THREE WESTERN SPECIES OF THE GENUS H/BENARIA WILLD. THEIR RELATIONSHIP AND CROSSABILITY. 3Y EMMY H. FISHER B. Com., University of Economics Vienna (Austria), 1922 THIS THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA. In presenting this thesis in partial fulfilment of the requirements f> an advanced degree at the University of British Columbia, I aqree that the Library shall make it freely available for reference and stud/. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department r>r by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia Vancouver 8, Canada ABSTRACT . Relationship and interfertility of Habenaria dilatata (lursh) Hook.,H. hyperborea (L.) R.Br, and H. saccata Greene was studied. Intraspecific and interspecific crosses were made. Chromosome counts of the three species showed 21 pairs of chromosomes in the cells, except for a small green-flowered population from Manning Park with n = 42 which was considered tetraploid and possibly of hybrid origin. These counts agree with earlier ones for the three species. Since creation of the genus Habenaria Willd» these species have been included under tribe Ophrydeae, which now has been changed to Orchideae, subtribe Orchidinae to conform with the rulings of the International Code of Botanical Nomenclature (1959)* In spite of apparently close relation• ships the species maintain their distinctness, even when growing sym- patrically, indicating barriers to outcrossing, or for plants growing in northerly regions, a lack of pollinators. Autogamous tendencies have therefore developed and H. dilatata and H. hyperborea are outcrossing or autogamous when the need arises. H. saccata seems to be self-sterile. Microsporogenesis in the species studied follows that of other orchids. The archesporial cell directly becomes the spore mother cell. All descendants of this cell stay together and divide together, forming a massula or pollen packet. Pollen mitosis results in the 2-nucleate pollen grain which is shed as such. The generative nucleus divides in the pollen tube, producing the 2 sperm nuclei, as the tube enters the ovary. Only the chalazal megaspore is functional. Three simultaneous divisions produce the monosporic, 8-nucleate Polygonum-type embryo sac. Fusion of the polar nuclei is the rule and takes place before fertilization. Triple fusion follows, hut the primary endosperm nucleus begins to degenerate usually before the r-ypote starts to divide. An haustorial susy ensor develops, which does not take part in the construction of the embryo proper. The mature embryo is an un differentiated body of 50-60 cells, suspended in the air-filled ca• vity of the reticulate testa. It takes from 3-4 weeks from polli• nation to saturation of the embryo. Intraspecific crosses were all successful. Interspecific cros• ses produced a higher percentage of seed with well developed embryos in IT. hyperborea x H. saccata crosses than in crosses between H. riil tata and either of the 2 other species. The tetraploid plants were successful both as seed and pollen parents. Regular meiosis would indicate allopolyploid origin. Artificial pollinations showed that gene flow is possible and that artificial crosses are easy to make. In nature isolating mec'r an isms must rrevent the species from losing their identity al• though hybridization may take place under favourable conditions. Control plants of H. dilatata and H, hyperhorea not emasculated and not protected showed a full seed set, indicating autocamy, whereas unpollinated H. sancata yielded only empty seed. TABLE OF CONTENTS. Page ABSTRACT ii-iii LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS vii HABENARIA DILATATA (PURSH) HOOKER).. vii INTRODUCTION 1-6 MATERIALS AND METHODS 7-12 OBSERVATIONS AND DIS.CUSSIONS ik- I. HABENARIA MORPHOLOGY 14-18 II. MORPHOLOGY OF H. DILATATA, H. HYPERBOREA AND H. SACCATA 19-23 III. MICROSPOROGENESIS AND THE MALE GAMETOPHYTE 2^-30 IV. MEGASPOROGENESIS AND THE FEMALE GAMETOPHYTE 31-53 V. CHROMOSOME COUNTS 5^-56 Problems encountered 57 Autogamy versus outcrossing 58-59 Diploidy versus polyploidy 60-63 Tables V.-IX 64-69 VI. GENERAL CONCLUSIONS 70-71 VII. SUMMARY 72 VII. BIBLIOGRAPHY 73-76 LIST OF TABLES. Table Page I. Classification of H. dilatata, H. hyperborea and H. saccata 5-6 II. Collection locations of the three species 10 III. Floret morphology 22 IV. Stages in ovule development at given times from pollination to maturity. 40 V. Chromosome counts 64 VI. Mean value of fertility in crosses made 66 VII. Crosses with H. dilatata as seed parent 67 VIII. Crosses with H. hyperborea as seed parent 68 IX. Crosses with H. saccata as seed parent 69 LIST OF FIGURES. Figure Pa 1 Map of collection locations in British Columbia . 12 2 Orchid flower diagrams 20 3 Front and side-view of Habenaria, longitudinal section of column in Habenaria species used 21 4 Development of anther and ovule in a perennating bud, showing next year's flower spike *^ 5 Microsporogenesis, the meiotic divisions ^7 6 Microsporogenesis continued , ^° 7 Microsporogenesis, pollen tubes ^9 8 -Microsporogenesis, continued ^0 4l 9 Megasporogenesis in H. hyperborea ho 10 Megasporogenesis in H. saccata 11 Megasporogenesis in_H. dilatata ^ 12 Megagametogenesis 13 Megagametogenesis, breakdown in the female gameto- ^ phyte 14 Megagametogenesis, mature embryo sacs ^ 47 15 /Fertiliaatinn 48 16 Fertilization, continued 4? 17 Embryology — • 5C 18 Embryology, continued ^ 51 19 Embryology continued ., 20 Mature embryos ^ 21 Polyembryony " 22-23 Chromosome counts 65 ACKNOWLEDGMENT . It is with gratitude that I wish to acknowledge the help and assistance Dr. Beamish has given me during this study and at all times I have been at the University of British Columbia. I also want to thank my other teachers, Dr. Cole, Dr. Maze, Dr. Marchant, Dr. Person and Dr. Schofield who have never failed to help when it was needed. Above all I owe thanks to my husband who has supported me in my studies, provided transportation for all my collecting trips and has suffered long and patiently during my years at the university. INTRODUCTION . Habenaria fcWilld. is a very large polymorphic genus comprising at least 500 species. Although mostly tropical with 100 species in India alone (Hooker 189*0 , the genus has some members in temperate re• gions of Europe, Asia and North America. Just a handful of species are present in Alaska, Greenland, Iceland and the Faroes. In North America there are 20 species and by far the largest number of them, and the showiest, are found in the East. Western Canada provides a home for 11 species, among them H. dilatata (Pursh) Hook.,H. hyperborea (L.) R. Br. and H. saccata Greene,which are the subjects of this study. Considering the great variability encountered within the genus, it is not surprising that complete agreement as to its taxonomic treatment has never been reached. The name Habenaria goes back to the Latin word "habena", a strap or thong. It was first applied by Linnaeus (1759) to a Jamaican plant which he called Orchis habenaria. Being aware of an inherent difference between the European genus Orchis and the Jamaican plant^ Willdenow (1805) created the new genus Habenaria Willd. and loosely circumscribed it to include all known habenarias. A few years later (l8l7) L.C. Richard clearly differentiated between the tropical Habenaria with long stigmatic projections and anther canals, and plants of more temperate regions with cup-like, concave stigmas and no stigmaphores, by establishing the genus Platanthera L.C. Richard for the latter. John Lindley (1835) recognized both new genera and several closely related ones, fitting them all into one tribe, Ophrydeae. Taxonomic confusion grew, as the number of species increased. It was then that George Bentham decided to re-unite the vast complex under Habenaria Willd. He outlined his ideas at a meeting of the Linnaean Society (l88l) and enlarged on them in his Genera Plan- tarum which he, together with Sir J.D. Hooker, published in 1883. He created a number of sections within the conglomerate genus, the two largest being Platanthera and Habenaria proper as sections 9 and 10. American botanists followed Bentham's lead (Gray 183*+, Ames 1908 and 1910, Correll 1950) and our North American species are now all grouped under Habenaria Willd. Rydberg (1901), who revised part of the genus as represented in North America north of Mexico, was much opposed to the conservative views of Bentham and again split the genus into many segregates, some of which may deserve only varie• tal status. On the other hand, Kraenzlin (1891-93), who painstakingly monographed the tropical habenarias, granted species status only to H. hyperborea, considering all others as mere varieties. Bentham's treatment of the genus is now generally accepted in North America (Ames 1910, 1924, Correll 1950, Szczawinski 1959, Hitchcock et al 1969) although many European botanists adhere to the segregate genus Platanthera. In spite of all taxonomic changes and upheavals the three species in question, H. dilatata, H. hyperborea and H. saccata have always been classified together in tribe Ophrydeae by the early botanists and recently under Orchideae, subtribe Orchidinae. According to Rydberg (1901), Correll (1950), Szczawinski (1959) and others, these species are closely related and form hybrid swarms under suitable conditions. On the other hand, large populations seem to retain their identity year after year, even when growing side by side. Therefore it seemed of interest to test their interrelation• ships experimentally. These tests entailed study of morphology, of pollen production and of seed development of the 3 species as found in nature, and comparison of the natural process with seed development after interspecific pollinations,, Leavit (1901) did the first embryological study on the genus, working with H.