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.