an evolutionary study of THE FERN ASPLKNIUM TUICHQMAHES. J. 1). LOVIS. -t Thesis submitted for the Degree of Doctor of Philosophy in the University of Leeds. Department of Botany 1958. VOLUME I : TEXT CONTENTS. Page Numbers ABSTRACT............................................... 1 FOREWORD............................................... INTRODUCTION.......................................... General Introduction........................... 7 Sources of live material....................... Taxonomic limits of the species aggregate.... GEOGRAPHICAL DISTRIBUTION........................... 11 Methods of investigation....................... Distribution of the species aggregate........ 13 Distribution of the individual cytotypeS.... Diploid oytotype.......................... li: Tetraploid cytotype....................... 18 Hexaploid cytotype........................ 2o AUTECOLOGY............................................. o A Introduction.................................... The special problem of the garaetophyte generation. The characteristics of the rock-crevice habitat... Autecology of the diploid cytotype........... go British Isles.............................. O C Austral..................................... i 8 Elsewhere.................................. „,r t * • Paise Numbers AUTECOLOGY (continued) Autecology of tue tetraploid cytotype...... 39 British Isles.......................... .... 43 Australia........................... 48 Elsewhere........................... Autecology of tiie hexaploid cytotype....... 5 8 COMPARATIVE MORPHOLOGY............................ 59 Qualitative differences..................... 5 9 Pinna shape.............................. ...... 60 Rhizome scale................... 63 Spore sculpture......................... * g Quantitative differences.................... Pinna size and pinna separation....... Spore size........................... 69 Annulus of the sporangium.............. 74 Stomata............................... Synopsis of morphological differences between the cytotypes........... 76 GENETICAL RELATIONSHIPS........................... Culture and hybridisation techniques....... Cultivation of adult sporophytes..... Germination and culture of gametophytes. 80 OO Hybridisation technique............... GENETICAL RELATIONSHIPS (continued) Page Numbers Cytogenetic investigation of the relationships of the cytotypes of As;;lenium trlchomanes s.l.. 85 Cytological and photographic techniques... 85 Cytology of the cytotypes................ 87 Experiments designed to test the hypothesis of autopolyploid origin of the tetraploid ___ 89 cytotype............................ Synthesis of hybrids between As j'lonium adulterinum and cytotypes of A.trichouanes, 94 with analysis of ........................ Synthesis of hybrids between cytotypes of As plenium t.ri chomanes, and analysis of meiosis.,100 Diploid x tetraploid1 ’H ................ ... 1^0 Diploid x hexaploid, .............. ..... 106 Tetraploid x hexaploid................. 107 ___ 107 Diploid x diploid................ 110 DISCUSSION AND CONCLUSIONS........................... Geographical distribution and ecology......... .... Ill Discussion......................... ...... 123 Conclusions........................... Genetical relationships of the three cytotypes. 125 .... 128 Discussion.......................... ........ 148 C onelusions........................... DISCUSSION AND CONCLUSIONS (continued) Page Numbers. Taxonomic conclusions....................... 152 SUMMARY............................................ 164 REFERENCES......................................... 168 APPENDICES......................................... 1. Diploid cytotype : List of chromosome counts..169 II. Diploid cytotype : Important determinations of herbarium material... 174 III. Tetraploid cytotype : List of chromosome counts. 178 IV. Tetraploid cytotype : Important determinations of herbarium material.... 188 V. Hexaploid cytotype : List of chromosome counts.. 193 SUPPLEMENT : ASPLENIUM ADULTERI................... 208 ACKNOWLEDGMENTS.................................... ABSTRACT. The aggregate species Asplenium trichom_ines includes three different cytotypes:- a diploid with n = 36 chromosomes, a tetraploid with n = 72, and a hexaploid with n = 108. Although the diploid and tetraploid cytotypes are widely though discontinuously distributed in both Northern and Southern Hemispheres, the hexaploid is known only from New Zealand. All three plants are distinguishable on morphological characters, and possess distinctive ecological preferences. Cytogenetic analysis of a series of artificial hybrids between different representatives of the cytotypes obtained from various parts of the world has demonstrated the close genetical inter-relationship of the entire species complex, and given information con­ cerning the nature of relationships existing between and within the three cytotypes. FOREWORD. Tlae bulk of the work described in this tnesis was carried out in the Department of Botany of the University of Leeds between July 1952 and June 1955, while homing a Nature Conservancy Research Studentship. Towards the end of this period, it became apparent that a study of the forms of As),leniua triouomunes present in Australia and New Zealand was crucial for • +• thp nroblem. Fortunately, an opportunity elucidation of the prooxeia. i o v in New Zealand, and xn occurred for personal study in new Via tjAT'fi soent at Canterbury consequence twelve months University College,n it i* o Lnrisucp hr i a tchurch, , New Zealand, from August 1955 to August 1956, while holding a New Zealand National Research Fellowship. It proved possible to make a brief visit to Victoria and New South Wales on the way out to New Zealand, and a longer stay of six weeks in Australia on the way back, in order to study the forms of Asolenium tricnomanes present in Australia in their native habitat. On return to the United Kingdom, the investigation was continued at Leeds, and is still in progress. INTRODUCTION, GENERAL INTRODUCTION. Prior to the commencement of this investigation, it had already been discovered by Wanton (22), that two cytological forms of Asrlonium trichomanes L existed in Britain. One form or cytotype* is a diploid, with n = 36 chromosomes, while the other, evidently much the commoner plant,is a tetraploid witn n = 72 chromosomes. The diploid had only been found at Aber Falls, in Caernarvonshire, and this one example was seen to be clearly morphologically very much alike the more common tetraploid cytotype, which had been determined from several localities scattered through the country. It was also known that both of these forms occurred on the continent of Europe, although again the diploid had only been found once, in the Auvergne in France. ♦Footnote. ovtotype ls used „ere and throughout this series is based. The term is not used in its restricted sense according to the definition of Valentine (34). diploid, tetraploid, and hexaploid chromosome races of Asulenium trichomanes s.l. are thus describe_____arif1 cvtotypes, although they have distinctive g e o g r a p --- ecological distributions, and according to the ^ e g o r ies or Valentine (34), would almost certainly correspond to diutriet coenospecies. (See also Clausen et al., 5J* . tl definition, use of the term coenospecies, would imply the demonstration that no gene exchange caH"occur between the^ three chromosome races, although in actuality . r only been established for these plants with a certain de^re of probability. The use of the term cytotype, in its less restricted sense has therefore been preferred. It was clearly very desirable that an effort should be made to find out more about the rarer diploid cytotype of Asplenium trichomanes, and especially about its relationship with the tetraploid, in view of their morphological similarity. Indeed the two cytotypes were evidently so nmeU alike in appearance that it seemed possible that the tetraploid was an autotetraploid derived from the apparently rarer diploid. Since no example of autopolyploidy had been demonstrated for any fern in a state of nature, it .as quite clear that a cytogenetical investigation of these two cytotypes ought to be undertaken, in order either to confirm or disprove the hypothesis of autopolyploidy in this case. Early in the investigation it was realised that the study would haveflimited value and interest if confined to European material, especially since As|;lenium tricao«m>g& has an extremely wide distribution throughout the world. Indeed, there is no other fern, unless it be Cvstopteris fragilis s.l., which has such a comprehensive range in the temperate regions of the world as has As|,lenium trichomanes. The possibility existed that by extending the investigation tc extra-European material, results of considerable evolutionary and phytogeographic importance might emerge from the work. Accordingly, efforts were made tc obtain material from various parts of the world, mainly from correspondents overseas. Fortunately, these efforts were largely successful, and it eventually became possible to consider the study as an investigation into the structure of the Asulenium trichomanes species complex over its entire distributional range. The investigation had been under way for some eighteen months when news was received from Mr. G. Brownlie, of Christchurch, New Zealand, of his discovery of the existence of a hexaploid cytotype of Asplenium trichomanes in New Zealand. Owing to his kind co-operation, material of this plant was subsequently included in this