Bobnicaljmmal of& k a n So&&
(2000),133: 285-302. With 5 figures
do? 10.1006/boj1.2000.0336,available online at http://www.idealibrary.com on
@
bl
Natural hybridization between knilla claviculata
(Wowright)Sw. and K barbellata Rchb.f.
(Orchidaceae):genetic, morphological, and
pollination experimental data
LENE ROSTGAARD NIELSEN
Botanical Institute, UniversiQ of Copetlhagen, Osh Farimagsgade 20, DK-1353
Copenhagen X, Denmark
Receivd3ub 1999; acceptedfor publication 3anuaty 2000
Vmniua clavinlutu (W.Wright) Sw. and K barbehtu Rchb.f. grow sympatrically in the southwestern part of h e r t o Rico. At three localities in the range of the two species, individuals
with flowers that appeared Merent from previously known species were found. To test
whether these were hybrids between !F clauiculata and K barbelh, 11 morphological floral
characters were recorded at seven populations and allozyme profiles of the three taxa
compared. Principal component analysis on the morphological characters gave three distinct
groups with minor overlaps. The first component axis reflects variation in overall size of the
flowers, while the second component reflects variation in the size of the distal aperture of
the labellum. AUozyme data revealed that the putative hybrids have a significant surplus of
heterozygotes (&= -0.65) as expected in a F, generation. Both data sets support the
hypothesis that the individuals are hybrids, which is further augmented by interspecific
pollination experiments between the putative parental species. This is the first case of natural
hybridization reported in the genus Vmilla.
Q ZOO0 The Linnean Society of London
ADDITIONAL, KEY WORDS:-haracter
germination - principal component analysis.
expression
-
enzyme electrophoreses -
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . .
Material and methods . . . . . . . . . . . . . . . . . .
Vmilla species . . . . . . . . . . . . . . . . . . . . .
Morphological data . . . . . . . . . . . . . . . . . .
Enzyme electrophoretic data . . . . . . . . . . . . . . .
Data analysis of morphological and enzyme electrophoretic data . . .
Pollination experiments . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . .
Morphology . . . . . . . . . . . . . . . . . . . . .
Genotypic compositions . . . . . . . . . . . . . . . .
Pollination experiments . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
0024-4074/00/070285+ 18 $35.00/0
285
.
.
.
.
.
.
.
286
286
286
289
289
291
292
292
292
295
298
299
30 1
30 1
0 2000 The Linnean Society of London
286
L. R. NIELSEN
JNTRODUCTION
Natural hybrids can be defined as the offspring between individuals from two
natural populations that are distinguishable based on one or more heritable characters
(Arnold, 1997; Harrison, 1990). Hybridization between such populations may be
avoided by either premating or postmating barriers. Premating barriers in plants
include, for example, differences in phenology or other ecological factors and in
floral structures or pollinator specificity (Van der Pijl & Dodson, 1966; Dressler,
1981; Gill, 1989; Grant, 1994; Arnold, 1997). Post-pollination barriers can be
incompatibility mechanisms (based on recognition molecules) (Arnold, 1997) or
barriers after fertilization such as zygote mortality, hybrid inviability or hybrid
sterility (Mayr, 1963).
Scientific investigations on natural hybridization can be traced back to Linnaeus
(Arnold, 1997). In recent years new technological methods, such as molecular
markers, have renewed interest in hybridization (Rieseberg & Ellstrand, 1993) and
reopened discussions on its possible significance. The origin of allopolyploids may
be the most significant evolutionary role of hybridization in plants (reviewed by
Soltis & Soltis, 1993). At the diploid level, a number of evolutionary consequences
caused by hybridization have been suggested, such as an increased genetic diversity
in the involved species (Anderson, 1948), the origin and transfer of adaptations
(Anderson, 1948; Stebbins, 1950), establishment of new ecotypes or species (Grant,
1981; Rieseberg, 1997), and breakdown or reinforcement of isolating mechanisms
(Ellstrand & Elam, 1993; Levin, Francisco-Ortega &Jansen, 1996; Rieseberg, 1997).
At a locality in Puerto Rico where three native species of Vanilla, R claviculata
(W.Wright) Sw., R barbellatu RchbX and R dilloniana H.B.Correll grow sympatrically,
plants were discovered with flowers that to some extent appeared to be intermediate
between the two species V claviculata and V barbellatu. As these two species overlap
in phenology and are possibly pollinated by the same bees (Nielsen & Ackerman,
unpubl.), it was questioned whether the deviating individuals could be hybrids of R
claviculatu and R barbellata.
In this study, the first natural hybrid in the genus Vanilla is confirmed using both
genetic and morphometric analyses and the reasons why hybridization may occur
in the studied species are discussed.
MATERIAL AND METHODS
Vanilla species
In Puerto Rico, R claviculata is known at localities ranging from Guajataca State
Forest in the north (Isabela district) to the Susua Forest Reserve in the south
(population CIS and CSU in Fig. 1). Vanilla claviculatu has been reported from Cab0
Rojo and Guanica in Ackerman (1995) but during the present study no specimens
were found at these sites. Vanilla barbellata was found in the southwestern to southern
part of the island where the Susua Forest Reserve is the northernmost locality and
shared with R claviculatu. In addition, a population of R barbellata was rediscovered
east of Puerto Rico on the small island Isla de Vieques. Vanilla claviculatu was found
in moist serpentine shrub forest while R barbellata was found in drier areas. The
NATURAL. HYBRIDIZATION IN V
.
i
1
.CSG
m
CIS
csu
.
BYA
XYA
Isla de Viequee
I
Caribbean Sea
10km
Figure 1. Localities of Vatlilfu claviGulata (beginning with C), K barbcllata (beginning with B) and the
putative hybrid (beginnimg with X) in Puerto Rico. See Table 1 for the population abbreviations.
Susua Forest Reserve, where both species occur, is geographically intermediate in
the range of K claviculata and I? barbellatu.
The long succulent viny stems entangle with the surrounding vegetation and some
individuals may be found throughout large areas. The leaves are scale-like and shed
early. The showy resupinate flowers are arranged spirally in the inflorescences. They
open sequentially, each flower lasting one day only. The petals and sepals are green
to olive-green in all three taxa. The basal margins of the labellum are adnate with
the lower part of the column; a character unique for the subtribe Vanillinae (Dressler,
1993). The free portion is basally involute and arching over the column so that the
labellum appears tubular. The anther contains four yellow, soft and granulose
pollinia and the stigmatic cavity is located below and ventral to the anther.
The three taxa are distinguished by their flowers: in K clauiculatu the labellum is
white, sometimes with two lateral red blotches (Fig. 2A). The free part of the
labellum is slender with the basal half containing numerous short fleshy hairs. The
labellum of K barbellatu is deep red with a yellow midrib and the tube formed by
the involute margins has a relatively wide distal aperture, making it appear trumpetshaped (Fig. 2B). The basal half contains no hairs. Flowers of the putative hybrid
have an almost white labellum but with light pink markings in the throat, only a
288
L. R.
~ELSEN
Figure 2. Flower of (.\ ) Jrmi!la clavicu!ata, (B)
r: barhel!ata and
(C ) the putative hybrid.
NATURAL HYBRIDIZATION IN VH.MlU
289
TABLE
1. List of abbreviations for populations, number of measured flowers, number of samples and
individuals analysed with enzyme electrophoresis
~
Taxon
Locality
Abbreviation
Measured
flowers
K barbellah
K cklviculah
Hybrid
Cabo Rojo
Penuelas
Ponce
Susua
Vieques
Yauco
Total
BCR
BPE
BPO
BSU
BVI
BYA
Campamento
Santana
lsabela
Maricao
San German
Susua
Total
CCS
Guanica
Susua
Yauco
Total
CIS
CMA
CSG
-
36
-
7
43
-
Sample size for
elertrophoresis
Defined
individuals
6
16
29
35
27
3
I16
12
24
25
17
3
87
6
5
18
29
16
21
89
6
csu
29
32
20
30
16
23
95
XGU
41
23
7
71
13
5
5
23
xsu
XYA
3
~
6
2
2
10
few are pure white (Fig. 2C). The shape of the labellum is simiiar to K barbellata but
it is generally larger and covered with numerous short hairs.
Morphological data
Morphological data were gathered from seven populations. The names of the
populations and their abbreviations are given in Table 1. Flowers were either
assigned to K claviculata or K barbellata by visual examination of floral characters
according to Ackerman (1 995). Flowers not fitting either species were classified as
deviant.
One qualitative and ten quantitative floral characters were recorded in the field
(Fig. 3 and Table 2). To reflect the variation within and between populations as
many flowers as possible were measured, but because of the entangling growth of
the lianas, flowers were only assigned to taxon and not to individual.
E n g m e ehchrphoretic data
Pieces of stem ( 40 cm) of K claviculata, K barbellata, and of hybrids were collected
from 14 populations at 11 different localities (Fig. 1). As the vegetative parts of the
taxa are very similar only material from flowering individuals was collected at
localities where more than one species were known to grow (based on Ackerman,
1995 and on herbarium material of knillu specimens kept at the herbarium of the
University of Puerto Rico, Rio Piedras). Clones with deviant flowers were found at
only three localities, where both K claviculata and K barbellata grew nearby. It was
N
L. R.NIELSEN
290
A
B
Figure 3. Illustration of the 10 quantitative characters in a general Wnilla flower. A, whole flower; dots
represent bristles along the midrib (particularly pronounced in K cluvinrlata). B, labellum seen from
above. See Table 2 for the descriptions of the characters.
TABLE
2. List of recorded floral characters
Number
I
2
3
4
5
6
7
8
9
10
11
Character
Width of inner aperture of labellum
Maximum overall width of labellum
Height of inner aperture of labellum
Maximum overall height of labellum
Maximum length of labellum
Length of the overlap of the two lateral lobes of labellum
Length of lateral petal
Width of lateral petal
Length of lateral sepal
Width of lateral sepal
Basal half of the labellum with or without numerous short hairs (not illustrated)
therefore assumed that distant populations where either R clauiculata or R barbellata
had been recorded flowering in previous years only consisted of a single species.
Samples were separated by at least 10 metres to prevent re-sampling of individuals
and only one sample was taken from each patch to avoid mixing of genotypes. The
stem pieces of presumed individuals were grown at the Botanical Garden of
Copenhagen.
NATURAL HYBRIDIZATION IN V M I U
29 1
Fresh pieces of stem tissue (0.125 cm3)were homogenized in a buffer from Corrias
et al. (1991) containing 2 mM EDTA, 200 mM ascorbic acid and 14 mM 2mercaptoethanol in a 0.2 M tris-HC1 buffer @H 7). The extracts were absorbed
onto wicks of Whatman no. 4 chromatography paper.
Seven interpretable systems polymorphic in at least one taxon were surveyed on
12% starch gels with two buffer systems: (1) Histidine-citrate consisting of a stock
solution of 0.13 M histidine titrated to pH 6.0 with citric acid (monohydrate,
approximately 0.03 M). The electrode buffer was based on a 1: 1 dilution of the
stock buffer, and the gel buffer from a 1:12 aqueous dilution of the stock (Ellstrand,
1984). Histidine-citrate resolved phosphoglucomutase (PGM, EC 5.4.2.2), malate
dehydrogenase (MDH, EC 1,1.1.37), isocitrate dehydrogenase (IDH, EC 1.1.1.42),
shikimate dehydrogenase (SKDH, EC 1.1.1.25) and UTP-glucose-1-phosphate uridylytransferase (UGUT, EC 2.7.7.9). (2) Sodium borate, pH 8.5/Tris-citrate, pH 7.8
buffer (Torres & Bergh, 1978)was utilized to resolve diaphorase (DIA, EC 1.6.99.1).
The electrode buffer consisted of a 1:l aqueous dilution of 0.60M boric acid,
adjusted to pH8.5 with NaOH and the gel buffer of a 1:9 dilution of a buffer
containing 0.165 M tris base titrated to pH 7.8 with citric acid. Weeden & Wendel
(1989) were followed when staining for PGM, MDH, SKDH, DIA and IDH,
excluding agar overlay, but with minor modifications for IDH and SKDH. When
staining for IDH only half the concentration of D,L-isocitric acid was used and for
SKDH the following recipe was applied: 50 mL Tris-HC1 pH 8.0, 90 mg shikimic
acid, 5 mg NADP, 15 mg MTT and 1.2 mg PMS. UGUT was stained as described
in Harris & Hopkinson (1976). When staining for MDH, three zones appeared with
two variable loci, Mdhl and Mdh3, where Mdhl codes for the most anodally moving
enzymes. The loci were numbered consecutively from the anodal end to the cathodal
end with the locus coding for the fastest enzyme being numbered 1. At each locus
the allozymes were named with numbers indicating their mobility relative to the
most common allele across taxa (designated 100).
Data anabsis of morphological and entyme electrophoretic data
As measured flowers could not be assigned to individuals, the morphological and
allozyme data were analyzed separately and no combination of the two data sets
was possible. Ranges, means, sample standard deviations and coefficients of variation
were estimated for each of the morphological characters. The characters were
compared between taxa with Sigmastat 1.O 1 (Sigmastat, 1992-1 994). In characters
where the measurements were normally distributed, a parametric one way analysis
of variance was applied. Otherwise, data were compared with a non-parametric
variance analysis (Kruskal-Wallis one way analysis of variance on ranks). If significant
differenceswere revealed, the taxa were tested pairwise with Student-Newman-Keuls
Method (for normally distributed data) or Dunn’s Method (when not normally
distributed).
In addition, the morphological data were subjected to a principal component
analysis using NT-SYS-PC (Rohlf, 1993). The 11 variables were standardized to
zero mean and variance one (the overall variance sums to 11). In the first analysis,
the distribution of the two parental species was tested using floral measures. Thereafter
all three taxa were included. The transformed values of the 11 characters of the
first two principal components were plotted along the two component axes.
292
L. R. NIELSEN
Before analysing the enzyme electrophoretic data, the multilocus genotypes of
the samples were examined. Individuals collected close to each other (with approximately 10 m between) that had the same multilocus genotype were pooled and
treated as one. Thereafter the allozyme profiles of the three taxa were compared.
For each individual the number of heterozygotic loci was counted and the median
values of each distribution of the three taxa was compared pair wise with a
Mann-Whitney rank sum statistic using SigmaStat 1.O 1 (Sigmastat, 1992).
Deviations from Hardy-Weinberg expectations within populations were quantified
with Wright’s F-statistics as described by Weir & Cockerham (1984). The standard
deviation of each estimate was obtained by jack-knifing over the populations.
Pollination experiments
T o verlfy whether the two species are interfertile, pollination experiments were
conducted over two seasons (1996 and 1997). Interspecific reciprocal pollinations
were carried out by tipping the anther cap of an open unpollinated flower with a
toothpick, removing the pollinarium and subsequently placing it on the stigma of
an open, unpollinated flower of the other species. The pollinarium of this flower
was then removed and placed on the stigma of the first flower. Reciprocal crossings
were performed similarly with flowers of the same species. Abortions were recorded
and the remaining fruits harvested at the end of each reproductive season. The
two pollination categories (interspecific versus intraspecific pollinated flowers) were
evaluated by comparing the proportion of aborted fruits in each class and by
comparing seed quality in the two groups. Prior to estimating the seed quality, a
sample of seeds per fruit was sowed on 5 cm Petri dishes with 6 ml half strength
(half of both the macro- and micronutrients)Murashige and Skoog minimal organics
media (MSMO) as described by Jergensen & Andersen (1998). Before sowing, the
seeds were surface sterilized for five minutes in 5% sodium hypochlorite with a drop
of detergent (Tween 80) per 15 ml sodium hypochlorite. The seeds were then washed
three times in sterile double destilled water and each sample sown on four Petri
dishes with 100-200 seeds per dish. Seed quality was estimated in two ways: by
counting the number of seeds with a viable embryo (out of 400 seeds), and by
examining approximately 400 seeds per fruit for germination after storage in darkness
at 32°C for 24 weeks. Because of fungus infections germination percentages could
not be scored in all replicates. In such cases, seed quality was only estimated as the
percentage of seeds containing viable embryos.
RESULTS
Morpholoa
The ranges of the quantitative characters of K claviculata and K barbellata overlap
(Table 3). However, all measurements are higher in K claviculata except for two
characters where there are no significant differences (characters 2 and 3 in Table
4). Also the measures of putative hybrid flowers overlap with the putative parentals,
although clear differences are found. In characters 1, 5 and 7 all three taxa are
Range
7-I9
I2-20
7-12
15-2I
32--47
11-20
36-5I
ll-I6
37--47
I0-14
Character
I
2
3
4
5
6
7
8
9
IO
II
12.72
I6.3I
8.75
17.56
41.84
15.75
42.63
13.78
42.28
11.81
100
Mean
1.92
1.94
1.08
1.39
3.11
2.30
3.I9
1.21
2.58
0.82
0
s
V. claviculaJa
15
II
12
8
7
15
7
9
6
7
0
C.V.(%)
9-14
20-27
8-I3
15-22
33--47
11-24
32--40
11-I6
32--40
IO-I5
-
Range
11.07
23.69
9.96
I8.I7
38.35
15.28
36.8I
13.6I
36.75
11.96
100
Mean
1.23
1.84
0.92
1.60
3.42
2.47
1.93
1.08
2.I6
0.85
0
s
Putative hybrid
II
8
9
9
9
16
5
8
6
7
0
C.V. (%)
8-I3
I2-20
6-10
14--19
32--40
5-16
25-39
10-14
30--41
8-13
-
Range
9.74
I7.I2
8.12
15.88
34.72
10.60
34.98
12.05
36.2I
10.5
0
Mean
1.05
1.91
0.98
1.42
1.98
2.99
2.29
0.97
2.55
0.85
0
s
v. bathe/lata
12
9
6
28
6
8
7
8
0
II
II
C.V.(%)
3. Range, mean, standard deviation (s) and coefficient of variation (C.V.) of morphological measurements. Sample size: Vamilla clavicuklta: n= 32, V.
barbellata: n=43 and hybrid: n=71. All characters in mm except for character ll (with or without hairs) which is presented as the percentage of flowers with
hairs
TABLE
t5
<.>0
~
z
~
2!
~5
t:l
~
......
~
:X:
~c::
~
L. R. NIELSEN
294
TABLE
4. Comparison of characters between knilh barbelfutu @), V clwicuhta (c) and the putative hybrid
(h). (none) indicates that none of the three taxa are equal at the 5% significance level in the given
character. Equal taxa are given in the parentheses. Characters of the hybrids are classified either as
I: intermediate, P parental or E: extreme. PC 1 and PC 2 are eigenvectors 1 and 2; characters with
a large contribution are in bold
Character
1
2
3
4
5
6
7
8
9
Equal
(ceO.05)
Hybrid status
PCI
(none)
I
E
E
P
I
P
I
P
P
P
P
0.546
0.426
0.598
0.696
0.787
0.785
0.794
0.656
0.724
0.665
0.832
@,c)
@>C)
(44
(none)
(ch)
(none)
(ch)
bh)
(44
(44
10
I1
PC 2
0.091
-0.759
-0.541
-0.317
0.488
0.054
0.489
-0.110
0.534
-0.215
-0.203
distinct with the deviants intermediate between K clauiculata and K barbellata (Tables
3 and 4).For characters 2 and 3 the deviant differs significantly from the two others.
The two measures are clearly higher, hence the putative hybrid is classified ‘extreme’
in these characters (Table 4). These differences may reflect a combination of the
labellum shape and size in K claviculatu and K barbellata: in K claviculata the labellum
is large ( 42 mm) but relatively narrow and slender while in K barbellata the labellum
is short ( 35 mm) with a relatively wide distal aperture. The putative hybrid has a
relatively large labellum with a wide distal aperture. Thus, the aperture and the
width in particular are larger than in the putative parental species. For the remaining
five characters (4,6, 8, 10 and 11) the means of deviant flowers equal one of the
putative parentals of which only one is equal to K barbellata. Hairs were discovered
in all flowers of both K clauiculata and the putative hybrid, but not in flowers of K
barbellata. Obviously, this character (scored as with or without hair) cannot be
intermediate.
The coefficients of variation are similar in all quantitative characters in all three
taxa (Table 3). Only one major exception is observed; in K barbellata there is a high
variation in the length of the overlapping parts of the lateral lobes of labellum.
The principal component analysis of apparently pure species resulted in two
distinct groups (Fig. 4).Flowers of K barbellata form a relatively dense group, while
the group of K claviculata is more dispersed especially along the second axis. When
all three taxa are included in the analysis, three groups are formed (Fig. 5). The
groupings of the two parental species show almost no overlap while overlaps are
discovered between the putative hybrid and the presumed parental species, particularly K claviculata. The first component axis is clearly the strongest vector
accounting for 48% of the total variance (Table 5). Five characters load heavier
than others (11, 7, 5, 6, and 9 in Table 4) do. These represent ‘hairiness’ and the
overall size of the flower. The one with highest contribution is character 11, which
is the only qualitative and mutual excluding character in the analysis. The second
component is weaker, but together with PC 1, they represent 65% of the total
variation (Table 5). The three most significant characters are 2, 3 and 4 (the sign
N
N
295
NATURAL HYBRIDIZATION IN VA.NIUA
1
0
0
oo
D
0.5 -
D
oo
,..,
D
JOD
D
0
oo
0
0
0
0
D
0
0
0
0
D
8
u
0 06l
0
D
D
D
DO D
-0.5 -
0
D
D
D
D DO
%
B
D
D
r:P
0
0
0
oo
oo
D
(
0
-1
-1.5
I
I
-1
-0.5
0
I
I
1.5
1
0.5
0
PC1
Figure 4. Principal component analysis: transformed values of the II characters of Vanilla barbella/4
and V. claviculal4 plotted along the first two component axes. (D) V. barbella/4; (0) V. clavicula/4.
1
0
co
D
o
D
0.5 I-
0
D
!Qo
~D
~
c9
D
D
D
B
riO 0 0
ouo
0
0
Do!
Do
9r
A
A
0
0
A
A A
A
A At:f.
D
0
o o 0 o
0
AAO
0
A
A
;p.
A
~
<t
A AA
A
~
A
A
A
Ao o
AA
0
A
AA
~ ~~ \Av~
A
A
-0.5 I-
00
0
ot&
A
0
0
0
A
A
A
AA
-1
-1.5
I
-1
I
I
-0.5
0
0.5
I
1
1.5
PC1
Figure 5. Principal component analysis: transformed values of the II characters of Vanilla barbella/4
and V. clavicula/4 and the putative hybrid plotted along the first two component axis. (D) V. barbella/4;
(0) V. clavicula/4; (.6.) hybrid.
in front is without significance); thus this component can be interpreted as representing
the size of the opening of labellum.
Geno!)lpic compositions
Analysing the multilocus genotypes of the putative hybrids revealed that resampling
had occurred. In 23 collected samples, 10 were identified as genetically distinct
L. R.NIELSEN
296
TABLE
5. Eigenvalues of eigenvecton 1-1 1 (PC1-PC 1 1). Percentage:
% of variance accounted for a given vector (PC1-1 1) and the cumulative values
PC
Eigenvalue
Percent
Cumulative
1
2
3
4
5
6
7
8
9
5.274
1.842
0.838
0.802
0.595
0.459
0.368
0.333
0.266
0.146
0.078
47.95
16.74
7.62
7.29
5.41
4.17
3.35
3.03
2.41
1.33
0.71
47.95
64.69
72.31
79.60
85.00
89.18
92.52
95.55
97.97
99.29
100.00
11
100
-
10
I1
Sum
individuals based upon the described criterion. In R barbellata 87 out of 116 samples
were identified as individuals and in R claviculata 89 of 95 samples (Table 1).
Multilocus genotypes of all identified individuals of the putative hybrid are presented
in Table 6. Of the parental species, only individuals from localities where presumed
pure individuals coexist with the hybrid are included (Yauco and Susua; in Guanica
only one individual of R barbellata was found and therefore not included). All alleles
found in the hybrid are also represented in the parental species. In Dia almost all
devinants are heterozygotic, a single-locus genotype which is very frequent in both
R c1aviCUlat.a and R barbellata. All deviants are monomorphic at two loci (Zdh and
Mdhl). At one of these loci (Mdhl), all putative parentals, except one, are also
monomorphic with the same allele represented. At Idh, variation is found in R
barbellata but allele Zdh"' is clearly more frequent than Zdh6'. At Mdh3 the allele
Mdh3"' is represented in seven putative hybrids. This allele is rare in I? claviculata
but common in R barbellata at both the Susua Forest Reserve and Yauco. Individual
7 (deviant),sampled at the Susua Forest Reserve, has an interesting allele composition
at Pgm. Pgm74 is only found in ?? barbellata while Pgm108is more frequent in R
claviculata. Most deviants are homozygotic at Skdh"' which is also very common in
the two hypothetically parental species (14 of 16 scored individuals in I? clauiculata
and in 16 of 27 scored in R barbellata). At Ugut,most individuals have the allele
composition Ugufoloo,
Uguf' is more frequent than Ugut"' in R claviculata, while
Ugut"' is the most common allele in R barbellata and Ugut"'is only found in two out
of 28 individuals.
Comparison of the distribution of individual heterozygosity in all populations of
R clauiculata and R barbellata showed that the median values do not differ significantly
(Mann-Whitney rank sum statistic; T = 5856.5; P= 0.47). The average number of
heterozygotic loci per individual is higher in the hybrid than in the two other taxa
(Table 7) and the heterozygosity distribution of hybrid individuals differs significantly
from both R barbellata and R clauiculata (Mann-Whitney rank sum statistic; T =
65 1.O; P= 0.007 and T = 588.5; P= 0.024, respectively). The high number of
heterozygotes is also reflected in the F,, values of the hybrids (Table 8). At three
polymorphic loci (Dia,Pgm,Ugut),the number of heterozygotes is clearly significantly
higher than expected (more than twice the standard deviation) and the overall level
NATURAL HYBRIDIZATION IN V%W.LA
297
TABLE
6. Multilocus genotypes of the putative hybrid, Vanilla barbellah and L! chuimlatu after pooling
individuals. Samples 1-10 are hybrids, with 1 - 6 collected from population XGU. Samples 7 and 8
from XSU and 9 and 10 from XYA. Samples 11-38 are L! barbellah, with 11-35 collected from
population BSU and 36-38 from BYA. Samples 39-59 are I? clauiculata, all from population CSU.
-indicates
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
100104
100104
100104
100104
100104
100104
100104
100104
104104
100104
100100
100104
100104
100104
100104
100100
100104
100104
104104
100104
100104
100104
100104
104104
100104
104104
100100
100100
104104
100100
100100
104104
100100
100100
100104
100104
100104
100104
100104
100100
100100
100100
100104
100104
100104
100104
100104
100104
100104
100104
100104
100104
100104
104104
100104
100100
100104
100104
104104
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
lOOI00
100100
60 100
100100
100100
~
100100
60100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
lOOI00
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100100
100114%
100100
100100
100100
missing values
100100
100114
1001 14
100100
100114
100100
100100
75100
100100
100100
100108
80 I00
80100
100108
80 100
100108
74108
7491
8091
8091
100100
100108
100100
100100
100100
100100
100100
100100
100100
100100
114114
100121
I14121
1001I4
121121
100121
100121
114121
114121
121 121
100114
100114
100114
1141I4
114121
114114
100114
75114
114121
100100
114121
100121
100121
121 129
114114
100100
1441I4
I141 14
75100
75 100
100100
100100
100121
75100
75100
7575
7575
75100
75100
7575
75100
100100
7575
75100
100114
91 100
100100
75 I00
75100
91 100
80 I00
74 100
100100
100100
80100
809 I
100100
100100
809 I
100100
100100
73100
73100
100100
__
88 I00
100100
88I00
100100
-
80100
-
91 100
100100
-
9191
80 100
80 100
100100
100108
80 100
809 I
100100
100100
100100
100100
80 I00
91 100
8091
100100
80108
80 100
91 100
9191
80 I00
80 100
9191
91 100
80 1 00
9191
100108
80100
9191
100100
100100
8091
91 I08
80100
100100
100100
100100
100100
100100
100100
73100
100100
100100
73100
88100
88I00
88I00
88 100
88 100
100100
100100
100100
100100
I00100
100100
100100
100100
100100
100108
100100
100108
100100
100100
100100
-
100100
-
100100
100100
-
100100
100100
-
90 I00
90 100
90100
90 100
90 I 00
90 100
7990
79100
90 I00
90 I00
100100
79100
90 100
79100
79100
79 I00
79 100
-
79100
79100
100100
100100
79 I00
100100
100100
79100
I00 I00
90 100
79 I00
100100
79 I00
100100
100100
100100
100100
100100
100100
90100
9090
7979
90100
79100
9090
9090
90100
90100
9090
9090
90100
9090
7990
90I00
7979
90100
9090
7990
9090
9090
L. R. NIEISEN
TABLE
7. Distribution of heterozygosity in Vanilla claziculala, V. barbellata, and the
putative hybrid. Sample size: total number of individuals without missing values.
Range: range of heterozygotic loci per individual. Average: average number of
heterozygotic loci per individual
I! clauitulata
Putative hybrid
74
0-4
2.5 I
1.07
10
78
2-5
3.40
0.84
1 -5
Sample sizr
Range
Average
Svandard deviation
P barbellah
2.46
0.99
TABLE
8.F-statistics of Vanilla barbellata, V. claviculata and their putative hybrid from Puerto Rico
'.P rlavinrlala
6,
L4ICUS
Dia
sd
(65)
6,
sd (Fid
- I .008
0.293
UP1
-0.182
0.069
-0.273
0.028
- 0. I00
0. I58
-0.003
0.181
0.504
0.096
0.149
0.046
0.092
0.081
-0.233
-0.533
0.121
-0.969
All loci
-0.01 9
0.054
-0.650
Idh
Aldhl
i\1dh3
pg.,
SMh
I? barbellala
Putative hybrid
6,
sd
(Fils
0.133
0.067
0
0.233
-0.020
-0.163
- 0.00 1
-0.126
- 0.047
- 0. I58
- 0.229
0.007
0.074
0. I44
0.072
0.07 1
0.088
-0.100
0.061
-
~
~
0.145
0.090
TABLE
9. Crossing experiments in Vanilla claviculala and V. barbellah
Recipient
I? rlauiculala
Donor
L! barbellah
t! rlavirulala
Non-aborted
Aborted
4
3
L! barbellala
Non-aborted
Alioried
10
2
(& = -0.65) is highly significant, strongly indicating that the hybrid-populations do
not show Hardy-Weinberg proportions.
Pollination experiments
Some fruits were aborted in both pollination classes (infraspecific and interspecific)
in K barbellata as well as K clauiculata (Table 9). There were no significant differences
in the number of aborted fruits in any of the t\;vo pollination classes in either species
(Chi square test for homogeneity [with Yates correction]; K barbellala: x' = 0.5, df=
2, P=0.48 and K claviculata: X2=0.09, df=2, P=O.76). In both species, the seed
quality was high in the non-aborted fruits, regardless of whether the fruits originated
NATURAL HYBRIDIZATION IN V f l l L f A
299
TASLE
10. Seed quality of non-aborted fruits from interspecific- and intraspecific-pollinations
lnterspecific crossing
embryos
Yo of seeds
germinating
Average
Range
Replicates
94
92-97
a
88
4-19
72-97
8
Average
Range
Replicates
91
89-94
4
% with viable
I? clauiculab
I? barbellab
Intraspecifir crossing
6
5
9
1
% with viable
embryos
95
88-99
10
% of seeds
germinating
10
0.2-42
8
14
3-5 1
7
from an intraspecific crossing or an interspecific crossing (Table 10). The range of
viable seeds of fruits from interspecific manipulations is within the range of viable
seeds of fruits from intraspecific crossings of both species. Germination is discovered
in seeds of fruits of outcrossed flowers in both interspecific and intraspecific crossings.
Thus, though the number of replicates is low, there seem to be no difference in
seed quality regardless of the crossing.
DISCUSSION
Both morphological and allozyme data support the hypothesis that the deviant
individuals are indeed hybrids between K claviculata and K barbelluta as also indicated
in Nielsen & Siegismund (1999).In terms of allozyme data, all alleles at the surveyed
loci in the putative hybrids occur in the parental species. Their genotypic compositions
seem to be combinations of the most common alleles in the parental species. In
some loci, for example Mdh3 and Ugut, one allele in the hybrid-genotype is common
in only one of the parentals. In addition, one individual has a unique allele
where the allele Pp7' is only found in K barbellata where it
combination, Pgm74108,
is very rare, while P p i U 8is very common in K claviculata. Because of the major
similarity in allozyme profile between the parentals, with most differences found in
allele frequencies, backcrossings are difficult to identify. However, the individuals
classified as deviants have a significant surplus of heterozygotes. This is both
revealed by the negative overall F,, value ( = -0.65) and when comparing genotype
distributions with the two putative parental species that are both known to accord
to Hardy-Weinberg proportions (Nielsen & Siegismund, 1999). Therefore, the
examined putative hybrids are most likely FI individuals where the genomes consist
of two sets of chromosomes; one of each parental species. As the allele frequencies
in K barbellata and K ckauiculata are different at the polymorphic loci, the possibility
of becoming homozygotic by an interspecific crossing is low. A higher level of
homozygotes would again be obtained in cases of either backcrossings or a F2
generation. Thus, an allozyme profile with a significant surplus of heterozygotes as
observed in the deviating individuals corroborates the theory that the individuals
are Fl hybrids.
Moreover, the morphological data set supports the hypothesis. When analysed
by a principal component analysis the taxa separate into three groups, though with
300
L. R.NIELSEN
minor overlaps. Of the 10 quantitative characters in the hybrids, five were classified
‘parental’, three ‘intermediate’, and two ‘extreme’. It has traditionally been believed
that hybrids express character intermediacy (Rieseberg, 1995; Rieseberg & Ellstrand,
1993). However, the finding of a mosaic of parentals, intermediate and extreme
characters in the Enilla hybrids agrees with a review by Rieseberg & Ellstrand
(1993). They examined 46 studies on morphological character expression in hybrids
and concluded that in the first generation, 45% of the characters were intermediate,
45% parental and 10% of the characters extreme. The high proportion of parental
characters discovered in hybrids (in the Enilla hybrid 6 of 11 characters, when
character 11 is included) may be explained by a simple genetic control of those
morphological characters that differentiates closely related taxa (Gottlieb, 1984;
Hilu, 1983). The expression of parental versus intermediate character states in
hybrids may thus depend on the genetic control of the particular character and of
interactions with the environment (Rieseberg, 1995).
In K barbellata and K claviculata, germination of seeds obtained from interspecific
crossings confirmed that no post-pollination barriers separate these species, thus
enabling hybridization in sympatric areas. The two species have almost synchronous
flowering times and are likely to share the same pollinator (Nielsen & Ackerman,
unpubl.). Hybrids were only discovered in the area where two parentals come into
contact and as K clauiculata was typically found in moist serpentine forests, while K
barbellata seemed to exploit dryer habitats, hybridization is presumably normally
avoided by spatial isolation because of minor differences in habitat preference. A
similar case was reported for Irishlua and Iris hexagona in Louisiana by Arnold,
Bennett & Zimmer (1990). They discovered that one species occurred in the
understorey environment associated with bayou margins while the other species
grew in open freshwater swamps. Hybridization between the two synchronous
flowering and pollinator sharing species was discovered where the areas came in
contact.
It is generally believed that the isolating mechanisms among sympatric outcrossing
orchids are caused by differences in floral structure, pollinator specificity and/or
ecological factors (Dressler, 1981; Gill, 1989; Grant, 1994; Van der Pijl & Dodson,
1966). Peakall et al. (1997) have further suggested that post-pollination barriers may
be poorly developed in the family as many cultivated orchids species have been
produced by artificially crossing different natural species. However,Johansen (1990)
discovered a comprehensive interspecific incompatibility system in Dendrobium and
a generalization may not be appropriate. In Kclaviculuta and K barbellatu both allozyme
profiles and morphology are similar and the species may be closely related, perhaps
with a relatively recent ancestor. It is therefore possible that potential barriers
separating the species have not been fully established.
In Puerto Rico, the area of overlap where hybridization was discovered is very
restricted and as the fruit set is low in the Enilla species (Nielsen & Ackerman,
unpubl.), the hybridization events may have no significant consequences. However,
counts on pollinaria removal and pollinia deposition of hybrid flowers in the 1996
flowering season at the Susua Forest Reserve showed that more flowers were visited
in the hybrids (34%) than in the parentals ( K cluuiculata 18% and K barbellatu 5%).
At another locality (Guanica Forest Reserve), the growth of a few hybrid individuals
is very extensive. They seem to dominate the piece of forest where the lianas grow,
and only one single individual of a parental species ( K barbellatu) was found at the
locality. Both flower and fruit production at this locality appear higher than in other
NATURAL HYBRIDIZATION IN VANILLA
30 1
Kznillu populations (pers. obs.), suggesting a relatively high fitness of the hybrids.
Additionally, naturally produced seeds can be germinated. Thus, the hybrid
K clauiculatu x K barbellatu may eventually become established in Puerto Rico.
ACKNOWLEDGEMENTS
I would like to thankJames D. Ackerman, University of Puerto Rico, Rio Piedras,
for his generosity and fruitful advice during the fieldwork in Puerto Rico. Thanks
are also due to the Puerto Rico Department of Natural Resources and Environment
for permission to work and camp in the Susua Forest Reserve. The forest ranger,
Wetsy Cordero, was helpful in many respects. Ruth Bruus Jakobsen was very
supportive in the laboratory. Hans R. Siegismund, Finn Rasmussen and Marianne
Philipp are thanked for many useful discussions and comments on the manuscript.
This research was supported by The Faculty of Science, University of Copenhagen
and by two grants, Hotelejer Anders Maansson og hustru Hanne Maanssons Legat
and Ingenirar Svend Fiedler og Hustrus Legat.
REFERENCES
Ackerman JD.1995. An Orchid Flora of herto Rico and the bigin Islands. New York The New York
Botanical Garden.
Anderson E. 1948. Hybridization in the habitat. Evolution 2: 1-9.
Arnold LM. 1997. Natural Hybridkation and Evolution. New York Oxford University Press.
Arnold LM, Bennett BD, Zimmer EA. 1990. Natural hybridization between 1ris>lva and
1. hexagona: pattern of ribosomal DNA variation. Evolution 44: 1512- 1521.
Corrias B, Rossi W, Arduino P, Cianchi R, Bullini L. 1991. Orchis longimmu Poiret in Sardinia:
genetic, morphological, and chorological data. Webbia 45: 7 1-101.
Dressler RL. 1981. zhe Orchids: Natural History and Classjriction. Cambridge, Massachusetts: Harvard
University Press.
Dressler RL. 1993. Phylogeny and Chszjication ofthe Orchid Famib. Portland, Oregon: Dioscorides Press.
Ellstrand NC. 1984. Multiple paternity within the fruits of wild radish, Raphanus sativus. American
Naturalist 123: 819-828.
Ellstrand NC, Elam DR. 1993. Population genetic consequences of small population size: implications
for plant conservation. Annual Re& of Ecology and Systematics 24: 2 17-242.
Gill DE. 1989. Fruiting failure, pollinator inefficiency and speciation in orchids. In: Otte D, Endler
JA, eds. Speciation and its Consegumces. Sunderland, Massachusetts: Sinauer, 458-48 1.
Gottlieb LD. 1984. Genetics and morphological evolution in plants. Amprican Naturalist 123: 681-709.
Grant V. 1981. Phnt Specktion. New York Columbia University Press.
Grant V. 1994. Modes and origins of mechanical and ethological isolation in angiosperms. h c e e d i q s
oftheNationa1 Academy ofSCiences USA 91: 3-10.
Harris H, Hopkinson DA. 1976. Handbook of Entyme Electmbhomk in Human CMetics. Amsterdam:
North Holland.
Harrison RG. 1990. Hybrid zones: windows on evolutionary process. Ogord Surveys in Evolutionaly
Biology 7: 69-128.
Hilu KW. 1983. The role of single-gene mutations in the evolution of flowering plants. Evolutionaly
Biologv 2 6 97-128.
Johansen B. 1990. Incompatibility in Dmdmbium (Orchidaceae).BotanicalJoumal ofthe Linnaean Sociep
103 165-196.
Jergensen BI, Andersen TI?.1998. Hardy and half hardy terrestrial orchids as potential new
potplants. A c b Hortkultura 454 195-205.
L. R. NlELSEN
302
Levin DA, Francisco-OrtegaJ,Jansen RK. 1996. Hybridization and the extinction of rare species.
Consmation Biology 1 0 1&16.
Mayr E. 1963. Animal Speciation and Evolution. Cambridge, Massachusetts: Belknap Press.
Nielsen LR, Siegismund HR. Interspecific differentiation and hybridization in Vanilla species
(Orchidaceae).Heredib 83: 560-567.
Peakall R, Bower CC, Logan AE,Nicol HI. 1997. Confirmation of the hybrid origin of Chilogloth
x prescottiana (Orchidaceae: Diurideae). 1. Genetic and morphometric evidence. Australian Journal
OfBotany 45: 839-855.
Rieseberg LH. 1995. The role of hybridization in evolution: old wine in new skins. American Journal
OfBotany 82: 944-953.
Rieseberg LH. 1997. Hybrid origins of plant species. Annwl Revim OfEcologu and syslm2atics 28:
359-389.
Rieseberg LH,Ellstrand NC. 1993. What can morphological and molecular markers tell us about
plant hybridization? Critical Reviews in Plant Scimces 12: 213-241.
Rohlf 8.1993. NTSYS-PC. Numerical Taxonomy and Multivariak Anabai $s!em. Setauket, New York
Applied Biostatistics.
Sigmastat. 1992. Usm Manual. Jandel Scientific.
Soltis DE,Soltis PS. 1993. Molecular data and the dynamic nature of polyploidy. Critical RpoierUs in
Plant Sciences 1 2 243-273.
Stebbins GL, Jr. 1950. Variation and Evolution in Plants. New York Columbia University Press.
Torres AM, Bergh BO. 1978. Isozymes as indicator of outcrossing among “Pinkerton” seedlings.
Earbook. Calijirnia Avocado Association 62: 103-1 10.
Van der Pijl L, Dodson CH. 1966. Orchid Flowers,their Pollination and Evolution. Coral Cables, Florida:
University of Miami Press.
Weeden IW,
Wendel JF. 1989. Genetics of plant isozymes. In: Soltis DE, Soltis PE, eds. Zsorymes in
Plant Biology. Portland, Oregon: Dioscorides Press, 6-72.
Weir BS, Cockerham CC. 1984. Estimating F-statistics for the analysis of population structure.
Evolution 3 8 1358- 1370.
- -