.
Fig. 4.
ffietaphase of
B. papyrifera, 2n = 56.
Fig. 5. ffietaphase of
B. papyrifera, 2n = 70.
Fig. 6.
metaphase of
B. papyrifera, 2n = 84.
53
Figure 3
Figure 5
Figure 4
Figure 6
10
55
5
111111118111110 III
11111111911111111
. :. 11111111111111
1111111111111'
A
10
15
.
.
.
10
~ : . 11111111111111
11111111111110
:
~
B
5
10
.
c
1IIIDIBIIIDDBl'J
11111111111111
D
15
10
: 1IIIIIIIIIBU8a
11111111111111
5
10
15
E
InUllIlIDUI
11111111111111
F
)
Figure 8.
Idiograms of trip1oid, tetrap10id
and pentap10id species of Betu1a.
G
B. caeru1ea-grandis (7AA)
H
B. species (119)
l
B. kenaica (91A)
57
10
5
0
5
10
G
15
10
5
....1
Q
0
&-
5
~
10
ODmgmDDDDDDDn06DcoœD~mmmDBCD
mg~nl~rnmDrnnDODOODg~BDDDGBmgm
Il
15
10
5
0
ODODconCODOOODOBOCCDacccocccooocceo
BBCDDODDCDaDDüODOOOnDDODC9COCCCOB~D
5
10
15
1
)
Figure 9.
Idiogram of a hexaploid species of Betula.
J
8. papyrifera (1)
)
59
5
o
crnec~rnmBsm~GDemOSDm~mœsaBaC~DceacceBmmDace
cmmB9mSœ~mBmmlmDmD~mmBmBmmCmmaaBe~aD~aacaB
5
10
15
20-
J
60
TABLE IV.
Karyotype analyses of the somatic chromosomes
for ten Betula species.
TCl = total complement length;
lis = long arm/short arme
For the 2n = 42, 56, 70 anà 84
chromosome taxa,
the length in microns has been given for
only the longest and shortest chromosome pair.
==========================================================
length
Chromosome
Species
in
%
TCl
lis
pair
microns
B. caerulea (6)
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
1.75
1.33
10.36
9.17
2.06
1.19
8.45
1.09
1.92
8.06
2.16
1.04
7.72
1.89
1.00
7.55
1.83
0.97
7.17
2.14
0.93
6.85
1.94
0.88
6.63
1.64
0.86
6.30
1.50
0.81
5.94
1.37
0.77
5.64
1.58
0.73
5.34
1.40
0.69
4.83
1.49
0.62
Average TCl = 25.74l!
g.
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
10.65
1.50
1.54
9.26
1.57
1.34
8.64
2.69
1.26
8.22
1.38
1.19
1.35
7.94
1.15
7.55
1.50
1.10
1.49
7.24
1.05
1.64
6.89
1.00
1.53
6.61
0.96
1.53
6.25
0.91
5.86
1.46
0.85
5.54
2.77
0.81
1.29
5.11
0.74
1.51
4.24
0.62
Average TCl = 28.97lJ
caerulea-grandis
(7)
(table continued)
61
TABLE IV.
(continued)
==========================================================
length
Chromosome
Species
TCl
in
lis
%
pair
microns
B.
caerulea-~randis
(7G)
B. eendula (21)
( table continued)
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
10.09
1.12
1.45
9.26
1.20
1.34
1.17
8.94
1.28
8.47
1.33
1.22
7.93
1.43
1.14
7.60
1.48
1.09
7.28
1.62
1.05
7.03
1.53
1.01
6.66
1.55
0.96
6.12
1.51
0.88
5.63
1.33
0.81
5.32
1.19
0.77
4.96
1.17
0.72
1.20
0.68
4.71
Average TCl = 28.721J
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
12.15
1. 61
1.75
1.33
1.42
9.87
9.11
1.42
1.31
8.48
1.40
1.23
8.04
1.65
1.17
1.60
7.38
1.07
1.65
6.95
1.01
6.68
1.37
0.97
6.19
1.42
0.90
5.69
1.33
0.82
5.47
1.30
0.78
5.18
1.42
0.74
4.76
1.15
0.67
4.06
1.36
0.58
Average TCl = 28.781J
62
TABLE IV.
(continued)
------------------------------------------------------------------------------------------------------------------Species
B. resinifera (110)
Chromosome
pair
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
B. populifolia (633)
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
(table continued)
% TCl
Lis
10.26
1.42
9.23
1.46
8.69
1.44
8.14
1.50
7.69
1.41
7.27
1.42
6.91
1.28
1.26
6.63
6.46
1.30
1.27
6.21
1.15
5.99
5.86
1.20
5.56
1.17
5.14
1.31
Average TCl =
length
in
microns
1.20
1.08
1.02
0.95
0.90
0.85
0.81
0.77
·0.75
0.72
0.70
0.68
0.65
0.60
23.29~
1.57
10.62
1.32
9.35
1.17
1.38
1.14
1.31
8.85
8.46
1.20
1.26
1.16
1.21
8.21
1.31
7.91
1.17
7.26
1.29
1.08
6.86
1.62
1.02
6.46
1.56
0.95
1.55
6.08
0.90
1.32
5.74
0.85
1.27
5.28
0.78
4.83
1.15
0.72
1.47
0.61
4.10
Average Tel = 29.53u
63
TABLE IV.
(con tinued)
---------------------------------------------------------Species
B. caerulea-9randis
(hybrid; 7AA)
Chromosome
pair
1
2
3
4
5
.6
7
8
9
10
Il
12
13
14
15
16
17
18
19
20
21
% TCL
6.86
6.28
6.09
5.91
5.74
5.55
5.37
5.25
5.08
4.93
4.75
4.60
4.39
4.30
4.22
3.97
3.71
3.57
3.32
3.21
3.09
lis
1.31
1.24
1.21
1.12
1.13
1.26
1.17
1.26
1.29
1.24
1.34
1.69
1.50
1.51
1.48
1.49
1.33
1.20
1.19
1.24
1.17
Length
in
microns
1.58
0.71
Average TCl = 46.09JJ
(table continued)
64
TABLE IV.
(continued)
==========================================================
length
Chromosome
Species
%
TCl
in
lis
pair
microns
B. papyrifera (119)
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
15
16
17
18
19
20
21
.. 22
23
24
25
26
27
28
5.90
5.42
4.98
4.78
4.55
4.35
4.18
4.07
3.94
3.80
3.73
3.63
3.47
3.41
3.33
3.25
3.14
3.05
2.98
2.94
2.90
2.81
2.77
2.70
2.63
2.55
2.45
2.35
1.37
1.28
1.23
1.27
1.28
1.37
1.39
1.3B
1.42
1.45
1.45
1.51
1.28
1.40
1.35
1.35
1.30
1.26
1. 24
1.37
1. 31
1.32
1. 36
1.24
1.26
1.16
1.09
1.20
Average TCl
(table continued)
1.62
0.64
= 55.00~
65
TABLE IV.
(continued)
-------------------------------------------------------------------------------------------------------------------Species
B. kenaica (91A)
Chromosome
pair
1
2
3
4
5
6
7
8
9
10
Il
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
% TCl
4.33
4.02
3.77
3.66
3.59
3.53
3.43
3.38
3.36
3.30
3.24
3.18
3.10
3.03
2.96
2.92
2.86
2.82
2.79
2.74
2.72
2.70
2.62
2.58
2.52
2.45
2.39
2.26
2.16
2.08
2.01
1.94
1.89
1.81
1. 72
1.30
1.40
1.20
1. 38
1.19
1.16
1.09
1.10
1.10
1.14
1.12
1.23
1.26
1.44
1.45
1.54
1.56
1.63
1.66
1.62
1.63
1.68
1.68
1.65
1.47
1.54
1.47
1.22
1.22
1.25
1.10
1.13
1.08
1.10
1.20
Average TCl
( table continued)
length
in
microns
lis
=
1.63
0.65
75.421.1
66
TABLE IV.
(continued)
---------------------------------------------------------Species
B. EaE:lrifera (1)
Chromosome
pair
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
% TCl
3.55
3.34
3.18
3.10
3.03
2.98
2.90
2.82
2.79
2.74
2.68
2.65
2.61
2.58
2.56
2.52
2.49
2.45
2.43
2.42
2.38
2.36
2.33
2.29
2.26
2.19
2.18
2.13
2.11
2.08
2.02
1.98
1.94
1.92
1.87
1.85
1.80
1.78
1.72
1.68
1.64
1.52
Average
Ils
length
in
microns
1.41
1.21
1.11
1.16
1.33
1.35
1.14
1.30
1.41
1.40
1.48
1.39
1.44
1.55
1.64
1.47
1.45
1.57
1.46
1.45
1.40
1.60
1.45
1.52
1.53
1.48
1.41
1.42
1.28
1.34
1.21
1.24
1.17
1.11
1.13
1.11
1.16
1.15
1.15
1.13
1.15
1.15
1.24
0.63
Tel = 79.2811
67
The formula used to calculate the percentage TCl was as
follows:
% TCl =
the total length of a chromosome pair
the total length of the chromosome complement
Based on the presence or absence of satellites, and
on the position of the centromere, the chromosomes could
be divided into the following groups:
1. SAT = satellite chromosome
2.
m = median chromosome, arm ratio between 1.00 and
1.28
3.
sm =
submedian chromosome, arm ratio between 1.29
and 2.17
4.
ST
= subterminal
chromosome, arm ratio from 2.18
upwards.
1. The karyological description
of the species
a. Diploid species
The six diploid species studied have been arranged
on the
basis of the morphological characters mentioned
above and are described below.
1. The SAT group
B. populifolia (633)
The average total complement length of this species
was 29.53p (Table IV, Figure 7E).
One pair of satellite
chromosome5 (pair number 3) with an arm ratio of 1.14
68
(Table IV) was observed.
The arm ratio was calculated
omitting the length of the satellite.
Of the remaining
thirteen pairs of chromosomes, eight were sub-metacentric,
five were metacentric.
The longest chromosome in this
complement was 1.57p (Table IV).
This was the only
chromosome measuring over 1.50p.
There were three chromo-
some pairs which varied in length from 1.25-1.50p, four
which varied in length from 1.00-1.25p, four between 0.75
and 1.00p and two which were less than 0.75p in length
(Table IV)~
2. The
sm
The shortest chromosome pair measured 0.61p.
group
AlI the remaining diploid species studied consisted
predominantly of submedian chromosomes.
i. 8. Eaerulea (6)
The total chromosome complement length of this
species was 25.74p with chromosomes ranging in size from
1.33p for the longest to 0.62p for the shortest (Table IV).
Four of the chromosome pairs were over 1.OOp in length.
Seven pairs varied in length from 0.75 to 1.00p and three
were shorter than
0.75~
in length.
AlI the chromosome
pairs of this complement were submetacentric (Figure 7A)
with arm ratios varying between 1.37 and 2.16 (Table IV).
69"
caerulea-grandi~
ii. B.
(7)
AlI the chromosome pairs of this complement were
submetacentric with the exception of the third and twelfth
chromosome pairs which were subterminal (Figure 7B).
Three of the chromosome pairs of this complement were over
in length, five ranged in length from 1.00 to
1.25~
four were between 0.75 and
measured less than
0.75~
1.OO~
0.62~.
g.
1.54~
The average length
and for the shortest,
The average length of the total chromosome com-
plement was
iii.
in length, and two
(Table IV).
for the longest chromosome was
1.25~,
28.97~.
caerulea-grandis (7G)
Plants of this accession number had a different
origin from those of B. caerulea-grandis (7).
B. caerulea-
grandis (7G) originated in Newton, N.S., while the
specimens of
g.
caerulea-grandis (7) were collected near
Charlottetown, P.E.I.
for
g.
The average total complement length
caerulea-grandis (7G) was
28.72~
value was very similar to that for
above.
g.
(Table IV).
This
caerulea-grandis (7)
The chromosome pairs could be separated into the
same groups on the basis of length.
As shown above for
8. caerulea-grandis (7), three of the chromosome pairs of
this complement were longer than
were between 1.00 and
and two were less than
1.25~,
0.75~.
1.25~
in length, five
four between 0.75 and
1.OO~,
The average length of the
70
longest chromosome was 1.45p, the shortest chromosome
0.68P.
The first three and the last three chromosome
pairs of this complement were submetacentric (Figure 7F)
having arm ratios which varied between 1.17 and 1.20
(Table IV).
iVe B. pendula (21)
The average
to~al
this species was 28.78p
chromosome complement length of
(Table IV).
This species possessed
the 10ngest pair of chromosomes of aIl the diploid species
(1.75P) and also the shortest (0.58p).
Chromosome pairs 2
and 3 were between 1.25 and 1.50p in length.
pairs 4 through 7 were between 1.00 and 1.25p •
Chromosome
Chromosome
pairs 8 to Il were between 0.75 and l.oop in length and
chromosome pairs 12, 13 and 14 measured between 0.50 and
0.75p.
AlI the chromosomes of this complement were sub-
metacentric (Figure 7C) with the exception of chromosome
pair 13 which was metacentric with an arm ratio of 1.15
(Table IV).
v. B. resinifera (110)
Of the six diplcid species studied, this species
had the shortest total chromosome complement length, 23.29p
(Table IV).
The average length of the longest chromosome
was l.20p and the average length of the shortest chromosome
was O.60p.
Three chromosome pairs were greater than 1.OOp
in length, six varied in length from 0.75 ta 1.00p, and
five were less than 0.75p.
Eight of the chromosome pairs
of this complement were submetacentric (Figure 70) with
arm ratios varying between 1.30 and 1.50 (Table IV).
The
remaining six chromosome pairs had their centromeres in a
median position, with arm ratios varying between 1.15 and
1.28.
b. Triploid species
B. caerulea-grandis (hybrid, 7AA)
The somatic chromosome number of this triploid
hybrid is 2n
= 42.
~.
caerulea-grandis (7AA) had a place
of origin (Windham, Vermont) different from either of the
two diploid specimens of
~.
caerulea-grandis (7 and 7G)
(see Brittain and Grant, 1967).
The average total com-
plement length of this species was 46.09p
(Table IV).
-This value is approximately 1.50 times the average total
complement length of the two diploid specimens (7 and 7G).
The length of the individual chromosomes ranges from
1.58p for the longest, ta 0.7lp for the shortest.
Nine
chromosome pairs of this species have their centromeres in
a median position (Figure 8G) with arm ratios varying
between 1.29 and 1.69 (Table IV).
The remaining twe1ve
chromosome pairs have their centromeres in a median
position with arm ratios varying between 1.26 and 1.12.
72
Thus, unlike the diploid species, which were predominantly
submetacentric mith respect ta location of the centromere,
the triploid species consists of predominantly metacentric
chromosomes.
c. Tetraploid species
B. species (119)
The soma tic chromosome number for this tetraploid
species of Betula is 2n
= 56.
The average length of the
total chromosome complement was 55.00p (Table IV).
This
is approximately 2.00 times the average total chromosome
complement length for the diploid species studied (27.51p).
The longest chromosome of this complement measured 1.62p
in length.
This value was slightly longer than the average
length for the longest chromosome pair of the diploid
species (1.47p).
The shortest chromosome measured 0.64p,
being quite similar ta the average length for the shortest
chromosome pair in the diploid species (0.62p).
Sixteen
of the chromosome pairs in this complement were submetacentric (Figure 8H) with arm ratios varying between 1.30
and 1.51 (Table IV).
Like the triploid species, the
tetraploid species had twelve chromosome pairs which had
their centromeres in a median position with arm ratios
varying between 1.09 and 1.28.
73
d. Pentaploid species
B. kenaica (91A)
Pentaploid species of Betula have the soma tic
chromosome number of 70.
The chromosome pairs in
B. kenaica (91A) were quite evenly distributed into two
groups on the basis of centromere position.
Eighteen of
the chromosome pairs were metacentric, the remaining
seventeen chromosome pairs were submetacentric (Figure 81).
The average total chromosome complement length was 75.42p
(Table IV).
This value is approximately 2.50 times the
average total complement length for the diploid species
studied (27.51p).
The average length of the longest
chromosome pair was l.63p.
The average length of the
shortest chromosome pair was 0.65p.
e. Hexaploid species
B. papyrifera (1)
Plants of this accession number have a soma tic
chromosome number of 84.
The average length of the
longest chromosome pair in this complement was 1.41p, the
shortest chromosome pair 0.63p (Table IV).
The average
total chromosome complement length was 79.28u, being approximately 3.00 times the corresponding value for the diploid
species (27.51p).
Chromosome pairs l, 2, 3, 6 and 28-42
had their centromeres in a median position (Figure 9J).
74
Chromosome pairs 4, 5, and 7-27 had their centromeres in a
submedian position.
Thus, the hexaploid species consists
of predominantly submetacentric chromosomes.
II. Statistical analyses
a. A comparison of the percentage total
complements (%TCL) and the long arm/
short arm (L/S) ratios of the six
diploid species
A comparison was made between the morphological
characteristics for the six diploid species.
An analysis
of variance was performed to test the statistical
significance of the values obtained for the %TCL and L/S
parameters.
Table V.
The results of the analysis are given in
The data show that there is no significant
difference between the means calculated for the %TCL
values between the diploid species.
However, an F test
shows that the means of the L/S values do differ
significantly between the six diploid species.
~.
The species,
caerulea (6), has the greatest mean value for L/S
(1.76).
The species, B. resinifera (110), shares the
lowest mean value for L/S (1.33) with B. populifolia
(633).
From the statistical analysis (Table V) it can be
seen that there is no significant difference in L/S
between B. caerulea-grandis (7G), B. pendula (21), and
B. caerulea-grandis (7), and between B. resinifera (110),
B. populifolia (633), ~. caerulea-grandis (7G), and
75
TABLE V. Analysis of variance and Duncan's test for (A) the
percentage total complement length (%TCL) and (B) the longshort arm ratio (LiS) for the diploid species of Betula
----------------------------------------------------------Source of
variation
A)
Species
Replications
Exp. error
Samp. error
Total
B)
Species
Replications
Exp. error
Samp. error
Total
Degrees
of
freedom
5
13
65
756
839
Sum of
squares
Mean
square
0.1093750
2384.133
66.14063
170.2695
2620.652
0.02187500
183.3948
1.017548
0.2252242
0.0971254
19.6460
7.76391
14.9477
100.129
142.486
3.929199
0.5972243
0.2299654
0.1324459
29.66643**
5
131
65
756
839
F value
**Significant at 1% level
Duncan's test (5% level)*
Species:
110
633
1.33
1.33
110 = B. resinifera
633 = B. populifolia
7G = B. caerulea-9randis
7G
11.
1.34
1.43
7
1.46
6
1.76
21 = !!. pendula
7 = B. caerulea-9randis
6 = B. caerulea
*Differences are significant between means lying on
different lines.
76
~.
pendula (21).
The species, ~. caerulea (6) differs
significantly (in Lis) from aIl the other diploid species
studied (Duncanls test in Table V).
b. A linear regression analysis of the total
complement lengths for diploid, triploid,
tetraploid, pentaploid and hexaploid
species
A linear regression was calculated for the total
chromosome complement lengths for the diploid, triploid,
tetraploid, pentaploid and hexaploid species studied.
is summarized graphically in Figure 10.
It
The linear
regression was analysed to determine if total complement
length does indeed vary in a linear fashion with chromosorne number.
Table VI.
The results of the analysis are presented in
The F value is not significant and therefore,
one assumes that a straight line satisfactori1y explains
the relationship between total complement length and chromosorne number in these Betu1a species.
TABLE VI.
Ana1ysis of the linear regression in Figure 10
==========================================================
Degrees
Sum of
mean
Source of
of
F value
squares
square
variation
freedom
Regression
l
1766.24
1766.24
Residua1
3
134.69
44.90
13.11
78
,...
::1
90
"""
80
c:
70
-.....
-=
8
-
60
E
50
c:
8
8
a.
E
0
u
--.
...
40
30
0
20
10
0
42
56
Clar •••••••
1 ••
14
28
70
84
la er (2.)
79
C. The Cytophotometric Comparison
of the Species
I. A comparison of the nuclear DNA content
of six diploid,
one triploid,
five
tetraploid, five pentaploid and five
hexaploid species of Betula
The relative nuclear DNA content was measured for
six diploid, one triploid, five tetraploid, five pentaplaid and five hexaploid species.
In this cytophotometric
study, ~. populifolia (633) was used as the standard
species with which ta compare the DNA absorbance readings
obtained for the twenty-two sample species.
The mean
values for the standard (633) DNA absorbances were subjected ta a one-way analysis of variance.
The results of
this analysis are given in Table VII.
TABLE VII.
Analysis of variance of the DNA absorbance
values for the standard species,
B. populifolia (633)
==========================================================
Source of
variation
Between series
Within series
Degrees
of
freedom
SUffi of
squares
ffiean
square
65
1254
120.12
286.06
1.85
0.23
F value
8.10**
**Significant at 0.01
ïhese results show that significant variations do
exist between the 66 (22 individual experiments, 3 series
per experiment) standard means.
Therefore, before any
80
further statistical analysis could be carried out, it was
'necessary to adjust the DNA absorbance values obtained for
the sample species in relation to those obtained for the
standard species.
The formula used to adjust the sample
means was as follows:
x
x
=a = the
(b
- Gm)
standardized value of the DNA content of
the 2C nucleus of the sample species
a
= the
original value of the 2C nucleus of the
sample species
b
=
the DNA value of the 2C nucleus of the standard
species
Gm
= the
grand mean of aIl 66 values obtained for
the DNA content of the 2C nucleus of the
standard species.
The twenty-two sample species along with their
adjusted DNA absorbances are listed in Table VIII.
The
relative DNA measurements are expressed in arbitrary units
and the mean values were obtained from an analysis of 20
nuclei from each series.
Individual histograms have been
compiled from the DNA density measurements obtained for
each species and these are presented in Figures 11-18.
The adjusted values for the sample DNA absorbances were
subjected to an analysis of variance (samples within samples;
see Snedecor, 1956).
The results of this analysis are
81
TABLE VIII.
Mean nuclear DNA values (in arbitrary units)
for 2C nuclei of Betula species.
The estimates are based
on an examination of 20 nuclei for each series.
==========================================================
DNA per 2C nucleus
Grand
Species
mean
Series 1 Series 2 Series 3
Diploids
B. caerulea (6)
B. caerulea-grandis
10.31
10.06
9.75
9.70
9.59
9.75
9.88
9.83
9.99
10.27
10.00
10.09
10.61
10.07
9.86
9.60
9.76
10.15
10.12
9.87
9.98
10.11
9.90
10.00
16.31
13.91
14.74
14.98
Tetraploids
B. papyrifera (11)
B. papyrifera (81)
B. species (119)
B. papyrifera (150)
B. species (329)
20.10
19.96
19.82
19.62
20.02
20.53
20.74
20.22
19.86
20.16
20.22
20.07
20.12
20.11
20.22
20.28
20.26
20.05
19.86
20.14
Pentaploids
B. papyrifera (4)
B. papyrifera (8)
B. eapyrifera (19)
8. kenaica (91A)
8. papyrifera (132)
24.85
25.11
22.48
24.86
25.11
24.89
25.06
24.89
25.59
26.31
25.16
25.66
25.71
25.04
24.68
24.97
25.52
25.22
25.16
25.37
(7)
B. caerulea-grandis
(7G)
B. pendula (21)
B. resinifera (107)
B. resinifera (110)
Triploid
B. caerulea-grandis
(hybrid; 7AA)
(table continued)
82
TABLE VIII. (continued)
==========================================================
DNA per 2C nucleus
Grand
Species
mean
Series 1 Series 2 Series 3
Hexap10ids
B. eae:lrifera (1)
B. eaE!:lrifera (15)
B. eaE!:lrifera (52)
B. eaE!:lrifera (53)
B. eae:lrifera (56)
22.48
22.46
22.38
22.44
22.33
21.24
21.77
22.38
21.80
22.82
21.40
22.51
21.48
21.50
21.76
21.32
21.86
21.53
21.58
21.73
)
Figure Il.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuclei of
Betula species.
A
B. caerulea (6), 2n = 28
B
B. caerulea-grandis (7), 2n
= 28
C
B. caerulea-grandis (7G), 2n
= 28
)
-~
84
25
20
15
10
5
0
A
9
10
Il
12
25
--•
0
B
1
::1
-..
c:
0
0
9
10
Il
12
.,
.,Q
&
•
z
C
·25
20
15
10
5
0
9
10
Il
DIA ia ar.itrarJ • aits
)
Figure 12.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuclei of
Betula species.
o
B. resinifera (107), 2n
= 28
E
B. resinifera (110), 2n
F
B. pendula (21), 2n = 28
= 28
86
25
20
15
·10
5
0
0
9
•
u
:::a
ID
-•..
.,
10
11
25
20
15
10
5
12
E
0
9
10
Il
12
.a
&
:::a
z
25
20
15
10
5
f
0
1
9
10
DI. i. .rlaitf.f' •• its
1'1
)
Figure 13.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuc1ei of
8etu1a species
G
8. caeru1ea-grandis (7AA), 2n
= 42
H
8. papyrifera (11), 2n = 56
l
8. papyrifera x 8. popu1ifo1ia (81)i
2n = 56
}
Figure 14.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuclei of
8etula species.
J
8. species (119), 2n = 56
K
8. papyrifera (150), 2n
= 56
L
8. species (329), 2n
= 56
90
J
20
10
18
20
22
-=
Q
g
K
=
-
20
0
a-
Q
10
-=E
z=
18
20
22
L
20
10
18
20
OKA
22
iD arbitrary
units
~
'.,
,. 'il
J
Figure 15.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuc1ei of
Betu1a species.
m
B. papyrifera
(4),
2n
=
70
N
B. papyrifera var. macrostachya (8),
2n
= 70
o
B. papyrifera (19), 2n
= 70
92
. \
M
20
10
21
23
25
27
--.
U
:1
CI
-..
N
0
20
•
11
..a
E
:1
Z
21
23
25
27
o
28
18
21
23
25
27
DI. ia ar.itrarJ aaits
)
Figure 16.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuc1ei of
Betu1a species.
P
B. kenaica (91A), 2n
=
70
Q
B. papyrifera (132), 2n
= 70
R
B. papyrifera (1), 2n
= 84
)
94
p
20
10
21
23 .
25
27
-CD
U
:1
Q
c::
ca
.,
20
~
.D
E
10
:1
-=
21
23
25
27
29
R
20
18
21
23
25
DI. ia • r.itr. rJ •• its
)
Figure 17.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuc1ei of
Betu1a species.
5
B. papyrifera (15), 2n
= 84
T
B. papyrifera (52), 2n
= 84
U
B. papyrifera (53), 2n
= 84
)
'\
96
·S
20
10
20
22
24
--•
Q
T
::a
c:
-..
0
•
-
~
20
10
&
Il
20
22
24
u
20
10
20
22
DI l i a
24
ar"itr~rJ
•• its
)
Figure 18.
Histograms of the distribution
of DNA amounts estimated in sixt Y 2C nuclei of Betula
species.
V
B. papyrifera (56), 2n = 84
j
;
.
-..
98
v
u
::a
c:
20
0
•
.ct
10
E
D
Z
20
22
24
DNA i a "a r bit r a r J
26
U
ait s
99
presented in Table IX.
The results show that significant
differences in DNA absorbance do exist between the species.
As may be se en in Table IX, it was possible ta arrange the
species sa that there was a gradual increase in the nuclear
DNA value between- them.
The sequence From lowest ta
highest DNA value is as follows:
B. caerulea (6),
g.
g.
resinifera (107),
g.
resinifera (110)
g. caeruleagrandis (hybrid; 7AA), g. ~rifera (150), g. species
(119), g. species (329), g. papyrifera (81), g. papyrifera
(11), B. papyrifera (52), g. papyrifera (53), ~. papyrifera
(56), B. papyrifera (1), g. papyrifera (15), g. papyrifera
(4), g. kenaica (91A), g. papyrifera (19), ~. papyrifera
(132), g. papyrifera (8).
From the Duncan's multiple
B. caerulea-grandis (7G),
g.
caerulea-grandis (7),
Eendula (21),
range test (Table IX) it may be seen that there is no
significant difference between the six diploid species.
Likewise, there is no significant difference between the
five pentaploid species.
The triploid species (7AA) is
significantly different From all other species and
therefore, stands alone.
It is interesting ta note that
the Duncan's multiple range test shows that there is no
significant difference between the DNA absorbance values
obtained for the five tetraploid species and those
obtained for the five hexaploid species.
Therefore, these
two levels of ploidy appear to be lumped together on the
100
TABLE IX.
Analysis of variance and Duncan's test of DNA
variation of 2C nuclei between diploid,
triploid, tetraploid, pentaploid and hexaploid species of Betula
==========================================================
Degrees
Sum of
Mean
Source of variation
of
F value
squares
square
freedom
Species
Between series
within species
Between observations
within series
21
44965.88
2141.23
44
152.00
3.46
1254
843.25
0.67
619.85**
**Significant a t o. Dl
Duncan's test (5% level )*
7
6
107
110
7G
21
9.83 9.88 9.90 10.00 10.09 10.11
7AA
14.98
150
119
329
81
Il
53
56
52
1
15
19.86 20.05 20.14 20.26 20.28 21.48 21.53 21.73 22.44 22.51
4
91A
19
132
8
24.97 25.16 25.22 25.37 25.52
*Differences are significant between means lying on
different lines.
Species:
7 = B.
6 = 8.
107 = 8.
110 = 8.
7G = B.
21 = 8.
7AA = 8.
caerulea-grandis, 2n=28 Il = B.
caerulea, 2n=28
52 = B.
resinifera, 2n=28
53 = 8.
resinifera, 2n=28
56 = 8.
caerulea-grandis, 2n=28
l = 8.
pendula, 2n=28
15 = 8.
caerulea-grandis, 2n=42
4 = 8.
(hybrid)
91A
8.
150 = B. papyrifera, 2n=56
19 = 8.
132
8.
119
8. species, 2n=56
8 = 8.
329 = 8. species, 2n=56
81 = B. papyrifera, 2n=56
=
=
=
papyrifera, 2n=56
papyrifera, 2n=84
papyrifera, 2n=84
papyrifera, 2n=84
papyrifera, 2n=84
papyrifera, 2n=84
papyrifera, 2n=70
kenaica, 2n=7o
papyrifera~ 2n=7o
papyrifera, 2n=70
papyrifera, 2n=7o
101
basis of their DNA content.
At least it would appear
that the hexaploid level is closer in DNA content ta the
tetraploid level than it is ta the pentaploid level.
II. The relationship between chromosome
number and DNA content for diploid,
triploid, tetraploid, pentaploid
and hexaploid species of Setula
The series of euploid Setula species is presented
in Table X along with the observed (given that 2n
= 28
is
represented by the value 1.00) and theoretical DNA absorbances.
It is evident that there is a correlation between
chromosome number and DNA absorbance for individuals with
somatic chromosome numbers of 2n
=
28, 42, 56 and 70.
The
mean amounts of relative nuclear DNA in arbitrary units
for the diploid, triploid, tetraploid and pentaploid levels
were found ta be 0.99, 1.47, 1.99 and 2.52, respectively,
relative ta 2n = 28 being given the value of 1.00.
The
relative number of chromosomes for these same euploid
levels is 1.00, 1.50, 2.00 and 2.50.
The results of a nt"
test performed on these data are presented in Table XI.
These results show that the observed DNA density values
obtained for the 28-, 42-, 56- and 70-chromosome plants do
not differ significantly from the expected DNA density
values.
A test of significance mas not performed for the
one triploid plant.
The DNA density value obtained for
this plant differs by only 2.00% From the expected value.
102
TABLE X.
DNA absorbance for different levels of ploidy in
Betula relative to the absorbance of the standard species,
g. populifolia (2n = 28) given the value of 1.00
==========================================================
Experimental
absorbance
Theoretical
absorbance
1.00
1.00
B. caerulea-~randis
B. pendula
B. resinifera
B. resinifera
7
7G
21
107
110
0.99
0.98
1.00
1.01
1.01
1.00
Triploids
B. caerulea-~randis
7AA
1.47
1.50
Il
2.04
2.00
81
119
150
2.00
2.02
1.94
1.98
2.00
2.00
2.00
2.00
2.47
2.54
2.49
2.51
2.50
2.50
2.50
2.50
2.50
2.50
Species
Diploids'
B. caerulea
B.
caerulea-~randis
Accession
number
6
1.00
1.00
1.00
1.00
Tetraploids
B. pap;trifera
B. pap;trifera x
8. populifolia
B. pap;trifera
B. pap;trifera
B. pap;trifera
329
Pentaploids
B. pap;trifera
B. pap;trifera
B. ·pap~rifera
B. kenaica
4
8
19
9lA
B. pap;trifera
132
(table continued)
103
TABLE X.
(continued)
===========================================================
Accession Experimental Theoretical
Species
number
absorbance
absorbance
Hexaploids
B. papyrifera
B. papyrifera
B. papyrifera
B. papyrifera
B. papyrifera
1
2.25
15
2.22
52
2.17
3.00
3.00
3.00
53
2.17
3.00
56
2.15
3.00
TABLE XI. "t" test for the relationship between chromosome
number and DNA density value for diploid, tetraploid and
pentaploid species of Betula
==========================================================
Euploid level
t value
Degrees of freedom
Diploid
0.4081
10
Tetraploid
0.2352
8
Pentaploid
0.0784
8
104
The five hexaploid species studied did not prove to
be consistent with the results obtained for the lower
levels of ploidy.
The mean relative nuclear DNA value in
arbitrary units for the 84-chromosome plants was found to
be 2.19 (relative to 2n
= 28
being given the value of
1.00), rather than the 3.00 value expected on the basis of
the ratio in chromosome number between the diploid and
hexaploid leveis (1.00:3.00).
A ut" test (Table XII)
showed that the observed DNA absorbance differed significantly from the expected DNA absorbance for 84-chromosome
plants.
In contrast to Grant (1969) this author found that
the observed DNA absorbance also differed significantly
from the expected DNA absorbance for 63-chromosome plants
(2.25) at the 0.05 level although not at the 0.01 level.
TABLE XII.
Ut" test for the relationship between chromosome number and DNA density value for hexaploid species of
Betula
==========================================================
Euploid level
t value
Degrees of freedom
Hexaploid
42.63**
8
3.16*
8
**Significant at 0.01
*Significant at 0.05
t value showing relationship between the observed DNA density for 84-chromosome
plants and that expected for a 63-chromosome plant.
)
Figure 19. The relationships between the
theoretically expected DNA absorbance (open circles)
and the observed DNA absorbance (closed circles)
for diploid, triploid, tetraploid, pentaploid and
hexaploid species of Betula.
The dotted lines are
an extrapolation to the theoretical chromosome
number calculated for the DNA density value observed
for the individuals with 84 chromosomes.
106
3
-
~
c::
::1
....
=--
2
ta
...
~
-
c::
III:
1
z:
Cl
14
28
42
Clar ••• s •• e
56
70
84
aa.ber (2a)
107
These results have been graphically illustrated in Figure
19.
The dotted line is an extrapolation from the observed
DNA density value for the 84-chromosome plants and falls
on a point between 56 and 70 on the axis depicting the
chromosome numbers.
Thus, it would appear that these
individuals with 84 somatic chromosomes have a DNA value
approximately equivalent to that of a theoretical plant
with slightly less than 63 chromosomes.
III. The relationship between chromosome
length and DNA content for diploid,
triploid, tetraploid, pentaploid
and hexaploid species of 8etula
A summary of the results showing the relationship
between total complement length (Tel), nuclear DNA content
and DNA content per micron of chromosome for the diploid,
triploid, tetraploid, pentaploid and hexaploid species of
Betula is shown in Table XIII.
The data show that DNA
content per micron of chromosome was very similar for the
diploid (with the exception of
~.
resinifera, 110),
triploid, tetraploid and pentaploid species with
approximate1y 0.350 units of DNA per micron of chromosome
length.
The hexaploid species showed a marked decrease in
DNA content per unit length of chromosome with only 0.283
units of DNA per micron of chromosome length.
This gives
a ratio of 1:1.24, respective1y, between plants with the
highest leve1 of ploidy and those at the lower eup10id
leveLs.
108
TABLE XIII.
The relationship between chromosome length
and DNA content in diploid,
triploid, tetraploid,
pentaploid and hexaploid species of.Betula.
The mean DNA
content per nucleus was calculated in relation to the
absorbance of the standard, g. populifolia (2n = 28)
==========================================================
mean length
of
mean DNA
chromosome
content per DNA content
per micron
Species
complement
nucleus in
of
(Tel)
arbitrary
chromosome
in microns
units
(li )
Diploids
B. caerulea (6)
B. caerulea-grandis
25.74
28.97
9.88
9.83
0.384
0.339
B. caerulea-grandis
28.72
10.09
0.351
B. pendula (21)
8. resinifera (110)
28.78
23.29
10.11
10.00
0.351
0.429
Triploid
B. caerulea-grandis
(hybrid; 7AA)
46.09
14.98
0.325
Tetraploid
g. species (119)
55.00
20.05
0.365
Pentaploid
g. kenaica (91A)
75.42
25.16
0.334
Hexaploid
~. papyrifera (1)
79.28
22.44
0.283
(7)
(7G)
DISCUSSION
A. Stomatal measurements
measurements of the length of the stomatal guard
cells (Table III) of Betula species were made to determine
if there is a difference in the size of the guard cells in
birch plants with different ploidy.
If, for instance, the
length of the guard cells in diploid plants was significantly different from the length of the guard cells in
species with higher ploidy, then perhaps one could
ascertain the true ploidy of an individual by measuring a
large number of the guard cells.
The results of the
present measurements show that there is a definite increase
in mean stomatal length with increasing chromosome number
(Figure 2).
However, there is a considerable overlapping
of the individual measurements between the different
levels of ploidy.
Therefore, unless an extremely large
number of guard cells is measured, their value in
determining the level of ploidy of an individual appears
to be limited.
more reliable methods must be used to
differentiate ploidy in the birches.
109
110
B. Karyotype Studies
When the karyotypes (Table IV) and idiograms
(Figure 7) of the six diploid taxa are compared, in
general they appear ta be quite similar morphologically.
They aIl consist of predominantly submetacentric chromosomes.
Their total complement lengths (Tel) aIl lie in
the range 23.29-29.53p, with an average of 27.51p.
Their
longest chromosome pairs range in length from 1.33 ta
1.75p with an average of 1.47p.
Their shortest chromosome
pairs aIl measure between 0.58 and 0.68p with an average
value of 0.62p.
There is a variation in the lengths of
the twenty-eight individual chromosomes between the six
di plaid species.
This variation is reflected in the total
complement lengths.
Although the cells were aIl analysed
in the metaphase stage, differences in degrees of coiling,
and therefore, in the amount of contraction of the
individual chromosomes could account for the observed
variation in lengths.
Perhaps it would be profitable ta
study a greater number of metaphase cells for each
individual species.
In looking at the chromosome data on the six diploids
more closely, certain differences become apparent and sorne
species are seen ta be more similar in chromosome morphology.
The species, B. populifolia (633) had the longest total
complement length of the six diploids (Table IV).
III
Betula resinifera (110) had the shortest total complement
length.
Betula caerulea-grandis (7 and 7G) possessed total
complement lengths of 28.97p and 28.72p, respectively.
Although these values are very similar, the species
Q. pendula (21) with a total complement length of 28.78p
appears to be more similar to Q. caerulea-grandis (7G)
th an the two caerulea-grandis specimens are to each other.
In an analysis of variance of the long arm to short
arm ratios
(Lis)
for each species (Table V) it was seen
that Q. caerulea-grandis (7 and 7G) and Q. pendula (21)
could be grouped together, while only Q. caerulea-grandis
(7G) and B. pendula (21) could be grouped with Q. populifolia (633) and B. resinifera (110).
Therefore, Q. caerulea-
grandis (7G) appears to be more closely related to Q. pendula
(21) (on the basis of chromosome morphology) than to
B. caerulea-grandis (7) as would be expected on the basis
of external appearance only.
Although Q. caerulea (6) had
values for total complement length, and for the length of
the longest and shortest chromosome pairs which were intermediate to the extremes of those found For these six
diploids, the analysis of variance of long arm to short
arm ratio showed it to be signiFicantly different From aIl
the other diploid species and therefore, in a class by
itselF.
112
This analysis of variance showed up the differences
in centromere positions between the six diploids.
g.
species,
The
populifolia (633) was the only species
possessing a pair of satellite chromosomes.
This fact was
not made apparent by the analysis of variance as the arm
ratios were calculated omitting any length which could be
attributed to the satellite.
some pairs in
g.
Of the remaining 13 chromo-
populifolia (633), 8 were submetacentric
and 5 were metacentric.
The species, B. resinifera (110),
had a karyotype very similar to
g.
populifolia with 8 sub-
metacentric and 6 metacentric chromosome pairs.
species,
g.
caerulea-grandis (7G), also had 8 submeta-
centric and 6 metacentric chromosome pairs.
karyotype of
The
g.
However, the
caerulea-grandis (7) again proved to be
different From that of ~. caerulea-grandis (7G).
The
latter had 12 submetacentric chromosome pairs with the
remaining two being subterminal.
It could be in a class
by itself as it was the only species which had subterminal
chromosomes.
The species,
g.
pendula (21), had 13 sub-
metacentric and 1 metacentric chromosome pair.
Betula
caerulea (6), although quite closely resembling B. pendula
(21), was in a class by itself.
It had no metacentric
chromosomes, aIl 14 pairs being submetacentric.
Four other taxa were studied, each at a different
level of ploidy: triploid, tetraploid, pentaploid and
113
hexaploid.
The triploid species studied,
~.
caerulea-
grandis (hybrid, 7AA), possessed 12 metacentric and 9
submetacentric chromosome pairs.
~.
The tetraploid species,
species (119), had the same number of metacentric
chromosome pairs (12), but had more submetacentric chromosome pairs (16).
The pentaploid species, ~. kenaica (91A),
had 17 submetacentric chromosome pairs, almost equivalent
to the tetraploid, but possessed more metacentric chromo-
-
some pairs (lS).
The hexaploid species, ~. papyrifera (1)
had a similar number of metacentric chromosome pairs (19)
when compared with the pentaploid, but had more submetacentric chromosome pairs (23).
On examination of the karyotypes of the triploid,
tetraploid, pentaploid and hexaploid taxa (Table IV) and
idiograms (Figures S-9) they do not appear to differ
grossly from the diploid taxa in chromosome morphology.
The total complement lengths differ in the ratio
1.67:2.00:2.72:2.91 instead of the 1.50:2.00:2.50:3.00
ratio expected.
It can be seen that total complement length
does increase with increasing chromosome number although
not in direct proportion to it.
Indeed, if different
genomes are involved in the evolution of this polyploid
series, one would not necessarily expect a strict proportionality between chromosome number and total length of
the chromosome complement as is suggested by consideration
114
of chromosome number alone.
Therefore, total complement
length of the 84-chromosome plants need not be 3.00 times
the total complement length of the 28-chromosome plants.
The longest chromosome pairs of these higher ploidy plants
show an average length of 1.51p in comparison with the
1.47p shown by the six dip10id species.
The shortest
chromosome pairs compare even more favorably in length:
0.65p for the higher ploidy species, 0.62p for the diploids.
From the foregoing, it appears that the species at
higher levels of ploidy do not differ to any great extent
in chromosome size or morphology from those at the diploid
level, and the differences shown could be within the
experimental limitations of the techniques used.
karyotypi~
More
studies should be carried out on plants at the
higher ploidy levels.
These, along with studies of
artificial crosses made at the diploid level, combined
with chromosome doub1ing, could possibly help in determining parental affinities at the polyploid levels.
C. Cytophotometric measurements- ,
Nuc1ear DNA Content
In various studies (Southern, 1967; Beçak
~
al.,
1967) cytophotometry has proved to be a useful tool in
differentiating between species having different levels of
p10idy.
Swift (1953) suggested that nuc1ear DNA increases
115
stepwise with ascending levels of polyploidy.
Hughes-
Schrader (1956) stated that polyploidy should be detectable
as multiple nuclear DNA values if the quantitative relationships in DNA values have not been masked by duplications
or deletions in the chromosomes.
Christensen (1966) and
Grant (1969) found that the nuclear DNA values did not
necessarily reflect the level of ploidy in some species of
Enchytraeus and Betula, respectively.
Grant (1969) found that the nuclear DNA content for
the 28-, 42-, 56- and 70-chromosome plants corresponded to
the values expected on the basis of chromosome number.
However, the 84-chromosome plants had a nuclear DNA content
corresponding to that expected for a 63-chromosome plant.
In an attempt to enlarge upon the work done by Grant, this
author has found that for the six diploid, one triploid,
five tetraploid and five pentaploid species studied, the
nuclear DNA content corresponded to the theoretical
expected values (Table X).
However, the five hexaploid
plants had a lower DNA absorbance th an that expected for
84-chromosome plants on the basis of chromosome number.
A nt" test showed that the observed DNA absorbance was
also significantly different from that expected for a
53-chromosome plant (at the 0.05 level, although not at
the 0.01 level).
An analysis of variance and Duncan's
mul tiple range test (Table IX) showed tha t al though the
116
observed DNA absorbance for the 84-chromosome plants did
not differ significantly From the observed DNA absorbance
for the 56-chromosome plants, i t did differ significantly
From the observed DNA absorbance for the 70-chromosome
plants.
Therefore, in nuclear DNA content at least, the
84-chromosome plants seed to be more closely related to
the 56-chromosome plants than to the 70-chromosome plants,
and perhaps have a DNA content equivalent to that of a
plant which would possess a chromosome number intermediate
to a 56- and a 63-chromosome plant.
Differences in nuclear DNA measurements between
closely related species imply that differences in chromosome structure may exist between the species.
Three
hypotheses have been advanced to explain this variation.
The first suggests that changes in nuclear DNA content are
caused by extensive longitudinal repetitions or accumulation of chromosomal units, that is, an increase or decrease
in chromosome length caused by duplications or deletions.
This theory is discussed by Gall (1963) and Keyl (1965).
The second hypothesis is concerned with lateral multiplicity of chromosome strands, and assumes that the
chromosome is multistranded (Christensen, 1966; martin and
Shanks, 1966; Rothfels et al., 1966; Schrader and HughesSchrader, 1956, 1958; Uhl, 1965).
The third hypothesis
deals with seriaI repetition of encoded DNA base sequences
117
by a "master-slave" process and is discussed by· Callan
(1967) and Whitehouse (1967).
8haskaran and Swaminathan (1960), in a cytolog~cal
study of diploid, tetraploid and hexaploid species of
Triticum, found that DNA content per unit length of chromosome was constant.
When this relationship was considered
for 8etula it was found that for the majority of species
nuc1ear DNA content was indeed constant per unit length of
chromosome.
However, the species
g.
resinifera (110),
which had the shortest total complement length of the six
dip10id species studied, had a higher DNA value in relation
to the total complement length (Table XIII).
have been reported by Nirula
et~.
Similar cases
(1961) and by Cheng
(1971) for Solanum nitidum and Lotus pedunculatus,
respectively.
Perhaps in this one dip10id species, and in
others not studied, there may be sorne basic change in
chromosome structure as compared to the other five diploid
species which do show a DNA content per unit length of
chromosome constant to that shown by the triploid, tetraploid and pentaploid species studied.
The majority of the
diploid plants, the triploid, tetrap10id and pentaploid
plants aIl showed a DNA content of 0.350 units per unit
length of chromosome.
The hexaploid plants showed a DNA
content of 0.283 units per unit length of chromosome.
This reduced DNA content per unit 1ength of chromosome of
118
approximately one-quarter is similar to that obtained by
Grant (1969) in a consideration of DNA content per chromosome.
Grant suggested that this reduced DNA value might
reflect a reduction in size of sorne of the chromosomes or
sets of chromosomes in the 84-chromosome plants, although
he found
~o
concomitant reduction in nuclear diameter as
one might expect with a reduction in chromosome size.
difference in chromosome size in sorne plants
A
animaIs
~nd
has proven to be correlated with DNA content in the nucleus
(Martin and Shanks, 1966; Rees et al., 1966; Rothfels et
al., 1966; Miksche, 1967).
As stated in part 8 of this
discussion, the size of the largest and smallest chromosome pairs at the higher levels of ploidy correspond to
the size of the largest and smallest chromosome pairs at
the diploid level.
The average total complement length
for the karyotypes analysed at the hexaploid level was
2.91 times the average total complement length for the
karygtypes of the diploid species as opposed to the 3.00
value expected on the basis of chromosome number.
these two values are in quite close agreement.
However,
Since the
value for the hexaploid level represented only plants of
one accession number, it is suggested that karyotype
analyses from plants of a number of different hexaploid
accessions be carried out to show whether there is a
119
variation in chromosome size between (1) different hexaploid accessions and (2) diploid and hexaploid species.
Darlington (1956) inferred that polyploidization resulted
in a decrease in chromosome size.
This reduction in
chromosome size between polyploid (with smaller chromosomes)
and diploid species was not necessarily a phylogenetic
reduction, but could simply arise From the fact that
diploids with smaller chromosomes gave rise to these
polyploids.
In part 8 of this discussion it was noted
that the total complement lengths of the karyotypes of the
triploid, tetraploid and pentaploid levels were 1.67, 2.00
and 2.72 times the total complement length of the diploid
level.
The values obtained for the triploid and penta-
ploid levels are higher th an the 1.50 and 2.50 ratios
expected on the basis of chromosome number, and yet, these
euploid levels had nuclear DNA contents equivalent to the
expected values on the basis of chromosome number.
How
does one explain the fact that the triploid and pentaploid
plants, while showing no increase in DNA content, show an
increase in total complement length of Il.3% and 4.8%,
respectively, above the expected values, while the hexaploid plants show a reduction in DNA content of approximately 25% per chromosome accompanied by a reduction in
total complement length of only 4.5%?
the
c~ie
In other words, in
of Setula there does not appear to be a deliberate
120
selection for chromosome complements with smaller chromosomes at the higher levels of ploidy.
This does not rule
out the possibility that diploids with smaller chromosomes,
due ta minute changes in chromosome size, could give rise
ta sorne of these higher ploidy plants.
In this study each
of the individual chromosomes was not analysed statistically.
Perhaps if this were done, minute differences in size would
be detected.
However, chromosome size is never exactly
precise and camera lucida drawings are only approximations.
They should be used with reserve in cases such as this
where small chromosomes and perhaps small differences in
size are under consideration.
Grant (1969) also suggested that since only a single
diploid species had been used as the "standard" for comparing the DNA absorbance values of the higher euploids,
the reduction in DNA content for the 84-chromosome plants
might have been directly related ta this specifie diploid.
However, this author used a different diploid species as
the standard in this set of experiments and obtained the
same results.
-~~ecies,
Grant (1969) felt that different diploid
although possessing the same chromosome number,
might vary in their chromosome morphology, and hence, DNA
absorbance value.
It has been shown here that although
small differences do exist between the chromosome morphologies of diploid plants (part 8), the DNA absorbance
values are very similar (Table X).
121
Therefore, the difference in nuclear ONA content
between the various euploid Betula species does not seem
to be associated directly with differences in chromosome
length and therefore, with longitudinal differentiation
(duplications, deletions, translocations, etc.) of the
individual chromosomes.
Could the observed reduction of
ONA content in the 84-chromosome plants be more closely
related to chromosome volume as has been shown for sorne
species (Oowrick and El Bayoumi, 1969; Fox, 1969)?
ODes
the presence or absence of heterochromatin affect the
nuclear DNA content?
Lima-de-Faria (1959) showed that
heterochromatic segments may contain two or three times
more DNA per unit area than euchromatin does.
Therefore,
no regular relationship could be found between DNA content
and chromosome length in species containing varying amounts
of heterochromatin.
On the other hand, Stebbins (1966)
stated that a positive correlation between chromosome size
and DNA content might possibly be related ta changes in
the amount of heterochromatin.
However, there has been no
visible evidence of heterochromatin in Betula chromosomes
in this study, hence, the role that heterochromatin may
play in the speciation of Betula is still to be investigated.
This study has not been concerned with the relationships of the birch species studied
~~.
Hence no
discussion of the taxonomy of the species is presented,
SUmmARY
1. An analysis of chromosome morphology and Feulgen cytophotometrie measurements of the nuclear deoxyribonucleic
acid (DNA) content of diploid, triplQid, tetraploid,
pentaploid and hexaploid species of 8etula mas carried
out to determine if there was any variation in chromosome morphology in the genus 8etula and to determine
the relationship between chromosome number, chromosome
size and nuclear DNA content.
2. measurements were made of the stomatal guard cells for
species at the diploid, triploid, tetraploid, pentaploid and hexaploid levels.
A definite increase in
mean stomatal length was noted with increase in chromosome number.
However, the high frequency of overlapping
of thg individual measurements between the different
euploid levels does not permit one to use mean stomatal
length in determining the ploidy of an individual plant.
3. Drawings mere made of ten karyotypes for each of six
diploid, one triploid, one tetraploid, one pentaploid
and one hexaploid plant.
The percentage total com-
plement lengths and the long arm to short arm ratios
were calculated From the karyotype measurements and
123
124
statistically analysed.
each species.
Idiograms were prepared for
Representative photographs of the karyo-
types for the diploid, tetraploid, pentaploid and
hexaploid species were taken.
4. A comparison of the chromosome morphology, including
the percentage total complement length and the long arm
to short arm ratio, for the six diploid species showed
that the species
(110),
g.
g.
populifolia (633), B. r~sinifera
pendula (21) and~. caerulea-grandis (7G) did
not differ significantly from one another in these
characteristics.
g.
The species,
s.
pendula (21) and
caerulea-grandis (7 and 7G) likewise did not differ
significantly from one another in these characteristics.
The species,
g.
caerulea (6) differed significantly
from aIl the other diploid species in long arm to short
arm ratio.
5. Setula populifolia (633) was the only species of the
diploids studied in which satellite chromosomes were
observed.
Setula caerulea-grandis (7G) was the only
species which possessed any subterminal chromosomes.
Setula caerulea (6) was the only species in which aIl
14 chromosome pairs were submetacentric.
6. The karyotypic measurements for the triploid, tetraploid, pentaploid and hexaploid species showed that the
values obtained for the lengths of their shortest and
125
longest chromosome pairs were very similar to those
obtained for the shortest and longest chromosome pairs
in the diploid species.
The total complement lengths
varied in the ratio 1.67:2.00:2.72:2.91 instead of the
1.50:2.00:2.50:3.00 ratio expected on the basis of
chromosome number.
The total complement lengths did
increase with increasing chromosome number, although
not in direct proportion.
7. The 2C DNA values of species at the diploid, triploid,
tetraploid, pentaploid and hexaploid levels were compared by using an integrating microdensitometer.
The
observed DNA density values for the 28-, 42-, 56- and
70-chromosome plants agreed with the expected DNA
density values.
The 84-chromosome plants showed a
ratio of 1.00:2.19 instead of the expected
1.00:3.~00
ratio expected for DNA density values between diploid
and hexaploid species.
The DNA absorbance for the 84-
chromosome plants was approxima tel y equivalent to that
expected for a plant with somewhat less th an 63 chromosomes.
8. The average DNA value per unit length of chromosome was
0.350 units for the 28-, 42-, 56- and 70-chromosome
plants.
The average DNA value per unit length of
chromosome mas 0.283 units for the 84-chromosome plants.
126
9. The total complement length for the 84-chromosome plant
was smaller than that expected, but in light of the fact
that the total complement lengths for the 42- and 70chromosome plants were larger than expected and yet
showed no corresponding increase in DNA content, i t did
not appear that the reduced DNA value in the 84-chromosorne plants was directly correlated with any change in
chromosome size, although this possibility was not
ruled out.
~
,
" . "-. .:, ,,"
,
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