Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue7.Aug2000:
Biochemical Systematics and Ecology 28 (2000) 651}663
Observations of high genetic variability
in the endangered Australian terrestrial orchid
Pterostylis gibbosa R. Br. (Orchidaceae)
I.K. Sharma*, M.A. Clements, D.L. Jones
Centre for Plant Biodiversity Research, Division of Plant Industry CSIRO, GPO Box 1600, Canberra, ACT,
2601, Australia
Received 2 August 1999; accepted 15 September 1999
Abstract
The genetic variation in all known populations of an endangered Australian native terrestrial
orchid Pterostylis gibbosa R.Br., was investigated with starch gel electrophoresis. A total of 16
isozyme loci were assayed. The percentage of polymorphic loci (P), the number of alleles per
locus (A), observed and expected heterozygosity at population levels were 69%, 2.21, 0.210,
0.261, respectively. The G value of 15% indicates that around 85% of variation resides within
4t
populations. Despite isolation by distance most alleles were distributed across most of the
populations. High genetic variability along with low population divergence may be the result of
recent population fragmentation or from extensive gene #ow maintained by seed and pollen
movement. To investigate whether poor seed viability contributed towards its rarity, an orchid
seed viability test using Fluorescein diacetate revealed high seed viability (range 68}90%).
Although endangered and restricted to only four geographical areas, P. gibbosa showed
a higher level of genetic variation than other orchids with larger populations. ( 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Orchidaceae; Allozyme; P. gibbosa; Electrophoresis; Genetic diversity
1. Introduction
The taxonomic history, typi"cation, distribution and habitat of the rare and
endangered Australian orchid Pterostylis gibbosa R.Br. was the subject of a recent
* Corresponding author.
E-mail address: ish.sharma@pi.csiro.au (I.K. Sharma)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 0 7 - 6
652
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
study (Jones and Clements, 1997). Currently its distribution is centred on Central
Coast of New South Wales with disjunct northern populations near Milbrodale on
the North Coast. Southern populations grow among shrubs in sparse open forest or
woodland dominated by melaleucas, whereas northern populations grow in open
forest dominated by eucalypts and native pines. The orchid plants survive hot dry
summers as a dormant subterranean tuber, produce a rosette of leaves after rains in
late autumn and #ower in spring. The survival of this species is threatened by urban
sprawl, habitat destruction and illegal collecting. Because of its rarity, this species has
been the subject of several conservation studies (Bradburn, 1984; Bradburn and
Tunstall, 1993; Muston, 1991), but no research has been carried out into its genetic
diversity or genetic structure. The present investigation also provides an excellent
opportunity to study the e!ect of small population size and population geographic
isolation on genetic variation in P. gibbosa.
Breeding structure is considered as one of the factors most strongly in#uencing the
genetic variation of plant populations and survival of a species in the long term (Falk
and Holsinger, 1991). Loss of variation can reduce the ability of a population to adapt
to changing environments (Hamilton, 1982) and could ultimately lead to extinction.
Among various ways of determining genetic diversity, isozyme electrophoresis has
been used routinely in plants to investigate genetic variation within and between
populations (House and Bell, 1994; Sonnante et al., 1996) and also to investigate the
genetic structure of rare and endangered taxa including orchids (Cosner and Crawford, 1994; Godt et al., 1995; Richter et al., 1994; Sharma and Clements, 1995). It
provides information on organisation of the gene pool in a large number of plant
species (Gottlieb, 1981) and has implications in understanding relationships within
populations of a taxon. Moreover such information is crucial for any sustainable
conservation program of a rare taxon, where a fundamental goal, in addition to
habitat preservation, is maintaining existing levels of genetic variation (Frankel and
Soule, 1981).
The present study aims to provide baseline genetic information which could be used
to develop appropriate conservation strategies by investigating allozyme diversity,
gene #ow and Nei genetic identity co-e$cient within and among 12 populations of
P. gibbosa. As an adjunct to this study, viability tests of seeds collected from various
localities was carried out to determine if poor seed viability was contributing to the
decline of this species.
2. Material and methods
Samples (255 in all) were collected randomly from 12 populations growing in four
geographical sites; Albion Park (55 samples), Yallah (66), Nowra (70) and Milbrodale
(64) (Table 1, Fig. 1). At the Albion Park site, leaves were collected from an area of
about 20]20 m; at Nowra four populations each about 100 m apart were sampled; at
Yallah three populations about 200 m apart, were sampled; at Milbrodale four
populations were sampled. Samples were collected in plastic bags and stored in the
refrigerator for a maximum of seven days prior to processing. One leaf per plant was
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
653
Table 1
Location and number of plants examined from 12 populations of Pterostylis gibbosa
Province
Populations
No. of individual examined
Albion Park
ALB
55
Yallah
YA1
YAW
YAE
25
26
15
Nowra
NO1
NO2
NO3
NO4
15
15
20
20
Milbrodale
ML1
ML2
ML3
ML4
20
12
16
16
Fig. 1. Map showing the collection sites and allozyme di!erentiation of Pterostylis gibbosa.
654
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
mashed in a small vial with a drop of extraction bu!er (phosphate bu!er pH 6.80,
dithiothreitol 1 mg/ml) and the extract absorbed onto a 2.5]10 mm chromatography
paper wick for starch gel electrophoresis.
Preparation of starch gel and running of electrophoresis was carried out using the
method described by Weeden and Wendel (1989). Gels were cut into three slices and
stained for di!erent enzymes. By utilising morpholine citrate (pH 6.1), Histidine (pH
8.0) and lithium bu!ers, eleven enzyme systems namely glucophospho isomerase
(GPI, E.C. 5.3.1.9) isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), 6-phosphogluconate
dehydrogenase (6PGD, E.C. 1.1.1.44), phosphogluco mutase (PGM, E.C. 2.7.5.1),
malate dehydrogenase (MDH, E.C. 1.1.1.37), uridine diphosphogluconic pyrophosphatase (UGP, E.C. 3.4.11.2), Shikimic acid dehydrogenase (SDH, E.C. 1.1.1.25),
alcohol dehydrogenase (ADH, E.C. 1.1.1.1), glycerate dehydrogenase (GLY, E.C.
1.1.1.29), (MR, E.C. 1.6.9.92) and GOT, E.C. 2.6.1.1 were assayed following the
staining procedures of Weeden and Wendel (1989).
Banding patterns on gels were interpreted and scored according to the Mendelian
genetic principal using known enzyme subunit structure with the fastest migrating locus of an enzyme numbered &1' and the next fastest as &2'. Similarly within
each locus the fastest migrating allele was designated &1' and the next fastest as &2' and
so on.
To assess genetic diversity the percentage of polymorphic loci (P), the allelic
richness (A), observed heterozygosity (H ), and gene diversity (H ) were calculated.
0
%
Wright's inbreeding coe$cient (F ) was used to measure deviations from random
*4
mating. To examine population genetic structure, the total genetic diversity (H ),
5
mean genetic di!erentiation within populations (H ), mean genetic di!erentiation
S
between populations (D ) and proportion of genetic di!erentiation between popula45
tions (G ) were calculated following Nei and Chesser (1983). Genetic divergence
45
among populations was also assessed using pairwise measures of Nei's unbiased
genetic distance (Nei, 1978). All parameters were calculated using the software
package BIOSYS-1 (Swo!ord and Selander, 1981). Analysis of variance (ANOVA)
was carried out using SAS (1988) to investigate whether values of A, H and H in
0
%
respective populations were signi"cantly di!erent from each other. Estimation of gene
#ow (Nm) between populations was carried out using Wright's (1951) method i.e.
(1!G /4G ).
45
45
Seed viability was tested following the procedure of Pritchard (1985) with the
following slight modi"cation: * seeds in a test tube were soaked in 0.5 ml of distilled
water along with 2 ll of detergent for about 20 h at room temperature. A drop of seed
suspension containing approximately 100 seeds were placed on a clean glass slide.
Excess water was absorbed with a "lter paper and a drop of 0.5% #uorescene
diacetate (FDA) was placed on the seed suspension before covering it with a glass
cover slip. After a gentle tapping (which ruptures the seed coat) FDA solution
was applied for about 10 min (depending on how quickly the FDA evaporates).
Seeds were then observed under the UV #uorescent microscope. Seeds with a bright
yellowish green embryo were considered viable. Seeds from 23 plants (15 from
Milbrodale, 3 from Yallah, 2 from Nowra and 3 from Albion Park were assayed for
seed viability.
Table 2
Allele frequencies at 16 loci in 12 populations of P. gibbosa
Populations
Albion Yallah
1
2
3
4
5
6
7
8
9
10
11
12
0.109
0.573
0.027
0.291
0.000
0.000
0.833
0.000
0.167
0.000
0.077
0.731
0.000
0.192
0.000
0.233
0.700
0.000
0.067
0.000
0.000
0.600
0.000
0.367
0.033
0.000
0.533
0.000
0.433
0.033
0.000
0.500
0.000
0.425
0.075
0.050
0.475
0.000
0.475
0.000
0.050
0.725
0.075
0.150
0.000
0.000
0.625
0.167
0.208
0.000
0.031
0.750
0.031
0.188
0.000
0.033
0.467
0.133
0.367
0.000
0.463
0.370
0.130
0.037
0.240
0.420
0.340
0.000
0.192
0.192
0.558
0.058
0.667
0.133
0.200
0.000
0.321
0.214
0.464
0.000
0.600
0.000
0.400
0.000
0.447
0.158
0.395
0.000
0.800
0.050
0.150
0.000
0.300
0.125
0.525
0.050
0.250
0.542
0.208
0.000
0.406
0.188
0.406
0.000
0.531
0.156
0.313
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.917
0.083
1.000
0.000
0.938
0.063
1.000
0.000
0.000
1.000
0.000
0.000
0.942
0.058
0.000
1.000
0.000
0.000
0.967
0.000
0.033
0.933
0.000
0.067
0.925
0.000
0.075
1.000
0.000
0.000
1.000
0.000
0.000
0.917
0.083
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.000
1.000
0.000
0.040
0.960
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.933
0.067
0.000
0.933
0.067
0.000
0.900
0.100
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.958
0.042
0.000
1.000
0.000
0.000
1.000
0.000
0.036
0.964
0.020
0.980
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.080
0.900
0.020
0.058
0.923
0.019
0.000
1.000
0.000
0.833
0.167
0.000
0.833
0.167
0.000
0.800
0.200
0.000
1.000
0.000
0.000
0.000
1.000
0.000
0.000
0.958
0.042
0.000
1.000
0.000
0.063
0.938
0.000
0.000
0.736
0.145
0.009
0.109
0.000
0.200
0.220
0.040
0.540
0.077
0.404
0.288
0.038
0.192
0.000
0.333
0.000
0.000
0.667
0.067
0.433
0.200
0.000
0.300
0.033
0.467
0.133
0.000
0.367
0.050
0.475
0.150
0.000
0.325
0.000
0.421
0.132
0.000
0.447
0.000
0.316
0.053
0.000
0.632
0.000
0.542
0.042
0.042
0.375
0.000
0.188
0.094
0.000
0.719
0.000
0.400
0.033
0.000
0.567
*continued
655
GPI
1
2
3
4
5
PGM
1
2
3
4
GLY
1
2
UGP-1
1
2
3
UGP-2
1
2
3
UGP-3
1
2
6PGD
1
2
3
MR
1
2
3
4
5
Milbrodale
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Locus
Nowra
656
Table 2*continued
Populations
Locus
ADH
1
2
3
IDH
1
2
3
SDH
1
2
3
4
MDH-1
1
2
3
MDH2
1
2
MDH-3
1
2
3
GOT-2
1
2
3
GOT-3
1
2
Nowra
Milbrodale
1
2
3
4
5
6
7
8
9
10
11
12
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.033
0.967
0.000
0.025
0.975
0.000
0.050
0.950
0.000
0.105
0.842
0.053
0.000
1.000
0.000
0.000
0.933
0.067
0.000
1.000
0.000
0.027
0.973
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.900
0.100
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.938
0.063
0.155
0.409
0.418
0.018
0.080
0.180
0.740
0.000
0.038
0.192
0.750
0.019
0.000
0.231
0.769
0.000
0.107
0.357
0.536
0.000
0.000
0.429
0.571
0.000
0.079
0.316
0.605
0.000
0.125
0.550
0.325
0.000
0.425
0.075
0.500
0.000
0.625
0.250
0.125
0.000
0.500
0.125
0.375
0.000
0.313
0.125
0.563
0.000
0.167
0.796
0.037
0.080
0.760
0.160
0.115
0.769
0.115
0.067
0.800
0.133
0.071
0.857
0.071
0.033
0.833
0.133
0.053
0.842
0.105
0.075
0.425
0.500
0.200
0.800
0.000
0.083
0.917
0.000
0.063
0.938
0.000
0.063
0.938
0.000
0.745
0.255
0.800
0.200
0.731
0.269
0.867
0.133
0.933
0.067
0.933
0.067
0.900
0.100
0.500
0.500
0.650
0.350
0.750
0.250
0.688
0.313
0.625
0.375
0.917
0.056
0.028
0.700
0.280
0.020
0.788
0.115
0.096
1.000
0.000
0.000
0.967
0.033
0.000
0.967
0.033
0.000
0.950
0.050
0.000
0.775
0.225
0.000
0.725
0.150
0.125
0.792
0.125
0.083
0.688
0.000
0.313
0.781
0.094
0.125
0.100
0.745
0.155
0.020
0.500
0.480
0.000
0.500
0.500
0.033
0.533
0.433
0.000
0.667
0.333
0.000
0.733
0.267
0.000
0.650
0.350
0.000
0.775
0.225
0.000
0.375
0.625
0.000
0.542
0.458
0.000
0.469
0.531
0.063
0.375
0.563
0.927
0.073
0.780
0.220
0.923
0.077
0.967
0.033
0.867
0.133
0.900
0.100
0.875
0.125
0.925
0.075
0.800
0.200
0.750
0.250
0.844
0.156
0.719
0.281
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Albion Yallah
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
657
Table 3
Measures of genetic diversity in 12 populations of P. gibbosa!
Geographical area
Population
A
P
H
0
H
%
F
*4
Albion
Park
Yallah
ALB, A
2.4
(0.3)
2.3
(0.2)
2.4
(0.3)
1.8
(0.2)
2.1
(0.2)
2.1
(0.2)
2.2
(0.2)
1.9
(0.2)
2.2
(0.3)
2.2
(0.2)
1.9
(0.2)
2.2
(0.2)
68.8
0.216
(0.062)
0.263
(0.071)
0.243
(0.068)
0.158
(0.062)
0.187
(0.051)
0.151
(0.050)
0.178
(0.049)
0.202
(0.058)
0.246
(0.059)
0.224
(0.053)
0.235
(0.064)
0.267
(0.075)
0.240
(0.061)
0.272
(0.058)
0.266
(0.061)
0.188
(0.056)
0.257
(0.063)
0.243
(0.054)
0.274
(0.059)
0.260
(0.063)
0.294
(0.058)
0.290
(0.058)
0.254
(0.062)
0.294
(0.064)
0.1
2.21
69.81
0.210
0.261
0.17
YA1, B
YAW, C
YAE, D
Nowra
NO1, E
NO2, F
NO3, G
NO4, H
Milbrodale
ML1, 1
ML2, J
ML3, L
ML4, M
Mean
75.0
68.8
50.0
75.0
81.3
81.3
62.5
68.8
81.3
62.5
75.0
0.03
0.08
0.15
0.27
0.37
0.35
0.22
0.16
0.22
0.07
0.10
!Note: (A) mean number of alleles per locus; (P) percentage of polymorphic loci; (H ) observed hetero0
zygosity; (H ) expected heterozygosity; (F ) "xation index. Standard error in parenthesis.
%
*4
3. Results
Eleven enzyme systems coded by 16 loci were scored: GPI, PGM, UGP-1, UGP-2,
UGP-3, GLY, 6PGD, MR, ADH, IDH, SDH, MDH-1, MDH-2, MDH-3, GOT-2,
GOT-3. Additional enzymes were assayed (SOD, peptidase) but were not included in
the analysis due to poor resolution. All loci exhibited a simple diploid banding
pattern. The number of alleles per locus ranged from two to "ve. The allele frequencies
observed at 16 loci is shown in Table 2. There were a number of unique alleles present
in a particular geographical area in low frequencies e.g. allele 3 of ADH and allele 3 of
IDH was present in Milbrodale site, allele 5 of GPI was present in Nowra, allele 3 of
SDH was present in Albion Park and Nowra areas (see Table 2). Twelve alleles can be
considered as unique alleles since their frequencies were less than 0.1.
Genetic variation values in each of the populations are presented in Table 3 and
there appeared to be little di!erence among populations with values of H which
%
ranged from 0.188}0.294 (mean 0.261) and values of P which ranged from 50}81%
658
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Table 4
Genetic diversity level among populations of P. gibbosa
Locus
D
45
H
5
G
45
H
4
s2
GPI
PGM
GLY
UGP-1
UGP-2
UGP-3
6PGD
MR
ADH
IDH
SDH
MDH-1
MDH-2
MDH-3
GOT-2
GOT-3
0.023
0.058
0.000
0.001
0.001
0.000
0.313
0.045
0.002
0.001
0.078
0.028
0.024
0.020
0.025
0.004
0.527
0.649
0.024
0.051
0.051
0.009
0.433
0.625
0.054
0.031
0.611
0.331
0.364
0.285
0.504
0.246
0.045
0.090
0.028
0.020
0.020
0.013
0.723
0.073
0.038
0.041
0.128
0.087
0.066
0.070
0.050
0.019
0.504
0.591
0.024
0.051
0.051
0.009
0.120
0.580
0.052
0.030
0.533
0.303
0.340
0.265
0.479
0.242
129.46!
130.46!
33.35!
57.45!
50.10!
11.99NS
398.82!
148.09!
55.80!
49.99!
149.23!
103.32!
37.58!
96.90!
78.61!
23.10"
Average
0.045
0.299
0.151
0.254
Note: D mean genetic diversity found between populations; H Total genetic variation; G proportion of
45
5
45
genetic di!erentiation between populations; H mean genetic diversity within populations;
4
!P(0.001.
"P(0.05.
(mean 69.81%). The mean number of alleles per locus (A) ranged from 1.8}2.4 (mean
2.21) and observed heterozygosity (H ) ranged from 0.158}0.267 (mean 0.210). Sim0
ilarly, at all four geographical areas, the mean values of A (range 2.07}2.40), H (range
%
0.240}0.283) and P (range 64}74%) when tested utilising ANOVA were not signi"cantly di!erent (Fig. 1).
Genetic diversity within and among populations is shown in Table 4. Total genetic
diversity (H ) was 0.299 with most of this variation partitioned within sub-populations
5
(H "0.254). The proportion between populations (G ) are ranged from 0 to 1 and
4
45
obtained by ratios between variation found among populations (D ) and the total
45
genetic variation (H ). G value in the present study ranged from 0.01 (UGP-3,
5
45
GOT-3) to 0.1 (SDH), with a mean value of G "0.15 indicating about 85% of
45
variation resides within populations. The higher G value indicate greater variability
45
between populations. Genetic diversity calculated from four geographical areas separately were found to be comparable (Fig. 1). Average Gene #ow (N ) of 6.51 suggests
.
that there is a moderate gene exchange among P. gibbosa population and this would
appear to be su$cient to prevent population di!erentiation.
The genetic identity coe$cient based on allele frequencies summarised over all loci
is calculated following the method of Nei (1978) and is presented in Table 5. The
highest genetic identity displayed was between populations NO1, NO2 and NO3 and
Population
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
*****
0.955
*****
0.968
0.990
*****
0.964
0.979
0.974
*****
0.929
0.937
0.943
0.927
*****
0.925
0.921
0.928
0.931
1.000
*****
0.932
0.935
0.942
0.934
1.000
1.000
*****
0.878
0.865
0.863
0.871
0.950
0.966
0.957
*****
0.944
0.982
0.979
0.971
0.911
0.897
0.912
0.844
*****
0.973
0.964
0.957
0.947
0.917
0.895
0.914
0.847
0.977
*****
0.946
0.978
0.968
0.971
0.913
0.900
0.912
0.850
1.000
0.983
*****
0.962
0.978
0.974
0.979
0.928
0.923
0.935
0.875
0.995
0.981
0.994
*****
ALB,
YA1,
YAW,
YAE,
NO1,
NO2,
NO3,
NO4,
ML1,
ML2,
ML3,
ML4,
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Table 5
Nei's genetic identity (I) for 12 populations of P.gibbosa
659
660
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
the lowest was between NO4 and ML1. The mean genetic identity value in all four
geographical areas were very similar ranging from 0.96}0.97.
To assess the hypothesis that poor seed viability might be contributing to this
species rarity, a seed viability test showed that the seed viability ranged from 68}90%
indicating substantial seed viability.
4. Discussion
A moderately high level of genetic variability was observed in P. gibbosa in
comparison to various studies carried out on various plant species in the last decade
or so (Karron, 1987; Hamrick and Godt, 1989; Hamrick et al., 1991; Soltis and Soltis,
1991). All these investigations emphasised the proposition that narrowly distributed
taxa have less genetic variation than widely distributed taxa, thus making them prone
to genetic drift leading to possible local extinction. Furthermore, Waller et al. (1987)
reported no polymorphism in the extreme endemic, Pedicularis furbishiae. In their
recent review Hamrick and Godt (1989) reported that the average genetic diversity
statistics for long lived herbaceous perennials with animal pollinated outcrossing
species with regional distribution were: percent polymorphic loci (P"49%), mean
number of alleles per locus (A"1.89), and genetic diversity H "0.131. By compari%
son, in P. gibbosa values of P"69.81%, A"2.21, H "0.210 and H "0.261 were
0
%
all higher than those reported by Hamrick and Godt (1989) and by Gottlieb (1981) for
comparable herbaceous plants. Results also indicated a high level of genetic variability compared with those reported for terrestrial orchids, e.g. Diuris sulphurea from
Australia (P"55%, A"1.71, H "0.169, H "0.180 (Sharma and Jones, 1996);
0
%
Gymnaedenia canopsea (P"56%, A"1.74, H "0.169, H "0.171) (Scacchi and
0
%
DeAngelis, 1989), Epipactis species (P"38%, A"1.63. H "0.141, H "0.14)
0
%
(Scacchi et al., 1987) and Orchis species (P"43%, A"1.63, H "0.141, H "0.149)
0
%
(Scacchi et al., 1990) from Europe. Although the populations of P. gibbosa from four
geographical regions were well separated (Albion Park, Yallah, Nowra and Milbrodale), they still maintained high levels of genetic variation and furthermore, no
signi"cant pattern of allozyme di!erentiation in terms of the value of A, P, and
H (Table 3) was observed at these sites.
%
The presence of high levels of genetic variation and relatedness is pointed to
a common, well distributed ancestry prior to its present decline due to mainly habitat
destruction for agriculture, urbanisation, bush"res, deliberately lit "res and over
collecting. Pterostylis gibbosa has the potential to extend its geographical range to
adjacent similar habitats as (a) seeds are wind blown (b) very high genetic variability
and (c) seed viability is also high. However, despite these obvious attributes P. gibbosa
is currently con"ned to only four relictual sites. This can be attributed to the suitable
ecological and microenvironmental factors being maintained in those areas. Furthermore the outcrossing nature of this species through specialised pollination system
(Dressler, 1981), high fecundity, wind dispersal of seeds and high level of gene #ow
could also be responsible for the presence of high genetic variation in P. gibbosa.
However, the presence of high genetic variation in endemic endangered taxa is not
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
661
unusual, and results supporting this have been recently reported for three species of
Coreopsis and endemic Delphinium viridescens (Cosner and Crawford, 1994; Richter
et al., 1994). Investigation by Ranker (1994) revealed high genetic variation in
geographically restricted species, reinforcing the need to consider factors other than
geographical distribution as an indicator of genetic variability (Soltis and Soltis,
1991). A high mean seed viability of 76% was found in P. gibbosa also emphasising the
fact that rarity in this species cannot be attributed to poor seed viability.
Gene #ow within populations via pollen and seed dispersal are the two major
contributors for genetic di!erentiation (Loveless and Hamrick, 1984). In P. gibbosa,
the two means of gene movement are (a) pollen transfer through specialised pollinators and (b) dispersal of wind blown seeds over larger areas. Another possible
contributing factor for long distance dispersal of seeds could be strong summer winds
which occur sometime in Oct}Nov at the time of seed capsule dehiscence. This adds
another dimension to seed dispersal over larger areas and prevents major population
di!erentiation development. An alternative hypotheses that all these populations
derived from a common ancestral stock and later spread to di!erent areas but still
maintaining the similar genetic structure due to similar evolutionary forces, ecological
characteristics prevailing in these sites cannot be ruled out. The estimated gene #ow
(N ) value of (6.51 migrants per population) were quite high in comparison to that
.
reported by Hamrick (1987) (N "1.15) for 16 outcrossing animal pollinated species.
.
This is responsible for high degree of genetic exchange which in turn reduces population di!erences and maintains a reasonable level of genetic similarity. While other
factors including genetic drift, founder e!ects, isolation from parent population and
selection pressure (Clegg and Brown, 1983) can contribute to genetic di!erentiation,
todate no evidence to support such factors has been observed in P. gibbosa.
The di!erentiation between populations is 15% (G "0.15) which is higher than
45
the values observed in other outbreeders (G "0.11) (Loveless and Hamrick, 1984),
45
lower than the means for outcrossing animal pollinated species (G "0.19) and is
45
comparable to species with wind dispersal seeds (G "0.14) (Hamrick and Godt,
45
1989). Furthermore, this value is similar also to values obtained with other outcrossing orchid species, e.g. Orchis palustris Jacq. (G "0.17) (Scacchi et al., 1990),
45
Cyperipedium calceolus L. (G "0.19) (Case, 1993), Pterostylis hamiltonii Nicholls
45
(G "0.14), P. rogersii E.Coleman and P. aw. alata (0.13) (unpublished data), all of
45
which are wind dispersed from seed. This suggests that the long distance wind
dispersal is a e!ective means of maintaining gene #ow and therefore genetic diversity,
between populations of plants (rare or common species alike). This appears to be true
even when the habitat requirements are restrictive and those regions are in widely
distributed sites.
Overall, results of this study suggest that because of the presence of high genetic
variability in small fragmented populations, high seed viability and the ability of seeds
to be wind borne, P. gibbosa has not been at risk under the present circumstances.
However action should be taken to ensure its long-term genetic viability by introducing and implementing appropriate conservation and management strategies. Of
particular signi"cance is the in situ conservation of the most diverse populations
namely YA1, YAW, NO4, ML1, ML2, ML3, ML4. Since the species appears to be
662
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
exinct from the original point of collection (Fig. 1), the protection of these remaining
sites is therefore paramount for the long-term survival of this species in the wild. To
create a complete framework for devising a conservation strategy exsitu conservation
should also be employed. This would include preservation of habitat and re-introduction of seedlings to colonise similar habitats. It is preferable to employ seed propagation from several individual as this avoids damage to the original plant and allows you
to produce large number of genetically di!erent seedlings within a relatively short
period of time. Furthermore, to ensure the long-term viability of these populations,
protection from invasive weeds, animals (feral goats, rabbits), and illegal collectors is
as crucial as the strategies for genetically depauperate population facing extinction.
Acknowledgements
We wish to thank Marion Garratt, Maggie Nightingale, Karina FitzGerald and Liz
Gregory for technical support and Geo!rey Robertson of NPWS (NSW) for his keen
interest and enthusiastic collection of samples.
References
Bradburn, G., 1984. Report on the proposed Tallawarra power project and its impact on Pterostylis gibbosa.
Report to the Electricity Commission of NSW by the Australian Native Orchid Society.
Bradburn, G., Tunstall, R., 1993. Census of Pterostylis gibbosa Illawarra Greenhood Orchid, Paci"c Power:
Yallah site. Report prepared for Paci"c Power.
Case, M.A., 1993. High levels of allozyme variation within Cyperipedium calceolus (Orchidaceae) and low
levels of divergence among its varieties. Syst. Bot. 18, 663}677.
Clegg, M.T., Brown, A.H.D., 1983. The founding of plant populations. In: Schonewald-Cox, C.M.,
Chambers, S.M., Macbryde, B., Thomas, L. (Eds.), Genetics and Conservation. Benjamin-Cummings,
Memlo Park, CA, pp. 216}228.
Cosner, M.E., Crawford, D.L., 1994. Comparison of isozymic diversity in three rare species of Coreopsis
(Asterceae). Syst. Bot. 19, 350}358.
Dressler, R.L., 1981. The Orchids, natural history and classi"cation. Harvard University Press, Cambridge,
MA.
Falk, D.A., Holsinger, K.E., 1991. Genetics and Conservation of Rare Plants. Oxford University Press.
Oxford, p. 283.
Frankel, O.H., Soule, M.E., 1981. Conservation and Evolution. Cambridge University Press, Cambridge.
Godt, M.L., Hamrick, J.L., Bratton, S., 1995. Genetic diversity in a threatened wetland species, Helonias
bullata (Liliaceae). Cons. Biol. 9, 277}280.
Gottlieb, L.D., 1981. Electrophoretic evidence and plant populations. Prog. in Phytochem. 7, 1}46.
Hamilton, W.D., 1982. Pathogen as causes of genetic diversity in their host populations. In: Anderson,
R.M., May, R.M. (Eds.), Population Biology of Infectious Diseases. Springer-Verlag, New York,
pp. 269}296.
Hamrick, J.L., 1987. Gene #ow and distribution of genetic variation in plant populations. In: Di!erentiation
Pattern in Higher Plants. Academic Press, New York.
Hamrick, J.L., Godt, M.L., 1989. Allozyme diversity in plant species. In: Brown, A.H.H., Kehler, A.I., Weir,
B.S. (Eds.), Plant Population Genetics, Breeding and Genetic Resources. Sinauer Associates, Sunderland, MA, pp. 43}63.
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
663
Hamrick, J.L., Godt, M.J., Murawski, D.A., Loveless, M.D., 1991. Correlation between species traits and
allozyme diversity: implications for conservation biology. In: Falk, D.A., Holsinger, K.E. (Eds.),
Genetics and Conservation of Rare Plants, Oxford University Press, New York, pp. 75}86.
House, A.P.N., Bell, J.C., 1994. Isozyme variation and mating system. In: Eucalyptus urophylla Blake, S.T.
(Ed.), Silvae Genetica Vol. 43, pp. 167}176.
Jones, D.L., Clements, M.A., 1997. Characterisation of Pterostylis gibbosa R. Br. (Orchidaceae) and
description of P. saxicola, a rare new species from New South Wales. The Orchadian 12, 128}135.
Karron, J.D., 1987. The pollination ecology of co-occurring geographically restricted and widespread
species of Astragalus (Fabaceae). Amer. J. Bot. 76, 331}340.
Loveless, M.D., Hamrick, J.L., 1984. Ecological determinants of genetic structure in plant populations.
Ann. Rev. Ecol. 15, 65}95.
Muston, R., 1991. Plan of management for the rare and endangered Illawarra Greenghood Orchid
(Pterostylis gibbosa) on property owned by Electricity Commission of NSW at Yallah. Report prepared
by Roslyn Muston and Associates, Fairy Meadow.
Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of
individuals. Genetics 89, 583}590.
Nei, M., Chesser, R.K., 1983. Estimation of "xation indices and gene diversities. Ann. Hum. Gen. 47,
253}259.
Pritchard, H.W., 1985. Determination of Orchid seed viability using #uorescein diacetate. Plant Cell
Environ. 8, 727}730.
Ranker, T.A., 1994. Evolution of high genetic variability in the rare Hawaiian fern Adenophorus periens and
implications for conservation management. Biol. Conser. 70, 19}24.
Richter, T.S., Soltis, P.S., Soltis, D.E., 1994. Genetic variation within and among populations of narrow
endemic Delphinium viridescens (Ranunculaceae) Amer. J. Bot. 81, 1070}1076.
SAS Institute Inc.1988. SAS/STAT user's guide, Release 6.03 edition, Cary, NC: SAS Institute Inc. 1028pp.
Scacchi, R., De Angelis, G., 1989. Isozyme polymorphism in Gymnadenia and its inferences for systematics
within this species. Biochem. Syst. Ecol. 17, 25}33.
Scacchi, R., Lanzara, P., De Angelis, G., 1987. Study of electrophoretic variability in Epipactis helleborine
(L.) Crantz, E. palustris (L.) Crantz and E. microphylla (Ehrh.) Swartz (fam. Orchidaceae). Genetica 72,
217}224.
Scacchi, R., De Angelis, G., Lanzara, P., 1990. Allozyme variation among and within eleven Orchis species
(fam. Orchidaceae) with special reference to hybridising aptitude. Genetica 81, 143}150.
Sharma, I.K., Clements, M.A., 1995. Evidence of low genetic diversity in Dendrobium malbrownii Dockrill
(Orchidaceae). The Orchadian 11, 459}464.
Sharma, I.K., Jones, D.L., 1996. An electrophoretic study of variability in Diuris sulphurea R. Br.
(Orchidaceae) in the Canberra region. J. Orchid Soc. India. 10, 9}24.
Soltis, P.S., Soltis, D.E., 1991. Genetic variation in endemic and wide spread plant species: examples from
Saxifragaceae and Polystichum (Dryopteridaceae). Aliso 13, 131}134.
Sonnante, G., Piergiovanni, A.R., Ng, Q.N., Perrino, P., 1996. Relationships of Vigna unguiculata (L.) Walp.,
V. vexillata (L.) A. Rich. and species of section Vigna based on isozyme variation. Genet. Resour. Crop
Evol. 43, 157}165.
Swo!ord, D.L., Selander, R.B., 1981. BIOSYS-1, A fortran program for the comprehensive analysis of
electrophoretic data in the population genetics and systematics. J. Heredity 72, 281}283.
Waller, D.M., O'Malley, D.M., Gawler, S.C., 1987. Genetic variation in the extreme endemic Pedicularis
furbishiae (Scrophulariaceae). Cons. Biol. 1, 335}340.
Weeden, N.F., Wendel, J.F., 1989. Visualisation and interpretations of plant isozymes. In: Soltis, D.S., Soltis,
P.S. (Eds.), Isozymes in Plant Biology. Dioscorides Press, Portland, Oregon, pp. 46}72.
Wright, S., 1951. The genetic structure of populations. Ann. Eugen. 15, 323}354.
Observations of high genetic variability
in the endangered Australian terrestrial orchid
Pterostylis gibbosa R. Br. (Orchidaceae)
I.K. Sharma*, M.A. Clements, D.L. Jones
Centre for Plant Biodiversity Research, Division of Plant Industry CSIRO, GPO Box 1600, Canberra, ACT,
2601, Australia
Received 2 August 1999; accepted 15 September 1999
Abstract
The genetic variation in all known populations of an endangered Australian native terrestrial
orchid Pterostylis gibbosa R.Br., was investigated with starch gel electrophoresis. A total of 16
isozyme loci were assayed. The percentage of polymorphic loci (P), the number of alleles per
locus (A), observed and expected heterozygosity at population levels were 69%, 2.21, 0.210,
0.261, respectively. The G value of 15% indicates that around 85% of variation resides within
4t
populations. Despite isolation by distance most alleles were distributed across most of the
populations. High genetic variability along with low population divergence may be the result of
recent population fragmentation or from extensive gene #ow maintained by seed and pollen
movement. To investigate whether poor seed viability contributed towards its rarity, an orchid
seed viability test using Fluorescein diacetate revealed high seed viability (range 68}90%).
Although endangered and restricted to only four geographical areas, P. gibbosa showed
a higher level of genetic variation than other orchids with larger populations. ( 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Orchidaceae; Allozyme; P. gibbosa; Electrophoresis; Genetic diversity
1. Introduction
The taxonomic history, typi"cation, distribution and habitat of the rare and
endangered Australian orchid Pterostylis gibbosa R.Br. was the subject of a recent
* Corresponding author.
E-mail address: ish.sharma@pi.csiro.au (I.K. Sharma)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 0 7 - 6
652
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
study (Jones and Clements, 1997). Currently its distribution is centred on Central
Coast of New South Wales with disjunct northern populations near Milbrodale on
the North Coast. Southern populations grow among shrubs in sparse open forest or
woodland dominated by melaleucas, whereas northern populations grow in open
forest dominated by eucalypts and native pines. The orchid plants survive hot dry
summers as a dormant subterranean tuber, produce a rosette of leaves after rains in
late autumn and #ower in spring. The survival of this species is threatened by urban
sprawl, habitat destruction and illegal collecting. Because of its rarity, this species has
been the subject of several conservation studies (Bradburn, 1984; Bradburn and
Tunstall, 1993; Muston, 1991), but no research has been carried out into its genetic
diversity or genetic structure. The present investigation also provides an excellent
opportunity to study the e!ect of small population size and population geographic
isolation on genetic variation in P. gibbosa.
Breeding structure is considered as one of the factors most strongly in#uencing the
genetic variation of plant populations and survival of a species in the long term (Falk
and Holsinger, 1991). Loss of variation can reduce the ability of a population to adapt
to changing environments (Hamilton, 1982) and could ultimately lead to extinction.
Among various ways of determining genetic diversity, isozyme electrophoresis has
been used routinely in plants to investigate genetic variation within and between
populations (House and Bell, 1994; Sonnante et al., 1996) and also to investigate the
genetic structure of rare and endangered taxa including orchids (Cosner and Crawford, 1994; Godt et al., 1995; Richter et al., 1994; Sharma and Clements, 1995). It
provides information on organisation of the gene pool in a large number of plant
species (Gottlieb, 1981) and has implications in understanding relationships within
populations of a taxon. Moreover such information is crucial for any sustainable
conservation program of a rare taxon, where a fundamental goal, in addition to
habitat preservation, is maintaining existing levels of genetic variation (Frankel and
Soule, 1981).
The present study aims to provide baseline genetic information which could be used
to develop appropriate conservation strategies by investigating allozyme diversity,
gene #ow and Nei genetic identity co-e$cient within and among 12 populations of
P. gibbosa. As an adjunct to this study, viability tests of seeds collected from various
localities was carried out to determine if poor seed viability was contributing to the
decline of this species.
2. Material and methods
Samples (255 in all) were collected randomly from 12 populations growing in four
geographical sites; Albion Park (55 samples), Yallah (66), Nowra (70) and Milbrodale
(64) (Table 1, Fig. 1). At the Albion Park site, leaves were collected from an area of
about 20]20 m; at Nowra four populations each about 100 m apart were sampled; at
Yallah three populations about 200 m apart, were sampled; at Milbrodale four
populations were sampled. Samples were collected in plastic bags and stored in the
refrigerator for a maximum of seven days prior to processing. One leaf per plant was
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
653
Table 1
Location and number of plants examined from 12 populations of Pterostylis gibbosa
Province
Populations
No. of individual examined
Albion Park
ALB
55
Yallah
YA1
YAW
YAE
25
26
15
Nowra
NO1
NO2
NO3
NO4
15
15
20
20
Milbrodale
ML1
ML2
ML3
ML4
20
12
16
16
Fig. 1. Map showing the collection sites and allozyme di!erentiation of Pterostylis gibbosa.
654
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
mashed in a small vial with a drop of extraction bu!er (phosphate bu!er pH 6.80,
dithiothreitol 1 mg/ml) and the extract absorbed onto a 2.5]10 mm chromatography
paper wick for starch gel electrophoresis.
Preparation of starch gel and running of electrophoresis was carried out using the
method described by Weeden and Wendel (1989). Gels were cut into three slices and
stained for di!erent enzymes. By utilising morpholine citrate (pH 6.1), Histidine (pH
8.0) and lithium bu!ers, eleven enzyme systems namely glucophospho isomerase
(GPI, E.C. 5.3.1.9) isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), 6-phosphogluconate
dehydrogenase (6PGD, E.C. 1.1.1.44), phosphogluco mutase (PGM, E.C. 2.7.5.1),
malate dehydrogenase (MDH, E.C. 1.1.1.37), uridine diphosphogluconic pyrophosphatase (UGP, E.C. 3.4.11.2), Shikimic acid dehydrogenase (SDH, E.C. 1.1.1.25),
alcohol dehydrogenase (ADH, E.C. 1.1.1.1), glycerate dehydrogenase (GLY, E.C.
1.1.1.29), (MR, E.C. 1.6.9.92) and GOT, E.C. 2.6.1.1 were assayed following the
staining procedures of Weeden and Wendel (1989).
Banding patterns on gels were interpreted and scored according to the Mendelian
genetic principal using known enzyme subunit structure with the fastest migrating locus of an enzyme numbered &1' and the next fastest as &2'. Similarly within
each locus the fastest migrating allele was designated &1' and the next fastest as &2' and
so on.
To assess genetic diversity the percentage of polymorphic loci (P), the allelic
richness (A), observed heterozygosity (H ), and gene diversity (H ) were calculated.
0
%
Wright's inbreeding coe$cient (F ) was used to measure deviations from random
*4
mating. To examine population genetic structure, the total genetic diversity (H ),
5
mean genetic di!erentiation within populations (H ), mean genetic di!erentiation
S
between populations (D ) and proportion of genetic di!erentiation between popula45
tions (G ) were calculated following Nei and Chesser (1983). Genetic divergence
45
among populations was also assessed using pairwise measures of Nei's unbiased
genetic distance (Nei, 1978). All parameters were calculated using the software
package BIOSYS-1 (Swo!ord and Selander, 1981). Analysis of variance (ANOVA)
was carried out using SAS (1988) to investigate whether values of A, H and H in
0
%
respective populations were signi"cantly di!erent from each other. Estimation of gene
#ow (Nm) between populations was carried out using Wright's (1951) method i.e.
(1!G /4G ).
45
45
Seed viability was tested following the procedure of Pritchard (1985) with the
following slight modi"cation: * seeds in a test tube were soaked in 0.5 ml of distilled
water along with 2 ll of detergent for about 20 h at room temperature. A drop of seed
suspension containing approximately 100 seeds were placed on a clean glass slide.
Excess water was absorbed with a "lter paper and a drop of 0.5% #uorescene
diacetate (FDA) was placed on the seed suspension before covering it with a glass
cover slip. After a gentle tapping (which ruptures the seed coat) FDA solution
was applied for about 10 min (depending on how quickly the FDA evaporates).
Seeds were then observed under the UV #uorescent microscope. Seeds with a bright
yellowish green embryo were considered viable. Seeds from 23 plants (15 from
Milbrodale, 3 from Yallah, 2 from Nowra and 3 from Albion Park were assayed for
seed viability.
Table 2
Allele frequencies at 16 loci in 12 populations of P. gibbosa
Populations
Albion Yallah
1
2
3
4
5
6
7
8
9
10
11
12
0.109
0.573
0.027
0.291
0.000
0.000
0.833
0.000
0.167
0.000
0.077
0.731
0.000
0.192
0.000
0.233
0.700
0.000
0.067
0.000
0.000
0.600
0.000
0.367
0.033
0.000
0.533
0.000
0.433
0.033
0.000
0.500
0.000
0.425
0.075
0.050
0.475
0.000
0.475
0.000
0.050
0.725
0.075
0.150
0.000
0.000
0.625
0.167
0.208
0.000
0.031
0.750
0.031
0.188
0.000
0.033
0.467
0.133
0.367
0.000
0.463
0.370
0.130
0.037
0.240
0.420
0.340
0.000
0.192
0.192
0.558
0.058
0.667
0.133
0.200
0.000
0.321
0.214
0.464
0.000
0.600
0.000
0.400
0.000
0.447
0.158
0.395
0.000
0.800
0.050
0.150
0.000
0.300
0.125
0.525
0.050
0.250
0.542
0.208
0.000
0.406
0.188
0.406
0.000
0.531
0.156
0.313
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.917
0.083
1.000
0.000
0.938
0.063
1.000
0.000
0.000
1.000
0.000
0.000
0.942
0.058
0.000
1.000
0.000
0.000
0.967
0.000
0.033
0.933
0.000
0.067
0.925
0.000
0.075
1.000
0.000
0.000
1.000
0.000
0.000
0.917
0.083
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.000
1.000
0.000
0.040
0.960
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.933
0.067
0.000
0.933
0.067
0.000
0.900
0.100
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.958
0.042
0.000
1.000
0.000
0.000
1.000
0.000
0.036
0.964
0.020
0.980
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.080
0.900
0.020
0.058
0.923
0.019
0.000
1.000
0.000
0.833
0.167
0.000
0.833
0.167
0.000
0.800
0.200
0.000
1.000
0.000
0.000
0.000
1.000
0.000
0.000
0.958
0.042
0.000
1.000
0.000
0.063
0.938
0.000
0.000
0.736
0.145
0.009
0.109
0.000
0.200
0.220
0.040
0.540
0.077
0.404
0.288
0.038
0.192
0.000
0.333
0.000
0.000
0.667
0.067
0.433
0.200
0.000
0.300
0.033
0.467
0.133
0.000
0.367
0.050
0.475
0.150
0.000
0.325
0.000
0.421
0.132
0.000
0.447
0.000
0.316
0.053
0.000
0.632
0.000
0.542
0.042
0.042
0.375
0.000
0.188
0.094
0.000
0.719
0.000
0.400
0.033
0.000
0.567
*continued
655
GPI
1
2
3
4
5
PGM
1
2
3
4
GLY
1
2
UGP-1
1
2
3
UGP-2
1
2
3
UGP-3
1
2
6PGD
1
2
3
MR
1
2
3
4
5
Milbrodale
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Locus
Nowra
656
Table 2*continued
Populations
Locus
ADH
1
2
3
IDH
1
2
3
SDH
1
2
3
4
MDH-1
1
2
3
MDH2
1
2
MDH-3
1
2
3
GOT-2
1
2
3
GOT-3
1
2
Nowra
Milbrodale
1
2
3
4
5
6
7
8
9
10
11
12
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.033
0.967
0.000
0.025
0.975
0.000
0.050
0.950
0.000
0.105
0.842
0.053
0.000
1.000
0.000
0.000
0.933
0.067
0.000
1.000
0.000
0.027
0.973
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.900
0.100
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.938
0.063
0.155
0.409
0.418
0.018
0.080
0.180
0.740
0.000
0.038
0.192
0.750
0.019
0.000
0.231
0.769
0.000
0.107
0.357
0.536
0.000
0.000
0.429
0.571
0.000
0.079
0.316
0.605
0.000
0.125
0.550
0.325
0.000
0.425
0.075
0.500
0.000
0.625
0.250
0.125
0.000
0.500
0.125
0.375
0.000
0.313
0.125
0.563
0.000
0.167
0.796
0.037
0.080
0.760
0.160
0.115
0.769
0.115
0.067
0.800
0.133
0.071
0.857
0.071
0.033
0.833
0.133
0.053
0.842
0.105
0.075
0.425
0.500
0.200
0.800
0.000
0.083
0.917
0.000
0.063
0.938
0.000
0.063
0.938
0.000
0.745
0.255
0.800
0.200
0.731
0.269
0.867
0.133
0.933
0.067
0.933
0.067
0.900
0.100
0.500
0.500
0.650
0.350
0.750
0.250
0.688
0.313
0.625
0.375
0.917
0.056
0.028
0.700
0.280
0.020
0.788
0.115
0.096
1.000
0.000
0.000
0.967
0.033
0.000
0.967
0.033
0.000
0.950
0.050
0.000
0.775
0.225
0.000
0.725
0.150
0.125
0.792
0.125
0.083
0.688
0.000
0.313
0.781
0.094
0.125
0.100
0.745
0.155
0.020
0.500
0.480
0.000
0.500
0.500
0.033
0.533
0.433
0.000
0.667
0.333
0.000
0.733
0.267
0.000
0.650
0.350
0.000
0.775
0.225
0.000
0.375
0.625
0.000
0.542
0.458
0.000
0.469
0.531
0.063
0.375
0.563
0.927
0.073
0.780
0.220
0.923
0.077
0.967
0.033
0.867
0.133
0.900
0.100
0.875
0.125
0.925
0.075
0.800
0.200
0.750
0.250
0.844
0.156
0.719
0.281
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Albion Yallah
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
657
Table 3
Measures of genetic diversity in 12 populations of P. gibbosa!
Geographical area
Population
A
P
H
0
H
%
F
*4
Albion
Park
Yallah
ALB, A
2.4
(0.3)
2.3
(0.2)
2.4
(0.3)
1.8
(0.2)
2.1
(0.2)
2.1
(0.2)
2.2
(0.2)
1.9
(0.2)
2.2
(0.3)
2.2
(0.2)
1.9
(0.2)
2.2
(0.2)
68.8
0.216
(0.062)
0.263
(0.071)
0.243
(0.068)
0.158
(0.062)
0.187
(0.051)
0.151
(0.050)
0.178
(0.049)
0.202
(0.058)
0.246
(0.059)
0.224
(0.053)
0.235
(0.064)
0.267
(0.075)
0.240
(0.061)
0.272
(0.058)
0.266
(0.061)
0.188
(0.056)
0.257
(0.063)
0.243
(0.054)
0.274
(0.059)
0.260
(0.063)
0.294
(0.058)
0.290
(0.058)
0.254
(0.062)
0.294
(0.064)
0.1
2.21
69.81
0.210
0.261
0.17
YA1, B
YAW, C
YAE, D
Nowra
NO1, E
NO2, F
NO3, G
NO4, H
Milbrodale
ML1, 1
ML2, J
ML3, L
ML4, M
Mean
75.0
68.8
50.0
75.0
81.3
81.3
62.5
68.8
81.3
62.5
75.0
0.03
0.08
0.15
0.27
0.37
0.35
0.22
0.16
0.22
0.07
0.10
!Note: (A) mean number of alleles per locus; (P) percentage of polymorphic loci; (H ) observed hetero0
zygosity; (H ) expected heterozygosity; (F ) "xation index. Standard error in parenthesis.
%
*4
3. Results
Eleven enzyme systems coded by 16 loci were scored: GPI, PGM, UGP-1, UGP-2,
UGP-3, GLY, 6PGD, MR, ADH, IDH, SDH, MDH-1, MDH-2, MDH-3, GOT-2,
GOT-3. Additional enzymes were assayed (SOD, peptidase) but were not included in
the analysis due to poor resolution. All loci exhibited a simple diploid banding
pattern. The number of alleles per locus ranged from two to "ve. The allele frequencies
observed at 16 loci is shown in Table 2. There were a number of unique alleles present
in a particular geographical area in low frequencies e.g. allele 3 of ADH and allele 3 of
IDH was present in Milbrodale site, allele 5 of GPI was present in Nowra, allele 3 of
SDH was present in Albion Park and Nowra areas (see Table 2). Twelve alleles can be
considered as unique alleles since their frequencies were less than 0.1.
Genetic variation values in each of the populations are presented in Table 3 and
there appeared to be little di!erence among populations with values of H which
%
ranged from 0.188}0.294 (mean 0.261) and values of P which ranged from 50}81%
658
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Table 4
Genetic diversity level among populations of P. gibbosa
Locus
D
45
H
5
G
45
H
4
s2
GPI
PGM
GLY
UGP-1
UGP-2
UGP-3
6PGD
MR
ADH
IDH
SDH
MDH-1
MDH-2
MDH-3
GOT-2
GOT-3
0.023
0.058
0.000
0.001
0.001
0.000
0.313
0.045
0.002
0.001
0.078
0.028
0.024
0.020
0.025
0.004
0.527
0.649
0.024
0.051
0.051
0.009
0.433
0.625
0.054
0.031
0.611
0.331
0.364
0.285
0.504
0.246
0.045
0.090
0.028
0.020
0.020
0.013
0.723
0.073
0.038
0.041
0.128
0.087
0.066
0.070
0.050
0.019
0.504
0.591
0.024
0.051
0.051
0.009
0.120
0.580
0.052
0.030
0.533
0.303
0.340
0.265
0.479
0.242
129.46!
130.46!
33.35!
57.45!
50.10!
11.99NS
398.82!
148.09!
55.80!
49.99!
149.23!
103.32!
37.58!
96.90!
78.61!
23.10"
Average
0.045
0.299
0.151
0.254
Note: D mean genetic diversity found between populations; H Total genetic variation; G proportion of
45
5
45
genetic di!erentiation between populations; H mean genetic diversity within populations;
4
!P(0.001.
"P(0.05.
(mean 69.81%). The mean number of alleles per locus (A) ranged from 1.8}2.4 (mean
2.21) and observed heterozygosity (H ) ranged from 0.158}0.267 (mean 0.210). Sim0
ilarly, at all four geographical areas, the mean values of A (range 2.07}2.40), H (range
%
0.240}0.283) and P (range 64}74%) when tested utilising ANOVA were not signi"cantly di!erent (Fig. 1).
Genetic diversity within and among populations is shown in Table 4. Total genetic
diversity (H ) was 0.299 with most of this variation partitioned within sub-populations
5
(H "0.254). The proportion between populations (G ) are ranged from 0 to 1 and
4
45
obtained by ratios between variation found among populations (D ) and the total
45
genetic variation (H ). G value in the present study ranged from 0.01 (UGP-3,
5
45
GOT-3) to 0.1 (SDH), with a mean value of G "0.15 indicating about 85% of
45
variation resides within populations. The higher G value indicate greater variability
45
between populations. Genetic diversity calculated from four geographical areas separately were found to be comparable (Fig. 1). Average Gene #ow (N ) of 6.51 suggests
.
that there is a moderate gene exchange among P. gibbosa population and this would
appear to be su$cient to prevent population di!erentiation.
The genetic identity coe$cient based on allele frequencies summarised over all loci
is calculated following the method of Nei (1978) and is presented in Table 5. The
highest genetic identity displayed was between populations NO1, NO2 and NO3 and
Population
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
*****
0.955
*****
0.968
0.990
*****
0.964
0.979
0.974
*****
0.929
0.937
0.943
0.927
*****
0.925
0.921
0.928
0.931
1.000
*****
0.932
0.935
0.942
0.934
1.000
1.000
*****
0.878
0.865
0.863
0.871
0.950
0.966
0.957
*****
0.944
0.982
0.979
0.971
0.911
0.897
0.912
0.844
*****
0.973
0.964
0.957
0.947
0.917
0.895
0.914
0.847
0.977
*****
0.946
0.978
0.968
0.971
0.913
0.900
0.912
0.850
1.000
0.983
*****
0.962
0.978
0.974
0.979
0.928
0.923
0.935
0.875
0.995
0.981
0.994
*****
ALB,
YA1,
YAW,
YAE,
NO1,
NO2,
NO3,
NO4,
ML1,
ML2,
ML3,
ML4,
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
Table 5
Nei's genetic identity (I) for 12 populations of P.gibbosa
659
660
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
the lowest was between NO4 and ML1. The mean genetic identity value in all four
geographical areas were very similar ranging from 0.96}0.97.
To assess the hypothesis that poor seed viability might be contributing to this
species rarity, a seed viability test showed that the seed viability ranged from 68}90%
indicating substantial seed viability.
4. Discussion
A moderately high level of genetic variability was observed in P. gibbosa in
comparison to various studies carried out on various plant species in the last decade
or so (Karron, 1987; Hamrick and Godt, 1989; Hamrick et al., 1991; Soltis and Soltis,
1991). All these investigations emphasised the proposition that narrowly distributed
taxa have less genetic variation than widely distributed taxa, thus making them prone
to genetic drift leading to possible local extinction. Furthermore, Waller et al. (1987)
reported no polymorphism in the extreme endemic, Pedicularis furbishiae. In their
recent review Hamrick and Godt (1989) reported that the average genetic diversity
statistics for long lived herbaceous perennials with animal pollinated outcrossing
species with regional distribution were: percent polymorphic loci (P"49%), mean
number of alleles per locus (A"1.89), and genetic diversity H "0.131. By compari%
son, in P. gibbosa values of P"69.81%, A"2.21, H "0.210 and H "0.261 were
0
%
all higher than those reported by Hamrick and Godt (1989) and by Gottlieb (1981) for
comparable herbaceous plants. Results also indicated a high level of genetic variability compared with those reported for terrestrial orchids, e.g. Diuris sulphurea from
Australia (P"55%, A"1.71, H "0.169, H "0.180 (Sharma and Jones, 1996);
0
%
Gymnaedenia canopsea (P"56%, A"1.74, H "0.169, H "0.171) (Scacchi and
0
%
DeAngelis, 1989), Epipactis species (P"38%, A"1.63. H "0.141, H "0.14)
0
%
(Scacchi et al., 1987) and Orchis species (P"43%, A"1.63, H "0.141, H "0.149)
0
%
(Scacchi et al., 1990) from Europe. Although the populations of P. gibbosa from four
geographical regions were well separated (Albion Park, Yallah, Nowra and Milbrodale), they still maintained high levels of genetic variation and furthermore, no
signi"cant pattern of allozyme di!erentiation in terms of the value of A, P, and
H (Table 3) was observed at these sites.
%
The presence of high levels of genetic variation and relatedness is pointed to
a common, well distributed ancestry prior to its present decline due to mainly habitat
destruction for agriculture, urbanisation, bush"res, deliberately lit "res and over
collecting. Pterostylis gibbosa has the potential to extend its geographical range to
adjacent similar habitats as (a) seeds are wind blown (b) very high genetic variability
and (c) seed viability is also high. However, despite these obvious attributes P. gibbosa
is currently con"ned to only four relictual sites. This can be attributed to the suitable
ecological and microenvironmental factors being maintained in those areas. Furthermore the outcrossing nature of this species through specialised pollination system
(Dressler, 1981), high fecundity, wind dispersal of seeds and high level of gene #ow
could also be responsible for the presence of high genetic variation in P. gibbosa.
However, the presence of high genetic variation in endemic endangered taxa is not
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
661
unusual, and results supporting this have been recently reported for three species of
Coreopsis and endemic Delphinium viridescens (Cosner and Crawford, 1994; Richter
et al., 1994). Investigation by Ranker (1994) revealed high genetic variation in
geographically restricted species, reinforcing the need to consider factors other than
geographical distribution as an indicator of genetic variability (Soltis and Soltis,
1991). A high mean seed viability of 76% was found in P. gibbosa also emphasising the
fact that rarity in this species cannot be attributed to poor seed viability.
Gene #ow within populations via pollen and seed dispersal are the two major
contributors for genetic di!erentiation (Loveless and Hamrick, 1984). In P. gibbosa,
the two means of gene movement are (a) pollen transfer through specialised pollinators and (b) dispersal of wind blown seeds over larger areas. Another possible
contributing factor for long distance dispersal of seeds could be strong summer winds
which occur sometime in Oct}Nov at the time of seed capsule dehiscence. This adds
another dimension to seed dispersal over larger areas and prevents major population
di!erentiation development. An alternative hypotheses that all these populations
derived from a common ancestral stock and later spread to di!erent areas but still
maintaining the similar genetic structure due to similar evolutionary forces, ecological
characteristics prevailing in these sites cannot be ruled out. The estimated gene #ow
(N ) value of (6.51 migrants per population) were quite high in comparison to that
.
reported by Hamrick (1987) (N "1.15) for 16 outcrossing animal pollinated species.
.
This is responsible for high degree of genetic exchange which in turn reduces population di!erences and maintains a reasonable level of genetic similarity. While other
factors including genetic drift, founder e!ects, isolation from parent population and
selection pressure (Clegg and Brown, 1983) can contribute to genetic di!erentiation,
todate no evidence to support such factors has been observed in P. gibbosa.
The di!erentiation between populations is 15% (G "0.15) which is higher than
45
the values observed in other outbreeders (G "0.11) (Loveless and Hamrick, 1984),
45
lower than the means for outcrossing animal pollinated species (G "0.19) and is
45
comparable to species with wind dispersal seeds (G "0.14) (Hamrick and Godt,
45
1989). Furthermore, this value is similar also to values obtained with other outcrossing orchid species, e.g. Orchis palustris Jacq. (G "0.17) (Scacchi et al., 1990),
45
Cyperipedium calceolus L. (G "0.19) (Case, 1993), Pterostylis hamiltonii Nicholls
45
(G "0.14), P. rogersii E.Coleman and P. aw. alata (0.13) (unpublished data), all of
45
which are wind dispersed from seed. This suggests that the long distance wind
dispersal is a e!ective means of maintaining gene #ow and therefore genetic diversity,
between populations of plants (rare or common species alike). This appears to be true
even when the habitat requirements are restrictive and those regions are in widely
distributed sites.
Overall, results of this study suggest that because of the presence of high genetic
variability in small fragmented populations, high seed viability and the ability of seeds
to be wind borne, P. gibbosa has not been at risk under the present circumstances.
However action should be taken to ensure its long-term genetic viability by introducing and implementing appropriate conservation and management strategies. Of
particular signi"cance is the in situ conservation of the most diverse populations
namely YA1, YAW, NO4, ML1, ML2, ML3, ML4. Since the species appears to be
662
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
exinct from the original point of collection (Fig. 1), the protection of these remaining
sites is therefore paramount for the long-term survival of this species in the wild. To
create a complete framework for devising a conservation strategy exsitu conservation
should also be employed. This would include preservation of habitat and re-introduction of seedlings to colonise similar habitats. It is preferable to employ seed propagation from several individual as this avoids damage to the original plant and allows you
to produce large number of genetically di!erent seedlings within a relatively short
period of time. Furthermore, to ensure the long-term viability of these populations,
protection from invasive weeds, animals (feral goats, rabbits), and illegal collectors is
as crucial as the strategies for genetically depauperate population facing extinction.
Acknowledgements
We wish to thank Marion Garratt, Maggie Nightingale, Karina FitzGerald and Liz
Gregory for technical support and Geo!rey Robertson of NPWS (NSW) for his keen
interest and enthusiastic collection of samples.
References
Bradburn, G., 1984. Report on the proposed Tallawarra power project and its impact on Pterostylis gibbosa.
Report to the Electricity Commission of NSW by the Australian Native Orchid Society.
Bradburn, G., Tunstall, R., 1993. Census of Pterostylis gibbosa Illawarra Greenhood Orchid, Paci"c Power:
Yallah site. Report prepared for Paci"c Power.
Case, M.A., 1993. High levels of allozyme variation within Cyperipedium calceolus (Orchidaceae) and low
levels of divergence among its varieties. Syst. Bot. 18, 663}677.
Clegg, M.T., Brown, A.H.D., 1983. The founding of plant populations. In: Schonewald-Cox, C.M.,
Chambers, S.M., Macbryde, B., Thomas, L. (Eds.), Genetics and Conservation. Benjamin-Cummings,
Memlo Park, CA, pp. 216}228.
Cosner, M.E., Crawford, D.L., 1994. Comparison of isozymic diversity in three rare species of Coreopsis
(Asterceae). Syst. Bot. 19, 350}358.
Dressler, R.L., 1981. The Orchids, natural history and classi"cation. Harvard University Press, Cambridge,
MA.
Falk, D.A., Holsinger, K.E., 1991. Genetics and Conservation of Rare Plants. Oxford University Press.
Oxford, p. 283.
Frankel, O.H., Soule, M.E., 1981. Conservation and Evolution. Cambridge University Press, Cambridge.
Godt, M.L., Hamrick, J.L., Bratton, S., 1995. Genetic diversity in a threatened wetland species, Helonias
bullata (Liliaceae). Cons. Biol. 9, 277}280.
Gottlieb, L.D., 1981. Electrophoretic evidence and plant populations. Prog. in Phytochem. 7, 1}46.
Hamilton, W.D., 1982. Pathogen as causes of genetic diversity in their host populations. In: Anderson,
R.M., May, R.M. (Eds.), Population Biology of Infectious Diseases. Springer-Verlag, New York,
pp. 269}296.
Hamrick, J.L., 1987. Gene #ow and distribution of genetic variation in plant populations. In: Di!erentiation
Pattern in Higher Plants. Academic Press, New York.
Hamrick, J.L., Godt, M.L., 1989. Allozyme diversity in plant species. In: Brown, A.H.H., Kehler, A.I., Weir,
B.S. (Eds.), Plant Population Genetics, Breeding and Genetic Resources. Sinauer Associates, Sunderland, MA, pp. 43}63.
I.K. Sharma et al. / Biochemical Systematics and Ecology 28 (2000) 651}663
663
Hamrick, J.L., Godt, M.J., Murawski, D.A., Loveless, M.D., 1991. Correlation between species traits and
allozyme diversity: implications for conservation biology. In: Falk, D.A., Holsinger, K.E. (Eds.),
Genetics and Conservation of Rare Plants, Oxford University Press, New York, pp. 75}86.
House, A.P.N., Bell, J.C., 1994. Isozyme variation and mating system. In: Eucalyptus urophylla Blake, S.T.
(Ed.), Silvae Genetica Vol. 43, pp. 167}176.
Jones, D.L., Clements, M.A., 1997. Characterisation of Pterostylis gibbosa R. Br. (Orchidaceae) and
description of P. saxicola, a rare new species from New South Wales. The Orchadian 12, 128}135.
Karron, J.D., 1987. The pollination ecology of co-occurring geographically restricted and widespread
species of Astragalus (Fabaceae). Amer. J. Bot. 76, 331}340.
Loveless, M.D., Hamrick, J.L., 1984. Ecological determinants of genetic structure in plant populations.
Ann. Rev. Ecol. 15, 65}95.
Muston, R., 1991. Plan of management for the rare and endangered Illawarra Greenghood Orchid
(Pterostylis gibbosa) on property owned by Electricity Commission of NSW at Yallah. Report prepared
by Roslyn Muston and Associates, Fairy Meadow.
Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of
individuals. Genetics 89, 583}590.
Nei, M., Chesser, R.K., 1983. Estimation of "xation indices and gene diversities. Ann. Hum. Gen. 47,
253}259.
Pritchard, H.W., 1985. Determination of Orchid seed viability using #uorescein diacetate. Plant Cell
Environ. 8, 727}730.
Ranker, T.A., 1994. Evolution of high genetic variability in the rare Hawaiian fern Adenophorus periens and
implications for conservation management. Biol. Conser. 70, 19}24.
Richter, T.S., Soltis, P.S., Soltis, D.E., 1994. Genetic variation within and among populations of narrow
endemic Delphinium viridescens (Ranunculaceae) Amer. J. Bot. 81, 1070}1076.
SAS Institute Inc.1988. SAS/STAT user's guide, Release 6.03 edition, Cary, NC: SAS Institute Inc. 1028pp.
Scacchi, R., De Angelis, G., 1989. Isozyme polymorphism in Gymnadenia and its inferences for systematics
within this species. Biochem. Syst. Ecol. 17, 25}33.
Scacchi, R., Lanzara, P., De Angelis, G., 1987. Study of electrophoretic variability in Epipactis helleborine
(L.) Crantz, E. palustris (L.) Crantz and E. microphylla (Ehrh.) Swartz (fam. Orchidaceae). Genetica 72,
217}224.
Scacchi, R., De Angelis, G., Lanzara, P., 1990. Allozyme variation among and within eleven Orchis species
(fam. Orchidaceae) with special reference to hybridising aptitude. Genetica 81, 143}150.
Sharma, I.K., Clements, M.A., 1995. Evidence of low genetic diversity in Dendrobium malbrownii Dockrill
(Orchidaceae). The Orchadian 11, 459}464.
Sharma, I.K., Jones, D.L., 1996. An electrophoretic study of variability in Diuris sulphurea R. Br.
(Orchidaceae) in the Canberra region. J. Orchid Soc. India. 10, 9}24.
Soltis, P.S., Soltis, D.E., 1991. Genetic variation in endemic and wide spread plant species: examples from
Saxifragaceae and Polystichum (Dryopteridaceae). Aliso 13, 131}134.
Sonnante, G., Piergiovanni, A.R., Ng, Q.N., Perrino, P., 1996. Relationships of Vigna unguiculata (L.) Walp.,
V. vexillata (L.) A. Rich. and species of section Vigna based on isozyme variation. Genet. Resour. Crop
Evol. 43, 157}165.
Swo!ord, D.L., Selander, R.B., 1981. BIOSYS-1, A fortran program for the comprehensive analysis of
electrophoretic data in the population genetics and systematics. J. Heredity 72, 281}283.
Waller, D.M., O'Malley, D.M., Gawler, S.C., 1987. Genetic variation in the extreme endemic Pedicularis
furbishiae (Scrophulariaceae). Cons. Biol. 1, 335}340.
Weeden, N.F., Wendel, J.F., 1989. Visualisation and interpretations of plant isozymes. In: Soltis, D.S., Soltis,
P.S. (Eds.), Isozymes in Plant Biology. Dioscorides Press, Portland, Oregon, pp. 46}72.
Wright, S., 1951. The genetic structure of populations. Ann. Eugen. 15, 323}354.