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Reproductive Biology |
Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, 1735 Neil Avenue, Columbus, Ohio 43210 USA
Received for publication May 23, 2002. Accepted for publication August 27, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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Key Words: habitat fragmentation • inbreeding • inbreeding depression • orchid • Orchidaceae • Platanthera leucophaea
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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). Many previously widespread species are now represented by few, isolated populations. The survival and evolution of fragmented populations depends on their evolutionary histories and the magnitude of future impacts. Thus, it is important to understand how habitat fragmentation impacts demographic and genetic processes in threatened species in order to maximize viability and growth within populations.
In naturally outcrossing species, a significant threat to population extinction is inbreeding, which can reduce individual fitness and produce genetically homogenous populations (Mills and Smouse, 1994
; Frankham, 1995
; Amos and Balmford, 2001
). For naturally outcrossing species, the magnitude of inbreeding depression is a likely predictor of persistence in populations that are small or have little access to interpopulation gene flow (Barrett and Kohn, 1991
; Lesica, 1993
; Newman and Pilson, 1997
; Amos and Balmford, 2001
). Inbreeding depression can result not only from self-fertilization but from mating between genetically similar individuals (i.e., biparental inbreeding). Thus, in flowering plants, any self-compatible species is potentially at risk of inbreeding depression.
The negative consequences associated with inbreeding result from increased homozygosity within inbred individuals, which comes about by the action of overdominance (Wright, 1977
) or partial dominance (Charlesworth and Charlesworth, 1987
). According to the overdominance hypothesis, heterozygous individuals are always more fit, and thus, it is reduced heterozygosity that results in loss of fitness. Alternatively, according to the partial dominance hypothesis, increased expression of and selection against recessive deleterious alleles during inbreeding cause a loss of fitness. Regardless of the genetic mechanism at work, inbreeding can increase the risk of extinction if increased homozygosity reduces reproductive output in naturally outcrossed populations.
The magnitude of inbreeding depression experienced by a species should be strongly influenced by the demographic and mating history of individual populations. Classical models of inbreeding depression predicted that a population with a history of bottlenecks and isolation would not be severely affected by repeated inbreeding because deleterious alleles in the homozygous condition are purged through repeated exposure to natural selection (Lande and Schemske, 1985
; Charlesworth and Charlesworth, 1987
; Barrett and Charlesworth, 1991
). In contrast, a population that once was large or experienced gene flow may be more likely to suffer greater losses initially should inbreeding become common. More recent theoretical work suggests, however, that purging may not be an effective or consistent force in reducing inbreeding depression in populations (reviewed in Byers and Waller, 1999
). The extent to which purging can reduce inbreeding depression will depend on a complex interaction of factors, including genetic effects such as dominance and epistasis, breeding system, population size, and the strength of natural selection acting on exposed alleles (Byers and Waller, 1999
). Once outcrossing is replaced by inbreeding as the predominant means of reproduction, populations may be on a path to extinction if individuals are unable to withstand the consequences of inbreeding depression.
In this study, the potential for inbreeding depression is examined in Platanthera leucophaea (Nuttall) Lindley (Orchidaceae), the eastern prairie white fringed orchid. This species was once frequently found in prairies and wetlands east of the Mississippi River, but loss of habitat, invasion of exotic species (e.g., Lythrum salicaria L. and Phalaris arundinacea L.), and over-collecting have caused significant population declines (USFWS, 2001
). Presently, P. leucophaea is a federally listed threatened species known from 59 populations in the United States (Illinois, Iowa, Maine, Michigan, Ohio, Virginia, and Wisconsin) and 12 populations in Ontario, Canada (USFWS, 2001
). Only six of the populations in the United States are considered highly viable. Because the majority of populations are critically small (i.e., fewer than 20 flowering plants) and fragmented, successful reproduction may be severely compromised in many of them. For example, small effective population sizes and isolation suggest that inbreeding may be common in many populations of P. leucophaea. Furthermore, estimates of the inbreeding coefficient, FIS, based on allozyme variation, indicate variable levels of inbreeding within populations (Cowden, 1993
; Wallace, 2002
). For example, Wallace (2002)
found significant heterozygote deficiency in five of seven populations surveyed (FIS = 0.74–1) but an excess of heterozygous individuals in two other populations (FIS = –0.043, –0.078). High FIS estimates were observed across four of the seven polymorphic loci analyzed, supporting the idea that inbreeding, not a Wahlund effect, is a more likely cause for the excessive homozygosity observed in populations. Although high FIS values can also result from genetic drift in small populations, a correlation between population size and diversity, which might be expected if drift causes fixation of alleles, was not found in P. leucophaea (Wallace, 2002
). Additionally, several other rare orchid species also show variable levels of inbreeding that are independent of population size (e.g., Wong and Sun, 1999
; Alexandersson and Agren, 2000
). Thus, the indirect interpretation of high levels of inbreeding based on FIS values seems appropriate and suggests that nonrandom mating has played a significant role in shaping population genetic structure in P. leucophaea. Given this variability in population genetic structure, the severity of inbreeding depression might also vary across populations. The goals of this study are to determine if inbreeding depression exists in P. leucophaea and to evaluate the extent to which the magnitude of inbreeding depression can vary with population size, genetic structure, and time.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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; Bowles, 1983
; Cuthrell, 1994
). Flowering plants produce 10–30 creamy white flowers that open sequentially from the bottom to the top over a period of 1–2 wk. The flowers produce an abundance of nectar in a long spur at the back of the flower as well as a strong, sweet fragrance at dusk. Upon visiting a flower, a hawk moth projects its proboscis down into the spur to drink the nectar. As the proboscis is withdrawn from the spur, it brushes against an anther sac and picks up a pollinarium (composed of a pollinium, stipe, and viscidium). The pollinium subsequently rotates about the viscidium to a point where it can contact the stigma of another flower. Darwin (1877)
believed this bending mechanism promoted outcrossing. A single pollinium is probably sufficient to fertilize the thousands of ovules in an ovary, but whole pollinaria are rarely deposited on single flowers (Neiland and Wilcock, 1995
). Rather, as pollinia are rubbed against the stigma, massulae (groups of pollen grains) are removed and stick to the stigma. Consequently, many flowers probably receive pollen from multiple donors because hawk moths regularly visit multiple flowers on an inflorescence and are frequently found carrying multiple pollinaria (Cuthrell, 1994
).
Several days after pollination, the external flower withers and the seeds develop within the ovary. The dust-like seeds are dispersed in the autumn by the wind when the capsule dehisces. The seeds contain only an embryo and a testa (seed coat). Seed germination is greatly aided by the presence of a mycorrhizal symbiont, and seedling survival is entirely dependent on the association (Stoutamire, 1996
; Zettler et al., 2001
; Bowles et al., 2002
). Germinated seeds remain parasitic on the mycorrhizae for some time before producing leaves. Additionally, roots of plants must be infected each spring before shoots reemerge. Despite the orchid's reliance on them, mycorrhizae are probably not species-specific or limiting because multiple species have been isolated from P. leucophaea (Curtis, 1939
; Zettler et al., 2001
). There is some evidence that seed germination is inhibited by light, but may be induced by high water levels (Stoutamire, 1996
; USFWS, 2001
). Under natural field conditions, seeds become infected during late spring or early summer, coinciding with the time when aboveground shoots reemerge (USFWS, 2001
; L. Wallace, The Ohio State University, unpublished data). The species probably does not maintain a large seed bank, relying instead on production of an overabundance of seeds from each flower each year (Stoutamire, 1996
).
Experimental design and statistical analysis
The potential for inbreeding depression was examined in two populations that differ in size, genetic structure, and community structure. The first population (hereafter referred to as the large population), near Lake Erie in northern Ohio (Sandusky County), is currently one of the largest known populations of this species. In 1996, more than 5600 flowering plants were observed in this population, but yearly censuses have shown many fewer flowering plants recently (J. Windus, Ohio Department of Fish and Wildlife, personal communication). The closest known population is approximately 2 km away. The large population occurs in an early successional lake plain prairie and is affected by fluctuating lake levels and heavy invasion of exotic species, including Lythrum salicaria and Phalaris arundinacea (USFWS, 2001
). Despite its large size, suitable habitat for this population is not abundant (USFWS, 2001
.) This large population contains very little allozyme variability (percentage of polymorphic loci = 8.33; HO = 0), and estimates of inbreeding are high (FIS = 1.0; Wallace, 2002
).
The second population (hereafter referred to as the small population) used in this study occurs in a late successional wet sedge meadow in north-central Ohio (Holmes County). It is typical of the size of many other extant populations in Ohio and Michigan where suitable habitat is limited (USFWS, 2001
). This population has had fewer than 50 flowering plants for the last 15 yr (J. Windus, Ohio Department of Fish and Wildlife, personal communication), but is within 10 km of two other P. leucophaea populations. While it also has low levels of polymorphic loci (8.33%), observed heterozygosity is slightly higher (HO = 0.021), and estimates of inbreeding are quite low (FIS = –0.078; Wallace, 2002
).
Experiments were carried out over 3 yr in the large population (1998–2000) and 2 yr in the small population (1999–2000) to evaluate the effect that different environmental conditions or genotype may have on the severity of inbreeding depression. In each population, plants were selected on the basis of having several unopened buds and/or fresh unpollinated flowers. Flowers were determined to be unpollinated by the presence of two pollinaria and the absence of visible massulae on the stigma. Every attempt was made to choose plants that were at the same stage of flowering. Unpollinated flowers and unopened buds were covered with net bags until they could be pollinated by hand. Of the plants that were suitable candidates, a sample was chosen for experimental manipulation (all flowering plants were used in the small population). Sample sizes were 34 plants in 1998, 50 plants in 1999, and 51 plants in 2000 in the large population. In the small population, sample sizes were 19 plants in 1999 and nine plants in 2000.
To control for genetic and maternal effects, each plant was treated as a block. On each plant, three flowers were chosen for one of the three pollination treatments: hand self-pollination, hand outcross pollination, or open pollination. Hand pollinations involved removing a single pollinarium from a flower with a metal dissecting needle and rubbing it on the stigma of a recipient flower. Before pollination, self-pollinaria were removed from flowers. Flowers that were selfed received pollen from their own anther (i.e., true selfing), while flowers that were outcrossed received pollen from an individual at least 2 m away. This species is not known to be clonal, but individual plants often grow in close proximity to one another. Thus, by choosing plants at this distance, the chances of effecting biparental inbreeding were reduced. Hand-pollinated flowers were covered by net bags until capsules matured. Additionally, in the large population in 1998, 21 flowers on a separate set of 21 plants were chosen as a test for autogamous pollination. The buds of these flowers were placed in net pollination bags and left bagged for the duration of flowering. Bagged flowers were periodically checked during the time between treatment and collection of capsules. In mid-September, mature, unopened capsules were collected and taken back to the laboratory where they were stored in desiccant at 4°C until further examination.
Reproductive success, and thus the potential for inbreeding depression, was estimated at the earliest possible stage in this species: at the level of seed set and seed viability. The percentage of capsules setting seed was determined for each treatment. Fruits were assumed to have set seed if viable seeds were found in a capsule. Each capsule and its seeds were weighed, and seed mass relative to total fruit mass was calculated. All of the seeds from a capsule were mixed and a subsample was taken for assessment of seed viability. Seeds were soaked at room temperature in a 5% chlorine bleach solution for 1.5 h, after which they were rinsed in distilled water, put into petri dishes with a 1% solution of triphenyltetrazolium chloride (TTC), and left in the dark at room temperature for at least 24 h. After treatment, seeds were rinsed onto filter paper and dried at room temperature until they could be easily removed from the paper onto a microscope slide. A minimum of 100 seeds (mean = 307) was examined per capsule at 100x under a light microscope to assess viability. A seed was considered viable if it contained a large, plump reddish-brown embryo, while a seed was judged inviable if it did not contain an embryo or contained a shriveled, uncolored embryo. (The TTC staining schedule worked inconsistently for seeds collected during 2000. Therefore, viability of seeds collected in 2000 was based primarily on the size and quality of the embryo.) These categories were easy to distinguish for the majority of seeds, but for the few ambiguous cases, the seeds were not included in the final count. The percentage of viable embryos in the subsample was subsequently determined.
Differences in the frequency of seed set among the three treatment groups were tested using a G test of independence (Sokal and Rohlf, 1995
) for each year of the experiment in each population. Differences in relative seed mass and percent seed viability among treatments were analyzed using a randomized complete block design and analysis of variance (ANOVA) in the large population for each year. Where necessary, data were arcsine transformed to meet the assumptions of ANOVA. Treatment was considered a fixed effect, and each plant was considered a block and treated as a random effect. Analyses were performed using the univariate analysis of general linear models in SPSS (version 10, 1999
) with the type IV sum of squares, as recommended for missing data within blocks. No block is missing data for more than one treatment. In the large population in 1998, seven data points are missing (two each in the self- and outcross-pollinated treatments and three in the open-pollinated treatment); in 2000, three data points are missing for the outcross-pollinated treatment. When significant differences were found, Bonferroni tests were used to discern where the differences existed (Sokal and Rohlf, 1995
). Due to small sample sizes in the small population, data for each year were analyzed with a nonparametric Kruskal-Wallis test (Zar, 1996
) performed in SPSS. Two data points are missing from the self- and outcross-pollinated treatments for the small population in 1998. There are no missing data for the 2000 data set.
Pearson's correlation (Sokal and Rohlf, 1995
) between total seed mass and seed viability was also calculated for each site and each year using SPSS. Lastly, the magnitude of inbreeding depression (
) was calculated for each site and each year according to the following formula: = (poutcross – pself)/poutcross, where pi represents the variable measured for each mode of pollination. According to this equation, estimates of inbreeding depression range from –1 to 1. Negative values result when selfed progeny are more fit than outcrossed progeny. A value of 0.5 is generally interpreted as evidence of a mixed mating system with intermediate levels of inbreeding depression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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Of the 21 buds that were bagged in 1998 in the large population, 15 were recovered at the end of the growing season, but none produced viable seeds. This result confirms previous observations that P. leucophaea requires a vector for pollination. However, it does not preclude the possibility of facilitated selfing in this species.
In the large population, the mode of pollination had a significant effect on seed mass and seed viability for both years (Table 1). However, the trends differed among years. In 1998, open-pollinated capsules were similar to hand-outcrossed capsules, but both had significantly higher seed mass and seed viability than hand-selfed capsules. In fact, there is more than a twofold difference in percent seed viability between open-pollinated (77%) or outcrossed (62%) capsules compared to selfed capsules (16%). In contrast to these findings, during the 2000 season, hand-pollinated capsules were relatively similar to one another in relative seed mass, but all three treatments differed significantly in the percentage of viable seeds per capsule. Seed viability was lower in both open-pollinated (59%) and hand-outcrossed (39%) capsules in 2000 compared to 1998, but it was slightly higher in the self-pollinated (23%) capsules in 2000. Nevertheless, seeds resulting from self-pollination still exhibit lower overall levels of viability than seeds resulting from outcross or open pollination.
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, measured for seed set or relative seed mass, are less than 0.30 in both populations in any year (Table 3). In contrast, estimates of , based on seed viability, range from 0.74 (1998) to 0.41 (2000) in the large population and from 0.49 (1999) to 0.17 (2000) in the small population.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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) and terrestrial orchid species (e.g., Ophrys insectifera [Darwin, 1877
]; Cypripedium calceolus [Nilsson, 1979
; Kull, 1998
]; Dactylorhiza lapponica [Neiland and Wilcock, 1995
], are usually attributed to a lack of pollinators (e.g., Snow and Whigham, 1989
; Zimmerman and Aide, 1989
; Ackerman and Montalvo, 1990
). Although the high levels of seed set (>50% Fig. 1) observed in P. leucophaea may seem anomalous, pollinators were frequently seen visiting flowers in the large population. High levels of seed set have previously been noted in this population as well (J. Windus, Ohio Department of Fish and Wildlife, personal communication). Successful seed production may have been aided by experimenter presence that reduced herbivory by insects and otherwise protected plants. Additionally, size and proximity to other populations may have acted to increase pollinator visitation rates in the populations included in this study. Nevertheless, because natural fruit set has been low (<50%) in other populations of P. leucophaea (Cuthrell, 1994
; J. Windus, Ohio Department of Fish and Wildlife, personal communication), isolation and small size may still be a strong deterrent to reproduction in this species (Washitani, 1996
; Steffan-Dewenter and Tscharntke, 1999
).
Frequent pollinator visitation in the large population also resulted in high levels of seed viability. However, open pollination was not expected to lead to increased levels of seed viability relative to outcross pollination performed by the experimenter. This difference may reflect disparity in how the flowers were pollinated, especially in the method of application and the amount of pollen applied to stigmas. Hand pollinations involved rubbing a single pollinium on the stigma of a recipient flower. The amount of pollen likely was not a limiting factor in the hand-pollinated treatment because a single pollinium contains more pollen than is needed to fertilize all of the ovules in an ovary (Neiland and Wilcock, 1995
; Light and MacConaill, 1998
). Many orchids have high pollen : ovule ratios, and the ratio in Platanthera chlorantha (Custer) Rchb., a morphologically similar species to P. leucophaea, is 24 : 1 (Neiland and Wilcock, 1995
). Perhaps, though, the large pollen load of an entire pollinium had a negative effect on seed production in hand-pollinated flowers. In most species with sectile pollinia, including P. leucophaea, massulae, rather than whole pollinia, are deposited on stigmas (Neiland and Wilcock, 1995
). Thus, the placement of a pollinium on the stigma may have led to pollen tube overcrowding in the style, thereby inhibiting access to all ovules in the ovary (Cruden, 1976
; Neiland and Wilcock, 1995
). In contrast, the placement of multiple massulae over several days by hawkmoths is probably less likely to lead to pollen overcrowding and may promote higher seed viability and multiple siring of seeds within a single ovary.
Magnitude of inbreeding depression
Orchids are widely known for their floral specialization and use of animal pollinators. Only 5–20% of species are capable of autogamous pollination (Catling, 1990
), but most species are self-compatible with pre-pollination barriers to self-fertilization (van der Pijl and Dodson, 1966
). As in many other outcrossing angiosperm species, though, inbreeding depression may also promote outcrossing in orchids by acting as a post-pollination barrier to self-fertilization. Pollination syndromes in orchids have been widely studied, but there are very few reports on the cost of inbreeding in orchids. When inbreeding depression has been studied, it is typically estimated from reproductive output (e.g., fruit set and seed viability) of the maternal plant, rather than survivability or reproductive success of the offspring. The results indicate a variety of responses to inbreeding in orchids. Species that are capable of self-fertilization via autogamy or facilitated selfing show little evidence of inbreeding depression (e.g., Peakall and James, 1989
; Ortiz-Barney and Ackerman, 1999
; Alexandersson and Agren, 2000
), while outcrossing species exhibit varying degrees of inbreeding depression (e.g., Nilsson, 1983
; Johnson, 1994
; Peakall and Beattie, 1996
; Vallius, 2000
; Borba, Semir, and Shepherd, 2001
; Luyt and Johnson, 2001
; Meléndez-Ackerman and Ackerman, 2001
). Results from this study, as well as previous work by Bowles et al. (2002)
, suggest P. leucophaea also experiences moderately high levels of early acting inbreeding depression.
Inbreeding depression was not severe at the level of seed set or seed mass (Table 3), a pattern that was consistent in both populations. These variables are probably less accurate measures of reproductive success than seed viability, though. Capsules were considered to have set seed if any viable seeds were present. Even though seed mass and percentage of viable seeds are significantly correlated, seed mass, like seed set, does not take into account those seeds that lack embryos. The difference in mass between a seed with an embryo and one without an embryo is miniscule and certainly undetectable with most balances. Thus, even in a bulk measure of seeds with and without embryos, it is probably difficult to distinguish an overall difference. The quality of seeds (e.g., embryo presence or germination) is expected to more accurately reflect differences in reproductive success, and P. leucophaea does show inbreeding depression for seed viability in both populations (Table 3). In general, estimates of for P. leucophaea are more similar to mean estimates of inbreeding depression for outcrossing angiosperm species ( = 0.49) than for selfing species ( = 0.22; Husband and Schemske, 1996
). Furthermore, the estimates of inbreeding depression in P. leucophaea are concordant with the floral biology, suggesting that outcrossing is the predominant means of reproduction. Nevertheless, the frequent presence of viable seeds in self-pollinated capsules (in some instances, the number of viable seeds was quite high) suggests that self-fertilization may be tolerated in some populations.
Variability in the expression of inbreeding depression
The strength of inbreeding depression is likely to vary across populations, owing to their unique evolutionary and demographic histories. While isolated populations of P. leucophaea may experience increased levels of inbreeding through geitonogamy or biparental inbreeding, the results of this study do not suggest that early acting inbreeding depression will be more severe in smaller populations relative to larger populations. On the contrary, the population, in which inbreeding is thought to be frequent, experienced higher levels of inbreeding depression as a result of self-fertilization. These data provide little evidence to support the classical view that repeated inbreeding reduces inbreeding depression in this population. Consequently, low levels of genetic heterozygosity combined with inbreeding depression suggest that this population, despite its current size, may be more vulnerable to extinction than other populations with more stable population histories. Although the large population is the largest known in Ohio presently, before 1992, fewer than 50 flowering plants had been observed (J. Windus, Ohio Department of Fish and Wildlife, personal communication). Thus, it is likely that the large population experienced a bottleneck or a recent influx of migrants from other populations. In contrast, the small population may have been historically small, experiencing relatively minor fluctuations in the number of reproductive individuals from year to year. Perhaps it has reached an equilibrium level of inbreeding and is less likely to be affected by inbreeding depression in the future. Results from this study as well as studies of population genetic structure (Wallace, 2002
) indicate that size (i.e., number of flowering individuals) alone is not an adequate predictor of population viability in P. leucophaea.
Temporal differences in the magnitude of inbreeding depression have been reported in other species (e.g., reviewed in Charlesworth and Charlesworth, 1987
; reviewed in Husband and Schemske, 1996
; reviewed in Byers and Waller, 1999
) and may be attributed to purging of deleterious alleles through repeated inbreeding (i.e., when decreases with increased inbreeding), the effect of differing environmental conditions on survival and reproductive success, or genetic variation among the individuals chosen for the experiment. Although purging of deleterious alleles may occur in populations of P. leucophaea with repeated inbreeding, it is impossible for such an effect to be seen during the 3-yr duration of this study because individual plants can grow for more than 3 yr before their first flowering (Bowles, 1983
). Additionally, seeds used to estimate inbreeding depression were not placed back into the population and therefore are not contributing to future generations. Thus, it would seem that some effect of the habitat and/or genetic make-up of the individuals used in the study accounts for the different levels of inbreeding depression seen across years.
An increase in inbreeding depression as a result of stress, which has been found in other species (Dudash, 1990
; Eckert and Barrett, 1994
; but see Johnston, 1992
; Groom and Preuninger, 2000
), was not readily apparent in this study. Although the effect of environmental stress was not directly tested in this study, rainfall levels varied across the three years of study in the large population. In 1999 and 2000, this population experienced extremely dry conditions, which resulted in death of aboveground shoots before fruit set in 1999. The majority of open-pollinated and outcross-pollinated flowers produced seed in 2000, but the percentage of viable seeds for these treatments was lower in 2000 than in 1998. In contrast, the percentage of viable seeds increased among the self-pollinated flowers in the large population, but the difference was only 7% (Table 1). While this finding suggests that inbreeding depression was not as severe in 2000 compared to 1998, it is noteworthy that self-pollinated flowers still produced significantly fewer viable seeds than outcross or open-pollinated flowers in both years of experimentation. In contrast, environmental conditions in the small population appeared more consistent over the two years of this study, but many fewer plants were seen flowering in 2000. Thus, reasons for the differences in inbreeding depression seen across years and populations are not easily explained and reflect the complex interaction of multiple environmental and biological factors on the expression of inbreeding depression.
Is inbreeding depression a significant threat to populations of Platanthera leucophaea?
Allozyme data strongly suggest inbreeding in many populations of P. leucophaea, irrespective of population size (Wallace, 2002
). Additionally, the results of this study indicate a negative consequence of inbreeding at the level of seed development. The relative importance of inbreeding via geitonogamy or matings between genetically similar individuals is not so clear, however. Studies on other orchids suggest biparental inbreeding is more likely than geitonogamy to lead to inbreeding depression. For example, Johnson and Nilsson (1999)
predicted that geitonogamy will occur in P. chlorantha only after a pollinator visits nine flowers. Geitonogamy was infrequent due to the short time a pollinator spent on each inflorescence and the length of time it takes for a pollinarium to bend into the correct position to contact a stigma (80 s). Furthermore, they suggested that deposition of massulae, rather than entire pollinia, promoted high levels of pollen carryover. Because of the strong resemblance to P. chlorantha in flower structure and pollination syndrome (i.e., both are nocturnally pollinated by moths), geitonogamy probably occurs infrequently in P. leucophaea as well. Pollen carryover could result in biparental inbreeding, though, if individuals within populations are genetically similar.
Even though inbreeding depression was observed at the level of seed production and seed development, the great abundance of seeds that can be produced in a single orchid ovary may compensate for reductions in seed viability in inbred flowers. Measures of inbreeding depression based on seed set or embryo viability, unfortunately, then, may say little about the long-term survival of inbred and outcrossed offspring. A few studies have demonstrated inbreeding depression at the level of seed germination. Bowles et al. (2002)
reported a significant and positive correlation between percentage of viable seeds per capsule and seed germination for P. leucophaea and suggested that inbreeding depression may have a cascading effect starting with fruit set and following through to later life stages. Unfortunately, the difficulty in germinating orchid seeds and growing seedlings severely hinders examination of inbreeding depression at later life stages in orchids, and I am unaware of any orchid studies in which offspring fitness has been considered beyond the level of seed germination.
Conservation implications
Management plans for rare, endangered, or threatened species designed with information from multiple sources, including population genetic structure, ecological requirements, and reproductive biology, offer the best hope of protecting these species. While much more is known about the life history of P. leucophaea than many other rare species, there are still many aspects of its life cycle that are not clear. The persistence of this species is dependent upon successful recruitment within populations. Given the small sizes and fragmented distribution of populations of P. leucophaea, seedling recruitment is likely to be severely limited by multiple factors, including pollinator service and suitable microsites for seed germination (Calvo, 1993
). The results of this study clearly suggest that early acting inbreeding depression exists in P. leucophaea and may be an additional threat to long-term survival of populations. Long-term studies that can identify critical life stages in seedling recruitment, which will also be the most difficult to study because they are the stages spent underground, will be invaluable for future conservation of P. leucophaea. This information will be particularly important if the creation of new populations or supplementation of existing populations become primary objectives in the conservation of this species. At this time, a cautious management plan would be one that strives to stimulate growth and stability within extant populations by promoting outcrossing within populations, increasing population sizes, and maximizing genetic variability.
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FOOTNOTES |
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2 Address for reprint requests (wallace.205{at}osu.edu
)
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSION LITERATURE CITED |
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20.05 [2] = 5.91)