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Department of Botany and Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong
Received for publication November 2, 1998. Accepted for publication February 12, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: allozymes • conservation genetics • Goodyera procera • Orchidaceae • orchids • pollination biology • RAPD
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Sanford, 1974
; Alphonso, 1975
; Hunt, 1984
; Heywood, 1985
; Dressler, 1993
), contributing up to 10% of all flowering plant species in the world (Dressler, 1981
). However, very little is known about the levels and patterns of genetic variation in species of the Orchidaceae. It is often difficult to make generalizations from the limited data on allozyme variability in orchid species (Case, 1994
). Data based on RAPD analysis are even more limited. This lack of data may be associated with the many difficulties in locating and studying orchid populations. Many terrestrial orchids defoliate and remain dormant after a flowering season such that only an inconspicuous bulb below ground or partly above ground can be found. Their sporadic occurrence makes it especially difficult to locate them because individuals are usually found in small colonies or scattered singly over wide areas (Rogaly, 1975
).
In spite of its small size (1070 km2), Hong Kong has heterogeneous topography and diverse habitats, which support very high floral diversity. In the Orchidaceae, more than 120 species representing 64 genera have been recorded (Hu, 1977
; Barretto and Saye, 1980
). Based on herbarium records, many natural populations have been destroyed or restricted to small sizes due to habitat destruction and fragmentation caused by urban development. Several studies have pointed out the significance of habitat destruction and overexploitation in the endangerment of wild orchids from different parts of the world (Alphonso, 1975
; Borromeo, 1975
; Pradhan, 1975
; Rogaly, 1975
). Notwithstanding the threat of local to global extinction, genetic diversity of orchid taxa has scarcely been documented. Comparative population studies using both allozyme and RAPD methods are needed to collect information on the levels and patterns of genetic diversity of wild orchids, which is a first step to facilitate their conservation. Knowledge about genetic diversity is the baseline for conservation (Geburek, 1997
). Such knowledge is essential for formulating comprehensive conservation plans (Hamrick, 1983
; Falk and Holsinger, 1991
; Loescheke, Tomiuk, and Jain, 1994
). Genetic discoveries can often provide novel, conservation-relevant insights (Avise, 1995
). Several aspects of conservation biology, such as loss of genetic diversity in conservation programs and restoration of threatened populations, can only be addressed by detailed population genetic studies (Hamrick and Godt, 1995
).
In plants, breeding systems have a profound effect on the genetic composition of natural populations (Hamrick, 1982
). The floral structure of orchids is generally specialized in a manner that prevents spontaneous self-fertilization and facilitates insect-mediated outcrossing (Dressler, 1981
; Sheehan and Sheehan, 1984
; Arditti, 1992
). However, panmixia is rather rare under natural conditions. Nonrandom mating can result from insect-mediated autogamy, geitonogamous selfing, and biparental inbreeding.
In this study, we investigated the breeding system of Goodyera procera (Ker-Gawl.) Hook by pollination experiments and field observations and inferred outcrossing rates in 15 populations based on fixation indices. The levels and patterns of genetic variation in these populations were documented by allozyme electrophoresis and random amplified polymorphic DNA (RAPD) analysis. Based on this information, optimum sampling strategies for its genetic conservation are recommended.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15 cm tall or up to 40 cm with inflorescence. The species grows in the warmer parts of the world with geographical distribution ranging from China and India to many countries of Southeast Asia (Seidenfaden and Smitinand, 1959
; Isaac-Williams, 1988
; Seidenfaden and Wood, 1992
). Small, white flowers, ranging from several to nearly a hundred on an inflorescence, usually open in spring from February to April in Hong Kong. The leaves wither and rot away at the end of the growing season. The plants inhabit primarily rock surfaces in stream beds at different elevations. At high elevations, some populations are found in shaded hillsides. The species was once abundant in Hong Kong, but its numbers have been greatly reduced in recent years because of over collection of this orchid for commercial gain (Barretto and Saye, 1980
). Fifteen populations of G. procera were sampled in several field sites (site locations are shown in Fig. 1). Fresh samples of young leaves were placed in moist paper towels in a cool ice chest during field collections and stored at 4°C until allozyme electrophoresis or DNA extraction.
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. Six of the 15 population sites were chosen for having larger numbers of flowering individuals at the time of the experiment. Five plants with pre-anthesis inflorescences were bagged at each site with a fine mesh plastic net to exclude pollinators. Pollination treatments were applied to ten flowers on each inflorescence. Same flowers are emasculated by removing pollinia from flowers. Hand pollinations were carried out placing pollinia onto stigmas with a pair of fine forceps. Fresh pollinia from a different population were used to effect xenogamy.
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. The following nine enzyme systems were resolved on 12.5% starch gels with two buffer systems, the histidine-tris-citrate system (Sun and Corke, 1992
) for resolving isocitrate dehydrogenase (IDH; E.C. 1.1.1.42), malate dehydrogenase (MDH; E.C. 1.1.1.37), phosphoglucomutase (PGM; E.C. 5.4.2.2), and phosphogluconate dehydrogenase (6-PGD; E.C. 1.1.1.44), and the "L" system of Shields, Orton, and Stuber (1983)
for resolving esterase (EST; E.C. 3.1.1.1), ß-glucosidase (ß-GLU; E.C. 3.2.1.21), glucose-6-phosphate dehydrogenase (P-6-GDH; E.C. 1.1.1.49), glucose-6-phosphate isomerase (PGI; E.C. 5.3.1.9), and leucine aminopeptidase (LAP; E.C. 3.4.11.1). Enzyme activity staining was carried out with the protocols of Wendel and Weeden (1989)
. Genetic interpretation of band patterns followed standard principles (Weeden and Wendel, 1989
; Wendel and Weeden, 1989
).
DNA isolation and PCR amplification
The protocol of CTAB total DNA isolation (Doyle, 1991
) was used to isolate genomic DNA from fresh leaves. After quantification with a fluorometer (Hoefer), a DNA sample solution (20 ng/µL) was prepared. A 25-mL amplification reaction contained 10 mmol/L Tris, 50 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L of each dNTPs, 0.4 µmol/L primer, 0.5 unit of Taq polymerase (Promega, Madison, Wisconsin) and 20 ng template DNA. PCR amplification was performed in a MJ Research PTC-100 thermal cycler for 45 cycles (1 min at 94°C, 1 min at 38°C, and 2 min at 72°C in each cycle). The amplification products were visualized by electrophoresis on 1.4% agarose gels followed by ethidium bromide staining.
Data analysis
Allozyme frequency data were analyzed by the computer program, POPGENE (Yeh et al., 1997
). Genetic parameters within populations, including the percentage of polymorphic loci (P), expected heterozygosity (He), and fixation index (F), were calculated. To estimate outcrossing rates for these populations, we used the fixation index F, with outcrossing rate t = (1 - F)/(1 + F). Nei's (1973)
total gene diversity (HT), coefficient of gene differentiation (GST), and Nei's (1972)
genetic identity (I) between populations were also computed at the species level. The coefficient of genetic differentiation among populations, GST, was used to estimate the level of gene flow, Nm (the number of migrants exchanged between local populations per generation), based on the relationship GST = 1/(4Nm + 1), where GST is Nei's (1973)
estimator of FST (Wright, 1951)
.
For RAPD analysis, bands were identified by an image analysis software for gel documentation (Molecular Analyst® /PC Version 1.2; Bio-Rad, Cambridge, Massachusetts). Smeared and weak bands were excluded. To estimate polymorphism parameters at both the population and species levels, the band presence/absence data matrix was analyzed within POPGENE (Yeh et al., 1997
) and adjusted for the fixation index (F), which was calculated based on allozyme data. An additional measure for partitioning genetic variation was obtained by the Shannon index (S) because it is relatively insensitive to the inability of RAPD in detecting heterozygous loci (Dawson et al., 1995
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Nei's genetic identities (I) between populations varied from 0.717 to 0.996 with an average of 0.909 ± 0.049. Based on the genetic identity matrix, a UPGMA dendrogram was constructed showing the relationships among these populations (Fig. 2). The total gene diversity (HT) in the species was estimated to be 0.151, of which 52% was distributed between populations (DST = 0.113 and GST = 0.523). The level of gene flow (Nm) was estimated to be 0.221 individual per generation between populations.
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; Gasperi et al., 1995
; Chan and Sun, 1997
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), but under natural conditions fruit set in the Orchidaceae is predominantly pollinator dependent. Artificial self-pollination and geitonogamous pollination, with fruit set of 92 and 94%, respectively, indicated that G. procera is highly self-compatible. The natural fruiting rate of G. procera (75%) is relatively high given the fact that fruit set in outcrossing orchids is generally low and limited by pollinator availability under natural conditions (Mendel, 1995
). Since agamospermy and spontaneous autogamy were negligible in G. procera, successful fruit set must be aided by pollen vectors. Thus pollinators were apparently not a limiting factor in the reproduction of G. procera. The high fruiting rate in Goodyera species has been noted (Dressler, 1993
). A recent review of fruit set in the Orchidaceae by Neiland and Wilcock (1998)
reported (1) a positive association between orchid rarity and lack of nectar reward to pollinators, and (2) that the provision of nectar as a reward to pollinators on average doubled the probability of fruit set. Understanding this type of causal relationship between pollinator visit, fruit set, and rarity is important for orchid conservation.
Tremblay (1992)
discussed the relationship between the specialization of the orchid flower with the number of pollinator species that visit the flower. Fewer pollinator species are expected to visit highly specialized species and vice versa. A wide range of pollinators would be expected to pollinate G. procera for it has an unspecialized floral structure. Bumble bee pollination has been reported for Goodyera species (Arditti, 1992
; Dressler, 1993
). Although we observed no pollinators during our daytime field collections of G. procera, the sparkling and shimmering appearance of the white flowers may attract pollinators at night. The differential fruiting success with inflorescence size may be related to the attractiveness of the spikes. The longer spikes with more flowers would be more attractive than the shorter ones. Murren and Ellison (1996) also found that fruit set is more frequent on multiflowered racemes than on racemes with fewer flowers.
There are practical difficulties in germinating orchid seeds for outcrossing rate estimation based on the progeny genotype arrays of families, as conventionally done in many studies of plant mating systems (e.g., Sun and Ritland, 1998
). Assuming the estimated fixation index in each population is entirely due to nonrandom mating, the level of outcrossing can be estimated based on the fixation index for each population (Sun and Corke, 1992
). Although this method may not be as accurate as the progeny-array approach, variation in the outcrossing estimates among populations of G. procera clearly indicated a strong environmental influence on the breeding system of the species. Differences in plant density, physical and genetical substructuring, and pollinator availability and behavior all result in variation in outcrossing rate among populations in many plant species (see review in Schemske and Lande, 1985
).
However, high inbreeding coefficients can also result from genetic drift in small populations even in the presence of random mating. Thus differences in population size may further contribute to the observed variation in t among populations of G. procera.
Genetic variation
At the allozyme level, relatively low genetic variation exists in G. procera, when compared with the average values for animal-pollinated outcrossing plant species (P = 50%, A = 1.99, H = 0.167; Hamrick and Godt, 1989
). Other outcrossing orchid species, such as Cypripedium calceolus (Case, 1994
), Orchis species (Scacchi, Angelis, and Lanzara, 1990
), and Spiranthes sinensis (Sun, 1996
) all showed high levels of genetic variation. Extensive genetic differentiation among populations of G. procera was detected (GST = 0.523), which is much higher than the reported average in outcrossing plant species (GST = 0.20; Hamrick and Godt, 1989
). Genetic identities between G. procera populations were also lower than the average reported for conspecific plant populations (I = 0.956; Gottlieb, 1981
). This level and pattern of genetic variation in G. procera are more comparable to predominantly selfing plant species than to those predominantly outcrossing species.
In comparison with allozyme diversity, higher levels of genetic variation were detected in G. procera at the RAPD level. However, the pattern of genetic diversity within and among populations was comparable between the two data sets. High estimates of GST based on RAPDs confirmed that natural gene flow between populations of G. procera was apparently limited, as indicated by allozyme data. Nei's genetic identity estimate based on RAPDs (I = 0.859 ± 0.038) also corroborates the allozyme estimate (0.909 ± 0.049), showing that a high level of population genetic divergence has occurred in the species. Our results are consistent with other comparative studies that reported that more variation was detected at RAPD loci than at allozyme loci, but the same pattern of population genetic structure was revealed by the two sets of data (e.g., Liu and Furnier, 1993
).
Due to the dominant nature of RAPD data, Hardy-Weinberg equilibrium is usually assumed for computing the allele frequencies at RAPD loci (e.g., Liu and Furnier, 1993
; Gasperi et al., 1995
; Waycott, 1995
). In the present study, the calculated RAPD allele frequencies were adjusted for fixation index, which was derived from allozyme data for the same populations, eliminating the need for making the assumption of Hardy-Weinberg equilibrium for each population. Comparing the gene diversity estimates adjusted for the departure from Hardy-Weinberg equilibrium with unadjusted estimates, only a slight decrease in gene diversity was observed in most populations after the adjustment. Our study appears to be the first to show that the assumption of Hardy-Weinberg equilibrium in the calculation of RAPD allele frequency does not significantly bias the gene diversity estimate even if significant inbreeding exists in a population.
A significant correlation between population size and genetic diversity has been documented by many researchers (e.g., Raijmann et al., 1994
; Godt, Johnson, and Hamrick, 1996
; Sun, 1996
). Populations of G. procera showed a wide range in size. However, no significant correlation was found between any parameters of genetic diversity (P, A, and H) and population size in the present study (data not shown). It has been reported that the species was once collected for use as aquarium plants (Barretto and Saye, 1980
). Some populations may have suffered a genetic bottleneck and recovered in subsequent generations, so that the existing variability and structure of populations could not be explained based on their present-day sizes.
Another major factor responsible for low genetic variation within most populations of G. procera could be inbreeding. Numerous flowers on a single inflorescence facilitate pollinator-mediated self-pollination and geitonogamous pollination. Like many other orchid species, G. procera can also reproduce vegetatively. After fruiting, the vegetative part of G. procera became senescent, one bud commonly emerging from the rhizome to replace the old plant. However, two or more buds from the same rhizome sometimes develop into mature plants and eventually proliferate into a clone. Biparental inbreeding would occur between the clonal individuals. In addition, population density of the plant could be very high at suitable habitats. The occurrence of intrafamily inbreeding is likely in dense populations (Hamrick, 1982
; Coates and Sokolowski, 1992
), leading to lowered gene diversity (Godt and Hamrick, 1996
). Furthermore, the unequal chance of transmitting genes among individuals may further decrease genetic variation. As the number of flowers and fruit set are highly heterogeneous among individuals within populations of G. procera, high variance in fertility among plants would reduce the effective population size.
Population clustering based on Nei's genetic identities generated from allozyme and RAPD data was quite different, and no correlation was found between genetic distance and geographic distribution among populations (Figs. 2, 3). A likely cause of this pattern is the lack of gene flow between populations, regardless of their geographical distances. Random genetic drift would lead to large gene differentiation in small and isolated populations. For example, differentiation in allele frequency between populations at mdh-1, lap-1, and ß-glu-2 was evident (allele frequency data not shown, but available upon request). Assuming no linkage among the allozyme and RAPD loci, stochastic processes would result in independent fixation of different alleles in different populations, which alone could play a very important role in reducing genetic variation within populations while increasing genetic divergence between populations of G. procera.
Gene flow greater than one individual exchanged per generation can prevent neutral alleles being fixed due to random genetic drift (Raijmann et al., 1994
; Godt and Hamrick, 1996
). The estimate of Nm was very low in the present study, about one migrant in every five generations. Geographical isolation is the most plausible cause of low gene flow in G. procera. The species' distribution is confined mostly to watercourses, although three populations (Tai Mo Shan, Victoria Peak, and Mountain Paker) were also found at high elevations. Because of the hilly topography of Hong Kong, two apparently closely located populations could actually be separated by mountain ridges. It is unlikely for pollinators to fly over the mountain barrier to enable interpopulation pollen transfer. Also, the wet habitat frequently associated with G. procera could further limit gene flow through wind-mediated seed dispersal.
Conservation consideration
A major cause of the decline of this species is the loss or disturbance of habitats and illegal collections. The habitats of G. procera are easily accessible to the public. For example, the Bride's Pool population is located next to a public barbecue site. The Shing Mun and Ng Tung Tsai populations are close to popular scenic spots that are frequently visited by hikers. The cumulative effect of anthropogenic disturbance on habitat degradation should not be neglected.
The ultimate goals of conservation are to ensure the continuous survival of populations and to maintain their evolutionary potential (Hamrick and Godt, 1995
). First, habitat destruction, which leads to the massive elimination of species, should be prevented. Community-based conservation strategy are necessary to protect rare plants (Sipes and Tepedino, 1995
), because pollinators are essential for the reproductive success of the plants. Although natural fruit set showed that pollinators were not limited in G. procera, further disturbance of the community may lead to a decrease in availability of pollinators. If pollinator abundance is reduced, outcrossing will be suppressed and genetic variability will also be reduced (Ohara et al., 1996
).
Information on existing genetic diversity is a prerequisite in designing suitable strategies for genetic conservation (Hamrick, 1983
; Falk and Holsinger, 1991
; Loeschcke, Tomiuk, and Jain, 1994
; Avise, 1995
; Hamrick and Godt, 1995
; Geburek, 1997
). A population with an effective size of 50 has been considered the minimum to retain sufficient allelic richness, while an effective size of 500 individuals is required to counteract the effect of genetic drift and permit evolutionary changes (Frankel, Brown, and Burdon, 1995
). Only a few populations of G. procera have a census size more than 50 individuals, and none reached 500. Immediate supplement of individuals should be undertaken for the populations that experienced severe bottlenecks, especially the Tung Chung and Mui Wo populations where less than ten individuals can be found. Increasing the number of individuals in a population would be effective in preventing further genetic loss after bottleneck and reduce the chance of local extinction by stochastic events.
Priority for genetic conservation was often set based on the level of genetic diversity and allelic uniqueness of populations as well as on the degree of gene differentiation between populations (Coates and Sokolowski, 1992
). The present study showed that most alleles were shared by several populations so that conserving a single large and genetically diverse population may have sampled most of the variation. However, plants from both habitat types (watercourse and hillside) should be sampled to conserve quantitative genetic differences. Although the GST value based on allozyme data suggested a high degree of differentiation between populations, the divergence is almost entirely due to differences in allele frequencies rather than unique alleles. However, RAPD data revealed several unique bands in some populations of G. procera. The genetic composition of small isolated populations may be fixed but significantly different from each other due to random genetic drift. Therefore, individuals should be sampled from as many populations as possible in order to capture most of the genetic diversity in the species for ex situ conservation.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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