SOME CYTOTAXONOMIC PROBLEMS IN THE FERN GENERA ASPLENIUM AND POLYSTICHUM. A Thesis submitted to the University of Leeds for the Degree of Doctor of Philosophy by ANNE SLEEP. Department of Botany, University of Leeds. July 1966. TABLE OF CONTENTS. Page. GENERAL INTRODUCTION ............................................. 1 METHODS ............................................................. 3 1. Collection of Material ...................................... 3 2. Cultural and Hybridisation Techniques ..................... 4 A. Cultivation of Adult Sporophytes: (i) Asplenium......................................... 4 (ii) Polystichum....................................... 5 B. Germination and Culture of Gametophytes .............. 6 C. Hybridisation Techniques ............................... 10 D. Cultivation of Young Sporophytes ...................... 12 3. Cytological Techniques: A. Meiotic Preparations ................................... 14 B. Mitotic Preparations: (i) Root tip sections ................................ 15 (ii) Root tip squashes ................................ 15 4. Spore and Scale Preparations ............................... 16 5. Photographic Techniques: A. Chrcmosome Photographs................................. 17 B. Chromosome Diagrams .................................... 18 C. Silhouettes: (i) Asplenium ......... ................................ 18 (ii) Polystichum....................................... 19 6. The Use of Colchicine ...................................... 20 Experimental data are given in Table I .............. 21 7. The Attempted Induction of Apogamy: A. Horticultural Techniques .............................. 21 B; Sterile Culture Methods ............................... 22 FART I. ASPLENIUM. P a g e . CHAPTER I. INTRODUCTION ................................... ....... 24 CHAPTER II. SOURCES OF MATERIAL................................... 26 Provenance of all material used is given in Table II .... ....... 26 CHAPTER III. THE SPECIES .................................... ....... 29 SECTION A. Cytology......................................... ....... 29 Chromosome counts listed in Table III ..................... ....... 29 SECTION B. Taxonomy, Distribution and Ecology ........... ....... 30 Table IV. Comparative morphology of members of the A. forisiense complex......................... 31 1. A. fontanum (L.) Bernh............................. ....... 38 2. The A. obovatum group: (a) A. obovatum Viv ............................... ....... 43 (b) A. billotii F. Schulze ...................... ....... 46 3« A. forisiense Le Grand................................... 51 4. A. macedonicum Kiimmerle .......................... ....... 56 5* A. ma.joricum Litard. ........... .................. ....... 68 6. A. petrarchae (Guerin) D.C ........................ ....... 69 CHAPTER IV. THE HYBRIDS..................................... 74 SECTION A. The Hybridisation Programme ................... 74 A list of the crosses attempted is given in Table V ..... 74 Table VI. List of successfully synthesised hybrids ..... 75 SECTION B. Results obtained ............................... 76 1. Hybrids within the A, forisiense complex........ 77 2. Hybrids involving A. ma.joricum ................... 97 3. Hybrids involving A, petrarchae .................. 113 4. Other hybrids involving A. billotii ............. 119 Page. CHAPTER V. THE INTERPRETATION OF THE CYTOGENETIC EVIDENCE 121 1. The A. forisiense complex ........................ ...... 121 2. A. ma.joricum....................................... ...... 125 3. Cytogenetic affinities of other tetraploids investigated with evidence of autoploidy in two European Asplenium species ................... ...... 130 CHAPTER VI. TAXONCMIC IMPLICATIONS ........................ ......136 PART II. POLYSTICHUM. CHAPTER VII. INTRODUCTION ................................... ...... 139 P. aculeatum ........................................... ......140 P. braunii ............................................. ...... 142 CHAPTER VIII. THE TAKA INVESTIGATED.......................... ...... 149 SECTION A. P. braunii...................................... ...... 149 T a x o n o m y ............................................... ...... 149 Distribution ........................................... ...... 151 E c o l o g y .............. .................................. ......154 SECTION B. Wild hybrids ................................... ...... I64 SECTION C. American species: P. munitum and P. acrostichoides .................... ....... 169 P. andersoni ........................................... ...... 1'70 CHAPTER IX. EXPERIMENTAL DATA.............................. 172 SECTION A. The Species: Cytology......................... 172 Chromosome counts listed in Table VII ..................... 172 SECTION B. The Hybrids..................................... 177 1. The Hybridisation Programme ...................... 177 Table VIII. List of successfully synthesised hybrids .... 179 2. Results obtained................................... 182 Cytology of synthesised hybrids .................. 182 Table IX. Cytology of wild hybrids ....................... 196 Page. CHAPTER X. DISCUSSION OP THE CYTOLOGICAL RESULTS ........... 199 Table X. Polystichum hybrids between supposedly unrelated species and the chromosome pairing they show .... 201 GENERAL DISCUSSION ................................................. 206 Table XI. Records of doubled gamete formation .............. 207 Table XII. Meiotic behaviour of diploid hybrids in European Asplenium ................................. 210 SUMMARY............................................................. 214 REFERENCES ......................................................... 218 APPENDICES: Asplenium: I. Additional locality details of species successfully incorporated into hybrids ...... 226 II. Hybridisation attempts: the numerical data .. 231 Polystichum: III. Provenance of species used in the present investigation ................................. 235 IV. Cytologically-examined wild hybrids in cultivation ................................... 239 V. Numerical data relating to the hybridisation attempts ....................................... 240 SUPPLEMENTS TO POLYSTICHUM: SUPPLEMENT I. A. Chromosome counts of some Japanese Polystichum species ........................... 246 B. Other Polystichum species counted during the course of the present investigation ......... 247 SUPPLEMENT II. Some further hybrids within the genus Polystichum ................................... 248 ACKNOWLEDGEMENTS ................................................... 252 LIST OF FIGURES. PART I. ASPLENIUM. Figure N o . Page N o . DISTRIBUTION MAPS: A. obovatum and A. billotii .................. ...... 3 44 A. fontanum, A. forisiense and A. macedonicum 6 61 A. petrarchae and A. ma.joricum.............. 19 73 SILHOUETTES OF SPECIES: A. fontanum ................................... ...... 1 36-37 A. obovatum ................................... ...... 2 41 A. billotii ................................... ......2a 42 A. forisiense ................................. ...... 4 49-50 A. macedonicum ................................ ...... 3 54-55 A. matjoricum .................................. 14 67 A. petrarchae ................................. 13 66 HABITAT PHOTOGRAPHS: A. forisiense ................................. Frontispiece 23 The A. forisiense complex .................... 7-12 62-65 A. ma.joricum.................................. 15-18 71-72 SILHOUETTES OF HYBRIDS: A. forisiense complex ........................ 20-28 83-95 Hybrids involving A. ma.joricum.............. 32-39 101-111 Hybrids involving A. petrarchae ............. 40-41 115-116 CYTOLOGY OF HYBRIDS: I A. forisiense complex ........................ 20a~31 84-96 Hybrids involving A. ma.joricum.............. 32a-39a 102-112 Hybrid involving A. petrarchae .............. 41a 117 EXPLANATORY DIAGRAMS: A. forisiense complex ........................ 42 124 A. ma.joricum.................................. 43 129 Other tetraploids ............................. 44 134 DIAGRAMMATIC INTERPRETATION OF RESULTS ......... 45 135 FART II. POLYSTICHUM. Figure No. Page No. DISTRIBUTION MAP: P. braunii .................... 47 150 SILHOUETTES OP SPECIES: P. braunii and P. ohmurae ..................... 46 147-148 The Taxa occurring in Britain................. 60 171 American diploids .............................. 59 168 HABITAT PHOTOGRAPHS: P. braunii......................................Frontispiece 138 P. braunii, P. lonchitis and wild hybrids .... 48-57 156-163 SILHOUETTES OF HYBRIDS: Wild hybrids from Floitental, Austria ........ 58 166 Synthesised hybrids ............................ 66 180-181 Individual pinnae of wild hybrids ............ 77 195 P. lonchitis x P. setiferum ................... 79 198 CYTOLOGY OF SPECIES: P. braunii ...................................... 6I-63 174-175 P. ohmurae ...................................... 64 176 CYTOLOGY OP HYBRIDS: Diploid hybrids ................................ 67-70 187-189 Triploid hybrids ............................... 71-75 190-194 Wild tetraploid hybrid ........................ 76 194 HYBRIDISATION POLYGON............................. 65 178 DIAGRAMMATIC SUMMARY OF RESULTS .................. 80 205 GENERAL INTRODUCTION. The basic aim of this research project is to attempt to obtain information concerning the phyletic affinities between species by means of a study of chromosome pairing in synthesised hybrids. Any evidence obtained is supplemented by a consideration of the morphology,