March 1997 North American Native Orchid Journal

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NORTH AMERICAN

NATIVE

ORCHID JOURNAL______________________________________Volume 3 March

Number 1 1997a quarterly devoted to the orchids of North America

published by the

NORTH AMERICAN NATIVE ORCHID

ALLIANCE* * * * * *

* * * * * *IN THIS ISSUE:The Genus Cypripedium


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NORTH AMERICAN NATIVE

ORCHID JOURNAL(ISSN 1084-7332)

published quarterly in

March June September Decemberby the

NORTH AMERICAN NATIVE ORCHID ALLIANCE,

Inc.

a group dedicated to the conservation and promotion of ournative orchids

Editor: Paul Martin Brown

Assistant Editor: Nathaniel E. ConardEditorial Consultants:

Philip E. Keenan

Stan FolsomProduction Assistant:Nancy A. Webb

The Journalwelcomes articles, of any length, of both a scientificand general interest nature relating to the orchids of NorthAmerica. Scientific articles should conform to guidelines such asthose in Lindleyanaor Rhodora. General interest articles and notesmay be more informal. Authors may include line drawings,and/or black and white photographs. Color inserts may bearranged. Please send all inquiries or material for publication tothe Editor at PO Box 772121, Ocala, FL 34477-2121 (mid June -

August: PO Box 759, Acton, ME 04001-0759).

1999 Membership in the North American Native Orchid Alliance, which includes a subscription to theJournal, is $26 per year forUnited States addresses, $29US in Canada and $32US otherforeign countries. Payment should be sent to Nancy A. Webb, 84Etna St. Brighton, MA 02135-2830 USA. Claims for lost issues orcancelled memberships should be made within 30 days.


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NORTH AMERICAN NATIVE

ORCHID JOURNAL

Volume 3 March

Number 1 1997

CONTENTS

NOTES FROM THE EDITOR

1

THE NEED FOR GLOSSARIESThe Slow Empiricist

3

THE GENUS CYPRIPEDIUMJohn W. Doherty

Guest Editor: William Steele

5

GLOSSARY

121

LOST & FOUND129LOOKING FORWARD

June 1997134

Color PlatesFig. 1 Cypripedium arietinum; Fig. 2 Cypripedium candidum p.105

Fig. 3 Cypripedium xandrewsii nm. andrewsii; Fig. 4Cypripedium xandrewsii nm. Favillianum p.105


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Fig. 5 Cypripedium parviflorum var. makasin; Fig. 6Cypripedium parviflorum var.pubescens p.107

Fig. 7 Cypripedium kentuckiense p.108

Fig. 8 Cypripedium montanum); Fig. 9 Cypripediumparviflorum var.pubescens flat-petalled form from Red Deer,

Alberta p.109Fig. 10 Cypripedium reginae; Fig. 11 Cypripedium reginae pale

form p.110Fig. 12 Cypripedium arietinum; Fig. 13 Cypripedium

xandrewsii Gunter Preusse CCM/AOS p.111

Fig. 14 Cypripedium xandrewsii ;Fig. 15 Cypripedium

parviflorum var.pubescens p.112Fig. 16 Cypripedium parviflorum var. makasin Sara Conds

Flower CCM/AOS p.113Fig. 17 Cypripedium reginae p.114

The opinions expressed in theJournal are those of the authors. Scientificarticles may be subject to peer review and popular articles will be examined

for both accuracy and scientific content.All drawings inthis issue are by Stan Folsom

Volume 3, number 1, pages 1-137; issued March 1, 1997.Copyright 1997North American Native Orchid Alliance, Inc.

COVER: Cypripedium reginae by Stan Folsom


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1

NOTES FROM THE EDITOR

This issue is a major departure from ourprevious issues, in that we are devoting the entire

Journal to one comprehensive work on the genusCypripedium. All of the North America species andmany additional species from other parts of the worldare included. To better understand the scientific termsin the article, the Slow Empiricist suggested that aglossary might be helpful. After careful consideration,and with the authors permission, I encouraged the

Slow Empiricist to supply this issue with such aglossary. You will find it at the end of the majorarticle, but I have included the Slow Empiricists

explanations at the conclusion of this introduction. Inaddition there are several longer footnotes to assist inclarifying some of the terms and concepts.

This issue would not have been possiblewithout the help of several people. Bill Steele

generously gave of his time as guest editor and wentthrough Johns manuscript in fine detail, making

several helpful suggestions. Stan Folsom and assistanteditor Nat Conard both spent considerable timehelping to edit and format the issue. Stans line

drawings have been used throughout as well as Johns

color photographs. It has been an enormous task but


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well worthwhile. I have not quite decided if onearticle is simpler to prepare for theJournal as opposedto the usual number of articles and authors the Journalincludes, but I enjoyed the challenge!

I trust you will find the work on the genusCypripedium elucidating and the glossary helpful.

Paul Martin Brown

15 Dresden Street

Jamaica Plain, MA 02130-4407

e-mail:[email protected]


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3

THE NEED FOR GLOSSARIES

The Slow Empiricist

A few articles back (Jargon? 2(3):228-238), Icovered many terms to help beginners understandsome of the words that the more scientific members ofthe orchid world use in their regular conversation. Asyou become more familiar with the language that is

used, the meanings become much clearer and aspecificity tightens the descriptions. When thefollowing splendid article came in about the entirerange of the genus Cypripedium in the world, andNorth America in particular, I was excited about thepossibilities for adding new knowledge to myunderstanding. As I read through the article, I realizedthat, to fully appreciate it, a glossary seemedinvaluable. I pulled out the words or terms that I

needed to understand better, let the computeralphabetize them, and used the built-in dictionary togive me precise definitions for the words. With PaulMartin Browns help, we further pulled out words andphrases to send to the author so he could explainexactly what he had in mind with his use of the terms.(Incidentally, he was thrilled that people wanted to


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THE GENUS CYPRIPEDIUM

a botanical and horticultural overview

John W. Doherty

Introduction

Terrestrial orchids are currently enjoying a

surge in popularity among horticulturists andorchidophiles around the world. This is due in largepart to increased accessibility to the ever expandinginformation base regarding this intriguing group.However, it is to advances in propagative technologythat must be given most of the credit for makingterrestrials so much more widely available. The*genus Cypripedium remains one of the most beloved,interesting, beautiful and sought after members of this

decreasingly exclusive group and it shall be thesubject of this paper.

A circumglobal genus of the subfamilyCypripedioideae, species can be found in the majorityof *temperate climes in the northern hemisphere, withthe exception of the Cypripedium irapeanum group,


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Doherty: The Genus Cypripedium

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Mexican natives, and the recently described C.subtropicum from Tibet. With as few as 30 differentspecies, or as many as 50, all remain *deciduousduring a pronounced cold period in the winter months.

The majority of species withstand below freezingtemperatures for a prolonged period. *Tropicalrelatives include the other slipper orchids, in the*genera Paphiopedilum, Mexipedium,Phragmipedium, and Selenipedilum.

Pollination in North American Cypripedium hasbeen extensively studied by Nilsson (1979), amongothers, who showed that it is achieved throughdeceptive floral lures, as opposed to floral rewards

which are conspicuously absent in this genus. Flowercolour and false nectar guides surely aid in deceptionof the various species of bees that are knownpollinators; however, the most intriguing adaptation isthe use of odour. Fragrance emitted by mature flowersis surely of a complex and incompletely understoodnature; however, it is likely that it is partiallycomposed of food-based attractants. Furthermore, ithas been found that a series of acetates dominates the

fragrance and bears significant chemical *homology tocertain bee *pheromones. Honeybees (Apis mellifera)have no fewer than 36 documented pheromones, withothers remaining unknown, each with a specificpurpose in the bee communication process. It isreasonable to assume that other bee species, and evenother genera, have a comparably complex method of


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communication. Besides foraging for food, bees alsoseek out potential future nest sites. Potential nest sitestend to be somewhat cavernous, as may the lip of aslipper orchid appear to be. These potential sites are

marked with a particular pheromone indicating such,which would then work to attract *conspecifics,prompting further investigation. It is suggested thatthis genus, and therefore perhaps other slipper orchids,have tapped into this behavioral pattern of these

bees as manifested by their floral structure as well asby the pheromonal composition of their fragrance.Such would truly be a marvel of evolutionaryprocesses, as several bee species have been shownresponsible for pollination: Bombus and Psithyrusashtoni queens, *halictid bees, Andrena spp. and

Lasioglossum spp., as well as Megachile (see Catlingand Catling, 1991, for an excellent review of alldocumented North American orchid pollinators), eachprobably with its own unique pheromone system.This adaptation would necessitate fine tuning at thespecies level (for which orchids are famous) to ensureefficacy of prolonged attraction to both the structureof the lip, and the pheromone-tainted fragrance.

Unfortunately, this tidy hypothesis reliespartially upon conjecture and definitely requiresfurther investigation, at a molecular level, of theorchids as well as of the bees in question, in order toconfirm pheromone/fragrance homology, in additionto the behavioral role of the pheromone in question.


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There are a great many misconceptionsregarding this genus as a whole; this paper is anattempt to clarify some of those misconceptions and

hopefully further the general understanding of thisgroup. To achieve this, there will be three sections:Part I will provide a brief *taxonomic overview of thegenus; Part II will give a brief explanation of*mycorrhizal fungus and its integral role in thedevelopment of these orchids; finally, Part III willlook at some current stances on propagativetechnology and *cultural practices.

This paper is by no means intended to act as a

definitive reference, but rather as a botanical,*mycological, and horticultural overview. Thereferences listed at the end of each section are felt tobe some of the best available, and are recommended toany reader with a specific interest in that area.

References

Catling, P.M., and V.R. Catling. 1991. Pollination in North

American orchids. Lindleyana. 6:187-210.

Nilsson, L.A. 1979. Anthecological studies on the ladys-

slipper, Cypripedium. Bot. notiser. 132:329-347.


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Part I

A Brief Taxonomic Overview

The most recent treatments of this genus are byCash (1991), and Perner (1996), who provide adetailed and thorough *infrastructure, into which theyhave, for the most part, neatly fit the various species.Cashs outline will be followed here. Species listedare those with which I have had some sort of first handexperience, preferably those that I have observed inthe wild. The kind of knowledge to be gained from

direct habitat observation cannot be surpassed by anynumber of books. For species not listed, readers aredirected to Cash (1991).

SubgenusCriosanthesCypripedium arietinum R. Brown (Fig. 1)

Rams-head ladys-slipper

This species has been the source of sometaxonomic controversy as it was once thought to differsignificantly enough from other species to warrant its

own genus, proposed to be Criosanthes. Reasons forthis exclusion include entirely unfused *sepals, a*spurred lip, and *anther-like *staminode. Thesefactors alone do not warrant a separate distinction;however, it has been suggested that its ephemeralbloom of only a day or two represents a significantevolutionary step in pollination biology and therefore


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Doherty: The Genus Cypripedium: Part I

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justifies taxonomic distinction. In reality, the bloom isnot at all ephemeral. Under cultivation, I have foundthat flowers remain open for up to a week underaverage to above average temperatures; even longer if

kept cool.

Cypripedium arietinum has a limited rangecentred about the Great Lakes region. The speciesoccupies two distinct habitats, the first being coolcedar swamps, often floored with sphagnum moss.Here it is usually found singly and infrequently.However, extremely large individuals can develop.The second habitat can be either sandy dunes orlimestone *barrens near shorelines, where offshore

breezes provide necessary cooling. It is in this habitatthat C. arietinum will develop huge colonies withindividual, smaller-statured and infrequently clumpedplants numbering into the hundreds.

Cypripedium plectrochilon Franchet is avicarious species1, native to a small region in Asia,

1 Cypripedium arietinum and Cypripedium plectrochilon are vicariousspecies. They are remarkably similar in nearly every respect, except that theyare found in drastically different parts of the world. They do NOT result fromconvergent evolution, as they most likely arose from common stock. Nor dothey exhibit divergent evolution, as they have not significantly diverged, orbecome different. Rather, they have evolved as parallel species, andmaintained their similarities, most likely due to very similar environmentalpressures experienced in each of their geographically distinct habitats.


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Cypripedium arietinum R. Brown

Rams-head ladys-slipper


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and is felt by some to be conspecific with C.arietinum. Sing-Chi (1983) and Perner (1995) havereviewed the taxonomic issues concerning this pair (aswell as another vicarious pair, C. flavum and C.

reginae) and feel C. plectrochilon to be sufficientlydifferent to warrant species level distinction. Perhapsthe closely matching distribution patterns, as well asvery similar ecological preferences shared by eachpair (but not between pairs), combined with therelatively primitive structure of these four species,suggest evolution from a common stock, whichformerly had a common *areal range. Furthermore,The modern distribution patterns of the vicarious

species are by no means fortuitous, but result fromsimilar migration patterns. (Sing-Chi, 1983). The*phytogeographic implication of these relationships isa fascinating topic on which much light could be shedthrough use of modern molecular genetic analyses.

SubgenusCypripedium *AllianceCypripediumCypripedium calceolus L.

Eurasian yellow ladys-slipper

Cash (1991) lists all of the North American

variants, as well as the Eurasian plants, under thisspecies. Sheviak (1994, 1995, 1996a, 1996b) hassince reclassified the North American species as aseparate *complex under C. parviflorum;consequently they shall be discussed under thatheading. Cypripedium calceolus, however, remainsthe name for the Eurasian species. This species is


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readily distinguished in the trade from its NorthAmerican relatives by its white staminode. Preferring*calcareous soils, this species can become locallyabundant and enjoys a variety of shaded or semi-

shaded habitats; however, the one common featureappears to be a preference for an alkaline *substrate.

Cypripedium candidum Muhl. ex Willd.2 (Fig. 2)

Small white ladys-slipperThis species is native to northeastern North

America and probably most commonly distributed inMichigan. Its reputation for sunny habitats is welldeserved as it is never found in much shade, unless atthe border of a mucky meadow that is gradually being

overgrown, where it will not flourish for much longer.Easily the most sun-loving of all our native species, C.candidum consequently has a high moisturerequirement. Water can often be found in a small holedug adjacent to healthy plants. Black, mucky soilseems to be preferred; however, it has also been foundin heavy *clay substrates. Native to prairie-typeenvironments, this species can thrive under controlledburn techniques used in management of prairie

conservation areas. If allowed to grow continually,prairie grasses can choke out thousands of plants to ahandful in a matter of one season. Carefully timed

2 Muhlenberg was the first to name this species but did not publish a validdescription. Willdenow was the first to validly publish the name according totheInternational Rules of Botanical Nomenclature. His name is precededby an ex to indicate that it was taken from Muhlenbergs description.


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burns can astonishingly replenish such populations inas fast a time.

It is suggested by Cash (1991) that this species

...is to all appearances an *alba (white flowered) formof the small yellow ladys slipper, Cypripediumcalceolus var. parviflorum [parviflorum var. makasinor parviflorum var. parviflorum]. Unfortunatelythere are a number of *morphological and ecologicaldifferences between the two that point to the contrary.The leaves ofC. candidum at flowering remain furledand held tightly around the stem. This may be in aneffort to reduce high *transpiration rates that wouldsurely be encountered at such an early stage in the

season where neighbouring grasses are not providingmuch-needed shelter from direct sun. The leaves ofC.

parviflorum are much more expanded at flowering. C.candidum is very rarely two flowered, whereas C.

parviflorum is frequently so. C. candidum does notproduce *albinistic flowers. Petals are oftenpigmented with browny greens; the interior of thepouch is often marked with red. It is seen, however,that plants in extremely high light conditions have

bleached looking flowers where normally pigmentedflowers are pure white and yellow, mimicking*albinism. The greatest reason to discount thishypothesis, however, is that of habitat differences.Neither species is ever prevalent in the others habitat.The only time one could be found in the habitat of theother is in an overlap situation, where a much shadier


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region, such as a woodlot, is found immediatelyadjacent to an open type prairie/meadow. Such apronounced habitat contrast is the strongest argumentagainst the alba-race hypothesis.

In addition to floral pigment, plant stature alsovaries according to levels of sunlight. The shortestplants are always found in sun, whereas taller plantsare found in shadier transition zones. This is not, asone might expect, genetically fixed; rather plant heightis an extremely variable characteristic highlydependent on a number of environmental variables,sunlight being one of them. This principle is not at allunique to this particular species, but rather a

characteristic that can be generalized to most of thegenus. The environmental factor certainly confoundstaxonomic treatises that attempt to use plant stature asa distinguishing character for a certain species,particularly in the Cypripedium parviflorum complex.There are, of course, limits to a plants potential height

response, not entirely discounting the use of height in*taxonomy.

Cypripedium xandrewsii Fuller3 (Fig. 3, Fig. 4)

is a natural *hybrid (*nothospecies) that occurs in thepreviously described situation where there is habitatoverlap between C. candidum and C. parviflorum andits varieties, var. makasin and var.pubescens. The net

3 When a hybrid has been given a name, this is indicated by placing an x(denoting multiplication) before the specific epithet, as in Cypripediumxandrewsii.


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result is a blend of both parents that can sometimesyield striking combinations, the most notable beingwhite-lipped multiple flowers with dark chocolatebrown petals, held above the foliage. There are,

however, all types of intermediates with pouch coloursranging from pure white to pale yellow, and petalsfrom yellow green to chocolate brown. Plant statureand *floral stance are comparably variable.Cypripedium xfavillianum is the previous name usedto describe the nothospecies resulting from a crossbetween C. candidum and C. parviflorum var.

pubescens before the yellow complex was reclassifiedto a single species, necessitating the use of a singlename. The correct current nomenclature for the

hybrids would be Cypripedium xandrewsii Fuller*nm. xandrewsii and Cypripedium xandrewsii Fullernm.favillianum (Curtis) Boivin. A third *nothomorphhas also been described, Cypripedium xandrewsiiFuller nm. landonii (Garay) Boivin (C. xandrewsiinm. xandrewsii x C.parviflorum var. makasin).

Cypripedium cordigerum D. DonOf all the Asian species, this high-elevation

native of India is most closely related to theCypripedium parviflorum complex. Its floral structureappears to be quite similar; however readers arereferred to Cash (1991) for specifics regarding habitatinformation and distribution.


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Cypripedium kentuckiense

Kentucky ladys-slipper


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Cypripedium kentuckiense C. Reed (Fig. 7)

Kentucky ladys-slipperCashs structure lists this as a *variety of

Cypripedium calceolus (C. parviflorum); however,

there are a number of morphological characteristicsthat distinguish it as a separate species in addition to,most importantly, its discrete habitat range preference.C. kentuckiense is a southern species, with its rangedipping south into Texas, with Kentucky actuallyrepresenting the northern limit of its distribution. It isa warm, long-season grower and cannot be forced intothe shorter seasons experienced up north; it is the firstup and last down. It can be found in shady locations,often near a source of running water, and apparently it

is often inundated with water. The ability to survivesubmergence alone likely constitutes reason enoughfor species distinction; however, floral characteristicslend further support.

Plants are always large statured. Flowers arealways very large, the smallest being as large as, if notlarger than, the biggest of the C. parviflorum complex.The pouch is, on average, the size of a chicken egg

and ranges in colour from butter yellow to pure white.There are seldom two flowers, and floral stance ischaracteristic. Stems often appear as though they arebowing in an effort to support the flowers. The*dorsal sepal is rarely vertical, usually drooping farover the pouch. Flowers give the impression that they


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are hanging. Petals can become quite long, thin, andcorkscrewed.

Cypripedium montanum Dougl. ex Lindl. Fig. 8)

Mountain ladys-slipperThis is a montane species with a specifichabitat: it prefers open woods, either moist or dry, athigh elevations. This results in its restricted range inthe Rocky Mountains in British Columbia south intoCalifornia. Plants are frequently two-flowered andsometimes three-flowered, with a white lip and petalsof a softer brownish green than that seen in C.candidum or the C. parviflorum complex.

Again, Cash (1991) suggests this to be an albarace ofCypripedium parviflorum. Unfortunately, thepreviously mentioned characteristics make thisimpossible. First of all, it is not albinistic, for thepetals show significant brown colour in addition tored/purple pigment inside the pouch. It is rarely singleflowered. However, most importantly, its specifichabitat preference distinguishes it from C.

parviflorum. There is occasional habitat overlap,

however, resulting in the production of C.xcolombianum Sheviak, a natural hybrid between thetwo.

One would imagine a white-flowered race of aparticular species to exist somewhere within the rangeof that species. Originating from a single albinistic


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mutant, differing from the rest of the populationsignificantly enough to warrant attention from adifferent pollinator, that individual would proliferateand eventually establish its own separate population.

Such cannot be said for Cypripedium montanum norfor C. candidum, as they both exhibit a high degree ofecospecificity such that their habitat of choice isnormally very different from that ofC. parviflorum. Itis possible, and likely probable, that these two speciesdid initially arise from an older precursor of the C.

parviflorum complex; however, they have beenthrough the evolutionary gears of *speciation longenough to become two very distinct and different taxa.

Cypripedium parviflorum Salisb.

Yellow ladys-slipper

Without a doubt this complex has attracted thegreatest amount of taxonomic attention of any in theentire genus. This is due in large part to its hugeNorth American distribution, and consequentvariability. It is likely, however, that the astoundingability of plants to change morphologically depending

on environmental conditions is the single greatestconfounding factor. Traditionally, classification isbased on characteristics such as plant stature, leaf sizeand stance, floral size, colour and morphology, andmost importantly with the slipper orchids, staminodeshape. In subspecies where this last characteristicremains fairly consistent throughout the entire range


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of this complex, one is required to rely on thepreviously listed features. Unfortunately, thesefeatures can vary tremendously within an individual.Plants from open, sunny situations tend to be shorter

statured, with smaller, narrower leaves and smaller,paler flowers (some may have larger flowers insunnier locations). Conversely, the same plant facingshady woodland conditions might have larger, broaderleaves on a tall stem. Flowers may be much larger anddarker coloured. This stunning environmentaladaptability has clearly provided this complex with astrong competitive advantage, perhaps explaining thewide geographic distribution. It makes excellentreproductive sense to be able to adapt to an

immediately open situation as the result of adisturbance, or a gradually shaded situation, as themajority of orchids are not found in *static, climaxstage habitats4. It is, therefore, the competitivestrength of this complex that weakens taxonomictreatments. Sheviak (1994, 1995, 1996a, 1996b)appears to be the first to demonstrate this principle in

4 A climax stage habitat is any habitat occupied by species characteristic ofthat stage, their most notable characteristic being the ability to survive in theshade of their parents until they are large enough to displace or replace

their forefathers. This is in particular reference to tree species. Herbaceousspecies, such as orchids, can be found in these habitats as well. Climax stagehabitats are very static (as opposed to dynamic) and represent the top of thehabitat evolutionary ladder, assuming some extreme force, such as a forest

fire, earthquake, or human encroachment does not act on the habitat andchange its balances.


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cultivated plants; his treatment of the complex isoutlined here.

Cypripedium parviflorum var. parviflorum

Southern small yellow ladys-slipperThis variety is restricted in range to the easternUnited States and is found in the driest habitat of thethree varieties. It has medium size flowerscharacterized by dark brown petals that tend to holdtheir colour along the entire length.

Cypripedium parviflorum var. makasin (Farwell)Sheviak (Fig. 5)

Northern small yellow ladys-slipperThis is the northern, smaller flowered variety

that prefers wetter sites, often in black, mucky soil.Again, the petals tend to be dark brown, perhapsbreaking towards the middle of the flower intoblotches, revealing the greeny-yellow basal colour ofthe petals.

Cypripedium parviflorum var. pubescens (Willd.)Knight (Fig. 6)

Large yellow ladys-slipperThis is the most variable of the three varieties.

It can have dark brown petals, however, usually not ofthe intensity seen in the other two varieties. Colourranges from pure yellow, to yellow green, to greenbrown. Flowers tend to be significantly larger. Plant


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stature has the greatest potential here to be large;however, very diminutive plants can also be found.

An apparently dwarf strain within the complex

found on windswept shorelines in Newfoundland wasat one point given the varietal status var .planipetalum, on account of its flat petals, thought tobe unique to that population. Based on the previouslydiscussed environmental adaptability of this complex,this varietal distinction has been abandoned. Anumber of sources have reported flat-petalled plantsfar removed from the Newfoundland populations, inplaces such as New York. Experience with cultivatedplants confirms this. Figure 9 shows a flat-petalled

plant, very similar to those thought to be var.planipetalum. This particular individual was collectedin Alberta amongst a normal-flowered population.Strangely enough, at the time of collection, it was nodifferent from any others in that population as itspetals were twisted. The flower came up flat-petalledthe following season, likely the result of an alteredenvironmental factor.

Please see Cash (1991) for additional membersof this alliance: Cypripedium henryi Rolfe, C.microsaccus Kraenzl.and C. shanxiense S.C. Chen.


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Cypripedium fasciculatum

Clustered ladys-slipper


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Alliance CorymbosaCypripedium fasciculatum Kellogg ex S. Watson

Clustered ladys-slipper

This native of the northwestern United States

can be found at high elevations in *coniferous forestsand shrubby thickets. Its two to four plus flowers aresmall, greenish-brown and, according to Fred Case,look like peeled grapes. They tend to droop, pointingtowards the ground, making observation difficult;however this is likely in an effort to attract specificpollinators. Successful pollination results insubstantial straightening and lengthening of the stem,thereby enhancing seed dispersal.

AllianceMacranthaCypripedium macranthos

5 Sw.Cypripedium macranthos is a widespread

species typifying a very large complex requiringfurther taxonomic clarification, as there are a numberof closely related species whose interrelationshipsremain to be fully worked out. A major barrier toclarification is a political one, as much of its range,along with related species, is within China. This

complicates and reduces the opportunity for fieldobservation. As a result, there is much to be learnedabout this entire group.

5 Cypripedium macranthos Sw. is a synonym for C. macranthum Sw. andthe preferred spelling. See Cribb (1994) and Perner (1966). PMB


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Preferring cool climates, Cypripediummacranthos can be found in high-light situations ineither deciduous shade or *montane grasslands. Onceconsidered a separate species, var. rebunense is a

closely guarded Japanese population of pure yellowflowers. A deep, solid, purple-red form is known asvar. hotei-atsumorianum. The Asian range of C.calceolus overlaps in some regions with C.macranthos; the natural hybrid between the two, C.xbarbeyi, occurs in these situations. Apparently thishybrid exhibits extensive variability, with coloursranging from pure white to deep wine red.

Please see Cash (1991) for remaining members

of this alliance: Cypripedium amesianum Schltr., C.corrugatum Franch., C. fasciolatum Franch., C.himalaicum Rolfe, C. lanuginosum Schltr., C.speciosum Rolfe, C. thunbergii Bl., C. tibeticum Kingex Hemal., C. ventricosum Sw., C. wilsoni Rolfe,andC. yunnanense Franch.

Alliance FlabellifoliaCypripedium acaule Ait.

Pink ladys-slipper; stemless moccasin flowerLikely the most *acid-loving of all our native

species, C. acaule has a very wide distribution fromnortheastern Alberta, into the Northwest Territories,over to the Atlantic coast and as far south as Georgia.There are two general and very different trends withrespect to habitat. The first is high and dry woods


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Cypripedium acaule

Pink ladys-slipper; stemless moccasin flower


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which are often sandy. The second habitat issomewhat boggy, usually with some type of sphagnumnearby. These two apparently divergent habitatsmaintain an acid medium as their common

denominator. White-flowered forms of this speciesexist and are most prevalent in the northeast.

Cypripedium farreri W.W. SmithThis large-flowered dwarf species *colonizes

limestone cliffs in the Yunnan province of China andperhaps represents an interesting evolutionary linkbetween members of the genus. Unlike othermembers of this alliance, a number of leaves areproduced alternately on a vertical stem, as opposed to

two *sub-*opposite leaves close to the ground (as inCypripedium acaule)or a short distance from it (as inC. formosanum and C. japonicum). In addition, theflowers completely lack the central *labellar fissurecommon to these three species. Interestingly enough,the pouch *orifice is toothed and somewhat *everted,reminiscent of C. guttatum, yet there is sufficient*infolding to link it to the alliance Cypripedium.These differences urge its inclusion in a different

alliance. However, it has been kept here for lack of amore appropriate place, as opposed to a distinction allits own.

Cypripedium formosanum Hay.Closely allied to C. japonicum, this species is

found in constant moisture on steep *shale slopes in


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Taiwan, where it grows in bright shade. Conditionsapparently are acid. The paired leaves are unique inthat they appear as though someone neatly trimmedthe ends off with a pair of scissors.

Cypripedium margaritaceum Franch.The paired leaves of this species closely hug the

ground and are marked with purple-brown spots, acharacteristic unique to this species and its closestrelative, Cypripedium lichiangense (previously C.daliensebefore Cribbs recent reclassification). It is amontane species preferring moist, well drained slopesin pine or mixed pine forests (Cribb & Chen 1994).

See Cash (1991) for remaining members of thisalliance: Cypripedium bardolphianum W.W. Sm. &Farrer, C. debile Reichb. f., C. ebracteatum Rolfe, C.elegans Reichb. f., C. japonicum Thunb., C.micranthum Franch., C. palangshanense Tang &Wang, and C. wumengense S.C. Chen.

Alliance GuttatumCypripedium guttatum Sw.

Spotted ladys-slipperCypripediumyatabeanum Makino

Yellow spotted ladys-slipperThere exists some confusion in regard to these

closely allied species. This can be largely attributed tothe natural hybrid formed between the two,Cypripedium xalaskanum P.M. Brown. Information


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recently provided by Paul Martin Brown, based onextensive field experience, has clarified this situation.Cypripedium guttatum is a small woodland species,producing one (or rarely two) white flowers with

cranberry blotching. Flowers are held above two orthree *alternate leaves. The underground *rhizomecan be quite extensive and wandering, producing alarge number of stems, or individual leaves over alarge area. In contrast, C. yatabeanum is a muchlarger-statured plant in every respect. Flowers arelarger and basically yellow, with rusty-brown to greenmarkings. Petals are shaped differently, appearingsomewhat pinched near the tips. In addition tomorphological differences, habitat ranges and

preferences are distinct as well. Cypripediumyatabeanum is primarily an Asian species, found inNorth America only on Kodiak and neighboringislands. It has apparently never been found on themainland, and prefers much sunnier locations.

Cypripedium xalaskanum, and the results of itssubsequent interhybridization, can be found on boththe Alaskan mainland and the islands. It exhibits the

entire range of intermediates between the two extremeforms, represented by the two different species.

Floral structure of these species is unique in thegenus. The front margins of the lip are polished andeverted, without any infolding characteristic of thegenus. This feature is very reminiscent of


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Cypripedium guttatum

Spotted ladys-slipper


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Cypripediumyatabeanum

Yellow spotted ladys-slipper


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Paphiopedilum, leading some to suggest that thisspecies might represent a possible evolutionary linkbetween the two genera. When considering*vegetative and geographic similarities, this linkage

becomes very unlikely. It is more reasonable toassume that this is an example of convergentevolution6.

Plant characteristics having a pronounced effecton reproductive strategies of a species, such as floraltraits, tend to be rather *dynamic as minor alterationscan have drastic outcomes on population trends. It is,therefore, highly unlikely that a dynamic feature of apopulation7 such as lip curvature would be

maintained over the extremely long evolutionaryperiod where vegetative changes might occur from thetropical to temperate plant habit (not temperate to

6 This is an evolutionary process whereby two organisms of differing originsevolve towards a similar feature. A good example of this is the eye of theoctopus in comparison to the human eye. The eye structure in an octopus isremarkably similar to that in humans. It is obvious that humans did notevolve from octopi and maintain this trait. Millions of years ago therespective ancestors branched off of the evolutionary path in response todifferent environmental pressures. At some point, both "lineages" were facedwith similar environmental pressures in some certain, particular aspect,resulting in the gradual development of very similar structures.

7 Some features of a population change very little over time, such as stemthickness (assuming constant environmental conditions!), leaf shape, or rateat which a particular enzyme functions. These can be loosely referred to asstatic or very slow moving traits. A dynamic trait is something that changesrelatively rapidly from generation to generation, such as floral colourpatterns. Dynamic traits are not as "fixed as static traits.


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tropical). If one were to search for relatednessbetween Cypripedium and other genera based entirelyon floral structure (a hazardous approach), it would bebest to look to members of the genus Phragmipedium,

as they exhibit very similar infolding of the lip soprevalent in Cypripedium. It is most advisablehowever, to look to tropical intermediates such as C.irapeanum or C. subtropicum and their relationshipwith other genera, including Selenipedilum,in order tounderstand fully the genetic organization within this

subfamily.

AllianceObtusipetalaCypripedium californicum A. Gray

California ladys-slipperThe most stunning feature of this yellow and

white species is its production of small flowers at*leaf bracts along the length of the stem, a featureunique in the entire genus. It has a very small range innorthern California and southern Oregon and is foundin open woods, usually near streams, often in the

company of the cobra lily (pitcher plant),Darlingtonia californica. Plants tend to form clumps

and exhibit very little floral variation.

Cypripedium dickinsonianum Hagsater

Dickinsons ladys-slipperCribb (1996) has apparently cleared up a degree

of confusion regarding this Central American groupthat has long been considered a single species, the


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Cypripedium californicum

California ladys-slipper


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Mexican yellow ladys-slipper, Cypripediumirapeanum Llave & Lex. Morphological andgeographic differences, however, pointed towards thedelineation of three different species: C. irapeanum,

C. dickinsonianum and the downy yellow ladys-slipper, C. molle Lindl. Distinctions between thethree are based on plant stature, leaf size and shape,floral differences and geographic distribution. Theentire group is found throughout southern Mexico andinto Guatemala and Honduras. C. irapeanum can befound in the drier parts of valleys, going without rainfrom October to May and experiencing summertemperatures from 30-35 C, dipping to less than 5 C inthe winter. It prefers clayey soils in pine/oak forests.The flowers of this group are truly stunning. Mediumsized to extremely large bright yellow flowers areproduced consecutively, atop showy stems that canvary in height from 30 cm to 1.5 m. Flowers are veryreminiscent of Paphiopedilum armeniacum, whereasflowering habit, unique in this genus, is characteristicof species in the Cochlopetalum group ofPaphiopedilum, such as P. primulinum or P.glaucophyllum.

Cypripedium passerinum Richards

Sparrows-egg ladys-slipperThis is a northerly species, found from Alaska

eastward into Quebec, remaining in the northernregions of the provinces along the way, with theexception of Alberta and British Columbia, where the


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cooler temperatures afforded by the increasedelevation of the Rockies permits its occurrence atlower latitudes. The plant can be found in bright,open clay woods, usually with some source of water

nearby, or in gravelly outwash plains. Flowers aresmall and self-pollinating, the only North Americanspecies in the genus with such a reproductive strategy.Consequently, there is very little variation within thespecies. *Ovaries are very swollen at blooming.Shoots are produced along a rhizome with a strongtendency to branch, and sprout from *dormant eyes.Frequently found accompanying this orchid in the

western part of its range is the small round-leaved

orchis,Amerorchis rotundifolia,often numbering intothe hundreds.

Cypripedium reginae Walter (Fig. 10)

Showy ladys-slipperThis moisture-loving native of the northeast is

often prevalent in the Great Lakes region. It can befound in boggy, open forest edges in *neutral toalkaline black mucky soils. Sun is a critical factor inthe *population ecology of this species. It seems to

require direct sun for only a portion of the day, as it isseldom found in constant sun or full shade. It prefersintermediate areas, such as boggy forest borders, orclearings within such a forest. It is this type ofenvironment that Cypripedium reginae will quicklycolonize, forming very large groups of stunningplants. There is a direct correlation between the


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amount of sun received and population size; as soonas its clearing or edge begins to grow in, the plants gettaller from season to season, until eventuallyindividuals begin to disappear completely. Copious

moisture is an additional requirement of this species,and it is often found accompanied by tamarack, Larixdecidua. This orchid is arguably the largest andshowiest of the North American species. Leaves arelong, broad, and relatively massive, resulting in veryhigh rates of transpiration. This physiological luxury,likely resulting in its increased overall stature, isafforded by its wet habitats. It is possible that theexceptionally *hirsute leaves are designed to be so inan effort to combat further transpirational water loss.

Fred Case (1987) suggests that deer play animportant role in the reproductive biology of thisspecies.

Firstly, they tend to graze tree branches nearforest edges. This allows for greater sun penetration,creating the optimal blend of sun and shade for C.reginae,which can be found in large numbers in such

situations. Secondly, C. reginae inhabitations tend toserve as winter deer yards, where the action of hoovesburies the orchid seed to an ideal depth forgermination. One to four flowers are produced, andwhite-flowered variants are known. Cash (1991) listsa variant, var. albolabium,lacking pigment in the lip.My experiences flowering numerous *clones of this


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species under cultivation makes such a distinctionquestionable. The flower seen in Figure 11, althoughslightly overexposed, could very easily be interpretedto be such a variant as there is indeed very little

pigment on the lip. This flower is from the same plantseen in Figure 10, but from the following season.There is no genetic difference between the two. Thedifference lies in the environmental conditions at thetime of flowering. The plant in Figure 10 experiencedwhat will be called a normal spring, in Windsor,Ontario, resulting in normal flower development.

The following season, that same plant experiencedanother normal spring, this time in Guelph, Ontario.

If anything, the plant was kept a little cooler during its

development as Guelph is usually cooler thanWindsor. The week before flowers were to open, theplant was brought to Windsor, which was thenexperiencing a heat wave, resulting in abnormallyhigh temperatures for development. The result is theflower in Figure 11. Extent of floral *pigmentationmay, therefore, be partly dependent on *ambient

temperature during floral development. The cooler the

temperature, the darker the flowers. That is not,however, to say that genetic factors do not play a role.They can indeed figure prominently into the extent ofpigmentation. The small plant in the bottom left ofFigure 17 illustrates this point, as all flowers seenexperienced the same environmental factors, however,that particular clone appears to be significantly darker.


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The principle of floral pigment intensity varying withenvironmental factors (including *soil *pH) is not anew one; however, this species appears to beespecially sensitive, a notion confirmed by other

growers.

8

Cypripedium flavum Ward is a vicariousspecies, with limited distribution in two regions ofAsia (Sing-Chi, 1983). Unlike the other vicarious pairdiscussed (C. arietinum and C. plectrochilon)there islittle question of the two being different species.Staminode shape and flower colour are quite different,eliminating any such concerns. Apparently, however,habitat preference and vegetative characteristics are

remarkably similar, confounding previous*herbarium-based analyses.

Please see Cash (1991) regarding remainingmembers of this alliance: C. flavum and C.subtropicum Chen & Lang.

8 White-flowered forms of Cypripedium reginae are correctly known asforma albolabium and occur sparingly within normally colored populations.These plants, in addition to having pure white flowers, have paler greenleaves and retain this coloration for the life of the plants. I too have seenpale colored flowers on plants that otherwise have had full colored flowers.Often these pale flowers appear to be white but upon close examination showslight traces of pink within the lip. Aside from producing a genetic sport, agiven plant does not change its color from year to year. Intensity of colorcertainly can be variable. PMB


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References and Recommended Reading

Case, F.W. 1987. Orchids of the Western Great Lakes Region.Cranbrook Institute of Science, Bulletin 48.

Cash, C. 1991. The Slipper Orchids. Timber Press, Portland,Oregon.

Cribb, P. 1996. The Genus Cypripedium in Mexico and CentralAmerica. North American Native Orchid Journal. 2: 3-

18.

Cribb, P. & S.C. Chen. 1994. Chinese spotted-leavedCypripediums. Orchid Review 102: 321-323.

Luer, C.A. 1975. The Native Orchids of the United States and

Canada Excluding Florida. New York BotanicalGarden. NY.

Perner, H. 1995. Winterharte Fraunschue (1): Cypripediumarietinum R. Brown und Cypripedium plectrochilum

Franchet.Die Orchidee 46(3): 103-108.

. 1996. Die Gattung Cypripedium L. in einer

stammesgeschichtlichen Ubersicht. Jour. Eur. Orch.

28(1): 84-107.

Sheviak, C.J. 1994. Cypripedium parviflorum Salisb. I: TheSmall-flowered Varieties. American Orchid SocietyBulletin. 63: 664-669.

. 1995. Cypripedium parviflorum Salisb. II: The Larger-

flowered plants and Patterns of Variation. American

Orchid Society Bulletin. 64: 606-612.


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. 1996a. The Cypripedium parviflorum complex in North America. North American Native Orchid Journal 2(4):

319-343.

. 1996b. Cypripedium hybrids in the Russian Far East: The

Red Influence. part 1. Orchids 65(11): 1170-1175. part2. Orchids 65(12): 1294-1299.

Sing-Chi, C. 1983. Two pairs of vicarious species ofCypripedium (Orchidaceae) from eastern Asia and

eastern North America. Brittonia. 35: 159-163.


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Part II

Mycorrhizal Fungus

Undoubtedly, the relationship orchids maintainwith their mycorrhizal partners is the most poorlyunderstood aspect of their entire biology. Research inthis area was quickly abandoned as Knudsonpioneered the techniques for *asymbiotic germinationof tropical orchids, completely eliminating any needfor mycorrhizal fungus in seed-sowing practices.

Interest was renewed in mycorrhizae as greaterattention was drawn to temperate terrestrial orchids,due in part to increased horticultural pressures, as wellas the ecological threats, which put the continuedexistence of these plants into question. *Temperateterrestrials are usually resistant to conventional seedpropagation techniques. Effort has therefore beenmade to understand better their mycological partnersand thus their reproductive biology, perhaps toprovide some of the keys to stubborn propagativetemperament.

In the orchid trade, both temperate and tropical,there are an astounding number of misconceptionsregarding mycorrhizal fungus. Rather than list andindividually address them, a brief overview of what is


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Doherty: The Genus Cypripedium: Part II

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currently known, with specific emphasis on temperateterrestrials, will be given.

Seed Germination

One of the most important characteristicfeatures of orchids is the minute seed they produce.Their small size effectively facilitates dispersal overlong distances by wind. One of the disadvantages ofhaving such a small seed, however, is the inability tostore significant amounts of nutrients. An orchid seedcan consist of as few as 50 different cells, with storagereserves being primarily proteins and *lipids. It hasbeen shown that in nature, seeds cannot *germinatewithout the help of a mycorrhizal fungus.

The fungus usually enters the seed through whatis called the suspensor end. The testa, or seed coat,completely envelopes the *embryo; with the exceptionof the point at which it was attached to the ovary wallthroughout its development. This hole in the seedcoat (Fig. 19) is at the suspensor end of the seed. It iswhere the fungus usually enters and first comes intocontact with the embryo. The suspensor end of the

embryo differentiates to form what is calledmycotrophic tissue. The opposite end of the embryo,called the chalazal end or chalazal pole, eventuallyforms *meristematic tissue which will then developinto the plant organs.


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Once the fungus has established this *interfacewith the embryo, germination begins. Technicallyspeaking, the fungus never is actually inside theindividual cells. To understand better this interface, a

plant cell will be simplified to the following: a squarewater balloon inside a rigid wooden box with holes inthe side. The water balloon is the cell itself, theballoon forming the cell membrane or plasmalemma,maintaining the cell contents in one place. Thewooden box represents the cell wall, providing the cellwith structural integrity. Without their rigid cellwalls, plants would be *amorphous blobs, like peoplewithout skeletons. The holes in the wooden boxrepresent what are called pit fields, or thin areas in the

cell wall. These permit intercellular communication,as well as the conduction of numerous substancesfrom one cell to another. When the fungus interfaceswith the cell, it never actually pierces the waterballoon. Rather, it inserts the equivalent of its armsand fingers into the holes in the side of the box. Thisgreatly alters the structure of the water balloon inside.However, it never actually breaks it. The more armsthe fungus inserts into the cell, the greater the surface

area between the two, allowing for more rapid andefficient exchange of materials.

As mycorrhizal fungus lacks a body (i.e., no

central unit more developed than another), it is said toconsist of many individual strands called *hyphae. Itis these hyphae that branch off in all directions (from


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no real centre) and act as arms of the fungus and

grow through the pit fields to interface with the cell.The extent to which hyphae are present within a cellcan vary. Sometimes structures called pelotons are

formed that consist of extensive hyphae forming acomplex coil within a single cell. This maximizesshared surface area and therefore exchange.

As the embryo germinates and grows, structurescalled rhizoids may be formed. They are simpleextensions of *epidermal cells and are formed on thebasal part of the *protocorm. Their function is tofacilitate mycorrhizal infection.

The germinated embryo will first grow into amass of relatively undifferentiated tissue called aprotocorm. Once of sufficient size, the meristematicregion of the protocorm will develop a shoot androots. The fungus remains in the mycotrophic tissuethroughout this entire development process, supplyingthe growing plant with an array of requiredsubstances.

The Exchanged MaterialsVery little is understood with respect to exactly

what chemical exchanges occur between the fungusand the orchid. Lipids in seed reserves can beconverted to starch and then glucose, providing thenecessary energy. It is suggested, however, that thefungus facilitates mobilization of these reserves that


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otherwise would not be possible. In effect, the fungusis actually providing the embryo with access to itsown reserves.

The portion of the fungus within the orchidtissue is only a small part of its whole. It remainsattached to its hyphal network within the soil. Thishyphal network functions as an extended root systemwith a large surface area, affording the germinatingorchid with tremendously greater access to soilnutrients than would otherwise be possible with itsown small surface area. Substances assimilated havebeen shown to include water, nitrogen, phosphorous,mineral salts, carbohydrates and other organics

(Rasmussen, 1995). In an elegant *in vitroexperiment, carbon and phosphorous *isotopes wereused to trace movement of these elements from thesurrounding medium into the fungus, where they weretransferred to the orchid (Smith, 1996; Alexander etal., 1984).

Very little is known about what the fungusreceives from the orchid. In fact, it is suspected that it

might not receive anything. This would, surprisinglyenough, make the orchid a *parasite on the fungus.Clarification of this issue will come with time asexperimental technology improves. Experiments, suchas that described, involving the tracing of materialsfrom the environment into the plant, are currentlymuch simpler to execute than those attempting to trace


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material originating within the plant and its suspectedtransfer to its inhabitant fungus.

Life Cycle of the Fungus

Once inside the embryo, it is likely that thefungus will remain within the plant for a number ofyears, perhaps in some cases for the entire life of theplant. The mycorrhizal interface previously described,however, is not a static relationship. Rather, it couldbe described as a recurring cycle, starting with initialinfection of the embryo. This infection leads to theformation of extensive hyphal connections, andperhaps peloton formation. These connections aresubsequently the victims of digestion by the plant,

followed by reinfection of adjacent cells by the samefungus to complete the cycle.

Peloton digestion is a process through which theplant utilizes its own digestive *enzymes to kill andassimilate the portion of the fungus within the cells.Non-absorbed remains of the fungus are *vacuolatedor compartmentalized within the cell where theyremain. *Chitin is a substance the fungus

manufactures as one of its own cell wall components(it also is the main component of insect*exoskeletons) and it cannot be digested by plantenzymes (nor by human enzymes). Consequently, it isa primary constituent of the vacuolated fungalremains. The digestion of the fungus is of particularbenefit to the orchid as it obtains a great number of


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chemical components from within the fungus.Unfortunately, the chemical makeup of assimilatedcomponents is quite poorly understood. It is likelythat somewhere within this chemical soup lie key

components that are integral to the orchidsdependence on the fungus. The purification andanalysis of such constituents would be a long, tedious,but extremely worthwhile process that would surelyserve to further our understanding of the biochemicalfactors affecting the interaction between fungus andorchid.

Besides entering the orchid at the seed stagethrough the suspensor end, mycorrhizal fungus can

also enter mature plants, presumably through roothairs. Figure 20 is a cross sectional view of a clearedroot ofSpiranthes odorata. The particular section ofroot chosen shows no signs of infection, which wouldbe seen as black regions within the cells as a chitin-specific stain was used. Figure 21 however, clearlyshows a region from the same root that has undergoneextensive colonization, as seen by the black bodieswithin the cells. Very few of these black bodies

represent living pelotons; the majority of them havebeen digested, nor were very many alive at the time ofpreparation. The actual hyphal coil can bedistinguished in living cells, as well as its hyphalconnection to adjacent cells (Figure 22). It can beseen that the only living coils are to be found at theedge of the mycorrhizal colony. Figure 23 shows the


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edge of the root with attached and cleared root hairs.The black lines within the hairs are a fungal hypha,presumably demonstrating their pathway into theroots. The problem, however, with this type of

evidence is that there is no direction implied in thephotograph. The hyphae could be coming into theroot from the soil, or out of the root into the soil.Experiments with live material have shown the formerto be more probable. Figure 25 shows a group of rootcortical cells pricked out9 and isolated, showing black,vacuolated fungal remains. It should be noted that inFigures 20-22, no colonization is observed within the*stele (the central cylinder of conductive tissue). Thisis an important aspect of the orchid/fungus

relationship. If the fungus were to venture into thestele, the result would be clogging of the conductivetissue (xylem and phloem). Consequently, the plantwould die in a fashion very similar to those plantsinfected by Verticillium wilt, a fungal *pathogen thatinfects plant roots and does indeed inhabit the stele,thereby causing a slow death by dehydration.

Host-fungus Specificity

There has been a recent change in the school ofthought concerning the specificity of mycologicalpartners. It was once believed that only a single

9 Cortical cells are the "filler" storage cells located in the roots, between theepidermis and vascular cylinder, or stele. Pricking them out is just theirremoval during dissection with a fine needle, or something similar.


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species of fungus was capable of forming this*symbiosis (perhaps more appropriately parasitism)with its orchid partner. It stood to reason, therefore,that for every orchid species there was a single

compatible species of fungus. Extensiveexperimentation has shown otherwise. Rather, asingle species of fungus can infect an array of orchidhosts. What varies sometimes, but not always, is theextent of its compatibility. Certain groups of fungus

may form a positive relationship with certain groups

of orchids.

This type of research has encountered numerousdifficulties including the formation of pure fungal

cultures. Mycorrhizal fungus can be very fussy invitro, as it tends to resist culturing. The only source ofmycorrhizal fungus until now has been infected plants.Successful isolations from the soil have not beenachieved. It is quite difficult to isolate a pure culturefrom a living plant. Very often plants are infectedwith other non-mycorrhizal entities, makingpurification very difficult. Sterilization of root tissuecannot be performed without killing the sought-after

fungus as well. As shown by previous photographs,living pelotons can be notoriously rare in some species(Curtis, 1939; Harvais, 1974; and Stoutamire, 1990),Cypripedium spp. in particular, making isolationnearly impossible. The problems encountered withfungal cultures could be attributed to either or both oftwo factors: (1) once within the plant, the fungus


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undergoes irreversible alteration making its in vitrogrowth extremely difficult, or (2) the fungus hasspecialized nutritional requirements that remain to beoptimized.

To complicate matters further, it has beensuggested that the fungus that infects seedlings may,in fact, be a completely different species from thatwhich infects mature plants (p. 241 in Rasmussen,1995). This stands to reason as the mode of entry ineach situation is very different. As anembryo/seedling, the orchid undergoes morphologicalchanges to facilitate infection. As a mature plant,there are very few, if any, such structures. Entry is

through the root hairs, a specialized cell designed foraltogether different purposes (water absorption).Colonization of mature roots could not be the result ofthe funguss originating from embryo infection andgradually moving through the plant and down into theroot *cortex. First of all, colonies found withinmature roots are not continuous with older portions ofthe rhizome, nor can the continuity of colonies betraced through the root with digested pelotons. In

addition, roots produced from mature plants arise*endogenously, from the *pericycle surrounding thestele. Therefore, the fungus that may already existwithin the rhizome never has access to newly formedroots. Infection of new roots, therefore, must be fromthe soil, and through a much less welcoming

pathway.


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This difference in mycological partners atdifferent stages of growth would easily explain whyinitial experiments, testing symbiotic germination by

using isolates from mature plants, met with littlesuccess. It is very clear that further research needs tobe done in this area.

One final point to be made is with respect to theability of the fungus to sustain itself in the soil withoutany apparent orchid partners nearby. Orchid seedcould be germinated in the wild in a situation wherethere are no single species of orchid within kilometres,yet the seed will be colonized. This gives rise to many

questions: Where did the fungus come from? Howcould it sustain itself in the soil? This group of fungican exist in the soil as *saprophytes; however, can itrely exclusively on this method of survival? How doorchids factor into this balance? Is there really amycorrhizal fungus per se, or is it some type ofubiquitous species that is irreversibly altered once incontact with the orchid? A thorough and clearunderstanding of terrestrial orchid biology cannot be

attained until these issues are addressed.

Specifics Regarding Cypripedium

Different orchids vary in their dependency ontheir mycological partners as mature plants (Sadovsky,1968; Rasmussen, 1995, Chapter 4). Without a doubtevery orchid needs its mycorrhizal fungus to reach a


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*photosynthetic stage where it can begin to providefor itself; however, once this stage is achieved itsdependence can change greatly. Some species remainhighly dependent on their fungi, perhaps represented

in the extreme by the *achlorophyllous species, suchas the phantom orchid, Cephelanthra austiniae. Theother end of the extreme, in my mind, is representedby the genus Cypripedium. As previously mentioned,living pelotons, as well as digested pelotons, arenotoriously difficult to find in mature roots. For lack

of sufficient material with Cypripedium, the fragrant

ladies-tresses, Spiranthes odorata had to be used forFigures 20-23, 25. It is the following experiment,which I conducted, that was found to be mostconvincing for *autotrophism in adult Cypripediumplants:

Numerous populations ofCypripedium parviflorum var.

pubescens were observed at various locations on the Bruce

Peninsula in Ontario. A few healthy plants were seen; however,nearly all plants encountered (at least 1000 in small groups, oras individuals over a large area) showed some degree of floral

*mutation or abortion, as well as leaf *necrosis. This afflictionwas not limited to individuals. What had clearly been at one

time sizable clumps were also affected, showing mutatedflowers and stems greatly reduced to a few leaves. Thesesymptoms were indicative of some sort of root pathogen.

Sample plants were collected and examined by an

Agriculture Canada plant pathologist. Remaining samples wereplaced under pot cultivation for experimentation. One pot was

treated with a systemic fungicide (until this point, a cultural


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taboo). The fungicide is toxic to mycorrhizal fungus. The otherpot was not treated.

The plant in the untreated pot died by the end of theseason, failed to make new growth for the following season andwas discarded. The plants in the treated pot came up the next

season with as many growths as the previous year, and producedtwo flowers on two of three stems. The analysis by the

pathologist revealed thatRhizoctonia solani was present, as wellas something he could not identify. Rhizoctonia solani is acommon root pathogen and likely the cause of the symptoms.

The unknown fungus was possibly mycorrhizal.These results were quite surprising. Depending on the

fungicide used, one would expect the treated individual to

immediately lose its mycorrhizal partner and therefore perish.Such was clearly not the case, putting into question the value of

the fungus to a mature plant. Admittedly, mammalian factors (a

squirrel) affected long term study of the treated plants. It wasimpossible to assess adequately things such as recolonization, or

the possibility that such a dramatic recouperation was in theshort term only. It would have been ideal to observe the plant inthe long term to see if its vigour was maintained.

The genus Cypripedium is not any differentfrom other genera during young stages with respect toits fungal dependency, as the first root (of exogenousorigin, i.e. from the protocorm cortex) is mycotrophic

from the beginning (p. 239 in Rasmussen, 1995).However, the following roots become less specializedfor mycotrophy as there are fewer root hairs formed(Rasmussen, 1995). The extent of mycorrhiza

declines from year to year, while the above groundorgans increase in volume. (Fuchs and Ziegenspeck,

1925 in Rasmussen, 1995).


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Two-year old roots show patches of infectionthat are mainly dead pelotons. These patches could bethe remains of temporary mycorrhizal infection during

the underground season; however, they wereinterpreted to be fortuitous and possibly pathogenic(Harvais, 1974).

Despite these findings, mature Cypripediumplants have been known to remain dormantunderground for one to five years (Gill in Rasmussen,1995). It is suggested that this might be impossiblewithout the aid of the fungus. It is possible that undersuch conditions, the plant may drastically increase its

dependence on the fungus. It is equally possible,however, that the plant is relying on the significantstored energy potential within the rhizome and rootsduring this time, eliminating the need for the fungusduring its dormant period.

The fact remains that the importance ofmycorrhizal fungus to mature plants is stillincompletely understood. Further experimentation

needs to be done on a larger and long term scale withmature plants.

References

Alexander, C., I.J. Alexander, and G. Hadley. 1984. Phosphate

uptake in relation to mycorrhizal infection. New

Phytologist97: 401-11.


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Curtis, J.T. 1939. The relation of specificity of orchid

mycorrhizal fungi to the problem of symbiosis. Am. J.

Bot. 26:390-9.

Harvais, G. 1974. Notes on the biology of some native orchidsof Thunder Bay, their endophytes and symbionts.Canadian Journal of Botany. 52:451-60

Rasmussen, H.N. 1995. Terrestrial orchids: from seed tomycotrophic plant. Cambridge University Press.

Sadovsky, O. 1968. Orchideen im eigenen garten. BLV

Munchen Basel Wien.

Smith, S.E. 1966. Physiology and ecology of orchid mycorrhizal

fungi with reference to seedling nutrition. NewPhytologist65:488-99.

Stoutamire, W.P. 1990. Eastern American Cypripedium speciesand the biology ofCypripedium candidum. Pp. 40-48 inNorth American Native Terrestrial Orchid Propagation

and Production Conference Proceedings, March 1989.Brandywine Conservancy, Chadds Ford, Pennsylvania.


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Part III

Propagation and Cultivation

Mature plants are readily propagated by

division, ideally in the fall or spring while growth is ata minimum; however, this can be accomplished quitesuccessfully at any time of the season assuming propercare is taken to minimize root damage. Roots as oldas three or four years remain active so should not besacrificed if possible. New roots are formed shortlyafter flowering at the base of the new growth. Singlebud or growth divisions should be avoided, but aresometimes unavoidable with the *stoloniferous

species such as Cypripedium formosanum or C.guttatum. Division is practical and effective for smallscale propagation, but is limiting because of the few*propagules produced over a relatively long period, aswell as their genetic uniformity.

Without a doubt, seed propagation is the mostdesirable method of propagation. Large numbers ofgenetically variable seedlings can be produced in ashort period of time. Unfortunately, this line of

research has historically been met with repeatedfailure or astronomically mediocre results. It is onlywithin the last decade that significant refinement oftechniques has been achieved so as to overcomepreviously insurmountable hurdles. We are not, asmany predicted years ago, on the verge of large scalecommercialization of this genus. Propagative


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mysteries remain, and further refinement of techniquesis necessary. However, the long and slow path hasfinally yielded some encouraging and viable results.

Numerous growers around the world haveunlocked the mysteries of propagating this genus fromseed and are consequently offering a vast array ofspecies and hybrid seedlings for sale (see Appendix Ifor a listing). Others have outlined their highlysuccessful procedures in excruciating detail for publicconsumption, specifically Bill Steele of Spangle CreekLabs (Steele, 1995, 1996) and Svante Malmgren, asurgeon in Sweden (Malmgren, 1993, 1996), as wellas Chu and Mudge (1996). Their work has provided

the basis for the following summary.

Considerations to be made when propagatingfrom seed can be loosely divided into seven differentcategories, each to be considered individually:(1) collection of seed,(2) sowing and pretreatment of mature seed,(3) cold stratification/dormancy requirements,(4) light conditions,

(5) substrate composition,(6) replating10(7) *deflasking and planting out.

10 Replating is the procedure of transferring in vitro growing plantlets to adifferent, sterile container. This is usually done to space out the seedlings;however, it can also be performed to change the nutrient medium or removeseedlings from an infected culture.


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(1) Collection of Seed

As with tropical orchids, seed of Cypripediumcan be sown as dry, mature seed, or as unripe,immature seed (greenpodding). Each method has its

own advantages. Greenpodding requires unripeseed pods to be harvested prematurely. Ideal harvestdate depends on a number of factors, including species(which will likely exhibit clonal differences), as wellas environmental conditions experienced duringripening. Harvest dates cited in the literature varyfrom four weeks after pollination to as many as eightor nine; five or six may be a good starting point.Unfortunately, optimal harvest dates must currently be*empirically determined as there is no known way to

consistently predict when the seed is ideal forgermination. Efforts have been made to correlateovary size and swelling in Cypripedium parviflorumto suitability for harvest (Light, 1989); howevergeneralizing to other species would not be appropriate.The major advantage to greenpodding is the

circumvention of seed dormancy. Dormancy factorsthat are incorporated into mature seed are not yetdeveloped in green seed. The ideal pod is one which

has been allowed to develop for as long as possiblewithout the onset of dormancy, which will greatlyreduce subsequent germination. The two majordisadvantages to greenpodding are the uncertain

timing of fruit collection and the need to flask it veryshortly thereafter. Green pods will not keep, makingtheir storage impossible and mailing to other parties


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Cypripedium parviflorum var. pubescens

large yellow ladys-slipper


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very difficult. Given that dormancy factors in matureseed can be overcome through various techniques,sowing and storing mature seed is usually the methodof choice.

Mature seed is harvested in the fall, ideally justbefore the pod cracks, thereby minimizingcontamination from exterior sources as well as seedloss to the wind. Sometimes this later harvest cannotbe avoided, and it is usually of no great consequence.

(2) Sowing and Pretreatment of Mature Seed

Whole green pods are surface-sterilized shortlyafter harvesting. Under *aseptic conditions, the pod is

then cut open and the immature seed scraped out andsown directly into culture vessels. Immature seedusually is still attached to the ovary wall, and is whiteto yellow in colour.

Mature seed differs significantly in itscolouring. It usually turns to some shade of brown;however, in some species its colour transformation isnot so pronounced and it appears very similar to the

juvenile yellow-white seed.As previously mentioned, mature seed contains

dormancy factors not found in immature seed.Unattended, these factors can severely inhibitgermination processes, sometimes completelyarresting them. This natural biochemical mechanismsurely acts as an environmental sensor to cue the plant


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to germinate at the most appropriate time; however,under artificial propagation regimes, control of theseprocesses is necessary. It has been found thatextended exposure to bleach before sowing greatly

reduces the action of these dormancy factors.Sterilization times for orchid seed have conventionallybeen in the 10-15 minute range for a 10% solution ofcommercial bleach, (i.e., 0.5% NaOCl); however,Anderson (1990) found by accident that leaving seedin the solution for two hours significantly enhancedgermination rates. Subsequent analysis showed somespecies to prefer soaking times of up to six hours!Malmgren (1993) has discovered this same principleand has found that treating the seed with acid before

bleach achieves the same result without the lengthysoaking time. It is likely that the brief acid treatmentweakens the seed coat, facilitating more effectiveaction of the bleach. Regardless of the technique, thenet effect is the oxidation of the dormancy factors bythe bleach and thus their relative inactivation. Inaddition to extensive bleaching of mature seed,germination can be expedited further in some speciesby adding a *cytokinin to the germination medium.

(3) Cold Stratification/Dormancy Requirements

Both mature plants and seedlings havedormancy requirements that have to be met. Ideally,mature plants should be given sub-zero temperaturesfor at least three months, if not longer. Colder-climatespecies such as Cypripedium guttatum and C.


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passerinum usually prefer longer and deeper cold.Seed of some species survives freezing, thoughseedlings, unlike mature plants, will die if frozen andtherefore must be kept between 0 and 5 C during

winter months. This is achieved in a cold frame,refrigerator, or similar setup.11

A great concern when vernalizing seedlings istiming. Seedlings will not leaf out until given anappropriate cold period. Until that point, the onlyshoot structure that forms is the small overwinteringbud, easily seen on mature plants in the fall. Ideally,plantlets with fully developed buds should bevernalized in the fall, concurrent with their natural

development cycle. This means that seed must besown at an appropriate time to allow for fulldevelopment of the seedlings prior to *vernalization,however, not too soon so as to force the plantlet towait too long before its required cooler temperatures.Seedlings are usually deflasked and sealed in airtightdishes or plastic freezer bags with a little extramoisture to prevent desiccation during vernalization.Again, determination of these procedures remains

empirical as considerable variations exist from speciesto species.

11 Seed of at least some species survives freezing quite well. Ive hadenhanced germination ofC. parviflorum seed from capsules left on the plantall winter and subjected to temperatures in the range of -15 C, as comparedwith refrigerator-stored seed maintained at 23 C. WS


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(4) Light Conditions

Seed must be placed in darkness immediatelyafter sowing. Light is not absolutely necessary untilplantlets complete with roots and bud have been

deflasked and vernalized; however, it can be given assoon as the first initial shoots are evident.

(5) Substrate Composition

Harvais (1982) was the first to extensivelyinvestigate the precise chemical requirements for seedgermination of Cypripedium reginae. His medium,therefore, was optimized for that particular species.Bill Steele (1995, 1996) has quite successfullyduplicated and even improved Harvais results, and

subsequently modified his recipe to accommodateother seemingly pickier species. Apparently theelement of interest is nitrogen. If provided in*inorganic form, notably ammonium nitrate, it can bedetrimental to many species. Steele has reduced itsconcentration in Harvais medium from 1400 mg/l to

500 mg/l with greatly improved results for a numberof species.

Svante Malmgren (1993, 1996) has alsoachieved success by supplying nitrogen in the form of*amino acids, a completely organic form. This couldbe correlated to a natural situation where the plantderives nutrients from digested fungal hyphae,containing *metabolized nitrogen in the form of aminoacids. The glaring absence of hyphae in roots of


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mature plants suggests that if the plant were to dependsolely on fungus as its nitrogen source, it would surelystarve. The reasons for improved germination andgrowth with reduced ammonium nitrate or complete

substitution are not completely understood; however,answers could lie in potential sensitivity to ammoniumnitrogen, or an optimal ratio of nitrate nitrogen toammonium nitrogen.

Additional components of Malmgrens medium

are *kinetin, pineapple juice (likely containing asubstance with plant growth regulator properties,probably analogous to the potato cubes used bySteele) and a basic vitamin solution. Readers are

directed to the references for specific formulations.

(6) Replating

Cytokinin in the germination medium will causeabnormal growth as the protocorms begin todifferentiate. Consequently, as soon as the first rootinitials are evident, developing protocorms must betransferred to fresh medium. It should be identical incomposition to the germination medium, with the

exception of kinetin, which must be excluded.

(7) Deflasking and Planting Out

The ideal time for deflasking is in the fall orwinter in preparation for vernalization. Attemptingvernalization on the flasking medium causes highmortality in some species, e.g., C. reginae.


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Choice of substrate for outplanting varies.Malmgren uses a combination of three parts woodssoil, one part peat and one part lime soil. Other

growers use

Documents

March 1997 North American Native Orchid Journal