Laddy's Slipper Orchids: Cypripedioideae
Orchids belong to the family of flowering plants known as Orchidaceae. This is one of the two most
diverse families of flowering plants on Earth, the other being the
Asteraceae (Daisy family). Which of the
two is actually the more diverse varies with time and opinion, according to latest species counts, but
most modern sources consider the orchid family to be the most diverse with an estimated 25 000 to 35
000 different species, versus about 20 000 for the daisy family. To some extent, this may depend on
how we define species, since in the flowering plant world the barriers between species are much more
blurred than they are in say the mammals, with plants hybridising much more freely and exhibiting a
greater variety of breeding systems. Apomixis ( a form of asexual reproduction in which new plants
develop parthenogenetically from the carpels without fertilisation) has also resulted in many
microspecies occurring in some genera of asteraceae, such as
Taraxacum and Hieraceum.

Among the orchid family there are five subfamilies based on morphological and genetic similarities:

      1. Epidendroideae, e.g.
Neottia, Listera, Phalaenopsis
      2. Orchidoideae. e.g. Dactylorrhiza
      3. Cypripedoideae, e.g. Phragmipedium
      4. Vanilloideae, e.g. Vanilla
      5. Apostasioideae, e.g. Apostasia, Neuwiedia

Here we shall discuss chiefly the subfamily Cypripedioideae, the Lady's Slipper Orchids. The current
genera for this subfamily are:

      1. Cypripedium
      2. Paphiopedilum
      3. Phragmipedium
      4. Mexipedium (1 species only)
      5. Selenipedium

Subfamilies 3, 4 and 5 have a different arrangement of stamens than the other two groups. Orchids
have monosymmetric flowers, meaning that they have only a single plane of symmetry, though those of
the Apostasioideae are only weakly monosymmetric and almost look radially symmetric like the flowers of
lilies. Lilies are monocotyledons, as are orchids, and both families have the classic momocotyledonous
trimerous symmetry and it is assumed that the more morphologically specialised flowers of orchids
evolved from a lily-like ancestor.
Paphiopedilum parishii by B.S. Williams, 1883.
Above: an historic botanical illustration of the slipper orchid: Paphiopedilum
parishii
(synonym: Cypripedium parishii; a synonym is an alternative Latin
binomen which is also accepted by the rules governing botanical nomenclature)
by B. S. Williams 1883,
www.biodiversitylibrary.org/pageimage/35714186.
Floral diagrams: lilly versus orchides
Phragmipedium
The diagram above shows floral diagrams for a typical lily (top), most orchids, such as epidendroids and
orchidoids (bottom left) and
Cypripedium lady's slipper orchids (bottom right). The white circles
represent stamens that fail to develop.

A typical lily flower consists of the basic monocotyledon bauplan: there are 5 whorls of three
appendages. The outermost three form the whorl of sepals, inside this is a whorl of three petals
(sometimes the sepals and petals are both referred to as tepals). Inside this are two whorls of three
stamens and inside this is the carpel, formed by the fusion of three carpels. Most orchids differ in the
following ways:

      1. Only one functional stamen reaches maturity, here assumed to belong to the outer whorl
      2. The stamen filament is fused to the style (a compound style formed from the three carpels)
      3. The uppermost petal develops into a distinctly different petal: the lip or labellum

The fusion of style and staminal filament gives rise to the column or
gynostemium. In reality this is a
complex structure and it is not clear to what extent undeveloped stamens contribute to it in different
genera. The pollen-containing anther is often partly enclosed in a cavity formed by an extension of the
column, called the
clinandrum, with the anther facing downwards above the receptive female stigma,
but usually separated from it by a beak-like projection of the column called the
rostellum, to prevent
self-fertilisation.

It is also not immediately obvious which of the 6 ancestral stamens develop into the functional stamen,
but is generally taken to be the median (abaxial or facing away from the main shoot axis) stamen of the
outer whorl. We return to this point shortly. In lady's slipper orchids the arrangement is different: there
develops a medial abaxial
staminode (infertile stamen) and two lateral abaxial fertile stamens.
Following
resupination, that is rotation of the flower through 180 degrees as it grows, which occurs in
most orchids, these stamens become adaxial, i.e. they come to face the shoot axis. Note that
resupination brings the labellum to the bottom of the flower, as a lower lip. The fact that all three
stamens are on the abaxial side, away from the shoot axis, suggests that the lateral two are from one
whorl, the median from another and the interpretation is that the staminode is all that remains of the
outer whorl, the laterals of the inner whorl.
In Cypripedium calceolus, at least, it has been stated that the
third stamen of the inner whorl forms the front of the column. Darwin considered two vascular bundles at
the sides of the labellum to be remnants of the two outer lateral stamens, but this has not been
confirmed.

More information about the nature of the stamens can be obtained by detailed microscopical study of
the developing flower. Such a study on
Neuwiedia and Apostasia from the subfamily Apostasioideae has
been carried out by Kocyan and Endress (2001).
Neuwiedia has three fertile stamens, which develop
abaxial initially, becoming adaxial after resupination. In this case, the median outer stamen is the first
stamen to appear. We know this is the outer whorl because the stamens of the outer whorl in monocots
generally develop opposite an accompanying sepal, the inner whorl are staggered and so lie opposite
the petals. Lateral inner stamens then appear, opposite the lateral petals.
Apostasia is similar, except
that the outer medial stamen is either missing or an infertile staminode. This suggests that our floral
diagram of Cypripedium is in fact correct: there is one medial outer staminode and two inner lateral
stamens, originally abaxial these become adaxial following resupination.
Orchid flower development
References, Further reading and Links

Banziger, H. 1996. The mesmerising wart: the pollination strategy of epiphytic lady slipper
orchid
Paphiopedilum villosum (Lindl.) stein (Orchidaceae). Bot. J. Linnean Soc. 121: 59-90.

Chen, L.; Ke-Wei Liu;  Xin-Ju Xiao; Wen-Chieh Tsai; Yu-Yun Hsiao; Jie Huang and Zhong-
Jian Liu. 2012. The Anther Steps onto the Stigma for Self-Fertilization in a Slipper Orchid.
PLoS ONE 7: e37478. www.plosone.org.

Dressler, R. and Pupulin, F. 2011. Phragmipedium Section Phragmipidium. The Long-Petaled
Tropical American Lady’s-Slipper Orchids. WWW.AOS.ORG.

Kocyan, A. and P. K. Endress, 2001. Floral structure and development of
Apostasia and
Neuwiedia (Apostasioideae) and their relationships to other Orchidaceae. Int. J. Plant Sci. 162
(4):847–867.

Li, P.; Y.B. Luo; P. Bernhardt; X.Q. Yang and Y. Kou, 2006. Deceptive pollination of the Lady’s
Slipper
Cypripedium tibeticum (Orchidaceae). Pl. Syst. Evol. 262: 53–63.

Pemberton, R.W. 2013. Pollination of slipper orchids (Cypripedioideae): a review.
LANKESTERIANA 13(1–2): 65—73.


Cypripedium calceolus

http://www.hardyorchidsociety.org.uk/HOS%201012/Pollination/Claessens%2010-2013.pdf

http://www.brc.ac.uk/plantatlas/index.php?q=plant/cypripedium-calceolus

Forrester, N.J. 2011. Inside the trap of a yellow lady's slipper orchid (
Cypripedium parviflorum
var.
pubescens): the effects of 'light windows' and flower orientation on the behaviour of a
native bee (
Andrena macra). Undergraduate Honors Theses. Paper 421.
http://publish.wm.edu/honorstheses/421

Ramsay, M.M. and J. Stewart, 1998. Re-establishment of the lady’s slipper orchid
(Cypripedium calceolus L.) in Britain. Orchid population biology: conservation and challenges,
S. Waite (ed.).
Botanical Joumal of the Linnean Society 126: 173-181.
Above: Paphiopedilum dianthum (photograph by Orchi, Wikimedia Commons)
Above: Development of the Neuwedia flower. The flower begins as a primordium, a bump of
cells on the tip of the flowering shoot, wider than long. As this primordium develops, primordia
of the various floral appendages appear, as bumps of cells, upon it. These rapidly increase in
height. The various stages are not drawn to scale: step F is about twice the diameter of stage
A. The outer lateral stamen primordia appear but are repressed and develop no further
(presumably their tissue becomes incorporated into the column). The closely related orchid
Apostasia develops in a similar way, except that the outer median stamen is either missing or a
staminode and the inner median stamen primordium appears instead of the outer lateral
stamens, but is similarly repressed. Based on the photomicrographs of Kocyan and Endress,
2001.
Phragmipedium
Phragmipedium is a genus of about 20 species from Central and South America. The drawing above is
of a hybrid of
Phragmipedium caudatum. Phragmipedium caudatum is characterised by the ribbon-like
extensions of its lateral petals which may extend by more than 2 inches (5 cm) a day and reach two feet
(24 inches, 60 cm) or more in length! This orchid is pollinated by hoverflies of a single species which
have been observed to follow the trailing petal 'tails' to the trap and so it has been hypothesised that the
tails release attractive odour to guide the hoverflies (Fowlie, 1972 cited in Dressler and Pupulin 2011).

Stefan Vogel, the famous pollination biologist, also alluded to this function for the long tail-like
appendages of these and similar plants, which may even trail on the ground. It should be noted that
some orchids (and other plants) utilise vibratile organs: appendages that vibrate in the breeze, often in
the form of projecting 'cilia', antennae or a hinged labellum, to attract flies by their motion. Some plants
have clusters of flowers that easily vibrate, perhaps resembling swarms of insects clustered around
potential food.


Despite mechanism to ensure cross-pollination, which increases the genetic fitness of a population,
there are times when it profits a plant to pollinate itself, particularly when plants are too widely scattered
or pollinators to scarce to guarantee cross-pollination. Some plants will encourage cross-pollination, but
resort to self-pollination if all else fails. Some
Paphiopedilum species are self-pollinating (autogamous).
Phragmipedium lindenii lacks the labellum and has three functional stamens (it is triandrous) with the
extra anther pressed against the stigma, to allow self-pollination. In
Phragmipedium reticulatum, the
stigma slowly grows sideways and backwards until it contacts the anthers: if they still contain pollinia
then these will be used in self-fertilisation.
Phragmipedium besseae has typical trap flowers with windows
to direct insects up and back towards the stigma and a stairway of hairs leading to the stigma. These
hairs allow the insect to gain a foothold when it is going the 'right way'.
Pollination of Lady's Slipper Orchids

Slipper orchids are so-called because the labellum, or lower lip-like petal, folds over itself to form a
pouch with a dorsal opening which acts as a trap for pollinators: the flower looking like a lady's
slipper, complete with ribbons (lateral petals). These plants use
deception, they promise a nectar
award when in fact there isn't one! They are self-compatible in almost all cases they require insects to
transfer the pollen. The rim of the pouch around the dorsal opening is generally slippery so that an
alighting insect soon falls in. Otherwise, the pollinator may fly in seeking nectar. The methods of
attracting the right pollinator in the first place varies with genus and species, as we shall see. Once
inside the trap, the insect tries to escape but the slippery walls of the trap and its infolded rim make this
very difficult. The insect is guided to
two lateral exit holes, one on either side of the column at the
back of the pouch, either by
translucent spots or windows in the pouch wall, as in Cypripedium
calceolus
, and/or by false nectar guides, on the inside of the labellum and staminode as in
Cypripedium calceolus. the escape route is tortuous: the insect must squeeze beneath the stigmas
receptive surface, depositing any pollen it may be carrying from a previous encounter. The papillae on
the stigma act as a brush to scrape off any pollen from the insect's back. Then the insect must pass
under the anthers, one of which guards each exit window, picking up pollen on its way out. Stiff hairs
may give the insect traction to help it walk the prescribed route (as in
Cypripedium calceolus).

Upon escape, the insect's instinct are to fly away, thus
, particularly as each flowering spike usually bears
a single flower,
any subsequent contacts are likely to be with new flowers, helping to ensure
cross-pollination. The one-way escape route also means that an insect can not back up and pollinate
the same flower. Insects which are too small will escape without giving up or receiving pollen. Insects
which are too large may never escape and die inside the trap!
Paphiopedilum (Venus Slipper)
Paphiopedilum is a tropical orchid genus of about 80 taxa (species and naturally occurring hybrids)
native to parts of Asia, including India, China and Southeast Asia. Some are terrestrial, living in humus
on the forest floor, others are epiphytes (epiphyte: growing attached to the aerial surface of other
plants) or lithophytes, growing on rocks on the forest floor. The shoot systems are
sympodial and the
roots are thick and fleshy. They are pollinated by hoverflies and typical of such plants they have green,
brown or maroon flowers with spots, stripes or hairs. The staminode is often large and yellow with
distinctive markings or bumps. The species of hoverfly utilised lay their eggs inside aphids and gravid
female hoverflies mistake the bumps or warts on the staminodes for aphids! They may even lay their
eggs on or in the labellum. During this process the hoverfly may fall inside the trap and effect pollination.
Some
Phragmipedium species also exploit hoverflies in this way.

Function of the Staminode

Banziger (1996) discovered that the staminode plays a major role in  the pollination of
Paphiopedilum
villosum
, an epiphyte. The staminode of slipper orchids can typically be seen as a leaf or shield-like
structure hanging down in front of the column behind the entrance to the trap. The shield-like staminode
glistens, perhaps resembling droplets of moisture or honeydew which lures the flies as they fly at the
staminode. The 'urine-like' odour of the flowers also helps lure flies to the trap. In the centre of the
staminode is a prominent tubercle or wart which flies alight on, mistaking it for a perch. The wart is
slippery and the flies slide off into the trap! However, in most slipper orchids the pollinators do not seem
to target the staminode, so it must have other functions.

Beauty is Threatened!

The same author was originally studying
Paphipedilum parishii and Paphiopedilum charlesworthii in
Thailand. Thailand has 10 or 11 species of slipper orchid, only China has more species. However, the
study populations were wiped out by poachers half-way through the study and so the study was
redirected towards
Paphiopedilum villosum which is harder for poachers to reach, growing as an
epiphyte high in the tree canopy. Give humanity a wondrous garden and it does not take them long to
reduce it to barrenness. Such is the consequence of 'economics' and resource imbalances resulting
from power imbalance in human societies. Species are disappearing faster than they can be studies.
This is the price slipper orchids pay for being so beautiful. Alas!

A Novel Pollination Mechanism

Paphipedilum parishii has an unusual mechanism for self-pollination. Although it has an apparently
functional trap, suggesting that it recently evolved from a cross-pollinating form, the only mode of
pollination appears to be self pollination, at least in populations studied by Chen
et al. (2012). As the
flower begins to open, the anther liquefies (a process requiring 24 hours) and then spreads, reaching
the stigma which draws the drop onto it's receptive surface (the anther 'steps on to the stigma'). This
presumably has something to do with the physical wetting properties of the stigma which draws the
anther across its entire receptive surface. Furthermore, no pollinators were seen visiting the flowers
which release no perceptible odour. In contrast, in
Paphipedilum dianthum, which is pollinated by
hoverflies, the anthers do not liquefy.
Cypripedium
Cypripedium calceolus is distributed from Europe, through Russia through to China and Japan. It is
pollinated by
Andrena bees which land on the lip only to slip in since the rim of the trap entrance is oily
and slippery. False nectar guides, consisting of tracts of white and crimson spots on the staminode and
veins of the labellum guide the bees to a potential food reward, whilst no reward is given (
food
deception
). Translucent spots also guide the bees to the exits. The mechanism is the usual
unidirectional route past stigma and anther, already described. Cypripedium calceolus also has a sweet
'orange' odour that presumably attracts pollinators either by mimicing suitable food and/or pheromones.
It has been suggested that in this orchid, as in certain other lady's slipper orchids that the plant may
release chemicals mimicking insect pheromones as a further lure. In
Cypripedium guttatum it has been
reported that bees land on the slippery staminode and then slide off into the trap.

Brood-site Mimicry

Cypripedium tibeticum occurs in the boreal zone in mountains in SW China. The flowers occur close to
the ground and have dark entrances to the traps which are notably inflated and are thought to mimic
mouse holes. Mouse holes are potential nesting sites for Bombus queen bees. These queens visited
the traps more often than worker bees and were a better size to act as effective pollinators. The
hypothesis is that the bees enter the traps searching for suitable nesting sites, enticed further by the
'sweet-fruity' odour these flowers produce.

Food Deception other than the Promise of Nectar (See review by Pemberton, 2013).

Cypripedium fasciculatum occurs in N. America and is wasp pollinated. It has a mushroom-like odour
which attracts parasitoid wasps that parasitise fungus flies.
Cypripedium fargesii is pollinated by the
hoverfly
Cheilosia which eats the spores of leaf fungi. Large hairy patches on the orchid's leaves
resemble leaf mould infection and the flower releases a fungus-like odour. Some species are
Cypripedium are pollinated by carrion flies, others by fruit flies (e.g.
Cypripedium bardolphianum and
Cypripedium micranthum which have dark flowers which smell of decaying fruit).

Rescue from the Brink?

Slipper-orchids have been endangered worldwide, both due to over-collection as a result of their beauty,
and due to habitat destruction. They are, like many orchids, slow growing.
Cypripedium calceolus may
not produce its first leaves for up to 4 years after germination and requires a further 6 to 10 years before
it produces its first flowers. However, individual plants are long lived, with many reaching past 30 years,
some passing 100 and one plant in Estonia was estimated to be 192 years old! This is based on an
analysis of the slender, branching underground stem or rhizome which branches each year, with each
branch eventually putting up an aerial shoot.


Cypripedium calceolus
is Britain's rarest plant (though it may not be alone in sharing this title perhaps
with the Ghost Orchid,
Epipogium aphyllum). In 1917 it was declared extinct. Although records suggest it
was never widespread in Britain, picking by collectors for haerbariums and gardens has been blamed for
its demise and in 1917 it was declared extinct in Britain!

However, in the 1930s one wild plant was discovered in Yorkshire: the last of its kind! In the 1970s
protection was applied as pieces of this last wild plant kept being taken (!). In its best year it has since
produced 20 flowers. A program of manual pollination began and the site contains some seedlings
produced by seed scattered from pods set by this method.

The first of these seedlings flowered in 1993.  Each year the plants are hand-pollinated and some pods
have their seeds scattered on the site, whilst others are sent to Kew Gardens where they are cultured in
the lab and three-year old seedlings planted out back in the wild. Typical of orchids,
Cypripedium
depends on a specific mycorrhizal fungus to supply nutrients to the tiny seed before it can successfully
germinate. (An orchid embryo consists of only 100 to 200 cells with minimal food reserves). In lab culture,
this barrier was overcome by  supplying a nutrient solution in place of the fungus. This project has been
generously sponsored by the Sainsbury's and is called the Sainsbury Orchid Conservation Project
(SOCP). See the review by Ramsay and Stewart (1998).

One consideration is the
genetic bottleneck that can result from inbreeding: insufficient genetic
variation would weaken the plants. To counter this, DNA tests were conducted on other plants remaining
in cultivation or planted artificially. One such plant in Lancashire is a foreign import, probably of
European origin. The aim is to maintain the genetic distinctiveness of the British population. Fortunately,
some plants in horticulture were shown to be closely related to the wild surviver, suggesting they are
likely descended from wild plants, and their pollen is also used along with the wild plants own pollen. The
resulting genetic diversity is apparently surprisingly high, which is good news. Pollen from foreign stock is
excluded. Seedlings have been introduced to a number of other sites, one of which is open to the public.

The wild plant is in a secret location and is well guarded.
Andrena bees visit occasionally but natural
pollination has not been seen to date. Perhaps the population is too small to effectively attract
pollinators, or perhaps it lacks suitable plants nearby which do offer bees a genuine reward. Only when
natural pollination occurs and the population(s) become self-sustaining can this rescue effort be truly
considered a success.