M.Sc. BIOLOGY
AB STRACT
SPECIES RELATIONSHIPS IN THE LOTUS CORNICULATUS GROUP (LEGUMINOSAE)
AS DETERMlNED BY KARYOTYPE AND CYTOPHOTOMETRIC ANALYSES
Rosa I-Jung Cheng
A~ analysis of chromosome morphology and Feulgen cytophotometric measurements of the nuclear deoxyribonucleic acid (DNA) content of L. corniculatus and related diploid species (L. alpinus, L. borbasii, L. filicaulis, L. japonicus, L. krylovii, L. pedunculatus, L. schoelleri,
~. tenuis), and L. coimbrensis of the L. aegeus group, was carried out.
The idiogram of L. coimbrensis differed markedly from those for the species of the L. corniculatus group which were considerably more uniforme Lotus pedunculatus was the only species in which chromosomes were observed bearing satellites. A comparison of the karyotypes of two accessions received as L. alpinus showed they were different taxa.
DNA values differed between the species, and in general, total complement lengths were correlated with DNA values. From the karyotype and DNA analyses it is considered that L. alpinus and L. borbasii would be putative species from which the tetraploid, L. corniculatus, could have arisen.
iv ,.
SPECIES RELATIONSHIPS IN THE LOTUS CORNICULATUS GROUP
(LEGUMINOSAE) AS DETERMlNED BY KARYOTYPE AND
CYTOPHOTOMETRIC ANALYSES
by
Rosa I-Jung Cheng
A thesis presented to the Facu1ty of Graduate Studies and Research in partial fu1fi11ment of the requirements for the degree of Master of Science
Bio1ogy Department McGi11 University Montreal March 1971
@ Rosa I-Jung Cheng 1971 M.Sc. Biology
Short title KARYOTYPE AND CYTOPHOTOMETRIC ANALYSES OF LOTUS SPECIES
Rosa I-Jung Cheng ACKNOWLEDGEMENTS
The author acknowledges her gratitude and appreciation to
Dr. W. F. Grant, Professor of Genetics, Department of Biology,
Macdonald Campus of McGi11 University, for his guidance throughout
the course of this study and for his help during the pre~aration of
the manuscript.
The author also expresses her appreciation to Professor J. D.
Burrage, Computer Center, McGi11 University and Mr. J. C. Miao for
[ their assistance in the statistical analyses; to Mr. Paul Choo-Foo
for printing the photomicrographs; to Miss Françoise Prieur for
typing the manuscript.
The author also thanks the National Research Council of Canada
for its financial assistance to Dr. Grant in sponsoring this research
project.
My speèial thanks are to my parents, Mr. and Mrs. T. Y. Cheng,
for their encouragement throughout the years of my studies.
i TABLE OF CONTENTS
Page ACKNOWLEDGEMENTS ...... i ABSTRACT •••••••••••••• ·...... iv LIST OF TABLES ...... v LIST OF FIGURES ...... vi
INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••••••• 1 LITERATURE REVIEW ••• ...... 6 A. The relationship between L. corniculatus and its related diploid species ...... -:...... 6 1. Morphological studies ...... 6 2. Biochemical studies in Lotus 8 3. Cytological studies ...... Il (1) Karyotypes and idiograms •••••••••••••••••••• Il (2) Chromosome pairing ••••••••••••••••• 13 4. Hybrid studies ...... 14 B. Cytophotometry ·...... 15 MATERIALS AND METHODS ...... 20 1. Plant material ·...... 20 2. Root tip squashes ...... 20 3. Preparation of karyotypes and idiograms 21 4. Cytophotometry ·...... 21 5. Statistic analysis ...... 24 RESULTS ...... 27 The karyotypes ...... 27 A. The karyological description of the species 28 (a) Diploid species 28 (b) Tetraploid species ...... 52 B. Statistical analysis ...... 52 (a) A comparison of the total complement length (TCL) and the long arm/short arm (LIS) ratio of the ten diploid species •••••••••••••• 52 (b) A comparison of the total chromosome length (long arm/short arm) between the ten diploid species ...... 54
ii page The cytophotometric comparison of the species ••••••••••••• 56 1. A comparison of the nuclear ,DNA content of three tetraploid accessions of L. corniculatus •••••••••• 57 2. A comparison of the nuclear DNA content values found for the 10 diploid species •••••••••••••••••• 57 3. A comparison of the nuclear DNA content between twelve Lotus hybrids •••••••••••••••••••••••••••••• 61 4. A comparison of the nuclear DNA content between six amphidiploids ••••••••••••••••••••••••••••••••• 64 5. The relationship between chromosome length and DNA content of the diploid species •••••••••••••••• 67 DISCUSSION ...... 87 A. Karyo type s tudies ...... 87 B. Cytophotometric measurements--Nuclear DNA content •••••• 90 1. L. corniculatus, the diploids and hybrids ••••••••••• 90 2. Amphidiploids ...... 92 . C. The relationship between chromosome length and DNA content of the diploid species ••••••••••••••••••••••••• 93 D. The basic change in chromosome structure ••••••••••••••• 94 E. Tracing the ancestor of the tetraploid species, Lotus cornicula tus ...... 95
SUMMARY ...... 99 LITERATURE CITED ••••••••••••••••••••••••••••••••••••••••••••••• 102
iii M.Sc. BIOLOGY .
ABSTRACT
SPECIES RELATIONSHIPS IN THE LOTUS CORNICULATUS GROUP (LEGUMINOSAE)
AS DETERMlNED BY KARYOTYPE AND CYTOPHOTOMETRIC ANALYSES
Rosa I-Jung Cheng
An analysis of chromosome morphology and Feulgen cytophotometric
measurements of the nuclear deoxyribonucleic acid (DNA) content of L.
corniculatus and related diploid species (L. alpinus, L. borbasii, L.
filicaulis, L. japonicus, L. krylovii, L. pedunculatus, L. schoelleri,
L. tenuis), and L. coimbrensis of the L. aegeus group, was carried out.
The idiogram of L. coimbrensis differed markedly from those for the
species of the L. corniculatus group which were considerably more
uniforme Lotus pedunculatus was the only species in which chromosomes
were observed bearing satellites. A comparison of the karyotypes of
two accessions received as L. alpinus showed they were different taxa.
DNA values differed between the species, and in general, total complement
lengths were correlated with DNA values. From the karyotype and DNA
analyses it is considered that L. alpinus and L. borbasii would be
putative species from which the tetraploid, L. corniculatus, could
have arisen.
~ .... -1.1--7, ;
iv LIST OF TABLES
Table Page
1. The species, hybrids, andamphidiploids studies, their accession number, source, and chromosome number ••••••••• 25
2. Karyotype analyses of the somatic chromosomes for ten Lotus species ...... 29
3. Mean values of the percentage total complement length (TeL) and the long-short ratio (L/S) •••••••••••••••••••• 53
4. Analysis of variance and Duncan's test of the mean total complement length for the 10 diploid species of Lotus 55
5. DNA values in arbitrary units for 2e nuclei of three accessions (tetraploid) of Lotus corniculatus from different sources ...... 58
6. Mean DNA nuclear values (in arbitrary units) for 2e nuclei of ten Lotus species ••••••••••••••••••••••••••••• 59
7. Analysis of variance and Duncan's test of DNA variation of the 2e nuclei between the diploid species of Lotus ••• 60
8. Mean DNA nuclear values, in arbitrary units, for 2e nuclei of twelve Lotus hybrids •••••••••••••••••••••••••• 62
9. Analysis of variance of the DNA values of 2e nuclei between Lotus h~brids ••••••••••••••••••••••••••••••••••• 63
10. Mean DNA nuclear values', in arbitrary units, for 2e nuclei of six Lotus amphidiploids ••••••••••••••••••••••• 65
Il. Analysis of variance and Duncan's test of the DNA values of 2e nuclei between amphidiploids •••••••••••••••••••••• 66
12. The relationship between chromosome length and DNA content of the diploid species of Lotus ••••••••••••••••• 68
v LIST OF FIGURES Page Figures 1-11. Soma tic chromosomes from root tip ce11s of Lotus dip10id species ...... 37 Figures 12-13. Idiograms of dip10id Lotus species ...... 49 Figures 14-16. Histograms of distributions of DNA amounts
in 2C nuc1ei of dip10id Lotus species ••••••••••••••• 70
Figures 17-20. Histograms of distributions of DNA amounts
in 2C nuc1ei of Lotus hybrids ••••••••••••••••••••••• 76
Figures 21-22. Histograms of distributions of DNA amounts
estimated in 2C nuc1ei of amphidip10ids ••••••••••••• 84
vi INTRODUCTION
The karyotype bas been recognized as a species character for
several decades by cytogeneticists who have used chromosome analyses
as a taxonomie tool for the elucidation of species relationships
(Stebbins, 1950). Karyotype analyses have been carried out mostly
on metaphase chromosomes, since in many species the chromosomes do not
lend themselves readily for study at other stages. The chromosome number and the morphological characteristics for each individual
chromosome of the genome or complement, including the length of
chromosome arms, the position of the centromeres, the number and size of satellites, the number and position of secondary constrictions, and other features such as heterochromatic regions, are recorded and characterize a karyotype for a species.
In general, karyotype analyses can detect only major structural changes which affect the chromosomes, and are not sufficiently sensitive
to detect small chromosomal changes such as minor rearrangements, duplications, or deletions, although these may have important genetical and evolutionary consequences. As a result, other techniques have been considered for the detection of quantitative changes to a genome. One su ch technique is that of cytophotometric analysis whereby the relative amount of deoxyribonucleic acid (DNA) present in a genome may be determined (Hughes-Schrader, 1951). From a comparison of the amount of
DNA present between the chromosome complements of species, it is possible to unmask evolutionary changes that may pass undetected from a simple comparison of karyotypes between species (Hughes-Schrader, 1953). 2.
An example of a study combining karyotype analysis and nuclear
DNA cytophotometry is that of John and Hewitt (1966) who compared the
karyotypes and the relative amounts of nuclear DNA between two groups
of species of short horned grasshoppers (Acrididae), in which one
group had 17 chromosomes, the other 23, but the total number of chromo-
some arms in both groups numbered 23. They found that significant
differences in the nuclear DNA content existed both within and between
some of the species of the 17 and the 23 chromosome groups even though
their karyotypes were similar. The 23 chromosome individuals had a
lower nuclear DNA content than the 17. This, coupled with the
existence of both acro- and telocentric chromosomes of these species
led the authors to refute the generally claimed karyotypic stability
of the acridids.
Studies in the plant kingdom have also shown how our knowledge of
species relationships in critical genera may be aided by combining karyotype and DNA cytophotometric analyses. In the genus Anemone,
Rothfels et al. (1966) studied 12 species which had the same chromosome number, and in which there were no conspicuous modifications in the
proportions of the karyotypes between species. However, they found a
two-fold range of DNA values between the different species and from these data they were able to de termine the relationships between the species.
Rees and Jones (1967a) presented evidence in Lolium to show that the evolution of the diploid species in this genus was associated with differences in nuclear DNA content, and that the DNA content of three inbreeding species was about 35% larger than that of three outbreeding 3.
! " , species. Likewise, Southern (1967) studying the species relationship in the genus Tulipa reported a difference in DNA values between the
diploid species which aided in classifying these species. DRA cyto-
photometry has also been used in conjunction with karyotype analyses
to study the phylogenetical relationships of species of Triticum (Rees
and Walters, 1965; Nishikawa, 1967; Nishikawa and Furuta, 1969). From
the DNA data, it has been shown that the tetraploid species, T. turkestanica,
has a 2e value (20.40) which is about twice that of its presumed diploid
progenitor T. biflora .(10.70). These few examples from the literature
have been chosen to show how karyotype analyses combined with Feulgen
cytophotometry have aided in determining species relationships in
different genera.
Tetraploid birdsfoot trefoil (L. corniculatus L., 2n = 4x = 24) has .'r-- ', " ,,' become one of the most important cultivated species of legumes in eastern
North America since its potentialities as a forage crop were first shown
in 1934 (MacDonald, 1946). In comp.arison with other polyploid crops
such as wheat, cotton, and tobacco, relatively few cytogenetic studies
have been carried out on Lotus, and hence, knowledge concerning this
genus has progressed much slower than others in this respect.
In this study, it was planned to combine both a karyotype and a cyto-
photometrie analysis of the tetraploid species, L. corniculatus and its
closely related diploid species in order to provide cytological information
on these species which could aid in determining their relationships and
ultimately be of value in a hybridization program.
In previous studies, karyotypes have been drawn for a few of these ( diploid species of Lotus, but idiogram data which is important for
comparing taxa have been published for only a few. Therefore, one
aspect of this study was to provide more data on the idiograms of the
diploid species.closely related to L. corniculatus and to de termine
their value in terms of species relationships and to see if these data
would provide information on the origin of the tetraploid species,
L. corniculatus. A comparison of DNA values between species has been
shown to be of value in elucidating species relationships, and so this
technique was used as a complementary study for the same purposes. Some
FI hybrids and amphidiploids of these diploid species which were available
for study were also analyzed cytophotometrically. No study employing
DNA cytophotometry has been carried out in the genus Lotus previously.
In summary, the studies reported in this thesis were planned as an
attempt:
First: To obtain as much information as possible on the karyotypes
of L. corniculatus and its closely related diploid species.
Second: To de termine the quantitative DNA relationships using
Feulgen cytophotometry to measure the DNA content of each diploid species
and L. corniculatus.
Third: To combine the knowledge obtained by these two cytological
techniques and to determine the possible phylogenetic relationships between
tetraploid L. corniculatus and its closely related diploid species.
From these analyses it may be possible to come to a better under-
standing of the ancestry of L. corniculatus and so help in determining
the polyploid nature of this species, that is, whether it is an auto- 5.
polyploid, or an allopolyploid, and the species concerned in its origine
In addition, some knowledge concerning the use of karyotype analysis
and Feulgen cytophotometry may be gained from this study for other
biosystematic problems in the genus Lotus.
(
l,~ ' 6.
LlTERATURE REVlEW
A. The re1ationship between L. cornicu1atus and its re1ated dip10id
species:
Within the 1ast three decades broad1eaf birdsfoot trefoi1, L.
cornicu1atus L., has emerged from an introduced weed to a high1y
successfu1 forage crop in northeastern. United States and Canada. As
a resu1t, there has been created a greater demand and interest in the
basic cytogenetic know1edge of this species and of those dip10id species
c1ose1y re1ated to L. cornicu1atus in the Lotus cornicu1atus group.
One aspect of interest has been the practica1 means for transferring
desirab1e characters from the dip10id species to the cu1tivated species
(Somaroo, 1970). Another feature with which this thesis is primari1y
concerned is the re1ationship of the dip10id species per ~ and their re1ationship to the origin of the tetrap10id L. cornicu1atus. There are severa1 ways to study the phy10genic re1ationships between L. cornicu1atus and re1ated species and these methods and the 1iterature concerned will be reviewed here.
l. Morpho1ogica1 studies
The ear1iest studies in Lotus consisted in comparing the morpho1ogica1 characteristics of the species, such as f10wer shape, number of f10rets per head, ca1yx size, 1eaf1et and stipule size, stem thickness and sty1e- shape in order to determine the re1ationship of the species (Brand, 1898;
Ott1ey, 1923). In the L. cornicu1atus group, Brand (1898) and Dawson
(1941) reported that L. tenuis and L. cornicu1atus had Many characters 7.
in common, but they distinguished these two species by the broader
1eaf1ets and stipules and the thicker stems which are possessed by
L. cornicu1atus. They attributed these differences to the dip10id-
tetrap10id re1ationships between these two species and considered that
L. tenuis was the dip10id progenitor of L. cornicu1atus.
It was postu1ated by Dawson (1941) that if L. cornicu1atus was an
autotetrap10id of L. tenuis, then the former might resemb1e the latter
in 1eaf and f10wer characteristics and in pollen diameter; in addition,
the supposed natura1 autotetrap1oid and the induced autotetrap10id might
a1so be cross-fertile. To ascertain these facts, Tome and Johnson (1945) by means of colchicine treatment, doub1ed the chromosome number of a plant of L. tenuis (2x) and found that the autotetrap10id L. tenuis (4x) did not resemb1e L. cornicu1atus (4x) in 1eaf or stipule shape. Crosses which they carried out between autotetrap10id L. tenuis and L. cornicu1atus gave a high percentage of pod set, but in no case was viable seed obtained.
They considered that chromosome differentiation must have occurred in L. cornicu1atus after it had become an autotetrap1oid from L. tenuis and
the resu1ting changes were sufficient to prevent the experimenta11y produced autotetrap1oid from setting seed when crossed with L. cornicu1atus.
Consequent1y, the evidence was not conclusive that L. cornicu1atus arose as an autotetrap10id from L. tenuis.
Severa1 years 1ater, another idea on the origin of L. cornicu1atus was advanced. In the Swiss Alps and in the Bohemian Carpathian mountains of Czechos10vakia, tetrap10id biotypes of L. cornicu1atus were found which were morpho10gica11y very simi1ar to another dip10id species in the Lotus 8.
cornicu1atus group, name1y, L. a1pinus (Favarger, 1953; Zertova, 1964).
Zertova considered that these L. cornicu1atus individua1s cou1d be
treated as one expression of an eco10gica1-morpho10gica1 variation of
the morpho10gica11y variable L. cornicu1atus.
In 1954, Larsen studied three species of the Lotus cornicu1atus
group occurring in Denmark, name1y, L. tenuis, L. u1iginosus and L.
cornicu1atus. He found that these species were not difficu1t to
distinguish in northern Europe, but in southern Europe, and especia11y
in southeastern Europe, these species were so morpho10gica11y high1y
variable that according to the f10ristic manua1s, it was often
extreme1y difficu1t to identify them individua11y. Since species
of Lotus were not cu1tivated in these areas, Larsen considered that
this extreme morpho10gica1 variabi1ity might be due to the formation of
hybrids between these species. Recent1y, Chrtkova-Zertova (1970) carried
out a morpho10gica1 study of another dip10id species c10se1y re1ated to
L. corniculatus namely, L. krylovii and she reported that L. krylovii
differed from L. cornicu1atus in several important morphological features.
II. Biochemica1 Studies in Lotus
It has long been known that the leaves of certain 1eguminous plants
contain a glucoside which yie1ds hydrocyanic acid (HCN) upon hydro1ysis
(Corkil1, 1940). Cyanogenesis has been of interest in forage plants for many years on account of its toxic effects to cattle (Finnemore and
Cooper, 1938). In genetic studies, this character has been used as a marker in the production of interspecific Lotus crosses (Grant ~ al., 9.
1962), and in systematic studies its presence, or absence, in plants
has served as a character to distinguish between taxa (Gibbs, 1963).
Cyanogenesis in L. cornicu1atus was first investigated by Armstrong
et al. (1912) and 1ater by Dawson (1941) and MacDonald (1946). These
authors found that different plants of birdsfoot trefoi1 vary in
concentration of hydrocyanic acid, name1y, from those plants 1acking
HCN to those strong1y positive for this character. A quantitative
reduction of HCN in Lotus species has a1so been shown to be associated
with a reduction in basic chromosome number, evo1utionary advancement
and geographic distribution of the species (Grant and Sidhu, 1967).
A comparison of the quantitative HCN reaction of dip10id species c10se1y
re1ated to the tetrap10id L. cornicu1atus has. been made in order to
determine whether any correlation exists between HCN reaction and the
putative ancestor, or ancestors, of L. cornicu1atus (Grant and Sidhu,
1967). These authors showed that three dip10id species, name1y, L.
japonicus, L. kry10vii and L. tenuis, have reaction intensities which
approached a mean concentration for that of L. cornicu1atus (Grant and
Sidhu, 1967).
The pheno1ic composition of the f10wers and 1eaves of a number of
dip10id species c10se1y re1ated to L. cornicu1atus has been determined by
paper chromatography and a species comparison made (Harney and Grant, 1963).
These authors found that the extracts of the f10wers of L. cornicu1atus and of six c10se1y re1ated dip10id species within the L. cornicu1atus
group contained eight identica1 compounds. In the 1eaves of the dip10id species of the L. cornicu1atus group, L. tenuis, and L. borbasii differed 10.
from the other species by only a single flavone and a single hydroxy- cinnamic acid. Lotus alpinus and L. japonicus possessed identical banding on their chromatograms; they contained leucocyanidin, quercetin, kaempferol, p-coumaric acid, sinapic acid and ferulic acid; L. pedunculatus contained the same leucoanthocyanidins and other phenolic compounds as
L. corniculatus. On this basis, Harney and Grant (1964) subdivided the species of the L. corniculatus group into three subgroups, namely:
1. L. corniculatus, L. caucasicus, L. pedunculatus.
2. L. alpinus, L. filicaulis, L. japonicus, L. krylovii, L. schoelleri.
3. L. borbasii, L. tenuis.
These latter two subgroups differed considerably in their phenolic content from that found for L. corniculatus. Evidence from the fluorescent compound data indicated that L. corniculatus was more likely to be an allotetraploid than an autotetraploid of L. tenuis (Harney and Grant,
1965) and the HCN content of these species showed also that species other than L. tenuis could be implicated (Grant and Sidhu, 1967).
From a thin-layer chromatographic study of fluorescent compounds in leaves of Lotus species, Grant and Zandstra (1967) found that L. tenuis and L. krylovii were very similar and had a coefficient of association of 75.86. The chromatogram of L. corniculatus differed only slightly from that of L. tenuis and L. krylovii and their coefficient of association was 64.52 and 66.67, respectively. Therefore, they suggested that L. krylovii should be included in studies aimed at investigating the progenitor, or progenitors, of the tetraploid species L. corniculatus. 11.
( III. Cytologica1 studies
Species of Lotus are adapted to a wide range of environmenta1
conditions and one might expect different cytotypes to exist in such cases (Love" and Love," 1961). Cyto1ogica11y this genus has two basic chromosome numbers, 6 and 7 (Senn, 1938; Grant, 1965). Lotus cornicu1atus
and its c1ose1y re1ated dip10id species a11 have a basic chromosome number
of six.
1) Karyotypes and Idiograms:
Most authors in their various studies on Lotus have mere1y reported
the chromosome number for the species under investigation. However,
severa1 authors, have made drawings of the chromosomes and have pub1ished
karyotypes for some of the species in the L. cornicu1atus group. These
are as fo11ows: L. cornicu1atus by Tschechow and Kartaschowa (1932),
Larsen (1954), Ujhe1yi (1960), Larsen and Zertova (1963), Gi10t (1965),
Przywara and Schmager (1967) and Zandstra and Grant (1968); L. tenuis by
Tschechow and Kartaschowa (1932), Gi10t (1965), Przywara and Schmager (1967)
and Zandstra and Grant (1968); L. a1pinus by Favarger (1953), Larsen (1954),
and Grant and Zandstra (1967); L. kry10vii by Larsen (1958) and Zandstra
and Grant (1968). In contrast, idiograms have been drawn for only two
species, name1y, L. tenuis and L. kry10vii (Zandstra and Grant, 1968). - 1 - These authors described the karyotype of L. kry10vii as fo11ows: "the
total complement (length) varied from 27.46 ~ to 41.14 ~, with an average
of 32.68 ~---this (species) had the longest total chromosome 1ength. The
1ength of individua1 chromosomes ranged from 3.46 ~ for the longest, to
1.97 ~ for the shortest. The chromosomes were a11 submetacentric with 12.
( pair 4 being nearly metacentric---no satellite chromosomes were observed."
For L. tenuis: "The total complement length varied from 22.04 II to 37.06 II ,
the average being 28.78 ll. The longest chromosome had an average length
of 3.72 II and the shortest, 1.56 ll. AlI the chromosomes were submeta-
centric---no satellite chromosomes were observed for this species." In
the case of L. corniculatus, it may be observed from their drawings of
the somatic chromosomes that most of the chromosomes were submetacentric
but some approached metacentricity and that there were no satellite
chromosomes.
After an analysis of the karyotype of L. tenuis, Przywara and
Schmager (1967) divided the chromosomes of this species into 4 groups
in the following manner:
Group A 6 chromosomes median centromeres
Group B 2 chromosomes submedian centromeres
Group C 2 chromosomes subterminal centromeres
Group D 2 chromosomes median centromeres and with satellites
In the case of L. corniculatus, they concluded from an analysis of
its karyotype that there were two pairs of chromosomes in this species
which corresponded with each pair of the chromosomes of groups A, Band C
of the karyotyp~ of L. tenuis. In addition, they found one pair of
chromosomes with satellites. However, they considered this latter
pair of chromosomes to belong to a group possessing longer chromosomes
than those of the D group in L. tenuis, and therefore, they considered
the chromosomes of L. corniculatus not to possess complete duplicate sets 13.
( of L. tenuis chromosomes.
2) Chromosome pairing:
Muntzing" ( 1936) early observed that " the presence of multivalents indicates autopolyploidy, the absence of multivalents indicates allopoly-
ploidy" and hence, tetraploid species in which multivalents were observed
have been considered to be of autopolyploid origine In studies of meiosis
of L. corniculatus, Dawson (1941) stated that although bivalents were
generally formed at metaphase l, quadrivalents did occur. Although the
quadrivalents were rare, Dawson considered that tetrasomic inheritance
could be taken as the chief criterion of autotetraploidy, and therefore,
he considered L. tenuis to be the diploid progenitor of L. corniculatus,
the latter having arisen from L. tenuis as an autotetraploid.
Wernsman et al. (1963, 1964) reported on some cytological analyses
of meiotic chromos omal behavior in L. corniculatus, in 4x L. tenuis and
in interspecific hybrids of these two species. From their study they
made the following four points:
(1) In L. corniculatus, usually 12 II's were observed at diakinesis,
although on the average there were 0.25 IV's per PMC.
(2) Autotetraploid L. tenuis had an average of 2.82 IV's per cell.
In plants of two "la ter generation" seed-lots of 4x L. tenuis, the frequency
of IV's had decreased from 2.85 IV's per cell to 2.68 and 1.65 IV's per
cell, respectively.
(3) In the interspecific hybrids of L. corniculatus X 4x L. tenuis
there were generally 12 II's at MI with only an occasional quadrivalent.
(4) Interspecific hybrids of 4~ L. tenuis X L. corniculatus were
Jf .... ij '. .""-~, 14.
easily backcrossed to both parents.
From a consideration of these points, Wernsman et al. (1964) interpreted that the occasional quadrivalent observed in the interspecific hybrids indicated at least a partial homology between the chromosomes of
L. corniculatus and L. tenuis. Further, since the univalent frequency was very low in the backcross progenies, the chromosomes from the genome of L. corniculatus must"contain a considerable degree of homology with the genome of L. tenuis. Thus, they believed there was a high degree of homology of the genomes of these two species. Therefore, they supported
Dawson's (1941) hypothesis that L. corniculatus had arisen as an auto- tetraploid from L. tenuis or its prototype.
IV. Hybrid Studies
Dawson (1941) carried out reciprocal crosses between L. tenuis and
L. uliginosus but failed to obtain any hybrid seed from a total of 425 flowers pollinated. Bent (1958) made reciprocal crosses between L. japonicus, L. tenuis, and L. uliginosus (all 2n = 12); but from all these crosses obtained only a single mature hybrid plant from the cross between L. uliginosus X L. tenuis.
By means of embryo-culture, Grant et al. (1962) produced 39 Fl hybrids from nine different cross-combinations between five diploid taxa of Lotus, closely related to L. corniculatus, namely, L. alpinus, L. japonicus, L. filicaulis, L. schoelleri and L. frondosus. From the morphological, cytological and genetical observations, L. alpinus, L. filicaulis and L. japonicus were considered to be more closely related to each other than to L. frondosus and L. schoelleri. In a study of reciprocal crosses between 15.
L. corniculatus and L. tenuis, Jaranowski and Wojciechowska (1963)
reported that embryo development in the cross in which L. tenuis was
used as the pistillate plant was very regular, but when L. corniculatus
was used as the pistillate plant, embryo, endosperm, and seed develop-
ment were irregular. Therefore, they suggested that L. corniculatus
was neither a natural autotetraploid of L. tenuis, nor an amphidiploid
with L. tenuis as one of the parents.
B. Cytophotometry:
By means of Feulgen cytophotometry, qualitative data on the amount
of nuclear DNA can be obtained for a species. This permits a comparison
of the nuclear DNA values between species. From such data, several
biological problems have been attacked in different organisms su ch as
information on chromosome structure and species relationships. While
no studies of this nature have been performed on species in the genus
Lotus, a brief review of some studies employing Feulgen cytophotometry
will be given here to indicate the type of data obtained and the
analyses drawn from such information.
Hughes-Schrader (1951, 1953) in comparing the nuclear DNA values of
several species of Mantidae showed that a quantitative DNA value of a
species complement can be used as lia cytotaxonomic tool in evaluating
evolutionary relationship among species whose karyotypes are not analysable
by the methods of comparative cytology." Since these studies, there has
been a growing body of evidence obtained from different organisms to show
that there is considerable variation in the quantity of nuclear DNA between
species and this information can be used to de termine species relationships r ... 16.
(McLeish and Sunderland, 1961; Key 1 , 1964, 1965; Rees et al., 1966;
Rothfels et al., 1966; Martin and Shank, 1966; Martin, 1966; John and
Hewitt, 1966). Certain closely related plant species which have the
same chromosome number have been shown to vary in the amount of nuclear
DNA between the species. The extent of such variation is often very
great, particularly among certain genera (Rothfels et al., 1966).
Christensen (1966) by means of cytophotometry studied the nuclear
DNA content of diploid and polyploid species of Enchytraeidae. The
total variation in relative DNA values from diploidy through the different
levels of ploidy for the 45 species ranged from 0.40 to 4.85 and for the
diploid species alone from 0.40 to 1.12. In contrast to two genera which
showed a wide variation in both chromosome number and DNA values, three
genera had only a small variation in both chromosome number but possessed
a wide variation in DNA value. Nine out of the 10 polyploid species
contained chromosome numbers in which the DNA value was a multiple of
those values found in related diploids or lower polyploids. Thus from
this study, it was possible to draw conclusions concerning the phylo
genetic relationships of the species based on quantitative Feulgen DNA
measurements.
In studying the evolution in the genus Allium by Feulgen cyto
photometry, Jones and Rees (1968) found a variation in nuclear DNA
values between species groups which was independent of any change in
basic chromosome number and polyploidy. Similar observations in Allium,
Lolium (Rees and Jones, 1967a) and Chironomus (Keyl, 1964, 1965) lead
these authors to the firm conclusion that, except for polyploidy, the
( variation in nuclear DNA content in higher plants and animaIs, is caused 17.
1 " by a lengthwise incorporation of chromosome segments such as those
which result from duplication or loss of segments. Rothfels et al.
(1966) and Rothfels and Heimburger (1968) have opposed the idea of
longitudinal repetition to account for an increase in DNA content in
their cytophotometric studies on species of Anemone, Drosophyllum,
and Drosera. They suggested a lateral increase in chromosome strands
to explain variations in DNA content although they found the increase
in DNA values to be non-genometric, that is the doubling of some of
the strands only.
Ullerich (1966) studied the DNA content of nuclei of three toad
(Bufo) species by Feulgen cytophotometry. He found these species aIl
possessed a similar karyotype, except B. bufo which had slightly larger
chromosomes than the other two species (B. viridis and B. calamita). The
relative total DNA values found per genome were: !. bufo, 1.49; !. viridis, 1.07; and !. calamita, 1.00. He considered that these interspecific
differences in the DNA content of these species were not a consequence
of differential polyteny but were caused during the evolutionary process
by a local increase in DNA in aIl the chromosomes of B. bufo and in the
largest chromo~ome of B. viridis.
Since polyploidy has not contributed greatly to the evolution and
speciation of gymnosperms, Miksche (1967) has explained differences in
DNA amounts between species of pine (Pinus) partly due to geographical
distribution of the species and partly due to species differentiation.
Another recent suggestion to explain the difference in DNA values
between species possessing similar karyotypes has been the idea of the
presence of different amounts of genetically inactive DNA present in the l8~
chromosomes (Dowrick and Bayoumi, 1969). Grant (1969) using an integrating microdensitometer measured the 2e values of birch (Betula) taxa with soma tic chromosome numbers of 28, 42, 56, 70 and 84. He found the DNA density values for the 42, 56 and 70 chromosome plants to be proportional to an increase in chromosome number, except for the
84 chromosome plants. He suggested there had been a selection for chromosome complements with smaller chromosomes at the highest level of polyploidy which could have occurred by hybridization of genomes with different chromosome sizes.
Microspectrophotometric determinations of nuclear DNA content have also been applied to studies on pathological materials. Bader (1953),
Lenchtenberger et al. (1954), Ojima et al. (1960, 1962), and lnui and
Oota (1965) have found that cells of malignant tissues possess a greater quantity of DNA than cells of normal tissue. Endoreduplicated cells in an X-irradiated culture of human leukocytes contained about twice as much DNA as diploid cells from the same culture (Bell, 1964).
The above studies employing cytophotometric methods are by no means complete but have been selected to give an idea of kinds of studies for which cytophotometric studies have been of value, especially in aiding in establishing evolutionary relationships of the species.
As bas been noted, no previous study employing Feulgen cytophotometry has been carried out in the genus Lotus. Since the diploid species closely related to L. corniculatus aIl have the same chromosome number (2n = 12), it gave an opportunity to compare the DNA values between these closely related species along With conventional karyotype analyses. The avail- 19.
abi1ity of F hybrids and some amphidiploids of these species a1so 1 gave an opportunity for comparing any changes in DNA values under
contro11ed experimenta1 conditions.
(
{' '--- 20.
MATERIALS AND METHODS
1. Plant material:
The species, hybrids, and amphidiploids used in this study are
listed in Table l, along with their sources and chromosome numbers.
II. Root tip squashes:
Root tips for cytologica1 examination were taken from potted
plants grown in a growth chamber, greenhouse, or in a co1d frame, part1y
depending on the season. The roots were fixed direct1y in Carnoy's
solution (3:1 a1coho1:acetic acid) for 20 minutes to 24 hours at room o temperature (20-22 C). After the roots were fixed, they were transferred
to 70 per cent a1cohol and stored in a refrigerator (5-70 C) unti1 needed.
In preparation for staining, the roots were washed for 15 minutes in ( running tap water, immersed in cold N HCl for 5 minutes, hydrolyzed for 8 minutes in N HCl at 60 o C, and then rinsed in disti11ed water for
approximately 5 minutes. For staining, the root tips were placed in
1eucobasic fuchsin (Feu1gen technique) for 2 hours. After this staining
period the root tips were washed for 15 minutes in each of three changes
of 502 water (5 ml of N HC1, 5 ml of 10% Na S 0 , 100 ml of distil1ed 2 2 5 water), and fina11y rinsed in distil1ed water. The stained root tips
were a1so stored in 70 per cent ethanol, or disti11ed water temporari1y,
in a refrigerator, until required. Maceration was carried out by p1acing
the root tips for 15-20 minutes in 4 per cent pectinase in order to dissolve
the pectic salt in the midd1e 1ame11a of the cel1 wal1s without the ce11
contents being undu1y softened.
For the preparation of the chromosomes for cytologica1 observation,
a stained root tip was p1aced on a c1ean microscope slide in a few drops 21.
of 45 per cent acetic acid. The root cap below the meristematic region,
as weIl as the portion of the root tip above this region, were cut off.
The meristematic region was then covered with a coverslip, and then
gently tapped by the sharp tipped end of a bamboo stick which aided in
the separation of the cells. The coverslip was then pressed with the
thumb under a few layers of fil ter paper. The slides were made semi
permanent by sealing with nail polish or paraffin wax. It was possible
to keep these temporary sI ides sufficiently long enough for a complete
examination of the slide including chromosome measurements and photo
graphing the appropria te figures.
III. Preparation of karyotypes and idiograms:
A karyotype study was carried out on ten diploid and one tetraploid Cl species. Before fixation, root tips were pretreated with 8-hydroxy quinoline which is a spindle anaesthetic used to suppress the division
of the nucleus by arresting mitosis at Metaphase and contracting the
chromosomes. By this means Many more cells in Metaphase were obtained
and the best were selected for analysis. A karyotype of the somatic
chromosomes for each species was made by drawing the chromosome complement
with the aid of a drawing apparatus. A total of ten Metaphase cells in
which the chromosomes were very weIl spread were drawn and measured.
Idiograms were constructed from short and long arm measurements of
the chromosome complements for each species.
IV. Cytophotometry:
For a comparison of the relative nuclear DNA densities between the
,. . species a standardized procedure was followed. One species, namely L. 22.
corniculatus, Acc. No~ B554, was used as the standard species by which
aIl of the others were compared. In the preparation of a slide for
cytophotometric examination root tips were colleçted at the same time
from potted plants of two accessions, one which was the standard species
(L. corniculatus) and the other was the material to be compared. Both
lots of root tips were processed in the same vial to avoid any possible
procedural changes. A 2.0 cm portion of root tip was collected for the
standard species and root tips of 1.0 cm in length were collected for
the material being compared. No pretreatment of the root tips was
performed prior to fixation for cytophotometric determinations (See
section III). In order to minimize any errors due to imperfections
between slides, root tips of the two species (standard and sample) that
were to be compared, were squashed adjacent to each other on the same
slide and covered with a thin coverslip (Size No. 0).
The relative absorption of nuclei was determined by using a type
GN 2 integrating microdensitometer (Barr and Stroud Ltd., Glasgow,
Scotland). This instrument incorporates a scanning device that
minimizes distributional error; extinction is summated as the scanning
progresses, so that a direct measurement of total absorption is provided
in arbitrary units for a single nucleus. Absorption was measured with
the integrating microdensitometer at a wavelength of 550~. For DNA
determinations, the chromosomes were centered within the appropria te
field stop aperture (depending on the area covered) for the absorption
measurement and three readings were made and recorded. In addition,
three readings of a suitable clear field (background) were also made at ( 23.
the same aperture. The relative absorption was obtaioed by subtracting the average of the three background readings from the average object readings. The te10phase stage (2e) was used for a11 measurements. 2e is customari1y designated for the DNA value of a nucleus before DNA synthesis has occurred. The total number of determinations was 60 chromosome fields (2 X 30 for each series) for each individua1 species.
A11 absorption measurements were made with a 45X oi1 immersion objective at an extinction of 1.0 and on the absorption range 20.
There are severa1 points which shou1d be noted:
1. Interphase nuc1ei were not used for measurement as it wou1d be difficu1t to know exact1y when DNA synthesis had occurred during this period and it wou1d be possible to obtain both 2e (nucleus prior to DNA synthesis) and 4e (after DNA synthesis) values from different interphase mlc1ei in the same slide.
2. The area'from which the ce11 was se1ected must be on1y one layer thick. Over1apping ce11s were not measured.
3. Since the time of hydro1ysis can influence the intensity of staining and consequent1y the intensity of relative absorption, therefore, the time of hydro1ysis must be fo11owed accurate1y.
4. The ce11s were se1ected for a cytop1asmic background of simi1ar density.
5. The constancy of performance of the integrating microdensitometer was obtained by testing the standard materia1 at ear1y prophase and a1so at te10phase. The resu1t of the relative absorption reading shou1d be the same as previous1y recorded for these stages or the difference 1ess 24.
( than 5 per cent if the machine had been running for a sufficient time
period to become stabilized.
v. Statistic analysis:
An analysis of variance was performed on the karyotype measurements
and the cytophotometric measurements of the species. For a comparison
of the significant differences between the species a ~ test, F test and
a Duncan's multiple range test were used. The mean and standard deviation
and standard error were calculated using an IBM computer.
( 25.
TABLE I. The species, hybrids, and amphidiploids studied, their accession
number, source,and chromosome number
Somatic Accession chromosome Species number* Source number
Diploids
L. alpinus Schleich. B77 Rocky limestone pasture at the 12 bottom of the valley of Emaney, Swiss Alps, about 1900 m altitude. Collector: C. Favarger.
L. alpinus Schleich. B324 Near Dahr el Baidar, 12 Lebanon, Collector: Mrs. W. S. Edgecombe.
L. borbasii Ujhelyi B255 Hillside, Kovacovske Kopce, at 12 Kamenica, South Slowakia, Czechoslovakia. Collector: A. Zertova.
L. coimbrensis Willd. B126 Origin Portugal. Seed from 12 Commonwealth Scientific and Industrial Research Organiza tion, Canberra, Australia.
L. filicaulis Dur. B37 Botanic Garden, Madrid, Spain, 12 U.S.D.A. Plant Introduction No. 51864.
L. japonicus (Regel) B129 River bank, Gifu, Japan. 12 Larsen Collector: Prof. I. Hirayoshi.
L. krylovii Schischk. B86 Hortus Botanicus Universitatis, 12 and Serge Uppsala, Sweden.
L. pedunculatus Cav. B193 U.S.D.A. Plant Introduction 12 No. PL 69-55. Source unknown.
L. schoelleri Schweinf. B166 Grassland Research Station, 12 Kitale, Kenya.
L. tenuis Waldst. B109 Origin Turkey. Seed from 12 et Kit. Australia, C.P.I. No. 23788
Table l Cont'd. 26.
TABLE l (Cont'd)
Somatic Accession chromosome Species number* Source number
Hybrids** L. japonicus X L. kry10vii JI{ 12
L. japonicus X L. fi1icau1is JF 12
L. japonicus X L. schoe11eri JS 12
L. japonicus X L. a1pinus JA 12
L. a1pinus X L. japonicus AJ 12
L. a1pinus X L. kry10vii AI< 12
L. a1pinus X L. fi1icau1is AF 12
L. a1pinus X L. schoe11eri AS 12
L. krl10vii X L. schoe11eri KS 12
'- - L. krl10vii X L. fi1icau1is KF 12
L. krl10vii X L. tenuis KT 12
Amphidip1oids**
L. krl1ov.:~.!. X L. fi1icau1is A/KF 24 L. krl10vii X L. schoe11eri A/KS 24
L. krl10vii X L. tenuis A/KT 24
L. japonicus X L. fi1icau1is A/JF 24
L. japonicus X L. schoe11eri A/JS 24
L. japonicus X L. a1pinus A/JA 24
Tetrap10id L. cornicu1atus L. B554 Izmir, Turkey. 24
B534 Svetozarevo, Yogos1avia. 24
B550 Izmir, Turkey. 24 Accession numbers of species correspond to the Lotus species collection * maintained in the Genetics Laboratory. ** The hybrids and amphidip10ids were produced in this 1aboratory by Dr. B. H. Somaroo. 27.
RESULTS
THE KARYOTYPES
Drawings of the karyotypes of the ten dip10id and the one tetrap10id
species of Lotus and representative photographs of the chromosome
complements are shown in Figures 1-11. Idiograms of the chromosomes
for the species are given in Figures 12-13.. Measurements of the
soma tic chromosomes of ten ce11s in which the metaphase chromosomes
were exceptiona11y we11 spread from root tips of plants of the same
accession number for each species are presented in Table II. The
numbers 1 to 6, representing the pairs of homo1ogous chromosomes,
have been arranged in descending order of the 1ength of the chromosomes,
with chromosome number 1 representing the longest pair of chromosomes
of the complement. The total complement 1engths (TeL) and arm ratios
(LIs) were ca1cu1ated as fo11ows:
L = 1ength of long arm S = 1ength of short arm
n = number of chromosomes in the ce11
N = number of ce11s used (in this case, N = 10)
The total 1ength of a chromosome L N (L + = 1 S)/N N The ratio of long arm to short arm L (L/S)/N = 1 N n The total 1eIigth of the chromosome complement L [L (L + S)l/N = 1 1 The formula used to ca1cu1ate the percentage TeL was as fo11ows:
% TeL = the total 1ength of a chromosome pair the total 1ength of the chromosome complement
Based on the location of the centromeres and on the presence, or
( ) absence, of satellites, the chromosomes have been divided into four 28.
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.
The karyotypes for aIl the diploid species of the L. corniculatus
group have six pairs of chromosomes. Before dealing with the statistical
treatment of the measurements, the different karyotypes for each species
will be discussed.
A. The karyological description of the species
(a) Diploid species
The ten diploid species have been arranged on the basis of the
morphological characteristics of their chromosomes and are described
below.
1) The SAT group.
L. pedunculatus (B193)
The length of total chromosome complements ranged from 20.00 ~ to
34.88 ~, with an average of 27.24 ~ (Table II; Figs. 1 and l2A). Two
pairs of satellite chromosomes were observed, namely, pair 2 and 4,
with arm ratios of 1.67 and 1.39, respectively (Table II). The arm
ratios were ca1cu1ated omitting any 1ength which might be attributed
to the satellite. The remaining four pairs of chromosomes had submedian
chromosomes (Fig. 1). The average length of the longest chromosome
was 3.64 ~ and the shortest 1.55 p (Table II). The first pair of
'~'l"~ ,t "..-~,
TABLE II. Karyotype analyses of the somatic chromosomes for ten Lotus species. TCL = total complement length; LIs = long arm/short arme Standard errors are given for the means of the measurements
Chromosome Length in Relative Species pair % TCL LIs micron length
L. pedunculatus (B193) 1 26.42±0.75 1. 75±0.05 3.64 100.00 2 l8.90±0.34 1.67±0.05 2.48 68.10 3 l6.28±0.26 1.58±0.08 2.24 61.72 4 l4.24±0.23 1.39±0.07 1.96 53.99 5 l2.80±0.24 1.42±0 .07 1.76 48.29 6 11.25±0.29 ' 1.39±0.04 1.55 42.49
Average TCL = 27.24 ~
L. krylovii (B86) 1 21.l3±0 .34 1.59±0.11 3.67 100.00 2 19.9l±0.28 1.58±0.09 3.46 94.22 3 l7.82±0.19 1.32±0.05 3.11 84.70 4 l5.47±0.44 1.62±0.11 2.66 72.51 5 l3.59±0.40 1.33±0.07 2.37 64.74 6 11. 75±0.30 1.36±0.09 2.04 55.65
Average TeL = 34.60 ~
Table II Cont'd
N ..\0 TABLE II (Cont'd)
Chromosome Length in Relative Species pair % TCL Lis micron 1ength
L. japonicus (B129) 1 24.16±0.64 1.64±0.07 3.44 100.00 2 18.99±0.46 1.36±0.08 2.69 78.16 3 16.49±0.14 1.38±0.05 2.34 67.99 4 15.19±0.32 1.29±0.06 2.16 62.71 5 13.38±0.31 1.33±0 .04 1.87 54.17 6. 11.53±0.28 1.30±0.07 1.64 47.57
Average TCL = 28.28 ~
L. a1pinus (B324) 1 24.92±0.56 1.90±0.10 3.01 100.00 2 18.20±0.33 1.40±0 .07 2.22 73.78 3 16.59±0.25 1.42±0.06 2.02 67.03 4 14.59±0.30 1.49±0.07 1.79 59.32 5 13.25±0.27 1.35±0.10 1.61 53.51 6 12.30±0.28 1.32±0.07 1.50 49.73
Average TCL = 24.27 ~ Table II Cont' d
w o TABLE II (Cont'd)
Chromosome Length in Relative Species pair % TCL Lis micron 1ength
L. a1pinus (B77) 1 24.65±0.68 2.04±0.11 3.62 100.00 2 18.60±0.37 1.84±0.17 2.74 95.53 3 16.64±0.29 1.52±0.11 2.46 67.90 4 14.92±0.09 1.30±0.08 2.20 60.72 5 13.18±0.10 1.39±0.09 1.94 53.65 6 11.61±0.25 1.18±0.04 1.77 48.82
Average TeL = 29.45 ~
L. borbasii (B255) 1 25.44±0.34 1.53±0.08 3.97 100.00 2 19.06±0.27 1.34±0.06 2.95 74.31 3 16.43±0.18 1.46±0.09 2.59 65.01 4 14.68±0.09 1.50±0.08 2.26 57.01 5 12.83±0.30 1.36±0.10 2.00 50.36 6 11.12±0.29 1.26±0.07 1.72 43.40
Average TCL = 30.99 ~
Table II Cont'd
...... UJ . . ~ /"""..,
TABLE II (Cont'd)
Chromosome Length in Relative Species pair % TCL LIs micron 1ength
L. fi1icau1is (B37) 1 22.88±0.33 1.64±0.06 3.42 100.00 2 18.90±0.38 2.33±0.07 2.83 82.86 3 17.42±0.28 1.50±0.09 2.62 76.58 4 15.52±0.31 1.33±0 .08 2.35 68.61 5 13.48±0.21 1.27±0.04 2.05 59.81 6 11.48±0.26 1.28±0.06 1.75 51.13
Average TCL = 30.02 ~
L. tenuis (B109) 1 24.43±0.42 1.65±0.07 3.31 100.00 2 18.81±0.27 1.43±0.10 2.56 77 .30 3 16.71±0.31 1.38±0.08 2.34 70.54 4 14.59±0.38 1.28±0.09 2.05 61.72 5 13.41±0.16 1.45±0.10 1.83 55.21 6 11. 78±0.25 1.44±0.08 1.63 49.08
Average TCL = 27.42 ~
Table II Cont'd
w N ------,
TABLE II (Cont'd)
Chromosome Length in Relative Species pair % TCL L/S micron 1ength
L. schoe11eri (B166) 1 24.03±0.51 1. 71±0.04 3.70 100.00 2 19.37±0.44 1.42±0 .07 2.94 79.54 3 16.59±0.31 1.42±0 .09 2.55 68.87 4 14.63±0.32 1. 21±0.04 2.24 56.11 5 13.49±0.21 1.22±0.04 2.07 60.51 6 11. 70±0.28 1.33±0.07 1.79 48.51
Average TCL = 30.56 ~
L. coimbrensis (B126) 1 26.16±0.52 2.62±0.06 3.48 100.00 2 22.33±0.40 3.07±0.11 2.98 85.73 3 15.48±0.21 1. 70±0.22 2.05 59.06 4 13.55±0.19 1.32±0.07 1.80 51.81 5 11. 75±0.23 1.28±0.06 1.55 44.68 6 10.49±0.28 1.39±0 .09 1.39 40.10
. Average TCL = 26.51 ~
Table II Cont'd
w ..w ~ '-=1
. TABLE II (Cont'd)
Chromosome Length in Relative Species pair % TCL Lis micron 1ength
L. corniculatus (B554) 1 13.24±0.27 1. 71±0.06 3.86 100.00 2 11.80±0.21 1.57±0.06 3.32 85.97 3 9.82±0.14 1.46±0.08 2.85 73.81 4 9.02±0.09 1.43±0.07 2.63 68.10 5 8.59±0.07 1.38±0.06 2.50 64.73 6 8.21±0.13 1. 41±0 .08 2.35 61.05 7 7.57±0.10 1.42±0.06 2.22 57.44 8 7.07±0.09 1.34±0.05 2.05 53.03 9 6.69±0.08 1.38±0.08 1.94 50.23 10 6.47±0.13 1.36±0.06 1.87 48.59 11 6.02±0.11 1.27±0.06 1.74 45.23 12 5.42±0.15 1.15±0.07 1.44 37.42
Average TCL = 57.50 ~
w ~ 35.
chromosomes were readily distinguished from the other five pairs, the
latter which were not markedly different from eachother and showed
only a graduaI decrease in size from the longest to the shortest pair.
2) The SM group consisted of species with predominantly submedian
chromosomes.
(i) L. krylovii (B86)
The total chromosome complements varied in length from 23.6 ~ to
43.86~, with an average length of 34.60 ~ (Table II). The length of
the individual chromosomes ranged from 3.67 ~ for the longest, to 2.04 ~
for the shortest. According to the size of the individual chromosomes,
this species, differed from the others in that it had 3 pairs of long
chromosomes, 2 pairs of medium long chromosomes and one small pair of ,- chromosomes. Of the three pairs of long chromosomes, pairs 1 and 2
were difficult to distinguish from each other; they had almost the
same long arm to short arm (LiS) ratio, with a total length difference
of only 0.2l~. In comparison with the other nine diploid species,
this species had the longest total chromosome length (34.60 ~). No
satellites were observed on any of the chromosomes (Figs. 2 and l2B).
(ii) L. japonicus (B129)
AlI of the chromosomes of this species were submetacentric with
the exception of the fourth chromoscme which was nearly metacentric
and had an arm ratio of 1.29. From measurements of the chromosome
lengths, the longest chromosome pair could be recognized easily from
the other chromosomes. The other chromosomes of the complement consisted Figures 1-3. Somatic chromosomes from root tip ce11s of Lotus ) dip10id species.
Figure 1. Metaphase of L. peduncu1atus, X ca. 1800.
Figure 2. Metaphase of L. kry1ovii, X ca. 1750. )
Figure 3. Metaphase of L. japonicus, X ca. 1800. 37.
[; ----;
1 • 1 1 1 i 1
1
u ... 2 37 .
. _. --_ .. _--- - .•.. _------.
. ., 1 j
1 : '"------_.__ --.:
----_.. __._------..
2
-_.. ------_ ...- ._'-_.'- " •f' .-JI , '1.. 3 ._- _._- 38.
of three pairs of medium sized chromosomes and 2 pairs of sma11 chromosomes
which had a1most the same arm ratios (Fig. 3). The 1ength of the total
chromosome complement ranged from 25.00 p to 32.11 p, with an average
TCL of 28.28 p (Table II). The average 1ength for the longest
chromosome was 3.44 p and the shortest chromosome 1.64 p (Table II).
No satellites were observed for any of the chromosomes in the complements
studied (Fig. 12C).
(iii) L. a1pinus (B324)
Plants of. this accession number had a different origin than those
of L. a1pinus (B77). Seed of L. a1pinus (B324) were co11ected near
Dahr el Baidar, Lebanon. This species had on1y one pair of long
chromosomes, but two pairs of chromosomes were medium long. In
addition, two pairs of chromosomes were just of medium 1ength, and
one pair was sma11. A1though the chromosomes of this karyotype a11
be10ng to the same metacentric group as L. japonicus and L. kry1ovii,
there were some differences between them in the arm ratio values of
the individua1 chromosomes. The chromosomes a11 had fair1y simi1ar
arm ratios with the exception of the longest pair of chromosomes. The
1ength of the total chromosome complements ranged from 21.26 p to 20.08 P
with an average TCL of 24.27 p (Table II). The average length of the
longest chromosome was 3.01 p and the shortest 1.50 p. This species
had the shortest total complement 1ength of the nine dip10id species.
No satellites were observed in any chromosome complements examined for
this species (Figs. 4 and 12D).
( Figures 4-6. Somatic chromosomes from root tip ce11s of Lotus ) dip10id species.
Figure 4. Metaphase of L. alpinus (B324), X ca. 2550.
Figure 5. Metaphase of L. alpinus (B77), X ca. 1700. )
'Figure 6. ~etaphase of L. borbasii, X ca. 2150.
) · 40.
f _____ • .._. .. •
4
,~ 1/ t J J., .. =- t ~~ " 5 ------... ---.-
.. ------, 6 - ~ - '"-. -...... -~--.- --_. -----._--_._._- .. - -. _._-- -.- --- 40.
'St .. ~ • " 1 ~ '" .... =- ~ _: " 4 ------
Il t. 1/ .. t J If,.J., =- 5 10--. _._. __ " .-.- -- -- .. __ .._-
--.11 "" 1/- .,." of.
" p 6 41.
( ) 3) This third group is composed of L. a1pinus (B77) and L. borbasii (B255)
These species had 5 pairs of submedian chromosomes and one pair of
median chromosomes. In this case, the chromosomes with median centro
meres were represented by the sma11est pair of chromosomes. The total
complement 1ength did not differ great1y between these two species,
the TeL of L. a1pinus being slight1y longer by 1.54~.
(i) L. a1pinus (B77)
Plants of this accession number had a different origin than those
of L. a1pinus (B324). Seed of L. a1pinus (B77) originated from a
rocky 1imestone pasture at the bottom of the valley of Emaney, about
1900 m., in the Swiss Alps. The total 1ength of comp1eme~~s for this
accession ranged from 23. 98 ~ to 34. 63 ~, wi th an average TeL of 29. 45 ~. Ci The longest pair of chromosomes having a total chromosome 1ength of
3.62 ~ cou1d be easi1y distinguished from the other chromosomes of
the complement. Two pairs of chromosomes were medium long, two pairs
were of just medium 1ength, and the sma11est pair of chromosomes had
a chromosome 1ength of 1.77~. This species, c1ose1y resemb1ed L.
borbasii according to ~imi1arity in karyotype (Table II) •. No satellites
were observed for any chromosomes in the complements studied (Figs. 5
and 12E).
(ii) L. borbasii (~255)
Of the ten dip10id species studied, the chromosomes of this species
were second longest in their total chromosome complement 1ength (Table II).
The TeL's varied from 22.11 ~ to 36.79 ~ with an average TeL value of
( 42.
30.99 (Table II). The average length of the longest chromosome was
3.97 ~ and the shortest chromosome was 1.72 ~ (Table II). The position
of the centromere of the shortest pair of chromosomes was nearly median, with an Lis ratio of 1.26 which is very similar to that for the shortest
chromosome of L. alpinus (B77). Chromosomes bearing satellites were
not observed in this species (Figs. 6 and l2F).
The remaining four diploid species differed sufficiently from
each other and from the above species to be considered separately.
4) Lotus filicaulis (B37)
The chromosomes of this species had centromeres in a wide range
of positions. The complement included 2 pairs of median chromosomes,
3 pairs submedian chromosomes, and one pair of chromosomes with sub
terminal centromeres. The Lis value of the subterminal chromosomes which belonged to the second longest chromosome pair in this species was 2.33 ~ (Table II). The total chromosome complement length varied from 23.74 ~ to 36.22 ~ with an average TCL of 30.02~. The length of
the longest chromosome in this species was 3.42 ~, the shortest chromosome was 1.75 ~ long. No satellite chromosomes were observed in this species
(Figs. 7 and l3G).
5) Lotus tenuis (Bl09)
This species had a karyotype very similar to L. alpinus (B77) and
L. borbasii except the four th pair of chromosomes was metacentric, rather than the shortest pair as in the case of these two species.
The total chromosome complement length varied from 21.14 ~ to 33.68 ~ with an average TCL of 27.42~. The average length of the longest Figures 7-9. Somatic chromosomes from root tip ce11s of Lotus ) dip10id species.
Figure 7. Metaphase of L. fi1icau1is, X ca. 1250.
Figure 8. Metaphase of L. tenuis, X ca. 2100.
Figure 9. Metaphase of L. schoe11eri, X ca. 2100.
) 44~
( < .,.x Il
7 .~------~-~
,"__ 0 __- _._----. CC -,. 1J\ "4- A ., 8 ------•
. " . :
,. le Il - ,. -(, Je ,. \~ Il ~ 9 ---- -_ ... _--- . ~ 44~
x
. . . 11"
7 .______--'-_._...J
• ------_._------_.
. .
,. te Il - ,. ~, Je \-\. "~ 9 - .-- -_. - _.. ; 45.
( chromosome was 3.31 ~ and the shortest chromosome 1.63 ~ (Table II).
No satellite chromosomes were observed in the karyotypes examined for
this species (Figs. 8 and 13H).
6) Lotus schoe11eri (B166)
The 1ength of the total chromosome complement for this species
varied from 22.56 ~ to 34.84 ~ with an average TeL of 30.56 ~ (Table II).
The 1ength of individua1 chromosomes range from 3.70 ~ for the longest
chromosome to 1.79 ~ for the shortest chromosome (Table II). The
chromosomes were a11 submedian with exception of pairs 4 and 5 which
were median, and had an L/S ratio of 1.21 and 1.22 ~, respective1y.
No satellite chromosomes were observed in the complements of this
species (Figs. 9 and 131).
7) Lotus coimbrensis (B126)
This species whi1e a European species is not a member of the L.
cornicu1atus group, but be10ngs to the L. aegeus section of the genus
Lotus and was inc1uded in this study in order to obtain some idea as
to the simi1arity and/or differences in karyotypes throughout the genus.
The chromosomes of this species possessed special characters which
separated it readi1y from the other dip10id species. Un1ike the species
in the L. cornicu1atus group, the first and second pairs of chromosomes
have subtermina1 centromeres and with L/S values of 2.62 and 3.07,
respective1y. Of the remaining four pairs of chromosomes, three pairs
have submedian chromosomes and one pair has median chromosomes (Figs. 10
and 13J). The chromosomes of this species have a total complement 1ength ... \ ( 1 Figures 10-11. Soma tic chromosomes from root tip ce11s of Lotus ) dip10id species.
Figure 10. Metaphase of L. coimbrensis, X ca. 2600.
) Figure 11. Metaphase of L. cornicu1atus, X ca. 1250.
/ 47.
1
,.. . 10
..... _.-----.,;--_.--""
1 47.
1
, ,~ f'
: : 10
" ." -.-_ ..• - . .._ ...... _- r Figure 12. Idiograms of dip10id Lotus species. \
A. L. peduncu1atus
B. L. kry10vii
c. L. japonicus D. L. a1pinus (B324)
E. L. a1pinus (B77)
F. L. borbasii 49. )1: ·1~lill. 111111 .: 1:111. ,1:111. 10 ! . :15: A B
.." 1: 111111 ,~.. 111111 ) :1: 1111 ." •• 1111
~ 15 C D '. 20 . . .
~ 10 .' .
" 5 111111. '., 11111 . 1: 1111 -,' 1111·",'·
\ 1 15 E F' 20 ) Figure 13. Idiograms of dip10id Lotus species. ..'
G. L. fi1icau1is
H. L. tenuis
l. L. schoe11eri J. L. coimbrensis
K. L. cornicu1atus
) 51.
10
o5 111111 111111. 5 III 1111. 10 ;:~ G 15 H
20
1:. 111111 '. 111111 ) : 1:"' 1111') 1111 .•
... 15 1 J 20 .
10 " ' . .. . : 111111111111 1: ..11111111111:
. 15
4' ::x:z:::s;x:a...... • ~ - 52.
) which ranged from 24.19 p to 32.72 p with an average TCL value of 26.5 p (Table II). The longest chromosome had an average length of 3.48 P
whereas the shortest chromosome had a length of only 1.39 p (Table II).
No satellite chromosomes were observed in this species.
(b) Tetraploid species
L. corniculatus (B554) The somatic chromosome number of this species is 2n = 24. The length of the total chromosome complement varied from 45.13 p to 75.45 p
with an average of 57.50 p (Table II). The length of the individual
chromosomes range from 3.86 p for the longest chromosome, to 1.44 p
for the shortest chromosome (Table II). The chromosomes were aIl
submedian with the exception o~ pairs Il and 12 which had chromosomes ) with median centromeres and with an LIs ratio of 1.27 and 1.15, respectively. No satellite chromosomes were observed in the metaphase
figures examined for this species (Figs. Il and l3K).
B. Statistical analysis:
(a) A comparison of the total complement lengths (TCL) and the long arml
short arm (LIS) ratios of the ten diploid species:
A comparison was made between the morphological .characteristics for
the six pairs of chromosomes in the ten diploid species. The values
obtained for the TeL and the LIs were tested for statistically signif
icance by means of a t test. The results of the t test are given in Table III by appending
letters to the means. Those means which do not differ significantly
from one another are followed by the same letter. For example, the -----.., '-----', 1 --'- '-,
TABLE III. Mean values of the percentage total complement 1ength (% TCL) and the long-short arm ratio (LIs)
Chromosome pair Species 1 2 3 4 5 6 L. peduncu1atus TCL 26.42 b* 18.90 ae 16.28 bd 14.24 b 12.80 b 11.25 ac (B193) LIs 1. 75 ac 1.67 b 1.58 ac 1.39 abd 1.42 a 1.39 a L. kry10vii TCL 21.13 c 19.91 c 17.82 e 15.47 ad 13.59 ab 11. 75 ad (B86) LIs 1.59 ad 1.58 bd 1.32 b 1.62 b 1.33 ab 1.36 ab L. japonicus TCL 24.16 ad 18.99 ac 16.49 bd 15.19 ad 13.38 ab 11.53 ad (B129) LIs 1.64 ad 1.36 cd 1.38 b 1.29 df 1.33 ab 1.30 ab L. a1pinus TCL 24.92 bde 18.20 a 16.59 bd 14.69 ab 13.25 ab 12.30 d (B324) LIs 1.90 ce 1.40 cd 1.42 ab 1.49 ab 1.35 ab 1.32 ab L. a1pinus TCL 24.65 bde 18.60 ae 16.64 ad 14.92 ad 13.18 ab 11.61 ad (B77) LIs 2.04e 1.84 b 1.52 abc 1.30 adf 1.39 ab 1.18 b L. borbasii TCL 25.44 bd 19.06 ae 16.43 bd 14.68 bd 12.83 ab 11.12 ac (B255) LIs 1.53 df 1.34 cf 1.46 abc 1.50 abd 1.36 ab 1.26 ab L. fi1icau1is TCL 22.86 a 18.90 ae 17.42 ae 15.52 a 13.48 a 11.48 a· (B37) LIs 1.64 ad 2.33 a 1.50 abc 1.33 acd 1.27 ab 1.28 ab L. tenuis TCL 24.43 de 18.81 ae 16.71 ad 14.59 ab 13.41 a 11. 78 ad (B109) LIs 1.65 acf 1.43 bdf 1.38 ab 1.28 adf 1.45 b 1.44 a L. schoe11eri TCL 24.03 ae 19.37 ec 16.59 ad 14.63 ab 13.49 a 11. 70 ad (B166) LIs 1. 71 acf 1.42 cd 1.42 ab 1.21 cf 1.22 b 1.33 ab L. coimbrensis TeL 26.16 b 22.33 b 15.48 c 13.55 c 11. 75 c 10.49 c (B126) LIs 2.62 b 3.07 e 1.70 c 1.32 adf 1.28 ab 1.39 ab *a,b,c,d,e,f: Mean value between species for each chromosome pair are significant at the 5% 1eve1 except when means are fo11owed by the same 1etter; comparisons are made between the same chromosome \.Il pair of the different species, not between chromosome pairs of the same species; see texte W 54.
() means of the TeL for the first pair of chromosomes of L. peduncu1atus,
L. kry10vii, L. japonicus, L. a1pinus (B324), L. a1pinus (B77), L.
borbasii, L. fi1icau1is, L. tenuis, L. schoe11eri and L. coimbrensis
are fo110wed by b,c,ad,bde,bde,bd,a,de,ae, and b, respective1y (read
vertica11y, not horizonta11y). This indicates that for chromosome 1
the difference between L. peduncu1atus, L. alpinus (B324, B77), L.
borbasii, L. coimbrensis, and between L. japonicus, L. a1pinus (B324~
B77), L. borbasii, L. coimbrensis, and between L. japonicus, L.
fi1icaulis, L. schoe11eri and between L. a1pinus (B324, B77), L. tenuis,
and L. schoe1leri, is not significant; other combinations are significant.
For chromosome 1, L. krylovii differs significant1y from a11 the other
species. This ana1ysis shows that these ten dip10id species do not
have identica1 karyotypes. From Table III, it may be seen that L.
coimbrensis differed in its 'karyotype from the other nine species in
that it had extremely different values for the means of the TeL and L/S
values except for chromosome 1. The TeL values of chromosome pairs
3, 4, 5, and 6 were sma1ler than those of a11 of the other nine dip10id
species. In the other extreme, chromosome pairs 2 and 3 had the
largest L/S values of any of the species.
(b) A comparison of the total chromosome length (long arm + short arm)
between the ten dip10id species:
The results of the comparison of the total chromosome lengths for
the ten diploid species are given in Table IV. The data show that
there is a significant difference in the total chromosome 1ength between ...,-...... \:,,'" v -.-c"
TABLE IV. Ana1ysis of variance and Duncan's test of the mean total complement 1ength for the 10
dip10id species.of Lotus
Source of Degrees of Sum of Mean F variation freedom squares squares value
Species 9 739.85 82.206 3.944** Error 90 1852.51 20.583 Total 99 2592.36 26.185
** Significant at 1% 1eve1 Duncan's test (5% 1eve1) * Species: B 324 B 126 B 193 B 109 B 129 B 77 B 37 B 166 B 255 B 86 24.27 26.51 27.24 27.42 28.28 29.45 30.02 30.56 30.99 34.60
B 324 '= L. a1pinus B 109 = L. tenuis B 37 = L. fi1icau1is B 126 = L. coimbrensis B 129 = L. J!Eonicus B 166 = L. schoe11eri B 193 = L. peduncu1atus B77 = L. a1pinus B 255 = L. borbasii B 86 = L. kry10vii
* Differences are significant between means 1yifig on different 1ines.
Ut Ut 56~
the diploid. species. The species, L. krylovii has the greatest total complement length (34.6 ~); in contrast, L. alpinus (B324) has the shortest TCL (24.27~). From the statistical analysis (Table IV) it may be seen that there is no significant difference in total complement length between L. schoelleri, L. borbasii, L. krylovii, between L. tenuis, L.• japonicus, L. alpinus (B77), L. filicaulis, L. schoelleri,
L. borbasii, between: L. coimbrensis, L. pedunculatus, L. tenuis, L. japonicus, L. alpinus (B77), L. filicaulis, L. schoelleri, and between
L. alpinus (B324), L. coimbrensis, L. pedunculatus, L. tenuis, L. japonicus (Duncan's test in Table IV). From Table IV it may be seen that the two different accession numbers for L. alpinus (B77, B324) belong to two different groups, the difference in TCL between them is
5 .18 ~.
THE CYTOPHOTOMETRIC COMPARISON OF THE SPECIES
As stated in the introduction, the purpose of this study was to gather more information concerning the relationships of the species of
Lotus, especially those in the L. corniculatus group. In this cyto- photometrie study, L. corniculatus (B554) has been used as the standard species with which aIl others were compared. Before carrying out the statistical analysis the values obtained for the species must be adjusted in comparison to the standard species. The formula used was as follows: x=aXb· x =. the standardized value of the DNA content of the 4 2C nucleus for the species under comparison a = the DNA value of the 2e nucleus of the species being compared b = the DNA value of the 2C nucleus of the standard species 57.
1. A comparison of thenuclear DNA content of three tetraploid accessions
of L. corniculatus:
Lotus corniculatus is a species widely distributed throughout the
world. The following experiment was carried out in order to compa~e
the DNA values obtained for three different accessions of this species.
The seed for these accessions originated from Samsun, Turkey (B554),
Svetozarevo, Yugoslavia (B534) and Izmir, Turkey (B550), respectively.
The values of the nuclear DNA content of the 2C nuclei determined for
- these accessions are given in Table V. The relative DNA measurements
are expressed in arbitrary units and the me an values were obtained
from an analysis of 30 cells for each accession. From the analysis of
the results it may,be seen that there was no significant differences
between these three different sources of L. corniculatusin regards to ) the DNA content per nucleus, the average values being 4.01, 4.00, and
4.01, respectively, for these accessions.
2. A comparison of the nuclear DNA content values found for the 10
diploid species:
The mean DNA value for the 10 diploid species are given in Table VI.
The DNA nuclear values are based on measurements of 60 nuclei for each
species. As may be seen in Table VI, it was possible to arrange the
species so that there was a graduaI increase in the nuclear DNA value
'between them with the exception of L. borbasii and L. krylovii which
had the same DNA value of 1.97. The sequence from lowest to highest
DNA value is as follows: L. coimbrensis, L. alpinus (B324), L. tenuis,
) 58~
C'.) TABLE V. DNA values in arbitrary units for 2C nuc1ei of three
accessions (tetrap1oid) of Lotus cornicu1atus from
different sources. Estimates are based on the means
of 30 nuc1ei for each accession number. Standard errors
are given for the means of the measurements.
Species DNA per 2Cnuc1eus Mean
L. cornicu1atus (B554) 4.01 ± 0.0820 4.01
L. cornicu1atus (B534) 4.00' 4.00
L. cornicu1atus (B550) 4.01 ± 0.0967 4.01
o 59.
~LE VI. Mean DNA nuc1ear values (in arbitrary units) for 2e nuc1ei
of Lotus species. The estimates are based on an exam-
ination of 30 nuc1ei for each series. Standard errors
are given for the means of the measurements
DNA per 2e nucleus Species Series 1 Series 2 Mean
L. coimbrensis (B126) 1.84±O.0395 1.66±0.0387 1.750
L. alpinus (B324) 1.93±O.0531 1. 66±0·. 0359 1.795
L. tenuis (B109) 1. 93±0.0924 l.79±0.0478 1.860
L. japonicus (B129) 1.85±O.0500 1.88±0.0415 1.865 L. schoe11eri (B166) 2.00±O.043 1. 91±0.0460 1.955 -, L. alpinus (B77) 2.00±0.0432 1. 93±0.0483 1.965 ') _/ L. fi1icau1is (B37) 2.00±O.0439 1.93±0.0404 1.968
L. borbasii (B255) 1. 94 ±O .0414 2.00±O.0443 1.970
L. kry10vii (B86) 1 1.97±O.0549 1.98±O.0399 1.970
L. peduncu1atus (B193) 2.24±0.0628 2.00±0.0497 2.120 ~ ( ') '-'
TABLE VII. Ana1ysis of variance and Duncan's test of DNA variation of the 2C nuc1ei between the
dip10id species of Lotus
Degrees of Sum of Mean F Source of variation freedom squares squares value
Between series 19 25.95493
Species 9 21.71173 2.4124 5.6853 ** Between series within species 10 4.24320 0.4243 5.8618**
Between species within series 580 41.96041 0.0724
** Significant at 1% 1eve1
Duncan's test (5% 1eve1) *
Species: B 126 B 324 B 109 B 129 B 166 B 77 B 37 B 255 B 86 B 193 1.750 1.795 1.860 1.865 1.955 1.965 1.968 1.970 1.970 2.120
* Differences are significant between means 1ying on different 1ines. B 126 = L. coimbrensis B 129 = L. J!Eonicus B 37 = L. fi1icau1is B 324 = L. a1pinus B 166 = L. schoe11eri B 255 = L. borbasii B 109 = L. tenuis B77 = L. alpinus B 86 = L. kr~lovii B 193 = L. ped_l!n~u~at~s 0'" 61.
L. japonicus, L. schoelleri, L. alpinus (B77), L. filicaulis, L. borbasii,
L. krylovii, L. pedunculatus with average values of 1.750, 1.795, 1.860,
1.865, 1.955, 1.965, 1.968, 1.970, and 2.120, respective~y. The value
of the first two species as weIl as the third and fourth and fifth to
ninth are very close. The last species, L. pedunculatus, had a somewhat
higher DNA content than the others.
It may be concluded from the analysis of variance that significant
differences exist between the diploid species (Table VII). However,
from the Dunc,an's test it may be seen that there is no significant
differences in DNA value between the following species: L. tenuis,
L. japonicus, L. schoelleri, L. alpinus (B77), L. filicaulis, L. borbasii,
L. krylovii, and L. pedunculatus and between L. coimbrensis, L. alpinus
(B324), L. tenuis, L. japonicus, L. schoelleri, L. alpinus (B77), L. ) filicaulis, and L. borbasii. The distribution of the amount of nuclear
DNA in 2e nuclei of the ten diploid species are presented by means of
histograms (Figs. 14-16).
AlI of the above data have shown that there exists some variation
in nuclear DNAcontent between the diploid species of Lotus. The
difference between the lowest and highest value being 0.370 units.
3. A comparison of the nuclear DNA content between twelve Lotus hybrids:
The relative DNA measurements expressed in arbitrary units for the
twelve hybrids are summarized in Table VIII. The overall range of the
mean DNA values for the individual hybrids was relatively small from
1.825 to 2.255 units. An analysis of variance of the data revealed that
) 62.
( ) TABLE VIII. Mean DNA nuclear values, in arbitrary units, for 2e
nuclei of twelve Lotus hybrids. The estimates are
based on an examination of 30 nuclei for each series •
. Standard errors are given for the means of the
measurements. Accession number, B77, was used in aIl
crosses involving L. alpinus.
DNA per 2e nucleus Species Series 1 Series 2 Mean
L. àlpinus X L. filicaulis 1. 72±0.0502 1.93±0.0499 1.825
L. japonicus X L. filicaulis 1. 77±0.05l8 2.00±0.05l3 1.885
L. alpinus X L. schoelleri 1.73±0.0437 2.07±0.0548 1.900 C) L. alEinus X L. krx:lovii 1. 76±0.0437 2.08±0.0530 1.920 L. fi1icaulisX. L~ schoelleri 1.98±0.0677 1.88±0.05l0 1.930 L. krYlovii X L. schoelleri 2.09±0.0751 1.82±0.0455 1.935
L. jaE0nicus X L. a1Einus 1.89±0.0544 1.92±0.034l 1.955
L. alEinus X L. japonicus 1.98±0.0461 1.94±0.0477 1.960
L. jaEonicus X L. krx:lovii 1. 90±0.0588 2.04±0.0655 1.970
L. krx:lovii X L. tenuis 1.87±0.0360 2.08±0.O501 1.975
L. jaE0nicus X L. schoelleri 2.l8±O.O632 2.09±O.O472 2.135
L. krx:lovii X L. filicaulis 2.34±O.O703 2.l7±O.O6l5 2.255 () ~
,_.-,f < .... / "----
!ABLE IX. Ana1ysis of variance of the DNA values of 2C nuc1ei between Lotus hybrids
Source of variation Degrees of Sum of Mean F freedom squares squares value
Between series 23 20.54514 0.8933
Hybrids 11 9.028717 0.8208 0.8553 Between series within hybrids 12 11.516432 0.9597 6.3221 ** Between hybrids within the same series 696 105.66246 0.1518
** Significant at 1% 1eve1
C'I W 64~
there was no significant differences between the hybrid DNA values
(Table IX). There were two further points which emerged from these
resu1ts. First, there was a significant dif~erence that occurred for
values between slides of the hybrid. Second1y, the hybrids L. krylovii
X L. fi1icau1is and L. japonicus X L. schoe11eri have a 2C value of
2.255 and 2.135, respective1y, which is slight1y higher than the
theoretica11y value of 2.0. The distribution of the.amount of nuclear
DNA in 2C nuc1ei of the 12 hybrids are presented in histograms (Figs. 17- 20).
4. A comparison of the nuc1ear DNA content between six amphidiploids:
The relative nuc1ear DNA measurements for the six amphidip10ids
are given in Table X. The DNA value for the tetrap10id L. cornicu1atus
was found to be 4.0 (Table III) and for the amphidip10ids five out of
the six had DNA values close to this value. The amphidip10id A/KT had
the lowest DNA value of 3.55.
The ana1ysis of variance of the DNA values per nucleus for the six
amphidip10ids is given in Table XI. The differences between the amphi
diploids was found to be significant. However, the Duncan's test showed
that there was no ·significant differences between certain amphidiploids,
namely, A/KS (L. kry10vii X L. schoel1eri), A/JA (L. japonicus X L.
alpinus), A/JF (L. japonicus X L. fi1icau1is), A/JS (L. japonicus X
L. schoe1leri)., and A/KF (L. kry10vii X L. fi1icau1is), and between
A/KT (L. krylovii X L. tenuis), A/KS (L. krylovii X L. schoe1Ieri),
A/JA (L. japonicus X L. a1pinus) and A/JF (L. japonicus X L. filicau1is).
The relative DNA measurements expressed in arbitrary units of the ) 65.
TABLE X. Mean DNA nuclear values, in arbitrary units, for 2e nuc1ei
of six Lotus amphidiploids. The estimates are based on
an examination of 30 nuc1ei for each series. Standard
errors are given for the Eeans of the measurements
DRA per 2e nucleus Species Series 1 Series 2 Mean
A/KT 3.41±O.O770 3.69±0.0611 3.550
A/KS 3.87±O.0780 3.80±0.0819 3.845
A/JA 3.84±O.1057 3.86±0.0904 3.850
A/JF 3.92±O.1l77 3.79±0.0744 3.855
. A/JS 4. 22±O .1257 3.85±0.1478 4.085
A/KF' 4.07±O.1257 4.22±0.1035 4.145 ) A/KT = (L. kry10vii X L. tenuis)2 2 A/KS = (L. kry10vii X L. schoelleri) A/JA = (L. japonicus X L. a1pinus)2 ' )2 A/ JF = (L. japonicus X L. filicaulis A/JS = (L. japonicus X L. schoelleri) 2 A/KF = (L. kry10vii X L. fi1icaulis) 2 .,.--...... " () ,-' "__._r' '---'
TABLE XI. . Ana1ysis of variance and Duncan' s test of the DNA values of 2e nuc1ei between amphi-
dip10ids
Degrees of Sum of Mean F Source of variation freedom squares squares value
Between series 11 94 •.83588 8.6214
Amphidip10ids 5 91.32208 18.2644 31.189**
Between series within amphidip10ids 6 3.51380 0.5856 1.525
Between amphidip10ids within series 348 133.60262 0.3839
** Significant at 1% 1eve1 Duncan's test (5% 1eve1) * Species: A/KT A/KS A/JA A/JF A/JS A/KF 3.550 3.845 3.850 3.855 4.085 4.145
* Differences are significant~ between means 1ying on different 1ines. A/KT = (L. kry10vii X L. tenuis) 2 A/KS = (L. kry10vii X L. schoe11eri) 2 A/JA = (L. japonicus X L. a1pinus) 2 2 A/JF = (L. japonicusX L. fi1icau1is), 2 CI\ A/JS = (L. japonicus X L. schoe11eri) CI\ A/KF = (L. kry10vii X L. fi1icau1is) 2 67.
six amphidip10ids are shown by means of histograms (Figs. 21-22).
5. The re1ationship between chromosome 1ength and DNA content of the
dip10id species:
A study of the re1ationship between total complement 1ength (TeL),
nuclear DNA content and the DNA content per micron of chromosome for
the dip10id speci~s are given in Table XII. The data indicate that
the mean DNA content per nucleus was not proportional to chromosome
1ength. Also, the DNA content per unit length of chromosome was not
constant per species. In genera1, the DNA value per micron of chromosome
increases with increasing chromosome length, except for the species,
L. coimbrensis and L. schoe1leri . (Table XII). The variation in mean
DNA content per unit length of chromosome ranged from 0.057 to 0.078 J units.
) ,--... ~ ",--..., i ' 1 ) '.~! ',,--,,' \--"
TABLE XII. The re1ationship between chromosome 1ength and DNA content of the dip10id species of Lotus
Mean 1ength of Mean DNA content DNA content Species chromosome complement (TeL) per nucleus in per micron of in micron (p) arbitrary units chromosome
L. a1pinus (B324) 24.27 1.795 0.074
L. coimbrensis (B126) 26.51 1.750 0.066
L. peduncu1atus (B193) 27.24 2.120 0.078
L. tenuis,(B109) 27.42 1.860 0.068
L. japonicu~ (B129) 28.28 1.865 0.066
L. a1pinus (B77) 29.45 1.965 0.067
L. fi1icau1is (B37) 30.02 1.968 0.066
L. schoe11eri (B166) 30.56 1.955 0.062
L. borbasii (B255) 30.99 1.970 0.064
L. kry10vii (B86) 34.60 1.970 0.057
0\ 00 .JO 69 ..
. ! . o 25 70 ... 20 ) 15 L.coimbrensis
10
5
1.0 2.0 3.0
-...... g ::t z: 20 ... 15 0 , 10 l.alpinus (B 324) . J II&: M 5 ID == =z: 1.0 2.0 3.0
...... 1"'- ~.- .------
15 10 L. tenui s
5
1.0 2.0 3.0 -'---- '------DNAIN ARBITRARY UNITS Figure 15.
Histograms of distributions of DNA amounts in 2C nuc1ei of
dip10id Lotus species
(
,. \J '20 72. 15 L. japonicus ) 10
5
1.0 2.0 3.0
-0 ___ ~. ______• ___ ---._.- -. -_ .. -' .. 25 20
15 L.schoelleri ,,'. 10 -... 1 .." 5 c.:» = z: ,1.0 2~0 3.0 .... c "--" ) 25 !ICII::.... m L.alpinus ( B 77 ) ::::1!5 = . z: 10
5 :. ~'..
1.0~ 2.0 3.0
-- .. ~. ~ .- -- --_ ... , .
L.filicaulis
1.0 2.0 3.0
DNA IN ARBITRARY UNITS Figure 16.
Histograms of distributions of DNA amounts in 2C nuc1ei of
dip10id Lotus species
) L.borbasii ) 10
5
1.0 2.0 3.0
\--_-:...... _----_._._- -_._------'- r .
25 -....lioI U 20 =» z L.kryloYÎÎ
&.1. Ct 10
1.0 2.0 3.0
... - ._--_.. -- .-' ...._._------.-.. _-_._-_.-- ---". 20
15 :: ~; , '. L. pedu ne ulat us
5
1.0 2.0 3.0
1 .D N A IN' ARBITRARY UIITS . \ '1... 1 1 1 i ()
Figure 17.
Histograms of distributions of DNA amounts in 2e nuclei of Lotus hybrids ()
) :76.•
) . --.-._------._.. ." .__ ._-
20
15 L.alpinus X 10 L.filicaulis
5
-... 1.0 2.0 3.0 g = z r------··
... 1 ct 1 .l~ L.japo~icus X 10 L.filicaulis 5
1.0 2.0 3.0
L.alpinus X L. schoell er i , • ! 5
1.0 2.0 3.0
L
DRA IN ARBITRARY UNITS Figure 18.
Histograms of distributions of DNA amounts in 2e nuclei of Lotus hybrids (J
) ?d
1 78. 1, 1 i - ) 1 i
L.alpinus X L.kryIOYi~
2.0 3.0
L. filicaulis X . L.séh·oelleri
2.0 3.0
------_._--_.•....• _._-•..
10 L.kryloYii X L.s,choelleri 5
D N AIN A R'8 1T RA RY UNI T S
.. ; ()
Figure 19.
Histograms of distributions of DNA amounts in 2C nuclei of
Lotus hybrids
) 80 ..
)
15
10 L.japonicus X - L.alpinüs 5
l' 1.0 2.0 3.0 ._------_. - ---
L.alpinus . X . L~ japonicus
) 3.0
L.japonicus X L.-krylovii
1.0 2.0 3.0
DNA IN ARBITRARY UNITS
) Figure 20.
Histograms of distributions of DNA amounts in 2C nuclei of
Lotus hybrids .82. Figure 21. Histograms of distributions of DNA amounts estimated in 2C nuc1ei of amphidip10ids
(L. kry10vii X L. tenuis) 2
(L. kry10vii X L. schoe11eri) 2 o
(L. japonicus X L. a1pinus) 2
,) 84.
! '1 ) 1 1
2.0
D N A IN ARBITRARY ONITS' Figure 22. Histograms of distributions of DNA amounts estimated in 2e nuclei of amphidiploids
2 (L. japonicus X L. filicaulis) 1
(L. japonicus X L. schoelleri) 2 ( .~
2 (L. krylovii X L. filicaulis)
,) 86.
)
2.0 ...-,., u =z ... Q ) ,.,AI: CG == 2.0 3.0. 6.0 =z
. 2.0 5.0
DNA IN ARBITRARY UNITS î 87.
-) DISCUSSION
A. Karyotype studies:
When the karyotypes and idiograms of the nine diploid taxa of
the L. corniculatus group, namely, L. alpinus (B77 and B324) , L.
borbasii, L. filicaulis, L. japonicus, L. krylovii, L. pedunculatus,
L. schoelleri, L. tenuis, are compared·~ the s.imilari ties in chromosome
morphology stand out more than their differences. In general, on
the basis of this karyological evidence, as weIl as the morphological
evidence (Somaroo and Grant, 1971)~ the author considers that it is
correct to maintain aIl these diploid species in the same taxonomie
group.
The analysis of the karyotypes showed that L. krylovii has the ) longest total complement length (TeL) of the diploid species in the L. corniculatus group. This would confirm the report of Zandstra
and Grant (1968) who showed that L. krylovii had the longest total
chromosome complement of the three species (L. krylovii, L. pedunculatus,
~. tenuis) that they studied in the L. corniculatus group. In this
study, the most obvious difference between the ~ryotypes of the
different species was for L. pedunculatus which possessed two pairs
of chromosomes with prominent satellites; nô SAT chromosomes were
found in any of the other species. Lotus pedunculatus was also studied
by Zandstra and Grant (1968) and there is good agreement in the
chromosome lengths (% TCL) of the idiogram presented by those authors
and the one drawn by the present author.
Seeds of accession numbers B77 and B324 were received from different 88.
) botanists as Lotus alpinus. There were certain obvious morphological
differences noted in flower·coloration and general growth habit which
differentiated the plants of these two accessions and hence, they were
treated as separate entries to de termine if there was any difference
between their karyotypes. From the chromosome analysis it was obvious
that the karyotypes belonged to two different gr.oups; B324 has aIl
submedian chromosomes, whereas B77 has one pair of chromosomes which
are median with the remainder submedian. However, in a comparison
of their total complement len~ths B324 has a'TeL of 24.27 p, whereas
the TeL of B77 .. is 29.45 p. These figures were statistically significant-
ly different. The cytophotometric analysis of the nuclear DNA of
these two accessions, likewise, showed there is considerable difference
between them which, likewise, was statistically significant. There-
fore, from the knowledge of the karyotype and DNA content differences,
the author considers that these two accessions do not belong to the
same species. In correspondence with Professor W. S. Edgecombe, of
the American University of Beirut, who collected the·seed of accession
B324, she believes after further examination of this material and a
survey of the tax~nomic literature, that the correct name for accession
B324 is Lotus·corniculatus var. brachyodon Boiss. and that this taxon
was incorrectly made synonymous with L. alpinus Schieich. Since L.
corniculatus has a soma tic chromosome number of 24, whereas B324 has
one of 12, it would ap~ear that B324 represents another new diploid taxon.
Thus, from the present study it is obvious that the accessions B77 and
B324 represent different taxa and cannot both be classified as L. alpinus. 82.
One dip10id species, L. coimbrensis, was chosen for study as it
be10nged to another section of the genus Lotus, name1y, the L. aegeus
group (Brand, 1898), which made it possible to compare this species
with those of the L. cornicu1atus group. The karyotype of L. coimbrensis
differs marked1y from those species of the L. cornicu1atus group in
that it possesses two pairs of long chromosomes which have the highest
arm ratios (Table III). From a comparison of the idiograms of the
different species, there are fair1y extensive differences in the
shape and relative size between the chromosomes of L. coimbrensis
and the otQer nine dip10id species of the L. cornicu1atus group.
Therefore, from ~his information we might conc1udethat whi1e there
is relative uniformity in karyotypes of the species within the L.
cQrnicu1atus group, this does not app1y to a11 the species of the ) genus. We might expect differences to exist between other 1ess
c1ose1y re1ated species, ~nd therefore, the karyotypes within the
genus probab1y'show much more variation than exhibited by the species
within the L. cornicu1atus group.
Within a species there was considerable variation in 1engths of
the individua1 chromosomes which ref1ected in the variation of total
complement 1engths. Although the ce11s ana1yzed were a11 in the
metaphase stage there was a considerable difference in contraction of
the chromosomes at this stage which cou1d account for the observed
variation in 1ength. Since the chromosomes of Lotus species are
sma11 with a tendency for sticking together, the writer suggests that
a greater number of metaphases be studied, a1though it is considered ) that the overall results may not differ from a smaller number as in this study carefully analyzed.
B. Cytophotometric measurements--Nuclear DNA content:
1. L. corniculatus, the diploids and hybrids
The cytophot~metric measurements of the nuclear DNA content of the
three different accession numbers of the tetraploid species, L. corniculatus
show that they are essentially the same. Since the accessions are from
different origins one might assume that there is little variation as
far as DNA content is concerned in L. corniculatus. The morphological
variability which is exhibited by different cultivars of this species
are probably genic in origin rather than due to any gross chromosomal
differences.
In regards to, the nuclear DNA content of the diploid species, the
cytophotometric study showed that although the species have the same
chromosome number their nuclear DNA values were not constant and they
varied in relative amounts from 1.75 units for the species with the
lowest nuclear DNA value to 2.12 units for the one with the highest
DNA value (table VI).
Other invest~gators have shown that species which have the same
chromosome number in some genera of both plants and animaIs also differ
significantly between' species in their nuclear DNA values, for example,
, ' ' species of Bufo (Ullerich, 1966) and Vicia (Martin 'and Shanks, 1966).
Rees et al. (1966) in a cytophotometric study of different species in
the genera Lathyrus, Vicia and Lolium, have shown that there are
differences of a similar magnitude between diploid species of Lathyrus, ) 91.
) a sevenfo1d difference between dip10id Vicia species and a similar but
sma11er variation between species of Lolium.
These authors suggested that a large change in chromosome size on
the dip10id 1eve1 wou1d resu1t in qualitative differences in nuc1ear
DNA.va1ues between species in which no polyp1oidy occurs, but at the
same time some of the "extra" DNA might be genetica11y "uninformative".
In the dip10id species of Chrysanthemum diffefences in nuc1ear DNA
values between the species have been shown to be associated with
chromosome 1ength, the cross-sectiona1 area, and the volume of the
chromosomes (Dowrick and Bayoumi, 1969). These authors found,that it
was not a1ways possible to corre1ate a change in'nuc1ear DNA values
direct1y to a change in chromosome size.
.... Whi1e thekaryotype of L. coimbrensis of the L~ aegeus section of' ) the genus Lotus was considerab1y different from those species of the
L. cornicu1atus group, the cytophotometric analysis showed that the
nuc1ear DNA value for this species was not significantly different
from many of the species of the L. cornicu1atus group. This non-
significant difference for L. coimbrensis might be attributed to
the 1imit of reso1ution of the technique used, however, it wou1d be
more like1y that the total amount of DNA present in the chromosomes
of this species'is comparable to that of species in the L. cornicu1atus
group and that the morpho1ogica1 changes as exhibited by the differences
in karyotypes is the resu1t of gross structural repatterning of the
chromosomes. .
The Iluclear DNA values for the Lotus hybrids in some cases was ) 92.
) slight1y higher than that for either parent and in other cases lower.
A few of the values approached that of one parent, but in general,
there was not a strictly additive re1ationship (Tables VI, VIII).
Whi1e the mean DNA values for the hybrids ranged from 1.825 to 2.255
units, the statistica1 ana1ysis indicated these differences these
differences to be non significant (Table IX).
2. Amphidip10ids
AlI the amphidip10ids arose directly from the treatment of the
hybrids with colchicine. Therefore, an additive and doub1ing re1ation-
ship for the nuc1ear DNA content might be expected between a given
amphidiploid and its parental species. On thisbasis the nuc1ear . 2 DNA value for the amphidip10id (L. kry10vii X L. schoe11eri) wou1d
be (L. krylovii, 1.970 + L. schoe11eri, 1.955, X 2) 3.93 units and
for the amphidip10id (L. japonicus X L. a1pinus)2 (1.865 + 1.965 X 2)
3.83 units (Tables VI, VIII, X). By doub1ing the observed value for
the hybrid L. krylovii X L. schoel1eri a value of 3.97 is obtained and
the actua1 value obtained for this amphidip10id was 3.85. In the case
of the hybrid L. japonicus X L. a1pinus, the ob~erved value when
doJb1ed is 3.91 and the ,value found for the amphidiploid was 3.85
(Tables VIII, ~). These findings are very c1ose'to the expected values'.
Therefore, it wou1d appear that hybridization and the subsequent
doub1ing of the chromosomes have not caused any appreciab1e change
in the amount of DNA per genome. Simi1ar resu1ts have been reported
in the Graminae for species of Triticum CRees and Walter, 1965) and
Avena (Yang and Dodson, 1970). 93.
) An exception to the above where there is a close re1ationship in the expected nuc1ear DNA values between parents, hybrid and the amphi-
dip10id was observed for the amphidip10id (L. kry10vii X L. tenuis) 2 •
The DNA value found for this amphidip10id was 3.55 units whereas that
ca1cu1ated for their parental species when added anddoub1ed wou1d be
3.83 units. This lower value for the amphidip10id might be accounted
for by experimenta1 error or technique, however, this is not considered
1ike1y in view of the other resu1ts.
C. The re1ationship between chromosome 1ength and DNA content of the
diploid species
Bhaskaran and Swaminathan (1960) in a cytophotometric study of
dip1oid, tetrap10id and hexap10id species of Triticum compared total
complement 1ength (TCL) at soma tic metaphase with nuc1ear DNA content
and found that the DNA content per unit 1ength of chromosome was
constant. When this re1ationship was considered for the Lotus species
(Table XII), it was found to ho1d true for most species with one major
discrepancy, name1y, for L. peduncu1atus. This species had the second
shortest TCL but had a much higher DNA value in relation to the total
complement 1ength. A simi1ar case has been reported by Niru1a et al.
(1961) for Solanum nitidum where they showed that this species with
the shortesf teL had a higher nuc1ear DNA content than the other species.
A question immediate1y arises as to whether there was some technica1
error in determining the nuc1ear DNA density value and in the'determination 94.
) of the chromosome measurements for L. pedunculatus. Since a recheck
of the karyotype drawings and chro~osome measurements showed no errors
and the techniques were maintained uniformily throughout the study,
the discrepancy in the results do not appear to be of a technical nature. Since the nuclear DNA' content for the species L. alpinus, L. borbasii,
L. filicaulis, L. j aponicus, L. krylovii and L. tenuis is direc tly propo.r
tional to the ratio of the total chromosome length, it may be speculated
that the variation between chromosome length and DNA content may be
explained by a ~asic change in chromosome structure.
D. The basic change in chromosome structure
Nuclear DNA measurements are of inte~est because of inferences they
may allow regarding chromosome structure. There are two hypotheses to
account for the variations of nuclear DNA among diploid species with
the same chromosome number. The first concerns extensive longitudinal
repetitions or accumulation of chromosomal units, that is, an increase
or decrease in chromosome length by duplications or deletions (Gall, 1963;
Keyl, 1965; Rees and Jones, 1967b). The second hypothesis is called
the total multiplicity hypothesis where the whole chromosome increases
in diameter and is referred to as "lateral mult1plicity" and assumes
the chromosome to be multistranded (Christensen, 1966; Martin and Shanks,
1966; Rothfels, et al., 1966; Uhl, 1965; Schrader and Hughes-Schrader,
1956, 1958). Wi~h longitudinal repeats, DNA values would be expected
to be more or less continuous in nature, whereas with chromosome poly
nemy, DNA values would be expected to be geometric in nature or at best 95.
estab1ish a quantized series of DNA values.
In the case of the striking differences in DNA content between the dip10id Lotus species which are associated with differences in chromosome
1ength (Table XII), it is considered that for most of the species longitudinal differentiation of the chromosomes (duplications, de1etions, translocations, etc.) wou1d account for the differences in DNA content between the species.
Lima-de-Faria ,(1959) has shown that heterochromatic chromosome segments may conta in two or three times more DNA per unit area than euchromatin and that no regu1ar re1ationshipbetween DNA content and chromosome 1ength may be obtained for species containing varying amounts of heterochromatin. However, Stebbins (1966) suggested that a positive corre1ation'between chromosome size and DNA content cou1d probab1y be re1ated to changes in the amounts ofheterochromatin. It is not certain,wh4t part heterochromatin p1ays in Lotus chromosomes as there has been no visible evidence of heterochromatin in Lotus chromosomes 'in this study nor has there been any report in the 1iterature of any heterochromatic regions in chromosomes ,for any species of Lotus.
E. Tracing the ancestor of the tetrap10id species, Lotus cornicu1atus
Of the many different methods used to determine the re1ationship of species, cytophotometry in which the nuc1ear DNA content may be compared between species, offers a usefu1 approach (Hughes-Schrader,
1958). In the wheats, cytophotometry provided new evidence on the dip10id source of' genomes comprising the cu1tivated po1yp1oid wheats 2.6."...
J (Rees, 1963; Rees and Walters, 1965). Since only a few amphidiploids were available for analysis, the
nine diploid species and the twelve hybrids provided the main data for
analyzing the relationship between the diploid species and the cultivated
tetraploid, L. corniculatus. From a comparison of the DNA values between
the diploids it would appear that L. schoelleri, L. alpinus (B77), L.
filicaulis, L. borbasii, and L. krylovii are more closely related to
the cultivated species than the other diploids (L. pedunculatus, L.
japonicus, L. tenuis, L. alpinus B324). If the DNA value for a diploid
species is doubled in order to make the value equivalent to a tetra
ploid species, then those diploids which would have'DNA values close
to that of L. corniculatus (DNA value 4.000) are L. krylovii (3.910),
L. alpinus (B77) (3.930), L. filicaulis (3.936), L. borbasii (3.940), ) and L. krylovii (3.940). These results would lend support to the
authors Chrtkova-Zertova (1970) and Favarger (1953) who have considered
L. krylovii and L. alpinus, respectively, as putative diploid ancestors
of L. corn1culatus on the basis of morphological evidence. Four of
the 12 hybrids had a DNA value (when doubled) which was close to that
of L. corn1culatus. These were the hybrids L. japonicus X L. alpinus
and its reciprocal L. alpinus X L. japonicus, L. japonicus X L. krylovii
and L. krylovii X L. tenuis with 2C DNA values of 1.9555, 1.960, 1.970,
and 1.975, respectively. Two of the parental species involved in
these crosses, are L. alpinus and L. japonicus. These species have
been considered by Somaroo (1970) to be progenitors of L. corniculatus on the basis of cytogenetic evidence. These data on nuclear DNA values 97.
) for the hybrids wou1d support such a view; however, considering the nuc1ear DNA value for L. japonicus, this species was.not one of the
dip10ids which was most c1ose1y re1ated to L .. cornicu1atus on this
basis.
There are, of course, precautions to be aware of in app1ying this
kind of data to the ana1ysis of species re1ationships both of a technica1
and factua1 nature. The technica1 precision is assumed to be carried
out uniformi1y for a11 materia1. Caution was used to fix, and prepare
materia1 to e1iminate any variation in staining by the Feu1gen procedure
since this wou1d be one source of error. In the ana1ysis of the resu1ts,
in certain cases differences were found between slides within the same
species which might be accounted ~or by variation in staining or
possib1y machine error, since one must wait for a. certain period for
the photomu1tip1iers to become stabi1ized in the integrating micro-
densitometer. It shou1d a1so be noted that nuc1ear DNA comparisons
between species is on1y one method for considering the re1ationship of
species.
From the ana1ysis of the karyotypes of the different species, it
is considered that L. aipinus (B77) and L. borbasii have cyto1ogica1
features in common with L. cornicu1atus. Both L. a1pinus (B77) and
L. borbasii have five pairs of chromosomes which are submedian and
one pair, the.shortest, that is median. Lotus cornicu1atus has 10
pairs of submedian chromosomes and the two shortest pairs of chromosomes
are median. Therefore, we cou1d specu1ate that the tetrap10id L.
cornicu1atus cou1d have arisen either as an autopo1yp1oid from either 98.
) of these two species or by means of allopolyploidy in which these
two species had initially hybridized. The karyotypes of the other
diploid species had features which distinguished them from L.
corniculatus. For example, the chromosomes of L. japonic~s aIl had
submedian centromeres. It is possible, however, that during the
evolutionary development of both these diploid species, and also
L. corniculatus, that chromosomal differentiation may have occurred
so that their chromosome structure could have radically diverged.
In considering both the DNA and karyotype data, in the author's
opinion, L. alpinus (B77) and L. borbasii may be considered putative
progenitors of L. corniculatus but whether the latter arose as an
autotetraploid, or as an allopolyploid, is not clear from the available
evidence from this study alone.
Further cytophotometric comparisons on the nuclear DNA content of
autotetraploid L. alpinus (4x), L. borbasii (4x) and the other species
of this group, and the amphidiploids and backcrosses with L. corniculatus
should be investigated. Once aIl these data have been obtained, it
might be possible to come to a final conclusion on the origin of this
tetraploid. ) SUMMARY
1. An ana1ysis of chromosome morpho10gy and Feu1gen cytophotometric
measurements of the nuc1ear deoxyribonuc1eic acid (DNA) content of a
number of dip10id species in the Lotus cornicu1atus group (L. a1pinus
Sch1eich., L. borbasii Ujh1eyi, L. fi1icau1is Dur., L. japonicus
(Rege1) ~arsen, L. kry10vii Schischk. and Serg., L. peduncu1atus Cav.,
L. schoe11eri Schweinf., L. tenuis Wa1dst. et Kit.) and one species,
L. coimbrensis Wi11d., be10nging to the L. aegeus group, was carried
out in order to determine the variation in chromosome morpho10gy in
the genus Lotus, and to determine if these techniques wou1d throw
further 1ight on the origin of the cu1tivated tetrap10id species, L.
cornicu1atus. In addition to the above species, the nuc1ear DNA .., ~ .. contents of a number of dip10id hybrids and amphidip10ids were a1so ../ determined.
2. Drawings were made of the karyotypes of each of the dip10id species
and the tetrap10id, L. cornicu1atus. The total complement 1engths and
long arm to short arm ratios were ca1cu1ated from the karyotype measure-
ments and statistica11y ana1yzed by means of an IBM computer. Idio-
grams were prepared for each species from the chromosome measurements.
Representative photographs of the karyotypes for each species were a1so
taken.
3. A comparison of the chromosome morpho10gy, inc1uding the total
complement 1ength and long arm to short arm ratios, for the six pairs
) 100.
of chromosomes of the diploid species of the L. corniculatus group
with the six pairs of chromosomes of L. coimbrensis of the L. aegeus
group showed that there were considerably more similarities among the
karyotypes for the species within the L. corniculatus than there were
between the species of the two groups. There were fairly exte~sive
differences in shape and relative size of the chromosomes·of L. coimbrensis
in comparison to those of the L. corniculatus group. Thus, karyotypic
variability would appear to exist between the different sections of the
genus Lotus.
4. Lotus pedunculatus was the only species of the L. corniculatus group
in.which chromosomes bearing satellites were observed.
s. The karyotypes and nuclear DRA values were considerably different for plants of the two collections received as L. alpinus (B77, B324).
One of these accessions (B324), although diploid corresponds to the , taxonomie description of L. corniculatus var. brachyodon Boiss.
6. Cytophotometric determinations of Feulgen absorption of nuclear DNA
contents of the species shoved, in general, that the species differed
in their DNA value from one another.
7. In general, total chromosome complement lengths (TCL) of the karyo-
types were positively correlated with nuclear DNA content. An exception was L. pedunculatus which bad the shortest teL but had a much higher
nuclear DNA value than expected. Such differences in correlation of
TCL and nuclear DNA values may be accounted for by differences in lQl~
) chromosome structure.
8. Nuclear DNA values for the amphidiploids had an additive and
doubling relationship of the parental species, but this relationship
was not as clear cut for the diploid hybrids. DNA values between
hybrids were not significantly different but were for the amphidiploids.
9. In"considering the karyotypes of the diploid species and L.
corniculatus along with the nuclear DNA values of these taxa, L.
alpinus (B77) and L. borbasii would qualify as putative species from
which the tetraploid, L. corniculatus, may have arisen.
)
) 102.
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