AMINO ACID AND SUGAR CONTENT OF THE NECTAR
EXUDATE FROM LIMODORUM ABORTIVUM (ORCHIDACEAE).
COMPARISON WITH EPIPACTIS ATROPURPUREA
NECTAR COMPOSITION
Maria Salomé S. PAIS Departame
ll
to de
Biologia Vegetal,
Faculdade de Ciêticias R. da EscolaPolitécnica,
1294 LisboaCodex, Portugal
H.J. CHAVES DAS NEVES Ana Maria P. VASCONCELOS Faculdade de Ciências eTec l1 ologia.
Universidade Nom de Lisboa2825 Monle da
Caparica, Portugal
SUMMARY
The amino acid content of nectary exudates from two
orchids,
L. nbortivum (spurnectary)
and E. ati!optirpiti-ea(open
nectary) were studiedby
means ofcapillary
gaschromatography.
Amino acids were analyzed as the
corresponding N-heptafluorobutyryl isopropyl
esters. For each aminoacid,
enantiomericcomposition
was achievedby
gaschromatography
of theN-pentafluoro- propionyl isopropyl
esters on acapillary
coated with the chiralliquid phase
Chirasil-Val.Comparison
of the amino acid contents of both nectars, as well as the enantiomericcomposition
of theparticular
aminoacids,
indicateevolutionary
differences of both nectaries.Gas
chromatographic analysis
of the nectar sugars, supports the conclusion that the open nectary ofEpipacti,r
atropllrpllreabelongs
to a more advancedevolutionary
typecompared
to the spur nectary of Gimoclonun o6ot!)t<m. The
possible
influence of the nectarcomposition
onpollinators
is discussed.INTRODUCTION
Floral and extrafloral nectaries from epiphytic and terrestrial orchids have been studied by different authors (M AURIZIO , 1959 ; B ASKIN and BLISS, 1969 ; A
RDITTI
et al., 1971). Particular attention has been paid
tothe sugar
content.Studies by P ERCIVAL (1961) using paper chromatography showed that Epi- pactis atrorubens, E. helleborine and E. palustris
are sucrosedominant. According
to J EFFREY
etal. (1970), the
nectarof orchids
canbe classified
asFructose (F),
Glucose (G) and Sucrose (S) dominant. Gas chromatography of the nectary
exudates from 20 orchid species revealed that fructose, glucose,
sucroseand
raffinose
aredominant
overother
mono-and oligosaccharides (B ASKIN and BLISS,
1969). Similar results
werereported for the sugar
contentof the
nectarof
Epipactis atropurpurea (PA I S and CHAVES
DASNEVES, 1980).
The first reports
onfree amino acid content of
nectars aredue
toZ IEGLER
(1956). BAKER and BAKER (1977) have intensively studied the amino acid
content
of
nectarexudates from different species. The role of the nectary exudate in attracting orchid pollinators has been emphasized since D ARWIN (1877), namely by
VAN DERP IJL and D ODSON (1966), P ERCIVAL (1961), BAKER
and BAKER (1973) and VoGEL (1983).
Our paper deals with the study of free amino acid and sugar
contentof the nectar exudate from Limodorum abortivum and its comparison with the nectar
exudate of Epipactis atropurpurea.
MATERIALS AND METHODS
1. Collection and
Storage of
NectarsThe nectary exudates from
Epipactis
atropurpurea and Limodorum abortivunt were collectedby
means of aglass capillary
from 30 to 40plants
until a volume of 3 ml was obtained. The nectar wasimmediately
frozen aftercollection, freeze-dried,
and stored under vacuum until furtheruse.
2. Materials
All solvents used were obtained from E. MERCK
(Darmstadt)
andpurified by
fractional distillation afterappropriate drying. Hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS), pyridine, ethanethiol, heptafluorobutyric anhydride (HFBA)
andpentafluoropropionic anhydride (PFPA)
werechromatographic grade
from Pierce Chemical Co.(R OCKFORD , ILL.)
and used without furtherpurification.
Standard amino acids were obtained fromSupelco
andcycloleucine
was
purchased
from AldrichEurope.
The solutions of HC1 inisopropyl
alcohol wereprepared
in a volumetric flaskby adding
theappropriate
amount ofacetyl
chloride(E. M ERCK , Darmstadt)
to
isopropyl alcohol,
in an ice bath. OV-101 was obtained from Alltech Associates.3. Instruments
Capillary
gaschromatography
wasperformed
with aPye
Unicaminstrument,
Model204, equipped
with asplitter
and a flame ionization detector(FID). Hydrogen
was used as carriergas, U = 25 cm/s,
split
ratio 1 : 60.Analysis
was carried out on a 25 m X 0.18 mrri! i.d. borosilicateglass capillary
coated with OV-101according
to the method describedby
SettoMSVxc et al.(1979).
Enantiomer
separations
were achieved on a 25 m X 0.25 mmglass capillary
coated with Chirasil- Val.Quantitative
calculations were carried out with aSpectra Physics computing integrator
ModelSP 4100.
Response
factors were calculated relative toN-heptafluorobutyryl cycloleucine isopropyl
ester as internal standard. GC/MS
experiments
were executed with a ShimadzuQP-1000
instrument.4. Isolation
of
Amino Acids andSugars from
L. abortivumThe initial volume of the nectar was reconstituted
by
addition of thecorresponding
amount of deionized water. From this solution a knownweight (0.640 g)
was transferred to a 10 ml volumetricflask,
0.100 ml of a 4mg/10
ml solution ofcycloleucine
were added and the volumecompleted
to 10 ml
by
addition of deionized water. The solution waspassed through
a column of the cationicexchanger
Dowex50 W-X 8,
the column was washed withapproximately
four times its volume of deionized water and the aqueous solutions collected andevaporated
todryness
in a rotary evaporator.Weight
of thesugar-containing
residue was 0.108 g(16.9 %).
The amino acids retained on the resin were eluted from the column with
approximately
four times its volume of a 4 M NH40H solution. The ammoniacal solution was collected andevaporated
to
dryness
in a rotary evaporator(0.022
g, 3.4%).
5. Isolation
of
Amino Acids andSugars from
E.atropurpurea
The freeze-dried nectar was treated as above. A
starting weight
of 0.549 g afforded 0.176 g of asugar-containing
residue(32 %)
and 0.036 g of an aminoacid-containing
residue(6.5 %).
6. Derivatization
of
theSugars
The sugars were derivatized to the
corresponding
TMS ethersby
reaction withN,O-bis (trimethylsilyl)
trifluoroacetamide(BSTFA)
indimethylformamide according
to apreviously
describedprocedure (P AIS
and CHAVES DASN EVES , 1980).
7. Derivatization
of
Amino AcidsThe residue
containing
the free amino acids was dissolved in a minimal amount of water and transferred to a volumetric flask and the volumecompleted
to 10 mlby
addition of deionized water. A. 0.250 to 0.500 mlaliquot
was transferred to a teflon-lined screw cap derivatizationflask,
the solventevaporated
under alight
stream ofnitrogen.
0.250 ml of 4 M HCI inisopropyl
alcohol and 0.010 ml of ethanethiol were added to the residue and heated at 110° for 30 min.in the closed vial. After
cooling
to room temperature, the solvent wasevaporated
under alight
stream of
nitrogen.
To the residue 0.200 ml ofdichloromethane,
0.100 ml of HFBA and 0.010 ml of ethanethiol were added. The flask was closed and heated at 150° for 15 min. Foranalysis
ofhistidine,
the residueresulting
from theevaporation
of the solvent under alight
stream ofnitrogen
was treated at 150’ for 20 min. with 0.250 ml of benzene and 0.050 ml of ethyl
pyrocarbonate.
The solution was concentrated to 0.005 ml and
directly
used for gaschromatographic analysis.
An identicalprocedure
was used for thepreparation
of theN-pentafluoropropionyl isopropyl
esters asrequired
for enantiomerseparation.
RESULTS AND DISCUSSION
As inherent constituents of
acell, amino acids represent
morethan 50 % of its dry weight, mainly
asbuilding blocks of proteins. They
are notonly
structural elements of the cell but also
act asvehicles of selective transport
or asmessenger substances, nutrients, intermediates in metabolism and biosynthetic
precursors. They play a major role in plant physiodogy and
areimportant metabolic
intermediates. Compared
toother methods, capillary gas chromatography offers
the advantages of high analysis speed, high sensitivity, high resolving power and great versatility of the instrument, together with its ability of on-line coupling
to a mass
spectrometer for rapid identification of unknowns. It requires, however,
the previous preparation of volatile derivatives (for
arevision
seeFRANK, 1985).
For many years, floral
nectars weretreated
asif they simply
were asolution of sugars in water. However, the
occurrenceof free amino acids in
nectars seems tobe universal among nectariferous plants, possibly of taxonomic value (BAKER and BAKER, 1973 a, b).
The amino acids of the
nectarsolutions
wereanalyzed
asthe corresponding N-heptafluorobutyryl isopro!pyl esters,
or asthe N-pentafluoropropionyl isopropyl
esters
for enantiomer separation. Histidine
wasanalyzed
asthe corresponding N&dquo;&dquo;-
ethoxycarbonyl derivative. All the amino acids could be separated in
asingle
run on a
WCOT column coated with OV-101 (Fig. 1). The results
aresummarized in Table 1. When the relative concentrations
areconsidered,
acharacteristic pattern of amino acids is obtained for each of the nectary types.
As
canbe
seenin Fig. 2, there
areconsiderable differences between the amino acid patterns of both
nectars.While in the open nectary of E. atropurpurea the
more
functionalized amino acids predominate, in the nectar of L. abortivum, which possesses
aspur nectary, the less functionalized amino acids dominate the amino acid spectrum. Although seasonal differences in the concentration of
aparticular amino acid have been observed during
athree year study of both
nectars, the described amino acid pattern
wasconstant and characteristic. A very
interesting result is the presence of D -amino acids in both
nectars.D -glutamic
acid is analyzed in both nectars with
ahigh relative enantiomeric concentration.
An unexpectedly high
amountof D -alanine
wasfound in the
nectarof E.
atropurpurea. D -cystein
wasexclusively present in the
nectarof Epipactis
atro-purpurea. In the
nectarof L. abortivum in the other hand, D -isoleucine has been
exclusively identified (Table 2).
A major difference
wasfound by comparison of the sugar
contentsof both
nectars.The
nectarsof Limodorum abortivum is largely dominated by
sucrose,with both fructose and glucose present in small amounts, mannitol, sorbitol and lactose being present only in
traces(Fig. 3). This
contrastsopenly with the
more
complex composition of the
nectarfrom E. atropurpurea (P AIS and C HAVES
DAS
N E V E S, 1980).
From
ourresults
wemay conclude that nectars of both species
arevery rich in amino acids (19 different amino acids for E. atropurpurea, 18 for L.
abortivum). The total amino acid-containing residue is 6.5 % of the total
nectar
weight for E. atropurpurea and 3.4 % for L. abortivum. Lysine and
histidine
aredetected only in E. atropurpurea. The
moresignificant difference
seems to
be correlated with the presence of D -amino acids. It is interesting
tonote
that while D -glutamic acid is present in significant
amountsin both
nectarstogether with
ahigh relative enantiomeric concentration of D -alanine (96.4 and 38.6 respectively for E. atropurpurea and L. abortivum), D -cysteine
wasexclu- sively detected in the former, while D -isoleucine could only be detected in the latter.
To the best of
ourknowledge,
noreport exists
onthe relative
amountsof D -amino acids in
nectarsfrom different species
orgenera. It will be interesting
to
check if differences in D -amino acids
cancontribute
totracing taxonomic relationships between genera or/and type of nectaries (spur
oropen nectaries).
According
toBAKER and BAKER (1973 b) higher amino acid
scores arerelated to increasing evolutionary advancement. If the amino acid concentration of nectars,
as
previously reported by BAKER and BAKER (1973 b)
canbe related
tothe evolution degree of the nectaries,
ourresults would
accountfor
aless evolved nectary in L. abortivum (6.3 mg/100 ml total amino acid concentration) when compared
tothe open nectary of E. atropurpurea (10.5 mg/100 ml). This
trend is in agreement with the results obtained in the analysis of both
nectars.The sugar-containing residue
amounts to32 % of initial
nectarweight from
E. atropurpurea and only
to16.9 % in the
nectarfrom L. abortivum. In this context, nectaries of L. abortivum may be less evolved than those of E. atropur- purea. Nectar from L. abortivum contains mainly
sucrose(Fig. 3), while that from E. atropurpurea is of the fructose-glucose type (PA IS and C HAVES
DASN EVES , 1980). H ARBORNE (1982), based
onthe sugar composition of nectars, suggested that, in angiosperms,
anevolutionary trend
canbe established from
nectarsof the
sucrosetype
to nectarsof the fructose-glucose type.
It has also been reported that the
nectarsof the
sucrosetype
aremainly
secreted by spur nectaries, while the
nectarsof the fructose-glucose type would originate from open nectaries (P ERC mAL, 1961). Our results
onLimodorum abortivum and Epipactis atropurpurea
nectars arein full agreement with this.
A correlation between the sugar
contentof nectaries and the kind of pollinators
has been suggested (P ERCIVAL , 1965 ; BAKER and BAKER, 1975). According
to
these authors, butterflies would prefer nectars containing 21-48 % sugar, while honey bees would prefer nectars with
asugar concentration range within 10
to74 %. On the other hand, bumble bees, would favor nectars the sugar concentration of which is in the range of 30 to 40 %. According
tothis point
of view, the nectars of both E. atropurpurea and L. abortivum would be suitable
for all three kinds of pollinators. The fact that the overall amino acid
contentof the nectar of L. abortivum is lower than in E. atropurpurea suggests that the
former may be preferentially collected by bees while the latter would be suitable for bees and butterflies. On this subject W ALLER (1972) has noticed that bees collect preferentially
nectarswith
sucroseconcentrations of 30
to50 %. This is the
casewith L. abortivum. According
tothe observations of G ODFER Y (1933)
the pollinators of L. abortivum
aredifferent kinds of bees. On the other hand,
bumble bees (G ODFERY , 1933) and honey bees have been described
aspollinators
of E. atropurpurea (W IEFELSP Ü TZ , 1970).
CONCLUSION
High resolution power, speed of analysis and high sensitivity of capillary
gas chromatography make this method the analytical tool of choice for the
analysis of complex mixtures. Capillary gas chromatography
wasthus used for
the determination of amino acids and sugars, after adequate der,ivatization, in the
nectarsof Epipacti.r atropurpurea and Limodorum abortivum. The former possesses
anectary of the open type in
contrastwith the spur type nectary of the latter. In agreement with observations of others
ourresults
seem toindicate
a
difference in the evolutionary stage between the orchids, L. abortivum belonging
to alower evolved type. According
toH ARB O RNE (1982), lower scoring
families tend
tobe bee-pollinated, bees being able
toobtain nitrogen from
sources
other than nectar, such
aspollen. Comparative discussion of the amino acid concentrations in both
nectarsclearly shows important quantitative and qualitative differences in amino acid composition. These differences
canbe correlated
tothe nectary type (open
orspur) in each case, in agreement with the observations of other authors. Major differences in sugar composition
werealso observed. While the
nectarof E. atropurpurea follows the fructose-glucose pattern,
aspreviously described, the
nectarof L. abortivuni belongs
tothe
sucrose
type.
Different kinds of bees have been described
aspollinators of L. abortivum
(GoDr.ERY, 1933), while bumble bees and honey bees
seem tobe pollinators of
E. atropurpurea. Our results suggest that systematic comparative studies of
nectarcomposition, by powerful analytical techniques
ascapillary gas chromatography
could prove invaluable in the study of pollinator-plant relations
aswell
asin the
chemotaxonomic
area.Received
for publication
inJuly
1985.Accepted for publication
inJanuary
1986.RÉSUMÉ
TENEUR EN ACIDES AMINÉS ET EN SUCRES DU NECTAR DE LIMODORUM ABORTIVUM
(ORCHIDACEA).
COMPARAISON AVEC LA COMPOSITION DU NECTAR D’EPIPACTIS ATROPURPUREA La teneur en acides aminés du nectar de 2
orchidées, Epipactis
atropurpurea(nectaire ouvert)
et Limodorum abortivum
(nectaire
àéperon)
a été étudiée parchromatographie
enphase
gazeuse sur colonnescapillaires, après
avoir été isolés paréchange
d’ions(Dowex 50 W-X 8).
Afin d’obtenir des dérivésvolatiles,
les acides aminés ont été transformés enN-heptafluorobutyryl isopropylesters.
Ceux-ci ont pu être entièrement
séparés
en 1 seul passagechromatographique
sur 1capillaire
enverre de 25 m X
0,18
mmimprégné
de OV-101(Fig. 1). L’analyse
a été réalisée par la méthode de l’étaloninterne, après
calcul des coefficients deréponse
individuels vis-à-vis de lacycloleucine
utilisée comme étalon interne. L’histidine a étéanalysée
sous forme du dérivéN’&dquo;’-ethoxycarbonyl correspondant.
Ces acides aminés ont été identifiés individuellement parcomparaison
avec des échantillonsauthentiques,
par les temps relatifs de rétention et parchromatographie
enphase gazeuse-spectrométrie
de masse(GCMS).
Les résultats sontprésentés
dans le tableau 1. Laséparation
des acides aminés D et L a été réalisée sur uncapillaire
de verre de 25 m X0,25
mmimprégné
d’unephase liquide
chirale de chirasil-val et la concentrationénantiomérique
relative a été calculée àpartir
de l’aire despics (Tabl. 2).
Un modèlecaractéristique
est obtenu pour chacun des types de nectar(Fig. 2).
Les acides aminés lesplus
fonctionnalisésprédominent
dans lenectar d’E. atropurpurea. Un résultat très intéressant est la
présence
dans les 2 nectars dequelques
acides aminés-Dqui
ne sont pas les mêmes àl’exception
de l’acideglutamique-D, présent
dans les 2 nectars avec unecomposition énantiomérique plus
ou moinséquivalente.
On a trouvé unequantité
élevée inattendue d’alanine-D dans le nectar d’E. atropurpurea. Une différencemajeure
entre les 2 nectars est la
composition
en sucres, déterminée par lachromatographie
gazliquide capillaire
des esters TMS. Le nectar de L. abortivum estlargement
dominé par le saccharose tandis que celui d’E. atropurpurea a unecomposition plus complexe (Pms
et CHAVES DASNEVES, 1980).
De ces résultats nous pouvons conclure que les deux nectars sont
qualitativement
riches en acidesaminés ;
19 acides aminés ont été identifiés chez E. atropurpurea et 18 chez L. abortiviiin.La concentration totale en acides aminés est
respectivement
de10,46
et6,32 mg/100
ml. Si les concentrations en acides aminés reflètent ledegré
d’évolution des nectaires(B AKER
and BAKER,1973 b),
alors nos résultatsindiquent
que le nectaire de L. abortivum est moins évolué que celui d’E. atropurpurea. Le fait que la teneur totale en acides aminés du nectar de L. nbortivum soitplus
faible que celle du nectar d’E. atropurpureasuggère
que lepremier
est récolté depréférence
par les
abeilles,
tandis que le second convient à la fois aux abeilles et auxpapillons.
La
puissance
de résolutionélevée,
la vitessed’analyse
et la haute sensibilité de la chro-matographie
enphase
gazeuse surcapillaires
permet d’introduire des méthodesplus précises
pour établir la teneur en acides aminés des nectars. Pour lapremière fois,
l’ensemble des acides aminés des nectars d’orchidées est établi en détail. L’utilisation de nouvellesphases liquides
chirales apermis
pour lapremière
fois d’identifier des acides aminés-D dans les nectars. Des études compara- tivessystématiques
de lacomposition
du nectar, à l’aide detechniques analytiques puissantes
tellesque la
chromatographie
enphase
gazeuse surcapillaires pourrait
se montrer trèsprécieuse
dans l’étude des relationspollinisateur-plante
et dans le domaine de la chimiotaxonomie.ZUSAMMENFASSUNG
AMINOSÄUREN- UND ZUCKERGEHALT DER NEKTARAUSSCHEIDUNG VON LIMODORUM ABORTIVUM
(ORCHIDACEAE).
VERGLEICH MIT DER NEKTARZUSAMMENSETZUNG BEI EPIPACTIS ATROPURPUREA Der
Aminosäurengehalt
von Nektarexsudaten der beiden OrchideenEpipactis
atropurpurea(offene Nektarien)
und Limodorum abortivum(Nektarsporn)
wurde mit Hilfe vonKapillar-
Gasflüssigkeits-Chromatographie
nach der Isolation durch Ionenaustausch (Dowex 50 W-X 8) unter- sucht. Um auchflüchtige
Derivateauffangen
zukönnen,
wurden die Aminosäuren inN-Heptafluoro- butyryl-Isopropylester
transformiert. Diese können mit einemchromatographischen Durchgang
in einer 25 m X0,18
mmi.d. Glaskapillare
beschichtet mit OV-101(Fig. 7)
isoliert werden. DieAnalyse
wurde mitCycloleucin
als internem Standarddurchgeführt.
Histidin wurde alsN’ m -Ethoxycarbonyl-
Derivatanalysiert.
Die Identifikation der individuellen Aminosäuren wurde durchVergleich
der rel.Retensionszeiten mit authentischen Proben und durch GCMS
durchgeführt.
DieErgebnisse
sind in Tab. 1zusammengestellt.
DieSeparation
von D- und L-Aminosäuren wurde durch eine 25m X 0,25
mmGlaskapillare
beschichtet mit der chiralenFlüssigkeitsphase
von Chirasil-Valdurchgeführt
und die relative enantiomerische Konzentration von den maximalen Flächenwerten errechnet(Tab. 2).
Ein charakteristisches Muster wurde fürjeden Nektarientyp aufgezeichnet (Fig. 2).
Die mehr funktionalisierten Aminosäuren dominieren im Nektar von E. atropurpurea. Ein sehr interessantesErgebnis
ist die Präsenz voneinigen
D-Aminosäuren in den beiden Nektaren.Diese sind unterschiedlich in
jedem
Nektar mit Ausnahme vonD -Glutaminsäure,
die in beiden in mehr oderweniger äquivalenter
enantiomerischerKomposition
vorhanden ist. Eine unerwartet hoheMenge
von D-Alanin wurde im Nektar von E. atropurpureagefunden.
Ein bedeutender Unterschied zwischen den beiden Nektaren wurde in derZuckerzusammensetzung gefunden,
diemit Hilfe der
Kapillar-Gasflüssigkeits-Chromatographie
der TMS Äther bestimmt wurde. Der Nektar von L. abortivum wird besonders von Sacharose dominiert imGegensatz
zu der mehrkomplexen Zusammensetzung
des Nektars von E. arroparpurea(P AIS
and CHAVES DASN EVES , 1980).
Aus diesen
Ergebnissen folgern wir,
daß beide Nektare reich an Aminosäuren sind. Für E. atropurpurea wurden 19 und für L. aboi-tiviim wurden 18 verschiedene Aminosäuren identifiziert.Die gesamte Aminosäurekonzentration war
jeweils 10,46
und 6,32(mg/100 ml).
Falls die Aminosäurekonzentration inBeziehung
zumEvolutionsgrad
der Nektarien gesetzt werden könnte(B
AKER
andB AKER , 1973 b),
würden unsereErgebnisse
einweniger
evoluiertes Nektarium bei L. abortivurre als bei E. atropiirpiireaaufzeigen.
Die Tatsache eines kleineren totalen Aminosäure-gehalts
im Nektar von L. abortivum als im Nektar von E. atropurpurea läßt daraufschließen,
daß ersterer vornehmlich von Bienengesammelt
wird während der andere Bienen undSchmetterlingen zugeschrieben
werden kann.Durch die hohe
Auflösung,
dieGeschwindigkeit
derAnalyse
und die hohe Sensitivität derKapillar-Gasflüssigkeits-Chromatographie
könnenpräzisere
Methoden derBestimmung
des Ami-nosäurengehalts
von Nektarienexsudateneingeführt
werden. Die Gesamtheit der Aminosäuren des Orchideen-Nektars wurde zum ersten Mal in Einzelheiten bestimmt. Durch den Gebrauch derneuen chiralen
Flüssigkeitsphasen
konnten zum ersten Mal die D-Aminosäuren in den Nektaren identifiziert werden.Systematische vergleichende
Studien derNektarzusammensetzung
durch effektiveanalytische
Techniken wieKapillar-Gaschromatographie
könnten für dieErforschung
von Bestäuber-Pflanze-Beziehungen
wie auf chemotaxonomischem Gebiet wertvoll werden.REFERENCES
A
RDITTI
J.,
I!OOPOWITZH.,
JEFFREYA.C.,
1971. - David’s reagent or the sugar content of orchid nectars. Am. Orch. Soc.Bull., 39,
1091-1092.BAKER
H.G.,
BAKERL,
1973 a. - Amino acids in nectar and theirevolutionary significance.
Nature, 241,
543-545.BAKER
H.G.,
BAKERI.,
1973 b. - Amino acidproduction
in nectar. In : HEYWOOD V.H.ed., Taxonomy
andEcology,
AcademicPress, London,
243-264.BAKER
H.G.,
BAKERL,
1975. - Nectar constitution andpollinator-plant
coevolution. In : GILBERT
L.E. and RAVEN P.H.
ed.,
Coevolutionof
animals andplants, University
TexasPress,
Austin,
100-140.BAKER
H.G.,
BAKERL,
1977. -Intraspecific
constancy of floral nectar amino acidcomplement.
Bot. Gaz., 138,
183-191.B
ASKIN
S.I.,
BLISSC.A.,
1969. -Sugar occurring
in the extrafloral exudates of orchidaceae.Phyto- chemistry, 8,
1139-1145.D
ARWIN
C.,
1877. - The various contrivancesby
which orchids arefertilized by
insects, 2nded.,
D.Appleton
&Co.,
New York.FRANK
H.,
JAEGERH.,
1985. -Capillary
gaschromatography
of amino acids. In : JAEGER H.ed.,
Glasscapillary chromatography
inmedicine,
clinicalchemistry
andpharmacology,
MarcelDekker,
NewYork,
73-124.G
ODFERY
M.J.,
1933. -Monograph
andiconograph of
native britishorchidaceae, Cambridge
Uni-versity
Press.H
ARBORNE
J.B.,
1982. - Introduction toecological biochemistry,
2nd ed. - AcademicPress, London,
NewYork,
57-65.J
EFFREY
A.C.,
ARDITTIJ.,
KOOPOWITZH.,
1970. -Sugar
content in floral and extrafloral exudates of orchids :pollination, myrmecology
andchemotaxonomy implication.
NewPhytol., 69,
187-195.M AURI
zio
A.,
1959. -Papierchromatographische Untersuchungen
anBliitenhonigen
und Nektar. Ann.Abeille, 4,
291-341.P
AIS
M.S.S.,
CHAVES DAS NEVESH.J.,
1980. -Sugar
content of the nectary exudate of Epipactis atropurpurea Rafin.Apidologie, 11,
39-45.P
ERCIVAL
M.S.,
1961. -Types
of nectars inangiosperms.
NewPhytol., 60,
235-281.P
ERCIVAL
M.S.,
1965. - FloralBiology.
- PergamonPress, Oxford,
New York.S
CHOMBURG
G.,
HUSMANNH.,
BORWITZKYH.,
1979. -Alquilpolysiloxane glass capillary
columnscombining high
temperaturestability
of thestationary liquid
and deactivation of the surface.Thermal treatment of dealkalinized
glass
surfacesby stationary liquid
itself.Chromatographia, 12,
651-660.V
ANDERPIIL
L.,
DODSONC.H.,
1966. - Orchidflowers.
Theirpollination
and evolution. -University
of MiamiPress,
CoralGlaves,
Florida.V
OGEL
S.,
1983. -Ecophysiology
ofzoophilic pollination
inphysiological plant ecology.
In : LANGE
U.L.,
NOBELP.S.,
OSMOND C.B. and ZIEGLER H.ed., Encyclopedia
ofplant physiology,
Vol. 12 C NewSeries, Springer Verlag, Berlin, Heidelberg,
560-624.W
ALLER
G.D.,
1972. -Evoluting
responses ofhoney
bees to sugar solutionsusing
an artificial flower feeder. Ann. Entomol. Soc.Amer., 65,
857-862.W IEFELSP Ü
TZ
W.,
1970. - f5ber dieBlutenbiologie
derGattung Epipactis.
Jb. Naturw.Ver., Wuppertal, 23,
53-69.Z
IEGLER
H.,
1956. -Untersuchungen
aber dieLeitung
und Sekretion der Assimilate.Planta, 47,
447-500.