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
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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. tridentata, now known as H. clavellata (Michx.) Spreng. and H. blephariglottis (Willd.) Hook., both Eastern species not known from British Columbia. In 1909 Brown worked on H. orbiculata (Pursh)
Torr. and H. integra (Nutt.) Spreng. The last embryological work on the genus was done by Swamy (19^6) who investigated nine species of
Indian habenarias, the most detailed work being done on H. platyphylla
Spreng. and H. rariflora A. Rich. Since then no embryological studies have been performed and no western species have ever been crossed artificially or investigated other than morphologically. Therefore there arose an excellent opportunity, not only to study the relation• ships of three western species, but to add to the knowledge of
Habenaria embryology in general.
Modern classification of the species used in this study is to be found in Table I. A partial list of those synonyms which indicate the confused relationshipsamong the species is included. Three varieties of H. dilatata are presently recognized, differentiated by length and shape of the spur. I have never encountered the extreme form of var. leucostachys bearing a spur 2 cm long, although this variety is sup• posed to be coastal and western (Correll 1950). H. dilatata var. albi- flora from the North and from the Rockies, with a short almost saccate spu: has not been found in British Columbia. Most often I have encountered plants with spurs 2-3 mm longer than the lip, which falls on the border line between H. dilatata var. dilatata and var. leucostachys.
They are referred to simply as H. dilatata in the present study. As the range of variation is extreme in the species, it is not always easy to differentiate between varieties which often approach each other. Table I. Classification of H. dilatata, H. hyperborea and
H. saccata based on Ames (1924), Dressier and Dodson
(I960), Hitchcock et al (1969) and Szczawinski (1959).
A partial list of synonymy indicates the confused
relationship of the species in question. Family. Crchidaceae. -Subfamily: Orchidoideae. Tribe: Crchideag. Subtribe: Crchidinae. Genus: "abenaria '••'il/'d. Species: H. dilatata (Pursh) "ook., F. hyperborea (L.) P. ~r., F. saccata Greene.
1. H. dilatata (Pursh) Hook. lF24 var. dilatata.
Synonyms: Crchis dilatata Pursh 1814, H. borealis Cham. lS28,
R. borealis var. dilatata Cham. l8?6,
Platanthera dilatata lindl. ex Bed lft33,
E' ££££iiif lindl. 1835, P. hyperborea var. dilatata Pchb.f. 1051, P. hyperborea var. conv-jllariaefolia Vrzl. l899,
H. dilatatiformis Pydb. l8Q7. Limnorchis dilatata Pydb. 1901,
— ' dilatata var. leucostachys (lindl.) .Ames 1910,
£• leucost - chys lindl. 1 351 H • leucostachys '"7ats. i860,
2* hyperborea var. leucostachys Krzl. 1899.
£.* dilatata var. al bif lora (Cham.) Correll 1943,
H. boreal is var. alH flora Cham. 1828.
2- hyperborea (L.) R.Er. l8l3
Synonyms: Orchis hyperborea L. 1767, 0. huronensis Nutt. l8l8,
H* huronensis Spreng. 1826,
H* borealis var. viridiflora Cham. 1828,
Platanthera hyperborea (L.) Lindl. 1835,
Limnorchi s hyperborea °ydb. 1900.
3- H. saccata Greene 18^5.
Synonyms: H. uracil is S. '-'ats. 1877,
Platanthera stricta Lindl. 1835,
H. stricta Pydb. l8°-7, limnorchis stricta Pydb. 19C0, MATERIALS AND METHODS.
Plants of all 3 species were collected during July and August of 1971i all collections being made in British Columbia (Table II,
Fig. 1). The plants were brought to the University of British Columbia, potted in a suitable soil mix and installed in a cold frame. As the plants arrived, their morphology was studied. Anther sqashes were made, which showed only pollen mitoses, as meiosis was past in these months. Ovaries were prepared for serial microtome sections and during the winter megasporogenesis, gametogenesis, fertilization and development of the embryo in the 3 species was observed.
Over the winter months the potted plants were protected with a layer of peat moss in a cold frame and in the spring of 1972 almost all emerged unharmed. Microsporogenesis and development of the pollen grain could then be followed and by mid-May the plants were ready for emasculation and pollination. The work had to be interupted at this stage, but was taken up again in November. All plants seemed to have flowered well and set abundant seed.
The plants were prepared for a second winter and in late March and early April 1973 it was found that more than a dozen had apparent• ly not survived. Almost all of the H. saccata plants did not emerge and were presumed dead. Therefore replacements had to be collected as soon as they could be located in May. A few new H. dilatata plants were also added.
The following approach to the project was adopted.
1. All three species were used as seed parents and reciprocal crosses
were made. 2. Intraspecific crosses were made. , A few plants of each species were emasculated and covered with .5 •
envelopes, to test the technique used.
4. A few plants of each species were emasculated and left open, un•
protected, to see if pollinators were present.
5. Finally some plants were left untouched and uncovered.
During April and May of 1973 chromosomes of the 3 species were counted in both anther cells and root tips. Glacial acetic acid-95% alcohol (1:3) was used as a fixative '.^nd the anther squashes stained with propionic carmine. The root tips were pretreated with 2-alpha-bromo naphthalene at 45 F for 4 hours or more to contract the chromosomes.
Root tips were then fixed as usual, hydrolyzed in IN HC1 for 10 minutes and stained according to the Feulgen reaction.
On May 16 the first cross was made and from then onward, almost daily, plants were crossed as they matured. The procedure was to emas• culate about half the florets of a spike, remove the rest of the spike and transfer a whole pollinium of the same or another species to each cup-like stigma. After pollination each spike was covered with a paper envelope.
To be effective the pollinium had to be at the right stage of maturity and turning yellow. It still had to be unbroken into "massulae" or pollen packets. If massulae had become detached from the pollinium when a bud was forced open, the floret had to be discarded as self- pollination might have taken place, starting 3-4 days after pollination an ovary was removed from each hand-pollinated spike at intervals for about 3-4 weeks and fixed in FAA (formalin-acetic acid-alcohol, in a ratio of 6:4:90 of 50% alcohol). Serial microtome sections were prepared in the usual manner. The stain used was at first saffranin-iron alum with tannin-orange G. Later saffranin-fast green was used exclusively as it gave better contrast and is certainly a better chromosome stain for this material.
Ovaries not removed for embryological studies were left on the spike to ripen naturally and when the capsules started to open the powdery seed was collected in small paper envelopes and left to dry.
To establish an approximate value for fertility of a cross the following procedure was adopted. Four ovaries were chosen at random from a spike and the seed removed. From each lot of seed k equal portions (a small probe full) were mounted on a microscope slide and 9 microscope fields examined in each portion. Ovules with large well formed embryos were considered fertile, empty ones sterile.
The values can be only approximate as there were slight differences in embryo size and amount of seed picked up on a probe. Some ovaries con• tained large amounts of seed, whereas a few were almost empty. Percen• tage of viable seed to the total number of seeds counted gave the mea• sure of fertility.
Voucher specimens of the plants used in this study have been deposited in the Herbarium of the University of British Columbia. Table II. Collection locations of H. dilatata, H. hyperborea
and H. saccata.
All collection sites were in British Columbia. H. dilatata (Pursh) Hook.
Vancouver area Ladner salt marsh No. 130
Manning Park Sumallo Lodge 101 Orchid Meadow 102a Orchid Trail 124 Allison Pass vicinity 129 Squamish area Erandywine Falls vicinity 106 " " No. 5 road 108 Whistler area (1971) 110 " " (1973) 132 Pemberton Meadows 118 Pemberton-D'Arcy 109 Mt. Currie area (1971) 111 ji (1973) 131 Garibaldi Park Taylor Cabin Trail 114
Black Tusk Meadows 123
Keremeos Mt. Apex 112
Braelorne McGillivray Pass 122 ************************************ H. hyperborea (L.) R.Br.
Enderby Mara Meadows 116, 117 Hunter's Range 137
Mt. Tatlow Mt. Tatlow Slope 125
Manning Park East of Blow Down area 103, 104 ***********************************
H. saccata Greene.
Manning Park Orchid Meadow . 102b Allison Pass vicinity 105
Squamish area Brandywine Falls 107
Garibaldi Park Taylor Cabin Trail 115
Braelorne McGillivray Pass 121
HarriF.nn Hemlock Valley 111, 133? 13^i 136
Liumchen Liumchen Valley 135, 139
V.I. Mallard Lake 128
Slocan Lake No. 8 Road 120
Kootenays Jewel Lake 119 Figure 1. Map of that section of British Columbia in which
collections were made.
Ik
OBSERVATIONS AND DISCUSSIONS.
I. HABENARIA MORPHOLOGY.
The genus Habenaria has apparently followed the general lines of orchid evolution, always geared to reduction for the sake of eco• nomy and to accommodation of insect pollinators (Dressier 196l,
Brieger 1970). The resulting organization is highly epgcialized, encompassing structures and terms not applied to other seed plants
(Figs. 2,3,4). The morphology of orchids has been studied in detail by early orchidologists (R. Brown 1813, Hofmeister 1849, Guignard
1886, Strassburger 1900 and others). More recently Schlechter (1926),
Swamy (19^9) and Brieger (1970) habe dealt with the subject. The following discussion on Habenaria morphology serves to introduce the structures and terms in use and is based on the findings of these workers, supplemented by my own observations.
The sepals.
The petaloid sepals form the outermost covering of the flower and are the least modified,,
The petals.
Of the 3 petals the median one has undergone the greatest changes.
It has become the lip or labellum and helps to attract insects and to guide them to nectar and pollen. As it has become heavier and larger it has brought on zygomorphy and also resupination, which is a twisting of the ovary by 180 degrees to bring the lip to the position of a convenient landing platform for insects. In the primordeal orchids the median petal was uppermost, as it is still in the bud of modern orchids before anthesi6 (Fig. 2-A). After anthesis the flower becomes resupinated as described above (Fig. 2-B). The staminal whorls.
Monandrous orchids, including Habenaria, have only one functional stamen, the median one of the outer whorl.
The column and polliniunu
In Habenaria as in other orchids the column is a fusion of stamens and style, the one functional stamen securely adnate to the top of this structure. The very short filament forms the attachment's of the anther to the column base and carries the sterile connective.
The connective, supplied with the vascular strand of the anther, separates the 2 anther cells. Within each anther cell is a pollinium consisting of 2 pollen masses. Each pollinium comprises thousands of pollen grains united into small packets or "massulae" and held together more or less firmly by viscin threads. Compound pollen grains are characteristic of the orchid family as a whole and are found as tetrads, as massulae in pollinia as in Habenaria, or just as pollinia. Each pollinium ends in a short, sticky, elastic stalk called a caudicle, whic consists of modified pollen grains. The caudicle is firmly attached
to the viscid gland or viscidium which is naked in Habenaria. The caudicles represent male tissue, whereas viscidia originate from female stigmatic parts. Polliniumi, caudicle and viscidium together are called the pollinarium.
Here again the trend to reduction and centralization is apparent and shows the high degree of adaptation to insect pollinators. Only, one insect visitor is necessary to carry a whole pollinarium to the next stigma and to fertilize all the ovules of its attached ovary.
Because the pollen grains have become united, not a single grain is
wasted if the pollinarium arrives safely on the next stigma. This ;"all or nothing" arrangement, perfected over long periods of time, seems to have worked out well for the Orchidaceae.
The stigma.
-*-n Habenaria the stigma is a shallow cup-like depression con• sisting of 3 stigmatic lobes, their lower parts firmly grown together and situated on the inner side of the column. In more primitive orchids all 3 lobes are fertile, in Habenaria pnly the 2 lateral ones are functional. The median lobe has become modified to form the rostellum, again a structure found only in the Orchidaceae. It is a tissue flap inserted between the anther and the stigma which helps to prevent self pollination. The viscidia, developed on the strap-shaped rostellum, help to bring about cross pollination by attaching themselves by means of a fast setting cement to a visiting insect. They are attached to the rostellum almost vertically and leave only a faint impression when removed. As the rostellum in the 3 species studied has only a slight middle fold, the viscidia are not far removed from each other. A large insect, fitting into the stigmatic cup, can remove both.Ilnsecifes entering a flower and touching a viscidium, carry off one or both pollinia to deposit them on the stigma visited next. The pollen germinates within 24 hours in the sugary solution produced by the stigma. The visible parts of the stigmatic lobes seem to swell and close after pollination. The perianth dries up and remains attached to the ovary.
The ovary.
The ovary is inferior, unilocular and composed of 3 fused carpels.
The placentae are parietal. The ovules situated on the placental ridges are very numerous and tiny. Being terrestrial, Habenariaestarts the development ojf the current year's ovules the previous summer (Fig. 4-2). The inter• val between pollination-fertilization and the maturation of the embryo is fyuch shorter than in epiphytic orchids, where the ovules are not initiated until pollen is placed on the stigma (Swamy 194-9, Brieger
1970).
The stem.
The stems of habenarias are hollow and each stem bears 6-8 sheathing leaves which become bracts toward the apex. Each floret is subtended by such a bract, which has conspicuous serrulate edges.
Being hollow,the stem breaks easily in the wind when not supported by other vegetation. Especially H. dilatata plants tend to become tall and spindly. Height of the stem is 20-120 cm or more. After maturation of the flower spike, the stem dies down and only the root system overwinters.
The flower spike.
The spikes are 8-40 cm in length and there is great variation in the number of florets on a spike, even within the same species.
Some spikes are lax, others more dense. There can be as many as 100 florets on a spike, but usually there are 20-40. The flowers open in succession, starting with the lowest ones. At any time only 4-6 flowers are open on a stalk, which is one way of preventing pollen of the same plant falling on a stigma lower down. Yet there are always some flowers open and ready should an insect arrive. Differences in the individual florets of the 3 species are summarized in Table III. Roots and perennating buds.
All members of the group possess a very short rhizome and
fusiform, finger-like roots. Overwintering takes place by means of a yearly developing adventitious underground bud (Fig. 4-1), which
contains all of next year's inflorescence and the foliage leaves. By
the time the flower spike is in full bloom, a new bud is formed near
the old stalk. In vigorous plants more than one bud may develop. During
the winter months the following year's flowering stalk develops slowly
underground and when the spike emerges in early spring, the spore mother
cells in the anthers are almost fully developed (Fig. 4-3,4), so that
the meiotic divisions follow in rapid order. The ovules have also
been initiated on the placental ridges (Fig. 4-2) and when the pollen
is applied in spring, fertilization and the formation of the embryo will take place in a relatively short time,,
The seeds.
Habenaria species like all other orchids develop tiny bu numerous
seeds. When ripe the seeds contain morphologically undifferentiated
embryos and lack endosperm. A mycorrhizal fungus is needed for the
germination and earliest nutrition of the seeds. The fungus which often
is a member of the Rhigoctonia group enters the embryo via the suspen-
sor and infects a number of cells nearest the suspensor. Sometimes the
embryo is destroyed by the fungus, but in most cases a certain balance
between fungus and embryo is reached. Habenaria seems to outgrow
its dependence on the symbiont in mature stages, as transplantation
is usually successful. Of the millions of seed produced in an ovary
only a few find a suitable substrate, habitat and the invading fungus
to be able to germinate and to grow into self-supporting plants. II. MORPHOLOGY OF H. DILATATA, H. HYPERBOREA AND H. SACCATA.
The three species are morphologically similar in features just described, but differ in the following ways (Table III).
The florets.
The variability within the three species is always so great that characters tend to overlap. Fragrance in particular is pronounced in some members of a species and missing in others. The flowers differ only slightly in size, more in colour, shape of the lip, length and
shape of the spur, width of the connective, attachement of the anther cells to the connective and shape of the viscidium. The shape of the
stem leaves, especially the lower ones, differs, some being more rounded, others more acute. H. saccata has more bract-like sheathing bases near the soil line.
The viscid glands of H. dilatata are shaped like a human foot, in the two other species they are round. Asa Gray stated that he would have followed Kraenzlin in recognizing only H. hyperborea as a good spe• cies, were it not among other things for the difference in the shape of the glands in the 2 species (Gibson 1905).
Attachement of the anther cells to the connective is also specific.
In H. dilatata the anther cells are parallel to each other and the connective between them is narrow. In H. saccata they are also parallel,
but the connective is wide. In H. hyperborea the anther cells are diver• gent toward the base of the anther. Figure 2. Orchid Flower Diagrams.
Figure A and B. Typical monandrous orchid flowers with dorsal-
ventral symmetry and 5 whorls of parts.
A Flower not resupinated, dorsal sepal toward axis of
inflorescence before anthesis.
B Resupinated flower after anthesis.
Si S2 lateral sepals; dorsal sepal.
Pi P2 lateral petals; labellum.
Aj median stamen of outer whorl, the only functional
one present in Habenaria.
B2 lateral stamens of inner whorl, present only as
vestigial structures.
Cj C2 functional stigmas; stigmatic lobe modified into
rostellum.
St. Stem
adapted from Brieger (1970).
Figure J>.
Front view (A) and side view (B) of Habenaria flower,
a = anther sac
b = bract
co = connective
1 = lip
o = ovary, resupinated in B
p = lateral petals
r = rostellum
s = lateral sepal
ds = dorsal sepal
st = stigma
sp = spur
Longitudinal section of column in Habenaria (C).
(adapted from brieger 1970).
c = caudicle
co = connective
It- filament
p = pollinium
r = rostellum
st = stigma
v = viscidium
Table III. Differences in floret morphology and in the
basal stem leaves. Colour, size of Species Lip florets Spur
rhombic- var. dilatata var. H. dilatata white, 8-15 mm var. lanceolate , leucostahys albiflora 5—8 mm same length as lip, 5-8 mm 1-2 cm half as often twice long as as long as lip lip
H. hyperborea j yellowish-green, linear to tapering filiform, 3 3-10 mm fleshy, slightly bent, 3-6 mm 2/3 of lip
H. hyperborea deep green, upper variable approach- cylindric, always half of petals j ing dilatata jllp, shorter than lip 4n white, 6-12 mm j A-8 mm
H. saccata yellowish-green, linear-elliptic, saccate, often 5-9 mm fleshy, didymous, A.5-7 mm 2.5-4 mm Figure 4. Development of anther and ovules from an adventitious
underground bud, sectioned in late August 1973,
showing next year's flowering spike enveloped by
stem leaves.
1 Longitudinal section of bud showing flower spike and
stem leaves (x 2800).
2 The immature anther showing sporogenous tissue (arrow)
blocked out in massulae (x 5000).
3 Developing ovules on placental ridges (arrow),
small spur (arrow) x 4500.
4 Massulae containing sporogenous tissue (x 5000).
III. MICROSPOROGENESIS AND THE MALE GAMETOPHYTE.
The onset of the meiotic divisions can be recognized by the enlargement of the spore mother cell nuclei (Fig. 8-1). Meiosis follows the usual pattern with homologous chromosomes paired and showing chiasmata, the chromosomes aligned at the equator in meta- phase and pulling apart in anaphase and telophase (Fig. 5-1,2,3,4,5).
The first meiotic division produces a dyad. No walls are laid down during this division. The second division is simultaneous in the nuclei of the dyad, resulting in a tetrad of microspores, still within the walls of the spore mother cell (Fig. 8-2). Only after the second division, when the nuclei have gone into a resting stage, are cell walls laid down by. inwardly directed furrows (Fig. 8-2).
The resulting haploid microspore tetrads are as a rule isobilateral
(Fig. 5-6). or tetrahedral (Fig. 8-3), but on occasion a linear row of spores is formed (Fig. 8-4) in massulae located in the narrowing end of the anther. All the cells of one massula divide simultaneously and so remain synchronized in development (Fig. 8-1, 2,3,4). Other massulae are in approximately the same stage. As the walls of the spores within a massula are thin, there can be interaction between spore nuclei, so that even chromosome-deficient ones can divide
(Barber 1942). This phenomenon explains why apparently no dead pollen grains have been found in the Orchidaceae (Barber 1942). The wall around the pollen packets is cutinized and does not allow interaction of nuclei of different massulae.
After a period of rest the first division of the microspores
(Fig. 6-1), aggregated in tetrads and massulae, results in the 2-nucleate pollen grain (Fig. 6-2) or male gametophyte within the anther. This division is characterized by a prolonged prophase, the chromosomes contracting strongly and becoming very round and widespread within the cell. This is a favourable period for chromo• some counts. Divisions in the tetrads are simultaneous and synchronized
(Fig. 5-6) and a large vegetative and smaller, but deeper staining generative nucleus is formed (Fig. 6-2). A clear area in the cyto• plasm can be seen separating the 2 nuclei though a cell plate i6 laid down at the time of the last division. A transitory wall is formed
between the 2 cells at their formation which, however, soon dis• appears (Swamy 194-9). The generative nucleus is at first lentil-shaped
and surrounded by very little cytoplasm (Fig. 6-2). Eventually it is
engulfed by the much greater amount of cytoplasm of the vegetative nucleus and is forced inward, away from the cell wall. This stage
constitutes the almost mature pollen grain as it is shed. When brought
upon a receptive stigma it germinates within 24 hours. At the time
of shedding the pollen grain has 2 wall layers (Fig. 6-3). When the
grain germinates the intine pushes through the exine and becomes
the pollen tube (Fig. 7-1). Usually the vegetative;In"u:o.leus^,;sifollowed
by the generative nucleus and the cytoplasm of the grain, enters
the lengthening tube and both are carried in the tube through the very
short stylar canal into the ovary. On the way the generative nucleus
divides, producing 2 gametes or sperm nuclei (Fig. 7-2,3). They are
oval in shape and stretched, having been forced through the narrow
neck of the ovary (Fig. 7-3). The 2 sperm nuclei seem to be surrounded
by a faint layer of cytoplasm. The vegetative nucleus gradually loses ita stainability and becomes very hard to see. When the stream of pollen tubes arrives in the ovary, it divides into 3 parts, each stream heading for one placental ridge. Here the assembled tubes divide again, some following each side of the ridge. Then one pollen tube enters one ovule along the funiculus and through the micropyle,, releasing the sperm nuclei into one synergid, or more rarely between the synergids.
I have compared the process of microsporogenesis in H. dilatata,
H. hyperborea and H. saccata to that in other members of the family, and find that the compound pollen grain is common to all with very few exceptions. However, the degree of adherence of the grains differs in the various tribes. The three western habenarias conform very closely to other members of the tribe Orchideae (Ophrydeae), with the tetrads in massulae or pollen packets, many massulae forming one pollinium (Heusser 1914, Swamy 1946).
On the other hand Tuschnjakova's study (19281) on Listera ovata reveals the adherence of the pollen grains in tetrads only. Listera, belonging to tribe Neottieae is believed to be less highly revolved and is usually classed before the Orchideae (Dressier & Dodson i960).
A third comparison was made with Eulophia epidendraea (Swamy 1942), a genus belonging to the highly evolved tribe Vandeae. Here the pollen grains adhere in complete pollinia, which is the highest step in adherence known and indicates high specialization. Figure 5» Microsporogenesis (The meiotic divisions).
1 H. saccata. Early prophase (leptotene-zygotene)
in spore mother cell (x 4-500).
2 H. saccata. Metaphase I (x 4500).
3 H. saccata. Anaphase I (x 4000).
4 H. saccata. Telophase I (x 4500).
5 H. hyperborea. Metaphase II (x 4500).
6 H. hyperborea. Synchronized pollen mitosis,
isobilateral tetrads (x 3000).
Figure 6. Microsporogenesis (continued).
1 H. dilatata. Synchronized pollen mitosis in tetrads
after the meiotic divisions (x kOOO).
2 H. hyperborea. Two-nucleate pollen grain, gn = gene•
rative nucleus, v| = vegetative nucleus, (x 3000).
3 H. dilatata. Two-nucleate pollen grain as it is shed.
Heavy outer wall layer noticeable (x 5000).
Figure 7. Microsporogenesis (pollen tubes from paraffin sections).
1 H. hyperborea. Five days after pollination,
pollen tube showing vegetative and generative nucleus
having reached the ovary. Arrows indicate the 2 nuclei,
(x 4000).
2 H. dilatata. Five days after pollination, generative nucleus
(arrow) dividing in the pollen tube, having reached the ovary,
(x 4000).
2 H. dilatata. Five days after pollination, 2 sperm nuclei
(arrow) in the pollen tube (x 4000).
Figure 8 . Microsporogenesis (continued).
1 H. hyperborea. Prophase of microspore mother cells
in massulae (x 2000).
2 H. dilatata. Finished tetrad of microspores
still within wall of mother cell (x 3000).
3" H. saccata. Pollen mitosis in tetrads, n = 21,
(x 3800,).
4. H. saccata. Linear tetrad of k microspores in
pollen mitosis, n = 21, (x 380O).
IV. MEGASPOROGENESIS AND THE FEMALE GAMETOPHYTE.
Megasporogenesis after successful crosses is so much alike in all 3 species that one generalized discussion is adequate. The ovules are initiated as microscopic dichotomously branching papillae developing from the placental epidermis. The young ovules consist of a linear row of 5-6 cells surrounded by an epidermis (Fig. 10-2).
The uppermost hypodermal cell in this row is the archesporial cell
(Fig. 10-1) which differentiates directly into spore mother cells'
(Fig. 9-D. The inner integument appears early (Fig. 10-3) and has surrounded the embryo sac by the time the megaspores are formed, leaving only a small opening at the micropylar end. It is 2-layered, whereas the outer integument which develops below the inner one a little later, consists of only one layer of cells. The outer integument has outgrown the inner one by the time the embryo sac is mature. The funiculus is the lowest part of the ovule and connects it to the pla• centa. The ovules are at first orthotropous, but soon become anatro- pous. At the onset of the reduction divisions the nucleus of the megasporocyte moves to the upper part of the cell (Fig. 10-2), en• larges and, going through the usual division stages (Fig. 9-2,3»
Fig. 10-3, Fig. 11-1), produces a dyad of cells (Fig. 10-4, 11-2).
Walls are laid down during megasporogenesis contrary to the situation in microsporogenesis and the chalazal cell is favoured as to size
(Fig. 10-4). The second division may take place only in the lower cell of the dyad or in both. In the first case the result is a row of 3 cells (Fig. 9-4, 11-3). In the second case both cells of the dyad divide to > Growth of the functional megaspore is very evident after the reduction divisions. In the process the 2 or 3 non-functional megaspores disintegrate (Fig. 12-1) and are pushed together, appearing as dark'-staining red caps on top of the young embryo sac in stained sections. Three .•suc.cfessi-ve^nuelear divisions (Fig. 12-1,2,4) produce the monosporic, 8-nucleate Polygonum-type sac (Fig. 14-1,2). After the first division the 2 nuclei are separated by a large vacu• ole. The next divisions are simultaneous, or nearly so, and no walls are laid down during the divisions. From the 4-nucleate stage to matu• rity the micropylar nuclei are always larger than the chalazal ones and often divisions of the latter seem to be delayed (Fig. 12-3) or do not take place (Fig. 13-2,4\ so that either 6- or 7-nucleate sacs arise. At this stage I have infrequently noted a very large spindle in the chalazal end of the sac instead of the usual two, indicating a fusion of the spindles, the resulting nuclei presumably being diploid instead of haploid and leading to a 6-nucleate sac. Apparently the advance of the pollen tube down the style affects the rate of embryo sac development, since the maturation of the sac is quicker when the pollen tube and style are of the same species. After intraspecific pollinations a mature embryo sac can be found from 6-7 days from the time pollen reaches the stigma. After interspecific pollinations it may take from 8-l4 days or more before the sac is ready for fertilization. Speed of development after pollination depends also on the freshness of the pollen, health and vigour of the seed parent and stage of maturity of the florets. The mature megagametophyte (Fig. 14-1,2) comprises the egg appa• ratus, the two polar nuclei and the four nuclei at the chalazal end of the sac. The two synergids are sister cells. The sister nu• cleus of the egg, the polar nucleus, moves toward the center of the embryo sac and is apparently not surrounded by a cell wall. The chalazal group arranges itself as the three antipodals and the chalazal polar nucleus, which moves up to the other polar nucleus and usually fuses with it before fertilization takes place. The embryo sac keeps on enlarging, still topped by the disintegrating spores. The nucellus and most of the inner integument have been resorbed earlier. The antipodals, or whatever remains of them, often stain darkly and begin an early degeneration. In some cases they are still visible after fertilization. The pollen tube enters the ovule via the micropyle and invades one of the synergids (Fig. 16-1) which is destroyed. The pollen tube usually empties its cytoplasm and the 2 sperm nuclei into the one synergid and from there the sperm makes its way toward the egg and the other toward the polar nuclei. At times the pollen tube empties its contents directly into the sac, advancing between the synergids (Fig. 15-2), both of which then remain unharmed. The re• sult is the same in both cases. One sperm always seems to move to• ward the egg cell, whereas the other is carried toward the fusion SUAleus (Fig. 15-2). The sperms are now round and smaller than the egg cell. The contents of the sac are often hard to see as fatty, dark- staining substances are brought into it by the ruptured pollen tube (Fig. 16-1.). At the time of fertilization starch grains are often present in the cell, in the fusion nucleus and in the one remaining synergid. Often one sperm can be seen closely eppressed to the egg cell (Fig. 15-1,2,3,), or already within it (Fig. 15-2, 16-1,2,3,) and I have observed fusion between egg and sperm nucleus in various stages of completion (Fig. 16-1,3). The polar nuclei are most often fused before fertilization (Fig. 15~2), at times only partially so (Fig. 15~3J 16-2). They are often closely accompanied by the sperm (Fig. 15~3)• I** triple fusion has taken place one can often see 3 nucleoli within the primary endosperm nucleus (Fig. 17-2, 18-2). If the polar nuclei fuse early the fusion nucleus becomes very large, often larger than the egg cell/ As a rule triple fusion does take place in the three species studied. However, the triploid endosperm nucleus usually begins to show signs of deterioration soon after fertilization. It becomes crescent-shaped (Fig. 17-1,2,3) and stains in .the same way as the disorganizing antipodals. On occasion the -endosperm nucleus, antipodals and one synergid are in good condition after the divisions in the zygote have started. The fertilized egg, the zygote (Fig. 17-1), grows considerably after fertilization and rests a few days before onset of the next divisions. The first division of the zygote is always transverse, producing a 2-celled proembryo (Fig. 17-2,3), consisting of the micropylar or basal, and the chalazal or terminal cell. The next division is in the basal cell, resulting in a 3-celled proembryo (Fig. 18-1), adding a middle cell. Next the terminal cell divides by a vertical wall (Figs. 17-4, 18-1, 19-1). The next division can be either in the terminal or basal cell, initiating in the latter case the suspensor which develops in all habenarias and will be discussed later. In the 4-celled proembryo 4 divisions follow, 2 in the terminal cells and 2 in the middle cell, producing 2 tiers of 4 cells each (Fig. 18-3). The next divisions produce 8 cells in each of these tiers to form the octant stage. The tiers originating from the middle cell divide again producing all the cells of the micropylar end of the ovary. Finally, divisions in the distal end result in groups of much smaller cells by formation of vertical, transverse and obliquejwalls. The cells of the upper part of the em• bryo are always larger than the rest and can be recognized as the descendants of the middle tier (Fig. 20-2,4). From the 5-celled stage (Fig., 19-2,5,4) divisions are irregular and hard to follow, but ultimately the mature embryo (Fig. 20 -1,2,4) consists of 50-60 morphologically undifferentiated cells. The terminal cell at all times contributes most to the mature embryo. The middle cell pro• duces the uppermost layers and the suspensor does not contribute to the formation of the embryo proper. The suspensor and its function. The divisions of the suspensor may start when the terminal cell is still undivided (Figs. 17-3, 18-2) or later, but are usually finished before the embryo is fully developed. All divisions are transverse and a row of 6-7 cells emerges, which, as the cells lengthen, pushes out of the embryo sac (Fig. 17-4, 20-1), the last cell implanting itself in the placenta (Fig. 20-2) and developing an haustorial function. The other cells presumably help in the trans• port of nutrients to the embryo. Occasionally a suspensor was seen which developed the usual number of cells, but did not push out of the embryo sac (Fig. 19-4). In such a case the embryo proper did not seem to develop fully. When the embryo matures and starch and aleurone grains fill the cells, the suspensor dries up. Sometimes part of the suspensor can still be seen in mature seeds. Polyembryony. Several times I have noticed two embryos in a young embryo sac, one embryo always being smaller than the other (Fig. 21-1,3). However, in mature seed with well developed embryos, at least a dozen seeds were found with two embryos within the seed coat, but both of the same size. According to Leavit (1901) and Swamy (19^9) who found multiple embryos in other Habenaria species, these embryos were arrived at by cleavage of the first embryo.Only one embryo survived (Swamy 19^9) as no supernumerary embryos were found in ma• ture seed. The multiple embryos which were seen in the present study (Fig. 21-1, 2,3) were apparently formed in the same way and could be noticed very early in the formation of the embryo (Fig. 21-1, 2), but some of these reached maturity. In Fig. 21-2 the splitting off of the second embryo can be seen right below the suspensor initial, but one embryo seems to be disorganizing. Several times in embryo sacs with already well advanced embryos a second pollen tube was observed, containing 2 sperm nuclei (Fig. 19-1,3). Ovary wall and placenta. In the course of ovule development, pollination and fertilization the placenta becomes hypertrophied and densely cytoplasmic. The sus- pensor becomes embedded in this rich storage area. When pollination has taken place the ovary becomes a fruit, swells and loses its green colour, the dried up periaath remaining attached. An unpollinated ovary stays green much longer, but does not mature and no swelling can be seen. In about 3-4 weeks after pollination the normal fruit is mature and the seeds are ready to be dispersed.(Fig. 20-J). According to Seshagiriah (194l) the growth hormones or auxins in the pollen tube and pollen stimulate the plastids present in the cells of the placenta and ovary wall to synthesize carbohydrates, which in the absence of endosperm, produce nutrients necessary for ordinary growth and development of the fruit and the seeds. The seeds. Similar to all orchid seed, those produced by the three Habenaria species are very light, very numerous and have morphologically un• differentiated embryos. The only food present is packed in the cells of the ovoid embryo (Fig. 20Gnly the outermost layer of the outer integument remains and becomes the transparent seed coat; all : the other layers of cells are absorbed or disintegrate. During matu• ration the cells of the outer integument lose their protoplasts and become transparent and reticulate. The embryo seems suspended in the air-filled cavity of the seed-coat, and at times a few cells of the suspensor are still visible. The unusual features of megasporogenesis and development of the femgla: gametophyte in Habenaria species studied are not without precedent. Orchid literature in general (Rolfe 1909, Poddubnaya- Arnoldi I960, Swamy 1949) stresses the fact that the influence of the pollen and pollen tube is necessary for the initiation ecf the ovules and that the ovules as a rule have not started to differentiate when the pollen arrives on the stigma. Therefore there are long delays between pollination and fertilization and between fertilization and the maturation of the embryo in all epiphytic and tropical orchids, the interval becoming longer with progressively higher evolution of the species in question. Habenarias being terrestrial and not as hiighly evolved as the epiphytes, do not need the presence of pollen for the initiation of their ovules. When investigating very young buds with the pollen not yet mature, inwsiiab^y I found ovules in the archespor'ial cell stage. Pollination had not yet taken place, but ovules had been initiated and a linear row of 5-6 cells,topped by the archesporial cell could be seen in scrapings from a living ovary. Even the slight bulge of the inner integument was present. Development would progress and mature ovules would result without pollination, but no embryos would be present if not fertilized at a later stage. After pollination development of the embryo sac is rapid, showing the spore mother cells in all stages of the meiotic divisions only 2-3 days after pollen has been applied in most ovaries. Reduction of the female gametophyte is a character inherent in the whole family and habenarias are no exception. This has been commented on by the various workers (W.H.Brown 1909, Afzelius 1916, Swamy 19^-9) who have dealt with the Orchidaceae. It is usually the group of antipodals which is reduced. Another peculiarity of the family is the suppression of endo• sperm which is the rule for most genera. Triple fusion does not take place in all orchids and without it the second sperm degenerates (Nawaschin 1900, Strassburger 1900, Sw3my 1949). Only a few orchids, among them Vanilla planifolia (Swamy 1949) and some Cypripedium species (Pace 1908) develop some endosperm tissue before degenera• tion sets in. The significance of the lack of endosperm is uncertain and some botanists have assumed that this is the reason for the undifferentiated orchid embryos (Coulter&. Chamberlain 1912). Others believe that endosperm does not develop because the primary endosperm nucleus is resorbed and used by the zygote immediately (Heusser 1914). But even orchid species which develop some endosperm have undiffe• rentiated embryos and the Podostomaceae have fully developed em• bryos and no endosperm development. As the suspensor has no part in the construction of the embryo proper -in Habenaria, the embryos were classed as belonging to the "Onagrad" type by Johansen (1950). The suspensor itself falls into type II according to Swamy (19*4-9). I have compared the development of three western habenarias to the Indian species of Swamy (1946) and cannot find any appreciable difference in microgametogenesis, megagametogenesis and embryology, except that in his species only 2 days are necessary from pollination to fertilization, whereas the Canadian plants take from 6-10 days. This difference might be explained by milder climate and higher tempe• ratures. It was observed in the previous century (Guignard 1886, Hofmeister 1849) that in higher evolved orchids the interval between pollination and fertilization and the maturation of the embryo is a longer one than in more primitive ugehe.ES. Schnarf (1931) tabulated AO the result of his studies in ovule development and seed maturation in the Orchidaceae. He gives a period from 8-l4 days from pollination to fertilization for Habenaria species. These figures correspond with my results. After all crosses, intra- or interspecific, some ovules began development and failed. The failure rate was much higher after inter- than intraspecific pollinations as evidenced by seed data (Table VI). The study of megagametogenesis and embryology showed no consistent pattern of degeneration, merely thet the process was slowed down after inter• specific crosses, especially in those where diploids were crossed with polyploids. However, there was some breakdown of cells in all crosses. Table V. Stages in ovule development at given times from pollination to maturity after crosses were made. 1 2-6 6-14 10-21 21 days day days days days onward Germina- megaspore developing zygotes, mature em• tion of mother embryo sacs, proembryos, bryos, pollen cells, mature sacs, embryos in maturing seed, grains in dyads, fertilization. all stages ripening massulae tetrads up to matu ovaries, on the linear and rity. dispersion of stigma. T-shaped. seed. Figure 9« Megasporogenesis in H. hyperborea. 1 Megaspore mother cell (arrow) in resting stage, 2 days after pollination (x4000). 2 Megaspore mother cell (arrow) in early prophase (leptotene-zygotene), 2 days after pollination (x 4000). 3 Megaspore mother cell (arrow) in anaphase, 4 days after pollination (x 4000). 4 Three megaspores, the 2 upper ones degenerating, the chalazal one (arrow), functional, 4 days after pollination (x 4000). Figure 10 Megasporogenesis in H. saccata, 1 Two developing ovules (arrows) before pollination at anthesis, one cell in division (x 4000). 2 Megaspore mother cell (arrow) before pollination at anthesis (x kOOQ). 3 Megaspore mother cell (arrow) in metaphase I, 6 days after pollination, inner integume nt visible (x 2500). 4 Dyad after first meiotic division, 8 days after pollination (x 3000). Figure 11. Megasporogenesis in H. dilatata. 1 Megaspore mother cell (arrow) in prophase, six days after pollination, inner integument clearly visible, (x 5000). 2 T-shaped tetrad, the upper dyad cell has divided by a ver• tical wall , spindle still visible; these 2 cells seem to be degenerating. The 2 lower spores both intact, (x 2500). 3 Three megaspores, the two upper ones degenerating, chalazal one (arrow) functional, 6 days after pollination, (x 4000). Figure 12. Megagametogenesis. H. hyperborea (4n) x H. dilatata. First division of embryo sac mother cell, 12 days after pollination, 2 degenerating spores topping the sac (x 5000). H. saccata x H. hyperborea (4n). Two-nucleate sac, 2 degenerating spores on top,, nucellus still intact, inner integument almost enclosing embryo sac (arrow), outer integu• ment visible (o.i.), 6 days after pollination (x 4000). H. hyperborea (4n) x H. dilatata. Three-nucleate sac, 7 days after pollination, the chalazal nucleus has not divided (x 5000). H. dilatata x H. hyperborea (4n). Four-nucleate sac, the 2 micropylar nuclei in the process of the next division, one of the chalazal nuclei partly sectioned out, these 2 cells show no signs of division (x 3000). Figure 13. Megasporogenesis continued, Breakdown in the cells of the female gametophyte. 1 H. hyperborea (4n) x H. dilatata. Two-nucleate embryo sac showing early breakdown in the chalazal cell (arrow). One degenerating megaspore on top of sac, 12 days after pollination (x 4000). 2 H. hyperborea (4n) x H. dilatata. Four-nucleate sac, 12 days after pollination, the lower cells in beginning disorganization., (x 4000). 3 H. hyperborea (4n) x H. dilatata. Five-nucleate sac. The 2 micropylar cells have divided, spindle still visible, 2 cells partly sectioned out; in the chalazal end only 2 cells visible, only one cell seems to have gone thEOUgh the second division, 14 days after pollination (x 3500). Figure 14. Megasporogenesis continued. Almost mature sacs. 1 H. hyperborea (4n), intraspecific, 7-nucleate embryo sac before fertilization, 1.6 days after pollination (x 5000). 2 H. hyperborea (4n) x H. dilatata. Eight-nucleate embryo sac before fertilization, 12 days after pollination. Four nuclei in chala• zal end of sac, polar nuclei not yet fused (x5000). Figure 15- Fertilization. 1 H. hyperborea, intraspecific. One well preserved synergid (s), egg cell (e) with sperm (arrow) touching it, polar nuclei (p) not quite fused, second sperm (arrow) touching one polar nucleus (x 3000). 2 H. hyperborea (4n) x H. dilatata. Two synergids (s) with pollen tube above large zygote (z), egg and sperm nuclei touching (arrow) within egg cell, 2 fused polar nuclei (2 arrows), second sperm (arrow) close to egg cell, 16 days after pollination (x 3000). 3 H. hyperborea, intraspecific. Large fertilized egg cell (e), 3 fusing nuclei below egg cell representing the primary endosperm nucleus (arrow), antipodal (a), 9 days after pollination (x 3000). 4 H. hyperborea, intraspecific. Pollen tube, one well preserved synergid (s), large zygote (z) with starch grains end large nucleus; fusing polars partly sectioned out (x 3000). Fertilization continued. H. saccata x H. hyperborea (4n). Pollen tube (p), sperm nucleus fusing with egg nucleus within egg cell (arrow), fusing polar nuclei with second sperm attached (arrows), one antipodal (a), 18 days after pollination (x 3000). H. hyperboreaA intraspecific. Egg and sperm fused (arrow), polar nuclei (p) only partly fused, second sperm (arrow) close to egg cell, triple fusion has not yet taken place, 9 days after pollination (x 3000). H. dilatata,intraspecific. Egg nucleus almost completely fused with sperm nucleus (arrow), 8 days after pollination (x 3000). 1 H. saccata, intraspecific. Large zygote, endosperm nucleus (arrow)crescent - haped, beginning degeneration, 19 days after pollination (x 3500). 2 H. hyperborea, intraspecific. Large terminal cell (arrow), basal cell partly sectioned out (b). Primary endosperm nucleus with 3 nucleoli (arrow), 12 days after pollination ( x 3500). 3 H. hyperborea (2n) x H. saccata. Two-celled proembryo, .degenerating primary endo• sperm nucleus (arrow), 1 synergid (s) and disorgani• zing antipodals (a), 19 days after pollination (x 3500). 4 H. dilatata x H. saccata. Four-celled proembryo, terminal cell has divided by a vertical wall (arrow) and one of the cells in division, 2n = 42, 22 days after pollination (x 3500). 1 H. hyperborea (4n) x H. saccata. Four-celled proembryo, terminal cell has divided by a vertical wall'( arrow), large fusion nucleus still in g-ood shape (arrow), 1 synergid (s), 2J days after pollination. Slow developing hybrid embryo, (x 3000). 2 H. dilatata intraspecific. Five-celled proembryo, degenerating endosperm nucleus with 3 nucleoli (arrow) indicating triple fusion, Ik days after pollination (x 3000), 3 H. hyperborea (2n) x H. dilatata. Transverse section of an embryo, showing one tier of cells in quadrant stage, the cells in divi• sion, 12 days after pollination (x 5000). 1 H. saccata x H. dilatata. Four-celled proembryo terminal cell in division (arrow), primary endosperm nucleus (e) degenera• ting, an extra pollen tube with 2 sperms(( 2 arrows) visible, 15 days after pollination (x 4000). H. hyperborea x H. saccata. Multi-celled embryo with suspensor, primary endo• sperm nucleus (e) crescent-shaped and partly covering the embryo; one cell of embryo in division, one nucleus of endosperm seems to be in prophase ? (arrow), 22 days after pollination (x 4000). H. hyperborea, intraspecific. Five-celled embryo, suspensor initial in prophase (arrow), endosperm nucleus degenerated, second pollen tube with 2 sperm (arrow) visible (x 4000). H. hyperborea (2n) x H. dilatata. Many-celled embryo, suspensor with 3 cells has not yet pushed out of the embryo sac, endosperm nucleus degenerated (arrow), 18 days after pollination (x 4000). Figure 20. Mature embryos. 1 H. saccata, intraspecific. Embryo with long suspensor, 19 days after pollination, (x 4000). 2 H. hyperborea (4n).:ix H. saccata. Mature embryo, suspensor implanted on placen• ta. Placental tissue disintegrating, 32 days after pollination (x3000). 3 H. saccata. intraspecific. Seed ready to be dispersed, 32 days after polli• nation, seed coatLsurrounding the embryo,(x 3000). Figure 21. Polyembryony 1 H. saccata x H. dilatata. One large proembryo, a second, much smaller embryo has split off and is in division; endosperm nucleus and 3 antipodals disintegrating, 10 days after pollination (x 3000). 2 H. dilatata x H. hyperborea (2n). Two embryos with common suspensor. The splitting off apparently occurred very early in development, the embryo on the left degenerating; endosperm disorganized, 21 days after pollination (x 3000). 3 H_. hyperborea (4n) x H. saccata. One almost mature embryo and a second much smaller one, consisting of 5 cells only, 32 days after pollination (x 3000). V. CHROMOSOME COUNTS AND CROSSES. Results. H. dilatata, H. hyperborea and H. saccata have 21 pairs of chromosomes in all but one of the populations checked in the present study. The one exception was a tall green-flowered popu• lation 103 (2-6) in Manning Park, where polyploids (n = 4-2) grew among diploids (n = 21). Counts are presented in Table V. Both chromosome numbers have been recorded previously, diploids frequently, (Humphrey 1954, Taylor and Mulligan 1968) and polyploids from northern areas (Harmsen 1943, L6ve 1968 and Ritchie 1956). In I969 F.C. Bent determined the chromosome number of 12 Habenaria species from Nova Scotia and lists H.. hyperborea var. huronensis as having 42 pairs of chromosomes, which should indicate that not only far northern plants are polyploids as has been suggested previously (Harmsen 1943). However, no other tetraploids have been recorded from southern British Columbia. The crossing program was carried but as outlined under "Materials and Methods". Results are discussed in the same order and are presented in summary in Tables VI, VII, VIII,.and IX. 1. Intraspecific crosses produced many seeds with large well formed embryos. Mean fertility was 60-90 % which would allow for a tremendous number of possible germinations (Table VI). 2. Of the spikes which were emasculated and protected all. but one dried up completely, indicating that the technique was effective. On only one flowering spike a few ovaries did swell slightly, suggesting that some pollen must have reached the stigma, probably during removal of the pollinia. 5. Each of the spikes on which florets were emasculated but left unprotected produced a few seed pods, indicating that polli• nators must have been present, but not very effective. The ovaries of one such spike produced only 6.6 % of seed with embryos. I have noted flies, bees and a great number of small greenish spiders scurrying in and out of the florets when disturbed and aphids to• ward the end of the summer, which probably were the prey the spiders were persuing. As spiders are carnivorous, pollen and nectar could not have been a great attraction, but in their wanderings in and out of the flowers, they might have been able to transfer a few loose pollen packets from one flower to the other and bring about fer• tilization. I have been unable to see night-flying moth which are supposed to be the usual pollinators of Habenaria ( van der Pijl & Dodson 1966). k. Plants of the three species left with neither emascu• lation nor cover did not all behave alike. H. dilatata and H. hyper• borea developed large numbers of capsules containing many seeds of which ?8 % had mature embryos in H. dilatata and 60.k % in H. hyper• borea . H. saccata produced only empty seeds (Table VI). 5. Results of interspecific crosses were not entirely consistent, even when the same 2 species were involved. However, some facts emerge. a) In reciprocal crosses it did not seem to matter in which direction the cross was made. However, it became apparent that some plants within a species showed higher fertility than others. Pla#fc 103-6, one of the tetraploids, was a failure both as a seed and a pollen parent, whereas a sister plant, 103-4 gave good results in both directions when crossed with another species (Tables VII and VIII). b) Data of crosses between H. dilatata and H. hyperborea are tabulated in Table VII. In 7 crosses with H. dilatata as seed parent the mean fertility was 62 % and probably would have been higher if seed of all the crosses had been .".available for sampling. Two pro• mising stalks were lost by breakage in spite of staking. One cross (101 x 117) did not produce sufficient seed to be counted, probably because the pollen parent was not a very vigorous plant. In one cross with very lov; percentage of apparently good seed, mixed pollen of the tetraploid plants was used which included pollen of plant 103-6. As mentioned above, this plant is a very poor parent and the poor result of cross 112a x 103 (Table VII) may be the result of having included this pollen in the mixture. c) Data of crosses between H. dilatata and H. saccata are shown in Table VII. Of the 3 crosses made, seed is available only from cross 110 x 133 with the low return of 25 %. However, cross 122 x 121 (Table VII) showed large well formed embryos in the paraffin sections, indicating that the 2 species will cross under favourable circumstances and with well established plant material. d ) H. hyperborea and H. dilatata, with the former as seed parent, were crossed 6 times and produced a mean of 53.4 % of good seed (Table VIII). H. hyperborea crossed with H. saccata gave consistently higher results (Table VIII). e) Crosses between H. saccata and the 2 other species with the former as seed parent gave inconclusive results mainly because so little seed was available for sampling. The reason for this will be discussed shortly. However in 3 crosses with H. hyperborea 63 % of good seed was harvested, which is far better than the results for crosses with H. dilatata. Problems encountered. Breakage of the flower stalks, even after staking, was a constant problem. The flower pots had to be lifted in and out of the cold frame and carried back and forth to my work area. The stalks are tall, hollow and very fragile, especially those of H. dilatata. The 2 other species, which are not quite so tall, did not break as frequently. The loss of plants over winter posed additional difficulties. With the exception of a few specimens, H. saccata had to be replaced in the spring of 1973. The new arrivals, having been recently disturbed, were not as well established and actively growing as the other plants. Furthermore, when they were ready for pollination, only stored pollen was available except when their own pollen was used for intraspecific crosses. Plantsppollinated with pollen one month old or older were always slow in development. Embryos took fully 25-28 days to mature, but seemed of good size. Here, too, breakage destroyed 3 stalks so that the success of the crosses can only be judgedi.from the paraffin sections. Unfortunately all ovaries of the last 3 crosses made with H. saccata as seed parent, were collected for sectioning, leaving none for seed counts. Plant 121, a H. saccata collected in 19?1, offers proof that this species will cross as well as any other, at least when arti• ficially pollinated if the plants are well established and active• ly growing. This plant was found entwined with H. dilatata (122) and as the plants could not be separated without damage, they were planted together and bloomed well for the last 2 summers. When reci• procally crossed' the seeds developed well. Autogamy versus outcrossing. As discussed in "Materials and Methods" (control 5, pg» 8", plants neither emasculated nor portected), H. dilatata and H. hyper- borea plants were very prolific in seed set. The regularity and orderliness of the seed pods on the spikes makes one think of self- pollination. It seems that even very efficient bees would miss a floret occasionally. Gray (1862) believed that H. hyperborea could be either autogamous or outcrossing and H. dilatata seems to follow the same mode of pollination if pollinators do not arrive in time and the spollen is ready to be shed. It seems that at a certain stage of maturity the pollen packets begin to break loose more easily and a gentle wind or a slight touch can remove them. Often, forcing . open a bud revealed free pollen packets in the flower and in the stigma. These pollen masses may have germinated on the stigma and may have fertilized some ovules of a flower, whereas the< rest-of •'the pollinium could have been carried off by an insect and fertilized another flower. Another pollinator could have brought pollen to the first flower, so that both systems can be at work at the same time. In H. hyperborea pollination can take place in the bud (Hagerup 1952b), as the anther thecae dehisce before anthesis. According to the same author (1952a), Habenaria species in the far North( (Faroes, Greenland) are almost always self-pollinated, as few insects are to be found in these wet and cold climates. For this reason plants with genes for autogamy have a better chance for survival. Autogamy, which imparts only the genetic make-up of one parent, may have been of special value to the species at certain times and in certain areas, as from a few windblown see^ds a new population may have arisen when finding a suitable habitat. This could be one of the many factors which have assisted the spread of the species from the South, following the retreating ice masses, to the Arctic Circle, always favouring wet habitats. As genes for outcrossing were retained, even an occasional cross pollination among species usually deprived of pollinators, may have helped to retain vigour and genetic plasticity. Because of plasticity and variability within the species they have been able to flourish in many different habi• tats, ranging from northern bogs and forests to southern mountain meadows and coastal salt marshes.which again must have affected their morphological characteristics and growth habits. H. saccata appears to be self-incompatible, as artificially pollinated plants respond well to pollen of other species, but plants left unpollinated and unprotected in the cold frame produced only empty seed (Table IX). Likewise, a very vigorous flower stalk, broken in transport and left to ripen naturally without access to pollinators produced only chaff, though in this case the failure to produce seed may have been due. to lack of nutrients rather than to absence of pollinators. According to van der Fiji and Dodson (1966) most outcrossing orchids have evolved complicated systems, both morphological and genetic, tq prevent self-pollination, self-steri• lity being one, and the opening of only a very few flowers at one time another. Both systems are operating in all 3 species examined. Diploidy versus polyploidy. The most interesting plants were certainly the members of the H. hyperborea complex. They came from 5 different populations scattered over southern British Columbia (Table :il) and, with the exception of one group already mentioned, were typical H. hyperborea plants with lax racemes and yellowish-green flowers and the chromosome count of 21 pairs established for the species. The exceptional green- flowered group was collected in Manning Park' in a very wet road• side ditch, intermixed with other weedy vegetation. The plants were about 2 ft tall, with stout stems and many-flowered racemes of deep- green florets all deliciously fragrant. Interspersed among the taller plants were more fragile green ones, to be mentioned again later^ and some ordinary H. dilatata plants. In gross morphology the small green plants differed from the larger ones only in size. It was not conclusively apparent until spring of 1973 that the population of stout green plants was tetraploid with 42 pairs of chromosomes in the cells. This count had also been deter• mined by Harmsen (19^3), by Ritchie (1956) and by Bent (1969),but not previously for southern British Columbia. The plants were at first thought to be green-flowered dilatatas, but further study seemed to place the population closer to H. hyperborea where it has been inclu^ ded in this paper. Hybrid origin of the polyploids suggested it- self, as the lip of the florets often approaches more closely that of H. dilatata (Table III), being variable in each plant examined. Also the upper, distal half of the petal is white, the lower half is green, and the lip is deep-green. The lip is fleshy as in typical H. hyperborea. In some florets the viscid glands also resemble those of H. dilatata, which makes the hybrid origin even more probable. As meiosis is regular (Fig. 22-3, 23-1) and the plants, with the exception of 103-6, seemed to cross well (Table VII), allopolyploid origin suggestd itself. The very disturbed habitat would favour the establishment of a hybrid population and an increase in chromo• some number often goes along with hybridity, especially in per• ennials (van der Pijl and Dodson 1969). Nevertheless the origin of the population is still a mystery, as no typical H. hyperborea plants were found in the imme• diate area. H. saccata as a possible parent was excluded from the start on morphological grounds. H. dilatata was present and could supply the genes of the one parent. What role did the more fragile green plants play in the population ? In contrast to the tall green plants they are diploids. Could they represent the primary hybrid ? Plant 104-2, one of the small green plants, was crossed with H. dila• tata and, to judge from the amount of seed produced (63 %), this cross was successful. The reciprocal cross was even more successful (71.5 %)• Meiosis in 104-2 is regular, a fact which casts doubt on it being the primary hybrid, as a fertile hybrid is not likely to pro• duce a fertile polyploid. As a whole the tetraploids were successful both as seed and as pollen parentr, again with the exception of plant 103-6. Some plants within a species seem compatible when crossed, while others repel foreign pollen by effective isolating mechanisms. The fact that the species have remained distinct, even when growing sympatrically may be related to this observation. In any case natural hybrids are not plentiful and except for intermediates in this population, none were found. The question remains where to place this small hybrid population. As an interesting sidelight to hybridity in the habenarias we find in Gray's New Manual of Botany (Fernald 1908) the following excerpts under Habenaria Willdo H. media (Rydb.) Niles: "Growing with H. hyperborea var. huronensis and H. dilatata and evidently a hybrid of them". Unfortunately the chromosome number of H. media is not known and we do not have H. hyperborea var. huronensis in our area. On the other hand Ames makes the following comment under "Notes on Habenaria"(1908); H. dilatata var. media (Rydb.) n.comb. (Limnorchis media Rydb.) " An examination of large quantities has convinced me, that Dr. Rydberg's Limnorchis media is simply a variety of H. dilatata, characterized by yellow-green flowers". The parentage of the Manning polyploid population is not known, but on the basis of morphology hybrid origin seems more probable than the population being just a green-flowered variety of H. dilatata. Only one other species is found in the immediate surroundings, but long distance dispersal by wind of the very light seed is common in the Orchidaceae and H. hyperborea has been found in other lo• cations in the Park. A few chance seeds of this species may have reacheai-'ithe area and finding a suitable habitat and a compatible species, may have produced some hybrid offspring. If such an event did take place, the original plants may not have been found or may have disappeared. Polyploidy often follows hybridization and results in allopolyploids which are fertile, as are the Manning plants. This fertility may go only as far as the polyploid generation and it may be that the primary hybrids from the seeds which I have produced by artificial pollination are partly or completely sterile. The perennial habit of Habenaria allows for vegetative reproduction, so that a small population of even a few hybrids can soon arise. As back crossing and introgression may be involved, morphological and probably genetic variability may result, to produce difficulties and confusion for the taxonomist. As the hyperborea influence in this small population seems stronger than the dilatata characteristics we may be justified in including this group in a H. hyperborea complex. It would take another summer's work dealing with just these plants to try to solve the problem. Unfortunately, when the area was visited in May of last year, a bulldozer had been through the ditch, wiping out most of the vegetation, but hopefully the plants will grow again from their root systems. Table V. Chromosome counts for H. dilatata, H, hyperborea and H. saccata, the species used in this study. 64a H. dilatata H. hyperborea H. hyperborea H. saccata n - 21 n - 21 n - 42 (4n) n - 21 101 Sumallo Lodge 104 Blow Down, 103 Blow Down, 111 Hemlock Valley Menning Perk Manning Park 102 Orchid Meadow 116 Mara Meadow 105 Allison Pass Menning Park Enderby Manning Park 112 Apex Kt.. 117 Mara Meadows 119 Jewel Lake Keremeos Enderby Kootenays j 118 Pemberton 120 Slocan Lake I Meadows Kootenays 1 124 Orchid Trail 121 McGillivray Manning Park Pass 125 Mt. Baker 1J0 Ladner salt- marsh 2n - 42 2n - 42 2n - 84 2n - 42 101 Sumallo Lodge 104 Blow Down 10J-6 Blow Down 105 Allison Pass Manning Park Manning Park Menning Park 112 Apex Mt. ' 104-2 Blow Down Hemlock Valley Keremeos Menning Park 135 Liumchen Val• ley •—1 Counts listed as "n" were determined from pollen mother cells in meiosis, those listed as "2n" from root tips. Figure 22. Chromosome counts made of. plcnts of H. dilatata, H. hyperborea and H. saccata . 1 H. dilatata, Metaphase I./n = 21 ' (x 3CC0). . 2 H. saccata Diakinesis, n ^ 21 (>'- ACCO). 2 H, hyperborea (An) n c A2 (x 40C0)„ . Figure 2^. Chromosome counts continued, 1 H. hyperborea (An). Camera lucida drawing from an irregular cnther with A pollen sacs, all A containing cells with A2 pairs of chromosomes. 2 H. hyperborea (diploid) Root tip count 2n - A2, (sketch), «\ myf *S * 1/ • • ® J* j1 Table VI. Mean values of fertility in crosses made. "Good" seed - seed with large well formed embryos. % of good seed - (supposed) fertility. Good seed x 100 •= % of fertility total seed counted "Good cross" - with many well formed embryos in the paraffin sections. CO I 1 emasculated H. dilatata H. hyperborea H. saccata open emasculated open pollinated Notes " cr* ~ v-r7 with cover intraspe-cif ic K. dilatata 78 % 66 % 62 % "good" seed = seed with 50 % ? dried up very few large well forced mean % of o 1 good cross, capsules erabrvos "pood" seed f no s~eed 2 crosses poor 2 crosses 4 crosses % of good eeed = fertilil (supposed' intraspecififc E. hycerborea O 53.4 % 9? % 7^.1 % dried up very few 60.4 % good seed x 100 = % capsules total seed counted •+ 6.6 % 6 crosses 2 crosses 2 crosses intraspecific H. saccata 27 % "good" cross = with 31.1 % 63 % 2 "good" dried up extremely numerous, well formed crosses, no few pods embryos in paraffin o value for 1 o: seed sections. t 5 crosses onl; no other seed 4 crosses Table VII. Crosses with H. dilatata as seed parent. All plants diploid except 103 (4n). Seed Pollen No. of peeds counted parent parent Penarks with with• total fmbryos out H. dila• open polli• open pollinations tata . nations 102a 611 306 917 66.6 lC2a-l 618 38 650 94 78 % mean 132-1 289 130 419 69 110-1 678 52 728 83 II* dilatata intrasnee i fic 130-3 110-4 471 288 759 62 110-4 130-3 422 239 661 69 66 H. hyperbo• interspecific rea 106-2 117 473 181 654 72.3 ovules smaller than usual. 101-3 117 stqred poll 1, no see'd. 112a-3 104-2 469 I 188 657 71.5 large embryos, pood cross 112 a-1 103 mixed 101 276 307 27.5 ji very little seed. pollen 112b 103-6 stalk broken no seed 506 173 679 74.5s ovules well deve- 130-3 103-4 I loped. 1 stalk broken, r o seed 3 sections show very 109-2 103-4 good development. 62 % mean saccata J interspecif ic. 110-3 133-3 173 509 682 25 only chaff 132-2 133 very j oor deve oprr.ent new plant,nopeed 122 121 excellf nt devel c pment, st; lk broker well established pi ants arprox. 5056 Table VIII. Crosses with H. hyperborea as seed parent. All plants diploid except 103 (An). Seed Follen No. of seeds counted % Perrarks parent parent with with• fertili• embryos out total ty H. hyperbo• open polli- Ct>en pollinations rea nations 117-1 451 356 807 56.9 prolific seed set 117-2 298 235 533 56 103-3 776 300 1.076 72 103-1 518 383 901 57.5 60.4 % mean H. hyperbo• intraspecific rea 103-3 103- 103x 633 19 652 97 prolific, well de• veloped seed 97 % mean H. dilatata interspecific 116 mixed pollen 448 307 755 59.5 117 112a-l 129 268 397 32 week seed parent 104-2 112a-3 472 278 750 63 good embryos,same % ase as in reci• procal cross, 103-4 mixed pollen 588 130 718 82 well formed embry• os 103-4 mixed pollen 3d develo ;ment, nc seed stalk broken I03-6 . 112b 243 546 789 31 >nly chaff, same re• sult as reciprocal cross 53.4 % mean H. saccata interspecific 103-3x 121 543 314 857 63.5 well developed seed 103-4 113 725 130 855 84.7 prolific seed set, large embryos. 7^ % mean Table IX. Crosses with H. saccata as seed parent. All plants diploid except 103 (An). Seed Pollen No. of seed counted Remarks parent parent with with• fertili• embryos out total ty H. saccata open polli• open pollinations nations 136 195 528 723 27 only chaff 27 % mean H. saceata intraspecif ic 13'*-3 very •rood development lar~e well developed embryos in sections 135-2 ^nixed pollen stored pollen us£d, slow flevelop- no seed, spike I, broken \ tnent H. dilatata interspecif ic. 105 112a-3 22? 403 630 31 small capsules with very little good seed 121 122 very good development no sped, stalk broken 133-1 mixed pollen (stalk broken,very slow development 133-2 110-3 good development no seed, all ova• ries sectioned 133-3 mixed pollen After 24 days large, well formed all ovaries sec- t ioned embryos 31 % mean H. hyperbo• interspecific rea 135-1 424 248 672 63 well developed '.' mixed pollen large embryos 10? slow de eloping, but norm 103-3 looking embryos all ovaries sec- t ioned 103-6 fertil zation after 18 days stalk broken 102b-l 63 ~/° mean emasculated 6.6 control nlant 134-2 open.without cover VI. GENERAL CONCLUSIONS. As indicated in "Materials and Methods" earlier,reciprocal crosses between H. dilatata, H. hyperborea and H. saccata were made. Besides the interspecific pollinations several intraspecific crosses within each of the three species seemeida-indicated and various controls were set up. The results of crosses and controls have been discussed in the previous chapter. It remains to point out some conclusions which seemed to arise from the present study. All pollinated florets produced seed pods. Some capsules be• came very large, especially the tetraploid ones, whereas the diploid H. hyper• borea plants had the smallest capsules and the least seed. A great deal of s seed seemed to ripen, all mature ovaries having produced some seed with well formed embryos. Empty seeds were found mostly after interspecific crosses and were foreshadowed by early breakdown in the cells of the embryo sac, noticeable from the 2-4 celled stage of the female gametophyte (Fig. 13- 1,2,3 ) onward, after arrival of the foreign pollen tube in the ovary. No difference could be detected in ovule and embryo development among the three species studied, whether pollinated naturally or artifi• cially. A comparison of ovule and embryo development in plants collected from the wild with development after interspecific pollinations gave no indication that hybrids develop in a different way from species, except for an increase in the amount of breakdown. In intraspecific crosses development from pollination to fertili- zation and from fertilization to maturation of the embryo is rapid. It is slower after interspecific crossings where, presumably, effec• tive isolating mechanisms function. The morphological differences between the species studied are slight, as are their habitat preferences; H. dilatata seems to prefer more open areas, often in full sun, whereas the two other species seem to seek out the protection given by other vegetation. Comparing my crosses with those made by Swamy (1948) on 9 Indian Habenaria species, with the work of Heusser (1914) on Hir- cinum himantoglossum and with that of Cocucci (1961) on a south- American orchid, all belonging to the tribe Orchideae, again I find similarities rather than differences. However, there is a slight difference between terrestrial orchids and epiphytes in ovule initiaa fcion:,-, which in the terrestrial habenarias takes place before arrival of the pollen, in epiphytic ones after arrival of the pollen. That gene flow exists and is in operation among the three Habenaria species is probable as indicated by the success of the artificial pollinations. The crosses seem to suggest that there is closer relationship between H. hyperborea and H. saccata than between either of these and H. dilatata. That the species are able to retain their discreetness in spite of their ability to cross can be explained by effective isolating mechanisms which are broken only occasionally. As a whole the three species studied ' follow closely the course of development found also in other members of their tribe. VII. SUMMARY. H. dilatata, H. hyperborea and H. saccata were crossed recipro• cally and intraspecific crosses were made within each species. Controls were set up to test the technique used, the presence of pollinators on the roof and the breeding system in operation in the three species. H. dilatata and H. hyperborea can be out-crossing or autogamous, depending on the early or late arrival of the insects. H. saccata is self- incompatible. Development of the embryos was studied after intra- and interspeci• fic crosses had been made. It was found that embryos of both species and hybrids show the same development stages, except for longer periods of time from pollination to fertilization and from fertilization to the maturation of the embryos in hybrid offspring. Comparing the development of artificially pollinated plants to those collected in the wild, I found only similarities. Seeds of mature ovaries of both intra- and interspecific crosses were counted and a mean value of fertility established. As expected, the intraspecific crosses yielded large amounts of seed with seemingly healthy, well formed embryos. Interspecific crosses gave better results when H. hyperborea was crosses with H. saccata. indicating closer relationship, then when H. dilatata was crosses with either of the two other species. 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